Ammonia nitrogen is a common contaminant in aquatic environments, and its potential toxicity to organisms has attracted extensive attention. However, few studies have comprehensively evaluated the negative impacts of ammonia stress on cold-water fish. In this study, liver, gill, and intestine specimens of Amur grayling (Thymallus grubii) from three treatment groups (control (0 mg/L), low ammonia (43.683 mg/L), and high ammonia (436.8 mg/L)), were collected for histological observation, biochemical examination, and transcriptomic, metabolomic, and intestinal microbiome analysis. Our results showed that excessive ammonia nitrogen blocked the normal immune function and compromised the integrity of liver and gill tissues through oxidative stress-mediated differential cell death pathways. Meanwhile, the multi-omics analysis revealed that ammonia exposure predominantly altered the carbohydrate, lipid, and amino acid metabolism modes. In addition, it was also demonstrated that ammonia nitrogen stress affected the composition of intestinal microbiota taxa. This study provides insights into the potential risks and hazards of ammonia stress on cold fish in natural waters and provides a reference for the environment control of the water quality in aquaculture.

Cold-water fish, particularly stenothermal species, are increasingly affected by rising temperatures driven by global warming. To explore the hepatic metabolism mode of Amur grayling under warming, Thymallus grubii was selected as the experimental model. Here, we measured the oxygen consumption rate (MO₂), energy metabolism enzymes, and transcription profiling in Thymallus grubii that was exposed to increased temperatures (9, 12, 15, 18, 21, and 24 °C) with the same flow velocity (34 cm/s). In this study, MO₂ initially increased and then decreased with rising temperature, with peak sensitivity between 12 and 15 °C (Q₁₀ = 5.30). Hemoglobin increased significantly at 12–18 °C but decreased in the 18–24 °C group (p < 0.05). Additionally, hepatic glycogen content (the amount of stored sugar in the liver, which serves as an energy reserve) also first increased and then decreased markedly (p < 0.05). Lactic acid in plasma and muscle contents increased, but creatine phosphate and glucose levels significantly decreased (p < 0.05). The result of transcriptome analysis showed that individuals in the 15 °C group could supply energy through glucose and amino acid metabolism. In contrast, individuals under 21 °C exposure could mainly supply energy through the lipid metabolism pathway. Our study underscores the vulnerability of Amur grayling to environmental temperature and identifies the instantaneous metabolic limit range, providing numerical limits (e.g., maximum river temperature) that managers can use to protect wild populations.