Deterimental Effects of Placement for Deep Freezing against Zonae Pellucida and Cell of Bovine Oocytes Filled in Plastic Straws

The deleterious effects against zonae pellucida and cell bovine oocytes filled into plastic straws frozen at the vertical or horizontal settings were compared. In the first experiment 1.320 bovine oocytes were suspended in 1.4M glycerol in Phosphate buffered saline (PBS), and in the second one 206 bovine oocytes were suspended in 1.4M glycerol and 0.3M sucrose in PBS. After the equilibration in 1.4M glycerol in PBS for 10 minutes, each plastic straw filled with the bovine oocyte was kept deep directly in an alcohol bath at -6℃ at the vertical or horizontal settings for 10 minutes. Thereafter, The straws were gradually cooled to -30℃ at a cooling rate of 0.3℃/min before stored in liquid nitrogen. To evaluate the morphological deformations incurred during the above manipulations each sample of frozen oocytes was thawed just before microscopic examinations at 100-200 fold magnification. Lost rates of oocytes frozen at the vertical and horizontal settins were 2% (13/628) and 0.4% (3/692) respectively. When straws were frozen at the vertical setting, the incidence of deformed zonae pellucida and cell was 31.1% (191/615) and 18% (111/615), and those were significantly higher (p<0.05) than 22.8% (157/689) and 10% (69/689) obtained with the straws frozen at the horizontal setting. However, no significant differences were discernible in the incidence of deformed oocytes between the vertical setting (5.8%

Frozen oocytes

straw setting

damage

zonae pellucida