The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MІІ stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum–free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serumcontaining and–free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFPexpressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and–free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (FIV-EGFP) lentiviral vector into perivitelline space of in vitro matured MІІ stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (P<0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (P<0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (P<0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of 100 μg Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (P<0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to 100 μg of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.