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Despite pigs\u2019 high relevance as a biomedical model, many aspects of EV biology remain poorly understood. EVs from boar seminal plasma (SP\u2010EVs) are relatively well studied, whereas epididymosomes remain largely uncharacterized. Therefore, this study aimed to isolate and characterize EVs from the caput, corpus and cauda regions of boar epididymis, as well as from SP, with their further precise analysis towards their interaction with sperm. We successfully obtained EVs from all studied fluids with sufficient purity. Importantly, our isolation protocol preserved the EVs\u2019 ability to interact with sperm, demonstrated by lipophilic dye staining and biotin labelling experiments, confirming precisely their interaction and cargo transfer to sperm cells. Well\u2010established EV markers, such as Alix and tetraspanins, were detected in the EVs, and additionally, phosphorylated, ubiquitinated and sialylated proteins were uniquely identified. 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