Identification of specific miRNAs in the seminal plasma extracellular vesicles from fertile and subfertile rabbit bucks. Identification of specific miRNAs in the seminal plasma extracellular vesicles from fertile and subfertile rabbit bucks

In 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; p2, 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.