Vitrification of equine expanded blastocysts

The cryopreservation of equine expanded blastocysts (\u3e300 um) has been largely unsuccessful primarily due to the low permeability to cryoprotectants and the large size of the equine embryo. Mechanical alternatives may provide means to overcome the capsule barrier and the relative large embryo size. In this regard, multiple experiments were performed in this study to evaluate different approaches of capsule puncture and blastocoele fluid extraction with the objective to develop a cryopreservation protocol for Day 8 equine expanded blastocysts. In the first experiment, twenty-four Day 8 expanded blastocysts were exposed to standard equine embryo vitrification solutions following one- or two-punctures. Mean pre-treatment embryo volume was not different across treatments. A reduction of 67% of embryo volume was achieved within 5 min of embryo puncture, blastocoele fluid extraction and exposure to VS1 for both treatments. Mean embryo volume was not different for one- and two-puncture treatments during exposure to VS1, VS2, VS3, DS and culture medium. Mean embryo volume was not different during in vitro culture at 24, 48 and 72 hours. Embryo growth rate at 72 hours culture was not different for one- or two puncture treatments (75 and 50%, respectively) (P=0.21). Capsule loss was not different for one- or two-puncture treatments (25% and 50%, respectively) (P=0.21). In the second experiment, twenty-six Day 8 expanded blastocysts were subjected to either capsule puncture, cryoprotectant injection and blastocoele fluid extraction (direct treatment) or capsule puncture and blastocoele fluid extraction (indirect treatment) prior to cryopreservation. Mean pre-vitrification embryo diameter for direct and indirect treatment groups was not different. A difference between the initial embryo volume and the embryo volume following warming of vitrified embryos in both indirect and direct introduction treatments was detected. Following in vitro culture there was no difference in mean embryo volume at 24, 48 and 72 hours. Re-expansion rate and subsequent growth at 72 hours of culture was not different for one- or two puncture treatments (69.2%). However, partial or total capsule loss was different (P=0.049) across treatments, with 69% of direct treatment embryos and 30.8% of indirect treatment embryos losing the capsule. A pregnancy rate of 83.3% was obtained following transfer of vitrified expanded blastocyst subjected to the indirect introduction technique