The Influence of Acute Cold Stress on Intestinal Health of the Juvenile Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

Sharp declines in temperature pose a significant risk for mass mortality events in the Chinese soft-shelled turtle (<i>Pelodiscus sinensis</i>). To assess the effects of acute cold stress on intestinal health, turtles were exposed to temperatures of 28 °C (control), 14 °C, and 7 °C for 1, 2, 4, 8, and 16 days. The results showed that acute cold stress at 14 °C and 7 °C induced time-dependent alterations in intestinal morphology and histopathology. The damage was more severe at 7 °C, characterized by inflammatory cell infiltration, lymphoid hyperplasia, and extensive detachment and necrosis across the villi, muscle layer, and submucosa. 16S rDNA sequencing revealed significant shifts in intestinal microbiota composition in the 7 °C group, dominated by <i>Helicobacter</i> and <i>Citrobacter</i>. Transcriptomic analysis identified differentially expressed genes (DEGs) that respond to acute cold stress and are involved in the Toll-like receptor signaling pathway (<i>Tlr2</i>, <i>Tlr4</i>, <i>Tlr5</i>, <i>Tlr7,</i> and <i>Tlr8</i>), the NOD-like receptor signaling pathway (<i>Traf6, Traf2</i>, <i>Casr</i>, <i>Rnasel</i>, <i>Pstpip1</i>, <i>Plcb2</i>, <i>Atg5</i>, and <i>Mfn2</i>), apoptosis (<i>Tuba1c</i>, <i>Ctsz</i>, <i>Ctsb</i>, <i>Kras</i>, <i>Hras</i>, <i>Pik3ca</i>, <i>Bcl2l11</i>, <i>Gadd45a</i>, <i>Pmaip1</i>, <i>Ddit3</i>, and <i>Fos</i>), and the p53 signaling pathway (<i>Serpine1</i>, <i>Sesn2</i>, <i>Ccng2</i>, <i>Igf1</i>, <i>Mdm2</i>, <i>Gadd45a</i>, <i>Pmaip1</i>, and <i>Cdkn1a</i>). Metabolomic profiling highlighted differentially expressed metabolites (DEMs) that cope with acute cold stress, such as organic acids (oxoglutaric acid, L-aspartic acid, fumaric acid, DL-malic acid, and citric acid) and amino acids (including L-lysine, L-homoserine, and allysine). The integrated analysis of DEGs and DEMs underscored three key pathways modulated by acute cold stress: linoleic acid metabolism, neuroactive ligand-receptor interaction, and the FoxO signaling pathway. This study provides a comprehensive evaluation of intestinal health in Chinese soft-shelled turtles under acute cold stress and elucidates the underlying mechanisms.