TRIS–Egg Yolk Extender Attenuates Seminolipid and Sphingomyelin Loss in Chilled Canine Semen: An LC–HRMS Lipidomic and CASA Study

This study combined lipidomic profiling and computer-assisted sperm analysis (CASA) to evaluate whether a TRIS–egg yolk extender preserves canine spermatozoa at both molecular and functional levels during 72 h of refrigerated storage. HILIC–ESI–HRMS was first used to distinguish sperm-derived lipids from extender-associated species. Lipid-focused feature filtering and multivariate analysis highlighted the seminolipid or sulfogalactosylglycerolipid SGG 32:0 as a major contributor to storage-related lipidomic variation. Together with three long-chain sphingomyelins, SM 46:5;O2, SM 48:5;O2, and SM 50:6;O2, SGG 32:0 was selected as a sperm-specific membrane marker. Targeted C30 reversed-phase LC–ESI–HRMS showed that these lipids progressively decreased during chilled storage, whereas their decline was significantly attenuated in extender-treated samples, with significant treatment × time interactions for all monitored species (p < 0.05). CASA confirmed the functional relevance of these molecular changes: in extender-treated semen, total motility was preserved during the first 24 h, whereas more evident impairment occurred at 48–72 h. Overall, these findings indicate that TRIS–egg yolk extender helps preserve canine semen by slowing the loss of sperm-specific membrane lipids and maintaining motility during chilled storage. SGG 32:0 may represent a candidate lipid marker for monitoring sperm membrane preservation.