The Tibetan sheep is an indigenous species of the Tibetan plateau and has been well adapted to high-altitude hypoxia. In comparison to lowland sheep breeds, the blood gas indicators have changed and the HIFs signaling pathway is activated in Tibetan sheep. These phenotypic and genetic alterations in Tibetan sheep are thought to be an important basis for adaptation to high-altitude hypoxia and variation in genes encoding the subunits that make up HIFs, such as HIF-1α can affect blood gas indicators. In this study, exons 9, 10, 12 of the HIF-1α gene were sequenced to find variations and 3 SNPs were detected, and these 3 SNPs were genotyped by KASP in 341 Hu sheep and 391 Tibetan sheep. In addition, 197 Hu sheep, 160 Tibetan sheep and 12 Gansu alpine merino sheep were used for blood gas indicators analysis. The results showed significant differences between the blood gas indicators of high-altitude breeds (Tibetan sheep and Gansu alpine merino sheep) and low-altitude breeds (Hu sheep), implying that the differences in blood gas indicators are mainly caused by differences in altitude. The haplotype combinations H2H3 and H1H3 were more frequent in the Tibetan sheep population, H2H3 increases O₂ carrying capacity by increasing hematocrit and hemoglobin concentrations; H1H3 makes O₂ dissociate more readily from oxyhemoglobin by decreasing partial pressure of oxygen and oxygen saturation. These results suggest that variants at the HIF-1α promote the ability of oxygen utilization in Tibetan sheep, which may underpin the survival and reproduction of Tibetan sheep on the Tibetan plateau.

Lymphatic enhancer factor 1 (Lef1) and distal-less homeobox 3 (Dlx3) are the transcription factors involved in regulating hair follicle development in mice, goats, and other animals. Their deletion can lead to hair follicle deficiency. In this study, hematoxylin–eosin staining (HE), real-time quantitative PCR (RT-qPCR), immunohistochemistry, and immunofluorescence were used to analyze the expression, location, and biological functions of Lef1 and Dlx3 in the lateral skin of Gansu Alpine Merino aged 1, 30, 60, and 90 days. The results revealed that the number of hair follicles decreased with age and was significantly higher at 1 day than in the other three age groups (p < 0.05). The mRNA levels of Lef1 and Dlx3 in the skin of 30-day old Gansu Alpine Merino were significantly higher than those in the other three age groups (p < 0.05). Protein expression of Lef1 and Dlx3 was lowest at 1 day (p < 0.05) and peaked at 60 days. Lef1 and Dlx3 exhibited a high density and strong positive expression in the dermal papillae; additionally, Dlx3 exhibited a high density and strong positive expression in the inner and outer root sheaths. Collectively, Lef1 and Dlx3 may facilitate the maturation of secondary hair follicles, which is mainly achieved through the dermal papillae and inner and outer root sheaths.

Endothelial PAS domain protein 1 gene (EPAS1) is a member of the HIF gene family. This gene encodes a transcription factor subunit that is involved in the induction of oxygen-regulated genes. Several studies have demonstrated that a mutation in EPAS1 could affect oxygen sensing, polycythemia, and hemoglobin level. However, whether EPAS1 mutation affects sheep oxygen metabolism is still unknown. Therefore, we explored the relationship between the variation of EPAS1 and oxygen metabolism in sheep. In this study, variations in ovine EPAS1 exon 15 were investigated in 332 Tibetan sheep and 339 Hu sheep by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. In addition, we studied the effect of these variations on blood gas in 176 Tibetan sheep and 231 Hu sheep. Finally, the mRNA expression of EPAS1 in six tissues of Hu sheep and Tibetan sheep living at different altitudes (2500 m, 3500 m, and 4500 m) was analyzed by real-time quantitative PCR (RT-qPCR). Four alleles (A, B, C, and D) were detected, and their distributions highly differed between Tibetan sheep and Hu sheep. In Tibetan sheep, B was the dominant allele, and C and D alleles were rare, whereas all four alleles were common in Hu sheep. Six single nucleotide polymorphisms (SNPs) were identified between the four alleles and one of them was non-synonymous (p.F606L). While studying the blood gas levels in Tibetan sheep and Hu sheep, one variant region was found to be associated with an elevated pO₂ and sO₂, which suggested that variations in EPAS1 are associated with oxygen metabolism in sheep. RT-qPCR results showed that EPAS1 was expressed in the six tissues of Hu sheep and Tibetan sheep at different altitudes. In addition, the expression of EPAS1 in four tissues (heart, liver, spleen, and longissimus dorsi muscle) of Hu sheep was lower than that in Tibetan sheep from three different altitudes, and the expression of EPAS1 was positively correlated with the altitude. These results indicate that the variations and expression of EPAS1 is closely related to oxygen metabolism.