There has been considerable disagreement and confusion on the concentration of progesterone in cow's milk. Previous studies in our laboratory (1) indicated that the concentration of progesterone during days 30-210 of pregnancy was approximately four times higher in milk (21.2 ng/ml) than in blood plasma (5.3 ng/ml). The samples were taken approximately 2 hr after the morning milking. Laing and Heap (2) have reported that the progesterone concentrations in the milk of non-pregnant and late pregnant cows were similar to those in plasma, whereas in early pregnancy the concentrations were apparently much higher in milk. However, in a subsequent study these workers (3) were unable to confirm the high concentrations in milk. The possible existence of progesterone metabolites which interfere with assay procedures has been considered (2) and differences in assay techniques among laboratories may also be a source of different findings.Another possible explanation for divergent results among laboratories may be differences in the method of obtaining milk samples. It is well known that the amount of fat in milk samples is dependent on the time during the milking process at which a sample was obtained (4). There are indications (5) that changes in milk progesterone and fat concentrations show a high positive correlation when an active corpus luteum is present. Thus some of the discrepancies among laboratories may be due to differences in the time or method of milk sampling.The present experiments were conducted to compare the effects of different progesterone antisera, purification procedures and determination methods (RIA vs. GLC) for the quantitation of progesterone in cow's milk. In addition, the effect of time of sampling during the milking process on progesterone concentration was studied.Materials and Methods. Experiment I . As part of a previous study, samples of milk (40 ml) were obtained from 8 cows on each of days 30, 60, 90, 120, 150, 180 and 210 of pregnancy and were stored at -20" (1). In the present project, samples were randomly selected from three cows on each of days 30, 120 and 210 after the samples had been stored for 10 mo. Progesterone concentration was quantitated for each sample by 3 assay methods: (a) radioimmunoassay after ether extraction and LH-20 chromatography (RIA-1) (b) radioimmunoassay after ether extraction, LH-20 chromatography and additional purification procedures (RIA-2), and (c) gas-liquid chromatography after initial processing identical to that used for RIA-2 (GLC).Tritiated progesterone (1, 2, 6, 7-3H progesterone, New England Nuclear; RIA-1, 1,000 cpm; RIA-2 and GLC, 10,000 cpm) was added to each sample to serve as an internal tracer for estimation of procedural losses. For RIA-1, the samples were assayed as reported previously (1).For RIA-2 and GLC, the following modifications were made prior to chromatography on LH-20 sephadex. After diethyl ether extraction the lipid was removed by a precipitation step. The dried extracts were dissolved in diethyl ether and 70 % methanol (1:4) and...
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