Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid

Abstract

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.