Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2’-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos M. Saini A , N. L. Selokar A,B , H. Agrawal A , S. K. Singla A , M. S. Chauhan A , R. S. Manik A and P. Palta A,C A Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India. B Animal Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India. C Corresponding author. Email: prabhatpalta@yahoo.com Abstract. We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2 0 -deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50 nM) and 5azadC (7.5 nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells. Additional keywords: cloning, development, embryo transfer, epigenetics, nuclear transfer, reprogramming. Received 28 May 2014, accepted 8 October 2014, published online 20 November 2014 Introduction Somatic cell nuclear transfer (SCNT), which has been suc- cessfully applied to several mammalian species for the pro- duction of cloned offspring, has a vast potential of being applied for the production of transgenic animals, fast multi- plication of animals with genetic value, conservation of endangered species, xenotransplantation, therapeutic cloning and disease modelling. However, in comparison to the more than 40% birth rate obtained with embryos produced by in vitro fertilisation, at ,5%, the rate of live offspring obtained from cloned blastocysts of most of mammalian species is very low (Campbell et al. 2007). This is primarily due to high embryo loss during pre- and post-implantation development. More- over, offspring suffer from a high incidence of abnormalities such as large offspring syndrome (LOS), short life span, severe placental deficiency, respiratory problems, prolonged gesta- tion and dystocia (Young et al. 1998; Yang et al. 2007). These abnormalities, which result in a low cloning efficiency, are believed to be due to incomplete or incorrect nuclear repro- gramming. A variety of epigenetic modulating agents have been used to treat somatic cells and cloned embryos of different mammalian species, to correct abnormal epigenetic marks and enhance nuclear reprogramming and developmental competence. CSIRO PUBLISHING Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14176 Journal compilation Ó CSIRO 2014 www.publish.csiro.au/journals/rfd