The Impact of Different Equilibration Methods on the Cryopreservation Effectiveness of Boar Semen

Boar semen cryopreservation is crucial for the porcine industry. The two-step (S2) is currently used for cryopreservation of boar semen. In this method, a cryoprotectant is added at 4°C to reduce its toxicity. Another method is the one-step (S1), which is easier, cheaper and reduces the need for equipment, but may increase the toxicity of cryoprotectant. This study compared S1 and S2 equilibration protocols, evaluating post-thaw total motility, viability, motion parameters, membrane integrity, antioxidant indicators, apoptosis and related gene expression. Results showed S2 had higher motility (p < 0.05), while S1 had greater average path velocity (p < 0.05), better plasma membrane integrity (p < 0.05), lower malondialdehyde (MDA) (p < 0.01), higher total antioxidant capacity (T-AOC) and catalase (CAT) (p < 0.01), lower superoxide dismutase (SOD) (p < 0.05) and reduced apoptosis (p < 0.05). S2 had higher Caspase-9, Bax and SOD-2 expression (p < 0.05). S1 shows superiority in membrane protection, antioxidant capacity and anti-apoptosis, despite slightly lower motility. It is a promising method for large-scale use with further refinement to enhance post-thaw motility.