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. SP 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˂0.05) 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. • Horse seminal plasma harbors a heterogeneous extracellular vesicles (sEVs) population. • Small and large sEVs display distinct phenotypic and compositional profiles. • Small sEVs exhibit higher expression of CD9 and HSP70 than large sEVs. • Ejaculates with good freezability contain a higher proportion of intact sEVs. • Seminal EVs may play a role in cryotolerance of horse sperm.
Barranco et al. (Sun,) studied this question.