Abstract
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.