Optimization of handmade cloning enhances pregnancy rates and live calf production in cattle.

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.