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The Qinghai-Tibet Plateau is a valuable genetic resource pool, and the high-altitude adaptation of Tibetan pigs is a classic example of the adaptive evolution of domestic animals. Here, we report the presence of Darwinian positive selection signatures in Tibetan pigs (TBPs) using 348 genome-wide datasets (127 whole-genome sequence datasets (WGSs) and 221 whole-genome single-nucleotide polymorphism (SNP) chip datasets). We characterized a high-confidence list of genetic signatures related response to high-altitude adaptation in Tibetan pigs, including 4,598 candidate SNPs and 131 candidate genes. Functional annotation and enrichment analysis revealed that 131 candidate genes are related to multiple systems and organs in Tibetan pigs. Notably, eight of the top ten novel genes, RALB, NBEA, LIFR, CLEC17A, PRIM2, CDH7, GK5 and FAM83B, were highlighted and associated with improved adaptive heart functions in Tibetan pigs high-altitude adaptation. Moreover, genome-wide association analysis revealed that 29 SNPs were involved in 13 candidate genes associated with at least one adaptive trait. In particular, among the top ten candidate genes, CLEC17A is related to a reduction in hemoglobin (HGB) in Tibetan pigs. Overall, our study provides a robust SNP/gene list involving genetic adaptation for Tibetan pig high-altitude adaptation, and it will be a valuable resource for future Tibetan pig studies., (© 2024. The Author(s).)

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.

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

The Tibetan pig is an endemic economic animal in the plateau region of China, and has a unique adaptation mechanism to the plateau hypoxic environment. Research into microRNAs (miRNAs) involved in the mechanism underlying hypoxia adaptation of Tibetan pig is very limited. Therefore, we isolated alveolar type II epithelial (ATII) cells from the lungs of the Tibetan pig, cultured them in normoxia/hypoxia (21% O 2 ; 2% O 2 ) for 48 h, and performed high-throughput sequencing analysis. We identified a hypoxic stress-related ssc-miR-141 and predicted its target genes. The target genes of ssc-miR-141 were mainly enriched in mitogen-activated protein kinase (MAPK), autophagy-animal, and Ras signaling pathways. Further, we confirmed that PDCD4 may serve as the target gene of ssc-miR-141. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to confirm the expression levels of ssc-miR-141 and PDCD4 , and a dual-luciferase gene reporter system was used to verify the targeted linkage of ssc-miR-141 to PDCD4 . The results showed that the expression level of ssc-miR-141 in the hypoxia group was higher than that in the normoxia group, while the expression level of PDCD4 tended to show the opposite trend and significantly decreased under hypoxia. These findings suggest that ssc-miR-141 is associated with hypoxia adaptation and provide a new insight into the role of miRNAs from ATII cells of Tibetan pig in hypoxia adaptation.

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.

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.

We compared the differences in testicular morphology and vascular anatomy between plateau-type Tibetan sheep and low-altitude Small-tailed Han sheep to explore any adaptations of Tibetan sheep to high altitudes and hypoxia factors. Testicular artery architecture specimens of plateau-type Tibetan sheep and lowland Small-tailed Han sheep were used to compare the physical parameters of the testis, testicular artery architecture, arterial diameter, testicular leaflets and the characteristics of epididymal head arterioles. The results showed that the weight, major axis, minor axis, overall length, caput major axis and caput minor axis in Tibetan sheep were larger than those of Small-tailed Han sheep; however, the caput major axis of epididymal head in Tibetan sheep was smaller than that of Small-tailed Han sheep. Compared with Small-tailed Han sheep, the testicular knot-like arteries of Tibetan sheep had denser spiral distribution and more small arteries, and the diameters of centripetal arteries, knot-like arteries, centrifugal arteries and epididymal microarterioles were also thicker. The 'spindle' indentation on the surface of the microarteriole of the head of the testicular leaflets and the epididymis in plateau Tibetan sheep was relatively shallow. The results showed that the physical testicular characteristics, testicular artery construction and arteriole surface structure of Tibetan sheep were adapted for the plateau environment., (© 2022 Wiley-VCH GmbH.)

In multicellular organisms, alternative splicing (AS) is central to the regulation of multiple biological processes. To further elucidate the adaptive strategy of AS in the lungs of Tibetan pigs in response to hypoxia, we identified and analyzed five basic AS types and 59,930 AS events in 18,179 genes. We found that approximately 65.10% of the total expressed genes underwent AS in the lungs of Tibetan pigs at a high altitude (TH). The frequencies of AS events were similar among the different groups (5.06-5.30 events in each gene on average). Skipped exons (SEs) were the predominant type of AS event, followed by mutually exclusive exons (MXEs), alternative 3' splice sites (A3SSs) and alternative 5' splice sites (A5SSs). Retained introns (RIs), the remaining type of AS event, showed lower frequencies. Further comparison analysis of differentially expressed genes (DEGs) and differentially spliced genes (DSGs) identified 2,209 differential splicing events in the above 18,000 expressed genes, including 918 increased and 1,291 decreased splicing events between the TH and Tibetan pigs at a low altitude (TL) groups. We identified 227 hypoxia-related genes involved in lung development that were differentially regulated through AS. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis clearly identified many DEGs and DSGs at high or low altitude. Seven pathways in the top 20 enriched KEGG terms overlapped for the DEGs and DSGs, including the chemokine signaling pathway, B cell receptor signaling pathway, and cytokine-cytokine receptor interaction, which exert many immunoregulatory and inflammatory actions critical to the lung under hypoxia. Twelve pathways overlapped in hypoxic DEGs and DSGs and included antigen processing, presentation and biosynthesis. ​GO analysis of the DEGs and DSGs among the four groups showed that numerous GO terms were enriched in the biological category, and the proportion of genes with downregulated expression was greater among 227 hypoxic genes than that of all genes. The results suggest that AS plays an essential role in the regulation of gene expression during hypoxia and that numerous genes involved in lung development are differentially regulated through AS., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tibetan pigs have exhibited unique characteristics from low-altitudes pigs and adapted well to the Qinghai-Tibet Plateau., Objectives: The current study was undertaken to investigate the hypoxic adaptation of heart in Tibetan pigs., Methods: The hearts of Tibetan pigs and Landrace pigs raised at high or low altitudes were compared using 3D casting technology, scanning electron microscopy and real-time quantitative PCR (qRT-PCR)., Results: We found that the ratio of the major axis to the minor axis and the density of the heart were significantly higher in Tibetan pigs than in Landrace pigs (p < 0.05). Tibetan pigs had larger diameters and higher densities of arterioles than Landrace pigs (p < 0.05), and these features have a similar variation with the expression of vascular endothelial growth factor (VEGF). The cardiac expression levels of hypoxia-inducible factor-1α (HIF-1α) and endothelial nitric oxide synthase (eNOS) were significantly higher in pigs reared at high altitudes than in those reared at low altitudes (p < 0.05). In contrast, Egl nine homolog 1 (EGLN1) had the opposite trend with respect to HIF-1α and eNOS and was related to red blood cell (RBC) counts. Notably, the expressions of erythropoietin (EPO) and endothelial PAS domain-containing protein 1 (EPAS1) were significantly higher in Landrace pigs kept at high altitudes than in the others (p < 0.05) and were associated with haemoglobin., Conclusions: These findings show that the regulation of the heart function of Tibetan pigs in a hypoxic environment is manifested at various levels to ensure the circulation of blood under extreme environmental conditions., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Due to their long-term colonization of and widespread distribution in plateau environments, yaks can serve as an ideal natural animal model for the adaptive evolution of other plateau species, including humans. Some studies reported that the lung and heart are two key organs that show adaptive transcriptional changes in response to high altitudes, and most of the genes that show differential expression in lung tissue across different altitudes display nonlinear regulation. To explore the molecular mechanisms that are activated in yak lung tissue in response to hypoxia, the mRNAs, lncRNAs and miRNAs of lung tissue from 9 yaks living at three different altitudes (3400 m, 4200 m and 5000 m), with three repetitions per altitude, were sequenced. Two Zaosheng cattle from 1500 m were selected as low-altitude control. A total of 21,764 mRNAs, 14,168 lncRNAs and 1209 miRNAs (305 known and 904 novel miRNAs) were identified. In a comparison of yaks and cattle, 4975 mRNAs, 3326 lncRNAs and 75 miRNAs were differentially expressed. A total of 756 mRNAs, 346 lncRNAs and 83 miRNAs were found to be differentially expressed among yaks living at three different altitudes (fold change ≥ 2 and P-value < 0.05). The differentially expressed genes between yaks and cattle were functionally enriched in long-chain fatty acid metabolic process and protein processing, while the differentially expressed genes among yaks living at three different altitudes were enriched in immune response and the cell cycle. Furthermore, competing endogenous RNA (ceRNA) networks were investigated to illustrate the roles of ceRNAs in this process, the result was also support the GO and KEGG analysis. The present research provides important genomic insights for discovering the mechanisms that are activated in response to hypoxia in yak lung tissue.