25 DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY TRANSFECTED OR NONTRANSFECTED FIBROBLASTS TRANSFER TO ENUCLEATED OOCYTES DERIVED FROM OVUM PICK-UP AND ABATTOIR OVARIES

Abstract

The objective of this study was to explore whether fibroblasts transfection and the source of oocytes – ovum pick-up (OPU) versus abattoir ovaries – affected the in vitro and in vivo developmental competence of somatic cell nuclear transferred (SCNT) embryos in buffalo. To this aim, the serum-starved ear fibroblasts were fused into enucleated oocytes derived from abattoir ovaries (Group 1) and OPU (Group 2). Furthermore, the enucleated buffalo oocytes derived from abattoir ovaries were also fused with pEGFP-N1 transfected ear fibroblasts, and the cloned embryos were enhanced green fluorescent protein (EGFP)-positive confirmed by fluorescence microscopy (Group 3). The reconstructed embryos cultured in Groups 1 to 3 were 262, 83, 120, respectively (5 replicates); and the data were analysed by one-way ANOVA (SPSS Inc., Chicago, IL, USA). As a result, the cleavage rate in Group 3 was significantly higher than that in Group 1 (75.0% v. 54.3%; P < 0.01), and the total blastocyst rate of reconstructed embryos in Group 3 (27.3%) was significantly higher than that in Group 1 (17.4%; P < 0.01) and Group 2 (24.4%; P < 0.05). The SCNT blastocysts were vitrified with 20% ethylene glycol + 20% dimethylsulfoxide + 0.5 M sucrose; the cryosurvival rates of SCNT blastocysts in the 3 groups were not different from each other (90.0%, 94.7%, 92.3%). Following culture, the cryosurvived blastocysts were transferred into synchronized local and crossbred buffaloes, with each recipient receiving 1 or 2 embryos. The pregnancy rates after transferring embryos derived from Groups 1 to 3 were not different from each other, and were 18.75% (3/16), 33.33% (4/12), and 26.67% (4/15), respectively. These results indicate that the oocytes derived from OPU can be enucleated as recipient cytoplasm and transfected fibroblast can be adopted as nuclei donor without decreasing the SCNT efficiency in buffalo.This research was supported by grants from the National Natural Science Foundation of China (31160456) and the Natural Science Foundations of China under Grant No. 0991011, No. 2011GXSFB018045).