Qianling Chen

This study aimed to explore the generational succession rules of rectal microbes and metabolites in crossbred dairy cows, and clarify the regulatory mechanism underlying generational differentiation of milk quality traits, so as to provide a theoretical basis for improving milk quality and realizing precision breeding of dairy cows based on hindgut microecology. In this experiment, 10 healthy cows in mid-lactation were selected from each of three consecutive generations (F₁, F₂, and F₃) of Holstein–Norwegian Red crossbred cattle, totaling 30 cows. High-throughput 16S rRNA sequencing combined with UPLC-MS/MS-based untargeted metabolomics was applied to systematically analyze the correlations between the rectal microbial community structure and metabolic profiles and milk quality indicators. The results indicated that milk quality traits exhibited obvious generational differentiation: daily milk yield increased gradually with generation and was significantly higher in F₃ cows than in F₁ cows (p < 0.05), while milk fat rate and milk protein rate showed an upward-then-downward trend and reached the highest level in the F₂ generation. The rectal microbial community underwent directional generational succession, with the relative abundance of Firmicutes continuously increased and Actinobacteriota gradually decreased, and the microbial functions were mainly specialized in carbohydrate and amino acid metabolism. Significant generational differences were also observed in rectal metabolic profiles, and the differential metabolites screened between F₁ and F₃ generations were primarily enriched in steroid hormone biosynthesis, nucleotide sugar metabolism, bile acid metabolism, and amino acid metabolism pathways. Multi-omics correlation analysis further verified that the core rectal microbial genera could regulate the lactation performance of the mammary gland by affecting the abundance of nutritional metabolic substrates and signaling molecules, with metabolites acting as critical mediators in the regulatory relationship between rectal microbes and milk quality traits. This study systematically characterizes the generational succession characteristics of rectal microbes and metabolites in crossbred dairy cows and identifies potential correlative mediating links of rectal metabolites between microbes and milk-quality traits, providing a novel theoretical reference for exploring hindgut microecology to regulate dairy cow milk quality and assist elite breeding.

The rumen epithelium of Tibetan sheep plays a critical role in energy metabolism and immune defense; however, its post-transcriptional regulatory mechanisms under high-altitude hypoxia stress remain unclear. In this study, we employed integrated mRNA and miRNA transcriptome sequencing to analyze the adaptive strategies of the rumen epithelium in Tibetan sheep at different altitudes. A total of 2183 differentially expressed genes (DEGs) and 135 differentially expressed miRNAs (DEmiRNAs) were identified. Functional enrichment analysis revealed that DEGs and their target genes were significantly enriched in immune-related pathways such as the NF-κB signaling pathway and cytokine–cytokine receptor interaction, as well as metabolic pathways including oxidative phosphorylation and branched-chain amino acid degradation. Integrated network analysis highlighted key regulatory pairs, including oar-miR-370-3p targeting PCK2 and IL1R2, and novel-miR-781 regulating PIK3R5, suggesting coordinated modulation between mitochondrial homeostasis and immune responses. Specifically, the upregulation of immune genes (CCL19, MADCAM1) and heat shock proteins at TS4500m indicates enhanced mucosal immunity and stress tolerance, while altered expression of metabolic genes reflects a shift in energy substrate utilization. These findings elucidate a complex mRNA-miRNA regulatory network that enables Tibetan sheep to maintain rumen epithelial integrity and energy balance under extreme high-altitude conditions, providing novel insights into the molecular basis of hypoxia adaptation in ruminants.

Tibetan sheep are an important livestock breed adapted to the extreme environment of the Qinghai–Tibet Plateau (QTP). Their energy metabolism and environmental adaptability are highly dependent on the rumen microbiome. However, systematic comparisons of the rumen microbiome, its functions, and the resistome between plateau-adapted breeds and lowland breeds remain insufficient. In this study, 6 Tibetan sheep (TS) and 6 Hu sheep (HS) were selected. All the selected sheep had a body weight of 34 kg (±0.5 kg) and an age of 1 year (±1 month) and were all managed under local traditional natural grazing (without supplementary feeding). Using metagenomics and metabolomics techniques, systematic comparative analysis was conducted on the differences in rumen microbial community structure, functions, resistome, and metabolites between the two breeds. Metagenomic analysis showed that at the phylum level, the abundance of Bacteroidetes in the rumen of TS was significantly higher than that in HS (p < 0.05); at the genus level, the abundance of Bacteroides in TS was also significantly higher (p < 0.05). Carbohydrate-active enzymes (CAZy) analysis indicated that the abundance of Glycosyltransferases (GTs) and Carbohydrate-Binding Modules (CBMs) in the rumen of TS were significantly upregulated (p < 0.05), while HS was rich in various Glycoside Hydrolases (GHs). Comprehensive Antibiotic Resistance Database (CARD) analysis revealed that more than 60% of the Antibiotic Resistance Genes (ARGs) in the rumen of HS were present at higher levels than those in TS. Metabolomics identified a large number of differential metabolites, among which metabolites such as 2E,6Z,8Z,12E-hexadecatetraenoic acid, Leukotriene F4, and Tenurin were significantly upregulated in the rumen of TS. Correlation analysis showed that rumen microbial flora and their metabolites jointly act to regulate rumen ARGs. Specifically, microorganisms including Firmicutes and Succiniclasticum had a significantly positive correlation with ARGs such as rpoB2 (p < 0.05), while differential metabolites like endomorphin-1 and Purothionin AII exhibited a significantly negative correlation with ARGs such as rpoB2 (p < 0.05). Therefore, compared with HS, the synergistic effect of the rumen microbial flora, their metabolites, and the resistome in TS is an important characteristic and strategy for their adaptation to the hypoxic environment of the QTP, and also contributes to the formation of their unique rumen resistome. Despite being reared in the same plateau environment, the rumen microbiome of HS still retains low-altitude characteristics, which are manifested as high GHs activity and high ARGs abundance.

Investigating the functional interactions between rumen microbial fermentation and epithelial mitochondrial dynamics/energy metabolism in Tibetan sheep at different altitudes, this study examined ultrastructural changes in rumen epithelial tissues, expression levels of mitochondrial dynamics-related genes (fusion: Mfn1, Mfn2, OPA1, Mic60; fission: Drp1, Fis1, MFF), and ketogenesis pathway genes (HMGS2, HMGCL) in Tibetan sheep raised at three altitudes (TS 2500m, TS 3500m, TS 4500m). Correlation analysis was performed between rumen microbiota/metabolites and mitochondrial energy metabolism. Results: Ultrastructural variations were observed across altitudes. With increasing altitude, keratinized layer became more compact; desmosome connections between granular layer cells increased; mitochondrial quantity and distribution in spinous and basal layers increased. Mitochondrial dynamics regulation: Fission genes (FIS1, DRP1, MFF) showed significantly higher expression at TS 4500m (p < 0.01); fusion genes (Mfn1, OPA1) exhibited altitude-dependent upregulation. Energy metabolism markers: Pyruvate (PA) decreased significantly at TS 3500m/TS 4500m (p < 0.01); citrate (CA) increased with altitude; NAD⁺ peaked at TS 3500m but decreased significantly at TS 4500m (p < 0.01); Complex II (SDH) and Complex IV (CO) activities decreased at TS 4500m (p < 0.01). Ketogenesis pathway: β-hydroxybutyrate increased significantly with altitude (p < 0.01); acetoacetate peaked at TS 2500 m/TS 4500 m; HMGCS2 expression exceeded HMGCL, showing altitude-dependent upregulation at TS 4500m (p < 0.01). Microbiome–metabolism correlations: Butyrivibrio_2 and Fibrobacter negatively correlated with Mic60 (p < 0.01); Ruminococcaceae_NK4A214_Group positively correlated with Mfn1/OPA1 (p < 0.05); WGCNA identified 17 metabolite modules, with MEturquoise module positively correlated with DRP1/Mfn2/MFF (p < 0.05). Conclusion: Altitude-induced ultrastructural adaptations in rumen epithelium correlate with mitochondrial dynamics stability and ketogenesis upregulation. Mitochondrial fission predominates at extreme altitudes, while microbiota–metabolite interactions suggest compensatory energy regulation mechanisms.

As high-quality sheep germplasm resources in China, Hu sheep are characterized by fast growth and development, high fecundity, and tolerance to drought and cold. Tibetan sheep, adapted to high-altitude environments, have developed strong environmental adaptability. To explore the differences in the interaction among rumen microbial flora, hepatic gluconeogenesis, and mitochondrial function between Tibetan sheep and Hu sheep in the Qinghai–Tibet Plateau, this study systematically compared and analyzed the rumen flora density, key enzyme activities related to hepatic gluconeogenesis and mitochondrial function, and the expression levels of related genes in Tibetan sheep and Hu sheep under identical feeding management conditions, followed by correlation analysis. The results showed that Hu sheep had significantly higher densities of Ruminobacteramylophilus (Ram) and Fibrobacter succinogenes (Fs) associated with starch and protein degradation (p < 0.01). The expression levels of Forkhead box O1 (FOXO1), pyruvate carboxylase (PC) activity, and adenosine triphosphate (ATP) content were also significantly higher than those in Tibetan sheep (p < 0.01). In contrast, Tibetan sheep had higher densities of Butyrivibrio fibrisolvens (Bf), Ruminococcus albus (Ra), Ruminococcus flavefaciens (Rf), etc., related to cellulose degradation (p < 0.01). The gluconeogenesis-related genes, Glucose-6-phosphatase catalytic subunit 1 (G6PC1) and phosphoenolpyruvate carboxykinase1 (PCK1), and the activities of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBPase) were significantly higher in Tibetan sheep than in Hu sheep (p < 0.01). Mitochondrial function-related genes Mitofusin-1 (Mfn1), Mitofusin-2 (Mfn2), subunit 6 of ATP synthase (ATP6), cytochrome b (Cytb), etc., also showed significantly higher expression in Tibetan sheep (p < 0.01). While no significant differences were observed in the contents of citric acid (CA), pyruvic acid (PA), glucose (Glu), etc. (p > 0.05). Correlation analysis indicated that rumen flora was associated with the key enzyme activities and gene expressions of hepatic gluconeogenesis and mitochondrial function to varying degrees. In summary, Tibetan sheep exhibit strong fiber degradation capacity, the efficient utilization of gluconeogenic intermediates, and mitochondrial oxidative phosphorylation (OXPHOS) ability, forming adaptive strategies for high-altitude environments. By contrast, Hu sheep show efficient protein and starch degradation capacity, thereby enhancing the supply of gluconeogenic precursors. It is indicated that when introducing Hu sheep to high-altitude areas, dietary intervention can be used to regulate rumen microorganisms, such as increasing fiber-decomposing bacteria or enhancing mitochondrial oxidative capacity, to counteract metabolic limitations induced by hypoxia.

Microbiota density plays an important role in maintaining host metabolism, immune function, and health, and age has a specific effect on the composition of intestinal microbiota. Therefore, the age-specific effects of age differences on the structure and function of the ileum microbiota in Tibetan sheep were investigated by determining the density of the ileum microbiota, the content of VFAs, and the expression levels of their transporter-related genes at different ages. The results showed that the contents of acetic acid and propionic acid in the ileum of Tibetan sheep in the 1.5-year-old group were significantly higher (p < 0.05) than those in other age groups, and that the contents of total VFAs were also significantly higher (p < 0.05) than those in other age groups. The relative densities of ileum Rf, Ra, and Fs were significantly higher in the 1.5-year-old group than in the other age groups (p < 0.05). The ileum epithelial VFAs transport-related genes AE2, MCT-4, and NHE1 had the highest expression in the 1.5-year-old group, and the expression of DRA was significantly lower in the 1.5-year-old group than in the 6-year-old group (p < 0.05). Correlation analysis showed that Cb, Sr, and Tb were significantly positively correlated with butyric acid concentration (p < 0.05) and negatively correlated with acetic acid, but the difference was not significant (p > 0.05); MCT-1, MCT-4, and AE2 were significantly positively correlated (p < 0.05) with acetic, propionic, and isobutyric acid concentrations; NHE1, NHE2, and MCT-4 were highly significantly positively correlated (p < 0.01) with Romboutsia and unclassified_Peptostreptococcaceae, while acetic acid was significantly positively correlated (p < 0.05) with NK4A214_group; Romboutsia, and unclassified_Peptostreptococcaceae were significantly positively correlated (p < 0.05). Therefore, compared with other ages, the 1.5-year-old Tibetan sheep had a stronger fermentation and metabolic capacity in the ileum under traditional grazing conditions on the plateau, which could provide more energy for Tibetan sheep during plateau acclimatization.

During the adaptive evolution of animals, the host and its gut microbiota co-adapt to different elevations. Currently, there are few reports on the rumen microbiota–hepato-intestinal axis of Tibetan sheep at different altitudes. Therefore, the purpose of this study was to explore the regulatory effect of rumen microorganism–volatile fatty acids (VFAs)–VFAs transporter gene interactions on the key enzymes and genes related to gluconeogenesis in Tibetan sheep. The rumen fermentation parameters, rumen microbial densities, liver gluconeogenesis activity and related genes were determined and analyzed using gas chromatography, RT-qPCR and other research methods. Correlation analysis revealed a reciprocal relationship among rumen microflora–VFAs-hepatic gluconeogenesis in Tibetan sheep at different altitudes. Among the microbiota, Ruminococcus flavefaciens (R. flavefaciens), Ruminococcus albus (R. albus), Fibrobactersuccinogenes and Ruminobacter amylophilus (R. amylophilus) were significantly correlated with propionic acid (p < 0.05), while propionic acid was significantly correlated with the transport genes monocarboxylate transporter 4 (MCT4) and anion exchanger 2 (AE2) (p < 0.05). Propionic acid was significantly correlated with key enzymes such as pyruvate carboxylase, phosphoenolpyruvic acid carboxylase and glucose (Glu) in the gluconeogenesis pathway (p < 0.05). Additionally, the expressions of these genes were significantly correlated with those of the related genes, namely, forkhead box protein O1 (FOXO1) and mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) (p < 0.05). The results showed that rumen microbiota densities differed at different altitudes, and the metabolically produced VFA contents differed, which led to adaptive changes in the key enzyme activities of gluconeogenesis and the expressions of related genes.

The gut microbiota is highly symbiotic with the host, and the microbiota and its metabolites are essential for regulating host health and physiological functions. Astragalus, as a feed additive, can improve animal immunity. However, the effects of Astragalus root powder on the rumen microbiota and their metabolites in lambs are not apparent. In this study, thirty healthy Hu sheep lambs with similar body weights (17.42 ± 2.02 kg) were randomly selected for the feeding experiment. Lambs were fed diets supplemented with 0.3% Astragalus root powder, and the rumen microbiota density and metabolome were measured to determine the effects of Astragalus on the health of lambs in the rumen. The results showed that the relative abundance of Butyrivibrio fibrisolvens (Bf), Ruminococcus flavefaciens (Rf), Succiniclasticum (Su), and Prevotella (Pr) in the rumen was increased in the Astragalus group (p < 0.01), and metabolic profiling showed that the metabolites, such as L-lyrosine and L-leucine, were upregulated in the Astragalus group (p < 0.01). KEGG functional annotation revealed that upregulated metabolites were mainly enriched in the pathways of amino acid metabolism, lipid metabolism, fatty acid biosynthesis, and bile secretion in the Astragalus group, and downregulated metabolites were enriched in the pathways of methane metabolism and other pathways. Correlation analysis revealed that butyric acid was positively correlated with Roseburia and Blautia (p < 0.05) and negatively correlated with Desulfovibrio (p < 0.05). Thus, by analyzing the interactions of Astragalus root powder with the density of rumen microorganisms and their metabolites in lambs, it was shown that Astragalus root powder could improve the structure of rumen microbiota and their metabolites and then participate in the regulation of amino acid metabolism, lipid metabolism, immune metabolism, and other pathways to improve the efficiency of energy absorption of the lambs.

The intestinal microbiota of ruminants is an important factor affecting animal production and health. Research on the association mechanism between the intestinal microbiota and meat quality of ruminants will play a positive role in understanding the formation mechanism of meat quality in ruminants and improving production efficiency. In this study, the fatty acid composition and content, expression of related genes, and structural characteristics of the ileum microbiota of ewes of Tibetan sheep at different ages (4 months, 1.5 years, 3.5 years, and 6 years) were detected and analyzed. The results revealed significant differences in fatty acid composition and content in the muscle of Tibetan sheep at different ages (p < 0.05); in addition, the content of MUFAs in the longissimus dorsi muscle and leg muscle was higher. Similarly, the expressions of muscle-related genes differed among the different age groups, and the expression of the LPL, SCD, and FABP4 genes was higher in the 1.5-year-old group. The ileum microbiota diversity was higher in the 1.5-year-old group, the Romboutsia abundance ratio was significantly higher in the 1.5-year-old group (p < 0.05), and there was a significant positive correlation with oleic acid (C18:1n9c) (p < 0.05). In conclusion, the content of beneficial fatty acids in the longissimus dorsi muscle and leg muscle of Tibetan sheep was higher at 1.5 years of age, and the best slaughter age was 1.5 years. This study provides a reference for in-depth research on the mechanism of the influence of the gut microbiota on meat quality and related regulation.