52 VITRIFICATION OF DAY-8 EQUINE EXPANDED BLASTOCYST: AN IN VITRO COMPARISON OF DIRECT VERSUS INDIRECT MECHANICAL INTRODUCTION OF CRYOPROTECTANT

Abstract

The cryopreservation of equine expanded blastocysts (>300 μm) has been largely unsuccessful primarily due to the low permeability of the embryo to cryoprotectants. This low permeability has been attributed to the acellular glycoprotein capsule that develops when an embryo approaches approximately 300 μm in diameter. Mechanical alternatives may provide a means to overcome the capsule barrier and the relative large embryo size to successfully cryopreserve equine embryos. The objective of this experiment was to compare re-expansion rates of vitrified equine expanded blastocysts following either direct or indirect mechanical introduction of cryoprotectants using a coaxial microinjection system (Dracula pipette). Twenty-six Day-8 expanded blastocysts were subjected to capsule puncture, cryoprotectant injection, and blastocoele fluid extraction (direct treatment) or capsule puncture and blastocoele fluid extraction (indirect treatment) before cryopreservation. The Dracula pipette incorporates the injection pipette within the holding pipette, facilitating aspiration of blastocoele fluid or injection of cryoprotectant in a single unit. A standard vitrification protocol using a final concentration of 3.4 M glycerol and 4.6 M ethylene glycol at cryopreservation was used. Vitrified embryo re-expansion was assessed following in vitro culture at 24, 48, and 72 h post-warming. Differences across treatments were analysed using the Student's t-test for re-expansion and the chi-squared test of independence for capsule loss. Pre-vitrification embryo mean diameter (mean ± standard error) for direct and indirect treatment groups were not different, 979 ± 85.6 μm and 912 ± 101.4 μm, respectively (P = 0.62). Post-vitrification embryo mean diameters were not different for the direct and indirect treatments (688 ± 63 and 662 ± 75 μm, respectively; P = 0.79). Following 72 h of in vitro culture, there was no difference in mean embryo diameter (1813.16 ± 209 μm v. 1383.88 ± 198 μm; P = 0.21), or re-expansion rates (69 v. 69%) for direct and indirect treatment groups, respectively. However, partial or total capsule loss was 69% (9/13) for direct treatment embryos compared with 30% (4/13) for indirect treatment embryos (P = 0.049). Results from this experiment demonstrate that capsule puncture and blastocoele fluid extraction before vitrification resulted in high re-expansion rates of Day-8 equine expanded blastocysts after warming. More importantly, the relatively large percentage of capsule failure when directly introducing cryoprotectant into the embryo may interfere with maternal recognition of pregnancy following embryo transfer. Nonetheless, based on the embryonic re-expansion rate of vitrified equine embryos following the indirect technique, we anticipate that a relatively high pregnancy rate can be obtained if this technique is used.