Background: The poultry industry experiences genetic losses due to recurring infectious diseases, necessitating effective preservation strategies. Nitric oxide plays a crucial role in male reproduction, and optimal NO (nitric oxide) levels may enhance sperm viability. This study investigated the effects of SNAP (S-nitroso-Nacetylpenicillamine) on the longevity of rooster sperm. Methods: Semen was diluted with Beltsville Poultry Semen Extender-I containing 0 or 25 μM SNAP and stored at 10°C. Sperm motility and acrosome integrity were assessed at 1, 3, and 7 days. NO levels were quantified by DAF-FM diacetate and AI trials were evaluated by fertility and hatchability. Results: On day 1, sperm motility in the SNAP 25 μM-treated group was significantly higher than in the control. NO quantification confirmed that SNAP-treated semen exhibited higher NO levels. For fertilization and hatchability assessment, hens were divided into two groups based on the presumed duration sperm resided in sperm storage tubules. Before artificial insemination, the sperm was preserved at low temperature (10°C) to maintain viability. Fertilization rates were significantly higher in the SNAP-treated group in both short-term and long-term SST storage conditions. However, hatchability was only significantly improved in the SNAP-treated group when fertilization occurred after extended storage. Conclusions: These findings suggest that NO enhances sperm viability and fertility in poultry semen stored at low temperatures. SNAP 25 μM enhances AI efficiency by maintaining sperm viability and extending fertilization potential. Further research is needed to refine NO-based fertility enhancement strategies for avian species.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Experimental animals and semen collection
    Reagents and semen extender composition
    Semen refrigeration and storage
    Sperm morphology and longevity analysis
    Acrosome length measurement
    Quantification of nitric oxide using DAF-FM diacetate
    Fertilization and hatchability assessment followingartificial insemination
    Statistical analysis
RESULTS
    Sperm motility and longevity analysis following SNAPtreatment
    Acrosome analysis of rooster sperm following SNAPsupplementation and low-temperature storage
    Quantification of nitric oxide (NO) using DAF-FMdiacetate
    Effect of low-temperature stored semen onfertilization and hatchability following artificialinsemination
DISCUSSION
CONCLUSION
REFERENCES

• Jae-Yeong Lee(Animal Genetic Resources Research Center, National Institute of Animal Science, RDA, Hamyang 50000, Korea)
• Yeoung-Gyu Ko(Animal Genetic Resources Research Center, National Institute of Animal Science, RDA, Hamyang 50000, Korea)
• Daehyeok Jin(Animal Genetic Resources Research Center, National Institute of Animal Science, RDA, Hamyang 50000, Korea)
• Se Young Lee(Animal Genetic Resources Research Center, National Institute of Animal Science, RDA, Hamyang 50000, Korea)
• Ga Yeong Lee(Animal Genetic Resources Research Center, National Institute of Animal Science, RDA, Hamyang 50000, Korea, Department of Animal Resources Science, Kongju National University, Yesan 32439, Korea)
• Bongki Kim(Department of Animal Resources Science, Kongju National University, Yesan 32439, Korea) Corresponding author
• Sung Woo Kim(Hanwoo Research Institute, National Institute of Animal Science, RDA, Pyeongchang 25340, Korea) Corresponding author