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β-hydroxybutyrate (BHBA) is utilized as precursors of de novo synthesized fatty acids in mammary gland of dairy cows.Butyric acid is absorbed and metabolized into BHBA by ruminal epithelium cells.However,perinatal dairy cows usually experienced a period of negative energy balance (NEB) induced by decreased dry matter intake and increased energy demand.Excessive NEB leads to lipid mobilization then results in high blood concentrations of non-esterified fatty acids, which further increased the concentration of BHBA in blood.Sterol regulatory element-binding protein-1c (SREBP-1c) and cell death-inducing DNA fragmentation factor-alpha-like effector α (Cidea) play a crucial role in lipid synthesis.Therefore, we hypothesized that BHBA could make SREBP-1c/Cidea pathway overactivity to increase milk fat synthesis in bovine mammary epithelial cells.The bovine mammary epithelial cells were treated with different concentrations of BHBA (0, 0.6, 1.2, 2.4 mmol/L) and transfected with adenovirus to silent expression of SREBP-1c.The effects of BHBA on the lipid synthesis in bovine mammary epithelial cells were investigated using qRT-PCR and Western-blotting.The results showed that BHBA could significantly increase the mRNA levels of SREBP-1c, fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACC-α) and Cidea and diacylglycerol transferase-1 (DGAT-1), with the highest levels in the 1.2 mmol/L BHBA-treated group.What s more, the protein levels of SREBP-1c, FAS, ACC-α and Cidea were also significantly increased in a dose-dependent manner.SREBP-1c silencing adenovirus significantly decreased mRNA and protein expression of SREBP-1c.SREBP-1c silencing significantly decreased the expression of FAS,ACC-α, Cidea and DGA T-1 in the 1.2 mmol/L BHBA+SREBP-1c silencing group compared with the 1.2 mmol/L BHBA group.The content of TG in the supernatant was significantly higher in the 1.2 mmol/L and 2.4 mmol/L BHBA group than in the control group.However, when SREBP-1c was silent, the content of TG was dramatically decreased in the 1.2 mmol/L BHBA+SREBP-1c silencing group compared with the 1.2 mmol/L BHBA group.BHBA treatment also increased the transfer of mature SREBP-1c and the fluorescence intensity in the nucleus was significantly higher in the 1.2 mmol/L BHBA group than in the control group.However, it was significantly decreased in the 1.2 mmol/L BHBA+SREBP-1c silencing group.The present data indicate that BHBA can promote fatty acids synthesis and significantly increase TG synthesis mediated by SREBP-1c in bovine mammary epithelial cells.