Seminal extracellular vesicles (EVs) include exosomes (ø 40–120 nm) and microvesicles (MVs, ø 120–1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs ( P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher ( P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.

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Background: Boar seminal plasma is rich in cytokines, which could influence the capability of spermatozoa to tolerate preservation.Objectives: To evaluate the involvement of boar seminal plasma cytokines in the changes experienced by boar spermatozoa during their storage, either in liquid or frozen state.Materials and Methods: In two separated experiments, semen samples from healthy and fertile boars were split in two aliquots, one centrifuged twice (1,500 ×g for 10 min) to harvest seminal plasma, whereas the other was either commercially extended (3 × 107 sperm/mL) and liquid-stored at 17°C during 144 h (n = 28, Experiment 1) or frozen-thawed using a standard 0.5 mL protocol (n = 27, Experiment 2). Sixteen cytokines were quantified using Luminex xMAP®. Sperm attributes (CASA-evaluated total and progressive motility; flow cytometry-evaluated sperm viability, production of intracellular H2O2 and O2•- and levels of lipid peroxidation in viable spermatozoa) were evaluated either at 0, 72, or 144 h of liquid storage (Experiment 1) or before freezing and at 30- and 150-min post-thawing (Experiment 2).Results: Multiple linear regression models, with Bayesian approach for variable selection, revealed that the anti-inflammatory TGF-β2, TGF-β3, IL-1Ra, and IL-4 and the pro-inflammatory IL-8 and IL-18, predicted changes in sperm motility for liquid-stored semen while the anti-inflammatory IFN-γ was included in the models predicting changes in all sperm attributes for cryopreserved semen.Conclusion: Specific boar seminal plasma cytokines would contribute to modulate the structural and metabolic changes shown by spermatozoa during preservation, either in liquid or frozen state.

Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein–protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.