Eosinophils from jennies form extracellular traps (EETs) in vitro in response to seminal plasma: A new perspective on uterine immune defense in donkeys

Jennies exhibit a post-insemination uterine inflammatory response characterized by the infiltration of polymorphonuclear cells (PMNs), primarly neutrophils, followed by eosinophils and basophils. Neutrophils are known to release neutrophil extracelular traps (NETs) to capture spermatozoa. Recently, eosinophil extracellular traps (EETs) have been identified in humans, and eosinophil infiltration into the uterus has been reported in jennies during estrus and diestrus, increasing after semen exposure. This study aimed to develop an in vitro model to evaluate EET formation in response to semen components. PMNs were isolated from jenny peripheral blood, using a standardized protocol. Eosinophils were separated using spectral flow cytometry cell sorter. Semen from three fertile jackasses was used to prepare treatments, including two negative controls (RPMI and Kenney solution), a positive control (A23187), and four experimental groups: seminal plasma (SP), spermatozoa (SPZ)-Kenney (K), SPZ-SP, SPZ-K-SP. Samples were analyzed at 0 and 3 h of incubation. EET formation was assessed by measuring changes in eosinophil nuclear fluorescence, stained with SYTOX Green, using a spectral flow cytometer. Confocal microscopy confirmed EET structures. At time 3, most of the analyzed parameters differed significantly (p < 0.05) between the treatment with seminal plasma and the negative control (RPMI). Notably, cell size already showed significant differences at time 0 (p < 0.05). A similar but less pronounced response was observed in the SPZ-SP and SPZ-K-SP groups, likely due to dilution of seminal plasma. No significant effect was seen with spermatozoa alone. These findings suggest that eosinophils rapidly release extracellular traps in response to seminal plasma, potentially modulating the post-insemination uterine immune response.