Abstract:

To explore the feasibility of bovine mammary epithelial cell-derived exocrine miRNAs in the early diagnosis of subclinical mastitis by studying exosomes and their miRNAs and mammary gland related lactation factors and the differential expression of exosomes miRNAs(bta-miR-146a,bta-miR-224)in milk,and to find new targets for the early diagnosis and treatment of subclinical mastitis,providing theoretical basis and practical guidance for the early prevention and treatment of cow mastitis.Therefore,20 μg/mL LPS was used to stimulate bovine mammary epithelial cells and construct an in vitro subclinical bovine mastitis cell model.Then,the differential expression of bta-miR-146a and bta-miR-224,along with milk genes in the exosomes derived from mammary epithelial cells were detected.Further,milk from healthy and diseased cows were collected to detect the differential expression of bta-miR-146a and bta-miR-224 in exosomes.The results showed that subclinical bovine mastitis cell models could be successfully established with 20 μg/mL LPS treatment for 24 h.In this model,the expression of ACSL1,an essential marker for decreased milk yield and quality,was significantly reduced.Exosomes derived from mammary epithelial cell culture media and milk were successfully extracted and identified,with the lipid bilayer structure of exosomes clearly visible under transmission electron microscopy,and vesicle sizes ranged between 30 and 150 nm.The expressions of bta-miR-146a and bta-miR-224 in exosomes derived from both mammary epithelial cells and milk were significantly reduced following 20 μg/mL LPS stimulation.In conclusion,bta-miR-146a and bta-miR-224 in milk-derived exosomes could serve as candidate factors for early diagnosis of bovine mastitis.

Key words: Cow, Mastitis, Exosome, bta-miR-146a, bta-miR-224