Measuring fecal testosterone in females and fecal estrogens in males: comparison of RIA and LC/MS/MS methods for wild baboons (Papio cynocephalus)

Abstract

The development of non-invasive methods, particularly fecal determination, has made possible the assessment of hormone concentrations in wild animal populations. However, measuring fecal metabolites needs careful validation for each species and for each sex. We investigated whether radioimmunoassays (RIAs) previously used to measure fecal testosterone (fT) in male baboons and fecal estrogens (fE) in female baboons were well suited to measure these hormones in the opposite sex. We compared fE and fT concentrations determined by RIA to those measured by liquid chromatography combined with triple quadropole mass spectrometry (LC/MS/MS), a highly specific method. Additionally, we conducted a biological validation to assure that the measurements of fecal concentrations reflected physiological levels of the hormone of interest. Several tests produced expected results that led us to conclude that our RIAs can reliably measure fT and fE in both sexes, and that within-sex comparisons of these measures are valid: (i) fTRIA were significantly correlated to fTLC/MS/MS for both sexes; (ii) fTRIA were higher in adult than in immature males; (iii) fTRIA were higher in pregnant than non-pregnant females; (iv) fERIA were correlated with 17β-estradiol (fE2) and with estrone (fE1) determined by LC/MS/MS in pregnant females; (v) fERIA were significantly correlated with fE2 in non-pregnant females and nearly significantly correlated in males; (vi) fERIA were higher in adult males than in immature males. fERIA were higher in females than in males, as predicted, but unexpectedly, fTRIA were higher in females than in males, suggesting a difference in steroid metabolism in the two sexes; consequently, we conclude that while within-sex comparisons are valid, fTRIA should not be used for intersexual comparisons. Our results should open the field to important additional studies, as to date the roles of testosterone in females and estrogens in males have been little investigated.

Keywords: Baboon; Estrogens; Fecal; LC/MS/MS; Method validation; Testosterone.

Fig. 1

Comparison of fT concentrations determined by RIA between ‘pregnant’ and ‘non-pregnant’ cycles. Each value represents the mean ± SE across female’s cycles, for each cycle day indexed from the onset of sex skin deturgescence, i.e., the first day that the sex swelling begins to diminish in size (Gesquiere et al., 2007). Data for non-pregnant females stop at day 17 because day 18 would be the first day of a new cycle (see Gesquiere et al. 2007 for further information).

Fig. 2

Profile across the pregnancy (mixed longitudinal and cross-sectional samples) of fT concentration. Each value represents the weekly mean ± SE across females.