Small Ruminant Research 105 (2012) 255–262 Contents lists available at SciVerse ScienceDirect Small Ruminant Research jou rn al h om epa ge: www. elsevier.com/locate/smallrumres Handmade cloned and parthenogenetic goat embryos – A comparison of different culture media and donor cells M.K. Jena, D. Malakar ∗ , A.K. De, S. Garg, Y.S. Akshey, R. Dutta, S. Sahu, A.K. Mohanty, J.K. Kaushik Animal Biotechnology Center, National Dairy Research Institute, Karnal 132001, India a r t i c l e i n f o Article history: Received 29 December 2010 Received in revised form 1 March 2012 Accepted 1 March 2012 Available online 27 March 2012 Keywords: Goat Manual cloning Karyotyping Parthenogenesis PCR Protrusion cone a b s t r a c t The aim of the present investigation was the production of handmade cloned and partheno- genetic goat embryos. One somatic cell was attached to an enucleated demi-oocyte and fused with another enucleated demi-oocyte with the aid of an electric pulse. Triplets were chemically activated with a Ca ionophore and 6-DMAP, and then cultured in three media. Blastocyst formation with fetal fibro cells was induced in RVCL (15.5 ± 4.2%), mSOF (11.0 ± 2.2%) and EDM (10.7 ± 2.4%) media, respectively (P > 0.05). Similarly, adult fibro- blast cells formation was also recorded in RVCL (14.1 ± 3.3%), mSOF (9.6 ± 2.7%) and EDM (9.7 ± 2.2%) media, respectively (P > 0.05). PCR analysis of the highly polymorphic MHC class II DRB gene of cloned embryos and donor cells, recorded similar bands. Zona and zona-free parthenogenetic embryos were also produced with the aid of a Ca ionophore and 6-DMAP, following culture in the three media. Zona parthenogenetic hatched blastocysts were recorded in RVCL (6.8 ± 0.9%), mSOF (1.2 ± 0.7%) and EDM (5.5 ± 0.7%) (P < 0.05) media, respectively. In the zona-free parthenogenetic blastocyst, formation was recorded in the RVCL (8.8 ± 0.9%), mSOF (5.6 ± 0.5%) and EDM (5.1 ± 0.8%) (P < 0.05) media, respectively. In conclusion, cloned and parthenogenetic goat embryonic development was higher in the RVCL medium and cloned embryos production was similar with both types of donor cells in this medium. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Somatic cell nuclear transfer (SCNT) is a technique for the faster multiplication of superior germplasm. “Dolly”, the first successful cloned sheep was obtained from a differentiated adult mammary epithelial cell and created a revolution in science (Wilmut et al., 1997). Willadsen (1986) also performed nuclear transplantation on sheep embryos, using single blastomeres (from 8- to 16-cell embryos) as donor cells. Further developments led to the establishment of SCNT for the production of cloned mam- malian embryos from various cell types, including fetal ∗ Corresponding author. Tel.: +91 9416741839; fax: +91 184 2250042. E-mail address: dhrubamalakar@gmail.com (D. Malakar). fibroblast cells (Keefer et al., 2001), granulosa cells (Wells et al., 1999), cumulus cells (Wakayama et al., 1998), and adult skin fibroblast cells (Wakayama and Yanagimachi, 1999). Subsequently different cloned animals have been produced by SCNT like, e.g. in cattle (Cibelli et al., 1998), goats (Baguisi et al., 1999), mice (Wakayama et al., 1998), pigs (Polejaeva et al., 2000), rabbits (Chesne et al., 2002), mules (Woods et al., 2003), horses (Galli et al., 2003), rats (Zhou et al., 2003) and dogs (Lee et al., 2005). The micromanipulator-based or traditional cloning technique, is a multi-step, time consuming and compli- cated procedure that requires expensive and sophisticated equipment, like, e.g. the micromanipulator, micropipettes and micro forge. Moreover, proper use of these tools requires highly skilled and qualified expertise. To over- come this problem micromanipulator free (handmade) 0921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2012.03.001