A radioimmunoassay for free estradiol-17.beta., conjugated estradiol-17.beta. or total (free + conjugated) estradiol-17.beta. in defatted milk of cows is described. Conjugated estradiol-17.beta. was hydrolyzed by enzymes of Helix pomatia juice. Estrogens were extracted with dichloromethane; no other purification step was required before radioimmunoassay because of the high specificity of the antiserum. Immunoprecipitation was used to separate bound and free estradiol-17.beta.. Concentrations measured were corrected for procedural losses on a per sample basis. The assays were shown to be accurate and specific. The sensitivity was 1.3pg/ml for the assay of free estradiol-17.beta. (5 ml of milk extracted) and 2.9pg/ml for conjugated or total estradiol-17.beta. (2 ml of milk hydrolyzed and extracted). Estrogens were measured in the milk of cyclic cows and in cows stimulated with pregnant mare serum gonadotropin (PMSG). A preovulatory increase was clearly observed. Whether or not the ovary was stimulated by PMSG, concentrations of estrogens were higher and the relative increase during the preovulatory peak was greater for conjugated estradiol-17.beta. than for the free form. The assay of conjugated or total estradiol-17.beta. in defatted milk should be a practical method for assessing preovulatory growth of follicles in cows.
