Flexible oocyte manipulation with delayed maturation and improved somatic cell nuclear transfer efficiency using...

Somatic cell nuclear transfer (SCNT) remains inefficient, limiting its practical use in cattle reproduction and research. This study investigated two complementary strategies to enhance handmade cloning (HMC): (i) holding bovine oocytes overnight and delaying maturation to enable a second round of SCNT, and (ii) using bovine-induced pluripotent stem cells (biPSCs) as donor nuclei for pluripotent stem cell nuclear transfer (PSCNT) to enhance developmental competence. Bovine oocytes were subjected to either conventional in vitro maturation (CONV; 20 h) or delayed maturation using a holding medium for 20 h before CONV (HOLD). Matured oocytes were used for SCNT, PSCNT, parthenogenetic activation (PA), or in vitro fertilization (IVF) as controls; HMC embryos were reconstructed using fibroblast or biPSC donor cells, activated, and cultured for 7 days. Results showed no significant differences between CONV and HOLD groups in oocyte maturation, recovery after stripping, survival after zona removal, or cleavage and blastocyst development after nuclear transfer. Fusion rates using fibroblasts were comparable between groups (42.6 ± 6.0 % vs. 50.3 ± 9.8 %), whereas biPSCs showing significantly higher fusion rates in CONV group (85.7 ± 8.2 % vs. 50.5 ± 8.8 %, P < 0.05). Among fused embryos, biPSC-PSCNT yielded higher blastocyst rates (33.3 ± 16.7 %) compared to fibroblast-SCNT donors (21.9 ± 12.6 %, P < 0.05). Across all reconstructed embryos, cleavage and blastocyst development were also greater with PSCNT (odds ratios 3.4 and 2.7 respectively). These findings indicate that delaying oocyte maturation offers flexible timing for SCNT without compromising competence. Moreover, PSCNT enhances embryo developmental outcomes, supporting its use as superior approach for advancing cloning efficiency and applications in reproductive biotechnology.