Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography–high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

Context: Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims: To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods: poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results: poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P < 0.05). Conclusions: poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm–oocyte interaction. Implications: The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm–oocyte interaction.

This systematic review examined research studies on extracellular vesicles (EVs) of the male reproductive tract in livestock species to summarize the research topics and methodologies used, key findings, and future directions. PubMed and Scopus were searched for time ranges up to 1 September 2024, and 1383 articles were identified. The application of screening and eligibility criteria resulted in the selection of 79 articles focusing on male reproductive EVs in livestock. Porcine and bovine male reproductive EVs were the most studied. A variety of EV isolation techniques were used, with ultracentrifugation being the most common. Characterization of male reproductive EVs in livestock was a weak point, with only 24.05% of the articles characterizing EVs according to MISEV guidelines. Inadequate characterization of EVs compromises the reliability of results. The results of 19 articles that provided a good characterization of EVs showed that male reproductive EVs from livestock species are phenotypically and compositionally heterogeneous. These papers also showed that these EVs would be involved in the regulation of sperm functionality. Research on male reproductive EVs in livestock species remains scarce, and further research is needed, which should include appropriate characterization of EVs and aim to find efficient methods to isolate them and assess their involvement in the functionality of spermatozoa and the cells of the female genital tract.

Seminal plasma (SP) contains a heterogeneous population of extracellular vesicles (EVs) recognized as key modulators of sperm function. However, the specific functional roles of each seminal EV (sEV) subset remain poorly understood. This study aimed to evaluate the interaction of two sized sEV subsets (small [S-sEVs] and large [L-sEVs]) with pig liquid-stored spermatozoa under different pH conditions and their effect on specific sperm functional parameters. Seminal EV subsets were isolated from SP samples using size exclusion chromatography and characterized following the MISEV2023 guidelines. Semen samples were incubated with each sEV subset or without sEVs (control) for 6 h at 37 ºC, 100 % humidity and 5 % CO₂ under different pH conditions (6.5, 7.0, or 7.5). Sperm functional parameters were assessed by flow cytometry (Cytoflex®S and LX, Beckman Coulter), under capacitating and non-capacitating conditions. Confocal microscopy revealed that both sEV subsets bound to and were internalized by spermatozoa as early as 30 min after incubation, regardless of pH. Flow cytometry revealed that both sEVs decreased reactive oxygen species production (P ≤ 0.0001), mitochondrial membrane potential (P ≤ 0.0001) and mitochondrial O₂^•⁻ levels (P ≤ 0.01) and increased apoptosis (active caspase-3) in viable spermatozoa (P ≤ 0.0001). However, the influence of sEV on acrosome integrity in viable sperm was time- and condition-dependent (P ≤ 0.05). This study showed that both S- and L-sEVs interact with porcine spermatozoa across a range of physiological pH conditions. This interaction is reflected by decreased oxidative stress and mitochondrial activity, as well as increased apoptosis in spermatozoa.

Background: Predicting male fertility in farm animals remains a challenge. Seminal plasma (SP) contains a high amount of heterogeneous seminal extracellular vesicles (sEVs), believed involved in reproductive processes and maybe key to understanding male fertility. Aims: To identify the sEV proteins that are differentially expressed between more and less fertile boars and that could be candidates for fertility biomarkers in boars used in artificial insemination (AI) programs. Materials and methods: Small (S) and large (L) sEV subsets from SP samples of AI boars with differences in fertility: high (H) or low (L) farrowing rate (FR) and large (L) or small (S) litter size (LS). The S‐ and L‐sEV subsets were isolated by size exclusion chromatography and characterized according to the Minimal Information for Studies of Extracellular Vesicles (MISEV2023) guidelines. Proteomic analyses (three biological replicates per fertility group and sEV subset) were performed using a Bruker timsTOF fleX™ instrument with data‐independent acquisition parallel accumulation‐serial fragmentation (diaPASEF) technology. Results: A total of 470 and 726 proteins were quantified in S‐sEVs and 1801 and 1834 proteins in L‐sEVs from FR and LS boars, respectively. Differentially expressed sEV proteins (log ₂ fold change ≥±1, p ≤ 0.05 and effect size d of Cohen >2.0) were found between the fertility groups: seven in S‐sEVs and 52 in L‐sEVs between H‐FR and L‐FR boars, and 47 in S‐sEVs and 52 in L‐sEVs between L‐LS and S‐LS boars. Many of these differentially expressed sEV proteins are involved in reproductive processes, particularly in sperm function and sperm‐zona pellucida binding, but also in embryo development and implantation. Conclusions: The sEV proteome differs between more and less fertile boars, with many of the differentially expressed proteins known as involved in reproductive processes. This would suggest that sEVs may be involved in male fertility and that some of the differentially expressed sEV proteins could be potential fertility markers for AI boars.

Aim: Pig seminal plasma (SP) contains oxytocin (OXT) at levels associated with the fertility outcomes of boars used for artificial insemination (AI). However, OXT easily volatilizes when circulating freely, making it difficult to maintain stability in AI seminal doses. The hypothesis is that OXT is stably carried in seminal extracellular vesicles (EVs). This study aimed to determine the following: (1) whether seminal EVs carry OXT and, if so, where they carry it; (2) the source of seminal EVs carrying OXT; and (3) whether the levels of OXT in seminal EVs are associated with the fertility of AI boars. Methods: Seminal EV samples were isolated by size-exclusion chromatography from entire ejaculates and ejaculate fractions: the first 10 mL of the sperm-rich fraction (SRF; SRF-P1), the remainder of the SRF (SRF-P2), and the post-SRF fraction. The OXT concentration was measured using a direct competitive immunoassay with AlphaLISA technology and an anti-OXT monoclonal antibody. Results: Seminal EVs carry OXT, primarily on the outer surface of the EV membrane, likely within the protein corona. The concentration of OXT in seminal EVs varied among ejaculate fractions (P < 0.0001). It was higher in SRF-P2 (3,017.68 ± 860.52 pg/mL of SP) and post-SRF (3,613.27 ± 935.08 pg/mL of SP) EVs than in SRF-P1 (1,675.15 ± 520.62 pg/mL of SP) EVs. The concentration of OXT in seminal EVs was associated with the fertility outcomes of AI boars. Higher concentrations were found in the seminal EVs of boars with a high farrowing rate and in those with a small litter size. Conclusion: Porcine seminal EVs carry OXT outside of membranes, and those originating from the accessory sex glands are particularly enriched. The association of OXT of seminal EVs with fertility is ambivalent: it enhances farrowing rate while potentially reducing litter size, likely via its effects on myometrial contractility, which facilitates sperm transport but may hinder embryo implantation.