ULTRASTRUCTURE OF IN-VITRO MATURED BOVINE OOCYTES AFTER CONTROLLED FREEZING IN 10-PERCENT GLYCEROL

The purpose of this study was to investigate the developmental capacit y of, and possible subcellular damage to, bovine oocytes upon freezing and thawing. Following in vitro maturation, bovine oocytes were dehyd rated in 10% glycerol, frozen, thawed, rehydrated, inseminated and fur ther cultured in vitro. The cleavage rate among control ova was 74% ve rsus only 3% among their frozen counterparts, In the control group 45% developed to blastocysts, whereas no blastocysts were obtained with f rozen/thawed oocytes. Oocytes were collected before freezing, after de hydration, cooling to -5 degrees C, seeding/thawing/rehydration and af ter freezing/thawing/rehydration and were prepared for transmission el ectron microscopy. In the seeded/thawed oocytes the vesicles of the oo plasm were peripherally dislocated and displayed some degree of conflu ence and variation in size. The frozen/thawed oocytes exhibited a high degree of heterogeneity in vesicle size and even more vesicles had be come confluent, The smooth endoplasmic reticulum (SER) and mitochondri a displayed pronounced dilation. Again, the vesicles were peripherally dislocated, and in more than half of the oocytes the oolemma was rupt ured and the perivitelline space was occupied by ooplasmic content. It is concluded that cryopreservation of in vitro matured bovine oocytes is associated with ultrastructural damages, which become evident afte r seeding and especially the subsequent freezing and which deprive the oocyte of its developmental capacity.