Abstract: 【Objective】 To investigate the effects of non-esterified fatty acid(NEFA) stress on mitochondria function and fatty acid metabolic processes of bovine skeletal muscle cells,and to provide reference for revealing the mechanism of nutritional metabolic diseases in cows in the perinatal period.【Method】 A NEFA-stressed bovine skeletal muscle cell model was established. After the optimal combination of concentration and time stimulation,mitochondrial morphological changes were observed by transmission electron microscopy,lipid deposition was observed by oil red O staining and BODIPY 493/503 staining,and mitochondrial membrane potential was detected by flow cytometry. The activities of catalase(CAT) and superoxide dismutase(SOD) and the content of malondialdehyde(MDA) were detected by the kit. The expression of genes related to mitochondrial function and lipid metabolism was detected by real-time fluorescent quantitative PCR.【Result】 Considering the effect of NEFA on survival rate and morphology of bovine skeletal muscle cells,1.5 mmol/L NEFA stimulation for 2 h was selected as the optimal dose-time combination for subsequent experiments. After 2 h stimulation with 1.5 mmol/L NEFA,the mitochondrial volume of bovine skeletal muscle cells became smaller,the matrix color was deepened,the mitochondrial ridge arrangement was disordered,and tended to be fused,even some of them were vacuolar. The number of cells with decreased mitochondrial membrane potential was greatly increased by 38.08% compared with the control group(stimulation 0 h) (P<0.05,the same below). Compared with the control group(stimulation 0 h),the content of MDA in the supernatant of cell culture was significantly increased by 1.38 times,while the intracellular CAT activity was extremely significantly decreased by 60.63%(P<0.01,the same below),and the activity of SOD was significantly decreased by 34.21%. Real-time fluorescent quantitative PCR detection results showed that after 2 h stimulation with 1.5 mmol/L NEFA,the relative expression levels of NRF1 and PGC-1α genes regulating mitochondrial gene replication and transcription,MFN2 gene regulating mitochondrial fusion,mitochondrial respiratory chain key enzyme genes(except COV gene), Patatin-like phospholipase domain 2 protein gene(PNPLA2) and long chain lipoic acid extension enzyme 6 gene(ELOVL6) were extremely significantly decreased, the relative expression of carnitine lipoacyltransferase 1B gene(CPT1B) was extremely significantly increased.【Conclusion】 High concentration of NEFA has lipid toxicity on bovine skeletal muscle cells,affecting mitochondrial morphology,function and membrane potential,causing mitochondrial dysfunction and abnormal cellular fatty acid metabolism,resulting in oxidative stress,and inducing abnormal lipid deposition.