Changes in pathogenicity of gut microbiota during fasting-induced molting in laying hens and their impact on spleen immune function.

Fasting-induced molting (FIM) leverages the natural molting mechanism of aging laying hens to enhance their egg production and egg quality. However, fasting may also increase the risk of pathogen infection and activate immune regulation in the spleen. Understanding the specific types of pathogen infections triggered during FIM and their impacts on the spleen's immune regulation mechanisms is crucial for enhancing the immunological resilience of poultry. A total of 90, aged 60 weeks, late-laying hens were randomly assigned to undergo FIM. During FIM, hens kept regular feed and 16 hours light initially. A 3-day fast with 8 hours light followed. Then, they gradually resumed water and feed, increasing from 30 g to 120 g daily, with light returning to 16 hours. This study investigates the effects of FIM on the gut microbiota and metabolites through metagenomic sequencing and metabolomics. Additionally, blood routine tests and ELISA assays were conducted to measure serum Ig concentrations. Real-time quantitative PCR and ELISA were used to detect the expression of inflammatory factors and immune cell markers in the spleen. The study also analyzed gene expression levels in the PI3K/AKT pathway, systematically exploring fasting's regulatory impact on gut-spleen immunity. Results showed that during FIM, the abundance of pathogens utilizing non-carbohydrates as an energy source initially increased and then decreased, while the abundance of pathogens relying on carbohydrates as an energy source declined. Correspondingly, with beneficial metabolites initially decreased before increasing, while harmful metabolites exhibited the opposite trend. During fasting, the concentration of eosinophilic leukocytes in serum significantly increased (P < 0.05), while the concentration of IgA significantly decreased (P < 0.05). In the spleen, significant increase in the expression of immune-related metabolites, including NF-κB, IFN-β, iNOS, IL-1β, and IFN-α, were observed, along with significant increases in the secretion of TNF-α, TGF-β, IL-1, and IL-8, suggesting a significant activation of the immune response. After refeeding, these factors in serum and spleen returned to pre-fasting levels. Notably, the PI3K/AKT signaling pathway was significantly activated during fasting, indicating that fasting exerts broad effects on the immune system via the PI3K/AKT pathway. This study holds significant theoretical and practical value for enhancing the immunity of molting laying hens, reducing pathogenic bacterial infections, and optimizing production performance.