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

Background Historically, cryopreservation of equine embryos >300 mu m 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 mu m 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 degrees 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 mu m, 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 mu m) 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 mu m is not a pre-requisite for successful vitrification. The is available in Spanish - see Supporting Information