Abstract
High-stocking-density (HD) environments can trigger systemic inflammatory responses, consequently impairing broiler growth. Given the broad anti-inflammatory properties of aspirin eugenol ester (AEE), this study investigated the effects of AEE supplementation on growth performance, immune organ indices, serum immunoglobulin levels, and hypothalamic inflammation-related markers in HD broilers. A total of 528 one-day-old male Arbor Acres (AA) broilers were randomly assigned to four groups: ND, HD, ND-AEE, and HD-AEE (ND, 14 birds/m2; HD, 22 birds/m2), with six replicate cages per treatment group over a 42-day experimental period. The results revealed that AEE significantly improved the growth performance of HD broilers. Immune organ indices, serum immunoglobulin levels, and the expression of spleen inflammatory factors was associated with the organismal inflammatory response, which manifested primarily during the late growth phase. On Day 35, AEE significantly suppressed (p < 0.05) the relative mRNA expression of p21-activated kinase 1 (PAK1) in the hypothalamus of HD broilers. On Day 42, AEE significantly reduced the relative mRNA expression of PAK1, p38 mitogen-activated protein kinase (p38MAPK), cyclooxygenase-2 (COX-2), prostaglandin E synthase 1 (mPGES-1), and interleukin-1β (IL-1β) (p < 0.05), while significantly elevating the relative mRNA expression of growth hormone-releasing hormone (GHRH) (p < 0.05). Collectively, these findings demonstrate that AEE mitigates high-density rearing-induced hypothalamic inflammation and is associated with downregulated mRNA expression of PAK1 and its downstream targets in the p38MAPK/COX-2 axis. This gene expression profile correlates with improved growth and immune function in high-density-stressed broilers, suggesting a potential regulatory link that requires further validation at the protein and functional levels.