Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells

Abstract

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C in a humidified atmosphere of 5% CO2 air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.