2011•Biology of ReproductionRequires access
Effect of DNA Methylation Inhibitors on the Developmental Competence of Cloned Buffalo (Bubalus bubalis) Embryos.
Ambikaprasanna Saha, Naresh Lalaji Selokar, Sudeepta Kumar Panda, Aman George, Musharifa Muzaffar, Satish Kumar, Ruchi Shrama, Manmohan Singh Chauhan, Prabhat Palta, Radhay Sham Manik, Suresh Kumar Singla
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Abstract
The most critical factor for the development of cloned embryo is the proper epigenetic reprogramming of the donor genome in recipient cytoplast by erasing the epigenetic marks of the donor nuclei. Low success rate of the nuclear transfer technique is mainly due to inappropriate reestablishment of DNA methylation patterns of the donor genome. The present study was carried out to investigate the effect of DNA methylation inhibitors like 5-aza-2'-deoxycytidine and zebularine on developmental competence of the zona-free cloned buffalo embryos. 5nM 5-aza-2'-deoxycytidine and zebularine were treated independently just after reconstruction of cloned embryos, during activation and in vitro culture. The efficiency of these inhibitors was determined in terms of cleavage rate, blastocyst rate and total cell number. The data were analyzed by one way ANOVA after arcsine transformation of percentage values. The experimental data shows that the cleavage rate for zebularine (92.6 ± 2.01), and 5-aza-2'-deoxycytidine (93.3 ± 1.40) was significantly higher (P<0.01) as compared to control (87.2 ± 2.25), respectively. Similarly the blastocysts rate was significantly higher (P<0.01) in zebularine treatment (60.9 ± 3.60) as compared to 5-aza-2'-deoxycytidine (53.5 ± 2.78), which is also significantly higher (P<0.01) than control (41.4 ± 1.81), respectively. Zebularine treatment found significantly higher (P<0.01) cell number in cloned embryos (324.6 ± 12.28) as compared to 5-aza-2'-deoxycytidine treatment (221.7 ± 3.74), and control (170.7 ± 13.38), respectively. When transfer of embryos from treatment of zebularine (32), 5-aza-2'-deoxycytidine (29) and control (14) to synchronized recipients (6, 8 and 4), only zebularine treated embryos established pregnancy in 4 recipients. Hence, zebularine is better epigenetically modifier than 5-aza-2'-deoxycytidine for cloned embryo production. Supported by grant No. 1(5)/2007-NAIP from ICAR, India. (poster)