Enzyme-linked Immunosorbent Assay Of The Fecal Progesterone For Early Pregnancy Diagnosis In Sows

Progesterone is a kind of steroid hormone that was produced mainly by corpus luteum after ovulation and the placenta during pregnancy. There is progesterone existing in blood, feces, urine, milk and saliva in sows, which can be used to determine concentration of progesterone for the purpose of assessing the ovarian functions, early pregnancy diagnosis and reproductive performance.According to the basic principle of the enzyme immunoassay, we have established ELISA method for detecting progesterone of porcine fecal and optimized the reaction condition. The coefficient of variation between microtiter plate intra-wells is 3.81%.Optimal coating concentration of antibody is 1:20000, and best working concentration of enzyme conjugate is 1:20000. The best blocking solution is 2% BSA (bovine serum albumin). The best antibody coating condition is 4℃ 12 hours and then 37℃ 2 hours again. Competition ELISA standard curve is Y=-10.71 X+62.751 (r2=-0.9947). The best reaction concentration of substrate is 0.4mg/mL. The best reaction dosage of hydrogen dioxide (30%H2O2) is 1.6μL/mL. The best reaction time of substrate is 37℃ 0.5 hour.According to Response curve-dose, we tested the correlated parameter about ELISA method by mathematic model A/AO-logC. The dose-responsive curve fitting equations is Y=-10.71 X+62.751 (r2=-0.9947); The sensitivity of progesterone determination was 12.13ng/g and its recovery rates were between 91～109.7%. The respective coefficients of intra-assay and inter-assay variation were 4% and 9.6%. Cross reactivities of progesterone antibody with estradiol, estriol, estrone was less than 0.01% and with testosterone rate was 0.35%.According to the assay of detecting progesterone in the porcine fecal, progesterone measurement has been used to check pregnancy in sows. The criteria for diagnosis of pregnancy based on the fecal progesterone level was positive for a progesterone level of= 150 ng/g and negative for a level of<150 ng/g. When fecal progesterone measurements were applied to early pregnancy diagnosis in 164 sows, the accuracy of diagnosis from day 21 to day 25 after the last mating was 95.9% for positive cases (138/146) and 72% for negative cases (13/18). Sow fecal progesterone enzyme immunoassay for early pregnancy diagnosis have a higher clinical applicability and it is significant to improve reproductive rates and farm economics.