Background: Heat stress (HS) poses a significant challenge to poultry production, causing oxidative stress, immunosuppression, and economic losses. N-acetylcysteine (NAC), exhibits antioxidant and anti-inflammatory properties, but its effects on immune organs and underlying epigenetic mechanisms in heat-stressed broilers remain unexplored. Methods: Forty male Arian broiler chicks were randomly assigned to four groups (n=10): control (thermoneutral), NAC (thermoneutral + 2 g/kg NAC), HS (cyclic heat stress at 34°C for 7 h/day), and HS+NAC (heat stress + 2 g/kg NAC). After 18 days, spleen, bursa of Fabricius, and thymus were collected for histological examination and HSP70 immunofluorescence. Serum was analyzed for oxidative stress biomarkers (MDA, SOD, GSH-Px, TAC), stress hormones (corticosterone, T3, T4), inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10), and miR-34a-5p expression. Results: Heat stress induced significant lymphoid depletion, follicular atrophy, and cortical thinning in immune organs, accompanied by elevated HSP70 expression, increased MDA and corticosterone, decreased TAC and T3, and upregulated pro-inflammatory cytokines and miR-34a-5p. NAC supplementation preserved immune organ histo-architecture, attenuated HSP70 overexpression, reduced MDA and corticosterone, restored TAC and T3, suppressed pro-inflammatory cytokines while enhancing IL-10, and downregulated miR-34a-5p expression (p < 0.05). Conclusions: Dietary NAC at 2 g/kg effectively mitigates heat stress-induced immune organ damage through integrated antioxidant, anti-inflammatory, hormonal, and epigenetic mechanisms, positioning NAC as a promising nutritional strategy for sustainable poultry production under heat stress conditions.
