Successful vitrification of manually punctured equine embryos.

Abstract

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. To develop a manual method of blastocoele collapse prior to vitrification using commercial products. In vivo experiment. 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≤560µm (n=10), punctured small (>300 to≤560µm; n=17) and large (>560µm; 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. Vitrified, manually punctured embryos≤560µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos≤300µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos>560µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. Limited group sizes. High pregnancy rates can be achieved by manually puncturing≤560µm equine embryos prior to their vitrification and subsequent warming in commercial media.