• Korean Journal of Animal Reproduction
• /
• v.23 no.4
• /
• pp.293-301
• /
• 1999

This study was to examine whether Hanwoo IVM/IVF/IVC blastocyst can be successfully survived in vitro/in vivo after vitrification and one-step dilution. For vitrification, blastocysts were serially exposed in glycerol (G) and ethylene glycol(EG) mixtures［10% (v Iv) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.］ which is diluted in 10% FBS added D-PBS. And then they were loaded in the straw, placed in cold nitrogen vapor for 3 min. and plunged into L$N_2$(－196$^{\circ}C$). One-step dilution within the straw was done in $25^{\circ}C$ and 36$^{\circ}C$ water for about 5 min. and 3 min., respectively. Recovered embryos after one-step dilution were cultured in cumulus cell mono-layered drop for 48 h or were transferred into recipient cows. When the embryo survival in vitro was assessed to re-expanded and hatched rates at 24 hand 48 h after one-step dilution, the results of vitrified group (85.4, 43.8%) was high, although these results were significantly lower than normal development (100.0, 63.3%) of control group, respectively (P＜0.001, P＜0.05). When in vitro survival of vitrified groups according to developmental stage was compared, the results of fast developed embryos (expanded blastocyst and early hatching blastocyst stage) were significantly higher than those of delayed developed one (early blastocyst stage) after one-step dilution (early hatching: 88.0, 48.0%: expanded: 81.1, 45.3%; early: 66.7, 14.3%) (P＜0.05). Also, in case of in vitro survival of vitrified groups according to embryo age (day 7, 8 and 9), when embryo age was younger, in vitro survival was significantly higher (day 7: 67.3, 34.5%; day 8: 76.9, 40.7%; day 9: 60.9, 23.9%)(P＜0.05). Finally, when in vivo development potential of vitrified and one-step diluted Hanwoo blastocysts was examined, 4 of 8 recipient (50%) cows became confirmed pregnant. These results demonstrated that our vitrification and one-step dilution technique can be applied easily and effectively on field trial without the equipment and embryological skills required for conservative dilution and transfer.

• Korean Journal of Animal Reproduction
• /
• v.27 no.1
• /
• pp.87-93
• /
• 2003

This study was conducted to investigate the effects of embryo development by fusion condition on the nuclear transfer with brindle coated cow's ear cells. Ear cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion condition with 1.9kv/cm, 2.0kv/cm, 2.1kv/cm each 10 and 20ug duration Nuclear transfer embryo were activeted with a combination of 5ug/ml and 1.9mM 6-DMAP (4min, 4h). Fusion rate was 51∼68% range among fusion condition (1.9, 2.0, 2.1kv/cm; 10, 20us). But, cytoplasm lysis rate was increased by higher electric condition (0∼51.8% range). Each parameter's cleavage and blastocyst formation rate were 1.9kv/cm for 10us (75.8 and 19.5%), 20us (69.8 and 48.6%), 2.0kv/cm for 10us (76.9 and 20.0%), 20 us (68.5 and 40.9%), 2.1kv/cm for 10us (70.5 and 44.2%), 20 us (68.5 and 27.0%). We compared the effectiveness of cloning for between brindle coated cow's ear cells and Hanwoo fetal fibroblast cells. There was no significant differences in the fusion rate and developmental rate to the blastocyst stage. After transfer of blastocysts derived from nuclear transfer embryos, pregnancy rates of the Hanwoo fetal fibroblast cells and brindle coated cow's ear cells were checked pregnant on day 60 as assessed by ultrasonography, 40% (2/5) and 15.8% (3/19), respectively. This studies conclude that brindle coated cow's ear cells have the developmental potentiality to term by nuclear transfer. These results demonstrate that the increased the field strength was to be profitable for development of blastocyst or reduce of cytoplasm's damage than increasing the pulse duration.