Vitrifying expanded equine embryos collapsed by blastocoel aspiration is less damaging than slow-freezing

The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300-550 μm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 μm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification.