Successful vitrification of manually punctured equine embryos

Background Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator‐mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. Objectives To develop a manual method of blastocoele collapse prior to vitrification using commercial products. Study design In vivo experiment. Methods Seventy‐nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator‐assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits containing, respectively, 2‐step concentrations of DMSO and ethylene glycol (7.5%–15% v:v) and decreasing concentrations of sucrose. After warming, embryos were transferred to recipient mares. Once validated, the commercial kits were used to vitrify and warm a further 39 embryos which were punctured manually using a microneedle, 2 (5%) were damaged during puncture and excluded; 20 more embryos were vitrified without puncture. Embryos were grouped as follows: non‐punctured ≤ 300µm (n = 10) and >300 to ≤560 µm (n = 10), punctured small (>300 to ≤560 µm; n = 17) and large (>560 µm; n = 10) and exposed to the equilibration solution (ES) in the kit for 6min. An additional group of punctured large embryos was exposed to ES for 8min (n = 10). For the initial warming step, embryos were exposed for 1min to the thawing solution at 42°C, before being moved to a dilution solution at room temperature. Results Vitrified, manually punctured embryos ≤560 µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos ≤300 µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos >560 µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. Main limitations Limited group sizes. Conclusion High pregnancy rates can be achieved by manually puncturing ≤560 µm equine embryos prior to their vitrification and subsequent warming in commercial media.