Untargeted metabolomics reveals changes in boar sperm and seminal plasma metabolites associated with sexual maturity

Abstract

Boars undergo physiological and biochemical changes in semen composition as they grow from puberty to sexual maturity. However, comprehensive metabolomic profiles of boar semen remain uncharacterised. Understanding metabolic alterations in semen during this period is important for optimising reproductive performance in breeding programs. The aim of this study was to characterise the semen metabolome as boars mature, utilising an untargeted metabolomic approach. Semen samples were collected from 15 Duroc boars at three developmental ages: ~ 7 months, 8.5 months, and 10 months. Sperm and seminal plasma were separated and analysed by hydrophilic interaction and reversed-phase liquid chromatography coupled with mass spectrometry to capture a wide range of metabolites. We identified a total of 4,491 features in boar semen, annotating 92 distinct metabolites. Amino acids, peptides and analogues constituted the most abundant components, followed by fatty acid esters. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) showed a clear separation between metabolomic profiles by age groups. PERMANOVA analysis of PCA scores confirmed statistically significant differences (P < 0.05) between younger (7 months) and more mature boars (8.5 months and 10 months). Pathway analysis identified porphyrin metabolism, taurine and hypotaurine metabolism, and glycerolipid metabolism as significantly enriched pathways in sperm, while glutathione and nitrogen metabolism were prominently enriched in seminal plasma. Using linear modelling, partial Spearman correlation and random forest analyses, we identified homoisovanillic acid as a key metabolite discriminating age groups in both sperm and seminal plasma. Additionally, L-glutamic acid, decanoyl-L-carnitine and N-(1,3-Thiazol-2-yl)benzenesulfonamide emerged as important sperm metabolites, while glyceric acid, myo-inositol, glycerophosphocholine, and several other compounds were identified as critical seminal plasma metabolites. This study provides a detailed characterisation of metabolic changes in Duroc boar semen during the transition from puberty to sexual maturity. Our findings enhance the understanding of reproductive development and could inform strategies to assess sexual maturity in breeding programs.