Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants.

To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development.

Post-warming hatching and birth of live calves following transfer of in vitro-derived vitrified water buffalo (Bubalus bubalis) embryos.

Viability of in vitro-derived vitrified-warmed preimplantation stage buffalo embryos were assessed in vitro and in vivo. Oocytes were collected from ovaries of slaughtered riverine buffaloes, matured and fertilized in vitro with frozen semen from riverine buffalo bull and cultured on cumulus cell monolayers. Resultant preimplantation stage embryos were cryopreserved by vitrification with ethylene glycol, ficoll and sucrose. Seventy-one frozen embryos were warmed in 0.5M sucrose and were further cultured in vitro for 72 h to assess hatching rate. On the other hand, 95 embryos were transferred non-surgically to riverine buffalo recipients to assess development competence in vivo through detection of pregnancy and birth of live calves. Hatching rate of 83.10% (59/71) was noted among embryos cultured in vitro. Pregnancy rate was 16.36% (9/55) while calving rate was 10.91% (6/55) after transfer of in vitro-derived vitrified-warmed embryos to recipient animals. Six healthy and normal calves with average birth weight of 38.75+/-3.55 kg were born from the transferred embryos. These results indicate the viability of vitrified in vitro-derived buffalo embryos and the potential application of in vitro embryo production and vitrification techniques for production and transport of buffalo embryos from germplasm-rich sources to guarantee genetic improvement in many parts of the world.