Varieties Of MiRNA In Milk Exosomes Of Dairy Cows In Early Pregnancy

Early pregnancy is a critical period for dairy cows to conceive and live.Changes in the physiological state of dairy cows at this stage,such as physical exercise,physiological regulation,and activity status,will change.In order to fully understand the early physiological stages of dairy cows,the accuracy of early diagnosis of dairy cows can record the physiological state of milk exosomes,participating in a wide range of milk activities,is a good biomarker,and its changes can be used as an important indicator of whether a dairy cow is pregnant or not.At present,there are many studies on exosomes in dairy cows during pregnancy,but most of the exosomes come from milk suction,placenta and fallopian,and the time is 21 days after artificial insemination.Regarding the situation of milk exosomes in early dairy cows,about early dairy cows in the study of milk exosomes,a large number of changes in dairy cow milk exosomes must be obtained first,but there is no research report yet.It is suitable for the extraction method of milk exosomes.Therefore,this study first used three established milk exosomes extraction methods,including rennet extraction,isoelectric point extraction,and EDTA extraction,to extract exosomes,and then morphology of the three exosomes.Comparison of structure,particle size,surface specific protein expression and other indicators,select and optimize a milk-derived exosomes extraction method.Newborns(D10,D15,D20,and D30)milk exosomes are biologically repeated for three periods each time.Using high-DO miRNA technology,analyze the composition and distribution of milk exosomes in dairy cows at different stages of pregnancy,and the changes in extrinsic miRNAs of pregnant women’s early milk,looking for possible markers,which are biomarkers for early pregnancy physiological research and birth diagnosis in cattle Provide new ways and references.The following results were obtained:1.According to the identification index of exosomes obtained by rennet extraction method,isoelectric point extraction method and EDTA extraction method.The three methods can be used to extract milk exosomes.The rennet extraction method can isolate a large number of milk exosomes with complete structure and high purity,which is more suitable for the extraction of milk-derived exosomes.2.Based on high-throughput sequencing technology,this experiment detected a total of2442 miRNAs from 15 milk exosomal miRNA groups of dairy cows at different stages of pregnancy,of which 335 were known miRNAs and 2107 were newly discovered miRNAs.There are 30 pairs of chromosomes distributed in dairy cows,and the most widely distributed on the X chromosome.3.The Venn diagram results show that a total of 18 miRNAs are expressed in 5 different gestational stages of dairy cows,bta-let-7a-5p,bta-let-7b,bta-mi R-30a-5p,bta-mi R-1307-R+1 and bta-mi R-11987-L-1-1ss8 TA are only expressed in dairy cows during pregnancy.4.The results of trend analysis showed that this experiment showed a total of 20 expression trends from 15 milk exosomal miRNA groups of dairy cows at different gestational stages,and a continuous upward trend was a significant enrichment trend.A total of 41 miRNAs were included,including bta-mi R.The expression of-30a-5p is high,the fold change is significant,and it is specifically expressed during pregnancy.11 miRNAs with high expression levels and significant differences were selected from 41 miRNAs that have a significant and continuous upward trend during different pregnancy periods,and compared with the d-0 days of change,the later q RT-PCR verification was carried out to verify the change pattern.The expression pattern is similar to the RNA sequencing result,indicating that the sequencing result is reliable.5.Target gene prediction,GO and KEGG pathway analysis results show that there is a significant and continuous upward trend and high expression level in 5 different pregnancy stages,and miRNAs with large changes are mainly enriched in cancer signaling pathways,endocytosis,and focal adhesion kinase Signal pathway,MAPK signal pathway,actin skeleton regulation,c AMP signal pathway,chemokine signal pathway,axon guidance,etc.The above results indicate that the rennet extraction method is more suitable for the extraction of milk-derived exosomes;the composition and content of miRNA in milk exosomes of dairy cows in early pregnancy have changed significantly,and bta-mi R-30a-5p can be used as potential biomarkers for pregnancy diagnosis.