| [Objective] Early pregnancy diagnosis is an important part of the sheep industry.At present,with the development of most farms in the direction of scale,the identification time of pregnant sheep has higher requirements.For many years,scholars have been seeking the fastest method of identification.Studies have shown that early pregnancy factors are the earliest established pregnancy indicators.Given the previous research on early pregnancy factors,blood is one of the detection pathways for early pregnancy factors.During early pregnancy,progesterone inhibits luteinizationand interferon(IFN)induces a stimulating factor that has a role in early embryos through the JAK-STAT pathway.The purpose of this trial is based on the IFN-induced JAK2-STAT1 pathway and the mechanism of action of progesterone on early maternal maternal and to compare the differences in the changes of early pregnancy factors and related progesterone levels in each period,in order to find the relevant factors for early diagnosis of pregnancy and its clinical diagnosis time.[Method] In this experiment,40 primiparous ewes were treated with chloroprostol sodium for the same period,and the test was performed 48-72 hours after the injection.Then select 30 ewes with the same estrus time for breeding.The test was carried out 14-17 days after breeding.After the test,6 sheep were found,and the remaining 24 ewes were pregnant ewes.The 24 pregnant ewes were divided into Group I and Group II,and Group I was used as the control group of the returning ewes.The number of sheep should be the same as the number of ewes returning,so Group I was not returned to the mother.For the 6 sheep,the number of Group II ewes is the remaining 18.Group III is 6 returning ewes.A total of 12 ewes in Group I and Group III were used for the determination of progesterone,IFN,and genes.Group II totaled 18 unreturned ewes for protein determination.Test 1: Fresh blood of healthy ewes at 0d,5d,15 d,20d,25 d after seeding was taken,serum was separated,and the content of progesterone and interferon was detected by ELISA.Test 2:The fresh blood of healthy ewes at 0d,5d,15 d,20d,25 d after breeding was taken,and mononuclear cells in blood were extracted.The early pregnancy factors Cox1,Cox2,Isg15,Oas1,Mx1 were screened by fluorescence quantitative(qRT-PCR)and detect the expression level of related genes.Trial 3: The fresh blood of healthy ewes 5d,10 d,13d,16 d,25d after the breeding was taken,the total protein in the blood was extracted,and the expression of related early pregnancy factor protein in blood was detected by Western blotting.[Results](1)Progesterone can maintain the maternal pregnancy status and reduce the content of prostaglandins.Cyclooxygenase is the rate-limiting enzyme of prostaglandins.Progesterone inhibits the production of cyclooxygenase-2,but has no significant effect on the expression level of cyclooxygenase-1.(2)Interferon increased the expression level of interferon-stimulated genes Isg15,Oas1,and Mx1 15 days before breeding by activating JAK2-STAT1 signaling pathway,and the expression level increased 15 days after breeding;The mRNA expression of Mx2 was decreased in the first 10 days after breeding,and the expression level was increased after 10 days of breeding.The protein expression levels of Isg15,Oas1 and Mx1 were the highest in 13 days of breeding.(3)By analysis and comparison,the Isg15 gene can show differences between groups after breeding 10,and the trend of the two groups is opposite after 15 days of breeding.The expression level of Isg15 protein was the highest in the 13 th day of breeding,and the expression level of the Isg15 protein was extremely significant at 10 and 16 days.Therefore,Isg15 can be used as an indicator for early pregnancy diagnosis at 13 days of gestation.[Conclusion] Progesterone has antagonistic effect on cyclooxygenase-2,but has no obvious effect on cyclooxygenase-1.Interferon induces the expression of interferon-stimulated genes through positive regulation of the JAK2-STAT1 signaling pathway.Because Isg15 factor can appear between groups in 10 days of breeding,and the trend of the two groups is opposite after 15 days of breeding,Isg15 can be used as an indicator for early pregnancy diagnosis. |
