In Vitro Fertilizing Ability of Bovine Oocytes Frozen-Thawed at Immature, Maturing, and Mature Stages

The effect of the nuclear stage during in vitro maturation (IVM) on the fertilizing ability of frozen-thawed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h, and were subjected to 2-step freezing in the presence of 10% ethylene glycol and 0.1 M sucrose. After thawing, the oocytes were additionally cultured to give 24 h for a total IVM period, and were inseminated with 3.5 to 5.0 × 106 cells/ml frozen-thawed spermatozoa for 20 h in TALP solution containing 20 μg/ml heparin. In vitro fertilization rates of oocytes frozen-thawed at 0, 6, 12 and 24 h of IVM were 40, 47, 49 and 54%, respectively, all of which were significantly lower than that of nonfrozen control oocytes (77%). A higher frequency of polyspermic fertilization was observed in the oocytes frozen-thawed at 24 h of IVM (53 vs. 18% in nonfrozen control oocytes). Lowering the sperm concentration during insemination from 5.0 × 106 cells/ml to 1.0 × 105 cells/ml did not decrease the rate of polyspermic fertilization in the frozen-thawed oocytes.