With 5 figures in the text) Measurement of urinary pregnanediol-3-glucuronide (PdG) provides a practical, non-invasive method for monitoring ovarian function and pregnancy in diverse mammalian species. To date, however, methods of laboratory analysis have relied on radio-immunoassay procedures which, because of their cost and the problems associated with the use and disposal of radioactive substances, have limited potential for routine application in zoos and other animal collections with limited laboratory facilities. The present paper reports the development of a non-isotopic enzyme-immunoassay for the measurement of urinary PdG in a wide range of exotic mammals and describes its application to reproductive assessment in the gorilla and rhinoceros.The assay is based on an homologous competitive binding system and utilizes antiserum raised against PdG-bovine serum albumen and alkaline phosphatase-PdG, prepared by the active ester technique, as conjugate. Sensitivity of the assay is between 12.5 and 25 pg/well and precision, expressed as the intra-and inter-assay coefficients of variation, is between 5.3 and 7.8% and 9.2 and 16.1 %, respectively. Serial dilutions of human, gorilla, orang-utan, mangabey, blackbuck, okapi, gaur, giant panda and Indian, black and white rhinoceros urine gave displacement curves parallel to that of the PdG standard, indicating the potential of the assay for multi-species application.The pattern of excretion of immunoreactive PdG during successive menstrual cycles and two complete pregnancies in a gorilla is described. Follicular phase levels of PdG were low (0.07-0.13 pg/mg creatinine (Cr)), in contrast to the high levels of excretion during the luteal phase (maximum values 1.5-2.1 pg/mg Cr). Mean cycle length was 29.6 1.5 days with a range of 26-34 (n = 5). The pattern of PdG excretion during pregnancy showed a slow increase during the first 60 days and a more rapid rise to values of 8-12 pg/mg Cr after 120 days, with no further consistent increase during the remaining period of pregnancy. Estimated gestation lengths were 280 and 256 days.The levels of immunoreactive PdG excreted during mid-to-late pregnancy varied considerably between the three species of rhinoceros, maximum values ranging from up to 10 pg/mg Cr in the Indian to 0.08 pg/mg Cr in the white rhinoceros. Despite this variation pregnancy was associated with elevated PdG excretion in all three species and each profile showed a marked fall in levels at the end of the gestation period. The results suggest potential for urinary PdG determination as a practical method of pregnancy detection in both Indian and African rhinoceros, although species differences in the relative importance of PdG as a progesterone metabolite probably exist.
The feasibility of monitoring ovarian function in scimitar-horned oryx (OFYX dammnh) by measurement of fecal 20a-progestagens was investigated. Fecal samples were collected daily or on alternate days over a 4-1 1 month period from five oryx during natural (n = 4) or synthetic PGF,, (cloprosteno1)-controlled (n = 1 ) cycles. Of the four oryx undergoing natural cycles, three had regular access to a vasectomised male, and mating dates were recorded. Ultrasonography was used to monitor changes in reproductive tract morphology in the female administered with cloprostenol. Neutral steroids were extracted from feces with methano1:petroleum ether (2: 1 viv) after first removing phenolic steroids with potassium hydroxide (1 M). The concentration of 20a-progestagens in the methanol phase was measured by enzymeimmunoassay. Excretion of 20a-progestagens in all females followed a cyclic pattern corresponding to the follicular and luteal phases of the ovarian cycle. Concentrations of fecal 20a-progestagens were on average twentyfold greater during the luteal phase compared with the follicular phase. Mean (t SD) ovarian cycle length, based on fecal progestagen profiles, was 24.4 * 2.2 days with mean (+SD) luteal and follicular phase lengths of 18.7 + 2.8 and 5.7 -C 1.6 days, respectively. Mating by a vasectomized male occurred when 20a-progestagen concentrations were still elevated or declining. Similarly, fecal progestagens did not return to follicular phase concentrations for 4-5 days after administration of cloprostenol, and a 4 day delay was observed between ovulation, as visualized by ultrasound scanning, and a rise in fecal 2Oa-progestagens. These data suggest a time lag of approximately 4 days between reproductive events and changes in fecal 20a-progestagen concentrations. We conclude that measurement of immunoreactive 20a-progestagen concentrations in feces has limited application for predicting ovulation or accurately timing inseminations because of delay in steroid excretion, but will enable noninvasive monitoring of ovarian cycles in scimitar-horned oryx for fertility assessment and for determining the outcome of artificial insemination programs.
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