Exploring the "Microbe-Metabolite-Gene" regulatory mechanism of compound probiotics on antioxidant function in heat-stressed broilers

Nutritional modification strategies have become pivotal in addressing heat stress in poultry farming. Probiotics are increasingly recognized as a sustainable additive by researchers. The enhancement of antioxidant capacity is critical for improving the overall health and productivity of broilers. However, the molecular mechanisms by which probiotics influence antioxidant functions in heat-stressed broilers remain unclear. Consequently, this study raised 400 one day old Arbor Acres broilers until 28 d. Among them, 300 broilers, showing no significant weight differences (P > 0.05), were randomly divided into three groups (control, heat stress, and heat stress with probiotics), each consisting of five replicates with 20 broilers per replicate. The heat stress conditions were maintained at 32 ± 1°C from 9:00 to 17:00, and at 21 ± 1°C during the remaining hours, identical to the control conditions. The heat stress with probiotics group received a basal diet supplemented with 10 g/kg of compound probiotics (Lactobacillus casei: Lactobacillus acidophilus: Bifidobacterium at a ratio of 1:1:2). All groups had ad libitum access to food and water over the 14-day experimental period. Results indicated that the compound probiotics enhanced growth performance and antioxidant capacity, increasing levels of glutathione peroxidase and catalase. Weighted correlation network analysis (WGCNA) identified Hub genes (e.g., PANK2, FAM167A, ABCG8, DYDC2), Hub metabolites (e.g., l-glutamine, ornithine), and Hub microorganisms (e.g., Burkholderia, Macromonas) that regulate antioxidant functions in heat-stressed broilers. By integrating the WGCNA results, we constructed a "microbe-metabolite-gene" regulatory network centered around co-enriched pathways, illustrating the interrelationships between molecules. Notably, NT5C1A, GDA,l-glutamine, and guanosine were notably enriched in purine metabolism, whereas ABCG8,l-arginine, allose, and deoxyguanosine were prominently enriched in ABC transporters pathways, highlighting their crucial involvement in orchestrating the antioxidant response to heat stress. This study elucidates the molecular mechanisms by which compound probiotics enhance antioxidant functions in heat-stressed broilers, offering a theoretical foundation for probiotic applications in poultry.