Seasonal and diel variation of hormone metabolites in European stonechats: on the importance of high signal-to-noise ratios in noninvasive hormone studies.

Most vertebrates living in seasonal environments show seasonal reproductive cycles and diel rhythms. The rhythmicity in behavior and morphology is accompanied by diel and seasonal patterns of hormone secretions. In small animals, the investigation of diel patterns of hormones has been hampered because repeated blood sampling is difficult and may influence subsequent measurements. A possibility to avoid these caveats is to investigate excreted hormone metabolites instead. Here, we describe the diel excretion patterns of testosterone and corticosterone metabolites in a small captive songbird during 4 seasons: winter, early spring (Zugunruhe), summer, and autumn molt. Our approach is quite unique, because the diel patterns of steroids have rarely--if at all--been investigated in the same individuals within several seasons. Small birds should be ideal to investigate diel patterns of hormone metabolites, because they defecate frequently enough to establish a diel profile. However, concentration measurements of hormone metabolites rely on the assumptions that hormone metabolites are placed into droppings of similar mass (the "dropping amount assumption") and are excreted in constant time intervals (the "constant interval assumption"). These assumptions were clearly violated in our study, as the dropping mass per time interval and the defecation intervals varied depending on the time of day and season. We thus used the rate of hormone metabolite excretion as an alternative measure to concentration. Both testosterone and corticosterone metabolites showed diel and seasonal rhythmicity. Furthermore, the diel pattern varied depending on season. Concentration and rate measurements gave similar results when the differences between hormone metabolite levels were large-for example, when testosterone metabolites were compared across seasons. When the differences were more subtle, though, the 2 measures did not always correspond well, indicating that the violation of the 2 basic assumptions affected the comparability of concentration measurements. We conclude that diel and seasonal comparisons of hormone metabolites potentially give biologically meaningful results, especially when rates instead of concentrations are measured. However, such studies require awareness of the limitations and pitfalls of noninvasive hormone measurements, a carefully designed experiment, and very cautious interpretation of the data.