CONICET | Buscador de Institutos y Recursos Humanos

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technology with potential for its application in agriculture, biomedicine, and biotechnology. However, the SCNT efficiency is low. Failure in embryo production by SCNT could be associated mainly with chemical activation treatments or the donor cell type. In this context, we compare the use of latrunculin A (LatA), instead of cytochalasin B during the activation with roscovitine (Rosco), versus the treatment of donor cells with demecolcine (D-cells) followed by activation just with Rosco to compare cloning efficiency. The aim of this study was to evaluate the in vitro developmental competence as well as the gene expression pattern of key genes (CDX2, OCT4, SOX2, and NANOG) in blastocysts obtained from the two treatments. To do this, cumulus-oocyte complexes were collected from cow ovaries obtained from slaughterhouses and were IVM for 21 h. After cumulus-cell removal, enucleation was performed as described by Gambini et al. (2014 PLoS ONE 9, e110998; https://doi.org/10.1371/journal.pone.0110998). The G0/G1 cells or D-cells were fused to the oocytes. For activation, reconstructed zygotes were treated with 5 μM ionomycin for 4 min followed by 5-h incubation into different randomly activation groups: D-cells + 50 μM Rosco (SCNT-Demec), G0/G1 cells + 50 μM Rosco/10 μM LatA (SCNT-LatA), and G0/G1 cells + 50 μM Rosco/5 μg mL−1 cytochalasin B (SCNT-Ctrol). Parthenogenetic controls were also included: Part-Demec, Part-LatA, and Part-Ctrol. Activated oocytes were cultured in synthetic oviductal fluid with amino acids medium until blastocyst stage. Rates of cleavage, morulae, and blastocysts were evaluated at Days 2, 5, and 7 of in vitro culture, respectively. Relative abundance of mRNA coding for the four genes was compared between SCNT-Demec, SCNT-LatA, SCNT-Ctrol, and IVF groups by RTqPCR. Data was analysed by Fisher´s exact test for in vitro culture (P