Enzyme immunoassays for water-soluble steroid metabolites in the urine and feces of Japanese macaques (Macaca fuscata) using a simple elution method.

Abstract

A simple, non-alcoholic extraction method for measuring estrogen and progesterone metabolites in excreta using enzyme immunoassays (EIAs) was developed in Japanese macaques. The obtained detection limits of EIAs using estrone conjugates (E1C), pregnanediol glucuronide (PdG), and estriol glucuronide (E3G) polyclonal antibodies with cross-reactivity to urinary and fecal steroid metabolites were 6.6 pg/ml, 2.1 ng/ml and 0.35 ng/ml, respectively. These assays allowed the determination of E1C, PdG, and E3G from the excreta with good reproducibility and accuracy. Thereafter, urine and fecal samples of two menstrual cycles and six pregnancies from eight female Japanese macaques were assayed. A typical increase in urinary and fecal E1C in follicular phase and PdG in luteal phase were shown during non-conceptive menstrual cycles. Urinary E3G levels also showed a preovulatory increase; however, fecal E3G levels were very low throughout the non-conceptive menstrual cycles. Levels of E1C and PdG in the urine and feces of pregnant females were gradually increased until parturition, while fecal E3G levels were low and reached detectable levels after the mid-pregnancy period. Although the extraction rate of estrogen and progestogen metabolites by our method was lower compared to those of the previous extraction method using an alcohol-containing buffer, our method was simple, and the correlation coefficients for the relationship between two methods were found to be statistically significant. The results presented here are of great practical value for a non-invasive method of monitoring ovarian function and pregnancy in Japanese macaques.