The objective of this study was to determine the effects of vacuum-cooled liquid nitrogen on the development of vitrified immature (germinal vesicle stage; GV) and mature (metaphase II; MII) bovine oocytes after re-warming. Liquid nitrogen was exposed to either atmospheric pressure or to a vacuum (300mm Hg for 45sec); the latter decreased the temperature of the liquid nitrogen to -200°C. Partially denuded oocytes were vitrified either just after selection (GV) or after 22 hours of in vitro maturation (MII) in TCM 199 medium + 10% of estrous mare serum. For vitrification, oocytes were firstly exposed to an intermediate solution (10% EG + 10% DMSO) for 30sec, followed by the vitrification solution (20% EG + 20% DMSO + 0.5M sucrose) for 20sec. Groups of three or four oocytes were loaded into an open-pulled-straw and directly plunged into liquid nitrogen. Oocytes were subsequently re-warmed by exposure to air (25°C) for 4sec, followed by 5 min exposure to decreasing concentrations (0.3 and 0.15M) of sucrose. Fertilization (Day 0) was done with 2 x 106 spermatozoa mL-1 (selected by a swim-up procedure) and incubated for 18 to 22 hours. Presumptive zygotes were cultured at 39°C in four-well dishes with SOFaaci medium, under 5% CO2 and saturated humidity. Cleavage (Day 2) and blastocyst rates (Day 8) were 33.9 and 4.2%, respectively, for GV stage oocytes at atmospheric pressure, 41.2 and 8.8% for GV oocytes under vacuum, 43.5 and 6.7% for MII oocytes at atmospheric pressure, and 53.6 and 10.6% for MII oocytes under vacuum. In conclusion, vacuum-cooled liquid nitrogen improved developmental rates of vitrified-thawed bovine oocytes.
vitrification; cryopreservation; oocytes; bovine; vacuum
