Developmental capacities of two-cell mouse embryos frozen by three methods.

The following three methods were evaluated in order to obtain a most efficient freezing protocol for the preservation of two-cell mouse embryos: (a) slow cooling and slow thawing in 1.5 M dimethyl sulfoxide, (b) slow cooling and fast thawing in 1.5 M propanediol (PROH), and (c) ultrarapid freezing and fast thawing in either 3.5 M DMSO or 3.0 M PROH. In the slow-cooling procedures (a and b) ice nucleation (seeding) was induced manually or automatically. With method a, only a slight difference, 51.8% for manual and 58.9% for automatic seeding, was observed in survival rates, while the development to blastocysts was significantly affected: 35.4% with manual and less than 10% with automatic induction (P less than 0.001). Method b gave high survival (86.2%) and developmental rates (69.0%) with manual seeding compared with automatic seeding (20.7 and 9.8%, respectively; P less than 0.001). Using protocol c, higher survival and developmental rates were obtained with DMSO (84.8 and 55.9%) than with PROH (39.8 and 19.4%, P less than 0.001). These results demonstrate that inducing nucleation manually is superior to the use of a highly sophisticated autoseeding system and that method b with manual seeding is most effective in preserving the developmental capacity of two-cell mouse embryos after freezing and thawing. There is evidence that this is also true of human embryo cryopreservation.