Enhanced propionate and butyrate metabolism in cecal microbiota contributes to cold-stress adaptation in sheep

During cold stress, gut microbes play crucial roles in orchestrating energy metabolism to enhance environmental adaptation. In sheep, hindgut microbes ferment carbohydrates to generate short-chain fatty acids (SCFAs) as an energy source. However, the mechanisms by which hindgut microbes and their metabolites interact with the host to facilitate adaptation to cold environments remain ambiguous. Herein, we simulated a winter environment (- 20 °C) and provided a rationed diet to compare the cold adaptation mechanisms between Hulunbuir and Hu sheep. Our findings show that cold exposure enhances SCFA metabolism in the sheep cecum. In Hu sheep, acetate, butyrate, and total SCFA concentrations increased, whereas in Hulunbuir sheep, propionate and butyrate concentrations increased, with a notable increase in total SCFAs. Notably, butyrate concentration was higher in Hulunbuir sheep than in Hu sheep under cold stress. Following cold exposure, the proinflammatory cytokine IL-1β levels increased in both breeds. In addition, Hu sheep showed increased IL-10, whereas Hulunbuir sheep exhibited elevated secretory IgA levels. The cecal microbiota responded differently, Hu sheep showed no notable changes in alpha and beta diversity, whereas Hulunbuir sheep exhibited considerable alterations. In Hu sheep, the abundance of fungi, specifically Blastocystis sp. subtype 4, decreased, and that of several Lachnospiraceae species (Roseburia hominis, Faecalicatena contorta, and Ruminococcus gnavus) involved in SCFA metabolism increased. Pathways related to carbohydrate metabolism, such as starch and sucrose metabolism, galactose metabolism, and pentose and glucuronate interconversions, were upregulated. In Hulunbuir sheep, the abundance of Treponema bryantii, Roseburia sp. 499, and Prevotella copri increased, with upregulation in pathways related to amino acid metabolism and energy metabolism. Cold exposure increased node connectivity within the symbiotic networks of both breeds, with increased network vulnerability in Hu sheep. Following cold exposure, the microbial community of Hulunbuir sheep showed a decrease in the influence of stochastic processes on community assembly, with a corresponding increase in the role of environmental selection. Conversely, no such shift was evident in Hu sheep. Further transcriptomic analysis revealed distinct regulatory mechanisms between breeds. In Hu sheep, protein synthesis, energy metabolism, and thermogenesis pathways were substantially upregulated. By contrast, Hulunbuir sheep showed considerable upregulation of immune pathways and energy conservation through reduced ribosome synthesis. Correlation analysis indicated that butyrate holds a central position in both networks, with Hulunbuir sheep exhibiting a more complex and tightly regulated network involving SCFAs, microbiota, microbial functions, and transcriptomes. Partial least squares path modeling revealed that cold exposure substantially altered the cecal microbiota and transcriptomes of Hulunbuir sheep, affecting SCFAs and cytokines. The findings of this study suggest that under cold exposure, Hu sheep enhance acetate fermentation and rely on tissue thermogenesis for adaptation. By contrast, Hulunbuir sheep exhibit changes in microbial diversity and function, leading to increased propionate and butyrate metabolism. This may promote physiological energy conservation and innate immune defense, balancing heat loss and enhancing cold adaptation.