The rumen is an important hallmark organ of ruminants and plays an important role in the metabolism and immune barrier of Tibetan sheep on the Plateau. However, there are few studies on rumen development and metabolism regulation in Tibetan sheep at different ages. Here, we comprehensively analyzed the immune function, fermentation function, rumen epithelial micromorphology and transcriptome profile of Tibetan sheep at different ages. The results showed that the concentration of IgG decreased and the concentration of IgM increased with age (p < 0.05), and the highest concentration of IgA was observed at 1.5 and 3.5 years of age. In terms of rumen fermentation characteristics, VFAs of 4-month-old lambs were the highest, followed by VFAs and NH₃-N of Tibetan sheep at 3.5 years of age. Hematoxylin-eosin staining and transmission electron microscopy section examination of rumen epithelial tissue showed that the rumen papilla width increased with age (p < 0.001), the thickness of the stratum corneum decreased, the cells in the stratum corneum showed accelerated migration and the thickness of the rumen muscle layer increased (p < 0.001). Desmosomal junctions between the layers of rumen epithelium increased at 1.5 and 3.5 years old, forming a compact barrier structure, and the basal layer had more mitochondria involved in the regulation of energy metabolism. RNA-seq analysis revealed that a total of 1006 differentially expressed genes (DEGs) were identified at four ages. The DEGs of Tibetan sheep aged 4 months and 6 years were mainly enriched in the oxidation–reduction process and ISG15-protein conjugation pathway. The 1.5 and 3.5-year-olds were mainly enriched in skeletal muscle thin filament assembly, mesenchyme migration and the tight junction pathway. WGCNA showed that DEGs related to rumen microbiota metabolite VFAs and epithelial morphology were enriched in “Metabolism of xenobiotics by cytochrome P450, PPAR signaling pathway, Butanoate metabolism pathways” and participated in the regulation of rumen epithelial immune and fermentation metabolism functions of Tibetan sheep at different ages. This study systematically revealed the regulatory mechanism of rumen epithelial development and metabolism in the plateau adaptation of Tibetan sheep, providing a new approach for the study of plateau adaptation.

Hypoxia-inducible factors (HIFs) play an important role in mediating the physiological response to low oxygen environments. However, whether the expression of HIFs changes with age is unknown. In the present study, the effect of aging on HIF-1α, HIF-2α, HIF-3α and VEGF expression in the heart and lung of 30 Tibetan sheep that were adapted to hypoxia was evaluated. The 30 sheep were subdivided into groups of 10 animals that were 1, 2 or 6 years of age. Immunohistochemistry for HIF-1α, HIF-2α, HIF-3α and VEGF revealed that the immunostaining intensity of VEGF protein in the heart and lung was significantly higher than the intensity of immunostaining against the HIFs (p < 0.05). HIF-1α and HIF-2α protein translocated into the nucleus of cardiac muscle cells. However, immunostaining for HIF-3α was restricted to the cytoplasm of the myocardial cells. Immunostaining for HIF-1α, HIF-2α, HIF-3α and VEGF was detected within alveolar macrophages. The concentration of HIF-1α and HIF-2α was higher in the lung of 1-year-old than 6-year-old sheep (p < 0.05). In contrast, HIF-3α and VEGF immunostaining was most prominent in the hearts of the oldest sheep. However, when RT-PCR was used to evaluate RNA within the tissues, the expression of all four studied genes was higher in the lung than in the heart in the 1-year-old sheep (p < 0.05). Furthermore, VEGF and HIF-3α gene expression was higher in the heart from 1-year old than 6-year old sheep (p < 0.05). However, in the lung, HIF-1α and HIF-2α gene expression was lower in 1-year old than 6-year old sheep (p < 0.05). We conclude that HIF-3α and VEGF may play be important in how the heart responds to hypoxia. Additionally, HIF-1α and HIF-2α may have a role in the adaptation of the lung to hypoxia. The expression of these proteins in alveolar macrophages suggests a potential role of these cells in the physiological response to hypoxia. These results are useful in understanding how age influences the hypoxia adaption mechanisms of the heart and lung and may help to better understand chronic mountain sickness that is commonly observed in Tibetan people living on the Qinghai-Tibetan plateau.

Plateau adaptation in animals involves genetic mechanisms as well as coevolutionary mechanisms of the microbiota and metabolome of the animal. Therefore, the characteristics of the rumen microbiome and metabolome, transcriptome, and serum metabolome of Tibetan sheep at different altitudes (4500 m, 3500 m, and 2500 m) were analyzed. The results showed that the rumen differential metabolites at 3500 m and 4500 m were mainly enriched in amino acid metabolism, lipid metabolism, and carbohydrate metabolism, and there was a significant correlation with microbiota. The differentially expressed genes and metabolites at middle and high altitudes were coenriched in asthma, arachidonic acid metabolism, and butanoate and propanoate metabolism. In addition, the serum differential metabolites at 3500 m and 4500 m were mainly enriched in amino acid metabolism, lipid metabolism, and metabolism of xenobiotics by cytochrome P450, and they were also related to microbiota. Further analysis revealed that rumen metabolites accounted for 7.65% of serum metabolites. These common metabolites were mainly enriched in metabolic pathways and were significantly correlated with host genes (p < 0.05). This study found that microbiota, metabolites, and epithelial genes were coenriched in pathways related to lipid metabolism, energy metabolism, and immune metabolism, which may be involved in the regulation of Tibetan sheep adaptation to plateau environmental changes.

The purpose of this study was to determine the mechanism of Astragalus activity on the immune function, rumen microbiota structure and rumen fermentation of early-weaned lambs. Thirty healthy early-weaned lambs with similar body weights (17.42 ± 2.02 kg) were selected for the feeding experiment. The control group (KB) was fed a basal diet, and the Astragalus group (HQ) was fed 0.3% Astragalus additive on the basis of a basic diet. The formal trial period was 60 days. The results showed that the concentrations of blood immunoglobulin A (IgA) and immunoglobulin M (IgM) in the HQ group were significantly higher than those in the KB group (P<0.05). Compared with the KB group, the concentrations of acetic acid, butyric acid and total volatile fatty acids (VFAs) in the HQ group were higher (P<0.01). The expression levels of the rumen epithelial-related genes MCT1, MCT4, NHE2 and ZO1 in the Astragalus group were significantly higher than those in the KB group (P<0.05). 16S rRNA analysis showed that at the phylum level, Bacteroidetes in the HQ group significantly increased (P<0.01); at the genus level, Prevotella (P<0.01) and Succiniclasticum (P<0.01) in the HQ group were found at significantly higher abundances than those in the KB group, and the results of microbiota gene and function prediction showed that “energy metabolism”, “glycan biosynthesis and metabolic” pathways were significantly enriched in the HQ group (P<0.05). As a feed additive, Astragalus can improve the immunity of early-weaned lambs, the structure of the rumen microbiota of lambs, and the fermentation capacity of the rumen.

The gut microbiota in Tibetan sheep's ruminant stomach varies across phenological phases, influencing nutrient absorption and adaptation. During the green grass period, acetic, propionic, and butyric acid concentrations in the rumen were higher than in the regreening and withered grass periods (P < 0.05). MCT1 expression peaked during regreening. Bacteroidetes dominated during the withered grass period (P < 0.01), with Rikenellaceae_RC9_gut_group and Prevotella most abundant in the rumen. Spatially, rumen VFA concentrations and MCT1 expression were significantly higher than in other stomachs (P < 0.05). Bacteroidetes, Prevotella and Rikenellaceae_RC9_gut_group were dominant across the ruminant stomach. Correlations between microbiota, VFAs, and transport genes highlight the interplay of fermentation, flora, and gene expression, crucial for Tibetan sheep's adaptation to the plateau environment.

The gut-brain axis is regarded as the “second brain” of the host. Gut microbiota and their metabolites affect intestinal homeostasis, function, and phenotype by regulating bidirectional communication between the gut and brain. This serves as a vital strategy for understanding how gut microbiota regulate nutrient metabolism and adaptability in animals. This study explored the metabolic mechanisms through which Tibetan sheep adapt to high-altitude environments via the rumen microbiota-gut-brain axis across different phenological periods (returning-green period, fresh grass period and withered grass period). By analyzing metabolic indicators, neurotransmitters, and gene and protein expression in serum, rumen, adipose, and hypothalamic tissues, we discovered that energy metabolism markers (creatine kinase, lactate dehydrogenase, glucose) and immunoglobulins (IgG, IgM) in the serum were significantly elevated during the fresh grass period (P < 0.05). In contrast, thyroid hormones T3 and T4 were at higher levels during the returning-green period (P < 0.05). The density of rumen fiber-degrading bacteria was higher during the returning-green period (P < 0.05). Meanwhile, the densities of Butyrivibrio fibrisolvens, Selenomonas ruminantium, and Treponema bryantii microbiota significantly during the fresh grass period and were positively correlated with isovaleric acid concentration (P < 0.05). Neurotransmitters (5-HT, DOPAC, 5-HIAA, and NE) were significantly elevated in both the rumen epithelium and hypothalamus during the fresh grass period (P < 0.05). The analysis of the cAMP-PKA-pCREB pathway showed that the genes and proteins of UCP1, PKA, and CREB1 were highly expressed in adipose tissue during the fresh grass and withered grass periods, and there significant negative correlations to specific microbiota (P < 0.05). In summary, Tibetan sheep adapt to high-altitude environments through the rumen microbiota-gut-brain axis, regulating metabolic and neurotransmitter changes to establish a unique metabolic adaptation mechanism.