The effect of the gonadotrophin-releasing hormone analog, buserelin, administered in diestrus on pregnancy rates and pregnancy failure in mares

Two trials involving 578 mares were performed to investigate the effect of a single intramuscular treatment of 40 microg buserelin, an analog of gonadotrophin releasing hormone, on pregnancy rate in mares. All mares were bred by natural mating and were allocated into pairs One mare in each pair was injected with buserelin either on Day 10 or 11 (Trial 1) or on Days 8 to 10 (Trial 2) after ovulation. Pregnancy status of mares was determined by transrectal ultrasonographic examination on Day 14 or 15 after the day of ovulation and was repeated between Days 28 and 30 of pregnancy. In Trial 1, buserelin treatment increased the pregnancy rate at Days 14 and 15 (72.5 vs 66.6%, P < 0.01). At the second pregnancy examination, pregnancy losses were lower in the treated group of mares (4.1 vs 7.4%; P < 0.05). In Trial 2, buserelin also improved the pregnancy rate (57.2 vs 53 5%; P < 0.05) at Days 14 and 15 Pregnancy losses between the first and second examinations were lower in the treated group of mares (6.5 vs 12.0%; P < 0.05). Buserelin increased pregnancy rates after breeding at the first estrus in both trials. In addition, buserelin treatment increased the pregnancy maintenance rate at Days 28 to 30.     

Use of buserelin to induce ovulation in the cyclic mare

Inducing ovulation in a cyclic mare is often necessary. For this purpose, hCG has been used commonly, but the response can be reduced after successive administrations. The aims of this study were to test the effectiveness of buserelin in hastening ovulation in estrus mares, and its influence on fertility; and to investigate the effect of treatment on LH secretion. Five crossover trials were designed to compare the effect of two treatments: buserelin (40 microg in 4 doses i.v. at 12 h intervals) vs placebo (Experiments 1 and 2); buserelin 40 microg (in 4 doses i.v.) vs 20 microg (Experiment 3); buserelin (4 doses of 20 microg i.v.) vs hCG (1 dose of 2,500 IU i.v.) (Experiment 4); or buserelin (3 doses of 13.3 microg at 6 h interval) vs hCG (Experiment 5). In Experiment 2, blood samples were taken hourly until ovulation, for LH measurements. In Experiment 1, buserelin treatment significantly hastened ovulation. Reduction of the dose by half (Experiment 3) did not alter the effectiveness. In Experiments 4 and 5, buserelin was as effective as hCG in inducing ovulation between 24 and 48 h after initiation of treatment. Buserelin treatment induced a rise in LH concentration during the 48 h period of the experiment, and LH concentrations before ovulation were significantly higher in buserelin treated cycles than in placebo cycles. These experiments demonstrated the usefulness of two new protocols of administration of buserelin, as an alternative to hCG for induction of ovulation. One hypothesis explaining the mechanism of action is that the persistant rise in LH concentration could modify the ratio of biological/immunological LH, as it occurs physiologically, thereby hastening ovulation.     

Effect of ovariectomy on pregnancy in mares.

Pony mares were bilaterally ovariectomized at different stages of pregnancy between Days 25 and 210. Abortion or fetal resorption occurred within 2 to 6 days after operations in all 14 mares ovariectomized between Days 25 and 45 and after an interval of 10 to 15 days in 9 of 20 other ovariectomized between 50 and 70 days. All 12 mares ovariectomized on either 140 or 210 days carried their foals to normal term. The termination of early pregnancy was preceded by a loss of uterine tone and of a palpable uterine bulge. The mean length of gestation in all mares in which pregnancy was not interrupted by ovariectomy was not significantly different from that in a group of contemporary control mares. Plasma progestagen concentrations dropped to less than 2 ng/ml after ovariectomy, whether or not pregnancy was maintained. Mares ovariectomized on Day 25 and injected with 100 mg progesterone daily for 10 or 20 days remained pregnant during treatment but showed a loss of uterine tone and the fetal bulge disappeared within 4 to 6 days after the end of treatment. Non-pregnant ovariectomized or intact seasonally anoestrous mares injected i.m. with 50 or 100 mg progesterone daily for 8 weeks showed changes in uterine tone, length and thickness similar to those occurring in mares during early pregnancy.     

Pregnancy rates, LH and progesterone concentrations in mares treated with a GnRH agonist

The aim of the study was to investigate the effect of the GnRH agonist Buserelin given on day 10 after ovulation on pregnancy rate and concentrations of progesterone and LH. Altogether 191 warmblood mares were used for two trials. Fresh or frozen/thawed semen from 27 stallions was used for A.I. In trial A 171 mares received either Buserelin (Receptal, Hoechst, Germany, 40 microg/animal) or 10 ml 0.9% NaCl (placebo). On day 16 after A.I. pregnancy diagnosis was performed by ultrasound scanning of the uterus. For statistical analysis, data were analyzed by a mixed model, with four fixed factors (treatment, type of spermatozoa, A.I. number, reproductive status of the mare) and a random factor (stallion). Least Square Means (LSM) for pregnancy rate were 46.0% in GnRH agonist treated mares and 36.4% in the control group (P=0.22). In trial B 20 lactating and cycling mares were used for endocrine studies. Blood samples were recovered for analyses of progesterone and LH from days 0 to 11. The mean progesterone concentrations increased continuously from days 0 to 8 after ovulation in both groups (GnRH group: from 0.81+/-0.48 to 5.47+/-0.48 ng/ml, control group: from 0.63+/-0.68 to 5.83+/-0.68 ng/ml). Moreover, the progesterone concentrations from days 9 to 11 were not different between the GnRH and the control group. In contrast to this LH concentrations were markedly influenced by the GnRH agonist. On day 10 LH concentrations were significantly higher in GnRH agonist treated than in placebo treated animals. From the data obtained from individual animals it can be concluded that GnRH agonist, given during luteal phase may have different effect on luteal function.     

Initiation of superovulation in mares 5 or 12 days after ovulation using equine pituitary extract with or without GnRH analogue

Cyclic mares were assigned to 1 of 3 treatments (n=15 per group): Group 1 received equine pituitary extract (EPE; 25 mg, i.m.) on Day 5 after ovulation; Group 2 received EPE on Day 12 after ovulation; while Group 3 received 3.3 mg of GnRH analogue (buserelin implant) on the day of ovulation and 25 mg, i.m. EPE on Day 12. Mares in each group were given 10 mg PGF(2)alpha on the first and second day of EPE treatment. The EPE treatment was continued daily until the first spontaneous ovulation, at which time 3,300 IU of human chorionic gonadotropin (hCG) were given to induce further ovulations. Mares in estrus with a >/=35 mm follicle were inseminated every other day with pooled semen from 2 stallions. Embryo recovery was attempted 7 days after the last ovulation. Follicular changes and embryo recovery during 15 estrous cycles prior to treatment were used as control data. During treatment, the number of follicles >/=25 mm was higher (P<0.05) for Day 5 than for Day 12 or control mares, but the number for Day-5 mares was similar (P>0.05) to that of mares treated with buserelin implants (Group 3). Initiation of EPE treatment on Day 5 resulted in a greater (P<0.05) number of ovulation (2.9) than on Day 12 (1.1) or in the control mares (1.3) but not in the buserelin-treated mares (1.8). The number of embryos recovered from mares in the Day 5 (1.2), Day 12 (1.0), buserelin (0.9) and control (0.9) groups was similar (P>0.05). The conclusions were 1) EPE initiated in early diestrus increased follicular development and ovulation and 2) treatment with GnRH analogue marginally improved response to EPE treatment.     

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.     

Dimethyl sulfoxide intrauterine therapy in the mare: effects upon endometrial histological features and biopsy classification

The effects upon equine endometrial histological features produced by 10 to 30% concentrations of dimethyl sulfoxide (DMSO) in sterile saline were compared with the effects of sterile saline (0.9% NaCl) alone as an intrauterine infusion therapy in 16 barren mares. No harmful histological changes were noted (P > 0.05) as a result of the therapy. Thirty percent intrauterine DMSO therapy produced a significant (P < 0.01) improvement (i.e., reduction of chronic inflammatory cell infiltrates and reduction of periglandular fibrosis) in endometrial biopsy classification in 18 of the 27 barren mares evaluated; whereas only 2 of 18 barren mares improved following intrauterine saline treatment in the control group. In subsequent breeding trials, the pregnancy rates following intrauterine therapy were no different between DMSO-treated mares and saline-treated control mares (P > 0.05); however, there was a trend toward a higher pregnancy rate following DMSO therapy. The inability to control for sire variability in the breeding trials may have contributed toward the result that no significant difference could be demonstrated in the pregnancy rates between treated and control mares.     

Effect of periovulatory prostaglandin F2alpha on pregnancy rates and luteal function in the mare.

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

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = −0.834, P < 0.05; day 35: r = −0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.     

Role of progesterone in mobility, fixation, orientation, and survival of the equine embryonic vesicle

Luteal progesterone was removed by an injection of prostaglandin F(2alpha) or bilateral ovariectomy on Day 12 of pregnancy in pony mares. The embryonic vesicle remained mobile in the uterus until loss occurred on Days 13, 13, 15, or 19 in four prostaglandin-treated mares and Days 15, 17, 19, or 26 in four ovariectomized mares. Exogenous progesterone given daily, starting on Day 12, maintained pregnancy until Day 40 in five of five prostaglandin-treated and three of four ovariectomized mares. During two-hour mobility trials on Day 14, embryonic vesicles in mares without luteal or exogenous progesterone (n = 9) moved to a different uterine segment less frequently (mean number of location changes per two-hour trial: 7.2 +/-1.0 vs 10.4 +/-1.1, P < 0.05) and were observed more often in the uterine body (14.9 +/-2.9 vs 8.9 +/-1.3, P < 0.10) compared to vesicles in mares with a progesterone influence (n = 15). Of mares that still had a vesicle present on Day 18, fixation occurred by Day 17 in all (12 12 ) mares under the influence of luteal or exogenous progesterone but failed to occur in the three mares that were not under progesterone influence. Progesterone replacement was started on Day 16 in three mares that received prostaglandin F(2alpha) on Day 12 and still had a vesicle on Day 16. The vesicle was maintained and continued to develop in all three mares, indicating that the vesicles were viable four days after PGF(2alpha) treatment. However, fixation tended to be delayed (P < 0.15) and orientation of the embryo proper was altered (P < 0.005) compared to mares that were continuously under the influence of progesterone. The results demonstrated the importance of luteal progesterone to mobility, fixation, orientation, and survival of the embryonic vesicle.     

The efficacy of different hCG dose rates and the effect of hCG treatment on ovarian activity: ovulation, multiple ovulation, pregnancy, multiple pregnancy, synchrony of multiple ovulation; in the mare

Despite the widespread use of hCG to advance ovulation in the mare there is little information on efficacy of dose rates and any contraindications of its use. This study aims to investigate the effect of dose of hCG on ovulation within 48h and the effect of hCG on: ovulation, multiple ovulation (MO), pregnancy, multiple pregnancy (MP) rates and synchrony of MO; additionally whether any seasonal effect is evident. Sequential ultrasonic scanning was used to monitor the occurrence of ovulation, within 48h of treatment, in 1291 Thoroughbred mares treated with either 750iu hCG or 1500iu hCG s.c. Ovulation rate, type (single ovulations (SO), MO, synchronous, asynchronous) and subsequent pregnancy were then monitored in 1239 Thoroughbred mares on a commercial stud over 3 years, 536 of which were treated with 750iu hCG at mating, all mares were also allocated into groups according to month of mating. No significant difference existed between the two dose levels of hCG and no significant difference existed between treated and untreated mares in overall ovulations (1.32 and 1.28 respectively), MO (31.7% and 27.7%), pregnancy (65.1% and 65.6%) or MP rates (10.8% and 11.8%). There was no significant association between month of year and pregnancy or MP rates for either treated or control mares, nor for MO for untreated mares. A significant (p<0.05) association was evident between month and MO in treated mares, MO being lowest in April (22.3%). 95.9% of treated mares multiple ovulated within 48h compared with 90.7% controls, a near significant difference. In conclusion this study demonstrates that: (i) hCG dose of 750iu s.c. is just as effective in inducing ovulation within 48h as 1500iu, (ii) 750iu hCG has no significant effect on ovulation, MO, pregnancy or MP rates; (iii) a significant (p<0.05) association exists between season and MO in hCG treated mares; (iv) a tighter synchrony (ovulation within 48h) of MO is evident in hCG treated compared with control mares (p=0.052).     

Dose dependent effect of GnRH analogue on pregnancy rate of repeat breeder crossbred cows

The aim of this study was to investigate the effect of treating repeat breeder dairy crossbred cows with different doses of GnRH analogue through i.m. at the time of artificial insemination, on pregnancy rates from their first service after treatment and overall pregnancy rates. One hundred and thirty seven crossbred dairy cows with a history of repeat breeding and eligible after 6-8 infertile services but clinically free of diseases were selected for the study. The animals were randomly divided into three groups. Group 1 (n = 55) cows were treated intramuscularly with each 20 microg Buserelin-acetate (Receptal, Hoechst Roussel Vet GmbH) at the time of artificial insemination. Group 2 (n = 40) cows were treated intramuscularly with each 10 microg Buserelin-acetate at the time of artificial insemination. Group 3 (n = 42) cows were treated intramuscularly with saline as control at the time of artificial insemination. The first service pregnancy rates in Groups 1-3 were 45, 25 and 17%, respectively. Similarly, the overall conception rates in Groups 1-3 were 87, 58 and 48%, respectively. The results indicated that the pregnancy rate in crossbred cows could be improved by the GnRH treatment. The higher dose of GnRH significantly increased (P < 0.05) the first service as well as overall pregnancy rate in a dose dependent manner in repeat breeder crossbred cow bred previously 6-8 times unsuccessfully.     

LH and testosterone responses to five doses of a GnRH analogue (buserelin acetate) in 12-month-old Thoroughbred colts

To determine the responsiveness of the pituitary-gonadal axis of peri-pubertal colts to GnRH, buserelin (0.5, 1, 5, 10 and 40 microg) was given to 13 male Thoroughbred yearlings ( n=3-8 colts per dose). Jugular venous blood samples were taken at -10, 0, 10, 20, 30, 40, 60, 120 and 180 min relative to buserelin administration. Increases (P < 0.05) in LH concentrations occurred in colts that received 5, 10, or 40 microg buserelin, but not in those that received 0.5 or 1 microg. Peak LH concentrations and mean area under the curve were higher (P < 0.05) in colts receiving 40 microg buserelin than in those that received 0.5 or 1 microg. Increases ( P< 0.05) in testosterone concentrations occurred in some, but not all, colts that received 1, 5, 10, or 40 microg buserelin. Neither peak concentration nor area under the curve of testosterone differed significantly among doses of buserelin. The percentage of horses that responded to the buserelin increased with increasing dose, with only the highest dose eliciting LH and testosterone responses in all colts. In conclusion, peri-pubertal colts exhibited a dose-response release of LH following buserelin treatment, but individual colts responded in an "all or nothing" manner, such that each either had an LH response or did not. Some colts that exhibited a significant LH response had no subsequent increase in plasma testosterone concentrations; perhaps the pituitary LH response may not have been great enough to stimulate the Leydig cells in these individuals.     

Oxytocin does not contribute to the effects of cervical dilation on progesterone secretion and embryonic development in mares

The aim of the present study was, to investigate the effects of oxytocin administration on Day 7 post-ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared to the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. At Day 7 after ovulation, mares had to undergo four treatments in a crossover design: (a) control, (b) oxytocin (10 IU i.v.), (c) carbetocin (280 microg i.m.) and (d) cervical dilation. On Day 13, all mares (8 of 8 mares) were pregnant on groups control, oxytocin and carbetocin and only 6 of 8 mares on group dilation. In one mare uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death by Day 11 occurred during dilation treatment. Another mare, which did not become pregnant during dilation treatment, developed uterine fluid accumulation and uterine edema from Day 10 to 14. Mean growth rates of the conceptuses did not differ among treatment groups and individual growth rates varied in a wide range from -0.1 to 0.8 cm per day. At Day 13, mean diameters of conceptuses yielded 1.4+/-0.1 cm in control group, 1.5+/-0.1 in oxytocin and carbetocin group and 1.3+/-0.2 cm in dilation group. Secretion of progesterone was not affected by treatments. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations of oxytocin, but onset and duration of peaks differed. Maximum concentrations after intramuscular application of carbetocin were obtained almost 20 min later when compared to intravenous administration of oxytocin. Duration of peaks after injection of the long-acting oxytocin analogue was more than three-fold longer than after administration of native oxytocin. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect progesterone secretion, pregnancy rate and embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: In the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm had no negative consequences on progesterone secretion and pregnancy outcome. However, cervical dilation caused uterine inflammation and subsequent luteolysis in two mares and early embryonic death in one of them. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare.     

Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone

The effect of treatment with a GnRH agonist, hCG or progesterone (P(4)) on corpus luteum function and embryonic mortality was investigated in buffaloes inseminated during mid-winter. Italian Mediterranean buffaloes (n=309) were synchronized using the Ovsynch with timed-AI program and mated by AI at 16 h (Day 0) and 40 h after the second injection of GnRH. On Day 5, buffaloes were randomly assigned to four groups: Control (no treatment, n=69), GnRH agonist (buserelin acetate, 12.6 microg, n=73), hCG (1500 IU, n=75) and P(4) (PRID without E(2) for 10 days, n=77). Progesterone (pg/ml) was determined in milk whey on Days 5, 10, 15 and 20 and pregnancy diagnosis was undertaken on Day 26 by ultrasound and Day 40 by rectal palpation. Treatment with buserelin and hCG increased (p<0.05) P(4) on Day 15 compared with controls (456+/-27, 451+/-24 and 346+/-28 pg/ml, respectively). Buffaloes treated with a PRID had intermediate P(4) concentrations (380+/-23 pg/ml). Embryonic mortality between Days 26 and 40 (22.9%) and pregnancies at Day 40 (48.9%) did not differ between treatments. A higher (p<0.01) P(4) concentration was found on Day 20 in pregnant animals compared with non-pregnant and embryonic mortality buffaloes, which did not differ. In summary, buserelin and hCG increased P(4) concentrations on Day 15 but this was not associated with a reduced incidence of embryonic mortality in buffaloes during mid-winter.     

Recovery Rate and Quality of Embryos from Mares Inseminated at the First Post-Partum Oestrus

The pregnancy rate is lower in mares inseminated at the first post-partum (p.p.) oestrus (40-50%) compared with pregnancy rates in subsequent oestrous cycles (55-65%). The causes of the lowered pregnancy rate are not fully understood. The aim of the present study was to examine if embryonic defects could be one of the reasons for lowered pregnancy rate. A total of 23 p.p. and 14 non-lactating control mares were flushed 7 days after detection of ovulation. Embryo recovery rate was 48% and 71% in p.p. and control mares, respectively (p=0.16). Embryos were photographed, measured, graded and stained with fluorescein diacetate to assess their viability. Thereafter embryos were bisected and stained with Hoechst 33342 to count the cell nuclei. Embryos in both groups were equally viable and the cell numbers were not significantly different. According to morphological evaluation all embryos were classified as excellent or good. Embryos aged 7.3 to 7.6 days (± 0.25 days) were smaller in the p.p. group than in the control group (p<0.05). Forty-seven (9/19) and 8% (1/13) of the uterine swabs, taken before the first insemination, yielded bacteria and neutrophils in p.p. and control mares, respectively. The amount of neutrophils and/or bacteria had no statistically significant effect on embryo recovery rate (p>0.10). Recovery of embryos was not related to histological findings in uterine biopsies taken after embryo recovery. Embryo recovery rate in p.p. mares (48%) was similar to previously reported foal heat pregnancy rates (40- 50%). Hence, early embryonic death in utero would not be the most likely reason for lowered pregnancy rate in mares inseminated at the first p.p. oestrus. Sperm transport and oviductal conditions by the time of the first p.p. oestrus would need to be studied to clarify the role of fertilisation failure as the cause of lower pregnancy rate in mares inseminated at foal heat.     

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).     

Ovulation induction in rabbit does submitted to artificial insemination by adding buserelin to the seminal dose

This study was aimed at determining if a GnRH analogue, buserelin, could be used for ovulation induction in rabbit does submitted to artificial insemination (AI) by intravaginal administration, by adding the hormone to the seminal dose. In a first experiment, 39 secondiparous experimental does (Hyplus strain PS19, Grimaud Frères, France, of about 30 weeks of age) were divided into 3 groups of 13 does each, which at the moment of AI received the following treatments, respectively: (1) control: an intramuscular injection of buserelin (0.8 microg/doe), (2) 8 microg/doe of buserelin added to the insemination dose, and (3) 16 microg/doe of buserelin added to the insemination dose. The experiment was done using 3 consecutive cycles at 42 day-intervals (n = 39). Four does from each of the 3 groups had blood taken at the fourth cycle for LH determination at 0, 60, 90, 120 and 150 min relative to AI. Kindling rates were 82% (28/34), 56% (29/36) and 85% (33/39), respectively for treatments 1, 2 and 3. In the does of groups 2 and 3, LH peaks were detected 60 min after AI, whereas in the does from group 1, the LH peak was detected 90 min after AI. Prolificacy was not different for the 3 treatments (average litter sizes ranged from 10.4 to 10.8). In a second experiment, 3 buserelin concentrations (8, 12 and 16 microg/doe) were used intravaginally and compared with the control treatment (0.8 microg/doe, via intramuscular). This experiment was done using 100 nulliparous rabbit does (Hyplus strain PS19, Grimaud Frères, France, of about 19 weeks of age) (4 groups of 25 does each) located on a commercial farm, to test if the previous results would be confirmed under field conditions. Kindling rates were no different (P < 0.05) for the 4 treatment groups [91.7% (22/24), 79.2% (19/24), 87.0% (20/23) and 87.5% (21/24) respectively for the control, 8, 12 and 16 microg of intravaginal buserelin], however, prolificacy was higher when using the maximal dose of intravaginal buserelin (11.7 vs. 9.4 for the control group). It was concluded that buserelin can be used for ovulation induction in rabbit does when included in the seminal dose, with similar AI results as those obtained when the hormone is administered intramuscularly.     

Timed artificial insemination in beef cattle using GnRH agonist, PGF2alpha and estradiol benzoate (EB)

The present work evaluated low-cost protocols for timed artificial insemination (TAI) in beef cattle. In Experiment 1, cycling nonlactating Nelore cows (Bos indicus, n=98) were assigned to the following groups: GnRH-PGF (GP) and GnRH-PGF-GnRH (GPG), whereas cycling (n=328, Experiment 2) or anestrus (n = 225, Experiment 3) lactating (L) cows were divided into 3 groups: GP-L, GPG-L and GnRH-PGF-Estradiol benzoate (GPE-L). In Experiment 4, lactating cows (n=201) were separated into 3 groups: GP-L, GPE-L and G/2PE-L. Animals from Experiment 1, 3 and 4 were treated (Day 0), at random stages of the estrous cycle, with 8 microg of buserelin acetate (GnRH agonist) intramuscularly (i.m.), whereas in Experiment 2 half of the cows received 8 and the other half 12 microg of GnRH (i.m.). Seven days later (D 7) all animals were treated with 25 mg of dinoprost trometamine (PGF2alpha, i.m.) except those cows from the G/2PE-L group which received only 1/2 dose of PGF2alpha (12.5 mg) via intravulvo-submucosa (i.v.s.m.). After PGF2alpha injection the animals from the control groups (GP and GP-L) were observed twice daily to detect estrus and AI was performed 12 h afterwards. The cows from the other groups received a second GnRH injection (D 8 in GPG-L and d9 in GPG groups) or one injection of estradiol benzoate (EB, 1.0 mg, D 8 in GPE-L group). All cows from GPG and GPG-L or GPE-L groups were AI 20 to 24 or 30 to 34 h, respectively, after the last hormonal injection. Pregnancy was determined by ultrasonography or rectal palpation 30 to 50 days after AI. In the control groups (GP and GP-L) percentage of animals detected in heat (44.5 to 70.3%) and pregnancy rate (20 to 42%) varied according to the number of animals with corpus luteum (CL) at the beginning of treatment. The administration of a second dose of GnRH either 24 (Experiment 2) or 48 h (Experiment 1) after PGF2alpha resulted in 47.7 and 44.9% pregnancy rates, respectively, after TAI in cycling animals. However, in anestrus cows the GPG treatment induced a much lower pregnancy rate (14.9%) after TAI. The replacement of the second dose of GnRH by EB (GPE-L) resulted in a pregnancy rate (43.3%) comparable to that obtained after GnRH treatment (GPG-L, 47.7%, Experiment 2). Furthermore, the use of 1/2 dose of PGF2alpha (12.5 mg i.v.m.s., Experiment 4) resulted in pregnancy rate (43.5%) similar to that observed with the full dose (i.m.). Both protocols GPG and GPE were effective in synchronizing ovulation in cycling Nelore cows and allowed approximately a 45% pregnancy rate after TAI. Additionally, the GPE treatment is a promising alternative to the use of GPG in timed AI of beef cattle, due to the low cost of EB when compared to GnRH agonists.