Sperm cryotolerance in boar semen emerges from integrated lipidomic architecture

Boar semen cryopreservation remains limited by pronounced inter-ejaculate variability in post-thaw sperm quality, which is largely attributed to intrinsic biological differences rather than to cryopreservation protocols alone. This study investigated whether ejaculate freezability and holding time (HT) modulate the lipidomic profiles of seminal plasma and spermatozoa, and how these changes relate to sperm cryotolerance. Ejaculates were classified as good or poor ejaculate freezers and sampled either immediately or after 24 h of HT at 17 °C prior to cryopreservation. Post-thaw sperm quality was assessed using functional parameters, and targeted lipidomic profiling was performed in seminal plasma and spermatozoa. Multivariate analyses revealed that ejaculate quality was the primary factor shaping lipidomic profiles, whereas HT exerted a modulatory, quality-dependent effect. Lipidomic differences were detectable in both seminal plasma and spermatozoa, but were more pronounced in spermatozoa, where good ejaculate freezers displayed a more homogeneous and consistently organized lipidome. Rather than isolated lipid alterations, cryotolerance was associated with coordinated changes across lipid classes involved in membrane organization and stability. Holding time promoted lipid remodeling predominantly in good ejaculate freezers, without altering the intrinsic freezability phenotype. In conclusion, sperm cryotolerance emerges as an integrated property of membrane lipidomic architecture and plasticity, highlighting lipidomic organization as a key determinant of cryogenic resilience and a promising target for improving boar semen cryopreservation strategies.