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Summary Background Historically, cryopreservation of equine embryos >300 μm gave poor pregnancy rates until researchers collapsed the blastocoele cavity and aspirated the blastocoele fluid prior to vitrification. Objective To determine if aspiration of the blastocoele fluid prior to vitrification is essential for post warming survival. Study design In vivo experiments. Methods Fifty embryos were recovered on day 7–8 and washed in holding medium (HM; M‐199HEPES + 20% FBS + antibiotics). Embryos were punctured using a micromanipulator mounted 30 μm biopsy needle; following this 28 had >90% of their blastocoelic fluid actively aspirated while the remaining 22 were not‐aspirated. Embryos were then vitrified using a two‐step process with increasing concentrations of DMSO and ethylene glycol (7.5–15% v:v), and 0.5 mol/L sucrose in the final solution before being loaded onto a Cryolock device and plunged into liquid nitrogen. The embryos were warmed by plunging the Cryolock tip into HM with 1 mol/L sucrose at 37°C. After 1 min, the embryos were transferred to HM + 0.5 mol/L sucrose at RT for 4 min before transfer into HM for a further 4 min prior to transfer to a recipient mare. Results Mean (±s.e.) embryo diameter was not significantly different between the punctured and punctured plus aspirated group (646.4 ± 61.7 vs. 754.8 ± 59.1 μm, respectively; P = 0.215). Nonaspirated and aspirated embryos gave pregnancy rates of 10/22 (45%) and 21/28 (75%) respectively (P = 0.061). Sub‐dividing embryos on the basis of size showed that vitrification of larger embryos (>550 μm) yielded a significantly higher pregnancy rate when they were aspirated vs. not‐aspirated (13/18 [72%] vs. 1/10 [10%], respectively; P = 0.006), whereas there was no difference for smaller embryos (8/10 [80%] vs. 9/12 [75%], respectively; P = 0.8). Main limitations Group sizes are limited. Conclusion Aspiration of blastocoele fluid from embryos ≤550 μm is not a pre‐requisite for successful vitrification. The Summary is available in Spanish – see Supporting Information

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.

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