摘要Background Exosomes are crucial mediators of intercellular communication.As a key component of milk,milk-derived exosomes are abundant in genetic cargo,particularly microRNAs(miRNAs),indicating their potential role in regulating mammary gland physiology.Therefore,this study aimed to investigate the specificity of miRNAs in milk-derived exosomes and their regulatory roles in lipid synthesis in bovine mammary epithelial cells(BMECs).Results Based on 17,838 DHI records showing a significantly higher milk fat percentage(MFP)in late lactation(4.24％±1.07％),10 high-(5.96％±0.26％,HMF)and 10 low-MFP(1.68％±0.23％,LMF)cows were selected during this stage for milk-derived exosome isolation and miRNA profiling.Exosomes isolated via differential ultracentrifuga-tion were verified as 50-150 nm vesicles expressing CD9,CD81,and TSG101.miRNA sequencing identified 1,320 differentially expressed miRNAs(496 upregulated and 824 downregulated)between the HMF_EXO and LMF_EXO groups.Uptake assays confirmed that BMECs internalized these exosomes,and qRT-PCR validation showed that miR-423-5p and miR-125b were significantly upregulated and downregulated in HMF_EXO-and LMF_EXO-treated BMECs,respectively.Functionally,exosomal miR-423-5p promoted intracellular lipid accumulation and TG synthesis in BMECs by targeting APOA5,whereas miR-125b inhibited lipolysis and fatty acid oxidation by repressing SLC27A1.Conclusions This study demonstrates that milk-derived exosomal miRNAs represent a novel mechanism for regu-lating milk fat synthesis.Specifically,miR-423-5p and miR-125b directly modulated lipid metabolism in BMECs via the miR-423-5p/APOA5 and miR-125b/SLC27A1 pathways.These findings provide new insights into the molecular regulation of milkfat synthesis and highlight the importance of exosome-mediated intercellular communication in the lactating mammary gland.