Transvaginal aspiration, oocyte maturation and production of blastocysts and a pregnancy via ICSI in an endangered breed: Baudet du Poitou donkey.

Two Poitou donkey jennies were presented for clinical oocyte recovery and embryo production via intracytoplasmic sperm injection (ICSI). Both jennies underwent transvaginal ultrasound-guided follicle aspiration on two occasions. Recovered oocytes were held overnight then placed into maturation culture, using standard methods for mare oocytes. On the first replicate for both jennies, the oocytes were divided into two groups; one group was denuded and examined at 30 h culture (standard culture duration for mare oocytes) and the second was denuded and examined at 36 h culture. No oocytes with polar bodies were observed at either time. The oocytes were maintained in maturation culture until 46 h, at which time oocytes with polar bodies were observed. Semen was then prepared; oocytes underwent ICSI approximately 48 h after being placed into maturation culture. On the second replicate for both jennies, oocytes were cultured for maturation for 42 h, then denuded and subjected to ICSI at 46 h. Sperm preparation, injection and embryo culture were performed as for mare oocytes. Blastocyst rates per injected oocyte were 8/19 (42 %) overall, being 4/12 and 4/7 for the first and second TVAs, respectively. Blastocysts were vitrified. Three blastocysts were warmed and transferred to Poitou donkey jenny recipients. One embryonic vesicle was visualized on ultrasonography on embryo Day 12, which increased in size on Day 13 but was not present when examined on Day 14. These results demonstrate that oocyte recovery and ICSI are efficient for production of Poitou donkey blastocysts. To the best of our knowledge, this is the first report of production of blastocysts via ICSI in the Poitou donkey, and the first report of transfer of ICSI-produced embryos in the donkey. Further work is needed on factors affecting pregnancy after embryo transfer in the donkey.

Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†.

Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47-79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.

Culture protocols for horse embryos after ICSI: Effect of myo-inositol and time of media change.

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0-5 embryo culture medium, using horse oocytes recovered by transvaginal aspiration. Experiment 2 was conducted to investigate effects of exclusion of a standard post-ICSI holding step (culture for 30-60 min in M199-based medium). Experiment 3 was conducted using oocytes recovered from abattoir-derived ovaries, to evaluate effects of earlier transition (Day 4 vs. Day 5) to the second-step medium and of media refreshment at different time points (Day 3 and/or Day 7) during embryo culture. In Experiments 1 and 2, there were no differences (P > 0.05) between groups in blastocyst development (Exp. 1, 36.7 % and 39.2 %; Exp. 2, 41.5 % and 44.6 %). In Experiment 3, blastocyst development was not different (P > 0.05) for embryos refreshed at both Day 3 and 7 (10.8 %) or only at Day 7 (26.6 %), or those transferred to second-step medium on Day 4 or Day 5 (20.6 % and 18.5 %). Knowledge of culture procedures compatible with blastocyst formation in vitro is valuable to laboratories starting to develop procedures for ICSI in horses.