This study aimed to assess the impact of serum concentrations and cell confluency levels during synchronization on the efficiency of ？ pig embryo cloning. In Experiment 1, the proportion of nucleated fibroblasts in the G0/G1 phase of the cell cycle was highest at serum concentrations of 0.2% and 0.3%, with no statistically significant difference between these two groups (88.75% and 85.12%, respectively; P > 0.05). Cleavage rates were maximized at serum concentrations of 0.1%, 0.2%, and 0.3%, yielding rates of 82.76%, 86.72%, and 85.06%, respectively. The blastocyst formation rate observed in the 0.2% group was significantly higher than that of 0%, 0.1%, 0.3%, 0.4%, 0.5%, and control groups (35.58% vs. 12.94%, 28.04%, 28.09%, 28.38%, 28.31%, and 18.41%, respectively; P < 0.05). Based on the results of Experiment 1, Experiment 2 utilized synchronized ？ pig fibroblasts cultured in 0.2% serum to examine how cell confluency influences cell cycle dynamics. The rate of fibroblasts with nuclei in the G0/G1 phase of 60% group was significantly lower than that of 80% and 100% groups (70.91% vs. 88.89% and 89.54%, respectively; P < 0.05). Moreover, no statistically significant differences were observed in the rate of cells in the G0/G1 phase between the 80% and 100% groups (P > 0.05). Among the groups studied, the 60% group demonstrated the lowest cleavage and blastocyst formation rates (69.34% and 20.94%, respectively). By contrast, no statistically significant difference in blastocyst formation was detected between the 80% and 100% groups (36.01% and 35.91%, respectively, P > 0.05). In conclusion, synchronizing ？ pig fibroblasts in 0.2% serum at 80% or 100% confluence markedly enhances the efficiency of cloned ？ pig blastocyst formation. The proportion of ？ pig fibroblasts with nuclei arrested in the G0/G1 phase exceeded 88%, while the rate of successful cloned ？ pig blastocyst formation surpassed 35%.
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