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.

Experimental crossover study on the effects of withholding feed for 24 h on the equine faecal bacterial microbiota in healthy mares.

BACKGROUND: An association between equine gastrointestinal disease causing colic signs and changes in faecal bacterial microbiota has been identified. The reasons for these changes and their clinical relevance has not been investigated. Withholding feed, which is an integral part of managing horses with colic, may contribute to the observed changes in the microbiota and impact interpretation of findings in horses with colic. Study objectives were, therefore, to determine the effect of withholding feed for 24 h on equine faecal bacterial microbiota in healthy mares to differentiate the effects of withholding feed from the changes potentially associated with the disease. RESULTS: Species richness and Shannon diversity (alpha diversity) were significantly lower at the late withheld (10-24 h post withholding feed) and early refed (2-12 h post re-feeding) time points compared to samples from fed horses (P < 0.01). Restoration of species richness and diversity began to occur at the late refed (18-24 h post re-feeding) time points. Horses having feed withheld had a distinct bacterial population compared to fed horses (beta diversity). Bacteroidetes BS11 and Firmicutes Christensenellaceae, Christensenella, and Dehalobacteriaceae were significantly increased in horses withheld from feed primarily during the late withheld and early refed time points. Bacteroidetes Marinilabiaceae and Prevotellaceae, Firmicutes Veillonellaceae, Anaerovibrio, and Bulleidia, and Proteobacteria GMD14H09 were significantly decreased in horses with feed withheld at late withheld, early refed, and late refed time periods (P < 0.01). Changes in commensal gut microbiota were not significant between groups. CONCLUSIONS: Withholding feed has a significant effect on faecal bacterial microbiota diversity and composition particularly following at least 10 h of withholding feed and should be taken into consideration when interpreting data on the equine faecal bacterial microbiota in horses.