Seminal extracellular vesicles and sperm freezability in Stallions

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

Anim Reprod Sci. 2026 Feb 26:289:108160. doi: 10.1016/j.anireprosci.2026.108160. Online ahead of print.
Authors
Isabel Barranco 1 , Pablo Martínez-Díaz 1 , Ana Parra 1 , Judith Diaz-Muñoz 2 , Patricia Panales 1 , Sergi Bonet 3 , Jordi Roca 4 , Jordi Miró 5 , Jaime Catalán 3
Affiliations

1 Department of Medicine and Animal Surgery, Faculty of Veterinary Science, University of Murcia, Murcia, Spain; International Excellence Campus for Higher Education and Research “Campus Mare Nostrum”, Institute for Biomedical Research of Murcia (IMIB-Arrixaca), University of Murcia, Spain; EV-lab, Faculty of Veterinary Science, University of Murcia, Murcia, Spain.
2 Unit of Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, Bellaterra, Cerdanyola del Vallès, Spain.
3 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain; Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, Girona, Spain.
4 Department of Medicine and Animal Surgery, Faculty of Veterinary Science, University of Murcia, Murcia, Spain; International Excellence Campus for Higher Education and Research “Campus Mare Nostrum”, Institute for Biomedical Research of Murcia (IMIB-Arrixaca), University of Murcia, Spain; EV-lab, Faculty of Veterinary Science, University of Murcia, Murcia, Spain. Electronic address: roca@um.es.
5 Unit of Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, Bellaterra, Cerdanyola del Vallès, Spain. Electronic address: jordi.miro@uab.cat.

PMID: 41764942
DOI: 10.1016/j.anireprosci.2026.108160

Abstract

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.

Keywords: Cryopreservation; Extracellular vesicles; Horse; Seminal plasma; Sperm.

Copyright © 2026 The Authors. Published by Elsevier B.V. All rights reserved.
Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no conflicts of interest. The funders had no influence on the contents of the manuscript.