Reduced embryo yield obtained from superstimulated ewes with low circulating AMH concentration is improved by lengthening the FSH treatment.

The aim of the present study was to evaluate: 1) the association between AMH, AFC, superovulatory response and embryo yield in sheep; and 2) the effect of FSH treatment length during superstimulation of the first follicular wave on ovarian response and embryo yield, particularly in ewes with low and high AMH. The experiment was performed on 63 Polled Dorset ewes that received an ovarian superstimulatory treatment during the first follicular wave (Day 0 protocol). Ewes were administered a total dose of 240 mg of FSH distributed in six (6-dose regimen, n = 30) or eight (8-dose regimen, n = 33) decreasing doses administered 12 h apart. On Day -9 (random stage of the estrous cycle) and Day 0 (day of the first FSH dose) ovarian ultrasonography was performed and blood samples were collected for AFC and AMH determinations, respectively. A weak positive correlation between AMH and small AFC (follicles <4 mm) was observed (r = 0.23; P = 0.07), and AMH concentration was positively correlated (r = 0.29; P < 0.05) with the number of corpora lutea (CL) determined at embryo collection (i.e., 6 d after insemination). The length of FSH treatment tended (P = 0.06) to affect the ovarian response, such that the number of CL was greater in 8-dose than 6-dose treated ewes, while no differences (P > 0.10) in embryo yield outcomes were observed. For further analysis, ewes were classified into low (<7 ng/mL) and high (>10 ng/mL) serum AMH. In high AMH ewes, there were no differences (P > 0.05) in the number of CL nor embryo yield between the 6-dose and 8-dose treatment (e.g., 7.8 ± 2.4 and 8.3 ± 2.5 transferable embryos, respectively; P = 0.92). Conversely, for low AMH ewes, fertilized ova and embryo yield were greater (P ≤ 0.05) for ewes receiving the 8-dose than the 6-dose superstimulatory treatment (e.g., 8.4 ± 2.8 vs. 2.7 ± 0.9 transferable embryos, respectively, P ≤ 0.05). In conclusion, embryo production in poor responding ewes with low low circulating AMH is improved by extending the superstimulatory treatment length from 6 to 8 FSH doses.

Serum progesterone concentrations during FSH superstimulation of the first follicular wave affect embryo production in sheep.

The aim of the present study was to evaluate the effect of serum progesterone concentrations during the superstimulatory treatment of the first follicular wave on fertilization rate and embryo development in sheep. A total of 71 Merino ewes received a superstimulatory FSH treatment during Wave 1 of ovarian follicular development (Day 0 Protocol), which was administrated under low progesterone concentrations typical of the early luteal phase (control group, n = 33) or under high progesterone concentrations induced by the administration of an intravaginal device from Day 0 to Day 3 containing 0.3 g progesterone (n = 38). Intrauterine insemination after FSH superstimulation was followed by uterine flushing 6 days later. Serum progesterone concentrations from Day 0 to 3 were greater in those ewes treated with progesterone (P < 0.05), while serum estradiol-17ß concentrations were not affected by the treatment. Although the mean number of corpora lutea per donor was not affected by the progesterone treatment, the number of collected ova and embryos was greater in progesterone treated than untreated ewes (6.6 ± 0.7 compared with 4.6 ± 0.9, respectively; P < 0.05). Furthermore, progesterone treatment increased fertilization rate (93.3% compared with 83.3%; P < 0.05) and the proportion of Grade 1 embryos (67.7% compared with 52.7%; P < 0.05) compared with the control group. In conclusion, oocyte fertilization rate and embryo quality are improved by high progesterone concentrations during FSH superstimulation, which suggests an important role of progesterone during preovulatory follicular development.

Day 0 protocol: superstimulatory treatment initiated in the absence of a large follicle improves ovarian response and embryo yield in goats.

A new superstimulatory protocol (Day 0 Protocol) to initiate FSH treatment in the absence of a large follicle was compared to a traditional protocol in goats. The Day 0 Protocol (n=44) consisted of pre-treatment with progesterone and eCG to synchronize ovulation and the emergence of Wave 1, with FSH starting 84 h after the end of progesterone exposure (i.e., soon after ovulation). The traditional protocol (n=46) consisted of 11 d of progesterone exposure, with FSH treatment beginning 2 d before the end of progesterone exposure. Treatment with FSH was initiated in the absence of a large follicle in 37/44 and in 6/46 goats in the Day 0 Protocol and traditional protocol, respectively (P<0.01). There was more CL in the Day 0 Protocol than in the traditional protocol (breeding season: 9.6+/-0.6 and 6.3+/-0.8, P<0.05; non-breeding season: 14.3+/-1.5 and 10.7+/-1.5; P<0.05). More Grades 1 and 2 embryos were recovered in the Day 0 Protocol than in the traditional protocol (breeding season: 4.8+/-0.7 and 1.8+/-0.5, P<0.05; non-breeding season: 5.6+/-1.1 and 3.5+/-0.7, P=0.07). Similarly, the proportion of embryos that were Grades 1 and 2 was higher for the Day 0 Protocol than for the traditional protocol (breeding season: 81/114, 71%, versus 16/43, 37%, P<0.05; non-breeding season: 118/203, 58% versus 95/205, 46%, P<0.05). In summary, the Day 0 Protocol, was effective in initiating superstimulatory treatment in the absence of a large follicle, and compared to the traditional protocol, induced a higher ovulation rate and better embryo yield in goats.

Semen deposition by cervical, transcervical and intrauterine route for fixed-time artificial insemination (FTAI) in the ewe.

Semen deposition through the cervix into the uterus is a difficult technique in ewes and represents the main limiting factor for insemination in this species. The objective of this study was to evaluate the pregnancy rate achieved with a new transcervical insemination method in comparison with conventional cervical and laparoscopic intrauterine techniques. A total of 586 multiparous Corriedale ewes were synchronized for fixed time artificial insemination (FTAI) performed by cervical, transcervical, or intrauterine route at 46-50 h or 52-56 h after progesterone device removal in a 3 × 2 factorial design. Pregnancy rate was affected by the insemination technique and by the moment of FTAI (P < 0.05), without interaction (P= NS). Overall, the fertility was improved as semen deposition was deeper and insemination was delayed. For transcervical insemination, pregnancy rate was intermediate (42.3%; P= NS) between cervical and intrauterine route (36.0% and 50.2%; P < 0.05), and was greater for those ewes inseminated beyond 4 cm into the cervix (60.0% versus 35.1% for insemination beyond or within 4 cm into the cervix, respectively; P < 0.05). Semen deposition beyond 4 cm into the cervix was achieved only in 28.8% of the females receiving transcervical insemination. This method was more time-consuming than cervical or laparoscopic insemination (11.4 ± 1.6 versus 85.5 ± 7.5 and 56.8 ± 5.6 ewes inseminated per hour, respectively; P < 0.05). In summary, greater pregnancy rate using FTAI is obtained when semen is placed into the uterus, which was achieved in all females only through laparoscopy. Further improvements are required for transcervical insemination to be applied in large-scale FTAI programs in Corriedale ewes.

Response of Corriedale ewes to the "ram effect" after priming with medroxyprogesterone, fluorogestone, or progesterone in the non-breeding season.

One hundred eighty-nine Corriedale ewes were used during the non-breeding season to study the "ram effect" stimulus after priming with progestogens. Intravaginal sponges containing either medroxyprogesterone acetate (MAP group, n = 49), fluorogestone acetate (FGA group, n = 49), or progesterone devices (CIDR group, n = 46) were inserted on Day-6 (Day 0 = introduction of the rams). Forty-five ewes were untreated and kept as a control group. On Day 0 the sponges were removed and rams provided with marking harnesses for oestrous detection were placed with the ewes. Onset of estrus was monitored until Day 25, and conception was determined by transrectal ultrasonography. Ewes came into heat during 4 periods: Days 0-3, 5-7, 17-20, and 21-23. The overall number of oestrus ewes were 29%, 53%, 35%, and 50% for the control, MAP, FGA, and CIDR groups, respectively (MAP and CIDR > control, p < 0.05). Control ewes presented oestrus only on Days 17-20 and 21-23. Oestrus in the progestogen-primed ewes was concentrated during Days 0-3 and 17-20, and some ewes came into oestrus on Days 5-7. There were no differences between different primings neither in oestrous response nor in conception rate. The conception rate from matings occurring on Days 0-3 was higher than on those occurring on Days 17-20. We conclude that MAP, FGA, and CIDR is equally effective in improving the response to the ram effect, and the pattern of oestrus in primed ewes was different than previously reported.

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 24h 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.

Prostaglandin F2alpha treatment associated with timed artificial insemination in ewes.

Traditional treatments of two prostaglandin F2 alpha (PGF2alpha) doses at 10-day intervals or more did not result in acceptable pregnancy rates in timed artificial insemination (TAI) programmes in ewes. An explanation might be the undefined time-period of the onset of oestrus and ovulation after the treatment. Recently a consistent interval to oestrus and ovulation was obtained by giving PGF2alpha at day 3 post-ovulation, i.e. when the largest follicle of the first follicular wave of the cycle was still growing. This can be achieved when a second dose of PGF2alpha is given 7 days after a first dose. In this work, we evaluated the synchronization of oestrus and determined which of three different moments of TAI was the most successful using a PGF2alpha (PG-7d) protocol in a large flock. A total of 436 nulliparous and multiparous ewes were treated with two doses of a PGF2alpha analogue (delprostenate 160 microg, i.m.) separated by 7 days. Onset of oestrus was recorded twice a day and a single cervical TAI with fresh undiluted semen was performed either at 42 h (n = 152), 48 h (n = 120), or 54 h (n = 164), after the second PGF2alpha dose without taking into account the oestrous response. Pregnancy rate was determined by transrectal ultrasonography 30 days after insemination. Onset of oestrus was detected in 308 of 328 and 89 of 108 multiparous and nulliparous ewes, respectively (p < 0.001), within 72 h after treatment. The distribution of the onset of oestrus did not differ between multiparous and nulliparous ewes and the highest proportion of ewes in oestrus was detected between 25 to 48 h (313/397) from the second PGF2alpha dose. The pregnancy rate in ewes inseminated at 42 h tended to be higher than those inseminated at 48 h (p = 0.09) and was higher than those inseminated at 54 h (p < 0.05) (56/152, 31/120, 37/164; respectively). Therefore, the use of the PG-7d protocol resulted in a very high synchronization of oestrus with the highest concentration (around 80%) between 25 to 48 h from the end of treatment. The best pregnancy rate (37%) was obtained after a single cervical TAI with fresh semen at 42 h.