A shortage of feed protein resources restricts poultry productivity. Key strategies to alleviate this problem include improvements to the structure of the gut microbiota by the appropriate intake of high-quality protein, improvements to the comprehensive protein utilization rate, and reducing the consumption of protein raw materials. In addition, damage to the environment caused by nitrogen emissions needs to be reduced. The aim of the study was to evaluate the effects of dietary protein levels on laying performance, host metabolism, ovarian health, nitrogen emissions, and the gut microbial structure and function of laying hens. In total, 360 hens at the age of 38 weeks were randomly allotted four treatments. Each of the groups consisted of nine replicates, with 10 birds per replicate, used for 12 weeks of study. Dietary protein levels of the four groups were 13.85 %, 14.41 %, 15.63 %, and 16.30 %. Results revealed that, compared with the 13.85 % crude protein (CP) group, the 15.63 % CP group experienced significantly enhanced final body weight, average daily gain, egg production, and egg mass. Compared with the 16.30 % CP group, the other groups' serum concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) were significantly reduced. Compared with the 16.30 % CP group, the 13.85 % and 15.63 % groups had increased CP utilization rates but reduced nitrogen emission rate, and daily per egg and per kilogram egg nitrogen emissions rose with increased dietary protein levels. Compared to the 13.85 % and 14.41 % CP groups, the 16.30 % CP group exhibited a significant increase in the expression of genes related to amino acids and carbohydrate metabolic pathways. According to the linear discriminant analysis effect size diagram, the predominant bacteria in the 15.63 % CP group (e.g., Subdoligranulum, and Ruminococcaceae_UCG-013) were significantly related to CP utilization. The results of this study emphasize that production performance is significantly reduced when protein levels are too low, whereas too high protein levels lead to gut microbiota imbalance and a reduction in the utilization efficiency of nutrients. Therefore, on the premise of ensuring the health of hens, the structure of the gut microbiota can be improved by appropriately reducing protein levels, which helps to balance the relationships among host health, productivity, resources, and the environment.
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