Su Rina, Ding Fangrong, Sorgog Moqir, Li Ling, Bai Suyilatu, Ritu Xiji, Nasenochir Narenhua, Fan Ruiwen, Herrid Muren
Grassland & Cattle Investment Co., Ltd., Hohhot, 010000, Inner Mongolia, China.
College of Biological Sciences, Chinese Agricultural University, Beijing, 100000, China.
Sci Rep. 2025 Apr 29;15(1):15032. doi: 10.1038/s41598-025-96511-8.
Handmade cloning (HMC) presents several advantages over conventional cloning, including higher throughput, cost-efficiency, and operational simplicity. However, a comprehensive analysis comparing embryo production rates and pregnancy outcomes in cattle has yet to be conducted. This study reveals that cytoplasts produced using the micropipette method exhibited higher quality than those created with a microblade, leading to improved cleavage and blastocyst rates. The fusion of one or double cytoplast did not significantly affect the developmental potential of reconstructed embryos, although blastocysts from single cytoplasts contained slightly fewer cells. Notably, HMC demonstrated higher pregnancy rates and live calf production efficiency compared to conventional cloning. Specifically, for 41 vitrified embryos from conventional cloning, the one-month post-transfer pregnancy rate was 41.4%, resulting in 6 healthy calves (14.6%). In contrast, HMC produced one-month pregnancy rates of 71.4% for fresh and 60.0% for vitrified embryos, yielding 6 (28.5%) and 4 (26.7%) healthy calves, respectively. The optimization of HMC emphasizes the micropipette method's potential for cattle cloning applications, including genomic selection and gene editing. Additionally, novel insights into the incompatibility issues between nuclear DNA and mitochondrial DNA (mtDNA) in cloned embryos are discussed.
手工克隆（HMC）相较于传统克隆具有诸多优势，包括更高的通量、成本效益和操作简便性。然而，尚未对牛的胚胎生产效率和妊娠结局进行全面比较分析。本研究表明，使用微量移液器法产生的细胞质体比用微型刀片产生的质量更高，从而提高了卵裂率和囊胚率。一个或两个细胞质体的融合对重构胚胎的发育潜力没有显著影响，尽管来自单个细胞质体的囊胚细胞数量略少。值得注意的是，与传统克隆相比，HMC表现出更高的妊娠率和活产犊牛生产效率。具体而言，对于41枚经传统克隆的玻璃化胚胎，移植后一个月的妊娠率为41.4%，产下6头健康犊牛（14.6%）。相比之下，HMC新鲜胚胎的一个月妊娠率为71.4%，玻璃化胚胎为60.0%，分别产下6头（28.5%）和4头（26.7%）健康犊牛。HMC的优化突出了微量移液器法在牛克隆应用中的潜力，包括基因组选择和基因编辑。此外，还讨论了关于克隆胚胎中核DNA与线粒体DNA（mtDNA）不相容问题的新见解。
