Research note: Efficient preservation of genome-edited chicken germplasm via N-methylacetamide-based semen cryopreservation.

The rapid development of genome-edited chicken lines for agricultural and biomedical use requires effective methods for long-term preservation. In birds, cryopreservation of oocytes and embryos is challenging due to the structure of the egg. This makes semen cryopreservation one of the most practical alternatives, however, this method has not been validated in genome-edited chickens. This study evaluated the feasibility of cryopreserving semen from genome-edited chickens using an N-methylacetamide (N-MA)-based protocol. Two genome-edited chicken lines were used in this study which are a DAZL-GFP germ cell reporter line and a RAG1 knockout line. Semen was stored for either one week or one month prior to assessing fertility and hatchability. The results demonstrated that cryopreserved semen from both genome-edited chicken lines maintained their fertilizing capacity, with no significant differences in fertility or hatchability compared to cryopreserved wild-type groups at either storage duration. Hatchability remained above 80% across all groups, indicating that semen cryopreservation did not significantly affect embryo development after successful fertilization. Furthermore, SYBR-14/PI staining revealed no significant differences in sperm viability between genome-edited and control groups, although both showed a significant decrease in viability compared to fresh semen. These findings demonstrate that N-MA-based semen cryopreservation provides a reliable and practical method for the preservation of genome-edited chicken lines.