385 Extracellular vesicle microRNAs in porcine seminal plasma reflect quality status

Abstract The pig industry relies heavily on artificial insemination as the primary breeding technique, using high-quality semen doses for genetic improvement. Semen consists of seminal plasma, a complex fluid that provides an optimal environment for sperm maturation, viability, and fertilization. Seminal plasma contains membranous structures called extracellular vesicles (EVs), facilitating intercellular communication. These seminal plasma-EVs (SP-EVs) contain microRNAs, which hold promise as predictive markers of semen quality. Thus, we hypothesized that the composition of SP-EVs might reflect the divergent quality status of corresponding spermatozoa. Therefore, this study aimed to explore the microRNA content of SP-EVs isolated from accepted (Passed) or rejected (Failed) boar semen for artificial insemination. Over 8 wk during the hot summer, raw semen was collected (n = 83) from sexually mature Duroc boars at a commercial boar stud (Prestage Farms, MS). Semen samples underwent sperm analysis to evaluate motility and morphology, classified as Passed or Failed quality based on predefined criteria (≥ 70% and < 70%, respectively). Seminal plasma was isolated through serial centrifugation, followed by SP-EV extraction using differential centrifugation. The isolated SP-EVs were characterized using transmission electron microscopy (TEM) for EV morphology and nanoparticle tracking analysis (NTA) for size and concentration. SP-EV miRNAs were extracted and identified via the Illumina NextSeq sequencing platform. Data were analyzed using SPSS software and significance was set at P < 0.05. Both TEM and NTA demonstrated that SP-EVs had a spherical structure with a diameter range of 50 to 300 nm. A total of 437 miRNAs were expressed in the Failed group and 443 miRNAs in the Passed group. MiRNA differential expression analysis revealed 28 downregulated and two upregulated miRNAs in the Passed group compared with the Failed group. Validated miRNA gene targets were associated with signaling pathways, including N-glycan biosynthesis, p53 signaling, pancreatic cancer, and apoptosis. These findings suggest that SP-EVs carry specific microRNAs potentially linked to boar semen quality. Thus, microRNAs from SP-EVs have the potential to serve as biomarkers to assess sperm quality, thereby providing insight into therapeutic approaches to address male infertility. Additionally, seminal plasma-EVs can be useful nanocarriers for further research interventions. Work supported by USDA-ARS grant# 6066-31000-015-00D