Comparison the effects of 5-Aza-2'-deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos.

Abstract

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5nM AzadC and 5nM zebularine compared with 2 and 10nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5nM zebularine than 5nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.