Hormetic dose-response of pure hydroxytyrosol in laying hens: Optimal dose for egg shelf-life extension and yolk docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid concentration

• Pure hydroxytyrosol (HT) shows a hormetic dose-response in late-phase laying hens. • A 20 mg/kg dose maximizes yolk DHA and preserves internal egg quality. • HT linearly reduces yolk lipid peroxidation (TBARS) for 30 days at 22°C. • Supranutritional doses (50 mg/kg) impair intestinal morphology and egg quality. • Multivariate analyses (PCA/HCPC) identify 20 mg/kg as the optimal biological dose. Hydroxytyrosol (HT) is a potent olive-derived antioxidant, but its dose-response effects on laying hen performance, gut health, and egg quality remain poorly defined. This study evaluated the effects of dietary supplementation with pure HT (0, 5, 10, 20, and 50 mg/kg) on productive performance, intestinal morphometry, oxidative stability, and egg quality in ISA Brown hens at 80 weeks of age. Performance traits and serum biochemical indicators were not influenced by HT supplementation ( P > 0.05). Regarding intestinal morphometry, villus height decreased linearly with increasing HT ( P < 0.001), suggesting a physiological threshold at higher doses. For egg quality, HT reduced yolk TBARS linearly after 30 days at 22 ± 4°C ( P < 0.001), while Haugh units showed a quadratic, hormetic response with best preservation at 10–20 mg/kg ( P < 0.001) and lower efficacy at 50 mg/kg. Docosahexaenoic acid (DHA) content in yolk was highest at 20 mg/kg ( P = 0.032), with a quadratic response for eicosapentaenoic acid (EPA) concentration (Quadratic effect: P = 0.016). Multivariate analysis (PCA/HCPC) confirmed distinct metabolic profiles and identified 20 mg/kg as the optimal biological threshold. In conclusion, 20 mg/kg pure HT is the optimal dose to extend egg shelf life and enrich eggs with n-3 PUFAs, whereas 50 mg/kg exceeds the physiological threshold, limiting intestinal morphology and egg quality. These results provide a clear dose recommendation for producing functional eggs with enhanced oxidative stability and DHA content under non-refrigerated storage and are particularly relevant for egg production systems in tropical and subtropical regions where non-refrigerated storage is common.