Cryopreservation Procedure Screening and Metabolomic Profiling of Age-Related Differences in Post-Thaw Semen Quality of Saanen Bucks

Semen cryopreservation is widely used to disseminate valuable genetics in dairy goats, yet cryoinjury can substantially compromise sperm function, and the influence of male age on cryotolerance remains incompletely understood. This study aimed to identify a practical cryopreservation working combination for Saanen buck semen and to characterize age group differences in sperm quality, redox-related traits, and metabolomic profiles. Cryopreservation conditions were sequentially screened using independently prepared pooled semen batches by evaluating water bath volume, equilibration time, semen collection method, liquid nitrogen vapor distance, and dilution strategy. Artificial vagina collection, a 200 mL water bath, 2.5 h equilibration, a 9 cm vapor distance, and two-step dilution showed favorable performance among the conditions tested and were selected as the working combination. Using these conditions, semen quality and post-thaw redox-related measurements were compared between young (10–14 months) and adult (≥24 months) bucks using three independently prepared pooled batches per age group. Adult group pooled semen exhibited better fresh and post-thaw sperm quality outcomes, with post-thaw motility of 66.36 ± 7.81% compared with 60.18 ± 9.81% in the young group, together with higher plasma membrane and acrosomal integrity. Post-thaw adult group samples also exhibited higher SOD and GSH-Px activities and lower DCFH-DA-derived oxidant-sensitive fluorescence and MDA levels. Untargeted metabolomics was subsequently performed on individually processed ejaculates from 10 bucks per age group. Although 26 metabolic features met the nominal fold-change, p-value, and VIP screening criteria, none remained significant after Benjamini–Hochberg false discovery rate correction, and the PLS-DA model showed limited predictive performance (Q2 = −0.22). Collectively, this study identifies a practical cryopreservation working combination for Saanen buck semen and reveals age group differences in post-thaw sperm quality and redox-related characteristics. The metabolomic findings remain exploratory and require validation in larger independent cohorts.