CATH-1 supplementation improves sperm quality and fertility of rooster semen during cold storage by reducing bacterial contamination.

The present study investigated the effects of antibiotic and antimicrobial peptide supplementation on semen quality, viable bacterial load, and fertility potential in Thai native chickens during cold storage. Semen samples were diluted in an extender supplemented with kanamycin (31.20 μg/mL) or CATH-1 (3.75 or 7.50 μg/mL) and stored at 5 °C for up to 72 h. Sperm quality parameters, viable bacterial load, and reproductive outcomes following artificial insemination were assessed at 0, 24, 48, and 72 h. Storage duration significantly affected all sperm quality parameters (P < 0.001), while fertility and hatchability were progressively lower across the sequential storage-time phases. Kanamycin and CATH-1 at 3.75 μg/mL consistently maintained higher sperm quality and reduced susceptibility to lipid peroxidation compared with the control (P < 0.05), with no significant difference between the two treatments. Both treatments effectively suppressed bacterial proliferation; however, viable bacterial load increased progressively beyond 24 h in all groups. Scanning electron microscopy revealed marked bacterial surface alterations following CATH-1 treatment, consistent with membrane-disruptive antibacterial activity. In contrast, CATH-1 at 7.50 μg/mL compromised sperm quality and reproductive outcomes from the initial time point onward, despite achieving the greatest bacterial suppression among all treatments. Across the sequential storage-time phases, fertility potential was progressively lower, although the kanamycin and CATH-1 (3.75 μg/mL) groups maintained higher fertility than the control during prolonged storage. These findings demonstrate a dose-dependent dual effect of CATH-1, with 3.75 μg/mL performing comparably to kanamycin and representing a promising antibiotic alternative in the context of One Health concerns in poultry artificial insemination programs.