Sperm motility is a key factor influencing male fertility and is associated with metabolic and lipid profiles across species. The aim of this study was to investigate the relationship between sperm motility and the seminal plasma lipid profile in Simmental bulls, and to identify key lipids potentially influencing sperm motility. Semen samples were collected from 26 healthy Simmental bulls with an average age of 4.9 years. Sperm quality was evaluated using computer-assisted sperm analysis (CASA). Based on motility, the samples were divided into two groups: high sperm motility (HSM > 65%) and low sperm motility (LSM < 65%). Compared to the LSM group, the HSM group exhibited significantly higher sperm viability, motility, straight-line velocity, beat-cross frequency, and sperm acrosome integrity, while the sperm malformation rate was lower ( < 0.05). Lipid profiles were determined using LC-MS/MS, and 40 differential lipids were identified by multivariate statistical analysis. Among them, 39 lipids were upregulated in the LSM group compared to the HSM group. They were primarily triglycerides and carnitines, mainly involved in four metabolic pathways related to glycerophospholipid and linoleic acid metabolism. Notably, PC (16:0/20:4; 14:0/18:3), LPC (22:4/0:0; 22:6/0:0), and PE (14:0/18:1; 18:1/20:3) were diagnosed with great accuracy (AUC > 0.7), which means they may serve as potential biomarkers for sperm motility.
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