Abstract: This study was conducted to investigate the effect of exogenous leucine supplementation on the proliferation of bovine myogenic cells and its mechanism. The effects of different concentrations of Leu on the proliferation of bovine myoblasts were examined by CCK-8 and EdU staining, and the optimal concentration of Leu was determined; transcriptome sequencing (RNA-Seq) was performed with the optimal concentration of Leu as the treatment group and 0 mmol·L^-1 Leu as the control group to screen the key pathways of Leu regulating the proliferation of bovine myoblasts. Key regulatory genes in the key pathway were screened using qRT-PCR and validated by adding inhibitors or activators. Addition of Leu increased bovine myogenic cell viability, with 0.5 mmol·L^-1 Leu significantly increasing myogenic cell viability and EdU positive rate (P<0.05). There was 1 290 differentially expressed genes between Leu group and control group, of which 688 were upregulated and 602 were downregulated. The 309 up-regulated genes annotated by KEGG enrichment analysis were involved in 47 significantly enriched pathways, among which the most enriched pathway related to skeletal muscle growth and development was the PI3K-AKT signaling pathway, containing 34 up-regulated genes. Six differentially expressed genes, including PIK3R1, FOXO3, IRS1, RPTOR, MTOR and MET, were randomly selected for qRT-PCR validation, and the results were consistent with the result of RNA-Seq. The addition of LY294002, an inhibitor of PI3K in the PI3K-AKT signaling pathway, blocked the proliferation-promoting effect of Leu on bovine myogenic cells. In conclusion, Leu can promote the proliferation of bovine myogenic cells through PI3K-AKT signaling pathway.

Key words: Leu, bovine, skeletal muscle, myoblast proliferation, RNA-Seq, PI3K-AKT