20 Comparative efficacy of conventional cloning and handmade cloning

In the livestock industry, somatic cell nuclear transfer (SCNT), commonly known as cloning, is a promising assisted reproductive technology (ART) that supports the rapid dissemination of elite genetics, replication of transgenic/gene-edited animals, and gene banking of proven phenotypes. Handmade cloning (HMC) has shown advantages over conventional cloning, including high throughput, cost-effectiveness, and ease of operation. Despite numerous reports on embryo production efficiencies across different species, a large-scale comparison of pregnancy rates and calf outcomes between these methods in cattle has not been conducted. This study aimed to compare the live calf production efficacy of conventional cloning and HMC to promote the broader application of cloning technology. Donor cells were sourced from a 45-day-old fetus and genotyped to calculate genomic estimated breeding values. In conventional cloning, cell nuclei were removed using a micromanipulator, while in HMC, nuclei of zona-free oocytes were removed using a fine glass pipette. Other aspects of embryo production, including media, were consistent between methods. From October to December 2023, 45 vitrified embryos produced via conventional cloning were transferred into recipients, resulting in pregnancy rates of 48.8% (21/45) at 1 month, 33.3% (15/45) at 3 months, and 20% (9/45) at 8 months. Concurrently, 21 fresh and 15 vitrified embryos produced by HMC were transferred to 36 recipients. Fresh HMC embryos resulted in pregnancy rates of 71.4% (15/21) at 1 month, 47.4% (10/21) at 3 months, and 23.8% (5/21) at 8 months, while vitrified HMC embryos showed pregnancy rates of 60% (9/15) at 1 month, 40% (6/15) at 3 months, and 26.6% (4/15) at 8 months. As of early July 2024, calving outcomes for the conventional cloning group included one early birth, two stillbirths, and two healthy calves from the remaining nine pregnancies at the 8-month test. For the HMC group, calving outcomes from the remaining nine pregnancies at the 8-month test included one healthy calf, with the remaining pregnancies ongoing. These results indicate that HMC demonstrated higher initial pregnancy rates compared with conventional cloning, although pregnancy rates decreased more significantly over time, whereas conventional cloning showed more stable pregnancy rates. Overall, the pregnancy rate was higher with HMC than with the conventional method. Considering the cost-effectiveness and large-scale operation, HMC offers an effective means for the commercial application of cloning technology.