Functional Analysis and Identification of MiRNAs Associated with Lipid Metabolism from Milk-Derived Exosomes

Background Exosomes have recently been considered as major players in cell-cell communication. Milk-derived exosomes contained abundant genetic cargos and the potential biological functions in the synthesis of milk fat remain poorly understood. Milk fat percentage is a crucial trait that influences dairy quality and consumer preference. This study focused on the regulatory role of milk-derived exosomal miRNAs in lipid synthesis within BMECs. Results Initially, based on 17,838 DHI milk production performance data collected from January 2021 to February 2023, compared with early lactation , mid-lactation from 100 to 199 days, late lactation from 200 to 299 days, duration lactation the milk fat percentage (MFP) of late lactation (4.24±1.07%) significant higher than other stages. The milk protein percentage during this stage (3.43±0.36) is significantly higher compared to other stages. The heritability estimates for milk fat and milk protein percentage show a positive correlation. Subsequently, 10 cows with high MFP （5.96±0.26 ) and ten cows with low MFP (1.68±0.23) were selected during the late lactation stage, and milk samples were collected for further analysis. Milk-derived exosomes isolated via differential ultracentrifugation exhibited a spherical vesicle structure ranging in size from 50 to 150 nm and were enriched in exosome-specific protein markers CD9, CD81, and TSG101. Through miRNA-seq, 1320 differentially expressed miRNAs were identified including 496 up-regulated and 824 down-regulated miRNAs by high-throughput miRNA sequencing. Furthermore, the exosome uptake experiment revealed that upon BMECs, exosomes predominantly localized in the cytoplasm.The results of exosomal q-PCR demonstrated that miR-423-5p and miR-125b were significantly up-regulated and down-regulated in HMF_EXO and LMF_EXO groups, respectively. Conclusions Milk-derived exosomal miRNAs associated with lipid metabolism can provide preliminary insights into the effects of exosomes on lactation mechanisms and potentially identify biomarkers for distinguishing molecular markers and loci related to high or low milk fat content in dairy cattle.