Ultrastructure of bovine in vitro-produced blastocysts cryopreserved by vitrification

The objective of this study was to examine ultrastructural aspects of bovine in vitro-produced blastocysts associated with cryopreservation by vitrification. Morphologically good embryos were used and treated with ethylene-glycol-based vitrification solution (VS). The untreated embryos had conventional fine structure. The post-warming embryos treated with direct exposure to VS (one-step procedure) showed cellular damage structurally by cryopreservation, which included loss of microvilli, disruption of the plasma membrane, mitochondrial changes and swelling of the endoplasmic reticulum. However, nuclei and junctional regions seemed to be resistant to cryoinjury. In contrast, the post-warming embryos pre-equilibrated with 10% ethylene glycol for 5 min and subsequent exposure to VS (two-step procedure) showed less damage than those treated by the one-step procedure. Post-warming embryos treated by the two-step procedure were cultured in vitro for 18 h. Some embryos survived and their structures re-formed to the former state, while other embryos showed serious injuries and could not reconstitute the blastocoele. Three post-warming embryos treated by the two-step procedure that survived after in vitro culture were transferred to three recipients and one of these resulted in pregnancy. These results indicate that cryopreservation by vitrification can damage membranous structures of the cells of bovine embryos, the extent and nature of this damage being dependent on the vitrification procedure.