Effects of the Interaction between Rumen Microbiota Density-VFAs-Hepatic Gluconeogenesis on the Adaptability of Tibetan Sheep to Plateau.

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.

Interactions of rumen microbiota and metabolites with meat quality-related genes to regulate meat quality and flavor of Tibetan sheep under nutrient stress in the cold season.

AIM: The meat of Tibetan sheep has a unique flavor, delicious taste, and superior nutritional value. However, the change of grass will lead to a change in meat quality. This study aimed to explore the potential regulatory mechanisms of microbial metabolites with respect to meat quality traits of Tibetan sheep under nutrient stress in the cold season. METHODS AND RESULTS: We determined and analyzed the longissimus dorsi quality, fatty acid composition, expression of genes, and rumen microbial metabolites of Tibetan sheep in cold and warm seasons. The shear force was decreased (P < .05), the meat color a*24 h value was increased (P < .05), and the contents of crude fat (EE) and protein (CP) were decreased in the cold season. Polyunsaturated fatty acids (PUFAs)-linoleic acid and docosahexaenoic acid increased significantly in the cold season (P < .05). The expressions of meat quality genes MC4R, CAPN1, H-FABP, and LPL were significantly higher in the warm season (P < .05), and the CAST gene was significantly expressed in the cold season (P < .01). The different microbial metabolites of Tibetan sheep in the cold and warm seasons were mainly involved in amino acid metabolism, lipid metabolism, and digestive system pathway, and there was some correlation between microbiota and meat quality traits. There are similarities between microbial metabolites enriched in the lipid metabolism pathway and muscle metabolites. CONCLUSION: Under nutritional stress in the cold season, the muscle tenderness of Tibetan sheep was improved, and the fat deposition capacity was weakened, but the levels of beneficial fatty acids were higher than those in the warm season, which was more conducive to healthy eating.

Astragalus additive in feed improved serum immune function, rumen fermentation and the microbiota structure of early-weaned lambs.

AIM: 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. METHODS AND RESULTS: 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). CONCLUSION: 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.

Interaction between Rumen Epithelial miRNAs-Microbiota-Metabolites in Response to Cold-Season Nutritional Stress in Tibetan Sheep.

Tibetan sheep are already well adapted to cold season nutrient stress on the Tibetan Plateau. Rumen, an important nutrient for metabolism and as an absorption organ in ruminants, plays a vital role in the cold stress adaptations of Tibetan sheep. Ruminal microbiota also plays an indispensable role in rumen function. In this study, combined multiomics data were utilized to comprehensively analyze the interaction mechanism between rumen epithelial miRNAs and microbiota and their metabolites in Tibetan sheep under nutrient stress in the cold season. A total of 949 miRNAs were identified in the rumen epithelium of both cold and warm seasons. A total of 62 differentially expressed (DE) miRNAs were screened using FC > 1.5 and p value < 0.01, and a total of 20,206 targeted genes were predicted by DE miRNAs. KEGG enrichment analysis revealed that DE miRNA-targeted genes were mainly enriched in axon guidance(ko04360), tight junction(ko04530), inflammatory mediator regulation of TRP channels(ko04750) and metabolism-related pathways. Correlation analysis revealed that rumen microbiota, rumen VFAs and DE miRNAs were all correlated. Further study revealed that the targeted genes of cold and warm season rumen epithelial DE miRNAs were coenriched with differential metabolites of microbiota in glycerophospholipid metabolism (ko00564), apoptosis (ko04210), inflammatory mediator regulation of TRP channels (ko04750), small cell lung cancer (ko05222), and choline metabolism in cancer (ko05231) pathways. There are several interactions between Tibetan sheep rumen epithelial miRNAs, rumen microbiota, and microbial metabolites, mainly through maintaining rumen epithelial barrier function and host homeostasis of choline and cholesterol, improving host immunity, and promoting energy metabolism pathways, thus enabling Tibetan sheep to effectively respond to cold season nutrient stress. The results also suggest that rumen microbiota have coevolved with their hosts to improve the adaptive capacity of Tibetan sheep to cold season nutrient stress, providing a new perspective for the study of cold season nutritional stress adaptation in Tibetan sheep.

Synergistic Responses of Tibetan Sheep Rumen Microbiota, Metabolites, and the Host to the Plateau Environment.

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.

Sex differences in rumen fermentation and microbiota of Tibetan goat.

BACKGROUND: The gut microbiota play an important role in maintaining host metabolism, the immune system and health, while sex, genotype, diet and health have specific effects on the composition of the gut microbiota. Therefore, to explore the sex differences in the structure and function of rumen microbiota in Tibetan goats, herein we analyzed sex differences in rumen fermentation parameters, rumen microbiota and the expression of genes related to VFA transport in Tibetan goats. RESULTS: The results showed that the contents of acetic acid and propionic acid in the rumen of TGM (Tibetan goat male) were significantly higher than those in TGFm (Tibetan goat female) (P < 0.05), and total VFAs was significantly higher in TGM than TGFm (P < 0.05). Expression of the VFA transport-related genes DRA, AE2, MCT-1, NHE1, and NHE2 in the rumen epithelium of TGFm was significantly higher than that in TGM. Analysis of the composition and structure of the rumen microbiota revealed significant sex differences. At the phylum level, Firmicutes and Bacteroidetes were the dominant phyla in Tibetan goats. In addition, Fibrobacteres and Spirochaetes had significantly greater relative abundances in TGFm than in TGM (P < 0.05). At the genus level, the relative abundance of Fibrobacter, Ruminococcus_1 and Pyramidobacter was significantly higher in TGFm than in TGM (P < 0.05). The functional prediction results showed that replication, recombination and repair, RNA processing and modification were mainly enriched in TGFm (P < 0.05). CONCLUSIONS: Correlation analysis revealed significant associations of some rumen microbiota with the fermentation product VFAs and VFA transport-related genes. We concluded that yearling TGM and TGFm have distinct fermentation and metabolism abilities when adapting to the plateau environment, which provides a certain sex reference basis for Tibetan goat adaptation to the plateau environment.

Response of Ruminal Microbiota-Host Gene Interaction to High-Altitude Environments in Tibetan Sheep.

Altitude is the main external environmental pressure affecting the production performance of Tibetan sheep, and the adaptive evolution of many years has formed a certain response mechanism. However, there are few reports on the response of ruminal microbiota and host genomes of Tibetan sheep to high-altitude environments. Here, we conducted an integrated analysis of volatile fatty acids (VFAs), microbial diversity (16S rRNA), epithelial morphology, and epithelial transcriptome in the rumen of Tibetan sheep at different altitudes to understand the changes in ruminal microbiota−host interaction in response to high altitude. The differences in the nutritional quality of forage at different altitudes, especially the differences in fiber content (ADF/NDF), led to changes in rumen VFAs of Tibetan sheep, in which the A/P value (acetic acid/propionic acid) was significantly decreased (p < 0.05). In addition, the concentrations of IgA and IgG in Middle-altitude (MA) and High-altitude Tibetan sheep (HA) were significantly increased (p < 0.05), while the concentrations of IgM were significantly increased in MA (p < 0.05). Morphological results showed that the width of the rumen papilla and the thickness of the basal layer increased significantly in HA Tibetan sheep (p < 0.05). The 16S rRNA analysis found that the rumen microbial diversity of Tibetan sheep gradually decreased with increasing altitude, and there were some differences in phylum- and genus-level microbes at the three altitudes. RDA analysis found that the abundance of the Rikenellaceae RC9 gut group and the Ruminococcaceae NK4A214 group increased with altitudes. Furthermore, a functional analysis of the KEGG microbial database found the "lipid metabolism" function of HA Tibetan sheep to be significantly enriched. WGCNA revealed that five gene modules were enriched in "energy production and conversion", "lipid transport and metabolism", and "defense mechanisms", and cooperated with microbiota to regulate rumen fermentation and epithelial immune barrier function, so as to improve the metabolism and immune level of Tibetan sheep at high altitude.

Rumen Epithelial Development- and Metabolism-Related Genes Regulate Their Micromorphology and VFAs Mediating Plateau Adaptability at Different Ages in Tibetan Sheep.

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.

Coevolution of Rumen Epithelial circRNAs with Their Microbiota and Metabolites in Response to Cold-Season Nutritional Stress in Tibetan Sheep.

This study explores the effects of the coevolution of the host genome (the first genome) and gut microbiome (the second genome) on nutrition stress in Tibetan sheep during the cold season. The rumen epithelial tissue of six Tibetan sheep (Oula-type) was collected as experimental samples during the cold and warm seasons and the study lasted for half a year. The cDNA library was constructed and subjected to high-throughput sequencing. The circRNAs with significant differential expression were identified through bioinformatics analysis and functional prediction, and verified by real-time quantitative PCR (qRT-PCR). The results showed that a total of 56 differentially expressed (DE) circRNAs of rumen epithelial tissue were identified using RNA-seq technology, among which 29 were significantly upregulated in the cold season. The circRNA-miRNA regulatory network showed that DE circRNAs promoted the adaptation of Tibetan sheep in the cold season by targeting miR-150 and oar-miR-370-3p. The results of correlation analysis among circRNAs, microbiota, and metabolites showed that the circRNA NC_040275.1:28680890|28683112 had a very significant positive correlation with acetate, propionate, butyrate, and total volatile fatty acid (VFA) (p < 0.01), and had a significant positive correlation with Ruminococcus-1 (p < 0.05). In addition, circRNA NC_040256.1:78451819|78454934 and metabolites were enriched in the same KEGG pathway biosynthesis of amino acids (ko01230). In conclusion, the host genome and rumen microbiome of Tibetan sheep co-encoded a certain glycoside hydrolase (ß-glucosidase) and coevolved efficient VFA transport functions and amino acid anabolic processes; thus, helping Tibetan sheep adapt to nutrient stress in the cold season in high-altitude areas.

Evaluation of the genetic diversity of six Chinese indigenous chickens.

OBJECTIVE: The extensive breeding of commercial chickens has led to a sharp decrease in the resources of many indigenous chickens, especially the indigenous chickens in the southeastern coastal region, which are on the verge of extinction, and the indigenous chickens in the northwestern region of China, which are also at risk. However, there are few reports on the evaluation of genetic diversity and conservation of genetic resources of indigenous chickens in remote areas in the Northwest of China. METHODS: In the present study, the genetic diversity and phylogenetic relationship of six indigenous chickens from different regions were studied based on variation in mitochondrial DNA control region (D-loop), and the degree of introgression from commercial breeds into these chickens was determined by the amount of haplotype sharing between indigenous and commercial breeds. RESULTS: Twenty-five polymorphic sites and 25 haplotypes were detected in 206 individuals. Principal component analysis showed that the Jingning chicken had the highest genetic diversity among the six indigenous chickens. According to the degree of introgression, the six indigenous breeds may be involved in haplotype sharing with commercial breeds, and the introgression from commercial chickens into the Haidong chicken is the most serious. CONCLUSION: The genetic uniqueness of indigenous chickens has been eroded, so it is necessary to consider the protection of their genetic resources. Phylogenetic analysis suggests that the six indigenous chickens have two major matrilineal origins: one from Yunnan or its surrounding areas in China and the other from the Indian subcontinent.