The Tibetan Plateau’s distinctive high-altitude environment, marked by extreme cold and reduced oxygen levels, presents considerable survival challenges for both humans and mammals. Natural selection has led to the accumulation of adaptive mutations in Tibetan pigs, enabling them to develop distinctive adaptive phenotypes. Here, we aim to uncover the genetic mechanisms underlying the adaptation of Tibetan pigs to high-altitude hypoxia. Therefore, we conducted a systematic analysis of 140 whole-genome sequencing (WGS) data points from different representing pig populations. Our analysis identified a total of 27,614,561 mutations, including 22,386,319 single-nucleotide variants (SNVs) and 5,228,242 insertions/deletions (INDELs, size < 50 bp). A total of 11% (2,678,569) of the SNVs were newly identified in our project, significantly expanding the dataset of genetic variants in Tibetan pigs. Compared to other pig breeds, Tibetan pigs are uniquely adapted to high-altitude environments, exhibiting the highest genetic diversity and the lowest inbreeding coefficient. Employing the composite of multiple signals (CMS) method, we scanned the genome-wide Darwinian positive selection signals and identified 32,499 Tibetan pig positively selected SNVs (TBPSSs) and 129 selected genes (TBPSGs), including 213 newly discovered genes. Notably, we identified eight genes (PHACTR1, SFI1, EPM2A, SLC30A7, NKAIN2, TNNI3K, and PLIN2) with strong nature selection signals. They are likely to improve cardiorespiratory function and fat metabolism to help Tibetan pigs become adapted to the high-altitude environment. These findings provide new insights into the genetic mechanisms of high-altitude adaptation and the adaptive phenotypes of Tibetan pigs.

In this study, the epididymal histology, caepididymal sperm physiological parameters, serum reproductive hormones, and antioxidant enzyme SOD levels of Tibetan sheep at a 2500 m and 3500 m altitude were compared by using a combination of transcriptome and metabolomics methods. This was conducted to investigate the effects of a high-altitude environment on spermatogenesis and the maturation of Tibetan sheep. The results showed that compared to the low-altitude group, the high-altitude group had a smaller epididymal lumen, thicker epididymal wall, significantly decreased sperm survival rate, and significantly increased sperm deformation rate, but no difference in sperm motility and sperm respiratory intensity. With increasing altitude, Tibetan sheep showed a decreasing trend in serum reproductive hormones (FSH and T), while the antioxidant enzyme SOD activity was significantly reduced. Transcriptomic analysis revealed 139 differentially expressed genes in the Tibetan sheep epididymis under high-altitude conditions. The SYCP2 gene is involved in multiple biological processes related to reproduction and plays an important role in the regulation of epididymal function and sperm quality in Tibetan sheep. Genes like ADCYAP1R1, CABP2, CALN1, and ATP6V1B1 can help maintain sperm viability and maturation by regulating the cAMP signaling pathway, calcium ion homeostasis, and cellular signaling. Metabolomic analysis found that the high-altitude group had increased adenosine content and decreased prostaglandin I2 content in the epididymis. These metabolites are involved in spermatogenesis, motility, fertilization, and early embryonic development. The integrated omics analysis suggests that Tibetan sheep adapt to the high-altitude hypoxic environment by regulating cAMP signaling pathway genes like ADCY and PRKACA, as well as metabolites like adenosine and prostaglandin I2, to maintain epididymal function and sperm motility. These genes and metabolites play an important role in maintaining normal epididymal function and sperm motility at high altitudes.

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress.