Development of Estradiol Enzyme Immunoassay and its Application of Monitoring Reproductive Status in Taiwan Buffalo（Bubalus bubalis carabanesis）

Abstract

Estradiol （E2） is derived from cholesterol through androgens by granulosa cells in female ovaries. E2 is necessary to maintain the development of oocytes in the estrous cycle via a positive feedback system, which induces luteinizing hormone （LH） surge and ovulation through the hypothalamic-pituitary events. The monitoring of E2 primarily reflects the activities of the ovaries. The Enzyme Immunoassay （EIA） is a common technique, by detecting the specific binding of antigen and antibody. Thus, it is necessary to develop a highly sensitive and rapidly detective E2 EIA system. The rabbit anti-E2 polyclonal antiserum was made from rabbits which against E2-17β-carboxymethyloxime : BSA. The sensitivity of this E2 EIA revealed 7.13 pg/mL, and the r-square value of the standard curve was 0.958~ 0.984. Except estrone （2.04%）, estriol （1.51%） and androstanolone （0.08%）, the cross-reactivity of other steroids and BSA were lower than 0.05%. The coefficient of variation of intra- （n=5） and inter-assay （n=5） in this system were 8.53% and 9.19%, respectively. The total experimental duration from incubation to assay was 1- 1.5 hours. To validate this E2 EIA system, different kinds of samples were tested, including bovine follicular fluid and follicle cell culture medium.he population of Taiwan Buffalo has decreased from 21,876 to 2,950 since 1990 to 2008. Therefore, it is urgent to conserve Taiwan Buffalos. In the previous experiment, the blood levels of progesterone （P4） and E2 were quite low and the difference between follicular and luteal phase was too small to distinguish. Other than the result above, we also used Taiwan Buffalo as our animal model to prove the E2 EIA validation. In our experiments, blood and fecal samples of three buffalo cows were collected to measure the concentrations of E2 and P4 by EIA for assessing the pattern of estrous cycle. In the follicular phase, the serum P4 and E2 level was 1.1 +/- 0.5 ng/mL and 39.9 +/- 10.0 pg/mL, respectively. The fecal P4 was 161.0 +/- 30.2 ng/g and fecal E2 was 49.9 +/- 14.1 ng/g. During the luteal phase, the serum P4 concentration was 4.4 +/- 1.0 ng/mL, serum E2 was 30.4 +/- 8.2 pg/mL, fecal P4 was 230.0 +/- 52.1 ng/g and fecal E2 was39.9 +/- 10.0 ng/g. Based on these data of hormones, the length of estrous cycle was estimated to be 19.8 +/- 0.7days, within the follicular and luteal phase were 5.0 +/- 0.6 and 14.8 +/- 1.2 days, respectively in buffalo cows.n this study, the E2 EIA system has many advantages such as highly sensitive and rapid reaction. We successfully used this system for measurement the level of E2 in the bovine follicular fluid and follicle cell culture medium, and monitoring of the Taiwan buffalo reproductive status. It is expected that this E2 EIA system would be a valid technique to further understand animal reproduction.