Role of seminal plasma extracellular vesicles and their miRNAs in black boar sperm cryopreservation

Context Seminal plasma extracellular vesicles (SP-EVs) interact directly with sperm and promote reproductive processes. SP-EVs and their microRNAs (miRNAs) exhibit cryoprotective properties. Aims This study investigated the potential of SP-EVs from KHAPS black pigs to improve the quality of cryopreserved boar sperm. Methods SP-EVs were isolated from seminal plasma by differential centrifugation and a commercial exosome isolation kit, then characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting for CD9 and β-actin to confirm vesicle purity and integrity. Purified SP-EVs (0,1,2,4 mg/mL) were supplemented into the semen extender during the equilibration stage before freezing, and selected miRNAs were transfected into spermatozoa using a liposome-based reagent. Key results SP-EVs significantly improved post-thaw sperm viability and acrosome integrity. Transfection with 100 nM miR-26a mimic further enhanced these parameters (P < 0.05). At the molecular level, miR-26a inhibition upregulated PTEN, whereas miR-423-5p inhibition decreased GSTM1 expression (P < 0.05). Conclusions These findings demonstrate that supplementation with SP-EVs and miR-26a during cryopreservation can enhance the post-thaw quality and survival of boar spermatozoa. Implications This strategy holds significant promise for improving the reproductive efficiency of frozen boar semen, thereby facilitating genetic resource preservation and supporting commercial artificial insemination applications.