Successful cryopreservation of bovine oocytes is a very important technology for research and commercial applications. However, the survival and development rate of vitrified-thawed oocytes is lower than non-vitrified oocytes. Hydroxypropyl Cellulose supplementation (HPCs) has extremely high viscosity, which permits transitions to a glassy state at low temperatures. This characteristics of HPCs have been reported to help the survival of human oocytes. In this study, we investigated the survival rate, fertilization rate and ROS levels to confirm the effect of cryoprotectant solutions with HPC for oocyte vitrification in bovine. For vitrification, bovine MII oocytes were pretreated with EG10 (added 0, 10, 50 and 100 ㎍/㎖ HPC) for 5 min, exposed to EG30 (added 0, 10, 50 and 100 ㎍/㎖ HPC) for 30 sec, and then directly plunged into LN2. Thawing was taken by 4-step procedures [1 M sucrose and 10% FBS added D-PBS (SFD) -> 0.5 M SFD -> 0.25 M SFD -> 0.125 M SFD] for 1 min, respectively. After thawing, oocytes were washed with TL-HEPES, incubated in a droplet of previous cultured IVM medium for 1 h to recover. IVF drop (44 ㎕) contained 10 vitrified-thawed oocytes with sperm concentration of 1 × 106 cells ㎖, and then 2 ㎕ heparin and 2 ㎕ PHE were added. At 2 days after IVF, cleaved embryos were cultured in CR1aa + 3 mg/mL FAF-BSA for 48 h and cultured in CR1aa + 10% FBS for 4 days. In the results, in vitro survival rate of bovine vitrified-thawed MII oocyte was significantly higher in 50 (85.5%) and 100 ㎍/㎖ (80.2%) HPC groups than 0 (71.2%) and 10 ㎍/㎖ (71.3%) groups (p<0.05). The ROS level was lower in 50 ㎍/㎖ HPC group than in control group. After in vitro fertilization, cleavage rate and blastocyst development rate were not significantly different among treatment groups. Therefore, these results indicated that HPC treatment has a positive effect on the survival of vitrified-thawed bovine oocytes.

This study was to analyse the usability of morphological evaluation of vitrified-thawed oocyte before somatic cell nuclear transfer (SCNT) using Oosight imaging system to show spindle. For the vitrification, in vitro matured bovine MII oocytes were treated by two-step freezing medium without (control group) or with 5 ug/ml cytochalasin-b (CCB group). In Exp. 1, after thawing, recovered oocytes in each treatment group were assessed by live image using Oosight imaging system or/and cytoskeletal protein image using immunostaining. In Exp. 2, in each treatment group the in vitro developmental potential of frozen-thawed bovine oocytes post evaluation using Oosight imaging system and then SCNT was investigated. The SCNT embryos were cultured in CR1aa medium supplemented with 10% FBS, 1 mM EGF and 1 mM IGF at 38.5 C in 5% O2 and 5% CO2 in air for 8 days. In Exp.1, the rates of in vitro survival, morphological good grade and spindle normality of CCB treatment group (91.1%, 54.2% and 55.5%) were better than those of control group (86.1%, 48.5% and 48.5%). After SCNT using vitrified-thawed oocyte, the rates of fusion, reconstructed embryos and blastocyst development were also high in CCB treatment group (66.6%, 36.4% and 3.0%) than control group (60.0%, 27.3% and 0%). These results demonstrated that the identification of morphological spindle image of the vitrified-thawed bovine oocytes using Oosight imaging system helps to predict the SCNT embryo quality.