Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows

Abstract

The purposes of this study were to develop an enzyme immunoassay (EIA) for determination of progesterone in unextracted whole milk and to apply it to pregnancy diagnosis. Paper fibers covalently coupled to antibody specifically and competitively bound 3H-progesterone and 11α-hydroxyprogesterone hemisucccinate-horseradish peroxidase and were stable for 9 mo at −20°C. The sensitivity, recovery, precision, and cross reactivity of the EIA and a radioimmunoassay (RIA) of milk progesterone were evaluated, and showed that this EIA was comparable to the RIA. Milk samples were collected once daily for one estrous cycle from ten lactating Holstein cows and the progesterone levels were determined by RIA. Milk progesterone in 70 samples measured by EIA were highly correlated (r = 0.90) with the values of RIA for the same samples. Milk samples for pregnancy diagnosis by EIA of milk progesterone were obtained daily from days 20 to 24 after 115 artificial inseminations of 85 lactating Holstein cows. Pregnancy diagnosis by EIA was confirmed by rectal palpation at 30 to 60 days after insemination or return to estrus. The accuracy based on single, two, three, four, and five consecutive samples was from 67.2 to 80.0%, 77.3 to 84.0%, 79.2 to 87.5%, 82.0 to 85.4%, and 85.4%, respectively, for pregnancy diagnosis; and from 95.0 to 98.3% for nonpregnancy.