1. Investigación

The objective of this investigation was to determine the ovarian response, fertility, and prolificacy of nulliparous sheep when compared to multiparous sheep after a short-term (7 days) CIDR/eCG treatment which was administered during the non-breeding season. All the multiparous sheep, whereas only 54% of the nulliparous ewes, showed signs of estrus. However, 81.8% of the multiparous sheep and 100% of the nulliparous ewes ovulated. Fertility was also low after short-term progesterone treatments during the anestrous season in maiden sheep (30.8 vs. 72.7% in multiparous ewes). Such results indicate significant differences in the response to CIDR/eCG protocols for induction and synchronization of estrus and ovulation between nulliparous and multiparous sheep during the non-breeding season.

This research aimed to evaluate whether the administration of hCG at the time of removal of a progesterone device may be effective at inducing estrus and ovulations in sheep during the natural seasonal anestrous, by comparing reproductive outputs (induction and duration of estrus, follicle development, ovulation, ovulation rate, and pregnancy rates) in ewes treated with eCG or only saline solution at the time of CIDR removal. Whereas results demonstrated no response in the control non-treated group, the largest rate of narrowly synchronized estrus signs and ovulations occurred in the eCG-group. The administration of hCG was effective at inducing estrus, promote follicular growth and a delayed yet significant ovulation (>84%) on day 10 after CIDR removal; moreover, an increased embryonic implantation rate was also observed. Moreover, if the hCG ewes remain exposed to active males for some days, said strategy could be adopted, aiming to induce pregnancies by natural mating in a short period of time.

Gonadotropin-releasing hormone (GnRH)–loaded chitosan nanoparticles (GnRH–ChNPs) were used at different doses and routes of administration to induce ovulation in rabbits as an attempt to improve artificial insemination (AI) procedures and outcomes. In this study, the characteristics (size, polydispersity, loading efficiency, and zeta-potential) of GnRH–ChNPs and the GnRH release pattern were determined in an in vitro study. A first in vivo study assessed the pituitary and ovarian response to different GnRH–ChNPs doses and routes of administration (two i.m. doses, Group HM = 0.4 g and Group QM = 0.2 g, and two intravaginal doses, Group HV = 4 g and Group QV = 2 g) against a control group (C) receiving bare GnRH (0.8 g). The HM, QM, and HV treatments induced an earlier LH-surge (90 min) than that observed in group C (120 min), whilst the QV treatment failed to induce such LH surge. The number of ovulation points was similar among treatments, except for the QV treatment (no ovulation points). A second in vivo study was consequently developed to determine the hormonal (progesterone, P4, and estradiol, E2) profile and pregnancy outcomes of both HM and HV treatments against group C. The treatment HM, but not the treatment HV, showed adequate P4 and E2 concentrations, conception and parturition rates, litter size, litter weight, and viability rate at birth. Overall, the use of GnRH–ChNPs allows for a reduction in the conventional intramuscular GnRH dose to half without compromising fertility. However, the addition of GnRH– ChNPs to semen extenders, although successfully inducing ovulation, has negative impacts on fertility. Thus, more studies are needed to explore this point and allow further adjustments.

Currently, there is an intense effort to find an alternative hormone to eCG to induce ovulation after estrus synchronization treatments in sheep. One of the proposed alternatives is based on the use of hCG, but the results are controversial since fertility rates are commonly affected. The present study aims to evaluate, therefore, the adequacy of hCG in protocols for the synchronization of estrus and ovulation. Ovarian follicle dynamics, occurrence of estrus behavior and subsequent ovulation, quality of corpora lutea, and pregnancy rate after controlled natural mating were assessed in two consecutive trials. The findings indicate that the low fertility rates reported for the protocols based on the administration of hCG for inducing ovulation during estrus synchronization in sheep may be related to a high occurrence of abnormal follicular growth patterns, disturbances, and retardments of ovulation and concomitant formation of follicular cysts in the treated females. These results preclude their practical application to induce ovulation concomitantly to estrous synchronization treatments.

The foreseen shortage of eCG for estrus synchronization in sheep makes necessary the development of alternative protocols. The aim of the present work was to evaluate the reproductive response of sheep in breeding season to the administration of GnRH using propylene-glycol as a cosolvent and the subcutaneous route for slowing and extending the release of GnRH, as well as the most adequate timing for such administration. In the present study, protocols based on a short-term CIDR treatment and a single subcutaneous dose of GnRH in propylene-glycol at 36 h after CIDR removal induced a similar ovarian response to protocols based on administration of eCG at CIDR removal or intramuscular GnRH in distilled water at 56 h after. In such protocol, 80% of the animals developed estrus in a narrow timing (75% between 36 and 48 h after CIDR removal), and all of them also ovulated in a narrow window (87.5% between 72 and 76 h after CIDR removal, with 62.5% between 72 and 76 h) and showed a similar ovulation rate and plasma progesterone concentrations at the induced estrous cycle. Hence, administration of GnRH in propylene-glycol may constitute an alternative to traditional protocols based on the administration of eCG.

The present study aimed to set up a short-term protocol for synchronization of follicular wave emergence in sheep, concomitant with estrus synchronization, which would improve ovarian response in assisted reproductive technologies. Administration of a single GnRH dose, concomitant with the insertion of a progesterone-loaded CIDR device, caused regression of gonadotrophin-dependent follicles 4 mm in all the GnRH-treated sheep and in around 80% of the controls treated only with CIDR (p < 0.05). Similar percentages of ewes lost all follicles (around 70%) or only the largest one (around 30%) in both groups. Hence, 54.1% and 70% of the sheep lost all large follicles and initiated a new follicular wave in the control and GnRH groups, respectively (p < 0.05). The remaining sheep showed follicles that were still not dependent of luteinizing hormone (LH). So, in fact, all the sheep had non-dominant follicles after treatment. In conclusion, a treatment including GnRH at CIDR insertion would o er a time- and cost-e cient protocol for inducing follicular turnover and synchronizing a new follicular wave at any stage of the estrous cycle.

The present study supports that 5-days short-term CIDR treatments without administration of eCG are equally effective for inducing estrus behavior, preovulatory LH discharge and ovulation in sheep than classical protocols based on 14-days treatments plus eCG at CIDR withdrawal. However, the implementation of a 5-days protocol without eCG for fixed-time artificial insemination would be adapted to a later timing of ovulation (p < 0.05).

The present study supports that short-term (7-days) protocols based on progestagen-impregnated sponges and progesterone-loaded CIDRs are equally effective to induce ovulatory response in sheep. There were no significant differences in the onset of estrus behavior (32.0±6.0 and 33.8±4.0h after device withdrawal for sponges and CIDRs, respectively; p > .05) and preovulatory LH discharge (5.1 ± 2.1 and 5.8 ± 3.3h after onset of estrus behavior for sponges and CIDRs, respectively; p > .05). These features are similar to previously described for classical long-term (12-14 days) treatments. Hence, short-term CIDR-based protocols may be implemented using the same time-intervals for insemination than sponge-based and long-term protocols.

The present study characterizes, for sheep, the occurrence and timing of the onset of estrus behavior and ovulation and the yields obtained (ovulation rate, progesterone secretion, and fertility) after Controlled Internal Drug Release (CIDR) insertion for five, six, seven, or fourteen days, with or without equine chorionic gonadotrophin (eCG) in ewes of the Segureña meat breed. All the treatments showed above 80% of females displaying estrus, but the onset of estrus was earlier and more synchronized when using eCG and, among groups with eCG, onset of estrus was earlier in the sheep treated for 14 days than in the short-term treatments (p < 0.05 for all). Administration of eCG after either short- or long-term treatments assured the occurrence of fertile ovulations in all the animals. Conversely, ovulatory success without eCG was found to be dependent on treatment length, with a high percentage of animals ovulating after five days of treatment (83.3%) and very low percentages after treatment for six or seven days (40% and 20%, respectively). Ovulation rate and progesterone secretion were similar among animals ovulating, but ovulation failures predetermined the fertility yields obtained in response to the treatments. Hence, the best results were found after treatment for 14 days plus eCG, and for 5 days without eCG (83.3 for both, p < 0.05 when compared to the other groups with different treatment lengths and with or without eCG).

The present study examined, for meat sheep (Segureña breed; 2–5-years old, mean body score of 3.5 ± 0.5), the timings of onset of estrus behavior, preovulatory luteinizing hormone (LH) surge and ovulation, and the ovulation rate and fertility obtained after insertion of controlled internal drug release (CIDR) devices for 5 days plus treatment with equine chorionic gonadotrophin (eCG; single dose at CIDR removal, n = 19 ewes) or gonadotrophin-releasing hormone (GnRH, either in a single dose at 56 h after CIDR removal, group CIDR-GnRH, n = 19 ewes; or in one dose at CIDR insertion and another dose 56 h after CIDR removal, group GnRH-CIDR-GnRH, n = 19 ewes). In all the ewes, the appearance of estrus behavior ranged between 84% and 90% and all females showing estrus signs had subsequent preovulatory LH peaks and ovulations. Onset of these events was earlier in the CIDR-eCG group than in the CIDR-GnRH and GnRH-CIDR-GnRH groups (p < 0.05). These differences were mainly determined by the onset of estrus behavior, since timing and intervals of LH peak and ovulation were similar among treatments. In fact, the range of ovulations was narrower in the GnRH-CIDR-GnRH group, which suggests better synchronization of follicular growth (p < 0.05). In conclusion, protocols with two doses of GnRH offer similar yields to eCG protocols.