Abstract
Objective: Protocols for somatic cell nuclear transfer (SCNT) remain technically challenging and inefficient. Demecolcine and nocodazole (Noc) have been used for chemically assisted enucleation to improve cloning efficiency; however, the procedure continues to be labor-intensive. This study aimed to use lysophosphatidic acid (LPA) to enhance Noc-mediated chemical enucleation (CE), thereby simplifying the SCNT procedure.Methods: Porcine MII oocytes were subject to CE by incubation with Noc and various concentrations of LPA for 1 h. Actin signals were evaluated to determine the optimal LPA. After treatment with Noc and 25 μM LPA for 1 h, spindle reformation was assayed following subsequent washout. Noc/LPA CE-treated oocytes were used for SCNT via direct microinjection of donor cells into the ooplasm. Reconstructed embryos were recovered for 2 h in PZM-5 prior to activation with ionomycin and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine. Activated reconstructed embryos were cultured in 6-DMAP for 4 h, then in chaetocin for an additional 20–24 h to promote demethylation and acetylation. Cleavage and blastocyst rates were evaluated on Day 2 and Day 6, respectively.Results: The culture of the matured oocytes in Noc/LPA for 1 h, the percentage of oocytes exhibiting actin signals increased significantly from 12.5% to 87.6% with the addition of 25 μM LPA. Although spindles reformed in 59.0% of oocytes 1 h after Noc/LPA washout, 83.9% of oocytes retained spindles enclosed by actin rings; enabling the direct microinjection of donor cells into the ooplasm within 30 min. Blastocyst development rates currently vary from 9.5% to 16.6% after 6 days of culture in PZM-5.Conclusion: Although the blastocyst rate requires further improvement, we have shown that enucleation of MII pig oocytes can be successfully achieved via Noc/LPA treatment. This protocol simplifies the SCNT procedure while ensuring consistent blastocyst development in pig in vitro maturation oocytes.