Mechanism of low temperature-induced intestinal damage in Danio rerio and the mitigating effect of alanylglutamine

Recent cold waves and abrupt seasonal temperature drops have significantly impacted aquaculture, slowing fish growth and causing mass mortality. The intestine plays a crucial role in digestion, nutrient absorption, metabolism, and physiological regulation. However, studies on the molecular mechanisms of fish intestinal response to low-temperature stress and methods to mitigate damage are limited. This study investigated the intestinal response of Danio rerio to low temperatures with three groups: a control group at 28 ​°C (CT28), a cryogenic group at 18 ​°C (ST18), and an extreme cryogenic group at 10 ​°C (LT10), with time points of 0, 12, 24, and 48 ​h. Results showed significant structural changes in the 18 ​°C group, with inflammatory infiltration starting at 24 ​h. Damage worsened in the 10 ​°C group, showing severe inflammation. Tunel staining revealed increased cell apoptosis with lower temperatures and longer exposure, peaking at 10 ​°C and 48 ​h following exposure (p ​< ​0.05). Alcian blue periodic acid-schiff stain revealed that lower temperatures promote an increase in the number of goblet cells. Metabolomics analysis compared intestinal metabolites between CT28 vs. ST18 and CT28 vs. LT10. Fifty-three metabolites were shared between the CT28_ST18 and CT28_LT10 groups in positive ion mode, and 16 in negative mode, with 22 and 9 metabolites showing linear changes. These 31 metabolites could serve as potential indicators for low-temperature stress in D. rerio. Specifically, eight metabolites from the lysophosphatidylcholine (LPC) group increased significantly, while antioxidant alanylglutamine (AGD) decreased. Supplementing the diet with 0.5% AGD reduced intestinal damage, and 1.0% AGD improved low-temperature tolerance. This study provides new insights into the molecular mechanisms of fish response to cold stress and lays the foundation for future research.