ASSAY OF FREE AND CONJUGATED ESTROGENS IN PERIPHERAL PLASMA OF CYCLIC COWS

Conjugated oestrone (E1), oestradiol-17β (E2β), and oestradiol-17β (E2α) were measured in the same plasma sample of cyclic cows. After enzymatic hydrolysis oestrogens were extracted with diethyl either and an initial chrmomatography on a micro-column of Sephadex LH 20 allowed the separation of E1 from both E2. As it is very difficult to separate the 2 epimers of E2, E2β was specifically oxidized to E1 so that following a second chromatography, the E2 fraction contained only E2α. Thereafter, the 3 oestrogens were quantified by radioimmunoassay. The method was validated for bovine plasma. As free oestrogens were not separated from the conjugated oestrogens prior to hydrolysis total E1, E2β and E2α (TE1, TE2β, TE2α) were assayed. TE1, TE2β and TE2α during the bovine cycle were at a higher level than the corresponding non-conjugated estrogens. A preovulatory increase was evident only for TE2β but neither its duration nor its relative increase were larger than of E2β.