show Abstracthide AbstractIn this study, the methods to isolate and identify extracellular vesicles (EVs) including exosomes, from the seminal plasma (SP) of 3 fertile (F) and subfertile (S) bucks have been developed. Additionally, we investigated whether specific miRNA abundance differences between F and SF bucks could serve as fertility biomarkers. Ultracentrifugation and size exclusion chromatography analysis have made it possible to isolate different SP-EVs concentrations (8.53x10^11 ± 1.04x10^11 and 1.84x10^12 ± 1.75x10^11 particles/ml of SP; p=0,008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; p=0.7422) in F and S males, respectively. Particle size was not significantly correlated with any kinetic parameter. Also, EVs have been identified by electron microscopy and their marker proteins by Western blot. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r=0.94; p<0.05) and with the percentage of immotile spermatozoa (r=0.88; p<0.05). A total of 18 miRNAs were differentialy expressed (FC>2, FDR<0.05) in F vs. S groups. SP from F and S males contains EVs with different miRNA cargo that could be used as biomarkers for male fertility. Overall design: 14 sexually mature male California x New Zealand white rabbits of 10-12 months of age were used. An ejaculate was collected from these animals twice a week with an artificial vagina for 1 month (8 ejaculates from each animal). The macroscopic quality of each ejaculate was assessed, discarding the ejaculates with abnormal colors (yellow or red) and low volumes (<0.2 ml). Sperm viability was also evaluated with the eosin staining test and the percentage of abnormal forms, concentration, and kinetic parameters (CASA program) were assessed, observing a minimum of 200 cells in each of the 3 replicates that were analyzed from each ejaculate. In the CASA system settings, a 70% straightness index (STR) was defined for progressive motility, and mean velocities (VAP) of <10 µm/s <25 µm/s <50 µm/s for slow, medium, and fast SPZ, respectively. From these analyses, 3 males of high and 3 of low seminal quality were chosen for Seminal Plasma-EVs (SP-EVs) isolation and their miRNA content identification.