Multiple stressors, including cold exposure, disrupt growth and intestinal homeostasis via TLR4/p38MAPK/NF-κB and...

Chronic and multifactorial stressors, including low-temperature exposure, represent major challenges in modern poultry systems. This study evaluated the effects of a chronic unpredictable mild stress (CUMS) model on growth performance and intestinal homeostasis in layer chicks. A total of 300 one-day-old chicks were randomly assigned to either a control (CON) group or CUMS group for a 5-week trial. CUMS significantly reduced body weight (BW) and feed intake (ADFI) while increasing the feed conversion ratio (F/G) (P < 0.05). Serum biochemical alterations indicated disrupted protein and lipid metabolism and elevated liver enzyme activity. CUMS markedly increased serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels (P < 0.05). Histological, ultrastructural and immunofluorescence analyses showed villus damage, reduced goblet cells, disrupted tight-junction proteins, and notable mitochondrial abnormalities, including swollen mitochondria, loss of cristae integrity, and increased ROS accumulation; ATP concentrations were significantly reduced across intestinal segments (P < 0.05). Mechanistically, CUMS suppressed humoral immunity and activated TLR4/p38MAPK/NF-κB signaling with increased pro-inflammatory and decreased anti-inflammatory cytokines (P < 0.05). Oxidative stress was evidenced by increased malondialdehyde (MDA) and reduced antioxidant enzyme activities (P < 0.05), alongside inhibition of the Nrf2/HO-1 pathway. Apoptosis was enhanced via mitochondrial and death receptor pathways, with reduced Bcl-2/Bax ratios and elevated cleaved Caspase-3 (P < 0.05). Additionally, the AMPK/SIRT1/PGC-1α pathway and mitochondrial biogenesis-related genes were significantly suppressed (P < 0.05). Overall, multi-stressor CUMS model compromises intestinal homeostasis and growth through convergent inflammatory, oxidative, apoptotic, and mitochondrial mechanisms.