Abstract
Knowledge on species’ reproductive biology is a fundamental pre-requisite of every conservation effort, but is often lacking. Sex steroids can provide valuable information for the assessment of reproductive success, whereas glucocorticoids are used to assess adrenocortical activity and stress-related bodily adaption. However, due to their perilous condition, access to animals is often difficult, which makes hormone measurement in non-invasively collected hair samples an attractive option. We determined cortisol, cortisone, corticosterone, testosterone and progesterone in Iberian lynx hair using enzyme immunoassay (EIA). Cross-validation was performed with high-performance liquid chromatography (HPLC) and high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Finally, we statistically evaluated the variations of sex steroids and glucocorticoids according to age, sex, origin, behavior and management. All steroids except corticosterone were detectable in Iberian lynx hair. Hair progesterone measured by EIA was overestimated by cross-reaction with 5α-dihydroprogesterone, a biologically active gestagene, and was highly correlated with HPLC-MS/MS results. Progesterone was higher in adult females compared to all other age-sex groups. Cortisol measured by EIA was overestimated due to antibody cross-reactivity with cortisone and was correlated to the sum of HPLC-MS/MS measurements for cortisol and cortisone. Cortisol was higher in females than in males measured by HPLC-MS/MS, but the EIA results were confounded by the lack of specificity. When using cortisol-cortisone and cortisol-dihydroepiandrosterone ratios, differences were noted between wild-caught and captive-bred lynxes. Additionally, longitudinal EIA measurements of an Iberian lynx after a wildfire showed an inversion of the cortisol-cortisone ratio that later subsided. These results validate the use of hair progesterone measurement for Iberian lynx reproductive monitoring and add to the growing evidence supporting the need for a more comprehensive approach to hair steroid measurement that accounts for local interconversion and co-regulation mechanisms.
Highlights
Conserving and restoring populations of wild species has become a global necessity in the attempt to slow down current extinction rates (Hoffmann et al, 2010; Pimm et al, 2014)
Cortisol was higher in females than in males measured by high-performance liquid chromatography (HPLC)-MS/MS, but the enzyme immunoassay (EIA) results were confounded by the lack of specificity
The analysis indicated a significant relation between the values obtained with the cortisol-3CMO-EIA and the HPLC-MS/MS values of cortisone (r = 0.84, P < 0.001) and cortisol + cortisone (r = 0.86, P < 0.001), but not with cortisol alone
Summary
Conserving and restoring populations of wild species has become a global necessity in the attempt to slow down current extinction rates (Hoffmann et al, 2010; Pimm et al, 2014). Questions related to species’ reproductive biology and physiology need to be answered promptly in order to ensure health, survival and reproduction in both captive and wild populations. Measurement of steroid sex hormones provides a valuable tool for understanding the reproductive biology and ecology of wild species, thereby supporting development of assisted reproductive techniques (ARTs) (Wildt and Wemmer, 1999; Schwarzenberger, 2007; Comizzoli et al, 2009; Jewgenow et al, 2017). Steroid hormone measurements have traditionally been performed using enzyme immunoassays (EIAs), which remain the most viable option for wide-scale daily application in field conservation, despite the availability of more specific yet more expensive and skilldemanding techniques such as (ultra) high-performance liquid chromatography ((U)HPLC) facultatively coupled to liquid chromatography-mass spectrometry (LC-MS)
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