The bidirectional differentiation potential of skeletal muscle satellite cells (SMSCs) enables them to differentiate into myofibers or intramuscular adipocytes, which affects meat quality in livestock. However, how insulin regulates ovine SMSC metabolism remains poorly understood. SMSCs were isolated from the longissimus dorsi muscle of 1-day-old Hu sheep, cultured, identified, and induced to differentiate with insulin. After induction, lipid droplet formation and the number of nuclei per cell were assessed, and samples were collected before adipogenic induction (No_AD) and after adipogenic induction (AD) for qPCR and whole-transcriptome sequencing. Immunofluorescence confirmed cells were positive for PAX7 and DESMIN. Bodipy, Oil Red O, and hematoxylin staining revealed lipid droplets and multinucleated cells. Sequencing and qPCR indicated that insulin promoted fatty acid uptake and utilization, inhibited adipogenic differentiation, and promoted myogenic differentiation. Integrated ceRNA analysis suggested that miR-2447-z and MSTRG.8123.1 may coordinate muscle development and lipid metabolism. In conclusion, under insulin induction, ovine SMSCs may undergo metabolic adaptation through the ceRNA network mediated by miR-2447-z and MSTRG.8123.1, exhibiting enhanced myogenesis, suppressed adipogenesis, and lipid droplet accumulation. These findings provide new insights into insulin-regulated SMSC metabolism, suggesting that leveraging the bidirectional differentiation potential of SMSCs to in-fluence muscle characteristics and fat deposition may be a feasible approach for im-proving meat production traits in sheep.

The proliferation and differentiation of myoblasts, as key regulatory processes for skeletal muscle growth, development, and in vivo balance, directly affect the economic value of livestock and poultry. Based on this, we examined the effects of Mettl21c (methyltransferase-like 21C) and carnosine (β-alanyl-L-histidine) on the proliferation and differentiation of ovine myoblasts. In this system, during the proliferation phase, Mettl21c overexpression reduced the EdU-positive cell rate and increased the apoptosis rate (p < 0.05), whereas interference had the opposite effect. These results indicate that Mettl21c inhibits myoblast proliferation and promotes apoptosis. At the transcript level, Mettl21c overexpression decreased Pax7 mRNA (progenitor marker) and increased Myf5, Myod1, and Myog mRNAs (MRFs, stage-specific myogenic markers). However, at the protein level, PAX7 increased while MYF5, MYOD1, and MYOG decreased. During the differentiation phase, interference with Mettl21c significantly reduced the cell fusion index and total myotube area (p < 0.05) while overexpression promoted these processes, indicating that Mettl21c promotes myoblast differentiation. At the same time, 1–2 mM carnosine was found to positively regulate myoblast proliferation, and carnosine increased the fusion index at 2 mM and total myotube area at 1–10 mM, which also positively regulated myoblast differentiation. However, Mettl21c perturbation altered the transcript levels of carnosine-metabolism genes (Carns1 and Cndp2), and carnosine treatment induced non-monotonic changes in Mettl21c mRNA expression. In summary, in primary ovine myoblasts, Mettl21c inhibits the proliferation of myoblasts and promotes their differentiation, while carnosine supplementation positively regulates both processes. Intracellular carnosine content and a functional interaction between Mettl21c and carnosine were not assessed in this study. These in vitro findings identify Mettl21c and carnosine as candidates for further studies of ovine myogenesis and muscle growth.

The genetic basis for Tibetan sheep adaptation to different high-altitude environments remains unknown. This study conducted whole-genome resequencing on 80 Tibetan sheep individuals from four major distribution areas on the Qinghai–Tibet Plateau. Based on the high-quality single-nucleotide polymorphisms (SNPs) obtained, an analysis of population-level genomic selection signals was performed. Population genomic analysis revealed that Tibetan sheep distributed across China originated in northern China but showed evidence of gene flow from South Asian sheep. Between populations from extremely high-altitude and mid-altitude regions, selection analyses identified five strongly positive selected genes (HIF1AN [Hypoxia Inducible Factor 1 Alpha Subunit Inhibitor], HBE1 [Hemoglobin Subunit Epsilon 1], HBE2 [Hemoglobin Subunit Epsilon 2], TNFAIP3 [TNF Alpha Induced Protein 3], RAD50 [RAD50 Double Strand Break Repair Protein]). These genes are associated with adaptation to hypoxia and intense UV radiation in high-altitude environments. Selection analyses between populations from extremely high-altitude and mid-altitude regions identified five strongly selected genes (HIF1AN [Hypoxia Inducible Factor 1 Alpha Subunit Inhibitor], HBE1 [Hemoglobin Subunit Epsilon 1], HBE2 [Hemoglobin Subunit Epsilon 2], TNFAIP3 [TNF Alpha Induced Protein 3], RAD50 [RAD50 Double Strand Break Repair Protein]) associated with hypoxia and intense UV radiation in high-altitude environments. Comparative genomic analyses of populations in cold and arid environments identified several candidate genes related to energy and water homeostasis, as well as hair development (TP53 [Tumor Protein P53], ATG101 [Autophagy Related 101], ATP12A [ATPase H+/K+ Transporting Non-Gastric Alpha2 Subunit], KRT80 [Keratin 80], KRT7 [Keratin 7]). Additionally, Tibetan sheep in the high-altitude arid deserts exhibit stronger adaptive selection for energy homeostasis and water utilization; meanwhile, the HIF-1 [Hypoxia Inducible Factor 1] signaling pathway helps counteract oxidative stress induced by extreme water scarcity in the plateau environment. Our study supports the hypothesis that Tibetan sheep originated in northern China and identifies distinct adaptive features in the Tibetan sheep genome corresponding to their habitats.

The lung is a key organ for oxygen uptake and gas exchange and plays an important role in adaptation to hypoxic environments at high altitudes. RNA modifications represent an important layer of epitranscriptomic regulation and participate in cellular processes by affecting RNA stability, splicing, translation, and degradation. In this study, the right lung parenchyma from low-altitude Hu sheep (LL), mid-altitude Tibetan sheep (ML), and high-altitude Tibetan sheep (HL) were used to investigate altitude-associated RNA modification profiles and m⁶A regulatory patterns. A UPLC-MS/MS platform was employed to quantitatively detect 55 RNA modifications, while RT-qPCR and Western blot analyses were performed to evaluate the expression of HIF1α, HIF2α, and m⁶A-related regulatory factors. The results showed distinct differences in lung RNA modification profiles among the three groups. Compared with the LL and ML groups, the HL group exhibited reduced m⁶A and m⁶Am levels, whereas several non- m⁶A modifications, including t⁶A, m¹G, m²G, m⁷G, m³C, and m⁵C, showed increasing trends. The expression levels of HIF1α and HIF2α were higher in the ML and HL groups than in the LL group. In addition, METTL3, METTL14, WTAP, and several YTH family reader proteins were downregulated in the HL group at both the protein and mRNA levels. These findings indicate that sheep lung RNA modification profiles and the m⁶A regulatory machinery differ among groups with different altitude-related adaptation backgrounds. Collectively, these findings reveal epitranscriptomic differences associated with altitude and breed in sheep lungs and provide a basis for further investigation of the role of m⁶A regulation in high-altitude adaptation.