ABSTRACT: Sexual maturation in boars impacts reproductive efficiency in swine production, yet the molecular mechanisms underlying this developmental transition remain poorly understood. This study aimed to investigate the transcriptomic changes in sperm from Duroc boars during sexual maturation, conducting a longitudinal analysis. The total RNA and miRNA profiles from the same individuals (n = 6) at puberty (7.24 ± 0.39 months) and sexual maturity (10 ± 0.40 months) were compared, identifying molecular signatures associated with reproductive development. Total RNA sequencing (Illumina NovaSeq-6000) and miRNA sequencing (Illumina NextSeq-500) were performed on all 12 paired samples (6 boars at 2 time points), followed by differential expression analysis using a paired statistical model in DESeq2 to account for repeated measures. Differential expression analysis identified 60 differentially expressed genes using stringent criteria (adj P < 0.05, |log₂FC| ≥ 0.5), with 65% upregulated in sperm of boars 10-months versus 7-months of age. Key upregulated genes included NCLN, RGS12, CIB2, FOXP4, PHC1, CDC25B and AKAP1, while HSP90AA1, EVI5, FSIP2, VDAC3, and ALMS1 were key downregulated genes. Furthermore, Gene ontology analysis revealed significant enrichment of 11 biological processes, mostly related to reproductive development, and four molecular functions (transferase activity, transferring phosphorus-containing groups, protein serine/threonine kinase activity, protein kinase activity and signal sequence binding). Additionally, our miRNA analysis identified six differentially expressed miRNAs (adj P < 0.05 & |log2FC| ≥ 0.5); ssc-miR-193a-5p, ssc-miR-574-3p, ssc-miR-126-3p, ssc-miR-196a, ssc-miR-210 were upregulated, and ssc-miR-338 showed downregulation in 10-months age. Integrated analysis of differentially expressed mRNA and predicted miRNA targets identified 18 miRNA-mRNA regulatory pairs, enriched in pathways related to cell cycle processes and chromatin binding, suggesting coordinated regulation across RNA biotypes in sperm during sexual maturation. Our study reveals coordinated transcriptomic shifts in boar sperm during sexual maturation. Most genes show increased abundance at 10 months of age, with enrichment in terms of reproductive development. Several miRNAs appear to regulate these changes through targeted mRNA interactions. These findings expand our understanding of the
