V.L. Estergreen

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

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 11 alpha-hydroxyprogesterone hemisuccinate-horseradish peroxidase and were stable for 9 mo at -20 degrees 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.

Positive identification of corticosterone and cortisol in jugular plasma of dairy cattle

Abstract Corticosterone and cortisol have been positively identified in jugular vein plasma in dairy cattle under normal dairy environment. The corticosteroids were extracted from plasma with dichloromethane and purified by solvent partition and paper chromatography. Quantitative analysis was carried out by isotope dilution and spectrophotometry. Criteria for identification Included solvent partition, Chromatographic mobility on paper and thin-layer, ultraviolet, sulfuric acid and infrared spectrum analysis. Other criteria included a maintenance of constant specific activity after chromatography of the free steroids and the steroid acetates each in three different chromatography systems, and non-separation of the steroid from internal radioactive standards throughout the solvent and Chromatographic partitions. The levels found in plasma from 18 lactating, non-pregnant dairy cows varied from 1.5 to 5.1 μg (mean = 3.0 μpg) corticosterone and 3.2 to 13.1 μg (mean 7.2 μg) cortisol per 100 ml. The ratio of cortisol/cortico-sterone varied from 0.85 to 5.94 with a mean of 2.40 for these dairy cows.

The in vivo metabolites of [14C] progesterone in bovine muscle and adipose tissue.

Muscle and adipose tissue were obtained from steers and dairy cows following subcutaneous administration of [14C] progesterone. Following extraction, purification and separation by column, thin layer and gas-liquid chromatography, various radioactive residues from these tissues were identified by their chromatographic mobility, crystallization to constant specific activity and mass spectra. Progesterone constituted 54% of free radioactivity extracted from muscle and 69 and 73% of radioactivity in the free and conjugated portions of extracts, respectively, from fat. Metabolites identified were: 5alpha-pregnane-3,20-dione, 9%, 0%, 0%, 20beta-hydroxy-4-pregnen-3-one, 8%, 11%, 3%; 3alpha-hydroxy-5beta-pregnan-20-one, 13%, 2%, 2%, 3alpha-hydroxy-5alpha-pregnan-20one, 3%, 3%, 6%; 20 alpha-hydroxy-5alpha-pregnan-3-one, 0%, 2%, 3%; of radioactivity in muscle (free) and fat (free and conjugated fractions), respectively. Tentatively identified in fat extracts by chromatographic mobility were: 20alpha-hydroxy-4-pregnen-3-one, 1%, 1% and 3beta-hydroxy-5beta-pregnan-20-one, 0%, 2% of radioactivity in free and conjugated fractions, respectively. The average concentration of steroid in these animals due solely to treatment, calculated from the specific activity of the [14C] progesterone administered, was 3.4 and 18.1 ng/g in muscle and subcutaneous fat, respectively.

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins in vitro was investigated. Tissue supernatants were incubated with [4-14C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37 degrees C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 +/- 14.2% in liver, 5.0 +/- 3.6% in subcutaneous fat, and 3.7 +/- 2.2% in kidney fat samples. Progestins identified in liver samples include 5 beta-pregnane-3 alpha, 20 alpha-diol (free and conjugate), 5 beta-pregnane-3 alpha, 20 beta-diol (free and conjugate), 3 alpha-hydroxy-5 beta-pregnan-20-one (free and conjugate), 3 beta-hydroxy-5 beta-pregnan-20-one (free), 5 beta-pregnane-3,20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3 alpha-hydroxy-5 beta-pregnan-20-one, 20 beta-hydroxy-4-pregnen-3-one, and 20 alpha-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.