Optimization of Embryo Culture Conditions for Increasing Efficiency of Cloning in Buffalo(Bubalus bubalis)and Generation of Transgenic Embryos via Cloning

Abstract

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

Improvement of Cloned Embryos Development by Co-Culturing with Parthenotes: A Possible Role of Exosomes/Microvesicles for Embryos Paracrine Communication

“…E mbryo culture environment is a crucial factor that affects embryo development in vitro, gene expression, and postimplantation development in the cultured mammalian embryos (Cheong et al, 2009;Wadhwa et al, 2009;Yamanaka et al, 2009a). The sensitivity of cloned embryos to culture environment was reported as higher than in vitro-fertilized or parthenogenetic (PA) embryos in terms of in vitro development (Yamanaka et al, 2009b).…”

Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos

“…In several studies, it has been shown that the use of growth factors such as EGF and IGF-I improves the rate of blastocyst formation in IVF (Moreira et al , 2002; Wasielak & Bogacki, 2007) and SCNT embryos (Wadhwa et al , 2009; Wang et al , 2012). EGF improves oocyte maturation and increases the developmental potential of embryos.…”

“…Limited information has been available concerning the structural composition of ICM and TE cells in NT bovine embryos (Koo et al , 2002) except for some studies with IVF, parthenogenetic and in vivo embryos in the cow (Iwasaki et al , 1990; De la Fuente & King., 1997; Van de Velde et al , 1999). Although several reports have been available regarding the effect of IGF-1 and EGF on cell proportion in IVF embryos (Makarevich & Markkula, 2002; Sirisathien et al , 2003; Block et al , 2008; Wadhwa et al , 2009; Sakagami et al , 2012), very limited data exist for a similar effect by growth factors on NT bovine embryos (Wang et al , 2012). The objective of the present study was to investigate the effect of supplementing in vitro maturation and embryo culture media with IGF-I and EGF on oocyte maturation, embryo development and cell allocation in the NT bovine embryos.…”