Preservation of Hamster Oocytes to Assay the Fertilizing Capacity of Human Spermatozoa

Between 70 and 80% of zona-intact hamster ova survived freezing after slow cooling (approximately 0.3 degrees C/min) to -80 degrees C in Medium PB1 containing 1.5 or 2.0 M-DMSO before transfer to -196 degrees C. After slow warming (approximately 8 degrees C/min), there was no difference in survival if the DMSO was diluted out by a slow stepwise or a rapid single addition of medium. When slow cooling was terminated at -40 degrees C by direct transfer to -196 degrees C, up to 75% of the ova survived rapid warming (approximately 500 degrees C/min) and rapid dilution if the medium contained 2.0 M-DMSO. The survival rates were calculated on the basis of the number of thawed ova which retained their normal morphological appearance after a 1 h incubation before removal of the zona pellucida with trypsin. All of these ova were penetrated after incubation with mouse spermatozoa, indicating that the freezing procedure per se does not adversely affect the penetration of frozen-thawed hamster ova by heterologous spermatozoa. There was no difference in the penetration rate of human spermatozoa into frozen (34%) or fresh (42%) oocytes when a Hepes-buffered Tyrode solution containing 30 mg BSA/ml and 2.0 M-DMSO was used as the freezing medium. However, fewer ova frozen in Medium PB1 containing 4 mg BSA/ml and 2.0 M-DMSO were penetrated by human spermatozoa (18%) compared with freshly collected ova (38%). Zona-free ova did not survive the freezing procedure as well as zona-intact ova. The survival of hamster oocytes stored at -196 degrees C offers a convenient means of supplying and transporting these ova for the assessment of the fertilizing capacity of human and other heterologous spermatozoa.