Cooling extender choice modulates the efficacy of freezing extenders for boar sperm cryopreservation

Boar semen cryopreservation is limited by high sperm sensitivity, with the cooling extender and holding time representing critical steps. However, the interplay between transport and freezing extenders remains poorly elucidated. This study evaluated the main effects and the interaction between two cooling extender (BTS and BSUI®) and two freezing extenders (TRIS® and BBB®) on post-thaw boar semen quality. Ejaculates from 10 boars were processed in a 2x2 factorial design (BTS/TRIS, BTS/BBB, BSUI/TRIS, BSUI/BBB). Sperm kinetics (CASA) and plasma membrane integrity (PMI) were assessed at pre-centrifugation, post-centrifugation, and post-thaw time points. At the post-thaw evaluation, no significant interaction was observed between transport and freezing extenders (P > 0.05), indicating that these factors act independently. Regarding the main effects, the BSUI® cooling extender resulted in superior kinetic performance compared to BTS (P < 0.05). Independently, the TRIS® freezing extender yielded higher motility and membrane integrity than BBB® (P < 0.05). Consequently, the sequential combination of BSUI/TRIS presented the highest absolute kinetic values (Total Motility 63.3%; Progressive Motility 36.1%) due to the cumulative effect of the most efficient extenders. In conclusion, the cooling extender influences post-thaw quality independently of the freezing extender. The BSUI/TRIS sequence optimized post-thaw kinetics through an additive effect. Therefore, protocol optimization should focus on selecting the most efficient extender for each isolated stage of the process.