Morphological changes of in-vitro-produced bovine blastocysts after vitrification, in-straw direct rehydration, and...

Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In-vitro-produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two-step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in-straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re-assembled in a central area forming a small blastocoele, cell adhesion structures were re-established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure.