The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5 alpha-pregnane-3 beta-ol-2O-one 3HS:BSA (5 alpha-20-one EIA) and 5 beta-pregnane-3 alpha-ol-2O-one 3HS:BSA (5 beta-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1-3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5 alpha-pregnane-3,20-dione, 5 alpha-pregnane-3 alpha-ol-2O-one, and 5 alpha-pregnane-3 beta-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3-7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 mu g/g) by week 11. During months 6-13 concentrations in the 5 alpha-20-one and in the 5 beta-20-one EIA (5-11 mu g/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5-3 mu g/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. (C) 1996 Wiley-Liss, Inc.
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