Development of an Optimized Zona-Free Method of Somatic Cell Nuclear Transfer in the Goat

Abstract

The purpose of this study was to develop an improved zona-free method of goat somatic cell nuclear transfer (SCNT) that has both ease of operation and efficiency. The main steps involved were: (1) optimization of in vitro oocyte maturation, (2) parthenogenetic activation of zona-free oocytes, (3) SCNT of zona-free anaphase II-telophase II (AII-TII) oocytes that subverted the need for long term UV-exposure of the oocytes, and (4) in vitro culture of groups of cloned embryos in wells in a highly efficient continuous serum-free embryo medium to the blastocyst stage before transfer to the recipients. Percentages of transgenic blastocyst production were 22.3 and 33.1% for adult and fetal cell lines, respectively. After transfer of cloned and transgenic blastocysts, 28.6 and 36.4% of the recipients were confirmed pregnant and 75 and 33.3% of the pregnancies resulted in the delivery of viable offspring, respectively. To our knowledge, this is the first report of successful live and survived birth of cloned and transgenic offspring through a whole procedure of in vitro oocyte maturation and embryo development to the blastocyst stage, and in this study the in vitro efficiencies of cloned and transgenic embryo production were higher than the available reports.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

“…According to Roth and Hansen (2004), inter-and intra-cellular components define how an oocyte will react to effects from the environment and high temperatures, even if within physiological ranges, potentially even being a stimulus to apoptosis in mammalian oocytes. A fact that corroborates this assertion is that the rate of development of parthenogenetic embryos to the morula and blastocyst stages obtained in our experiment was 18.3%, a low value for oocytes previously selected for the presence of the 1st PB extrusion and cytoplasm morphology when compared with studies by Apimeteetumrong et al (2004) and Nasr-Esfahani et al (2011), who obtained 42.3 and 54.9% of parthenogenetic development to the morula and blastocyst stages, and to the blastocyst stage, respectively. The animal response to climatic elements and factors may play an influence in the results of this study, resulting in a low overall efficiency of cloning under our conditions.…”

“…Although cloning by SCNT is well established in goats, with birth rates similar to those found in other species, there are no reports of cloned goats born in the tropics of the world, between parallels 30 o N and 30 o S, with all cloned goats born in countries under temperate or subtropical climates (Baguisi et al, 1999;Keefer et al, 2001;Reggio et al, 2001;Ohkoshi et al, 2003;Lan et al, 2006;Chen et al, 2007;Folch et al, 2009;Akshey et al, 2010;Colato et al, 2011;Liu et al, 2011;Nasr-Esfahani et al, 2011;Wells et al, 2011;Meng et al, 2012;Zhou et al, 2013;Yuan et al, 2014;Feng et al, 2015;Hosseini et al, 2015;Zhang et al, 2015;Yang et al, 2016a;Bai et al, 2017). This fact may be associated with the low productive and reproductive indexes of goat herds in tropical countries, such as in the Brazilian Semi-Arid region, where the annual birth rate in goats does not exceed 20% (Guimarães, 2006).…”

Epigenetic modification does not determine the time of POU5F1 transcription activation in cloned bovine embryos

“…To confirm fibroblast phenotype, somatic cells at passage 2 were used for immunostaining against anti-vimentin (for fibroblasts) and anti-pancytokeratin (for epithelial cells), as described previously [13]. Confirmed fibroblasts were plated in 6-well culture dishes (TPP, Switzerland) containing modified Eagle medium F-12 (DMEM/F-12) plus 10% fetal calf serum (FCS) until subconfluence (2×10 5 ).…”

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

“…Also, standardization is easier, with the possibility for future automation. So far, HMC has been successfully established in cattle (Vajta et al, 2004), pig (Du et al, 2007), horse (Lagutina et al, 2007), goat (Nasr-Esfahani et al, 2011), sheep (Zhang et al, 2013), and water buffalo (Saha et al, 2013).…”