Somatic cell nuclear transfer (SCNT) is a pivotal assisted reproductive technology and serves as a cornerstone in embryonic engineering research [1]. In animal husbandry, the synergistic convergence of cloning with transgenic techniques establishes an innovative pathway for targeted breeding and genetic enhancement of core animal populations, thereby helping to overcome the limitations of conventional husbandry practices. However, incomplete nuclear reprogramming in donor cells leads to reduced embryonic developmental potential and abnormal development in cloned animals, thereby restricting the practical application of this technology in agricultural production [2].

Unlike naturally fertilized embryos, cloned embryos undergo a process of resetting the transcriptional program of differentiated somatic cells to a totipotent state, which remains largely undefined. Researchers have employed buffalo fetal fibroblasts [3], porcine fibroblasts [4] and Holstein bovine ear fibroblasts derived from different individuals [5] to generate cloned embryos and related animals. The results showed that donor cells derived from different individuals significantly influenced cloning efficiency and the cloned animals. Further molecular investigations reveal that fibroblasts with higher cloning efficiency exhibit increased energy metabolism [6]. Mitochondria, as the "energy factories", play a crucial role in regulating metabolic transitions [7]. During the development of cloned embryos, donor cell mitochondria inevitably contribute to mitochondrial heterogeneity within the embryo, which in turn affect its developmental potential [8], [9], [10]. However, the underlying mechanisms governing this process remain unclear.

Early research on cloned embryos indicates that uncertainties in mitochondrial functionality may influence developmental potential. Compared with parthenogenetic embryos, cloned embryos exhibited a significant decrease in mitochondrial membrane potential, elevated levels of reactive oxygen species (ROS) and cell apoptosis, and a significant reduction in the mitochondrial-related protein MFN1, indicating the crucial role of mitochondria in embryonic development [11]. The addition of mitochondrial antioxidants (Mito-Q) during IVM and IVC was beneficial in reducing ROS levels and enhancing mitochondrial membrane potential, thus improving embryonic development potential [12]. Furthermore, enhancing mitochondrial function in donor cells-such as by adding antioxidants to reduce mitochondrial ROS and increase mitochondrial membrane potential [13], [14]-boosts the expression of mitochondria-related genes and promotes mitochondrial ATP production and membrane integrity, thereby contributing to the developmental potential of cloned embryos. Studies have shown that enhancing mitochondrial function in oocytes, donor cells or embryos significantly increases the developmental potential, further underscoring the critical role of mitochondria in the development of cloned embryos.

Numerous studies have optimized cloning efficiency by regulating the epigenetic modification levels of donor cells to influence their chromatin state[15], [16], [17], [18]. Current research has revealed that mitochondrial metabolic remodeling is involved in the molecular mechanisms of early embryo development through epigenetic modifications, thereby influencing the developmental potential of cloned embryos [19]. For example, mitochondrial TCA cycle-derived acetyl-CoA serves as an enzyme cofactor in nuclear epigenetic machinery, by modulating histone acetylation dynamics [20]. S-adenosine methionine (SAM), synthesized from methionine and ATP, acts as a methyl donor, directing histone and DNA methylation to regulate chromatin remodeling in cells [21], [22]. Mitochondria and the substrates produced during metabolic processes are inextricably linked. Research by Ying et al. demonstrates that the mitochondrial permeability transition pore (mPTP)-mediated elevation of PHF8 and the cofactor α-KG regulates H3K9me3 and H3K27me3 methylation landscapes, thereby influencing the nuclear reprogramming efficiency of cells [23]. These studies further underscore the critical role of mitochondria as a bridge in epigenetic modifications. However, the specific mechanisms by which mitochondria participate in chromatin remodeling and influence the developmental potential of cloned embryos remain to be elucidated.

Previous work in our laboratory has demonstrated that variation in cloning efficiency among different individuals arises from differences in the energy metabolism, epigenetic modifications, and chromatin structure of donor cells [6]. However, how the mitochondrial function of donor cells influences cloning efficiency requires further investigation. The current data indicate that optimizing mitochondrial function, metabolic remodeling, and chromatin remodeling in donor cells can effectively enhance cloning efficiency, further highlighting the importance of mitochondria in early cloned embryo development. Therefore, elucidating the relationship between mitochondrial function in donor cells and chromatin remodeling will aid in screening donor cells with high cloning efficiency, offering new directions for improving cloning success.