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.

Lower blastocyst quality after conventional vs. Piezo ICSI in the horse reflects delayed sperm component remodeling and oocyte activation.

PURPOSE: The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. METHODS: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between injection techniques and between sham-injected and sperm-injected oocytes among time periods. RESULTS: Blastocyst rates were 39 and 40%. The nucleus number was lower, and the nuclear fragmentation rate was higher, in blastocysts produced by Conv. Cortical granule loss started at 0H after both sperm and sham injection. The acrosome was present at 0H in both ICSI treatments, and persisted to 18H in significantly more Conv than Piezo oocytes (72 vs. 21%). Sperm head area was unchanged at 6H in Conv but significantly increased at this time in Piezo; correspondingly, at 6H significantly more Conv than Piezo oocytes remained at MII (80 vs. 9.5%). Sham injection did not induce significant meiotic resumption. CONCLUSIONS: These data show that Piezo ICSI is associated with more rapid sperm component remodeling and oocyte meiotic resumption after sperm injection than is conventional ICSI, and with higher embryo quality at the blastocyst stage. This suggests that there is value in exploring the Piezo technique, utilized with a non-toxic fluorocarbon ballast, for use in clinical human ICSI.

Determination of equine fetal sex by Doppler ultrasonography of the gonads.

REASONS FOR PERFORMING THE STUDY: The identification of fetal sex in horses by location of the genital tubercle between 55 and 70 days of pregnancy is hampered by the large amount of allantoic fluid, extensive fetal movements and the extremely long umbilical cord; however, reliable results have been achieved by ultrasonographic evaluation of the fetal gonads at 110-150 days of pregnancy. OBJECTIVES: The aim of this study was to diagnose the sex of equine fetuses using B-mode and/or colour Doppler transrectal ultrasonography in fetuses of different ages. STUDY DESIGN: Cross-sectional study comparing 2 methods of determining fetal sex. METHODS: The evaluations were performed in 86 mares at 90-180 days of pregnancy using transrectal B-mode and colour Doppler mode ultrasonography. Fetuses that had gonads with a homogeneous texture and a thin central longitudinal echogenic line were considered to be male. Females were identified by the presence of gonads with a central circular echogenic structure surrounded by a hypoechogenic external halo. RESULTS: Using B-mode ultrasonography, it was possible to determine the sex of 75% of the males, while determination of sex based on differences between the medullary and cortical layers of the ovary allowed 91.1% of females to be correctly identified. Using Doppler ultrasonography, 100% of males were successfully identified, while characteristic vascularisation of the female gonad could be detected in 98% of the evaluated female fetuses. CONCLUSIONS: Colour Doppler ultrasonography combined with B-mode ultrasonography allows the determination of fetal sex with greater accuracy than B-mode ultrasonography alone, particularly for the identification of the male gonad. The use of Doppler ultrasonography enables the identification of sex in older fetuses.