Vitrification of immature bovine oocytes by the microdrop method

This study was conducted to determine the optimal vitrification conditions for immature bovine oocytes using the microdrop method. In experiment 1, the optimal pre-equilibration period for microdrop vitrification was examined. The maturation rate of vitrified oocytes with a 3 min first pre-equilibration period (41.1%) was higher than that of vitrified oocytes with a 0 min first pre-equilibration period (21.4%), and the values of those with a 1 (33.9%) or 5 min (27.4%) first pre-equilibration period were intermediate. The value for a 1 min second pre-equilibration period (44.4%) was significantly higher (P<0.05) than those for a 0.5 (28.6%) and 2 min (21.4%) second pre-equilibration period. In experiment 2, the distribution of microtubules in matured oocytes was investigated. There was no difference among the first pre-equilibration times in terms of the rates of normal spindles in vitrified oocytes. However, this value was significantly higher (P<0.05) in the 1 min group (52.8%) compared with the 0.5 (16.7%) and 2 min groups (12.3%). In experiment 3, we investigated the developmental capacity of immature bovine oocytes vitrified under optimal pre-equilibration conditions (3 min and 1 min for the first and second pre-equilibrations, respectively). Although the total fertilization rates were significantly lower (P<0.05) in the vitrified oocytes (65.6%) compared with the control oocytes (92.4%), there was no difference in the rate of normal fertilization (2PN) between the vitrified (78.6%) and control (82.0%) oocytes. Cleavage and blastocyst rates were significantly lower (P<0.05) in vitrified oocytes (55.7 and 2.3%) than in control oocytes (84.4 and 34.7%). Thus, these results indicated that immature bovine oocytes can survive after microdrop vitrification and subsequently can be cultured to mature oocytes capable of undergoing fertilization in vitro and developing into blastocysts.