Vitrification of immature porcine oocytes: effects of lipid droplets, temperature, cytoskeleton, and addition and removal of cryoprotectant.

Three experiments were conducted to investigate the effects of single-step and stepwise exposure to and removal of cryoprotectant, of temperature, and of a cytoskeletal relaxant on the development of germinal vesicle porcine oocytes to the M-II stage. In experiment I, noncooled cumulus-oocyte complexes (COCs) were treated using single-step/stepwise exposure to ethylene glycol (EG) and removal at 23 or 42 degrees C. Stepwise exposure to EG and dilution at 42 degrees C were found to have a positive effect on the COC developmental rate. In experiment II, also without cooling, COCs were treated with Cytochalasin B at 42 degrees C using single-step and stepwise protocols of exposure to and removal of EG. No effects of Cytochalasin B were noticed on the COCs pretreated for vitrification but not cooled. In experiment III, COCs were divided into two treatment groups and one control group. Group 1 COCs were vitrified by stepwise exposure to EG at 42 degrees C and direct plunging into liquid nitrogen. Warming was carried out by 5-s immersion in a water bath at 50 degrees C, and the cryoprotectant was removed by exposure to a graded series of sucrose solutions at 42 degrees C. Group 2 COCs were vitrified using the same procedure but following pretreatment with Cytochalasin B. Group 3 COCs were control (untreated) oocytes. After rewarming and passage through the sucrose series, the experimental COCs were cultured for 48 h. The results of cultivation (progression to MII stage) suggest that the gradual saturation/removal of cryoprotectant, elevated temperature, and pretreatment with cytoskeletal inhibitor Cytochalasin B have a positive effect on vitrification of GV-porcine oocytes.