Overcrowding Stress in Livestock Production Alters Gut Microbiota Composition and Neuronal Nitric Oxide Synthase (nNOS) Expression in nNOS-HiBiT Knock-in Mouse Model

Abstract Overcrowding stress in livestock farming is a significant concern for animal health and livestock products such as meats, milk, and eggs. It affects gut health by altering microbiota and regulating neuronal nitric oxide synthase (nNOS). This study aimed to investigate the effects of overcrowding stress on the gut microbiota composition and nNOS expression. We generated an nNOS-HiBiT knock-in mouse model using the HiBiT system, a highly sensitive tool for accurately quantifying gene expression. Overcrowding stress was induced by housing twenty mice per cage (MPC20) and compared with a control group of two mice per cage (MPC2). Overcrowding stress increases nNOS levels in the hypothalamus and ileum and serum corticosterone levels. Gut microbial composition differed between the control and overcrowding stress-induced groups in the ileum, cecum, and colon. Specifically, Bifidobacterium and Akkermansia decreased in all three regions of MPC20, whereas Helicobacter in the ileum and colon and Parasuterella in the cecum increased in MPC20. Notably, Bifidobacterium consistently decreased when nNOS and corticosterone expression were used as covariates under overcrowding stress. These regional variations reflect the differential impact of overcrowding stress on the intestinal tract, indicating complex interactions through nNOS expression within the brain-gut-microbiome axis. Importantly, the addition of probiotic feed, particularly those containing Bifidobacterium, may counteract these decreases, leading to enhanced gut health and improved quality of livestock food products. This study enhances our understanding of the correlation between overcrowding stress and the gut microbiota, providing valuable data for improving the management environment in livestock farming.