The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo (Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without.
The
protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at -7℃, slow cooled (0.3℃/min) to -30℃ before plunging to -196℃. The
oocytes
were
thawed at 23-25℃ air (20sec/150sec) and/or 33-35 water (10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay (SPA) system (Chang et
al,
1990) using fertile men semen sample.
One-step equilibration showed slightly higher survival rate (83.9% vs. 71.0%) and fertilization rate (83.9% vs. 71.0%) compared with four-step (p>0.05). And two-step thawing (air & water exposing) of oocytes frozen after 1-step equilibration
showed
significantly higher survival rate (96.3%) than one-step thawing at air (85.2%) or water (65.0%) only (p<0.05). Therefore, by the modified method (1-step equilibration & 2-step thawing), Zona-intact (ZI) and Zona-free (ZF) oocytes were frozen and
thawed. ZI-oocytes showed significantly higher survival rate (95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes (P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also
significantly
higher fertilization rate (92.4±8.9% vs. 63.7±18.5%) and higher mean number of penetrated sperm (6.2±4.2 vs. 3.9±3.3) than ZF-oocytes, but not higher than control (fresh oocytes; 99.3±2.4%, 8.4±4.2) (P<0.001). Conclusively, this modified
method
will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.
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