Dynamic Changes Of Peripheral Blood Kissspeptin And MiRNA Screening Of Exosome Source Differences In Dairy Cows During Early Pregnancy

Holstein cows are the highest milk producing and most numerous cows in China,and are also a high quality dairy breeding resource in Ningxia and surrounding areas.Improving the fertility of Holstein cows in Ningxia can therefore increase the stock and milk production of Holstein cows in Ningxia,which is an important guarantee for the economic benefits of dairy farms.To investigate the changes in plasma Kissspeptin concentration and plasma exosomal miRNA in early gestation,it is important to investigate the reproduction of Holstein cows.In this study,plasma was collected from four uninseminated cows at 16d,19d,22d,25d and 28d from 26 cows that were regularly inseminated at the same time,and the differences in the levels of Kissspeptin,progesterone,oestrogen and other immunosuppressive factors were measured by ELISA.In addition,blood was collected from 16d,19d,22d and 25d from four uninseminated cows and 26 cows that were regularly inseminated at the same time.Leukocytes were extracted from the blood and the expression of the early pregnancy factors ISG15,MX1,MX2 and OAS1 was determined by fluorescence quantification(RT-qPCR).Finally,plasma was collected from four un-inseminated cows and 26 cows that were regularly inseminated at the same time for their 16d,19d and 22d blood separations.Using highthroughput sequencing technology,the changes in plasma exosomal miRNAs of cows at different periods of pregnancy were analysed to find possible markers,providing a new way and reference for early pregnancy physiological research and pregnancy diagnostic biomarkers in cows.The main results obtained from the experiment were as follows:1.There were no significant differences in plasma Kissspeptin levels between cows in the pregnant and uninseminated groups.Kissspeptin levels in cows in the pregnancy group were higher than Kissspeptin levels in cows in the uninseminated group at 16 d,19 d,22 d,25 d and 28 d after artificial insemination,but there were no significant differences in Kissspeptin levels at any of the time periods.The progesterone concentrations of cows in the pregnancy group increased with the number of days after artificial insemination,and the progesterone concentrations of cows in the pregnancy group were higher than those of cows in the uninseminated group at 16 d,19 d,22 d,25 d and 28 d after artificial insemination,with the plasma progesterone concentrations of cows in the pregnancy group being significantly higher than those of cows in the uninseminated group on day 22(P<0.05)and plasma progesterone concentrations of cows in the pregnancy group being significantly higher than those of cows in the uninseminated group on days 16 and 28.Plasma progesterone concentrations were significantly higher in the pregnant group than in the uninseminated cows on days 16 and 28(P<0.01).In addition,the levels of interferon-inducible genes ISG15,MX1,MX2 and OAS1 in the leucocytes of the pregnant cows increased with the number of days after insemination.The levels of ISG15 and MX1 were significantly higher in the 19 and 22 d gestation groups than in the uninseminated cows(P<0.05)and the levels of MX2 and OAS1 were significantly higher in the 25 d gestation group than in the uninseminated cows(P<0.05).2.A total of 64 differentially expressed miRNAs were detected from plasma exosomal miRNAs at 16d,19d and 22d in pregnant and un-inseminated cows,which were useful for early pregnancy diagnosis.The results of GO and KEGG enrichment analysis indicated that the differentially expressed miRNA target genes in the 16d and 16d gestational groups mainly targeted metabolic processes,kinase activity and protein binding,mainly in the metabolic pathway,mTOR signaling pathway,MAPK signaling pathway and HIF-1 signaling pathway;the differentially expressed miRNA target genes in the 19d and 19d gestational groups mainly targeted positive regulation of transcription,keratin fibrils,protein binding and cyclin regulator activity,mainly in the VEGF signaling pathway and The KEGG enrichment pathway is mainly involved in the regulation of oxytocin signaling pathway,MAPK signaling pathway,insulin signaling pathway,cAMP signaling pathway and calcium signaling pathway and calcium signalling pathways.