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Palme, R. (2005). Measuring fecal steroids: guidelines for practical application. Ann N Y Acad Sci, 1046, 75–80.
Abstract: During the past 20 years, measuring steroid hormone metabolites in fecal samples has become a widely appreciated technique, because it has proved to be a powerful, noninvasive tool that provides important information about an animal's endocrine status (adrenocortical activity and reproductive status). However, although sampling is relatively easy to perform and free of feedback, a careful consideration of various factors is necessary to achieve proper results that lead to sound conclusions. This article aims to provide guidelines for an adequate application of these techniques. It is meant as a checklist that addresses the main topics of concern, such as sample collection and storage, time delay extraction procedures, assay selection and validation, biological relevance, and some confounding factors. These issues are discussed briefly here and in more detail in other recent articles.
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Palme, R., Rettenbacher, S., Touma, C., El-Bahr, S. M., & Mostl, E. (2005). Stress hormones in mammals and birds: comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples. Ann N Y Acad Sci, 1040, 162–171.
Abstract: A multitude of endocrine mechanisms are involved in coping with challenges. Front-line hormones to overcome stressful situations are glucocorticoids (GCs) and catecholamines (CAs). These hormones are usually determined in plasma samples as parameters of adrenal activity and thus of disturbance. GCs (and CAs) are extensively metabolized and excreted afterwards. Therefore, the concentration of GCs (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantages of easy collection and a feedback-free sampling procedure. However, large differences exist among species regarding the route and time course of excretion, as well as the types of metabolites formed. Based on information gained from radiometabolism studies (reviewed in this paper), we recently developed and successfully validated different enzyme immunoassays that enable the noninvasive measurement of groups of cortisol or corticosterone metabolites in animal feces. The determination of these metabolites in fecal samples can be used as a powerful tool to monitor GC production in various species of domestic, wildlife, and laboratory animals.
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Thiel, D., Jenni-Eiermann, S., & Palme, R. (2005). Measuring corticosterone metabolites in droppings of capercaillies (Tetrao urogallus). Ann N Y Acad Sci, 1046, 96–108.
Abstract: The capercaillie (Tetrao urogallus), the largest grouse species in the world, is decreasing in numbers in major parts of its distribution range. Disturbances by human outdoor activities are discussed as a possible reason for this population decline. An indicator for disturbances is the increase of the glucocorticoid corticosterone, a stress hormone, which helps to cope with life-threatening situations. However, repeated disturbances might result in a long-term increase of the basal corticosterone concentration, which can result in detrimental effects like reduced fitness and survival of an animal. To measure corticosterone metabolites (CMs) noninvasively in the droppings of free-living capercaillies, first an enzyme immunoassay (EIA) in captive birds had to be selected and validated. Therefore, the excretion pattern of intravenously injected radiolabeled corticosterone was determined and 3H metabolites were characterized. High-performance liquid chromatography (HPLC) separations of the samples containing peak concentrations revealed that corticosterone was extensively metabolized. The HPLC fractions were tested in several EIAs for glucocorticoid metabolites. The physiological relevance of this method was proved after pharmacological stimulation of the adrenocortical activity. Only the recently established cortisone assay, measuring CMs with a 3,11-dione structure, detected an expressed increase of concentrations following ACTH stimulation. To set up a sampling protocol suited for the field, we examined the influence of various storage conditions and time of day on concentrations of CMs.
Keywords: Adrenocorticotropic Hormone/administration & dosage/analysis/metabolism; Animals; Circadian Rhythm; Corticosterone/administration & dosage/*analysis/*metabolism; Feces/*chemistry; Female; Freezing; Galliformes/*metabolism; Male; Reproducibility of Results; Sex Factors; Temperature; Time Factors; Tritium/diagnostic use
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Baltic, M., Jenni-Eiermann, S., Arlettaz, R., & Palme, R. (2005). A noninvasive technique to evaluate human-generated stress in the black grouse. Ann N Y Acad Sci, 1046, 81–95.
Abstract: The continuous development of tourism and related leisure activities is exerting an increasingly intense pressure on wildlife. In this study, a novel noninvasive method for measuring stress in the black grouse, an endangered, emblematic species of European ecosystems that is currently declining in several parts of its European range, is tested and physiologically validated. A radiometabolism study and an ACTH challenge test were performed on four captive black grouse (two of each sex) in order to get basic information about the metabolism and excretion of corticosterone and to find an appropriate enzyme-immunoassay (EIA) to measure its metabolites in the feces. Peak radioactivity in the droppings was detected within 1 to 2 hours. Injected (3)H-corticosterone was excreted as polar metabolites and by itself was almost absent. A cortisone-EIA was chosen from among seven tested EIAs for different groups of glucocorticoid metabolites, because it cross-reacted with some of the formed metabolites and best reflected the increase of excreted corticosterone metabolites, after the ACTH challenge test. Concentrations of the metabolites from fecal samples collected from snow burrows of free-ranging black grouse were within the same range as in captive birds. The noninvasive method described may be appropriate for evaluating the stress faced by free-living black grouse populations in the wild, particularly in mountain ecosystems where human disturbance, especially by winter sports, is of increasing conservation concern.
Keywords: Adrenocorticotropic Hormone/metabolism; Animals; Bird Diseases/*metabolism; Conservation of Natural Resources; Corticosterone/*metabolism; Ecosystem; Feces/*chemistry; Female; Galliformes/*metabolism; Immunoenzyme Techniques/methods/veterinary; Male; Reproducibility of Results; Stress/metabolism/*veterinary; Tritium/diagnostic use
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Mostl, E., Rettenbacher, S., & Palme, R. (2005). Measurement of corticosterone metabolites in birds' droppings: an analytical approach. Ann N Y Acad Sci, 1046, 17–34.
Abstract: Fecal steroid analyses are becoming increasingly popular among both field and laboratory scientists. The benefits associated with sampling procedures that do not require restraint, anesthesia, and blood collection include less risk to subject and investigator, as well as the potential to obtain endocrine profiles that are not influenced by the sampling procedure itself. In the feces, a species-specific pattern of metabolites is present, because glucocorticoids are extensively metabolized. Therefore, selection of adequate extraction procedures and immunoassays for measuring the relevant metabolites is a serious issue. In this review, emphasis is placed on the establishment and analytical validation of methods to measure glucocorticoid metabolites for a noninvasive evaluation of adrenocortical activity in droppings of birds.
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