81 IN VITRO MATURATION, FERTILIZATION AND DEVELOPMENT OF BOVINE IMMATURE OOCYTES CRYOPRESERVED BY VITRIFICATION WITH STEPWISE EXPOSURE USING A NYLON MESH

Abstract

Vitrification of bovine immature oocytes has been reported using an open pulled straw, but with limited success. In a previous report, we developed an alternative material (nylon mesh) for vitrification of large quantities of oocytes and embryos. This study was conducted to demonstrate effects of components of a cryoprotectant and a protocol of exposure for bovine immature oocytes on their subsequent in vitro maturation, fertilization and development after cryopreservation by vitrification using a nylon mesh. Bovine oocytes at the germinal vesicle stage were collected from 2–5 mm follicles in ovaries, and cumulus-oocytes complexes (COCs) were randomly assigned to treatment groups. Before vitrification, COCs were exposed to the cryoprotectant, which was composed of 40% ethylene glycol, 18% ficoll and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40) by single step or stepwise exposure. Forty COCs were transferred onto a nylon mesh (0.5 cm²), which was then plunged directly into liquid nitrogen. After thawing in warm medium, vitrified COCs were in vitro-matured, fertilized and cultured. After culture for in vitro maturation, the rates in the oocytes reaching to metaphase II were 64.1% and 63.1% in the stepwise exposure to EFS40 or EFT40, respectively, which was significantly higher (P < 0.05) than the corresponding rates after a single step (22.6% and 10.0%, respectively). There was no significant effect of the two sugars on in vitro maturation after single or step-wise equilibration. Transmission electron microscopy revealed that the cytoplasm of oocytes equilibrated in a single step had many vacuoles and broken mitochondria, while oocytes equilibrated in a step-wise manner had significantly fewer abnormalities and were similar to untreated controls. Cleavage rate of thawed oocytes after IVMFC was significantly higher after stepwise exposure to EFS40 or EFT40 than that after single step exposure (37.7% and 22.2% v. 20.8% and 0%, respectively, P < 0.05). Step-wise equilibration of oocytes in EFT40 was dramatically detrimental: no cleaved embryos developed to blastocysts after a single step exposure to either vitrification solution, or stepwise exposure to EFT40. However, blastocysts were obtained following stepwise exposure to EFS40 (8%). These results suggest that stepwise equilibration and vitrification on a nylon mesh minimizes structural damage to the organelles of immature oocytes and facilitates successful cryopreservation.