Identification And Analysis Of Milk Exosomes MiRNAs In Cows With Milk Fat Depression

To meet the energy needs of high-yielding cows,grains and oils are often added to the diet in dairy farming.This feeding pattern increases the incidence of low milk fat syndrome in dairy cows,causing economic losses to dairy production.Conjugated linoleic acid is a polyunsaturated fatty acid containing conjugated double bonds.CLA has been found to inhibit the synthesis of mammary fat in cows,causing MFD in cows.Exosomes refer to functional vesicles secreted by different types of cells.Exosomes have been found to play an important role in disease diagnostic markers and lipid metabolism.Therefore,CLA was used to induce MFD generation in dairy cows and extract and analyze the differentially expressed miRNAs in milk exosomes,which provided basic data for the application of milk exosome miRNAs in dairy cow MFD.(1)Cow experiment: The experiment selected 12 healthy Holstein lactating cows and randomly divided into control group and experimental group,cows fed basal diet as the control group(Con group),cows fed basal diet +400 g/d CLA as the experimental group(CLA group),the trial lasted for 10 days,milk was collected on days 0,2,4,6,8,9 and 10 for milk yield and milk composition determination,and exosomes were extracted and identified by ultracentrifugation on day 10.Expression profiling of bovine milk exosomes was analyzed using high-throughput sequencing to screen differential miRNAs.The results showed that CLA had no significant effect on milk yield,milk protein and lactose content of dairy milk with the increase of feeding days,but the milk fat content decreased significantly,indicating that CLA successfully induced MFD formation in dairy cows.Milk exosomes were observed by transmission electron microscopy,and it was found that they were cup-disc-shaped,with exosome particle size distribution between 30-150 nm,and western blotting detected exosome marker proteins TSG101 and CD63,indicating that milk exosomes were successfully extracted by ultracentrifugation.High-throughput sequencing of miRNAs in bovine milk exosomes showed a total of 46 differential miRNAs,of which 13 were significantly upregulated and 33 were significantly down-regulated.Through GO analysis,it was found that 291 candidate target genes were enriched in the lipid metabolic process,indicating that differentially expressed miRNAs may play a role in lipid metabolism.The target genes of significantly different miRNAs were predicted by miRanda,Targetscan and PITA,and the miRNAs and their target genes mainly involved in fat metabolism were screened out.(2)Cell test: In vitro cultured cow mammary epithelial cells,transfected and screened three differential miRNAs(miR-126-5p,miR-935,miR-2284c),RT-PCR detected the expression of miRNA in cells,and the results showed that all three miRNAs were significantly expressed.Target gene promoters were constructed,and the cells were co-transfected with different miRNAs.The activity of target gene promoters was detected by double luciferase.The results showed that miR-126-5p significantly inhibited the promoter activity of target gene SCD,and miR-935 significantly inhibited the promoter activity of target gene PPARGC1 A.miR-2284 c had no significant effect on the promoter activity of target gene SREBF1.To sum up,this experiment treated dairy cows with CLA,extracted milk exocrine body for high-throughput sequencing,and screened miR-126-5p,miR-935,and miR-2284 c miRNAs involved in MFD.In vitro cell experiments confirmed that miR-126-5p and miR-935 can inhibit the promoter activity of target genes.These results lay the foundation for milk exosomal miRNAs as diagnostic markers for MFD in dairy cows.