Metagenomic and Metabolomic Analysis of Intestinal Excrement Differences Between Natural Hatching and Artificial Peeling out of the Shell in Nipponia nippon

The Nipponia nippon is a critically endangered species, and its breeding efforts are of vital importance for its conservation. Although artificial shell removal is sometimes employed in current breeding programs to increase survival rates, it may also have unknown impacts on chicks’ development. To investigate the influence of artificial shell removal on the gut microbiota composition in Nipponia nippon, metagenomic sequencing and untargeted LC-MS/MS analyses were performed. Samples from the early, mid, and late stages of natural hatching (ZE, ZM, ZL) and artificial shell removal (RE, RM, RL) were compared. Results indicated that the natural hatching groups formed a unique, highly diverse, and stable community by the late stage (ZL). Conversely, artificial peeling caused the microbial community succession to stagnate at an intermediate state. The RL group experienced a sharp decline in alpha diversity and a significant enrichment of opportunistic pathogens, such as Edwardsiella, Clostridium, and Fusobacterium. Functionally, the microbial community in the RL group remained in a stage of expanding basic functions rather than reaching an advanced equilibrium state. Metabolomic analysis confirmed this developmental arrest, revealing abnormal accumulations of organic acids, such as citric acid, and indole derivatives in the RL group. This indicates metabolic dysregulation, stress, and altered microbial–host chemical signaling. Furthermore, the significant biomarker Edwardsiella was strongly correlated with multiple differential metabolites in the RL group. Ultimately, these results indicate that artificial peeling intervention disrupts environmental adaptation and induces metabolic alterations in the intestinal development of the Nipponia nippon chicks.