Equine fetal genotyping via aspiration of yolk-sac fluid at 22-28 days of gestation.

Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22-28 of gestation. Fluid (0.56-1.02 mL) was recovered from five of seven mares. Four of the five mares maintained pregnancy. One pregnancy was electively terminated at Day 75; the other three mares delivered healthy foals. Extraction of DNA from the fluid sample followed by direct PCR allowed the highest rate of determination of fetal alleles. Fetal genotype was correctly determined in three samples, and for 14/19 alleles in one sample. In Exp. 2, we evaluated whether recovery of more fluid (up to 1.6 mL), and fractionation of the sample, would minimize maternal DNA contamination. One of four mares maintained pregnancy. Evaluation at informative loci showed no difference in maternal contamination among fractions. We determined that mares can maintain pregnancy after aspiration of yolk-sac fluid, and that fetal genotype can be accurately determined from the sample obtained. Further work is needed on factors affecting maintenance of pregnancy after the procedure. The ability to access the yolk sac in early pregnancy opens the door to novel potential clinical and research applications.

Intrafollicular oocyte transfer in the horse: effect of autologous vs. allogeneic transfer and time of administration of ovulatory stimulus before transfer.

PURPOSE: To assess meiotic and developmental competence after transfer of immature cumulus-oocyte complexes (COCs) to the preovulatory follicles of mares (intrafollicular oocyte transfer (IFOT)). METHODS: In Experiment 1, mares received an ovulatory stimulus at IFOT. Thirty hours later, COCs were recovered from the follicle, and mature oocytes underwent ICSI and embryo culture. In Experiments 2 and 3, autologous vs. allogeneic COCs were used. The mares were inseminated and embryos were recovered. In Experiment 3, the ovulatory stimulus was administered 9 h (autologous) and 15 h (allogeneic) before IFOT. In Experiment 4, only allogeneic COCs were used; the ovulatory stimulus was administered 9 or 15 h before IFOT. Excess embryos (autologous) and parentage-verified embryos (allogeneic) were considered IFOT-derived. RESULTS: In Experiment 1, 36/54 IFOT oocytes (67%) were recovered, of which 56% were mature, vs. 49% of in vitro matured oocytes (P > 0.1). After ICSI, blastocyst rates were 25% and 18%, respectively (P > 0.1). In Experiment 2, 0/6 autologous and 2/6 allogeneic IFOT yielded IFOT-derived embryos. In Experiment 3, 0/7 autologous and 2/5 allogeneic IFOT yielded IFOT-derived embryos. The proportion of mares yielding IFOT-derived embryos was lower after autologous vs. allogeneic IFOT (0/13 vs. 4/11; P < 0.05). In Experiment 4, 1/8 9-h and 1/7 15-h IFOT yielded IFOT-derived embryos. CONCLUSIONS: Transferred oocytes mature within the follicle and can maintain developmental competence. Allogeneic IFOT was more efficient than was autologous IFOT. The time of ovulatory stimulation did not affect embryo yield. The IFOT procedure is still not repeatable enough to be recommended for clinical use.