Pregnancy and twinning rates in Thoroughbred mares following the administration of human chorionic gonadotropin (hCG)

AIM: To determine the effect of hCG administration to cycling Thoroughbred mares, on pregnancy and twinning rates and the number of serves in the treated cycle. METHODS: A retrospective case control approach was conducted involving 2119 mare ovulatory cycles, on 1110 mares over a 7-year period. Data were collected by 1 of the authors during routine stud work at 3 commercial Thoroughbred farms in the Waikato region of New Zealand. The hCG (1500 IU) was administered by intravenous injection to selected mares 24 h before the expected time of breeding. Mares were scanned for pregnancy (singleton or twins) 14 days after the onset of dioestrus or detection of ovulation. Multilevel logistic regression analyses were used to identify the risk factors associated with the outcomes of interest while simultaneously controlling for possible confounding factors. RESULTS: Treatment with hCG tended to improve the odds of pregnancy (p=0.06), produced a 3-fold increase in the odds of twins (p<0.001), and increased the odds of a mare having a single serve in the treated ovulatory cycle (p=0.036). The first ovulatory cycle of a season in which a mare was bred was associated with a lower odds of pregnancy (p=0.02), and a lower odds of twins (p=0.003), when compared with subsequent cycles. Lactating mares were less likely to be diagnosed with twins (p=0.005), and were more likely to have a single serve (p<0.001), in any one ovulatory cycle than non-lactating mares. CONCLUSIONS: This report supports the role of hCG as an important therapeutic tool in veterinary management of broodmares for optimal reproductive performance. Mares treated with hCG must be managed in the knowledge that they have an increased likelihood of twins.     

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.     

Relationship between pharmacological induction of estrous and/or ovulation and twin pregnancy in the Thoroughbred mares

The aim of this study was to evaluate the possible relationship of pharmacological induction of estrous and/or ovulation with the occurrence of twin pregnancies in Thoroughbred mares. Out of 680 mares, 356 received one of the following treatments during the estrous cycle in which they became pregnant: injection of 0.5mg of cloprostenol at the ultrasonographic detection of a CL (n=86); injection of 5000 IU human chorionic gonadotropin (hCG) immediately before mating (n=221); injection of 0.5mg of cloprostenol at the ultrasonographic detection of a CL plus injection of 5000 IU hCG immediately before mating on cloprostenol-induced estrous (n=49). The other 324 mares, not treated for induction of estrous or ovulation in the estrous cycle resulting in pregnancy, were used as control group. The occurrence of twin and single pregnancies in treated and control mares underlines that the percentage of twin pregnancy in treated mares (16.6%) was statistically significantly higher (P<0.0001; odds ratio, OR=2.87) than the percentage of twinning in the control group (6.5%). Comparison of the occurrence of twins between treatments revealed a statistically significant difference between mares treated with hCG alone compared to animals given prostaglandin F2alpha (PGF2alpha) plus hCG. The results show a statistically significant difference for each treatment compared to controls, with the least difference (P<0.05; OR=2.18) for the comparison between hCG treatment group and controls, a significance of P<0.01; OR=3.05 for the comparison between PGF2alpha treatment and controls, and a highly statistically significant difference (P<0.0001; OR=6.37) for the comparison between PGF2alpha plus hCG-treated animals and controls.     

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.     

Treatment of transition phase mares with progesterone intravaginally and with deslorelin or hCG to assist ovulations

Over four years, four investigators in the Northern Hemisphere treated 413 privately owned transition phase mares between late February and early April, for the purpose of breeding such mares early in the season. Mares received an intravaginal device (CIDR-B) carrying 1.9 g progesterone, for about 12 days. Thereafter mares forming preovulatory follicles >30 mm were either treated with a short acting implant releasing the GnRH analog deslorelin (Ovuplant™) or with 1,500—2,500 IU hCG, or not. Follicle sizes were determined with ultrasonography at admission to the study (i.e. day of CIDR-B insertion), at intervals during treatment, at device removal and in 24 (to 48) hour intervals thereafter to determine the time for treatment to induce and accelerate ovulation and to ovulation, respectively. Pregnancies were determined by ultra-sonography between Days 14 to 18 after breeding, mostly 12 to 14 days after ovulation. Based on the size of the largest follicle at admission, mares were grouped into Classes with a ollicle diameter of 10 mm or less in Class I, and mares with follicles 11-20 mm, 21-30 mm and >30 mm in Classes II, III and IV, respectively. Overall, 80.2% of all mares responded to treatment with estrus and 80.7% ovulated. For mares in Classes I to IV, the rate of mares bred and becoming pregnant was 53.4% and 66.7%, 65.6% and 58.7%, 87.5% and 52.3%, and 75.0% and 52.0%, respectively. The overall pregnancy rate was 55.6% for the first breeding in response to treatment. Mares not assisted with Ovuplant or hCG were bred at a significantly lower rate (<0.0001) and the pregnancy rate was lower, 44.4% vs. 54.2% and 60.5%, respectively. Treatments with Ovuplant or hCG ensured ovulation rates of 96.0 and 84.9% versus 53.3% in unassisted mares overall. Follicle diameters increased significantly with CIDR-B in situ, and progressed after device removal to >30 mm within 4.0 days and to ovulation 5.3 days. Those mares in Class I responding to treatment (ca 60%) did not differ from Class II to IV mares in almost all the parameter evaluated. Significant differences were seen in the UK in response to treatment between years for the percentage of mares showing heat, ovulated, were bred and became pregnant.     

Follicular dynamics after treatment with hCG for ovulation induction in mares

In this study the use of hCG for induction of ovulation is described. Factors such as follicle diameter at the time of administration of hCG (3000 IE hCG i.v.), follicular growth after hCG and the rate of double ovulations were evaluated. A total of 168 mares presented for artificial insemination were used. In 249 estrous periods hCG was given to mares exhibiting standing estrous when a minimum follicle diameter of 30 mm and a well developed edema of the endometrium could be detected by ultrasonography. In nine estrous periods ovulation occurred within 24 hours after hCG. The majority of mares (216; 86.7%) ovulated 24 to 48 hours after hCG and in 24 cases ovulation was delayed beyond 48 hours. Follicle size at the time of hCG administration (30-34 mm, 35-39 mm, > or = 40 mm) had no influence on the percentage of mares ovulating 24 to 48 hours after hCG (89.2%, 87.9%, and 83.7%, respectively). Double ovulations could be observed in 17.7% of estrous periods. The one cycle pregnancy rate was not influenced by follicle size (small 45.9%; medium 41.6%; large 47.5%). Repeated treatments with hCG during successive estrous cycles within one year did not influence the rate of responding to hCG. Mares in standing estrous respond well to hCG if a minimum follicle size of 30 mm and a well developed endometrial folding is present.     

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.     

Effect of purified equine follicle-stimulating hormone on follicular development and ovulation in transitional mares

Horse owners want to have their mares bred as early as possible in the breeding season after February 1. Numerous medical treatments, such as progesterone, dopamine antagonists, and gonadotropin-releasing hormone have been administered to anestrous or transitional mares in an attempt to induce follicular development. Some of these treatments are ineffective or impractical, so there is a need in the horse industry to develop alternative techniques to stimulate follicular development and ovulation early in the breeding season. Twenty transitional mares were assigned to one of two treatment groups. Mares in group 1 (n = 10) served as untreated controls, and mares in group 2 (n = 10) were administered 12.5 mg of purified equine follicle-stimulating hormone (eFSH) (Bioniche Animal Health USA, Inc., Athens, Ga) intramuscularly twice daily for a maximum of 15 consecutive days. Mares were considered to be in transition when the diameter of the largest follicle was ≥25 mm. Once one or more follicles >35 mm were detected, eFSH treatment was discontinued and human chorionic gonadotropin was administered intravenously. The percentage of mares ovulating during the 15-day observation period was compared by means of chi-square analysis. The interval to ovulation and the number of ovulations per mare were compared between the two groups by Student t test. In 8 of 10 mares treated with eFSH follicles developed and ovulation occurred during the 15-day observation period, compared with 0 of 10 control mares. Interval from onset of treatment to ovulation was 7.6 ± 2.4 days for these eight mares. The eight mares were treated for an average of 5.2 ± 1.3 days with eFSH. Thus, the eFSH treatment was effective in advancing the first ovulation of the year in transitional mares.     

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.     

Comparison of HCG, buserelin and luprostiol for induction of ovulation in cycling mares

Sixty nonlactating light-horse mares were used to compare the efficacy of hCG, buserelin (a GnRH analog) and luprostiol (a PGF₂α analog) for induction of ovulation in cycling mares. Mares were assigned to 1 of 4 treatments: 1) controls; 2) 40 μg buserelin IM at 12 hr intervals during estrus until ovulation; 3) 7.5 mg IM luprostiol; and 4) 3,300 IU hCG. Treatments were given once a mare obtained a ≥35mm follicle and had been in estrus ≥2 days. Both buserelin and hCG shortened (p<0.05) the interval from treatment to ovulation compared to controls; whereas, luprostiol failed to hasten ovulation. Number of follicles ovulated was similar among all 4 groups. Although buserelin and hCG were equal in their ability to induce ovulation, an average of 3.8 injections of buserelin was required for hastening of ovulation.     

A Single Subcutaneous Administration of Buserelin Induces Ovulation in the Mare: Field Data

The aim of the present study, was to evaluate whether a single subcutaneous administration of the GnRH analogue buserelin could induce ovulation in the mare during the breeding season. Two studies were carried out under field conditions. In Experiment 1, 90 cycles of trotter mares aged 2-7 years, were assigned to a buserelin group (Bus1) or to a control group (Control), in the presence of a >/=35 mm pre-ovulatory follicle, with uterine oedema and a relaxed cervix. Ten mares were assigned to the two groups for 32 cycles in Bus1 and 52 cycles in Control, two mares received only Bus1 for three cycles, and one mare was assigned to Control for three cycles. Mares treated with buserelin received 6 ml of SUPREFACT s.c. (1.05 mg/ml buserelin acetate), and no treatment was given in Control. The ovulation rate between 24 and 48 h was higher (p < 0.0001) in Bus1 (31/35) than in Control (15/55). In Experiment 2, the condition of inducing ovulation with 6 ml SUPREFACT s.c. (Bus2) or 1500 ui human chorionic gonadotropin were identical to the first study. Forty-nine mares of ages 2-21 years, were used for 120 cycles, 56 cycles were assigned to Bus2, and 64 cycles were induced with 1500 IU human chorionic gonadotropin i.v. No significant difference was found in the ovulation rate on day 2 (38/56, 50/64), or in the fertility rate (19/48, 26/57). No negative effect of the treatment was observed with repeated buserelin administration in either study during the breeding season. We can conclude that a single administration of buserelin induces ovulation in the mare, under suitable conditions for veterinary practitioners.     

Effect of Deslorelin and/or Human Chorionic Gonadotropin on Inducing Ovulation in Mares During the Transition Period Versus Ovulatory Season

The objective of this study was to compare the rate of ovulation when deslorelin and/or human chorionic gonadotropin (hCG) was administered in mares in both the transition period and the ovulatory season. A total of 200 Paint Horses, Quarter Horses, and crossbred mares were used during the transition season (July to September) and the ovulatory season (October to February) of the southern hemisphere. The animals were divided into four groups. In the control group (n = 72), mares received 1 mL of saline; in deslorelin group (n = 171), 1.5 mg of deslorelin was administered by intramuscular (IM) injection; in hCG group (n = 57), 1,667 IU of hCG was administered IV; and in hCG + deslorelin group (n = 438), 1.5 mg of deslorelin (IM) and 1,667 IU of hCG (IV) were administered. The drugs were administered after follicles ≥35 mm in diameter were identified and grade III uterine edema was observed. At 48 hours after application, ultrasonography was performed to detect ovulation. During the transition period, the ovulation rates were 4.3% (control), 78.6% (deslorelin), 50% (hCG), and 73.3% (hCG + deslorelin). During ovulatory season, the ovulation rates were 16.4% (control), 68.8% (deslorelin), 60% (hCG), and 73% (hCG + deslorelin). There was no significant difference (P > .05) in the ovulation rate between the groups or the periods, except that the control group was lower than all others. Furthermore, both hCG and deslorelin are viable options for inducing ovulation during the transition period before ovulation season.     

hCG‐Induced Ovulation in Thoroughbred Mares Does Not Affect Corpus luteum Development and Function During Early Pregnancy

Our aim was to compare Corpus luteum (CL) development and blood plasma concentration of progesterone ([P₄]) in thoroughbred mares after spontaneous (Control: C) or human chorionic gonadotrophin (hCG)‐induced ovulation. Lactating mares (C = 12; hCG = 21) were daily teased and mated during second oestrus post‐partum. Treated mares received 2500 IU hCG i.v. at first day of behavioural oestrus when dominant follicular size was >35, ≤42 mm and mated 12–24 h after. Control mares in oestrus were mated with dominant follicular size ≥45 mm. Dominant follicle before ovulation, CL and gestational sac were measured by ultrasound and [P₄] by radioimmunoassay (RIA). Blood sampling and ultrasound CL exams were done at days 1, 2, 3, 4, 8, 12, 16, 20, 25, 30, 35, 40, 45, 60 and 90 after ovulation and gestational sac from day 12 after ovulation in pregnant (P) mares; non‐pregnant (NP) were followed until oestrus returned. Data analyses considered four subgroups: hCG‐P, hCG‐NP, C‐P and C‐NP. Preovulatory follicular size was smaller in hCG mares than in C: 39.2 ± 2.7 mm vs 51.0 ± 1.8 mm (p < 0.0001). All hCG mares ovulated 24–48 h after treatment and presented similar oestrus duration as controls. C. luteum size in P mares showed the same pattern of development through days 4–35, presenting erratic differences during initial establishment. Thus, on days 1 and 3, CL was smaller in hCG‐P (p < 0.05); while in hCG‐NP, CL size was greater than in C‐NP on day three (p = 0.03). Corpus luteum size remained stable until day 90 in hCG‐P mares, while in C‐P a transient and apparently not functional increase was detected on days 40 and 45 (p < 0.05) and the decrease from day 60 onwards, made this difference to disappear. No differences were observed in [P₄] pattern between P, or between NP subgroups, respectively. So, hCG‐induced ovulation does not affect CL development, neither [P₄] during early pregnancy. One cycle pregnancy rate tended to be lower in hCG mares while season pregnancy rates were similar to controls.     

Evaluation of human chorionic gonadotropin as a replacement for gonadotropin-releasing hormone in ovulation-synchronization protocols before fixed timed artificial insemination in beef cattle

Two experiments were conducted during 2 yr to evaluate differences in ovulation potential and fertility in response to GnRH or hCG. In Exp. 1, 46 beef cows were given 100 microg of GnRH or 500, 1,000, 2,000, or 3,000 IU of hCG. Ovulation incidence was not different between GnRH and any of the hCG doses, indicating that ovulatory capacity of at least 500 IU of hCG was equivalent to GnRH. In Exp. 2, beef cows (n = 676) at 6 locations were assigned randomly to a 2 x 3 factorial arrangement of treatments. Main effects were: 1) pre-timed AI (TAI) treatment (GnRH or hCG) and 2) post-TAI treatment (saline, GnRH, or hCG) to initiate resynchronization of ovulation in previously inseminated cattle. Blood samples were collected (d -21 and -10) to determine progesterone concentrations and assess cyclicity. Cattle were treated with a progesterone insert on d -10 and with 100 microg of GnRH or 1,000 IU of hCG. A PGF(2alpha) injection was given at insert removal on d -3. Cows were inseminated 62 h (d 0) after insert removal. On d 26 after first TAI, cows of unknown pregnancy status were treated with saline, GnRH, or hCG to initiate a CO-Synch protocol. Pregnancy was diagnosed 33 d after first TAI to determine pregnancies per AI (P/AI). Nonpregnant cows at 6 locations in yr 1 and 1 location in yr 2 were given PGF(2alpha) and inseminated 56 h later, concurrent with a GnRH injection. Five weeks later, pregnancy diagnosis was conducted to determine pregnancy loss after first TAI and pregnancy outcome of the second TAI. Injection of pre-TAI hCG reduced (P < 0.001) P/AI compared with GnRH, with a greater reduction in cycling cows. Post-TAI treatments had no negative effect on P/AI resulting from the first TAI. Serum progesterone was greater (P = 0.06) 7 d after pre-TAI hCG than after GnRH and greater (P < 0.05) after post-TAI hCG on d 26 compared with saline 7 d after treatment in association with greater frequency of multiple corpora lutea. Compared with saline, injections of post-TAI GnRH and hCG did not increase second insemination P/AI, and inconsistent results were detected among locations. Use of hCG in lieu of GnRH is contraindicated in a CO-Synch + progesterone insert protocol. Compared with a breeding season having only 1 TAI and longer exposure to cleanup bulls, total breeding season pregnancy rate was reduced by one-third, subsequent calving distribution was altered, and 50% more AI-sired calves were obtained by applying 2 TAI during the breeding season.     

Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares

The effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19°45′45′S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post‐ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35‐mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35‐ to 40‐mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively – one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low‐dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.     

First Service Pregnancy Rates Following Post‐AI Use of hCG in Ovsynch and Heatsynch Programmes in Lactating Dairy Cows

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 μg of GnRH agonist, i.m. (day 0) starting at 44 ± 3 days in milk (DIM), and 500 μg of cloprostenol, i.m. (day 7). In O group, cows received 100 μg 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), method₁ (O, H and C) and method₂ (hCG and saline) showed that all factors, except method₁, 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).     

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.     

Serum thyroxine concentrations and pregnancy rates 15 to 16 days after ovulation in broodmares

OBJECTIVE: To determine whether serum thyroxine (T4) concentration was associated with pregnancy rates 15 to 16 days after ovulation in mares and to determine whether thyroid hormone supplementation would enhance fertility in mares. DESIGN: Cohort study. ANIMALS: 329 clinically normal broodmares. PROCEDURE: Mares were examined 15 to 16 days after ovulation to determine whether they were pregnant; blood samples for determination of serum T4 concentration were collected at the same time. Sixty mares were receiving thyroid hormone supplementation prior to the study because of low serum T4 concentration (< 16 microg/dl) prior to breeding. RESULTS: Serum T4 concentration ranged from 4.5 to 53.9 mg/dl. Forty (12%) mares had low (< 16 microg/dl) concentrations, 283 (86%) had normal concentrations, and 6 (2%) had high (> 45 microg/dl) concentrations. Two hundred thirty-one mares were pregnant 15 to 16 days after ovulation. A significant association between serum T4 concentration (low, normal, or high) and pregnancy (yes or no) was not detected, and logistic regression analysis indicated that serum T4 concentration was not significantly related to pregnancy. Of the 269 mares not receiving thyroid hormone supplementation, 187 were pregnant, and of the 60 mares receiving thyroid supplementation, 44 were pregnant. There was no significant relationship between thyroid hormone supplementation and pregnancy status. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that serum T4 concentration in mares is not significantly associated with pregnancy 15 to 16 days after ovulation. Results also suggest that supplementation of mares that only have low T4 concentrations is not indicated or likely to be beneficial.     

The effect of systemic administration of cloprostenol on ovulation in mares treated with a prostaglandin synthetase inhibitor

Prostaglandins (PGs) are essential to trigger the cascade of events that degrade the extracellular matrix of follicles leading to follicular rupture and ovulation. In mares, systemic administration of flunixin meglumine (FM), a PG synthetase inhibitor, blocks ovulation by inducing luteinized unruptured follicles (LUF). In the rat, the administration of PGF(2α) (PGF) and PGE restored ovulation in indomethacin treated animals. The mares were treated with FM 0, 12, 24 and 36 h after human chorionic gonadotrophin (hCG) administration to induce experimentally LUF (n = 15) or were left untreated (controls, n = 5). In addition, 250 μg of cloprostenol were administered intravenously to the mares 33, 35 and 36 h (CLO 33, n = 5) or 48, 49 and 50 h (CLO 48, n = 5) after hCG. One group was treated with FM but not with cloprostenol (FM-control, n = 5). The ovulation rate, follicular diameter and progesterone concentration were compared amongst groups. The ovulation rate at 48 h was higher (p < 0.05) in the controls (100%) than in the FM-control (0%), CLO 33 (0%) or CLO 48 (20%) mares. All but one FM treated mares developed LUF by 48 h after hCG administration. Two LUF collapsed between 48 and 60 h and 72 and 84 h in one mare from FM-control and from the CLO 33 group each, respectively. Progesterone concentration was significantly higher (p < 0.05) in the control mares than in any of the FM treated mares 5, 9 and 13 days after hCG. In conclusion, FM administered during the periovulatory period blocked ovulation in the mares. In contrast, the administration of cloprostenol, a PGF analogue, in the previously FM treated mares failed to restore ovulation.     

影响马胚胎移植成功率的关键因素分析

旨在探讨影响马胚胎移植效率的几种关键因素。本研究统计了国内北京马场、河北马场和山东马场2013-2018年胚胎移植数据,3个马场供体马数量分别为15、21和25匹,受体母马数量分别为56、50和75匹。所有母马年龄为3~12岁。统计供体马冲胚时间对胚胎回收率的影响;胚胎日龄对移植后受体马妊娠率的影响;供、受体母马排卵同期化程度对移植后妊娠率的影响;受体母马居住移植基地时间对移植后妊娠率的影响。结果显示,母马在配种季节注射前列腺素（PG）+GnRH类似物或PG+hCG诱发排卵,发情周期分别为（14.5±0.8）和（14.3±1.1）d,显著低于对照组的（（20.5±2.6）d,P<0.05）;排卵后第8天冲洗子宫的胚胎回收率均高于第7天,但差异不显著;8日龄胚胎移植后受体马的妊娠率均高于7日龄,差异不显著;供体母马排卵比受体母马早1 d时,胚胎移植后的妊娠率最高;受体母马在移植基地居住时间大于1年时,移植后妊娠率高于居住时间小于0.5年的受体马。根据以上结果,本研究得出如下结论,PG与hCG或GnRH类似物联合使用可缩短母马发情周期,母马排卵后第8天的胚胎回收率和移植后妊娠率较高,胚胎移植时选择居住时间大于1年且排卵时间比供体晚1 d的母马作受体。