Influence of reproductive stage at PRID insertion on synchronization of estrus and ovulation in mares

In the present study, we investigated the effects of reproductive status, size of follicles and plasma progesterone concentrations of mares at PRID insertion on the efficacy of the treatment, estrous cycle patterns, plasma concentrations of progesterone and LH. The progesterone-releasing device (PRID) was administered intravaginally to 28 Haflinger mares for 11 days at different reproductive stages: anestrus (n=6), estrus (n=11) and diestrus (n=11). Plasma concentrations of progesterone at insertion (Day 1) of PRID differed among treatment groups (anestrus: 0.2-0.6 ng mL(-1), estrus: 0.2-0.5 and diestrus: 1.6-10.8 ng mL(-1); P<0.001). Total secretion of progesterone (area under curve (AUC)) during treatment period revealed highest values in diestrus (38.2+/-3.1 ng mL(-1)h(-1)) followed by estrus (25.1+/-2.7) and anestrus (21.0+/-0.4 ng mL(-1)h(-1); P<0.05). Progesterone area under curve (AUC) was positively correlated with initial progesterone concentrations (R=0.5; P<0.05), but it did not correlate with the interval from PRID removal to ovulation. Plasma concentrations of LH during treatment period, were significantly lower in anestrous mares (184.6+/-28.6 ng mL(-1)h(-1)) when compared to estrous and diestrous mares (349.7+/-53.3 and 370.5+/-40.3 ng mL(-1)h(-1); P<0.05). Follicular size at PRID insertion had no effects on the intervals from PRID removal to subsequent estrus and ovulation. Follicle diameters at removal of PRID were significantly correlated with the interval from coil removal to estrus (R=-0.55, P<0.05) and ovulation (R=-0.72, P<0.0004) in cyclic mares. In anestrus 0 of 6 (0%) mares, in estrus 5 of 11 (45.5%) and in diestrus 6 of 11 (54.5%) mares ovulated within a defined interval of 1 day before to 1 day after mean interval from PRID removal to ovulation. In cyclic mares, response to treatment was significantly higher when compared to anestrous mares: almost all mares responded with estrus and ovulation independent from the stage of the estrous cycle at the start of treatment. However, accuracy of synchronization was still unsatisfactory. In cyclic mares, the plasma progesterone concentrations at insertion of PRID seem to be more important for the efficacy of the treatment than the assignment to estrous cycle stages.     

Temporal interrelationships among luteolysis, FSH and LH concentrations and follicle deviation in mares

The effect of altered LH concentrations on the deviation in growth rates between the 2 largest follicles was studied in pony mares. The progestational phase was shortened by administration of PGF2alpha on Day 10 (Day 0=ovulation; n=9) or lengthened by daily administration of 100 mg of progesterone on Days 10 to 30 (n=11; controls, n=10). All follicles > or = 5 mm were ablated on Day 10 in all groups to initiate a new follicular wave. The interovulatory interval was not altered by the PGF2alpha treatment despite a 4-day earlier decrease in progesterone concentrations. Time required for growth of the follicles of the new wave apparently delayed the interval to ovulation after luteolysis. The FSH concentrations of the first post-ablation FSH surge were not different among groups. A second FSH surge with an associated follicular wave began by Day 22 in 7 of 11 mares in the progesterone group and in 0 of 19 mares in the other groups, indicating reduced functional competence of the largest follicle. A prolonged elevation in LH concentrations began on the mean day of wave emergence (Day 11) in the prostaglandin group (19.2 +/- 2.2 vs 9.0 +/- 0.7 ng/mL in controls; P<0.05), an average of 4 d before an increase in the controls. Concentrations of LH in the progesterone group initially increased until Day 14 and then decreased so that by Day 18 the concentrations were lower (P<0.05) than in the control group (12.9 +/- 1.6 vs 20.2 +/- 2.6 ng/mL). Neither the early and prolonged increase nor the early decrease in LH concentrations altered the growth profile of the second-largest follicle, suggesting that LH was not involved in the initiation of deviation. However, the early decrease in LH concentrations in the progesterone group was followed by a smaller (P<0.05) diameter of the largest follicle by Day 20 (26.9 +/- 1.7 mm) than the controls (30.3 +/- 1.7 mm), suggesting that LH was necessary for continued growth of the largest follicle after deviation.     

Effects of ovarian input on GnRH and LH secretion immediately postovulation in pony mares

The potential involvement of ovarian factors in regulating GnRH and LH postovulation was studied in ovarian intact (Group 1; n=3) and ovariectomized (OVX; Group 2; n=3) mares (OVX within 12 hr of ovulation). Blood samples were collected every 10 min for 6 hr from jugular vein (JV) and intercavernous sinus (ICS) during estrus and on Day 8 postovulation for LH and GnRH analysis. Additionally, JV samples were collected twice daily (12-hr intervals) for 30 days for LH and progesterone (P4) analysis. A significant treatment x day effect (P<0.0001) describes declining plasma LH concentrations in intact mares, and regression analysis indicated that response curves were not parallel (P<0.001). Plasma LH concentrations remained elevated in OVX mares. LH increased further in OVX mares by Day 8 post-OVX (P<0.06), reflecting the increased (P<0.07) LH episode amplitude. GnRH decreased from estrus to Day 8 in both groups reflecting an effect of sampling period (P<0.03). GnRH episode amplitude declined (P<0.08) from estrus (62.8+/-3.1 pg/mL) to Day 8 (46.3+/-3.1 pg/mL) in OVX mares, but not in control mares (intact estrus, 36.5+/-6.4; intact Day 8, 37.5+/-7.3; OVX estrus, 62.8+/-3.1; OVX Day 8, 46.3+/-3.1 pg/mL). In conclusion, we propose that postovulatory LH decline requires ovarian feedback in mares, and that OVX alters GnRH secretory dynamics such that LH concentrations does not decline postovulation and, in fact, is further elevated with time after OVX.     

Progesterone and estradiol in biodegradable microspheres for control of estrus and ovulation in mares

Progesterone and estradiol 17-beta in poly (DL-lactide) microspheres were used to control estrus and ovulation in mares after luteolysis was induced by prostaglandin F(2)infinity. Mares were given a single intramuscular injection of biodegradable poly (DL-lactide) microspheres, 1 day following prostaglandin treatment, containing no hormones (control), 0.625 g progesterone and 50 mg estradiol (low dose), 1.25 g progesterone and 100 mg estradiol (medium dose), or 1.875 g progesterone and 150 mg estradiol (high dose; n=15 mares per group). Mares treated with the low dose had significantly longer intervals (P<0.05) to estrus and ovulation than the control mares; however, low dose mares had shorter intervals (P<0.05) to estrus than high dose mares and shorter intervals to ovulation than medium and high dose mares. Regression analysis indicated that the medium dose was sufficient for maximizing interval to ovulation while the high dose maximized interval to estrus. All groups of mares exhibited similar (P>0.05) post-treatment estrus lengths. A clinical response scoring system based on synchrony of both estrus and ovulation within a treatment group was also used to measure the effectiveness of treatments on control of estrus and ovulation. Clinical response scores did not differ (P>0.05) among treatment groups. Mares were randomly assigned for insemination at the beginning of the first post-treatment estrus. Rates for embryo recovery performed by uterine lavage 7 days post-ovulation did not differ (P>0.05) among groups. Concentrations of serum progesterone increased in mares receiving progesterone and estradiol microspheres. At 10 to 14 days post-injection of microspheres, progesterone concentrations were higher (P<0.05) and remained above 1 ng/ml in the mares receiving the high dose. Progesterone concentrations were also higher (P<0.05) on Days -3 to -1 (Day 0 = day of post-treatment ovulation) in mares receiving the high dose when compared to control mares. Gonadotropin concentrations were suppressed (P<0.05) in the medium and high dose groups.     

Response to GnRH on day 6 of the estrous cycle is diminished as the percentage of Bos indicus breeding increases in Angus, Brangus, and Brahman x Angus heifers

Angus (n=6), Brangus (5/8 Angus x 3/8 Brahman, n=6), and Brahman x Angus (3/8 Angus x 5/8 Brahman, n=6) heifers exhibiting estrous cycles at regular intervals were used to determine if the percentage of Bos indicus breeding influenced the secretory patterns of LH in response to a GnRH treatment on Day 6 of the estrous cycle. Heifers were pre-synchronized with a two-injection PGF(2 alpha) protocol (25 mg i.m. Day -14 and 12.5 mg i.m. Day -3 and -2 of experiment). Heifers received 100 microg GnRH i.m. on Day 6 of the subsequent estrous cycle. Blood samples were collected at -60, -30, and -1 min before GnRH and 15, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min after GnRH to determine concentrations of serum LH. Estradiol concentrations were determined at -60, -30, and -1 min before GnRH. On Day 6 and 8, ovaries were examined by ultrasonography to determine if ovulation occurred. On Day 13, heifers received 25 mg PGF(2 alpha) i.m. and blood samples were collected daily until either the expression of estrus or Day 20 for heifers not exhibiting estrus to determine progesterone concentrations. There was no effect (P>0.10) of breed on ovulation rate to GnRH as well as size of the largest follicle, mean estradiol, and mean corpus luteum volume at GnRH. Mean LH was greater (P<0.05) for Angus (7.0+/-0.8 ng/mL) compared to Brangus (4.6+/-0.8 ng/mL) and Brahman x Angus (2.9+/-0.8 ng/mL), which were similar (P>0.10). Mean LH peak-height was similar (P>0.10) for Brangus (13.9+/-3.4 ng/mL) compared to Angus (21.9+/-3.4 ng/mL) and Brahman x Angus (8.0+/-3.4 ng/mL), but was greater (P<0.05) for Angus compared to Brahman x Angus. Interval from GnRH to LH peak was similar (P>0.10) between breeds. As the percentage of Bos indicus breeding increased the amount of LH released in response to GnRH on Day 6 of the estrous cycle decreased.     

Deslorelin on Day 8 or 12 postovulation does not luteinize follicles during an artificially maintained diestrous phase in the mare

Practical estrus synchronization schemes are needed for mares. The Ovsynch synchronization protocol for cattle involves the administration of gonadotropin-releasing hormone (GnRH) to induce ovulation or luteinization of dominant follicles during the luteal phase and prostaglandin 7 days later to cause regression of any luteal tissue and development of a preovulatory follicle. An Ovsynch-type synchronization program potentially could be developed for horses if luteinization or ovulation of diestrous follicles occurred in response to GnRH treatment. The objective of this study was to determine if administration of the GnRH agonist, deslorelin acetate, on Day 8 or 12 postovulation would induce luteinization or ovulation of diestrous follicles in the mare. The model used was cycling mares maintained in an artificial luteal phase by administration of a synthetic progestin following prostaglandin-induced luteal regression. On the day of ovulation, 21 light horse mares were randomly assigned to one of three groups: (1) no GnRH, altrenogest from Days 5 to 15 postovulation with prostaglandin on Day 15; (2) GnRH on Day 8, altrenogest from Days 5 to 15 with prostaglandin given on Day 6 to induce luteolysis of the primary corpus luteum, an implant containing 2.1mg of deslorelin acetate inserted on Day 8 and removed on Day 10, with a second prostaglandin treatment on Day 15; (3) GnRH on Day 12, altrenogest from Days 9 to 19, prostaglandin on Day 10, a deslorelin acetate implant injected on Day 12 (subsequently removed on Day 14), and a second dose of prostaglandin administered on Day 19. Follicular development was monitored every other day from Day 5 until a 30-mm sized follicle was observed, and then daily to detection of ovulation. Serum progesterone concentrations were determined daily for 12 consecutive days. Progesterone concentrations in Group 1 remained elevated until approximately Day 12 postovulation. Prostaglandin administration on Day 15 resulted in complete luteolysis in all seven mares. In Group 2, progesterone concentrations in six of seven mares declined to baseline after prostaglandin treatment. No increase in serum progesterone was noted in any of the six mares that were given GnRH on Day 8, including three mares that had diestrous follicles > or =30mm in diameter at the time of treatment. Similarly, progesterone concentrations in six of seven mares in Group 3 declined to baseline after prostaglandin and there was no increase in progesterone after administration of GnRH on Day 12. No ultrasound evidence of luteinization or ovulation of diestrous follicles were noted after GnRH administration in any mares of Group 2 or 3. In conclusion, administration of the GnRH agonist deslorelin acetate to mares failed to induce luteinization or ovulation of diestrous follicles. Consequently, the Ovsynch program (as used in cattle) has little efficacy for synchronization of estrus in mares.     

Effects of luteinizing hormone on superovulatory and steroidogenic responses of rat ovaries to infusion with follicle-stimulating hormone

Immature female rats were infused s.c. continuously over a 60-h period with a partially purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH activity 4.2 x NIH-FSH-S1 and luteinizing hormone (LH) activity 0.022 x NIH-LH-S1. High rates of superovulation were observed in rats receiving 1 U FSH/day, with 69 +/- 11 oocytes/rat recovered as cumulus-enclosed oocytes from oviducts on Day 1 (equivalent to the day of estrus). Addition of LH to the FSH, at dosages equivalent to 2.5-100 micrograms/day NIH-LH-S1 equivalents (2.5-100 mU) resulted in a dose-related inhibition of superovulation, reaching a nadir of 15 +/- 7 oocytes recovered from rats receiving 50 mU LH/day together with 1 U FSH/day. At the two highest LH doses, 50 and 100 mU/day, ovulation was advanced so that 12 +/- 3 and 15 +/- 4 oocytes, respectively, were recovered from oviducts of these rats flushed on the morning of Day 0, compared to none in rats infused with FSH alone. Ovarian steroid concentrations (ng/mg) observed on the morning of Day 0 in rats infused with FSH alone were progesterone, 0.50 +/- 0.13; testosterone, 0.16 +/- 0.08; androstenedione, 0.06; and estradiol, 0.23 +/- 0.05. On the morning of Day 1, ovarian progesterone concentrations in rats infused with FSH alone had risen to 3.30 +/- 0.33 ng/mg, whereas concentrations of testosterone, androstenedione, and estradiol, had fallen to essentially undetectable levels. Addition of LH to the FSH infusion resulted in dose-related increases, on Day 0, of all four steroids up to a dosage of 25 mU LH/day. At higher LH dosages, Day 0 ovarian concentrations of androgens and estradiol fell markedly, while progesterone concentrations continued to increase. Histological examination of ovaries revealed increases in the extent of luteinization of granulosa cells in follicles with retained oocytes on both Days 0 and 1 in rats infused with 25 and 50 mU LH/day together with 1 U FSH/day, compared to those observed in rats receiving FSH alone. These findings indicate that the elevated progesterone levels on Day 0 and inhibition of ovulation observed at these LH doses were due to premature luteinization of follicles, thus preventing ovulation. At lower LH doses, no sign (based on histologic or steroidogenic criteria) of premature luteinization was evident, suggesting that the decreased superovulation in these rats was due to decreased follicular maturation and/or increased atresia rather than to luteinization of follicles without ovulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Continuous administration of low-dose GnRH in mares II. Pituitary and ovarian responses to uninterrupted treatment beginning near the autumnal equinox and continuing throughout the anovulatory season

We tested the hypothesis that continuous subcutaneous treatment with low-dose GnRH, administered to mares from late September/early October through March, would prevent the development of seasonal anovulation. Quarter Horse mares (n=20) were stratified by age and body condition score and assigned randomly to either a saline control (n=9) or a GnRH (n=11) treatment group. Gonadotropin-releasing hormone was delivered continuously via osmotic minipumps, with sham pumps placed in control mares. Initial pumps were inserted on Day 3 following ovulation or during the follicular phase if the next anticipated ovulation did not occur by 9 October. Delivery rate of GnRH was 2.5 microg/h (60 microg/day) for the first 60 days, followed by 5.0 microg/h (120 microg/day) thereafter. Pumps were replaced every 30 days. Eighty and 100% of all mares had become anovulatory by 1 November and 1 December, respectively, and remained anovulatory through the end of February. Neither serum concentrations of LH throughout the study nor total releasable pools of LH in March differed between groups. Although control mares that exhibited ovulatory cycles after study onset had greater (P<0.05) mean concentrations of LH during the follicular phase and metestrus compared to GnRH-treated mares, neither size of ovulatory follicles nor interovulatory intervals differed between groups. Serum concentrations of FSH were not affected by treatment, but were lowest (P<0.05) from November through January. Continuous infusion of low-dose GnRH, beginning soon after autumnal equinox and continuing until just after vernal equinox, failed to prevent the occurrence of or to hasten transition from seasonal anovulation.     

Pulsatile administration of gonadotropin-releasing hormone advances ovulation in cycling mares

Cycling standardbred mares were infused with saline or 20 micrograms gonadotropin-releasing hormone (GnRH) in a pulsatile pattern (one 5-sec pulse/h, 2 h or 4 h) beginning on Day 16 of the estrous cycle. Although serum concentrations of luteinizing hormone (LH) increased significantly earlier in all three GnRH-treated groups (within one day of the initiation of infusion) compared to saline-infused controls, there were no differences in peak periovulatory LH concentrations among treatments (overall mean +/- SEM, 8.98 +/- 0.55 ng/ml). The number of days from the start of treatment to ovulation was significantly less in mares infused with 20 micrograms GnRH/h (mean +/- SEM, 2.9 +/- 0.6 days after the initiation of treatment, or 18.9 days from the previous ovulation; N = 7) compared to mares treated with saline (5.9 +/- 0.3 days, or 21.9 days from previous ovulation; N = 7) or 20 micrograms GnRH per 4 h (5.4 +/- 0.9 days or 21.4 days from previous ovulation; N = 5). Although mares infused with 20 micrograms GnRH/2 h ovulated after 4.3 +/- 0.7 days of treatment (Day 20.3; N = 7), this was not significantly different from either the control or 20 micrograms GnRH/h treatment groups. Neither the duration of the resulting luteal phase nor the length of the estrous cycle was different between any of the treatment groups (combined means, 14.7 +/- 0.2 days and 21.3 +/- 0.4 days, respectively). We conclude that pulsatile infusion of GnRH is effective in advancing the time of ovulation in cycling mares, but that the frequency of pulse infusion is a critical variable.     

Persistent ovarian follicles in dairy cows: a therapeutic approach

Anestrus is common during the postpartum period in high-producing dairy cows. In a previous investigation, we were able to diagnose persistent follicles of 8 to 12 mm in anestrous cows. This report describes 2 consecutive studies. The objectives of the first were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate 2 therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with the Ovsynch protocol. For Study 1, anestrous cows were considered to have a persistent follicle if it was possible to observe a single follicular structure > 8 mm in the absence of a corpus luteum or a cyst in 2 ultrasonographic examinations performed at an interval of 7 d. At diagnosis (Day 0), cows were assigned to 1 of 3 treatment groups. Cows in Group GnRH/PGF (n=17) were treated with 100 microg GnRH i.m., and 25 mg PGF2alpha i.m. on Day 14. Cows in Group PRID (n=18) were fitted with a progesterone releasing intravaginal device (PRID, containing 1.55 g of progesterone) for 9 d and were given 100 microg GnRH i.m. at the time of PRID insertion, and 25 mg PGF2alpha i.m. on Day 7. Cows in Group Control (n=18) received no treatment. The animals were inseminated at observed estrus and were monitored weekly by ultrasonography until AI or 5 weeks from diagnosis. Blood samples were also collected on a weekly basis for progesterone determination. The mean size of persistent follicles on Day 0 was 9.4 +/- 0.04 mm. Progesterone levels were < 0.2 ng/mL during the first 35 d in 16 of 18 Control cows. Cows in the PRID group showed a lower persistent follicle rate (16.7% < 70.6% < 88.9%; P < 0.0001; PRID vs GnRH/PGF vs Control, respectively); a higher estrus detection rate (83.3% > 29.4% > 11.1%; P < 0.0001) and a higher pregnancy rate (27.8% > 5.9% > 0%; P = 0.02). For the second study, 145 cows with persistent follicles were randomly assigned to 1 of 2 treatment groups: cows in Group Ovsynch (n=73) were treated with 100 microg GnRH i.m. on Day 0, 25 mg PGF2alpha i.m. on Day 7, and 100 microm GnRH i.m. 32 h later. Cows in this group were inseminated 16 to 20 h after the second GnRH dose (Ovsynch protocol). Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1, and were inseminated 56 h after PRID removal. Cows in the PRID group showed a higher ovulation rate (84.8% > 8.2%: P < 0.0001); a higher pregnancy rate (34.2% > 4.1%; P < 0.0001) and lower follicular persistence rate (22.2% < 63%; P < 0.0001) than those in Ovsynch. Our results indicate that persistent follicles affect cyclic ovarian function in lactating dairy cows. Cows with persistent follicles can be successfully synchronized and time inseminated using progesterone, GnRH and PGF2alpha but show a limited response to treatment with GnRH plus PGF2alpha.     

Reproductive hormone secretions in pony mares subsequent to ovulation control during late winter

Beginning in December, pony mares were placed under a schedule of increasing light. Starting in February, onset of estrus was checked by daily teasing with a stallion. Mares were randomly assigned to one of three treatments (6 mares per group) administered in March. Treatments were: Group I — 75 mg progesterone injected intramuscularly every day for 10 days in combination with a 1.25 mg injection of PGF₂α on day 7 of progesterone treatment and a 2,000 IU injection of HCG on day 2 of estrus; Group II — a norgestomet ear implant inserted for 10 days in combination with 1.25 mg PGF₂α given 7 days after insertion and 2,000 IU HCG administered on day 2 of estrus; and Group III — same as II except that 2 mg of GnRH rather than HCG were administered on day 2 of estrus. Blood plasma for radioimmunoassay of progesterone, LH and estradiol was collected from the first day of treatment until 14 days after the end of estrus. Also in March, 6 mares were bled daily from the first day of estrus until subsequent estrus or day 21 (control estrus). Although estrus was detected in all mares, 14 of 18 mares ovulated subsequent to treatments and four of the six control estrus mares ovulated. Only among HCG treated mares was the ovulation rate higher (P < .05) than it was in the control estrus group. The interval from last progesterone injection or norgestomet implant removal to estrus did not differ between treatment groups. Concentrations of estradiol and LH were increased for several days around the time of ovulation and tended to be positively correlated with each other. In the mares that did not ovulate, concentrations of LH and estradiol appeared to be lower than in mares that ovulated. In summary, progestins in combination with PGF₂α and increasing light will synchronize estrus in mares during late winter and HCG will hasten ovulation in some mares.     

Ovarian activity and plasma concentrations of progesterone and estradiol during pregnancy in jennies

The objectives of this study were to determine ovarian activity (with ultrasound) and plasma concentrations of progesterone and estradiol during pregnancy in jennies. There was considerable ovarian activity during the second month of pregnancy. Secondary corpora lutea (total of 2 to 7 per jenny) were formed (mainly by luteinization) starting on Day 41.8 +/- 1.0 (range Days 38 to 46; ovulation = Day 0). The echogenicity of the primary and secondary corpora lutea gradually decreased during pregnancy. Plasma progesterone concentrations increased between Days 0 and 10 (0.9 and 19.9 ng/mL, respectively), gradually decreased to Day 30 (12.1 ng/mL), increased between Days 30 and 40 (plateau, at approximately 17 ng/mL), gradually declined from Days 110 to 160 (nadir of approximately 6 ng/mL), and remained nearly constant until increasing again just before parturition. Plasma estradiol concentrations increased gradually from Day 65, peaked (1.2 ng/mL) on Day 165 (> or = 1 ng/mL on Days 150 to 210), and decreased thereafter, with very low concentrations during the last 20 d before parturition. Ovarian function and hormone profiles were generally similar to those previously reported during pregnancy in mares.     

Ovarian follicular development and hormone concentrations in inseminated dairy cows with resynchronized estrous cycles

The objective was to investigate ovarian follicular development and hormone concentrations in previously inseminated cows with estrous cycles resynchronized with various resynchronization treatments. Lactating dairy cows were treated with a previously used intravaginal progesterone releasing device (IVD) for 7d (EB+IVD 7+EB, n=15) or 8d (EB+IVD 8+EB, n=16), starting 13d (Day 13) after a first estrus (Day 0) and AI. Estradiol benzoate (EB; 1mgim) was given at device insertion and 24h after removal. Other cows were given the same treatment as the EB+IVD 8+EB cows, but were not treated with EB at IVD insertion (IVD 8+EB, n=11). There were no differences (P>0.05) between EB+IVD 7+EB and EB+IVD 8+EB treatments for follicle dynamics and plasma progesterone concentrations during treatment. Based on a comparison between the IVD 8+EB treated cows and the pooled results of the EB+IVD 7+EB and EB+IVD 8+EB treated cows, EB at device insertion increased the number of follicular waves between Days 13 and 20 (mean+/-S.E.M.; 2.3+/-0.14 vs 2.7+/-0.10, P=0.033), delayed emergence of follicles that were dominant or emerging on Day 20 (17.2+/-0.36 vs 14.1+/-0.65d, P<0.001), reduced diameters of dominant or emerging follicles on Day 20 (9.0+/-0.58 vs 12.7+/-0.59, P<0.001), and reduced plasma progesterone concentrations by 0.85+/-0.44ng/mL (P=0.059) during treatment. Furthermore, comparison of the IVD 8+EB to the EB+IVD 8+EB treated cows demonstrated that treatment with EB at device insertion also reduced the diameter of ovulatory follicles (14.2+/-0.58 vs 19.0+/-0.71mm, P=0.001), delayed emergence of ovulatory follicles (17.0+/-0.32 vs 13.5+/-1.26, P=0.020), and reduced the interval from emergence to ovulation (7.0+/-0.32 vs 10.5+/-1.26d, P=0.020). We concluded that administration of EB altered ovarian follicular dynamics and tended to reduce plasma progesterone concentrations during treatment with an IVD that was used to resynchronize estrous cycles. However, use of a 7-d compared to an 8-day treatment with an IVD did not significantly affect follicle dynamics nor plasma progesterone concentrations during treatment.     

Frequency of luteinizing hormone pulses in cattle influences duration of persistence of dominant ovarian follicles,follicular fluid concentrations of steroids,and activity of insulin-like growth factor binding proteins

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.     

Induction of ovulation in the acyclic postpartum ewe following continuous, low-dose subcutaneous infusion of GnRH

Pituitary and ovarian responses to subcutaneous infusion of GnRH were investigated in acyclic, lactating Mule ewes during the breeding season. Thirty postpartum ewes were split into 3 equal groups; Group G received GnRH (250 ng/h) for 96 h; Group P + G was primed with progestagen for 10 d then received GnRH (250 ng/h) for 96 h; and Group P received progestagen priming and saline vehicle only. The infusions were delivered via osmotic minipumps inserted 26.6 +/- 0.45 d post partum (Day 0 of the study). Blood samples were collected for LH analysis every 15 min from 12 h before until 8 h after minipump insertion, then every 2 h for a further 112 h. Daily blood samples were collected for progesterone analysis on Days 1 to 10 following minipump insertion, then every third day for a further 25 d. In addition, the reproductive tract was examined by laparoscopy on Day -5 and Day +7 and estrous behavior was monitored between Day -4 and Day +7. Progestagen priming suppressed (P < 0.05) plasma LH levels (0.27 +/- 0.03 vs 0.46 +/- 0.06 ng/ml) during the preinfusion period, but the GnRH-induced LH release was similar for Group G and Group P + G. The LH surge began significantly (P < 0.05) earlier (32.0 +/- 3.0 vs 56.3 +/- 4.1 h) and was of greater magnitude (32.15 +/- 3.56 vs 18.84 +/- 4.13 ng/ml) in the unprimed than the primed ewes. None of the ewes infused with saline produced a preovulatory LH surge. The GnRH infusion induced ovulation in 10/10 unprimed and 7/9 progestagen-primed ewes, with no significant difference in ovulation rate (1.78 +/- 0.15 and 1.33 +/- 0.21, respectively). Ovulation was followed by normal luteal function in 4/10 Group-G ewes, while the remaining 6 ewes had short luteal phases. In contrast, each of the 7 Group-P + G ewes that ovulated secreted progesterone for at least 10 d, although elevated plasma progesterone levels were maintained in 3/7 unmated ewes for >35 d. Throughout the study only 2 ewes (both from Group P + G) displayed estrus. These data demonstrate that although a low dose, continuous infusion of GnRH can increase tonic LH concentrations sufficient to promote a preovulatory LH surge and induce ovulation, behavioral estrus and normal luteal function do not consistently follow ovulation in the progestagen-primed, postpartum ewe.     

Changes in patterns of luteinizing hormone secretion before and after the first ovulation in the postpartum mare

To determine whether luteinizing hormone (LH) secretion during the first estrous cycle postpartum is characterized by pulsatile release, circulating LH concentrations were measured in 8 postpartum mares, 4 of which had been treated with 150 mg progesterone and 10 mg estradiol daily for 20 days after foaling to delay ovulation. Blood samples were collected every 15 min for 8 h on 4 occasions: 3 times during the follicular phase (Days 2-4, 5-7, and 8-11 after either foaling or end of steroid treatment), and once during the luteal phase (Days 5-8 after ovulation). Ovulation occurred in 4 mares 13.2 +/- 0.6 days postpartum and in 3 of 4 mares 12.0 +/- 1.1 days post-treatment. Before ovulation, low-amplitude LH pulses (approximately 1 ng/ml) were observed in 3 mares; such LH pulses occurred irregularly (1-2/8 h) and were unrelated to mean circulating LH levels, which gradually increased from less than 1 ng/ml at foaling or end of steroid treatment to maximum levels (12.3 ng/ml) within 48 h after ovulation. In contrast, 1-3 high-amplitude LH pulses (3.7 +/- 0.7 ng/ml) were observed in 6 of 7 mares during an 8-h period of the luteal phase. The results suggest that in postpartum mares LH release is pulsatile during the luteal phase of the estrous cycle, whereas before ovulation LH pulses cannot be readily identified.     

Comparison of the effects of two GnRH antagonists on LH and FSH secretion,follicular growth and ovulation in the mare

The effects of two GnRH antagonists were tested in order to delay and/or synchronise ovulation in mares. Five mares received Antarelix (0.01 mg.kg(-1)), 5 mares received Cetrorelix (the same dose), 5 mares (control mares) received the vehicle intravenously, twice daily, for 8 days from the day the largest follicle reached 22 mm following prostaglandin administration. Ovulation was postponed in all mares injected with Antarelix (19.4 +/- 1.2 days after the beginning of the treatment) and in 2/5 mares injected with Cetrorelix (20 +/- 1 days) vs. 6.2 +/- 0.4 days in control mares. During the treatment, LH concentrations were strongly depressed in Antarelix and in Cetrorelix mares (1.6 +/- 0.1 and 3.8 +/- 0.5 ng.mL(-1) respectively vs. 21 +/- 2.5 ng.mL(-1) in control mares). In the 3 Cetrorelix mares which ovulated during the treatment. 2 initiated their LH surge at this moment. FSH concentrations were not affected in Antarelix or in Cetrorelix mares during the treatment (11.4 +/- 1.3 and 7.9 +/- 0.8 ng.mL(-1) respectively vs. 10.5 +/- 0.8 ng.mL(-1) in control mares). In conclusion, Antarelix seems more efficient than Cetrorelix for postponing ovulation in mares. The role of LH in antral follicular development before the preovulatory stage is confirmed.     

Steroid concentrations in follicular fluid aspirated repeatedly from transitional and cyclic mares

The objectives of the present study were to determine follicular progesterone (P4) and estradiol-17beta (E2) in transitional mares and to compare follicular steroid concentrations between transitional and cyclic mares. Follicles > 8 mm were aspirated under transvaginal ultrasound-guidance 4 times at 3 to 4 day intervals (T1-T4) in Norwegian pony mares during vernal transition. During the breeding season, follicular aspirations were conducted in each mare on Day 6, Day 14 and Day 18 after ovulation of 3 separate estrous cycles (Day of ovulation = Day 0). Plasma and follicular fluids were analyzed for P4 and E2 with ELISA and RIA, respectively. Plasma P4 concentrations remained below 1 ng/mL throughout T1-T4, while the follicular P4 concentrations increased significantly to cyclic levels after the first transitional aspiration. Plasma E2 concentrations similarly remained at low levels during the course of the transitional aspirations, while the follicular E2 concentrations increased gradually over the 4 aspirations to cyclic concentrations. The mares ovulated on average 9.8 +/- 1.6 (mean +/- SEM) days after the last transitional aspiration, and 16.6 +/- 0.2, 11.3 +/- 1.5 and 23.2 +/- 4.4 days after aspirations conducted on Day 6, 14 and 18, respectively. The present study demonstrates that in the transitional mare newly developing follicles exhibit biosynthesis of P4 and E2. Furthermore, an increase in follicular steroid concentrations is not necessarily reflected in the peripheral steroid concentrations.     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.     

Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus x Bos taurus, and Bos taurus heifers

The objective of this study was to evaluate the effects of treatment with an intravaginal progesterone-releasing device (CIDR) and estradiol benzoate (EB) on follicular dynamics in Bos indicus (n=23), Bos taurus (n=25), and cross-bred (n=23) heifers. To assess the influence of reduced serum progesterone concentrations during 8 days of treatment with a progesterone-releasing device on follicular dynamics, half of the heifers received PGF at CIDR insertion (Day 0; 3 x 2 factorial design). Mean (+/-S.E.M.) serum progesterone concentrations during CIDR treatment varied (P<0.05) among genetic groups: B. indicus (5.4+/-0.1 ng/mL), B. taurus (3.3+/-0.0 ng/mL), and cross-bred (4.3+/-0.1 ng/mL). Maximum diameter of the dominant follicle (DF) was smaller (P<0.01) in B. indicus heifers (9.5+/-0.5 mm) than in cross-bred (12.3+/-0.4 mm) or B. taurus heifers (11.6+/-0.5 mm). B. indicus experienced lower (P<0.01) ovulation rate (39.1%) than did B. taurus (72.7%) and cross-bred (84.0%). Heifers treated with PGF on Day 0 had lower (P<0.05) serum progesterone concentrations during progesterone treatment. The PGF treatment on Day 0 increased (P<0.01) the diameter of the DF (11.9+/-0.4 mm vs. 10.5+/-0.4 mm). Moreover, greater (P=0.02) ovulation rates (78.8 vs. 54.0%) occurred in heifers treated with PGF on Day 0. In summary, B. indicus heifers had greater serum progesterone concentrations, smaller DF diameter, and a lower ovulation rate compared to B. taurus heifers. Prostaglandin treatment on the day of CIDR insertion reduced serum progesterone during treatment, and resulted in increased maximum DF diameter and ovulation rate.