Biological transcomplementary activation as a novel and effective strategy applied to the generation of porcine somatic cell cloned embryos

Abstract

A novel method termed the biological transcomplementary activation (B-TCA) has been recently utilized for the stimulation of porcine oocytes reconstituted by somatic cell nuclear transfer (SCNT). The use of cytosolic components originating from fertilized (FE) rabbit zygotes as the stimuli for the B-TCA of SCNT-derived pig oocytes appeared to be a highly efficient strategy applied to promote the in vitro development of cloned embryos, leading to a significant improvement in the blastocyst yield (43.6%) compared to the yields achieved using the standard protocol of simultaneous fusion and electrical activation (SF-EA; [31.3%]) or the protocol of delayed electrical activation (D-EA) independent of extracellular Ca2+ ions (0%). The FE rabbit zygote cytoplast-mediated B-TCA resulted in the increased blastocyst formation rate of porcine cloned embryos as compared to the B-TCA triggered by either cytoplasts isolated from pig parthenogenotes (PAs; [27.8%]) or rabbit PA-descended cytoplasts (0%). A considerably lower percentage of blastocysts containing apoptotic and/or necrotic (annexin V-eGFP-positive) cells were obtained from the SCNT-derived oocytes stimulated by the FE rabbit zygote cytoplast-based B-TCA (22.2%) compared to those stimulated using the SF-EA protocol (35.1%). In contrast to the B-TCA induced by FE rabbit zygote cytoplasts, apoptosis/necrosis incidence decreased totally among the cloned pig blastocysts that developed from reconstituted oocytes undergoing the porcine PA cytoplast-evoked B-TCA. In conclusion, the FE rabbit zygote cytoplast-mediated B-TCA turned out to be a relatively effective strategy for the in vitro production of porcine blastocyst clones of higher quality compared to those created using the standard SF-EA approach.