Nicotinamide mononucleotide enhances porcine sperm quality by activating the SIRT3-SOD2/ROS pathway and promoting oxidative phosphorylation

Sperm quality is crucial for animal reproduction but is often deteriorates due to oxidative stress. Preserving functionality during storage by protecting it from oxidative stress is crucial for enhancing the success of assisted reproductive technologies and maintaining fertility. Nicotinamide mononucleotide (NMN), a precursor of Nicotinamide adenine dinucleotide (NAD⁺), acts as a potent antioxidant. However, its role and regulatory mechanism in porcine sperm liquid preservation in vitro are remains unclear. In this study we demonstrated that NMN supplementation in the semen diluent significantly improved porcine sperm quality during storage at 17 °C by increasing NAD⁺ concentrations and decreasing the reactive oxygen species (ROS) concentrations in sperm. Subsequent analysis revealed that NMN, upon conversion to NAD⁺ within sperm, exerted antioxidant effect by upregulating the mitochondrial protein sirtuin 3 (SIRT3), resulting in the downregulation of superoxide dismutase 2 (SOD2) acetylation. This pathway mitigated oxidative stress. Additionally, NMN also enhanced sperm oxidative phosphorylation (OXPHOS) thereby increase adenosine triphosphate (ATP) production concentrations via activating SIRT3, without affecting sperm glycolysis. Furthermore, the semen diluent supplemented with 50 μg/mL NMN resulted in improved reproductive outcomes, including the birth of healthier piglets and a reduction in stillbirths rates following artificial insemination (AI). Taken together, NMN improves porcine sperm quality through SIRT3-activating the SOD2/ROS axis and oxidative phosphorylation. Our findings advance understanding of sperm physiology and metabolism and provide a new avenue promising approach for developing innovative semen diluents for liquid preservation in animal reproduction biology in vitro.