Niacin Improves Cryopreserved Equine Sperm Quality and Gene Expression: An Artificial Intelligence Assisted Evaluation

Niacin acts as an antioxidant that protects cells from oxidative damage. This study evaluated the effects of adding niacin to the equine semen freezing extender on sperm quality and gene expression after cryopreservation. Ejaculates from ten stallions were frozen using the INRA 96 extender (control) or extenders supplemented with 10- and 20-mM niacin. After thawing, sperm were analysed for motility, kinematics, viability, membrane integrity, mitochondrial potential, lipid peroxidation, nitrite, hydrogen peroxide, malondialdehyde and reactive oxygen species (ROS) concentrations, DNA integrity, sperm binding to bovine oviduct explants, and expression of apoptosis related (BCL2, BAX), mitochondrial (ROMO1), sperm binding (SPACA3) and DNA repair (OGG1) genes. Data were tested for normality (Shapiro-Wilk) and analysed by randomised block ANOVA followed by Tukey's test (p < 0.05). Machine learning algorithms (Logistic Regression, MLP, XGBoost, KNN and SVM) with SHAP analysis ranked the most influential parameters associated with sperm quality. The addition of 10 mM niacin improved sperm motility, mitochondrial activity, and the number of sperm bound to bovine oviduct explants, while reducing ROS levels and expression of BAX and ROMO1, and increasing BCL2 and SPACA3 genes. The 20 mM treatment also enhanced sperm binding and upregulated SPACA3 expression compared to the control. However, 20 mM niacin showed lower binding activity than 10 mM. Machine learning identified sperm binding to oviduct explants and SPACA3 expression as the most influential variables for classifying samples. In conclusion, both niacin concentrations improved equine cryopreserved sperm quality, although 10 mM showed superior antioxidant, anti-apoptotic, and functional effects, representing optimal supplementation level.