A comparative analysis of the efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle embryos at pronuclear and blastocyst stages

The effectiveness of three cryopreservation protocols (slow freezing, short equilibration vitrification and long equilibration vitrification) on in vitro-derived cattle embryos at expanded blastocyst and pronuclear stages was compared. 199 expanded blastocysts of good quality were assigned randomly into four treatment groups [control, non-cryopreserved (fresh, unfrozen); and the three cryopreservation methods]. The re-expansion of the cryopreserved blastocysts after 24 h in vitro culture was similar to that of the fresh control group. However, the hatching rate of expanded blastocysts after 48 h culture was significantly less for the slow freezing group (31/47; 66.0%) than for both the short equilibration vitrification (46/51; 90.2%) and long equilibration vitrification groups (42/50; 84.0%). Denuded presumptive zygotes at the pronuclear stage (14-18 h post-insemination) were assigned randomly to the same four treatment groups and, following thawing, embryos were assessed for their capacity to cleave and to develop into a blastocyst. Overall, cleavage rates of cryopreserved zygotes were significantly less than those of the fresh control. The blastocyst formation rate of slow-frozen zygotes (4/81; 4.9%) was significantly less than that of zygotes subjected either to short equilibration vitrification (18/82; 22.0%) or long equilibration vitrification (16/74; 21.6%). All cryopreservation groups showed rates of blastocyst formation that were significantly less than that of the fresh control (51/92; 55.4%). Collectively, our findings indicate that vitrification is the preferred technology to cryopreserve in vitro-derived cattle embryos at expanded blastocyst and pronuclear stages. Moreover, short equilibration vitrification technology can improve outcomes and be more efficient by taking less time to perform.