Pituitary responsiveness to GnRH in mares following deslorelin acetate implantation to hasten ovulation

The present experiment characterized the pituitary responsiveness to exogenous GnRH in the first 10 d after ovulation following commercially available deslorelin acetate implantation at the normal dosage for hastening ovulation in mares. Twelve mature, cyclic mares were assessed daily for estrus and three times weekly for ovarian activity starting May 1. Mares achieving a follicle at least 25 mm in diameter or showing signs of estrus were checked daily thereafter for ovarian characteristics. When a follicle >30 mm was detected, mares were administered either a single deslorelin acetate implant or a sham injection and then assessed daily for ovulation. On d 1, 4, 7, and 10 following ovulation, each mare was challenged i.v. with 50 microg GnRH, and blood samples were collected to characterize the LH and FSH responses. The size of the largest follicle on the day of treatment did not differ (P = 0.89) between groups. The number of days from treatment to ovulation was shorter (P < 0.001) by 2.0 d for the treated mares indicating a hastening of ovulation. The size of the largest follicle present on the days of GnRH challenge was larger in the treated mares on d 1 (P = 0.007) but smaller on d 10 (P = 0.02). In addition, the interovulatory interval was longer (P = 0.036) in the treated mares relative to controls by 4.4 d. Concentrations of FSH in plasma of the treated mares were lower (P < 0.05) than control concentrations from d 3 to 12; LH concentrations in the treated mares were lower (P < 0.05) relative to controls on d 0 to 5, d 7, and again on d 20 to 23. Progesterone values were the same (P = 0.99) for both groups from 2 d before ovulation though d 23. There was an interaction of treatment, day, and time of sampling (P < 0.001) for LH and FSH concentrations after injection of GnRH. Both the LH and FSH responses were suppressed (P < 0.009) in the treated mares relative to controls on d 1, 4, and 7; by d 10, the responses of the two groups were equivalent. In conclusion, deslorelin administration in this manner increased the interovulatory interval, consistently suppressed plasma LH and FSH concentrations, and resulted in a complete lack of responsiveness of LH and FSH to GnRH stimulation at the dose used during the first 7 d after the induced ovulation. Together, these results are consistent with a temporary down-regulation of the pituitary gland in response to deslorelin administered in this manner.     

Comparison of two dosage regimens of the GNRH agonist deslorelin acetate on inducing ovulation in seasonally anestrous mares

This study investigated the efficacy of two dosage regimens of a potent GnRH analogue (GnRHa), deslorelin acetate, in inducing ovulation in seasonally anestrous mares. Forty-five seasonally anestrous mares were randomly assigned according to follicular size to one of three treatment groups: control, increasing GnRHa dose, and constant GnRHa dose. Treatment began on February 28 and continued until ovulation or for a maximum of seven treatments. Mares were palpated every other day until a 35 mm follicle was detected, then every day until ovulation or regression of the follicle occurred. Blood samples were taken from five randomly chosen mares in each treatment group and analyzed for LH levels.Twenty percent of mares in both deslorelin treatment groups ovulated, while no control mares ovulated during the treatment period. There was no difference in the number of mares that ovulated between treatment groups. Four of the six mares that ovulated were in transitional anestrus at the initiation of treatment, while only two were in deep anestrus.Concentrations of LH were greater (p=0.0008) in both GnRH-treated groups than in the control mares. Concentrations of LH did not differ between the two GnRH-treated groups until day 12 of treatment, when mares treated with a constant dosage had higher (p=0.0358) levels of LH than those treated with an increasing dosage. It is possible that administration of larger amounts of the GnRH agonist lowered the sensitivity of the pituitary to stimulation by GnRH.Deslorelin acetate did stimulate follicular growth and ovulation in a limited number of anestrous mares. Further investigation into the potential of this short-term implant to shorten the onsent of the breeding season is recommended.     

Clinical experience with deslorelin (ovuplantTM) in a kentucky thoroughbred broodmare practice (1999)

Palpation records of 155 Throughbred broodmares maintained on one of seven farms (3–80 mares per farm) that were administered deslorelin on one or more estrous cycles (204 treated cycles) during the 1999 breeding season were retrospectively examined. Some deslorelin-treated mares were also treated with hCG (2500 units intravenously), or had no ovulation-inducing drugs administered, during different estrous cycles of the same season. Most mares were treated with an ovulation- inducing drug after returning to their resident farm following breeding and were subsequently examined by transrectal ultrasonography daily until ovulation was confirmed, and again 13–14 and 15–16 days after ovulation for determination of pregnancy status.Per-cycle pregnancy rate for all 155 mares bred was 53%, and for all deslorelin breeding was 57%. Per-cycle pregnancy rates for mares ovulating 0–1 days, 1–2 days, and 2–3 days after treatment with deslorelin did not differ (P>0.05). Forty-six mares received more than one treatment during the breeding season, yielding 115 breedings (estrous cycles) for comparison of pregnancy rates among treatment. Per-cycle pregnancy rates for these mares did not differ among treatments (P>0.10).No differences due to treatment were detected in mean interval to ovulation (P>0.10). Mean interovulatory interval was longer for deslorelin-treated mares than for untreated or hCG treated mares (P>0.01). Eighty percent (80%) of deslorelin-treated mares had interovulatory intervals of 18–25 days, and 19% had interovulatory intervals>25 days. Ninety-seven percent (97%) of untreated or hCG-treated mares had interovulatory interovulatory intervals>25 days. More deslorelin-treated mares had extended (>25 days) interovulatory intervals than hCG- or nontreated-mares (P>0.05). In this group of Thoroughbred mares, it appeared that season (month) and management (farm) factors had only minor effects on the incidence of extended interovulatory intervals following use of deslorelin.     

Efficacy of Deslorelin Acetate (SucroMate) on Induction of Ovulation in American Quarter Horse Mares

Equine clinicians rely on ovulation induction agents to provide a timed ovulation in mares for optimal breeding management. Numerous studies have been performed on the efficacy of human chorionic gonadotropin (hCG) to induce ovulation in the mare, but limited clinical data are available for the new deslorelin acetate product SucroMate. This study was designed to evaluate the efficacy of SucroMate (deslorelin) in comparison with hCG to induce ovulation. American Quarter horse mares (n = 256) presented to Colorado State University for breeding management were used in this study. Mares received either deslorelin or hCG when a follicle ≥35 mm was detected by transrectal ultrasound in the presence of uterine edema. Ultrasonographic examinations were subsequently performed once daily until ovulation was detected. Deslorelin was administered to 138 mares during168 estrous cycles, and hCG was given to 118 mares during 136 estrous cycles. Mares administered deslorelin had a similar (P < .05) higher ovulation rate (89.9%) within 48 hours following drug administration than mares administered hCG (82.8%). There are no effects of season or age on ovulation rates in either treatment group. Twenty-one mares administered deslorelin and 11 mares administered hCG were monitored by transrectal ultrasound every 6 hours to detect ovulation as part of a frozen semen management program. Average intervals from deslorelin or hCG administration to ovulation were 41.4 ± 9.4 and 44.4 ± 16.5 hours, respectively. Results of this study indicate that SucroMate is effective at inducing a timed ovulation in the mare.     

Testosterone propionate treatment of stallions: Effects on secretion of luteinizing hormone and follicle stimulating hormone in daily samples and after administration of gonadotropin releasing hormone

Ten stallions were used to determine if the stallion responds to administration of testosterone propionate (TP) with an increase in follicle stimulating hormone (FSH) secretion after administration of gonadotropin releasing hormone (GnRH) as has been previously observed for geldings and intact and ovariectomized mares. Five stallions were treated with TP (350 μg/kg of body weight) in safflower oil every other day for 11 days; control stallions received injections of safflower oil. The response to GnRH (1.0 μg/kg of body weight) was determined for all stallions before the onset of treatment (GnRH I) and at the end of treatment (GnRH II). Blood samples were also withdrawn daily from 3 days prior to treatment through GnRH II. Treatment with TP decreased (P<.10) concentrations of FSH in daily blood samples. However, treatment with TP did not affect (P>.10) the GnRH-induced secretion of FSH. Concentrations of luteinizing hormone (LH) decreased (P<.05) in daily blood samples averaged over both groups of stallions and were lower (P<.10) in TP-treated stallions than in controls during the latter days of treatment. We conclude that TP administration to stallions does not alter the FSH response to GnRH as has been observed for geldings and for mares of several reproductive states.     

Effects of deslorelin acetate implants in horses: single implants in stallions and steroid-treated geldings and multiple implants in mares

Three experiments were performed to test the following hypotheses: 1) stallions and/or progesterone-estradiol-treated geldings could serve as models for the effects of a single implant of the GnRH analog, deslorelin acetate, on LH and FSH secretion by mares; and 2) multiple implants of deslorelin acetate could be used as a means of inducing ovarian atrophy in mares for future study of the mechanisms involved in the atrophy observed in some mares after a single implant. In Exp. 1, nine light horse stallions received either a single deslorelin implant (n = 5) or a sham injection (n = 4) on d 0. In Exp. 2, 12 geldings received daily injections of progesterone on d -20 through -4, followed by twice-daily injections of estradiol on d -2 to 0. On the morning of d 0, geldings received either a single deslorelin implant (n = 6) or a sham injection (n = 6). Daily injections of progesterone were resumed on d 2 through 15. In Exp. 1, plasma LH and FSH were elevated (P < 0.05) in the treatment group relative to controls at 4, 8, and 12 h after implant insertion. In the treated stallions, FSH was decreased (P < 0.05) on d 3 to 13, and LH was decreased on d 6 to 13. In Exp. 2, plasma LH and FSH were elevated (P < 0.05) at 4,8, and 12 h after deslorelin implant insertion. Plasma LH was suppressed (P < 0.05) below controls on d 2 to 7, 9, and 11 to 15; plasma FSH was suppressed (P < 0.05) on d 4 to 15. In Exp. 3, 21 mares were used to determine whether multiple doses of deslorelin would cause ovarian atrophy. Mares received one of three treatments: 1) sham injections; 2) three implants on the first day; or 3) one implant per day for 3 d (n = 7 per group). Treatment with multiple implants increased (P < 0.05) the interovulatory interval by 14.8 d and suppressed (P < 0.01) LH and FSH concentrations for approximately 25 d; no mare exhibited ovarian atrophy. In conclusion, after an initial short-term increase in LH and FSH secretion, deslorelin implants caused long-term suppression of both gonadotropins in stallions as well as in geldings treated with progesterone and estradiol to mimic the estrous cycle. It is likely that either of these models may be useful for further study of this suppression in horses. Although multiple implants in mares suppressed gonadotropin secretion longer than a single implant, the lack of ovarian atrophy indicates that the atrophy observed after a single implant in previous experiments was likely due to the susceptibility of individual mares.     

Deslorelin acetate (Ovuplant) therapy in cycling mares: effect of implant removal on FSH secretion and ovarian function

Following induction of ovulation with deslorelin acetate (Ovuplant), gonadotrophin concentrations are reduced in the subsequent cycle, leading to increased interovulatory intervals in some mares. This study determined whether implant removal after 2 days prevented the decrease in gonadotrophin concentrations and follicular growth during the ensuing cycle. Twenty-four mares were randomised equally into 3 groups. Group 1 ovulated spontaneously, Groups 2 and 3 received the deslorelin implant to induce ovulation. Two days after treatment, the implant was removed from Group 3. On Day 10 postovulation, FSH was lower (P = 0.009) in Group 2, but not different between Groups 1 and 3. Follicular diameter on Day 14 was less (P<0.05) in Group 2 (19.0 +/- 2.1 mm) than in Groups 1 and 3 (36.6 +/- 2.5 and 30.5 +/- 2.0 mm, respectively). Interovulatory interval was longer (P<0.05) for Group 2 (25.8 +/- 2.9 days) compared to Groups 1 and 3 (18.5 +/- 0.7 and 19.4 +/- 0.3 days, respectively). Removal of the deslorelin implant eliminated the decreased FSH secretion and the increased interovulatory interval associated with implant administration. Therefore, it is recommended that the implant be removed after ovulation is detected to prevent the occurrence of a prolonged interovulatory interval.     

Testosterone administration to mares during estrus: duration of estrus and diestrus and concentrations of LH and FSH in plasma

To study the possible role of ovarian androgens in regulation of follicle stimulating hormone (FSH) secretion in the cycling mare, five mature, intact mares were treated with testosterone (20 micrograms/kg of body weight) daily during estrus; five control mares received safflower oil on the same schedule. Mares were teased for estrus and samples of jugular blood were drawn daily through one full estrous cycle. Concentrations of FSH in plasma were measured by a newly developed radioimmunoassay based on anti-ovine FSH serum and radioiodinated equine FSH. Testosterone treatment during estrus had no effect on duration of estrus, diestrus or the total cycle. Concentrations of FSH in plasma during estrus were unaffected by testosterone treatment. However, FSH concentrations in testosterone-treated mares were elevated (P less than .05) compared with controls during mid-diestrus (d 6 through 11). The magnitude and timing of the LH peaks were unaffected by treatment, as was the day on which the first elevated progesterone concentration occurred. These data are consistent with a model of FSH secretion in which ovarian androgens cause an accumulation of FSH in the pituitary during estrus in preparation for the surges that occur in FSH secretion during diestrus.     

Clinical experience with native GnRH therapy to hasten follicular development and first ovulation of the breeding season

Breeding records of 48 Thoroughbred and Standardbred mares treated with native GnRH (500μg im, bid) during February—April, 1999 or 2000, on 7 farms in central Kentucky were retrospectively examined. Treated mares were classified as being in anestrus or early transition (n=42; if no signs of estrus occurred within 31/2 weeks and the largest follicle remained ≤25 mm in diameter or the first larger follicle(s) of the season regressed without ovulating), or were classified as being in late transition (n=6; if follicular growth achieved 30-40 mm diameter but ovulation had not yet occurred during the breeding season). Thirty-eight mares (38/48; 79%) ovulated in 13.7 ± 7.4 days. Interval to ovulation was negatively associated with size of follicles at onset of native GnRH therapy (P < 0.01). Per cycle pregnancy rate was 53% (19/36 mares bred). Ovulation inducing drugs were administered to 32 of the native GnRH treated mares (2500 units hCG intravenously, n = 20; deslorelin implant [Ovuplant™] subcutaneously, n=12), while 6 mares were not administered any additional drugs to induce ovulation. Per cycle pregnancy rate did not differ among mares treated only with native GnRH (2/5 mares bred; 40% PR), mares treated with native GnRH plus hCG (12/19 mares bred; 63% PR), or mares treated with native GnRH plus Ovuplant™ (5/12 mares bred; 42% PR) (P > 0.10). Additional treatment with either hCG or Ovuplant™ did not alter mean follicle size at ovulation or interovulatory interval (P > 0.10). The proportion of interovulatory intervals > 25 days was not different between mares receiving no additional treatment to induce ovulation (0/4; 0%) compared to mares receiving hCG to induce ovulation (3/8; 38%) (P > 0.10), but the proportion of interovulatory intervals > 25 days was greater for mares receiving Ovuplant™ to induce ovulation (5/7; 71%) compared to mares receiving no additional treatment to induce ovulation (P < 0.05). The proportion of mares with extended interovulatory intervals (i.e., > 25 days) did not differ between mares with follicles < 15 mm diameter (4/8, 50%) and those with follicles > 15 mm diameter (3/11, 27%) at onset of native GnRH treatment (P > 0.10). While concurrent untreated controls were not used in this study, the 79% response rate to twice daily administration of native GnRH is in agreement with other reports using pulsatile or constant infusion as methods of administration, confirming therapy can hasten follicular development and first ovulation of the breeding season. As with previous reports, follicle size at onset of treatment is an important determinant of interval from onset of native GnRH therapy to ovulation. Use of hCG or Ovuplant™ did not enhance ovulatory response in native GnRH treated mares. Use of Ovuplant™ during native GnRH therapy may increase the incidence of post-treatment anestrus in mares not becoming pregnant.     

Superovulation in cycling mares using equine follicle stimulating hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation.In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation.Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares.In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3).In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Introduction

Induction of multiple ovulations or superovulation has been an elusive goal in the mare. Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates.[1 and 2] Superovulation also has been suggested as a critical requirement for other types of assisted reproductive technology in the horse, including oocyte transfer and gamete intrafallopian transfer. [2 and 3] Unfortunately, techniques used successfully to superovulate ruminants, such as administration of porcine follicle stimulating hormone and equine chorionic gonadotropin have little effect in the mare. [4 and 5]The most consistent therapy used to induce multiple ovulations in mares has been administration of purified equine pituitary gonadotropins. Equine pituitary extract (EPE) is a purified gonadotropin preparation containing approximately 6% to 10% LH and 2% to 4% FSH.[6] EPE has been used for many years to induce multiple ovulations in mares [7, 8 and 9] and increase the embryo recovery rate from embryo transfer donor mares. [10] Recently, a highly purified equine FSH product has become available commercially.The objectives of this study were to evaluate the efficacy of purified eFSH in inducing superovulation in cycling mares and to determine the relationship between ovulation rate and pregnancy rate or embryo collection rate in superovulated mares.

Materials and methods

Experiment 1

Forty-nine normally cycling mares, ranging in age from 3 to 12 years, were used in a study at Colorado State University. Group 1 (control) mares (n = 29) were examined daily when in estrus by transrectal ultrasonography. Mares were administered an implant containing 2.1 mg deslorelin (Ovuplant, Ft. Dodge Animal Health, Ft. Dodge, IA) subcutaneously in the vulva when a follicle 35 mm in diameter was detected. Mares were bred with frozen semen (800 million spermatozoa; minimum of 30% progressive motility) from 1 of 4 stallions 33 and 48 hours after deslorelin administration. The deslorelin implants were removed after detection of ovulation.[11] Pregnancy status was determined at 14 and 16 days after ovulation.Group 2 mares (n = 10) were administered 25 mg of eFSH (Bioniche Animal Health USA, Inc., Athens, GA) intramuscularly twice daily beginning 5 or 6 days after ovulation was detected. Mares received 250 g cloprostenol (Estrumate, Schering-Plough Animal Health, Omaha, NE) intramuscularly on the second day of eFSH treatment. Administration of eFSH continued until a majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Mares were subsequently bred with the same frozen semen used for control mares, and pregnancy examinations were performed as described above.Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting 5 or 6 days after ovulation and were administered 250 μg cloprostenol on the second day of treatment. Mares were randomly selected to receive either a deslorelin implant (n = 5) or 2500 IU of human chorionic gonadotropin (hCG) intravenously (n = 5) to induce ovulation when a majority of follicles reached a diameter of 35 mm. Mares were bred with frozen semen and examined for pregnancy as described above.

Experiment 2

Sixteen cycling light-horse mares were used during the physiologic breeding season in Brazil. Reproductive activity was monitored by transrectal palpation and ultrasonography every 3 days during diestrus and daily during estrus. On the first cycle, mares were administered 2500 IU hCG intravenously once a follicle 35 mm was detected. Mares were subsequently inseminated with pooled fresh semen from 2 stallions (1 billion motile sperm) daily until ovulation was detected. An embryo collection procedure was performed 7 days after ovulation. Mares were subsequently administered cloprostenol, and eFSH treatment was initiated. Mares received 12 mg eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time hCG was administered. Mares were inseminated and embryo collection attempts were performed as described previously.

Statistical analysis

In experiment 1, 1-way analysis of variance with F protected LSD was used to analyze quantitative data. Pregnancies per ovulation were analyzed by x² analysis. In experiment 2, number of large follicles, ovulation rate, and embryo recovery rate were compared by Student,'s t-test. Data are presented as the mean S.E.M. Differences were considered to be statistically significant at p < .05, unless otherwise indicated.

Results

In experiment 1, mares treated with 25 mg eFSH twice daily developed a greater number of follicles 35 mm in diameter (p = .001) and ovulated a greater number of follicles (p = .003) than control mares (Table 1). However, the number of pregnancies obtained per mare was not significantly different between the control group and the group receiving 25 mg eFSH (p = .9518). Mares treated with 12 mg eFSH and administered either hCG or deslorelin to induce ovulation also developed more follicles 35 mm (p = .0016 and .0003, respectively) than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles (p = .003) than control mares, whereas the number of ovulations for mares receiving eFSH followed by deslorelin was similar to that of control mares (p = .3463). Pregnancy rate for mares induced to ovulate with hCG was higher (p = .0119) than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of controls (p = .692). Pregnancy rate per ovulation was not significantly different between control mares (54.5%) and mares treated with eFSH followed by hCG (52.9%). The lowest pregnancy rate per ovulation was for mares stimulated with 25 mg eFSH and induced to ovulate with deslorelin. The mean number of days mares were treated with 25 mg or 12 mg of eFSH was 7.8 ± 0.4 and 7.5 ± 0.5 days, respectively. Overall, 80.0% of mares administered eFSH had multiple ovulations compared with 10.3% of control mares.     

Effects of dihydrotestosterone administration with and without estradiol pretreatment on gonadotropin secretion in ovariectomized pony mares

Twenty ovariectomized pony mares were used to determine if dihydrotestosterone propionate (DHTP) administration, with or without estradiol benzoate (EB) pretreatment, would have the same effects on follicle stimulating hormone (FSH) and luteinizing hormone (LH) secretion as testosterone propionate (TP) administration. All mares were given an initial injection of gonadotropin releasing hormone (GnRH) to characterize their LH and FSH response, and then two groups of mares (n = 4/group) were administered EB (22 micrograms/kg of body weight), two groups were administered vehicle (safflower oil) and a fifth group was administered TP (175 micrograms/kg of body weight) daily for 10 days. Following a second injection of GnRH, one group of EB-treated mares and one group of oil-treated mares were administered DHTP (175 micrograms/kg of body weight) daily for 10 days; the other EB- and oil-treated mares were administered oil and the TP-treated mares were continued on the same dose of TP for 10 days. A final injection of GnRH was then given. Treatment with EB increased (P less than .01) concentrations of LH in daily blood samples and increased (P less than .05) the LH response to exogenous GnRH. Administration of TP or DHTP reduced (P less than .05) both daily LH concentrations and the LH response to exogenous GnRH. Concentrations of FSH in daily blood samples were reduced (P less than .05) and the FSH response to exogenous GnRH was increased (P less than .05) by administration of EB alone, DHTP alone or TP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of the gnrh agonist deslorelin acetate for inducing ovulation in mares relative to age of mare and season

Deslorelin acetate (Ovuplant™, Fort Dodge), a GnRH agonist, is commonly used to induce ovulation in cycling mares. Although its efficacy in hastening ovulation has been previously reported, the effects of age of mare and month of administration on percent of mares responding and interval to ovulation have not been studied.Data was gathered from reproduction records of 376 mares receiving deslorelin acetate at the Equine Reproduction Laboratory, Colorado State University, from 1995 to 1999. Age of mare, date of administration, size of largest follicle at treatment, and interval to ovulation were recorded. Age of mare was categorized into five groups: 2–4, 5–9, 10–14, 15–19, and greater than or equal to 20 years. Date of administration was divided into four groups: March and April, May and June, July and August, and September and October.A higher (p < 0.05) percentage of mares aged 10–14 (98.5%) ovulated in response to deslorelin acetate than mares aged 2–4 or 5–9 (90.2% or 91.0%, respectively) or mares aged 15–19 or ≥ 20 (87.9% or 83.8%, respectively). Mares ≥ 20 had the lowest ovulation rate (83.8%). However, mares ≥ 20 that responded to deslorelin acetate had a shorter (p < 0.05) interval from treatment to ovulation (1.7 ± 0.1 days) than mares 2–4 and 5–9 years of age (1.9 ± 0.1 and 1.9 ± 0.0 days, respectively).Deslorelin acetate was more effective in inducing ovulation in the July and August (95.4%) (p < 0.01) and September and October (95.7%) (p = 0. 04) than in the March and April (81.1%). Mares treated in May through October also experienced shorter (p < 0.05) intervals to ovulation than mares treated in March and April.     

Attempts at Superovulation of Mares With Porcine Follicle Stimulating Hormone and Recombinant Equine Follicle Stimulating Hormone

Attempts to superovulate mares have been disappointing and expensive. Conflicting data exist on the effectiveness of porcine follicle stimulating hormone (pFSH) as a superovulatory treatment for horses. Recently, a recombinant equine FSH (reFSH) has become available with covalently linked alpha and beta subunits, which results in a longer half-life than endogenous FSH. The purpose of this study was to compare doses of pFSH and reFSH for superovulating mares. Twenty-nine mares received injections of 25, 50, 100, or 150 mg pFSH or 0.5 mg reFSH 2 times per day. Mares were used up to three times, with their second reproductive cycle serving as an untreated control. All treated mares were administered cloprostenol on the second day of treatment and given 2,500 IU of human chorionic gonadotropin 24 to 38 hours after the majority of large follicles were >30 mm. Mares with untreated control cycles also received cloprostenol, but deslorelin was used to induce ovulation. No response from superstimulation treatments differed (P > .1) from those of controls; mean ovulations per cycle ranged from 0.85 to 1.31; mean embryo recovery rates ranged from 0.66 to 1.08. Two of the eight mares treated with reFSH failed to ovulate. Porcine FSH was ineffective at inducing multiple ovulations at any dose. Although previous studies of reFSH yielded high ovulation rates, further research is needed to establish optimal protocols and to determine the cause of failed ovulations.     

Effects of gonadotropin-releasing hormone infused in a pulsatile or continuous fashion on serum gonadotropin concentrations and ovulation in the mare.

Studies were conducted to compare continuous vs pulsatile i.v. infusion of GnRH on serum gonadotropin concentrations and ovulation in seasonally anestrous mares and in cycling mares. Anestrous mares (Exp. 1) received no treatment (control; n = 3), 2, or 20 micrograms of GnRH/h continuous infusion (CI) (n = 4 and n = 6, respectively), or 20 micrograms of GnRH/h pulsatile infusion (PI) (n = 5). After initiation of GnRH infusion, serum LH levels increased earlier, and to a greater extent, in the PI group than in other groups (P less than .05). In contrast, serum FSH concentrations did not differ among groups. The number of days to development of the first 35-mm follicle was not different among GnRH treatment groups; however, mares receiving PI ovulated on d 9.4 of treatment, 2.8 d earlier than those receiving 20 micrograms of GnRH/h CI (P less than .05). Mares given 2 micrograms of GnRH/h CI failed to ovulate spontaneously after 16 d of treatment, but each one ovulated within 2 to 4 d after injection of 2,000 IU of hCG on d 16. Control mares did not ovulate or show any significant follicular development throughout the experiment. Cycling mares (Exp. 2) received no treatment (control; n = 6), 20 micrograms of GnRH/h CI, or 20 micrograms of GnRH/h PI (n = 4) beginning on d 16 of an estrous cycle (d 0 = day of ovulation). Serum LH concentrations in all groups increased after initiation of treatment; however, on the day of ovulation LH concentrations were lower in the CI group than in the PI or control groups (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive performance of commercial broodmares after induction of ovulation with HCG or Ovuplant™ (deslorelin)

Soon after Ovuplant™, the sustained-release implant containing the gonadotropin releasing hormone (GnRH) agonist deslorelin, was approved for commercial use in the United States for induction of ovulation in mares, anecdotal field observations were reported that some Ovuplant™—treated mares that did not become pregnant experienced a delayed return to estrus and prolonged inter-ovulatory interval. Although those observations have been subsequently confirmed, further data on how mares respond to Ovuplant™ compared to human chorionic gonadotropin (hCG) during the post-treatment period is needed. The objective of this study was to further evaluate the clinical use of Ovuplant™ by comparing the reproductive performance of commercial broodmares treated with hCG or Ovuplant™. This retrospective study was completed by examining the 1999 reproductive records of 106 mares treated with hCG during 134 estrous cycles and 117 mares treated with Ovuplant™ during 151 estrous cycles. There were no differences (P > 0.10) in follicle size at the time of treatment (39.4 ± 0.5 vs. 38.9 ± 0.5 mm), interval from treatment to ovulation (2.2 ± 0.1 vs. 2.2 ± 0.1 days), proportion of mares that failed to ovulate after treatment (3.0 vs. 4.6 %), or per-cycle pregnancy rate (47.7 vs. 51.4 %) between hCG-and Ovuplant™-treated mares, respectively. The interval from ovulation to return to estrus (25.8 ± 1.3 vs. 15.5 ± 0.6 days) and the inter-ovulatory interval (30.4 ± 1.5 vs. 20.8 ± 0.6 days) were longer (P<0.001) for Ovuplant™-compared to hCG-treated mares, and the proportion of non-pregnant mares that failed to return to estrus within 30 days after ovulation (31.4 vs. 1.5 %) was higher (P<0.001) for Ovuplant™-compared to hCG-treated mares, respectively. For Ovuplant™—treated mares, follicle size at the time of treatment tended (P<0.1) to be smaller for mares that failed to return to estrus within 30 days compared to mares that returned to estrus within 30 days (37.1 ± 1.1 vs. 40.1 ± 0.6 mm, respectively). Also, the average date of ovulation during the calendar year was later (P < 0.05) for Ovuplant™—treated mares that failed to return to estrus within 30 days compared to those that returned to estrus within 30 days (May 15 ± 4 vs. April 30 ± 4 days). The results of this study confirm previous reports that although the ovulatory response and fertility were not different for hCG- and Ovuplant™—treated mares, mares treated with Ovuplant™ that did not become pregnant had a significantly delayed return to estrus and prolonged inter-ovulatory interval. Based on recently published information, it appears this effect is due to Ovuplant™—induced down-regulation of the pituitary gland, which suppresses subsequent follicular growth and development. This study also demonstrated that follicle size and/or season may influence the probability that Ovuplant™—treated mares would experience a delayed return to estrus/ovulation; therefore, further work is needed to determine whether these or other factors are related to this specific outcome following Ovuplant™—treatment.     

Testosterone effects on gonadotropin response to GNRH: cows and pony mares

Effects of testosterone propionate (TP) treatment on plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) before and after an injection of gonadotropin releasing hormone (GnRH) were studied using ovariectomized cows and pony mares. An initial injection of GnRH (1 microgram/kg of body weight) was followed by either TP treatment or control injections for 10 (cows) or 11 (ponies) d. A second GnRH injection was administered 1 d after the last TP or oil injection. Concentrations of LH and FSH were determined in samples of plasma taken before and after each GnRH injection. Control injections did not alter the response to GnRH (area under curve) nor the pre-GnRH concentrations of LH and FSH in ovariectomized cows or ponies. Testosterone treatment increased (P less than .01) the FSH release in response to GnRH in ovariectomized mares by 4.9-fold; there was no effect in cows, even though average daily testosterone concentrations were 59% higher than in pony mares. Testosterone treatment reduced the LH release in response to GnRH by 26% in ovariectomized mares (P less than .05) and by 17% in ovariectomized cows (P approximately equal to .051). These results are consistent with a model that involves ovarian androgens in the regulation of FSH secretion in the estrous cycle of the mare, but do not support such a model in the cow.     

Acceleration and Timing of Fertile Ovulation in Cyclic Mares with a Deslorelin Implant

In a blinded trial, the effectiveness and safety of 2.2 mg of the GnRH analog deslorelin acetate, administered in a short–term implant (STI) to normally cycling mares in estrus with a dominant ovarian follicle of 30 mm in diameter or larger, were evaluated, using a placebo implant as a negative control. A total of 39 mares received treatments at admittance with pre–randomized implants containing either 2.2 mg or 0 mg deslorelin. Mares were teased daily and examined rectally with ultrasound at 24 h intervals to determine time to Ovulation and duration of estrus. The number of breedings and the pregnancy rate at 18 (±3) and 38 (±3) days were recorded, as were systemic side effects and local reactions at the implantation sites. Pregnancies resulting from breedings during the treatment estrus and/or from breedings during the next estrus were followed and the early and late pregnancy loss rate, the number of pregnancies going to term and of live–born foals was recorded.Mean follicle diameter at treatment was not significantly different between the deslorelin and placebo treatment group with 41.6 mm and 40.8 mm, respectively. Treatment with deslorelin STI reduced the time interval to Ovulation significantly from 69.5±25.48 h to 42.7±12.35 h (p<0.001). The percentage of mares having ovulated within 48 h rose from 26.3% to 95.0%, respectively, for placebo and deslorelin STI (p<0.001). As a consequence, the duration of estrus in days and the percent of animals requiring more than 1 breeding were significantly reduced in deslorelin treated animals from 5.4 days to 4.6 days, and from 55.6% to 5.0%, respectively (p=0.009 and =0.001). The percent of mares pregnant from breedings at the treatment estrus (65.0% versus 44.4%) or the next estrus (83.3% versus 92.3%) was satisfactory and similar for deslorelin and placebo treated mares (p>0.005), and in 70.0% and 66.7% of these once or twice bred mares did pregnancies go to term and live foals were born. kw|Keywords|k]GnRH     

Effects of active immunization against gonadotropin releasing hormone on gonadotropin secretion after ovariectomy and testosterone propionate administration to mares

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) conjugated to bovine serum albumin (BSA) to study the involvement of GnRH in luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion following ovariectomy (OVX) and after administration of testosterone propionate (TP). Five mares immunized against BSA served as controls. Immunizations were started on November 1, and OVX was performed in June (d 1). All mares were treated with TP from d 50 to 59 after OVX. On the day of OVX, concentrations of LH were lower (P less than .05) in GnRH-immunized mares than in BSA-immunized mares and were generally nondetectable; FSH concentrations were reduced (P less than .05) by 50% in GnRH-immunized mares relative to BSA-immunized mares. In contrast to BSA-immunized mares, plasma concentrations of LH or FSH did not increase after OVX in GnRH-immunized mares. The LH response to GnRH analog (less than .1% cross-reactive with GnRH antibodies) on d 50 was reduced (P less than .05) by 97% in GnRH-immunized mares relative to BSA-immunized mares, whereas the FSH response was similar for both groups. Treatment with TP for 10 d reduced (P less than .01) the LH response and increased (P less than .01) the FSH response to GnRH analog in BSA-immunized mares, but it had no effect (P greater than .1) on the response of either gonadotropin in GnRH-immunized mares.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dihydrotestosterone benzoate administration on gonadotropin secretion in ovariectomized pony mares

Eight long-term ovariectomized pony mares were treated with either dihydrotestosterone (DHT) benzoate (400 micrograms/kg body weight) in safflower oil or an equivalent amount of oil every other day for 21 d to determine the effects of DHT on follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in blood samples drawn once daily and after administration of three successive injections of gonadotropin releasing hormone (GnRH). The GnRH injections were given at 4-h intervals on the day following the last DHT or oil injection. Treatment with DHT benzoate did not alter (P greater than .10) concentrations of FSH or LH in daily blood samples relative to controls. The FSH and LH response, assessed by areas under the GnRH curves, decreased (P less than .05) from the first to third injection of GnRH when averaged over both groups of mares. There was no effect of DHT treatment on FSH response to GnRH. There was an interaction (P less than .05) between treatment and GnRH injection for LH areas; areas decreased (P less than .05) for DHT-treated mares from the first to third GnRH injection but were unchanged for control mares. It seems that DHT alone cannot mimic the stimulatory effects of testosterone on FSH production and secretion as observed in previous experiments with ovariectomized and intact mares. Moreover, because intact mares have been shown previously to respond to DHT treatment with an increase in GnRH-induced FSH secretion, it appears that some mechanism is lost in long-term ovariectomized mares, making them unresponsive to DHT treatment.     

Efficacy of altrenogest administration to postpone ovulation and subsequent fertility in mares

A recent report suggested administration of altrenogest during the follicular phase could postpone ovulation. Based on these results, two questions were generated. We first hypothesized that by initiating a altrenogest treatment earlier in the estrous cycle, a greater and/or more consistent delay in ovulation would result. Second, we hypothesized that exposure to elevated progestin concentrations might alter viability of the ovulatory follicle and oocyte. The focus of the first experiment was to determine if initiation of altrenogest treatment at different stages of the estrous cycle would yield a more predictable time to ovulation, whereas the second experiment was designed to determine whether mares receiving altrenogest during estrus had compromised fertility. In the first experiment thirty mares of mixed light breed, ranging in age from 5-15 years, were randomly assigned to one of three groups. The two treated groups received altrenogest (0.088 mg/kg of body weight) for two days once a follicle of 30 or 35 mm in diameter was detected. Control mares were not treated. Mares treated with altrenogest whether initiated at the detection of a 30 or 35 mm follicle demonstrated similar (P>.05) day to ovulation interval when adjusted to 35 mm (5.4 and 5.6 days, respectively). Both treated groups demonstrated a delayed interval (P<.05) when compared to control (3.9 days). Thirty-six mares of similar breed and age, were randomly assigned to two groups for use in the second experiment. All mares were monitored daily via transrectal ultrasonography from the time a 35 mm or greater follicle was detected until ovulation. Treated mares received daily doses of altrenogest (0.088 mg/kg of body weight) for two days once a follicle of 35 mm or greater was detected. Control mares received no treatment. Fertility data were collected from mares inseminated every other day with 500 million motile spermatozoa from one of two stallions with proven fertility. Pregnancy data were collected via transrectal ultrasonography at days 12, 14 and 16 post-ovulation. Ovulation data were collected from 27 control cycles and 26 treated cycles. Contrary to previous reports and Experiment 1, no difference (P=0.35) was noted between groups with respect to days to ovulation. Control mares averaged 4.14 days and treated mares averaged 4.7 days to ovulation from initial detection of a 35 mm follicle. Fertility data were also similar (P=0.8) between control and treated mares (66.6% and 61.5% per cycle, respectively). Interestingly, a greater number (P=0.017) of treated cycles (5/26) resulted in follicular regression than did control cycles (0/27). While these data suggest that this dosage of altrenogest may not postpone ovulation, it did appear related to increased incidence of follicular regression. Fertility was unaffected, however, in those mares that ovulated. Further studies are needed in which initiation at different stages of estrus and different doses of altrenogest are used.