Characterization of Seminal Plasma Extracellular Vesicle MicroRNAs and Their Association with Boar Semen Quality...

Boar fertility is negatively affected by subfertility and elevated temperatures, which alter seminal plasma (SP) composition and reduce semen quality. Extracellular vesicles (EVs) in SP transfer microRNAs (miRNAs) to sperm and may influence sperm function. This study aimed to identify SP-EV microRNAs associated with differences in boar semen quality during the summer season. Semen collected from Duroc boars was evaluated and classified as Passed (≥70%) or Failed (<70%) based on sperm quality parameters. SP-EVs were isolated and characterized, and small RNA sequencing was performed to profile miRNA content. SP-EVs ranged from 90 to 200 nm, with concentrations of 4.33 × 10¹⁰ particles/mL in the Passed group and 1.85 × 10¹¹ particles/mL in the Failed group. Western blotting confirmed the presence of EV surface markers CD9, CD63, and CD81. A total of 446 unique miRNAs were identified, with 28 downregulated and two upregulated miRNAs in Passed compared with Failed SP-EVs. Additionally, functional enrichment analysis revealed that target genes of upregulated miRNAs were involved in sperm-related biological processes and PI3K-Akt, regulation of actin cytoskeleton, and ErbB signaling pathways. These findings demonstrate that SP-EV miRNAs reflect physiological responses to changes in environmental conditions and may contribute to the regulation of boar semen quality.