Synergistic effect of L-carnitine, sodium pyruvate and casein supplementation in Tris-based extender on freezeability of Barbari buck semen

BACKGROUND: Post-thaw quality of frozen buck sperm is strongly influenced by the choice of diluent and antioxidant supplementation. L-carnitine, sodium pyruvate and casein enhance sperm cryopreservation by providing antioxidant protection, stabilizing membranes, supporting mitochondrial energy metabolism and preserving DNA integrity during the freeze–thaw process. OBJECTIVE: This study aimed to assess the combined effect of L-carnitine, casein and sodium pyruvate addition in semen extenders on cryopreserved buck semen survivability, in vitro sperm parameters and oxidative stress at various months and seasons in a tropical area. MATERIALS AND METHODS: A total of 12 adult Barbari bucks were used in this study. Semen was cryopreserved using an extender supplemented with L-carnitine (2mM), sodium pyruvate (1mM), and casein (0.068g). Post-thaw semen was evaluated for volume, sperm concentration, mass motility, progressive motility, viability, plasma membrane integrity (HOST), DNA integrity, capacitation and apoptosis status, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and superoxide dismutase (SOD) activity. RESULTS: Supplementation significantly improved post-thaw sperm motility, viability, plasma and acrosomal membrane integrity, mitochondrial function, and DNA stability while reducing apoptosis and cryocapacitation compared to the control group. Seasonal variations were observed, with semen collected during September–November and the rainy season showing superior parameters, followed by the transition from rainy to winter. The antioxidants effectively mitigated oxidative stress and preserved mitochondrial and DNA integrity throughout the freeze–thaw process. CONCLUSION: The inclusion of L-carnitine, sodium pyruvate, and casein in extenders significantly improved post-thaw buck sperm quality by preserving motility, membrane integrity, mitochondrial function, and DNA stability while reducing oxidative stress and apoptotic-like changes. These findings highlight the potential of this formulation to enhance the fertilizing capacity of cryopreserved semen across seasonal variations.