Production of HanWoo(Bos taurus coreanae) Fetuses Following Interbreed Somatic Cell Nuclear Transfer.

Abstract

ABSTRACT. The possibility of producing HanWoo (Bos taurus coreanae) calves from transferable bovine embryos, obtained by interbreed nuclear transfer using Holstein cytoplasts and surrogates, was investigated. As donor nuclei, HanWoo fetal fibroblasts were used. Cells were induced into quiescence by serum deprivation for 4-7 days before nuclear transfer. In vitro matured Holstein oocytes were enucleated, and single donor cell was placed into the perivitelline space of enucleated oocyte. After reconstruction, the embryos were fused, activated and cultured. On day 7, the embryos that developed to the blastocyst stages were transferred into Holstein recipient cows on day 6 to 7 of estrous cycle (estrus=0). The reconstructed embryos were successfully fused (58.8%; 47/80), cleaved (91.5%; 43/47), and developed to blastocysts (29.8%; 14/47). Eleven blastocysts were transferred into 5 Holstein recipient cows. Two recipients were pregnant, confirmed by ultrasonography at day 60 of gestation. But, one of them was opened between on day 80 to 100 of pregnancy, and the other had a stillbirth on day 255. The stillborn calf was physically normal, and we couldn't find any evidence of anomaly. These results show that cells derived from HanWoo somatic cell lines can be reprogrammed by interbreed nuclear transfer and develop subsequently in vivo as well as in vitro. KEY WORDS: HanWoo (Bos taurus coreanae), interbreed, nuclear transfer.J. Vet. Med. Sci. 63(9): 945-948, 2001 Production of cloned animal by somatic cell nuclear transfer has recently been developed. The birth of live offspring derived from embryos reconstructed with somatic cells have been reported in several species; sheep [10], cattle [1,3,8], and mouse [7].Animal cloning technology can be used to preserve endangered mammalian species by interbreed or interspecies nuclear transfer. The success of interbreed nuclear transfer was reported in last surviving cow of the Enderby island cattle breed [9]. HanWoo (Bos taurus coreanae) inhabits only in the region of Korea peninsula and becomes endangered animal in the bovine species. To produce HanWoo more efficiently, we developed in vitro production and embryo transfer system and produced HanWoo twin calves from the Holstein recipient cows [5]. Due to the relatively small size of HanWoo calves, we could obtain twins easily without dystocia or other problems during the pregnancy.In this study, we used Holstein recipient cytoplast for the production of HanWoo nuclear transfer embryos, and transfer them into Holstein cows to obtain cloned HanWoo offspring. MATERIALS AND METHODS Preparation of fetal fibroblasts:Fibroblast cells were obtained from HanWoo conceptuses on Day 50 to 60 of pregnancy. The head, extremities and internal organs were removed before tissues were cut into small pieces, and cells were dispersed by exposure to 0.25% (w/v) trypsin solution (Gibco BRL, Grand Island, NY). Cells were cultured in Dulbeco's minimum essential medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS, Gibco BRL). For each p...