Successful vitrification of manually punctured equine embryos

Abstract

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.

Keywords: blastocoele collapse; capsule; embryo; horse; micromanipulation.