Isolation and characterization of seminal extracellular vesicles subsets and their impact on sperm freezability in stallions

The ability of spermatozoa to withstand cryopreservation differs between stallions. While the underlying mechanisms of these differences are not fully understood, seminal plasma (SP) may play a crucial role in modulating sperm cryotolerance. Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs), nanometer-sized membrane particles that can transfer biomolecules to sperm modulating their function. This study aimed to isolate and characterize two sized-sEV subsets-small (S-) and large (L-)-from stallion SP, and to evaluate their involvement in sperm freezability. Seminal plasma samples (n = 10) were collected from 10 stallions, which were divided according to sperm freezability into: good (GFE, n = 5) and poor freezability ejaculates (PFE, n = 5). The sEV subsets were isolated using a size-exclusion-chromatography (SEC)-based procedure, and characterized according to MISEV2023, including morphology, concentration, particle size distribution, purity (albumin assessment) and presence of canonical EV-markers (CD9, CD63, and CD81, and HSP70). Results showed that stallion SP contains a heterogeneous population of sEVs, with S- and L-sEVs exhibiting distinct phenotypic and compositional profiles. The S-sEVs displayed a higher expression (P<0.01) of CD9 and HSP70 (mean±SD; 47.0%±10.5% and 51.0%±6.5%, respectively) than in L-sEVs (35.7%±7.5% and 39.0%±1.8%, respectively), suggesting functional differences between subsets. Interestingly, GFE contained higher (P<0.05) CFSE+ particles (intact sEVs) in S- and L-sEVs (66.4%±13.4% and 72.2%±5.0%, respectively) compared to PFE (45.1%±7.8% and 37.8%±9.5%, respectively). In conclusion, our study establishes an SEC-based procedure suitable for isolating two sized-sEV subsets in SP stallions that differ in the canonical EV-markers expression. The higher abundance of intact sEVs in GFE suggests their involvement in sperm freezability.