122 THE SURVIVAL RATE AND EMBRYONIC QUALITY OF BOVINE PARTHENOGENETIC BLASTOCYSTS POST-VITRIFICATION CRYOPRESERVATION

Abstract

Cryopreservation of oocytes and embryos is very useful to conservation of animal genetic resources. Recently, parthenogenesis has received considerable attention as a tool for the production of stem cells. Oocytes and embryos undergo considerable morphological changes and functional damage during cryopreservation, and the survival rate is highly depending on species and developmental stage of oocytes and embryos. The aim of this study was to investigate the survival rate and embryonic quality of bovine parthenogenetic blastocysts post-vitrification cryopreservation. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were aspirated from 2-mm to 8-mm visible follicles with a 5-mL syringe. The COC were matured in vitro for 22 h in bicarbonate-buffered TCM199 media supplemented with 1 mg mL^-1 of FSH, 10 mg mL^-1 of LH, 1 mg mL^-1 of 17-βiestradiol, and 10% FBS. After in vitro maturation, cumulus cells were removed from COC, oocytes with first polar body were activated by 5 μM ionomycin for 5 min and 2 mM 6-DMAP for 4 h. Subsequently, oocytes were co-cultured with bovine fetal fibroblast cells in SOF media supplemented with amino acids (1% NEAA and 2% EAA), 4 mg mL^-1 of BSA, and 10% FBS at conditions of 38.5°C and 5% CO₂ for 7 to 9 days. The good expanded blastocysts were selected and refrigerated in different vectors [glass micropipettes (GMP) and straws] and same vitrification solution (VS, 20% EG + 20% DMSO). Blastocysts were exposed to VS, loaded on vectors, and plunged into liquid nitrogen within 25 s. After two days refrigeration, vitrified blastocysts were thawed in air for 10 s and placed into 0.25 M sucrose solution for 1 min and 0.15 M sucrose solution for 5 min. Then, the blastocysts were cultured in the SOF medium same as above. Our results showed that when VS was 20% EG + 20% DMSO, the hatching rate (65%) of blastocysts loaded into GMP was significantly higher (P < 0.05) than that (19%) of blastocysts loaded into straws post-vitrification. Meanwhile, vitrified and nonvitrified blastocysts were fixed and stained for differential cell counting as described by Thouas GA et al. 2001 Reprod. Biomed. Online 3, 25-29). By the differential staining, the total cells of nonvitrified parthenogenetic bovine blastocysts were 102.7, which was higher (P > 0.05) than that (86.7) of vitrified blastocysts. Also, no significant difference (P > 0.05) was seen on ratios between vitrified blastocysts (ICM/TE = 0.22) and nonvitrified blastocysts (ICM/TE = 0.25). Our results indicated that a glass micropipette vector was much better than a straw in vitrification cryopreservation of bovine parthenogenetic blastocysts and caused less damage to blastocyst cells. This study lays the foundation for further research to increase the survival rate of vitrification cryopreservation of bovine embryos.

This work was supported by the grant from national support plan, China, No. 2007BAD55B03; corresponding author: Ziyi Li.