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Jugular vein blood samples collected from 5 Holstein-Friesian cows during the luteal phase of the estrous cycle were incubated at 4 C, 22 C, and 37 C for 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours. Blood samples were centrifugated after each incubation period, and plasma was frozen for later progesterone assay. Mean progesterone concentration in blood samples incubated at 4 C, 22 C, and 37 C decreased from 5.7 ng/ml at 0 hour to minimum concentrations of 3.5 ng/ml at 12 hours, 0.9 ng/ml at 12 hours, and 1.0 ng/ml at 8 hours, respectively. The first progesterone concentration which was significantly different from the initial value (0 hour) was at the 8-hour incubation period for blood samples incubated at 4 C and at the 1-hour incubation period for blood samples incubated at 22 C and 37 C. Progesterone concentration decreased similarly when blood samples were collected into heparinized tubes or EDTA tubes and incubated at 22 C for 0, 6, and 12 hours. Incubating harvested plasma or serum for 0 to 6 hours at 37 C had no effect on progesterone concentrations (7.9 and 8.2 ng/ml for plasma, and 6.9 and 6.6 ng/ml for serum.

The nature of progesterone decline in samples of bovine blood as affected by whole blood incubation time and temperature was investigated in 3 experiments. In 2 experiments, progesterone concentrations were determined in plasma, in hemolyzed (by freezing and thawing) blood, and in washed blood cells and blood cell washes before and after 8 hours of incubation at 22 C to study the effect of RBC on detected progesterone. In the third experiment, the effect of alteration in RBC metabolism on progesterone degradation by these cells was studied by using potassium oxalate-sodium fluoride as anticoagulant. Results of these experiments indicated that (a) progesterone was significantly reduced because of whole blood incubation time and temperature, (b) lost progesterone was not detectable on or in blood cells indicating chemical degradation of progesterone, (c) intact blood cells were needed to obtain the decrease in the progesterone, (d) a small amount of circulating blood progesterone was associated with blood cells, and (e) degradation of bovine blood progesterone by blood cells was dependent on the glycolytic process. Potassium oxalate-sodium fluoride anticoagulant and refrigeration provided the best progesterone results when blood samples were not processed immediately after collection.