Study On The Expression Of Early Pregnancy Factors In Sheep Reproductive Organs And The Detection Technology Of Early Pregnancy

Early pregnancy factor(EPF)is one of the earliest growth factors detected in the serum of mammals after fertilization.it has been used in early pregnancy diagnosis and embryo quality monitoring in female mammals.With the rapid development of large-scale and industrialization of sheep industry,the application of early pregnancy diagnosis technology to evaluate the pregnancy efficiency of livestock,especially ruminants with relatively low lambing rate,is very important to improve reproductive efficiency and economic benefits.Five healthy and healthy ewes of 3 months old(before sexual maturity),6 months old(sexual maturity)and 12 months old(after sexual maturity)were selected to study the difference of EPF localization and m RNA expression in ovary,fallopian tube and uterus by immunohistochemical method and real-time fluorescence quantitative method,in order to reveal its role in regulating sheep reproduction.In this experiment,the blood,vaginal collection and cervical mucus of small tail Han sheep were studied by enzyme linked immunosorbent assay(Elisa).Five pregnant ewes were selected on the 0th,5th,10 th,15th,20 th,25th and30 th day after insemination,and the concentrations of early pregnancy factor in peripheral serum,vaginal collection and cervical mucus were detected.The differences of progesterone and early pregnancy factor in each body fluid in each period of one month were compared in order to find the best clinical diagnosis time and collection site with early pregnancy factor as pregnancy detection index.to show whether the early pregnancy factor is more convenient than the traditional detection method.Through the above research,the following results are obtained:1.Immunohistochemical method was used to study the expression and localization of EPF protein in three main reproductive organs of sheep at different months.The results showed that EPF protein was expressed in uterine horn,ovary and fallopian tube of 3-month-old,6-month-old and 12-month-old sheep.The expression of EPF m RNA in uterine horn,ovary and fallopian tube of sheep at different ages was studied by real-time fluorescence quantitative PCR.The results showed that EPFm RNA was expressed in three main reproductive organs of sheep at different ages,and the expression levels were different.The main results were as follows:(1)The expression of EPF gene m RNA was different in sheep of the same month and different reproductive organs.There was significant difference in the expression of EPF gene m RNA in ovary and uterine horn of 3-month-old sheep(P<0.01),but there was no significant difference between ovary and uterine horn(P>0.05);and there was no significant difference in EPF gene m RNA expression among three reproductive organs of 6-month-old sheep(P>0.05);The expression of EPF gene m RNA in ovary,uterine horn and fallopian tube of 12-month-old sheep was significantly different(P<0.01),but there was no significant difference between ovary and uterine horn(P>0.05).(2)there were differences in the expression of EPF gene m RNA in the same reproductive organs at different ages.The expression of EPF gene m RNA in uterine horn was significantly different between 3-month-old and 6-month-old and12-month-old(P<0.05),but there was no significant difference between 3-month-old and 6-month-old and 12-month-old(P>0.05);The expression of EPF gene m RNA was significantly different between 3-month-old and 6-month-old and 12-month-old(P<0.01),but not between 6-month-old and 12-month-old(P>0.05).;The expression of EPF gene m RNA in ovary was significantly different between3-month-old and 6-month-old III and 12-month-old(P<0.05),but not between6-month-old and 12-month-old(P>0.05).2.The expression of early pregnancy factor and progesterone in blood,cervical mucus and vaginal samples of sheep on the 0th,5th,10 th,15th,20 th,25th and 30 th day of mating were studied by double antibody sandwich enzyme-linked immunosorbent assay(ELISA).The results showed that there were significant differences in the expression of progesterone and early pregnancy factor in blood,vaginal collection and cervical mucus of sheep on the 10 th day of mating(P < 0.01),and there was no significant difference in the expression of progesterone and early pregnancy factor in blood,vaginal collection and cervical mucus of sheep with the same mating days(P>0.05).There was a significant correlation between progesterone and early pregnancy factors within 1 month of mating(P < 0.01).