Metabolomic Differences Associated with Egg Yolk and Candidate Additives for Boar Sperm Cryopreservation

Egg yolk (EY) protects boar sperm during cryopreservation, but the small molecules involved remain poorly characterized. Duroc boar semen was studied at Sichuan Agricultural University, Chengdu, China, under institutional animal care approval 20230163. Sperm processed with or without EY were compared using widely targeted ultra-performance liquid chromatography–tandem mass spectrometry. Of 1334 detected metabolites, 439 differed in abundance (variable importance in projection > 1; nominal p < 0.05): 18 were more abundant and 421 less abundant with EY. These differences mainly involved glycerophospholipids, sphingolipids and amino acid derivatives. Sixteen of 17 sphingolipid candidates were less abundant, whereas the feature annotated as ceramide Cer(d18:0/19:1) was more abundant. Metabolite set enrichment analysis highlighted phosphatidylinositol signalling, and myo-inositol abundance was lower with EY. Supplementation with myo-inositol, ceramide Cer(d18:1/18:0) or D-erythro-dihydrosphingosine (SPH; sphinganine, d18:0) improved motility under selected conditions in EY-containing extenders. Combined supplementation was assessed only for motility and produced the highest observed mean among the formulations tested across multiple processing stages. Flow-cytometric analyses of individual compounds showed higher JC-1 Q2 and FITC-PNA-negative/PI-negative fractions. Mean oxidant-sensitive fluorescence within the fluorescence-positive sperm population was also lower with each compound. The descriptive metabolomic comparison may reflect contributions from residual EY and differential cell survival. These findings identify candidates for further evaluation; their efficacy in chemically defined extenders and effects on fertility require separate validation.