The development of an enzyme‐immunoassay for urinary pregnanediol‐3‐glucuronide and its application to reproductive assessment in exotic mammals

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 μg/mg creatinine (Cr)), in contrast to the high levels of excretion during the luteal phase (maximum values 1.5–2.1 μg/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 μg/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 μg/mg Cr in the Indian to 0.08 μg/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.