TRIS-Egg Yolk Extender Attenuates Seminolipid and Sphingomyelin Loss in Chilled Canine Semen: An LC-HRMS Lipidomic...

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.