Optimization of electroporation conditions for sperm cells of Jiangxi Kangle chicken (Gallus gallus)

Gene editing in chickens (Gallus gallus domesticus) is used in global poultry industry, such as enhancing economic traits of poultry, breeding for disease resistance and developing vector vaccines, improving poultry welfare, improving the safety of poultry egg consumption, and producing pharmaceutical proteins in eggs, and so on. At present, gene editing in poultry relies on the traditional in vitro or in vivo primordial germ cell (PGC) pathways, and gene editing efficiency is much lower. In vitro gene editing of sperm, or sperm transfection assisted gene editing (STAGE), is a promising approach to improve gene editing efficiency, and electroporation is a safe, economical and simple method for sperm transfection. The purpose of this study was to electroporate Cy-3 fluorescein-labeled DNA into Jiangxi Kangle chicken (Gallus gallus) sperm cells to screen the optimal electroporation conditions through evaluating sperm dynamics parameters using a computer-assisted sperm analysis system (CASA), investigated sperm cell membrane/acrosome integrity, mitochondrial membrane potential and foreign DNA uptake efficiency by fluorescence microscopy and flow cytometry under different combination of electroporation parameters. The results showed that under the optimized electroporation conditions (100 V, 5 ms, 5 pulses), the DNA uptake efficiency was up to 57.1%. Notably, no significant changes were observed in key fertilization-related parameters (progressive motility, acrosome integrity, mitochondrial membrane potential), while only total motility and cell membrane integrity showed a significant but moderate decrease (P < 0.05). These results indicate that the optimized conditions cause minimal damage to sperm function and maintain fertilization potential.