Seminal plasma extracellular vesicles modulate low-fertility bull sperm function beyond miRNA cargo transfer

Bull fertility is a multifactorial trait influenced by molecular determinants beyond conventional semen parameters. Seminal plasma extracellular vesicles (EVs) mediate intercellular communication and may modulate sperm function through the transfer of regulatory cargo, including microRNAs (miRNAs). This study investigated (i) differences in miRNA profiles of spermatozoa and seminal plasma EVs from proven- and low-fertility Holstein-Friesian bulls, and (ii) the functional and molecular effects of co-incubating low-fertility spermatozoa with EVs derived from proven- or low-fertility bulls. Extracellular vesicles were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. Sperm kinetics were assessed by computer-assisted sperm analysis. Small RNA sequencing was performed to profile miRNA cargo in spermatozoa and EVs. Although conventional semen parameters did not significantly differ between fertility groups, EV concentration was significantly higher in proven-fertility bulls. Co-incubation of low-fertility spermatozoa with seminal plasma EVs from proven-fertility bulls was associated with a distinct kinematic profile compared with untreated spermatozoa and spermatozoa incubated with EVs from low-fertility bulls. miRNA profiling identified 16 differentially expressed miRNAs between proven- and low-fertility spermatozoa, whereas EV miRNA cargo did not differ significantly between groups. Notably, incubation alone was associated with a larger number of miRNA changes, while co-incubation with proven-fertility EVs was associated with fewer changes in the sperm miRNA profile. These findings suggest that the observed effects of seminal plasma EVs on low-fertility bull spermatozoa cannot be explained by fertility-specific EV miRNA cargo and may involve other EV-associated mechanisms not investigated in the present study.