Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation.

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

Periovulatory administration of firocoxib did not alter ovulation rates and mitigated post-breeding inflammatory response in mares.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a therapeutic option for the treatment of inflammation. However, negative effects of non-selective NSAIDs for treatment of mares with endometritis have been described, including delayed uterine clearance and impairment of ovulations. Firocoxib is a specific cyclooxygenase-2 (COX-2) inhibitor and has the ability to act in the uterus of mares. We investigated the effects of firocoxib on ovulation rate, numbers of polymorphonuclear neutrophils (PMNs), and COX-2 protein levels in the endometrial tissue of susceptible mares after insemination. Two experiments were conducted. In experiment 1, twenty mares were evaluated in two consecutive estrous cycles broken into the following groups: Control - no pharmacological interference; Treatment - mares were treated with 0.2 mg/kg of firocoxib orally. The treatment began on the day of ovulation induction, and firocoxib was administered until one day after artificial insemination (AI). Ovulation was induced with 1 mg of deslorelin acetate and the mares were inseminated 24 h after the injection. Ovulation was confirmed 48 h after induction, and embryos were collected eight days after ovulation. Experiment 2: Nine mares susceptible to persistent mating-induced endometritis (PMIE) were artificially inseminated. The mares were examined with ultrasound and inseminated with fresh semen in two consecutive cycles, control and treated, in a cross-over study design. The amount of intrauterine fluid was measured, and endometrial samples were collected 24 h after AI. The number of PMNs was determined by endometrial cytology and biopsy, and COX-2 labeling in endometrial samples was evaluated by immunohistochemistry. Firocoxib treatment did not induce ovulatory failure or affect embryo recovery rate in Experiment 1. In Experiment 2, firocoxib treatment reduced inflammation after AI in mares as evidenced with results regarding PMN numbers/percentage and endometrial COX-2 staining. In conclusion, the proposed treatment with firocoxib reduced endometrial inflammation in mares susceptible to PMIE after breeding, with no adverse effects.

Uterine clinical findings, fertility rate, leucocyte migration, and COX-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after AI.

Persistent mating-induced endometritis (PMIE) results in decreased fertility in horses, thereby causing a significant impact in the horse market. Platelet-rich plasma (PRP), a modulator of the inflammatory response, has been largely used in veterinary medicine. Here, we investigated the effects of PRP on uterine inflammation, conception rate, endometrial polymorphonuclear neutrophil (PMN) migration, and COX-2 protein levels in the endometrial tissue. Thirteen PMIE-susceptible mares were used for artificial insemination (AI). The mares were inseminated with fresh semen in three consecutive cycles in a cross-over study design. The following cycle classifications were used: control cycle, no pharmacological interference; pre-AI, 20 mL of PRP was infused 24 h before AI; and post-AI, 20 mL of PRP was infused four h after AI. Follicular dynamics were monitored daily by transrectal ultrasound. When a follicle larger than 35 mm was detected, ovulation was induced with deslorelin acetate (1 mg, im). AI was performed 24 h after ovulation induction. Intrauterine fluid (FLU) was evaluated by ultrasonography before and 24 h after AI. PMNs in uterine cytology (CYT) and biopsy (HIS) were also observed before and 24 h after AI. Pregnancy was determined within 14 days after ovulation. Number of COX-2 positive cells was evaluated by immunohistochemistry. Both PRP treatments resulted in a decrease of PMNs in the CYT after breeding when compared to controls. FLU did not differ between cycles; however, the conception rates were significantly higher in the PRP mares. Mares positive for endometritis decreased in both treatment groups, and a more intense positive COX-2 labeling was observed in the control group when compared to the two treatment groups. In conclusion, PRP beneficially reduces inflammatory response in PMIE mares independent of when treatments were administered, thus increasing chances of successful pregnancy.