Ovarian response in sheep superovulated after pretreatment with growth hormone and GnRH antagonists is weakened by failures in oocyte maturation

The administration of growth hormone (GH) or GH plus GnRH antagonists (GnRHa) in sheep allows the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries and may be useful to increase yields obtained in embryo programmes. The objective of the current study was to evaluate the ability of follicles recruited in response to treatment with GH and GnRHa to grow in response to exogenous follicle stimulating hormone (FSH) and the competence of their oocytes to resume meiosis. Seven females were treated with two doses of GnRHa (days 0 and 3) and three doses of 15 mg of GH (days 3, 4 and 5). Thereafter, this group and a second group (n = 7) were treated with three doses of 1.5 ml of FSH 12 h apart. A third group (control; n = 4) did not receive GH/GnRHa or FSH. The mean number of follicles aspirated on day 7 was higher in ewes treated with GH and GnRHa prior to the stimulation with exogenous FSH than in ewes treated with FSH without pretreatment and in untreated control sheep (20.4 +/- 2.6 vs 17.7 +/- 3.9 and 11.5 +/- 0.8, p < 0.05 and p < 0.01, respectively). The number of recovered cumulus-oocyte complexes after follicular aspiration was higher in the GH/GnRHa + FSH group (8.7 +/- 0.9 vs 6.8 +/- 1.3 in FSH group, n.s., and 4.5 +/- 0.8 in control, p < 0.05), but there were no differences found in the resumption of meiosis (63.1 +/- 9.5% for GH/GnRHa + FSH vs 79.5 +/- 6.3% for FSH and 60.0 +/- 8.8% for control). These results indicate that GH and GnRHa would be useful to increase the number of gonadotrophin-responsive follicles in the ovary, but adjustment of later FSH treatment allowing further development of follicles may be necessary prior to its use in superovulatory protocols.     

Follicular wave status at the beginning of the FSH treatment modifies reproductive features in superovulated sheep

In the current study we investigated whether the developmental status of the two largest follicles (LF1 and LF2) at the time of administration of the first two doses (0 and 12 h) of FSH of a superovulatory treatment influences periovulatory events and embryo yields in sheep. A larger size of LF1 was negatively correlated with embryo recovery (r=-0.608 for 0 h and r=-523 for 12 h, p<0.05), fertilization (r=-0.464 for 12 h, p<0.05) and viability (r=-0.775 for 12 h, p<0.005). Embryo viability rates were also lower when a higher difference between LF1 and LF2 (r=-0.839 for 0 h and r=-0.761 for 12 h, p<0.01) and a smaller size of LF2 (r=0.877 for 0 h and r=0.622 for 12 h, p<0.01) were observed. This indicates the existence of a limit in the follicular size that will be able to give rise a viable embryo. Conversely, a larger size of LF2 at the time of administration of the first two FSH doses was correlated with reduced recovery rates (r=-0.884 for 0 h and r=-0.706 for 12 h, p<0.01), whilst a decreasing size of LF1 and LF2 was correlated with an increased ovulation rate and recovered embryos. The dominance effect appeared to affect the timing of the preovulatory LH surge. Ewes with a higher difference between LF1 and LF2 displayed earlier LH surges (r=-0.420 for 0 h and r=-0.401 for 12 h, p<0.05) which were related to a higher number of non viable embryos (r=-0.777, p<0.05). The fact that superovulatory yields were affected by, both LF1 and LF2 supports the hypothesis of co-dominance effects in sheep.     

Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, C(max), and both the area under the plasma concentration-time curve extrapolated to the infinite, AUC(inf), and to the last moment of sampling, AUC(last)) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 x 1.32 mg, 2 x 1.10 mg, and 3 x 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7+/-0.6 versus 10.0+/-0.4 in N and 9.8+/-0.6 in M, P<0.05 for both) and embryos (7.9+/-0.8 versus 4.3+/-0.4 in N, P<0.05, and 6.7+/-0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54+/-0.08 ng/ml for R, 0.45+/-0.05 ng/ml for N and 0.35+/-0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4+/-2.2), M (18.9+/-1.5, n.s.) and N sheep (14.1+/-1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.     

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.     

Restoration of endocrine and ovarian function after stopping GnRH antagonist treatment in goats

We have tested if the high number of unfertilized ova and degenerated embryos found in superovulated goats previously treated with GnRH antagonist can be related to a prolongation of gonadotrophin down-regulation and/or alterations in follicular function during the period of administration of the superovulatory treatment, around 4 days after the end of the antagonist treatment. A total of 15 does were treated with intravaginal progestagen sponges and daily injections of 0.5mg of the GnRH antagonist Antarelix for 6 days, while 5 does acted as controls receiving saline. During the antagonist treatment, the mean plasma LH concentration was lower in treated than control goats (0.5 +/- 0.2 versus 0.7 +/- 0.5 ng/ml, P < 0.0005 ); however, the FSH levels remained unaffected (0.8 +/- 0.4 versus 0.8 +/- 0.5 ng/ml). In this period, treated does also showed an increase in the number of small follicles with 2-3 mm in size ( 10.7 +/- 0.7 versus 8.4 +/- 0.6, P < 0.05), and a decrease in both the number of follicles > or =4 mm in size ( 5.0 +/- 0.3 versus 6.8 +/- 0.5, P < 0.005) and the secretion of inhibin A (120.9 +/- 10.7 versus 151.6 +/- 12.6 pg/ml, P < 0.05). After cessation of the antagonist treatment, there was an increase in LH levels in treated goats from the day after the last Antarelix injection (Day 1), so that LH levels were the same as controls on Day 3 (0.6 +/- 0.1 versus 0.6 +/- 0.2 ng/ml). However, there were even greater numbers of small follicles than during the period of antagonist injections (15.4 +/- 0.6 in treated versus 8.9 +/- 0.7 in control, P < 0.0005 ). Moreover, the number of > or =4 mm follicles and the secretion of inhibin A remained lower in treated goats (3.9 +/- 0.3 follicles and 84.4 +/- 7.0 pg/ml versus 5.4 +/- 0.5 follicles, P < 0.05 and 128.9 +/- 14.2 pg/ml, P < 0.05 ). These results indicate that pituitary secretion of gonadotrophins is restored shortly after the end of antagonist treatment, but activity of ovarian follicles is affected.     

Ovarian response,embryo production and hormonal profile in superovulated goats treated with insulin

The influence of insulin on ovarian response and embryo production was investigated in 30 mixed breed goats, divided randomly into three equal (n=10) groups. Goats in Group 1 (control) were superovulated using 20 IU FSH i.m. in six divided descending doses, i.e. 4/4, 3/3 and 3/3 IU at 12 h interval for three consecutive days and were not given insulin treatment. Goats in Group 2 (insulin pretreatment) were pretreated with long acting purified bovine insulin 0.2 IU/kg body weight per day s.c. on Days 7, 8 and 9 of the estrous cycle prior to initiation of superovulatory treatment as in Group 1. Animals in Group 3 (insulin cotreatment) were treated as in Group I, but in addition received long acting purified bovine insulin 0.2 IU/kg body weight per day s.c. as a cotreatment along with the first, third and fifth FSH treatments on three consecutive days. Total ovarian response (corpus luteum and unovulated large follicle (UOLF)) was significantly (P<0.05) higher in insulin pretreatment (17.90+/-3.08) than in the cotreatment (11.50+/-2.34) and control (11.90+/-1.87) groups. The number of UOLF was significantly higher (P<0.05) in the insulin pretreatment (10.2+/-1.67) than the cotreatment (4.9+/-1.14) and control (3.6+/-1.09) groups. The mean transferable quality of embryos did not differ significantly among treatments. Progesterone concentration on the day of PGF(2)alpha treatment was not different (P>0.05) between the insulin treatment groups (5.28+/-0.79; 5.30+/-0.66 ng/ml). Estradiol-17beta concentration was significantly (P<0.05) higher on the day of PGF(2)alpha treatment in both the insulin treatment groups (36.67+/-6.40; 34.33+/-4.33 pg/ml) as compared to the control group (20.00+/-2.73 pg/ml). There is ample evidence to indicate beneficial effect of insulin on folliculogenesis and steroidogenesis in superovulated goats.     

The effect of a GnRH antagonist on endocrine and seminal parameters in stallions

Relatively little is known about endocrine control of reproduction in the stallion, but gonadotropins are thought to be central in regulating spermatogenesis and libido. The ability to effectively antagonise GnRH, and thereby gonadotropins, is therefore important both in further investigations of hormonal control of reproduction in stallions, and for clinical applications. In the present study four pony stallions were treated with a potent GnRH antagonist, Antarelix. Their libido, seminal parameters, and hormonal profiles were compared with those recorded before administration of the antagonist. Plasma concentrations of gonadotropins, testosterone and estradiol decreased by 48 h after antagonist administration, with estradiol and FSH being most consistently suppressed, and remained at reduced concentrations for 4 weeks. Spermatozoal motility, numbers and morphology were not significantly affected by treatment, but increasing numbers of round spermatogenic cells were seen in the ejaculate as the trial progressed. Libido was assessed by the time taken for the stallions to regain an erection in the presence of a mare after ejaculation (refractory period). The refractory period increased significantly after treatment. When the stallions were castrated 8 weeks after antagonist treatment, histological evidence of testicular degeneration was present. We concluded that use of this antagonist showed promise as a valuable research tool in modulating changes in circulating hormone concentrations in stallions. Reversibility of the effects on libido and testicular changes need further investigation.     

Effects of the Booroola Fec gene on ovarian follicular populations in superovulated Romanov ewes pretreated with a GnRH antagonist

Endocrine control of follicular growth was studied in mature Romanov ewes carrying (RF+) or not carrying (R+2) the Booroola Fec gene during an oestrous cycle after gonadotrophin-dependent follicles were suppressed by treatment with an antagonist of GnRH (Antarelix, 0.5 mg per day) and superovulatory treatment was administered. The left ovary was removed after 10 days of treatment (saline or Antarelix) and the right ovary was removed at the end of the superovulatory treatment. Ewes of both genotypes treated with Antarelix had lower plasma LH concentrations than did controls from day 0 to day 10. The inhibitory effect of Antarelix on LH concentration increased with day of treatment. The variability in FSH concentrations during the initial 10 days was reduced by Antarelix treatment in both genotypes. Plasma FSH concentrations were higher in RF+ ewes than in R+2 ewes. In both genotypes, FSH concentrations varied significantly with day of treatment, with the lowest concentrations at day 8 and the highest concentrations at day 5. RF+ ewes had a greater total and atretic number of antral follicles 0.62-1.12, 1.12-2.00 and 2.00-3.00 mm in diameter (classes 2, 3 and 4) than did R+2 ewes before and after superovulatory treatment. After superovulatory treatment, the total number of atretic and non-atretic follicles > 3.00 mm in diameter (class 5) increased in both genotypes. Superovulatory treatment also increased the number of total and atretic class 4 follicles in RF+ only. Conversely, superovulatory treatment decreased the mean number of class 3 follicles in both genotypes, while the number of atretic follicles was decreased only in R+2 ewes. Antarelix treatment significantly reduced the percentage of follicles > 2.00 mm in diameter in RF+ but not in R+2 ewes. Antarelix treatment before superovulatory treatment increased the total number of class 4 follicles in both genotypes but the increase was more significant in RF+ than in R+2 ewes. These results indicate that Antarelix pretreatment favours a greater superovulatory response in Romanov ewes carrying the Fec gene because ovulatory follicles are recruited from a wider range of follicular size classes.     

Follicular growth and regression during the 8 days after hypophysectomy in sheep

Ewes were hypophysectomized on Day 0 and ovariectomized 1, 2, 4 or 8 days later. There was no effect of hypophysectomy on the overall population of follicles greater than 0.8 mm in diameter during the time studied. However, the growth of healthy follicles greater than 2 mm in diameter was prevented by Day 2. Turnover of follicles was very active in the ovaries of hypophysectomized ewes as shown by peaks in the proportion of follicles in early atresia at Day 4 and in advanced atresia at Days 1 and 8. By Day 8, most of the measures of the population of follicles 0.8 to 2 mm in diameter were back to the values of Day 0 ewes. The mitotic index of the granulosa cells of the healthy follicles exhibited a similar pattern with a nadir at Day 2 followed by a return at Days 4 and 8 to values similar to Day 0 ewes. Ink-marked preovulatory follicles underwent a steady decrease in their histological size after hypophysectomy and this was associated with time-related changes in the health status of these follicles. By Day 1, 4 out of 7 follicles were still healthy while at Days 2, 4 and 8, all follicles were in advanced, late and collapsing atresia respectively. There was no evidence of an ability of PMSG (1000 i.u.) to rescue large follicles in advanced atresia (48 h after hypophysectomy). Furthermore, at 24 h after hypophysectomy, only 2 out of 5 follicles were maintained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular growth, ovulation and embryo recovery in dairy cows given FSH at the beginning or middle of the estrous cycle

Variability in the superovulation response is an important problem for the embryo transfer industry. The objective of this study was to determine whether FSH treatment at the beginning of the cycle would improve the ovulation rate and embryo yield in dairy cows. Twenty-eight postpartum cyclic dairy cows were allocated at random to 4 treatment groups (A, B, C and D). Group A cows (n = 10) received FSH (35 mg) at a decreasing dose, starting on Day 9 (Day 0 = day of estrus) for 5 days followed by PGF(2alpha) (35 mg) on Day 12. Cows assigned to Groups B, C and D (n = 6 cows each, respectively) were given 35 mg FSH at a decreasing dose from Days 2 to 6 followed by PGF(2alpha) on Day 7. Group C and D cows received PRID inserts from Day 3 to Day 7. Cows in Group D additionally received 1000 IU hCG 60 hours after PGF(2alpha) treatment. Ovaries were scanned daily using a real time ultrasound scanner from the beginning of FSH treatment until embryo recovery, to monitor follicular development, ovulation and the number of unovulated follicles. Embryos were recovered from the uterus by a nonsurgical flushing technique 7 days after breeding. There were no differences (P>0.01) in the number of follicles > 10 mm at 48 hours after PGF(2alpha) treatment among the 4 groups. The mean numbers of follicles were 10.6 +/- 1.2, 9.3 +/- 1.3, 12.2 +/- 1.3 and 15.0 +/- 2.9 for Groups A, B, C and D, respectively. A significantly (P<0.001) higher number of ovulations was observed and a larger number of embryos was recovered in Group A than in the other groups. The results of this study indicate that superovulation with FSH at the beginning of the cycle causes sufficient follicular development but results in very low ovulation and embryo recovery rates.     

Ovulatory response and embryo yield in superovulated holstein heifers given a priming dose of FSH-P at day 2 of the estrous cycle

Mature Holstein heifers were given either a priming dose of follicle-stimulating hormone-pituitary (FSH-P, 10 mg) or saline on Day 2 of the estrous cycle, or no pretreatment. All animals were subsequently given a decreasing dose superovulatory treatment of FSH-P beginning between Days 8 and 14, coupled with an injection of prostaglandin F2a to induce luteolysis. Pretreatment with FSH-P had no effect on the total superovulatory response or on the number of transferable embryos collected at Day 7 of gestation. Comparison of the results of our study with previous reports in the literature may suggest that FSH-priming early in the cycle may be advantageous in promoting superovulation only when the superovulatory response of the population of animals is otherwise weak.     

Interruption of the canine estrous cycle with a low and a high dose of the GnRH antagonist, acyline

To test the efficacy and clinical safety of a low and high dose of the GnRH antagonist, acyline, on estrous cycle interruption and anovulation in female dogs, 20 proestrous (<3 d) bitches were randomly assigned to one of the following pharmacological protocols (given sc): acyline 110 μg/kg (ACY-L; n = 6); acyline 330 μg/kg (ACY-H; n = 8); or placebo (PLACE, n = 6). The animals were monitored (clinical and vaginal cytology examinations) daily for 60 d. Blood samples for serum progesterone serum concentrations were collected 14 d after treatment to determine if ovulation had occurred. Appearance of side effects and days to the onset of the first spontaneous estrous cycle after treatment were also recorded. In both ACY groups, but not the PLACE group, estrous cycles were interrupted after treatment (P < 0.05). The interval from treatment to estrus interruption in ACY-L and ACY-H groups was 3.0 ± 0.6 and 3.2 ± 0.2 d, respectively (LSM ± SEM; P > 0.05). In the PLACE bitches, physical, behavioral and cytological proestrus slowly progressed to estrus and diestrus. Ovulation was absent in all ACY, but not in PLACE bitches (P < 0.05). None of the females manifested side effects related to the treatments (P > 0.05). Spontaneous return to a normal estrous cycle during the study period occurred in all ACY (ACY-L 19.5 ± 2.7 d vs ACY-H 24.8 ± 2.0 d; P > 0.05), but in none of the PLACE bitches (P < 0.05). In conclusion, acyline efficiently, safely and reversibly interrupted an early phase of the estrous cycle in bitches by preventing ovulation.     

Control of ovulation with a GnRH agonist after superstimulation of follicular growth in buffalo: fertilization and embryo recovery

The potential to use a GnRH agonist bioimplant and injection of exogenous LH to control the time of ovulation in a multiple ovulation and embryo transfer (MOET) protocol was examined in buffalo. Mixed-parity buffalo (Bubalus bubalis; 4-15-year-old; 529 +/- 13 kg LW) were randomly assigned to one of five groups (n = 6): Group 1, conventional MOET protocol; Group 2, conventional MOET with 12 h delay in injection of PGF2alpha; Group 3, implanted with GnRH agonist to block the preovulatory surge release of LH; Group 4, implanted with GnRH agonist and injected with exogenous LH (Lutropin, 25 mg) 24 h after 4 days of superstimulation with FSH; Group 5, implanted with GnRH agonist and injected with LH 36 h after superstimulation with FSH. Ovarian follicular growth in all buffaloes was stimulated by treatment with FSH (Folltropin-V, 200 mg) administered over 4 days, and was monitored by ovarian ultrasonography. At the time of estrus, the number of follicles >8 mm was greater (P < 0.05) for buffaloes in Group 2 (12.8) than for buffaloes in Groups 1(8.5), 3 (7.3), 4 (6.1) and 5 (6.8), which did not differ. All buffaloes were mated by Al after spontaneous (Groups 1-3) or induced (Groups 4 and 5) ovulation. The respective number of buffalo that ovulated, number of corpora lutea, ovulation rate (%), and embryos + oocytes recovered were: Group 1 (2, 1.8 +/- 1.6, 18.0 +/- 13.6, 0.2 +/- 0.2); Group 2 (4,6.1 +/- 2.9, 40.5 +/- 17.5, 3.7 +/- 2.1); Group 3 (0, 0, 0, 0); Group4 (6, 4.3 +/- 1.2, 69.3 +/- 14.2, 2.0 +/- 0.9); and Group 5 (1, 2.5 +/- 2.5, 15.5 +/- 15.5, 2.1 +/- 2.1). All buffaloes in Group 4 ovulated after injection of LH and had a relatively high ovulation rate (69%) and embryo recovery (46%). It has been shown that the GnRH agonist-LH protocol can be used to improve the efficiency of MOET in buffalo.     

Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 °C, 5% CO₂, and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH + 10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH + 5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14 ± 0.074 and 0.15 ± 0.076 vs. 0.46 ± 0.078 and 0.50 ± 0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.     

Progesterone treatment, FSH plus eCG, GnRH administration, and Day 0 Protocol for MOET programs in sheep

The effect of various superstimulatory treatments on the number of corpora lutea, fertilization rate, and embryo yield was studied in sheep. Overall, data from 708 Merino donors and 4262 embryos were analyzed in four experiments. In Experiment 1, varying intervals of progesterone treatment (5 to 14 d) before follicle-stimulating hormone (FSH) administration did not significantly affect the proportion of responding donors, the mean number of corpora lutea, or the mean number of recovered and transferable embryos per donor. In Experiment 2, a single dose of equine chorionic gonadotropin (eCG, 200 or 300 IU) combined with the FSH treatment (i.e., given at CIDR removal) reduced the number and the quality of embryos compared with that for not giving eCG (P < 0.05). In Experiment 3, one dose of gonadotropin-releasing hormone (GnRH) given 24 h after CIDR removal improved the number of transferable embryos compared with that for not giving GnRH (P < 0.05). In Experiment 4, the new superstimulatory Day 0 Protocol, which includes starting FSH treatment at the emergence of Wave 1 (i.e., soon after ovulation, in the absence of a large follicle), improved ovarian response, with a tendency to produce more embryos compared with that for the Traditional Protocol. In summary, this study, analyzing data from various pharmacologic treatments, allows an improvement from four to eight transferable embryos per treated donor in multiple ovulation and embryo transfer programs in sheep.     

Embryo production and endocrine response in ewes superovulated with PMSG, with or without monoclonal anti-PMSG administered at different times

Mature nonlactating Altamurana ewes (n = 168) were synchronized in the seasonal anestrus period with FGA-impregnated intravaginal pessaries for 12 d. In Experiment 1, 48 ewes were divided into a 3 x 4 factorial design for anti-PMSG monoclonal antibody (AP) bioassay test. Concomitant injections of PMSG (1000, 1500, 2000 IU) and AP (0, 1, 2, 3 microl/IU PMSG) were given, and ovarian response was evaluated by laparoscopy. In Experiment 2, 120 ewes were divided into 8 experimental groups (n = 15 per group). The ewes treated with 1000 or 1500 IU PMSG at -24 h from sponge removal were given AP intravenously at 50 h after pessary withdrawal, 12 or 24 h after the onset of estrus, while the controls did not receive AP. Blood samples were collected from ewes (n = 6) treated with 1500 IU PMSG with or without anti-PMSG. Ovarian response and embryo production were evaluated on Day 7 after sponge removal upon laparotomy. It was found that 1 microl AP was effective in neutralizing 1 IU PMSG. No significant differences in serum concentrations of progesterone were observed among the groups of superovulated ewes. Estradiol-17 beta levels were reduced following AP treatment 12 h after the onset of estrus. At a lower dosage of superovulatory treatment (1000 IU PMSG), AP injected at 12 or 24 h after the onset of estrus significantly lowered large follicles (P < 0.01) and increased the rate of ovulation (P < 0.05). Moreover, embryo production showed a more than two-fold increase (P < 0.01) of viable embryos following AP injection at 12 or 24 h after the onset of estrus (3.2 to 3.3 vs 1.3, with vs without anti-PMSG). It is concluded that superovulatory treatment with 1000 IU PMSG plus AP administered at a fixed time after the onset of estrus may improve ovarian response and the yield of viable embryos in ewes.     

Individual variations in FSH release after a GnRH challenge and relationship with semen output in young bulls

Blood samples collected from 54 Montbéliarde young post-pubertal bulls were assayed for FSH. In the first trial, 0.25 mg GnRH was given to each of 25 bulls at 12 months of age. In a second group of 29 bulls, each received 20 mg dexamethasone followed by 0.25 mg GnRH 5 h later. Based on measurements of semen output, the bulls were classified into three categories: good, medium and poor semen producers. The total FSH response was significantly different among individuals (P less than 0.05) and repeatable (r = 0.45) only after the combined treatment. The mean total responses did not differ significantly between the 3 categories of semen producers, but individual FSH responses were significantly and negatively correlated to quantitative and qualitative semen production characteristics (r = 0.4-0.7). It was concluded that measurement of FSH after a combined dexamethasone-GnRH challenge permits the demonstration of a significant relationship between FSH release and semen production in individual bulls.     

Effect of season and duration of FSH treatment on embryo production in sheep

Thirty-six mature Manchega ewes were used in two experiments to determine the effect of season and of 2- or 3-d FSHp treatment on the ovulation rate and number of transferable embryos produced. During the breeding season, estrus was synchronized with FGA (30 mg for 13 d). Begining 48 or 24 h before sponge removal, each ewe received two daily injections of 4-4-3-3-1-1 or 5-5-3-3 mg of FSHp. Concurrently with the two last injections both groups were administered 100 mug of LH. Ewes were tested for estrus and 6 or 7 d later were laparotomized and surgically flushed to recover embryos. The number of corpora lutea (CL), the total number of embryos and of viable embryos were recorded. Six months later (nonbreeding season) the design was repeated, with each ewe receiving the opposite treatment to that received in the fall. Response in ovulation rate and number of viable embryos did not differ between seasons. Mean (SEM) numbers of observed CL and embryos recovered were higher (P<0.001) with the 3-d treatment (8.7+/-5.8 and 7+/-4.8) than with the 2-d treatment (5.8+/-3.2 and 4.4+/-3) when pooled over the two seasons. The mean number of transferable embryos was higher (P<0.01) with the 3-d (4.2+/-3.9) than with the 2-d treatment (2.5+/-2.3).     

Fixed-time AI protocols replacing eCG with a single dose of FSH were less effective in stimulating follicular growth, ovulation, and fertility in suckled-anestrus Nelore beef cows

The aim of the present study was to evaluate the effects of a single treatment with FSH on diameter of the largest follicle and on conception rates of suckled Bos indicus beef cows submitted to timed artificial insemination (TAI). Four hundred fifty-six suckled anestrous Nelore beef cows at 30-60 days postpartum were assigned to treatments. At the first day of the estrous synchronization protocol (Day 0), all cows received a progesterone-releasing intravaginal device plus 2mg of estradiol benzoate. On Day 8, cows were assigned to blocks according to the diameter of the largest follicle and then allocated to one of three treatment groups (Control, FSH, or eCG) within each block. Simultaneously to progesterone device withdrawal on Day 8, cows in the eCG treatment group (n=150) received 300 IU of eCG and cows in FSH treatment group (n=153) received 10mg of FSH, and Control cows (n=153) did not receive any additional treatment. Additional treatments with 150 μg of cloprostenol and 1mg of estradiol cypionate (EC) were also administered concurrently to progesterone device removal in all cows on Day 8. Two days later (D10), TAI and ovarian ultrasonic examinations to evaluate follicle size were performed in all cows. On Day 12, a subset of cows (n=389) were submitted a second ultrasonic exam to confirm ovulation. Final follicular growth (mm/day) was less (P=0.006) in both Control (0.95±0.11) and in FSH-treated cows (0.90±0.10) than in eCG-treated cows (1.40±0.13). Interestingly, there was a treatment-by-BCS interaction in ovulation results (P=0.03), in which, eCG treatment increased percentage of cows having ovulations with a lesser BCS. Similarly, there was a treatment-by-BCS interaction for conception (P=0.04), where the eCG treatment increased fertility in cows with a lesser BCS. In conclusion, FSH failed to stimulate final follicular growth, ovulation, and conception rate in sucked-anestrous beef cows submitted to TAI as effectively as eCG. However, physiological effects of eCG seem to be more evident in cows with a lesser BCS.