Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with beta-mercaptoethanol and hemoglobin

Abstract

In preliminary experiments, the treatment of donor somatic cells with b-mercaptoethanol (ME) or hemoglobin (Hb) improved in vitrodevelopment of bovine cloned embryos. This study was subsequently evaluated whether the exposure to Hb and/or ME during in vitro-maturation or embryo culture could further promote the development of embryos cloned with ME-treated donor cells. A prospective, randomized study was conducted and, embryo development, cell number, and apoptosis in blastocysts were monitored. A significant (P<0.05) effect was found after the combined treatment of cloned embryos with Hb (1 mg/ml) and ME (10 mM); the development of morulae (53 vs. 35%) was greatly improved, which resulted in enhanced blastocyst formation (38%). However, cell number and apoptosis in blastocysts were predominantly affected by ME rather than Hb; a significant increase in total cell number of blastomeres (142–154 vs. 123 cells/embryo), inner cell mass (ICM) (39–41 vs. 27), and trophectoderm (TE) (103–114 vs. 98), and the ratio of ICM to TE cell number (0.26–0.27 vs. 0.22) was found. Also, the apoptosis index indicating the ratio of apoptotic cell to normal blastomere number was greatly reduced after ME treatments (0.85 vs. 0.056–0.069). When embryos cloned with ME-treated cells were cultured in HbþME-containing medium, any of the treatments to recipient oocytes before enucleation did not further promote the development. In conclusion, combined treatment of cloned embryos with HbþME not only improved in vitro-development but also decreased blastomere apoptosis. The use of ME-treated donor cells and the culture of cloned embryos in HbþMEcontaining medium yielded the optimal results for promoting the production of blastocysts with improved quality.