Analysis of factors contributing to the efficiency of the in vitro production of transgenic goat embryos (Capra hircus) by handmade cloning (HMC)

Cloning by Somatic Cell Nuclear Transfer (SCNT) still is challenging and inefficient. Recently, the handmade cloning (HMC) procedures have been successfully applied to livestock species. The aim of this study was to compare the effect of distinct oocyte sources (in vivo vs. post-mortem) and the final cytoplasmic embryo volume (similar to 85% or 2 x 50%) on fusion rates and on the developmental potential of Day-1 or Day-7 cloned transgenic goat embryos produced by HMC procedures. Cloned embryos were reconstructed by HMC using skin fibroblast donor cells established from a transgenic goat. Oocytes were obtained in vivo by laparoscopic oocyte recovery (LOR) from hormonally stimulated females or post-mortem from slaughterhouse ovaries from nonstimulated goats, resulting in no differences in the number of aspirated follicles, cumulus-oocyte complexes (COCs), and viable COCs per goat. However, the COC recovery rate was higher for slaughterhouse ovaries (86.0%) than for LOR (73.0%). Also, cytoplasmic volume (similar to 85% vs. 2 x 50%) had no effect on fusion rates after embryo reconstruction. Using slaughterhouse ovaries for cloning, a total of 18.0% (27/150) and 12.7% (19/150) of the in vitro-cultured embryos developed to the compact morula and blastocyst stages on Day 7. However, no recipients became pregnant on Day 30 following the transfer of Day-1 or Day-7 embryos. In conclusion, the use of slaughterhouse ovaries was as valuable to supply oocytes for the production of cloned goat embryos by HMC as the in vivo approach. The HMC was proven a simple alternative for the production of cloned transgenic goat embryos. (C) 2012 Elsevier B.V. All rights reserved.