Investigating the role of the bacterial metabolite ectoine on immune regulation within the bovine female reproductive tract.

Ectoine, a compatible solute produced by extremophilic bacteria, is widely used for mucosal protection due to its cytoprotective and anti‑inflammatory properties. However, its effects within the female reproductive tract and its interaction with sperm‑associated immune responses remain unknown. This study provides the first assessment of ectoine's immunomodulatory activity in bovine cervical and uterine tissues and its influence on sperm-polymorphonuclear neutrophil (PMN) interactions. Cervical and uterine explants from follicular‑phase heifers (n = 5) were pre‑incubated with 0, 0.5, 5, or 50 mM ectoine prior to stimulation with lipopolysaccharide (LPS) or sperm. Cytokine concentrations (IL‑8, IL‑6, and IL‑1β) were measured by ELISA, and sperm-PMN binding and sperm motility following PMN co‑incubation were evaluated. Baseline cytokine secretion was higher in uterine versus cervical explants (P < 0.05), indicating distinct tissue‑specific immune profiles. Ectoine alone did not alter basal cytokine secretion (P > 0.05). LPS increased IL‑8, IL‑6, and IL‑1β secretion from cervical explants and IL‑8 and IL‑1β in uterine explants (P < 0.05), and ectoine did not modify these responses (P > 0.05). Sperm elevated IL‑1β release from uterine explants (P < 0.01), while IL‑6 and IL‑8 remained unchanged; this response was also unaffected by ectoine (P > 0.05). Ectoine did not influence sperm-PMN binding or sperm motility in the presence of PMNs (P > 0.05). Collectively, this study provides the first reproductive‑tract safety evaluation of ectoine, demonstrating that it does not disrupt constitutive or stimulus‑induced inflammatory signaling nor interfere with sperm-immune interactions. These findings support ectoine's safe incorporation into semen preservation and assisted reproductive technologies.