The interaction between luteotrophic and luteolytic agents in controlling progesterone production by the marmoset corpus luteum in the late luteal phase/early pregnancy was investigated at the local level in vivo using a perfusion cannula system. Perfusion of the prostaglandin F2 alpha (PGF2 alpha) analogue, cloprostenol (0.5 microgram/ml), resulted in an immediate fall in progesterone production. This response was not sustained in two out of five corpora lutea but pregnancy was terminated in all animals exposed to PGF2 alpha. Perfusion of human chorionic gonadotrophin (hCG) (4 micrograms/ml) alone significantly stimulated progesterone secretion but there was no response to hCG when the corpus luteum had previously been perfused with PGF2 alpha. Perfusion with hCG together with PGF2 alpha prevented a fall in progesterone secretion. The results suggest that the luteolytic action of PGF2 alpha in the marmoset may be to prevent luteotrophic support of the corpus luteum. Melatonin (860 pmol/l), perfused either with PGF2 alpha or after PGF2 alpha, stimulated progesterone production. The ability of melatonin to influence progesterone production by the primate corpus luteum may therefore be by both a direct luteotrophic action and the prevention of luteolysis. Application of the perfusion system in order to investigate the ability of deglycosylated hCG to antagonize the action of hCG at the corpus luteum showed the necessity of testing pure preparations of hormones.

Dispersed marmoset luteal cells were incubated for 2 h and progesterone production measured after exposure to hCG, cloprostenol, dibutyryl cAMP, PGF-2 alpha, PGF-2, adrenaline or melatonin. The cells were studied on Days 6, 14 and 20 after ovulation in conception and non-conception cycles. Luteal cells from Day 14 non-pregnant marmosets were compared with human luteal cells taken in the mid-luteal phase. All the treatments stimulated progesterone production including cloprostenol, which is luteolytic when administered to the marmoset in vivo, but the degree of response varied with the stage of the cycle or pregnancy and between marmoset and human luteal cells. In the marmoset, overall analysis of the effect of the treatments showed that, on Day 6 after ovulation, there was no significant effect of any of the treatments in cells from pregnant or non-pregnant animals. In contrast, luteal cells from non-pregnant animals on Day 14 showed a significant response to the treatments (F (8,41) = 2.79, P less than 0.0145) whereas cells from pregnant Day-14 animals were responsive; in cells from pregnant animals, the control production of progesterone was high and already equivalent to the levels stimulated by the treatments. By Day 20, cells from pregnant animals produced lower control concentrations of progesterone than did those on Day 14 and there was a significant overall effect of the treatments (F (8,33) = 3.78, P less than 0.003). These results show that the marmoset CL gains responsiveness to treatment between Days 6 and 14 after ovulation in the non-pregnant cycle. In pregnancy, on Day 14, 2 days after attachment of the embryo, the high control concentrations of progesterone and absence of response to treatment suggest that an embryo message may have affected the CL, providing an endogenous stimulus.

Our objective was to determine whether rates of luteolysis or pregnancy differed in lactating dairy cows of known progesterone status and either known or unknown luteal status after either cloprostenol or dinoprost was injected as part of a timed-insemination program. In Experiment 1, 2358 lactating dairy cows in six herds were given two injections of PGF(2 alpha) 14 d apart (Presynch), with the second injection given 12 to 14 d before the onset of a timed AI protocol (Ovsynch). Cows (n=1094) were inseminated when detected in estrus after the Presynch PGF(2 alpha) injections. Cows not inseminated (n=1264) were enrolled in the Ovsynch protocol and assigned randomly to be treated with either cloprostenol or dinoprost as part of the timed-AI protocol. In cows having pretreatment concentrations of progesterone>>or= 1 ng/mL and potentially having a functional corpus luteum (CL) responsive to cloprostenol (n=558) or dinoprost (n=519), dinoprost increased (P<0.05) luteal regression from 86.6 to 91.3%. Despite a significant increase in luteolysis, pregnancies per AI did not differ between luteolytic agents (dinoprost=37.8% and cloprostenol=36.7%). Fertility was improved in cows of both treatments having reduced concentrations of progesterone at 72 h and in cows showing signs of estrus. In Experiment 2, an ovulation-resynchronization program was initiated with GnRH or saline in 427 previously inseminated lactating dairy cows of unknown pregnancy status in one herd. Seven days later, pregnancy was diagnosed and nonpregnant cows were blocked by number of CL and assigned randomly to be treated with cloprostenol or dinoprost. Compared with cloprostenol, dinoprost increased (P<0.05) luteal regression from 69.1 to 78.5%, regardless of the number of CL present or the total luteal volume per cow. Pregnancies per AI did not differ between dinoprost (32.8%) and cloprostenol (31.3%). Although dinoprost was more effective than cloprostenol at inducing luteolysis in lactating dairy cows exposed to an Ovsynch or ovulation-resynchronization protocol, resulting fertility did not differ between products.

The initial events in prostaglandin F2 alpha-(PGF2 alpha)-induced luteolysis were studied in pregnant mare serum gonadotropin/human chorionic gonadotropin-(PMSG/hCG)-treated rats with luteinized ovaries. Injection with a potent PGF2 alpha analog (cloprostenol, 5 micrograms/ml) induced functional luteolysis, as assessed by plasma levels of progesterone and 20 alpha-dihydroprogesterone. At 0.5 and 3 h after cloprostenol administration the luteolytic effect was also evident as a reduced response of luteal adenylate cyclase to all stimulatory agents tested, LH, isoproterenol, fluoride, guanylylimidodiphosphate and forskolin. 24 h after cloprostenol the response to all agents, except to LH, had returned to normal. This general and transient block of the luteal adenylate cyclase system indicates that a common factor, possibly the stimulatory guanine nucleotide binding protein (Ns), is involved in the mechanism of action of PGF2 alpha. To test this hypothesis, we measured the functional coupling of the Ns protein to the beta-adrenergic receptor in luteal membranes. Binding competition curves showed a marked shift to the right in membranes prepared from rats injected with cloprostenol 0.5 and 3 h before membrane preparation, while at 24 h after cloprostenol the shift had disappeared. The total number of beta-adrenergic receptors was, however, not affected by the cloprostenol treatment. Computer analysis of the data indicates that, at 0.5 and 3 h after cloprostenol treatment, there was a reduced number of high affinity binding sites, 38 and 41%, respectively, compared to 53% for control membranes. The cellular mechanism for this action of PGF2 alpha on the Ns protein remains to be elucidated.

OBJECTIVE

The receptor-coupled signal transduction systems present in isolated human choroidal melanocytes (HCOMs) were investigated.

METHODS

[(3)H]-inositol phosphates ([(3)H]-IPs) generated in the cells were measured by ion-exchange chromatography. cAMP generated in the cells was quantified using an enzyme immunoassay.

RESULTS

Initially, HCOM cells were challenged with a relatively high concentration (e.g., 1 µM-1 mM) of a variety of pharmacological agents in order to determine which functional receptors were present in these cells. Full concentration-response pharmacological studies were subsequently conducted on endothelin receptors. While a number of prostaglandins (PGs) (e.g., PGD(2), PGE(2), PGF(2α), cloprostenol, latanoprost acid, U-46619), histamine, carbachol, bombesin, and arginine-vasopressin were essentially inactive at stimulating the phosphoinositide (PI) hydrolysis response, endothelin-1 (ET-1) potently and efficaciously generated [(3)H]-IPs. Concentration-response studies yielded the following potency (EC(50)) and efficacy (E(max) relative to ET-1) data: ET-1 EC(50) = 3.4 ± 1.4 nM, E(max) = 100%, n = 3; BQ-3020 (ET(B) receptor-selective agonist) EC(50) = 13 ± 4 nM, E(max) = 73 ± 2%, n = 3). The effects of ET-1 on [(3)H]-IPs production were blocked by the ET(B) receptor-selective antagonist, BQ-788 (IC(50) = 10 ± 5 nM, n = 3), while the ET(A) receptor-selective antagonist (BQ-610) was essentially inactive. In the adenylyl cyclase (AC) assay, while isoproterenol (10 µM), ET-1 (1 µM) and PGE(2) (10 µM) stimulated cAMP production, numerous other PGs (e.g., PGD(2), PGF(2α), PGI(2), latanoprost, latanoprost acid, U-46619 and BW245C [all at>> 10 µM]) were inactive.

CONCLUSIONS

It is concluded that HCOMs express functionally active ET(B) receptors that mediate the production of [(3)H]-IPs. Additionally, HCOMs generate cAMP in response to ET-1, PGE(2), and isoproterenol. These data may have relevance to the melanogenic activity of HCOM cells.

Injection of steroid-free bovine follicular fluid (bFF; 2 X 5 ml s.c. 12 h apart) into anoestrous ewes lowered plasma FSH concentrations by 70% and after 24 h had significantly (P less than 0.01) reduced the number of non-atretic follicles (greater than or equal to 1 mm diam.) without influencing the total number of follicles (greater than 1 mm diam.) compared to untreated controls. Hourly injections of FSH (10 micrograms i.v. NIH-FSH-S12) for 24 h did not influence the number of non-atretic follicles but did negate the inhibitory effects of bFF on follicular viability. Hourly injections of FSH (50 micrograms i.v., NIH-FSH-S12) + bFF treatment for 24 h significantly increased the total number of non-atretic follicles, and particularly the number of medium to large non-atretic follicles (greater than 3 mm diam.) compared to the untreated controls (both P less than 0.01). The 10 micrograms FSH regimen (without bFF) significantly increased aromatase activity in granulosa cells from large (greater than or equal to 5 mm diam.; P less than 0.01) but not medium (3-4.5 mm diam.) or small (1-2.5 mm diam.) follicles compared to controls. The 10 micrograms FSH + bFF regimen had no effect on granulosa-cell aromatase activity compared to the controls. However, the 50 micrograms FSH plus bFF regimen increased the aromatase activity of granulosa cells from large, medium and small non-atretic follicles 2.6-, 8.3- and greater than or equal to 11-fold respectively compared to that in the control cells. Ewes (N = 11) that ovulated 2 follicles had significantly higher plasma FSH concentrations from 48 to 24 h and 24 to 0 h before the onset of a cloprostenol-induced follicular phase (both P less than 0.01) than in the ewes (N = 12) that subsequently ovulated one follicle. Hourly FSH treatment (1.6 micrograms i.v., NIAMDD-FSH-S15) for 24 h but not for any 6 h intervals between 48 and 24 h or 24 and 0 h before a cloprostenol-induced luteolysis also resulted in significant increases (P less than 0.05) in the number of ewes with 2 ovulations.(ABSTRACT TRUNCATED AT 400 WORDS)

Treatment of ewes with steroid-free ovine follicular fluid (oFF) during the follicular phase of the oestrous cycle results in the immediate inhibition of the ovarian secretion of oestradiol, inhibin and androgens. An experiment was conducted to determine whether this effect of oFF was due to inhibin, or to direct inhibition of ovarian function by other factors in oFF. Eight ewes in which the left ovary and vascular pedicle had been autotransplanted to a site in the neck were studied during the breeding season. Luteal regression was induced in all animals by injection of cloprostenol (100 micrograms i.m.; PG) on Day 10 of the luteal phase. The animals were divided into two groups (n = 4) and treated with either steroid-free oFF (oFF; 3 ml s.c.; 3.2 microgram p1-26 alpha inhibin/ml) or steroid-free oFF in which the inhibin content had been reduced by greater than 90% (IFoFF; 3 ml s.c.; 0.3 microgram p1-26 alpha inhibin/ml) by affinity chromatography, 24 and 36 h after PG. Samples of ovarian and jugular venous blood were collected at (i) intervals of 4 h from 16 h before until 120 h after PG and (ii) intervals of 10 min from 48 to 52 h after injection of PG to investigate the pattern of pulsatile secretion of ovarian hormones. All ewes had previously been monitored during a normal PG-induced follicular phase. Injection of oFF resulted in an increase (P less than 0.05) in the concentration of inhibin in jugular venous plasma and a profound (P less than 0.001) and prolonged decrease in the peripheral concentration of follicle-stimulating hormone (FSH). Injection of IFoFF had no significant effect on peripheral concentrations of inhibin or FSH in the first 24 h after treatment; thereafter inhibin concentrations fell (P less than 0.01) progressively until 40 h and then increased (P less than 0.01) until 72 h after treatment. In both treatment groups, however, within 24-36 h of treatment the concentration of FSH increased 5-10-fold (P less than 0.001) to a peak that occurred within 48-60 h and then declined to basal concentrations within 72-84 h of treatment. The concentration of luteinizing hormone (LH) in jugular venous plasma increased in both groups after treatment (P less than 0.01), although the rise after injection of oFF only started after 24 h. Thereafter, there was a progressive increase in the concentration of LH, peaks occurring 48-60 h after treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

The uterine effects of oxytocin, the prostaglandins dinoprost and cloprostenol as well as clenbuterol, ergometrin, xylazine and Utrorale were investigated in 8 cows during late pregnancy, parturition and early puerperium (until 4th day p. p.). Uterine motility was measured by means of pressure microsensors and electrodes which were surgically implanted 3 to 4 weeks before parturition. Hysterograms were characterized by means of pressure amplitude, frequency and duration of uterine contractions and also by electromyography. Oxytocin (2-5 IE) given intravenously always provoked strong uterine contractions until the 4th day p.p. From the prostaglandins examined during early puerperium only dinoprost (15 mg i.v.) produced uterotonic effects, while the synthetic analogue cloprostenol (0.25 mg i.v.) had a weak stimulatory activity only on day 1 p.p. Both prostaglandins were ineffective when injected intramuscularly. Clenbuterol (0.3 mg i.v.) a beta 2-mimetic compound effectively induced long lasting tocolysis during parturition, which could be abolished by oxytocin. Xylazine (10 mg i.v.) was able to significantly increase uterine motility during late gestation. Following intravenous administration of ergometrin (1 and 10 mg), bunitrolol (1-16 mg) and Utrorale (0.1-4 ml) including its compounds oleum sabinae, oleum terebinthinae, balsamum copaivale and Styrax no uterokinetic activity was recorded at any time.

The aim of this study was to investigate the effect of puerperal uterine disease on histopathologic findings and gene expression of proinflammatory cytokines in the endometrium of postpuerperal dairy cows; 49 lactating Holstein-Friesian cows were divided into two groups, one without (UD-; n = 29) and one with uterine disease (UD+; n = 21), defined as retained fetal membranes and/or clinical metritis. General clinical examination, vaginoscopy, transrectal palpation, and transrectal B-mode sonography were conducted on days 8, 11, 18, and 25 and then every 10 days until Day 65 (Day 0 = day of calving). The first endometrial sampling (ES1; swab and biopsy) was done during estrus around Day 42 and the second endometrial sampling (ES2) during the estrus after synchronization (cloprostenol between days 55 and 60 and GnRH 2 days later). The prevalence of histopathologic evidence of endometritis, according to the categories used here, and positive bacteriologic cultures was not affected by group (P>> 0.05), but cows with uterine disease had a higher prevalence of chronic purulent endometritis (ES1; P = 0.07) and angiosclerosis (ES2; P ≤ 0.05) than healthy cows. Endometrial gene expression of IL1α (ES2), IL1β (ES2), and TNFα (ES1 and ES2) was higher (P ≤ 0.05) in the UD+ group than in the UD- group. In conclusion, puerperal uterine disease had an effect on histopathologic parameters and on gene expression of proinflammatory cytokines in the endometrium of postpuerperal cows, indicating impaired clearance of uterine inflammation in cows with puerperal uterine disease.

This study evaluated the feasibility of using an embryo transfer protocol in an alpaca farm in Canada. Alpaca donors and recipients were synchronized with 2 doses of gonadotrophin-releasing hormone (GnRH), 12 days apart. In donors (n = 5), superstimulation was induced with follicle stimulating hormone (FSH) given daily (40 mg) for 5 days beginning 2 days after the second GnRH treatment. Cloprostenol was given on the last day of FSH, the donors were bred 2 days later, embryos were collected 7 days after breeding. In recipients (n = 8), the second dose of GnRH was given the day before donor mating, and embryos were transferred on the day of donor collection. On average (± SEM), 5.2 ± 1.4 corpora lutea were detected and 2.5 ± 1.2 transferable embryos were collected in the donors. A mature corpus luteum was detected in 6/8 synchronized recipients and a single embryo was transferred to each. One recipient alpaca became pregnant and delivered a healthy baby 349 days after embryo transfer. This is the first report of successful embryo transfer in alpacas in Canada.

A total of 253 cows was treated using 500mug cloprostenol intramuscularly. Of those, 129 cows were treated for failure to exhibit an observed oestrus, 124 for being negative at pregnancy diagnosis examination; all possessed a corpus luteum as detected by rectal palpation. One hundred and sixty nine (66.8 per cent) were detected in oestrus within eight days, and of these 159 were served. Eight-seven (54.7 per cent) of them conceived. Sixteen cases of luteinised ovarian cysts were also treated with a resultant 14 conceptions occurring at a mean interval of 19-4 days. It is condisered that the time of the induced oestrus is not predictable enough to warrant "fixed time" insemination. The success of treatment will be related to: (1) The efficiency of oestrus detection on the farm (2) The normal conception rate on the farm (3) The accuracy of the rectal palpations.

Lactating dairy cows (n = 667) at random stages of the oestrous cycle were assigned to either ovsynch (O, n = 228), heatsynch (H, n = 252) or control (C, n = 187) groups. Cows in O and H groups received 100 microg of GnRH agonist, i.m. (day 0) starting at 44 +/- 3 days in milk (DIM), and 500 microg of cloprostenol, i.m. (day 7). In O group, cows received 100 microg of GnRH (day 9) and were artificially inseminated without oestrus detection 16-20 h later. In H group, cows received 1 mg oestradiol benzoate (EB) i.m., 24 h after the cloprostenol injection and were artificially inseminated without oestrus detection 48-52 h after the EB injection. Cows in C group were inseminated at natural oestrus. On the day of artificial insemination (AI), cows in all groups were assigned to subgroups as follows: human Chorionic Gonadotrophin (O-hCG) (n = 112), O-saline (n = 116), H-hCG (n = 123), H-saline (n = 129), C-hCG (n = 94) and C-saline (n = 93) subgroups. Cows in hCG and saline subgroups received 3000 IU hCG i.m. and or 10 ml saline at day 5 post-AI (day 15), respectively. Pregnancy status was assessed by palpation per rectum at days 40 to 45 after AI. The logistic regression model using just main effects of season (summer and winter), parity (primiparous and pluriparous), method(1) (O, H and C) and method(2) (hCG and saline) showed that all factors, except method(1), were significant. Significant effects of season (p < 0.01), hCG and parity (p < 0.01), and a trend of parity and season (p < 0.1) were detected. A clear negative effect of warm period on first service pregnancy rate was noted (p < 0.01). The pregnancy rate was the lowest in the H protocol during warm period (p < 0.05). Treatment with hCG 5 days after AI significantly improved pregnancy rates in those cows that were treated with the H protocol compared with saline treatments (41.5% vs 24.8%; p < 0.01). O and H were more effective in primiparous than in pluriparous cows (46.1% vs 29.9%; p < 0.1 and 43.6% vs 24.6%; p < 0.01). First service pregnancy rates were higher in primiparous hCG-treated than in pluriparous hCG-treated cows (57.9% vs 32.3%; p < 0.01). The pregnancy rate was higher for the hCG-treated cows compared with saline-treated cows during warm period (37.9% vs 23.6%; p < 0.001).

An experiment was performed to evaluate the association between the antral follicle count (AFC) plus body condition score (BCS) and the pregnancy rate in Bos indicus undergoing timed artificial insemination (TAI). A total of 736 Nelore cows with BCSs ranging from 2 to 4 received a conventional protocol for TAI. On a random day of the estrous cycle (Day 0), all cows received an intravaginal P4 device and an intramuscular (i.m.) injection of 2.0 mg estradiol benzoate. On Day 8, the P4 device was removed, and 150 μg sodium D-cloprostenol, 300 IU equine chorionic gonadotrophin and 1.0 mg estradiol cypionate were administered by i.m. injection. TAI was performed 48 h after P4 device removal, and pregnancy diagnosis was performed by ultrasonography after 30 days. On Day 0, all cows were examined by ultrasonography to determine the AFC by counting the number of follicles >3 mm in diameter that were present in both ovaries and to evaluate the BCS (scale of 1-5). The cows were then classified based on their AFCs as those with low (≤10 follicles), intermediate (11-29 follicles) and high AFC (≥30 follicles). Furthermore, cows were classified as having low (≥2.0 to ≤ 2.9) and high (≥3.0 to ≤ 4.0) BCSs. The AFCs and BCSs were analyzed using the generalized linear model, and the pregnancy rate was assessed with the binary logistic regression model (P ≤ 0.05). The pregnancy rate was influenced (P < 0.05) by AFC and BCS classification and by interactions (P = 0.034) between these factors. Cows with a low AFC exhibited higher a pregnancy rate than did cows with a high or an intermediate AFC (57.7% ^a, 47.9% ^b and 49.7% ^b, P = 0.008). Low BCS resulted in a higher pregnancy rate than did high BCS (55.2% vs. 50.4%, P = 0.008). Cows with a high BCS and a low AFC had a higher pregnancy rate (P < 0.05) than did those with a high BCS and an intermediate or a high AFC (59.8%^a, 48.0%^b, and 38.0%^b, respectively). An interaction (P < 0.05) was observed between the AFC and BCS, and the pregnancy rate decreased significantly in cows with an AFC > 30 and a BCS between 3 and 4. In conclusion, AFC and BCS classifications influence the pregnancy rate of Bos indicus beef cattle subjected to TAI. In addition, an important interaction between these factors was observed, namely, the lowest pregnancy rates were found in cows with high BCSs and high AFCs.

In previous studies, we demonstrated that the administration of a luteolytic dose of cloprostenol, followed by 750 IU hCG plus 3 mg estradiol benzoate (EB) 12 h later, synchronized estrus in cows in the luteal phase. Most cows were ready for service 48 h after the beginning of treatment. The objectives of this study were to evaluate the reproductive performance of lactating dairy cows treated with this method of estrus synchronization and to determine the effect of decreasing the hCG-EB dose on synchronization and pregnancy rates after timed AI. Data were obtained from cows first inseminated within an interval of 45 to 70 d postpartum. A total of 2,472 lactating dairy cows in their first to second lactation period were assigned to 4 groups. Cows estimated to be in the luteal phase by rectal palpation were treated with 500 mcg, im, of cloprostenol and assigned to 1 of 3 groups to be intramuscularly injected with hCG-EB 12 h later at the following doses: Group 1 (n=626), 250 IU of hCG and 1 mg of EB; Group 2 (n=592), 500 IU of hCG and 2 mg of EB; and Group 3 (n=664), 750 IU of hCG and 3 mg of EB. Cows displaying natural estrus were inseminated to serve as controls (n=590). The synchronized cows were inseminated 48 h after cloprostenol injection, and control animals visually determined to be in natural estrus during the morning or afternoon were inseminated the following morning. Pregnancy diagnosis was performed by rectal palpation at 34 to 40 d postinsemination. All synchronized cows showed estrous activity within 24 to 36 h after cloprostenol treatment and were considered to be ready for service 48 h after this treatment. There was a significant effect of treatment on the pregnancy rate, either to first AI or to 2 rounds of AI. The pregnancy rate in response to first or second rounds of AI was similar to control rates for cows in Groups 1 and 2, and lower than control rates in Group 3. Cows in Group 1 showed a higher pregnancy rate to first AI than those in Group 3 (P<0.0001), and a higher pregnancy rate to second AI rounds than cows in Groups 2 (P<0.02) and 3 (P<0.0001). The number of cows returning to estrus was unaffected by treatment. However, treatment significantly decreased (P<0.01) the time of return to estrus as the hCG-EB dose increased. These findings indicate that the lowest dose of hCG-EB treatment tested gave the overall best pregnancy results among the treated groups. Furthermore, the synchronization protocol used in this experiment allows effective AI management of lactating dairy cows without the need for estrus detection.

The relationship between expression of inhibin mRNA and ovarian secretion of estradiol (E2) and immunoactive inhibin was investigated at midluteal phase and throughout the follicular phase of the sheep estrous cycle. At laparotomy, timed samples of ovarian blood were collected and ovaries were removed from 39 Scottish Blackface ewes (ovulation rate 1.3 +/- 0.1) on Day 10 of the luteal phase or 24, 48, 60, 72, or 84 h after injection of cloprostenol (PG; 100 micrograms) on Days 10-12. Ovaries were removed and fixed for in situ hybridization using 35S-labeled antisense riboprobes transcribed from inhibin alpha, beta A, and beta B cDNAs. LH, E2, and inhibin concentrations were determined by RIA. On the basis of peripheral LH levels and the presence of estrogen-active follicles (E-A;>> or = 3 mm in diameter secreting>> 1 ng/min E2) or recent ovulations, animals were grouped as follows: presurge (24 or 48 h post-PG; LH < 5 ng/ml; n = 7), midsurge (with E-A; LH>> 5 ng/ml; n = 6), late surge (large follicle not E-A; LH>> 5 ng/ml; n = 4), postsurge (large follicle not E-A; LH < 5 ng/ml; n = 7), and postovulation (n = 10). As expected, E2 secretion by the "active" ovary (containing preovulatory follicle) tended to increase with follicular development such that secretion was maximal at midsurge and then declined. E2 secretion by the "inactive" ovary was low at all stages. Immunoactive inhibin, in contrast, was secreted in substantial quantities by both ovaries, although secretion from active ovaries was higher at all stages (p < 0.05). Effects of stage on secretion were not significant, but immunoactive inhibin secretion from active ovaries was high in postsurge animals when E2 secretion was very low. Hybridization for inhibin mRNA was specific for granulosa cells of antral follicles. While most sheep in the luteal (4 of 5), presurge (2 of 3), and midsurge groups (5 of 5) had at least one inhibin-positive large follicle (expressing both alpha- and beta-subunit mRNA), none were present between the LH surge and ovulation (late and postsurge groups). Inhibin mRNA was undetectable in midcycle CL, but 4 of 10 recent ovulations hybridized weakly with the alpha probe and one very weakly with the beta A probe. The mean number of inhibin-positive large follicles per animal (in those having at least one) was 1.3 +/- 0.15 (n = 15 ewes).(ABSTRACT TRUNCATED AT 400 WORDS)

Dairy (Bos taurus) heifers and cows (n = 40) in a tropical environment were treated during mid-luteal phase using either SUPER-OV(R) or OVAGEN to induce superovulatory response after synchronization of the superovulatory estrus with a synthetic progestagen and cloprostenol (PG). Estrous cattle were inseminated twice using frozen-thawed semen, and embryos were recovered nonsurgically, on-farm, 7 d later. Between initiation of gonadotrophin treatment and recovery of embryos, 4 blood samples per animal were collected from 26 animals for determination of plasma progesterone (P4) concentration. Two (5%), 28 (70%) and 10 (22%) of the animals were observed in estrus 1.5, 2 and 2.5 to 3 d after PG, respectively. There was no difference (P = 0.7) in the number of palpable CL between animals treated with SUPER-OV (7.6 +/- 1.0; n = 18) and those treated with OVAGEN (7.9 +/- 1.1; n = 22). There was also no significant difference (P>> 0.05) between Jersey vs Ayrshire breeds or heifers vs cows in the ovarian response as estimated by the number of palpable CL. However, a higher proportion of Ayrshire cattle and donors treated with OVAGEN yielded a higher total number and viable/transferable embryos than Jersey and SUPER-OV-treated cattle. There was a significant (P < 0.05) correlation between the number of CL and total number of embryos (r = 0.65); the number of transferable embryos was also significantly related to the total number of embryos per recovery (r = 0.85; P < 0.05). For 15 animals with normal P4 profiles, the mean (+/-SEM) plasma P4 concentration was 14.4 +/- 0.8, 0.5 +/- 0.2, 5.4 +/- 1.1 and 39.4 +/- 3.0 nmol L at initiation of gonadotrophin treatment, superovulatory estrus and Days 3 and 7, respectively. The mean (+/-SEM) interval between a PG injection given after embryo recovery and the induced estrus was 7.1 +/- 0.7 d (range 3 to 14 d) and the length of the superovulatory cycle was 24.1 +/- 3.2 d (range 12 to 35 d).

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150microg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n=12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n=11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.

The objective of this research was to determine if PGF2alpha-induced milk letdown (ML) is an accurate indicator of luteolysis, allowing cows to be synchronized to begin the Ovsynch protocol (GnRH-7d-PGF2alpha-2d-GnRH-24h-AI) at the most beneficial time of the estrous cycle (days 5-9), and determine if this would improve pregnancy rate (PR). Lactating Holstein cows between 55 and 70 days in milk were used to evaluate the ML test and PR after the Ovsynch protocol, when initiated on the basis of the test result (PROSYNCH). PROSYNCH cows (n = 60) had one teat cannulated to test for ML and were treated with 500 microg cloprostenol, PGF2alpha analogue (PG). Cows with ML were started on Ovsynch 10 days later, and those without started 3 days later. Cows in the control group (OVSYNCH, n = 64) were injected with physiological saline and observed for ML. This group was started on Ovsynch 10 days after saline treatment. Milk samples were collected thrice weekly to determine progesterone concentrations. ML indicated luteolysis with a sensitivity of 98% and a specificity of 60%. The positive and negative predictive values were 83 and 92%, respectively. Pregnancy rates were 48% for PROSYNCH and 52% for OVSYNCH (P = 0.72). When data from both groups were combined, PR was greater in cows that started the Ovsynch protocol in stage 2 of the estrous cycle (days 5-9, 67%) than all other stages (stage 1: days 1-4, 35%; stage 3: days 10-16, 45%; stage 4: days 17-21, 42%; P < 0.01). The proportion of animals with ovulation after GnRH#1, luteolysis after PGF2alpha, and ovulation after GnRH#2 were all greater in the PROSYNCH group (77% versus 55%, P < 0.02; 83% versus 66%, P < 0.03; 97% versus 84%, P < 0.03, respectively). Therefore, the ML test indicated luteolysis with sufficient precision to time the initiation of the Ovsynch protocol between days 5 and 9 of the cycle, however, this did not alter PR compared to starting the protocol randomly throughout the cycle. Initiating the Ovsynch protocol between days 5 and 9 of the cycle increased PR, and improved the efficacy of each injection.

Luteolysis is a key event in cattle reproduction. A standard dose of exogenous PGF(2α) will induce full luteolysis in the majority of cows with a matured corpus luteum (CL). However, this will not occur in cows with a CL <5d old. To date, it is not known whether a larger dose will have a more potent luteolytic effect in cows during early diestrus. The objective of this study was to characterize the effect of 2 doses of d-cloprostenol (150 and 300 μg) on the progesterone concentration, luteal diameter, and ovulation rate in nonlactating dairy cattle 96 to 132 h postovulation. Twenty nonlactating dairy cows were included in the study. Each cow received 2 treatments of d-cloprostenol in 2 consecutive cycles: a standard dose of 150 μg and a double dose of 300 μg. The cows were allocated randomly to 1 of 4 groups (5 cows in each group) according to the age of the CL at the time of treatment: 96, 108, 120, and 132 h. The exact time of ovulation was known within 12h, because of twice per day ultrasound examination. The CL diameter and progesterone concentration were measured before treatment (d 0) and 2 and 4d after treatment. Within each CL age group, the effect of d-cloprostenol dose on luteolysis was determined. More cows treated with double dose tended to have full luteolysis compared with the standard dose (8/10 vs. 4/10, respectively). This effect was only apparent in cows with CL of 120 and 132 h but not in earlier CL. The interval from treatment to ovulation was shorter (3.3 ± 0.1d) in cows treated with a double dose than in cows treated with the standard dose (4.5 ± 0.4d).

This study was designed to assess endocrine changes associated with termination of mid-term pregnancy after use of two different protocols. For this purpose we compared the effects of aglepristone (AGL) alone and in combination with cloprostenol (CLO) on serum concentrations of progesterone (P4), estradiol (E2) and relaxin (RLN) measured at short-term intervals during the abortion period in bitches. Fourteen pregnant bitches between day 25 and 32 of gestation were used in the study. In the AGL group (n=7), aglepristone was administered solely (10mg/kg body weight (BW), subcutaneously, once daily on two consecutive days) whereas in the AGL-CLO group (n=7), aglepristone (dosage as in AGL group) and cloprostenol (1μg/kg BW, subcutaneously, same with aglepristone) were combined. All pregnancies were successfully terminated 5.2±1.6 days after initiation of treatments, which was significant in both groups (P>0.05). At the time of the start of abortion (SA) and the end of abortion (EA), the mean P4 concentrations were 26.6±7.3 and 12.0±6.4ng/ml in AGL group, and 2.7±0.7 and 0.9±0.1ng/ml, in AGL-CLO group, respectively (P<0.01). Serum E2 concentrations were significantly higher (P<0.05) in AGL group at 42, 48, 54h and SA after initiation of treatment. In the AGL-CLO group, serum RLN concentrations did not significantly change from the initiation of treatment to EA (P>0.05). However, markedly higher RLN concentrations (P<0.05) were observed in the AGL group at 48h (1.5±0.7ng/ml) and at SA (1.6±0.5ng/ml). The results of the present study indicate that changes in the hormonal concentrations affect the mechanism of abortion in different ways. Further in depth studies investigating changes in the expression of hormone receptors inside the ovary, endometrium and placenta might be helpful to our understanding of the endocrinological differences observed in this study.

This study evaluated the effect of two doses of prostaglandin at different intervals on reproductive parameters of crossbred ewes. In Experiment 1, 30 ewes received two doses of 120 μg cloprostenol at 7 (G 7 days), 9 (G 9 days), or 11.5 (G 11.5 days) days apart. Ultrasound assessments were performed from the first and second cloprostenol administration for 5 days or ovulation detection. Estrus signs were checked by a teaser male. Plasma progesterone concentration was measured before each cloprostenol dose. In Experiment 2, 95 ewes were allocated into the same treatments and after the second dose, ewes in estrus were mated. At 30 days after breeding, pregnancy diagnosis was conducted and prolificacy was evaluated at lambing. In Experiment 1, at the first cloprostenol administration, 50% of ewes had an active CL and all showed estrus. At the second administration, 66.7% of ewes had an active CL and one did not present estrus. There was no difference (P>> 0.05) after the second dose for as follows: overall estrous response (90%), interval from cloprostenol administration to estrous onset (42.0 ± 4.9 h), estrus duration (31.5 ± 2.1 h), ovulation rate (100.0%), and number of ovulations (1.5 ± 0.3). In Experiment 2, both pregnancy and prolificacy rates were similar (P>> 0.05) for G 7 days (73.3; 145%), G 9 days (75.9; 125%), or G 11.5 days (75.9; 145%), leading to an overall pregnancy rate of 75.0% (66/88) and prolificacy rate of 137%. Therefore, the three treatments proposed were able to promote high pregnancy and prolificacy rates in crossbred ewes.

In this study we compared the effect of dinoprost and cloprostenol on changes of corpus luteum blood flow during luteolysis. Ten nonlactating cyclic ewes were synchronized with double PGF2α injections 11 days apart. At Day 10, the animals were classified into 2 groups and received the third dose of PGF2α after confirmation of the presence of a mature CL. The first group received (12.5 mg/im) dinoprost and the second group received (250 μg/im) cloprostenol. A color Doppler ultrasound scan was performed by the same operator according to the following timeline: 0, 0.5, 1, 2, 4, 6, 12, and 24 hours, then every 24 hours until Day 4). The size, morphology, and blood flow of the CL was evaluated during the regression. The results showed that regression of the CL did not differ between the dinoprost and cloprostenol groups. There was no significant effect on diameter of the CL in both groups, though the size of the CL decreased gradually and slowly. Pretreatment progesterone concentration did not differ between groups. The results showed that the nitric oxide level was significantly increased within half an hour after the dinoprost treatment, and was significantly decreased in the cloprostenol group after half an hour. The blood velocity was increased significantly half an hour after the dinoprost treatment and it was decreased in the cloprostenol-treated group. In conclusion, both cloprostenol and dinoprost affect CL by controlling the nitric oxide level and blood supply of the CL via different mechanisms to induce luteolysis.

Twelve Hereford cross Friesian cows received subcutaneous implants containing 6 mg norgestomet and intramuscular injections of 5 mg oestradiol valerate and 3 mg norgestomet. Six of the cows also received 0.5 mg cloprostenol eight days later and all implants were removed on day 9. When treatment was commenced between days 3 and 5 of the ovarian cycle, luteal function was not prevented although the luteal phase was shortened in some cases. When treatment was commenced between days 8 and 14 of the cycle progesterone concentrations remained above basal levels for five to seven days. Cows with corpora lutea that were given cloprostenol underwent rapid luteolysis. It is concluded that oestradiol valerate does not control luteal function adequately, particularly if administered early in the cycle, and this may explain failure of oestrus synchronisation in some cases. Administration of prostaglandin 24 hours before norgestomet implant removal may improve the degree of oestrus synchronisation in groups of cyclic cows.

In 22 silent heat cows with functional corpus luteum the blood progesterone concentrations and appearance of oestrus after administration of Estrumate were examined. 2 ml Estrumate (500 micrograms cloprostenol) were given intravaginally to 12 cows and intramuscularly to 10 cows respectively. Progesterone concentrations declined 32 hrs after application to levels below 3, 18 nmol/l in all animals. Within 10 days 66.6% of the cows after intravaginal administration of Estrumate and 70% after intramuscular administration came into oestrus. The results indicate that intravaginal application of Estrumate in silent cows with functional corpus luteum induced luteolysis and oestrus identical with intramuscular application.