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Author |
Heistermann, M.; Palme, R.; Ganswindt, A. |
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Title |
Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis |
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Journal Article |
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Year |
2006 |
Publication |
American journal of primatology |
Abbreviated Journal |
Am. J. Primatol. |
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Volume |
68 |
Issue |
3 |
Pages  |
257-273 |
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Keywords |
11-Hydroxycorticosteroids/*analysis; Adrenocorticotropic Hormone/pharmacology; Anesthesia; Animals; Corticosterone/analysis; Feces/*chemistry; Glucocorticoids/*analysis; Haplorhini/*metabolism; Hydrocortisone/analysis; Hypothalamo-Hypophyseal System/drug effects/physiology; Immunoenzyme Techniques/*methods; Pituitary-Adrenal System/drug effects/physiology; Species Specificity |
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Abstract |
Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used. |
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Department of Reproductive Biology, German Primate Center, Gottingen, Germany. mheiste@gwdg.de |
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0275-2565 |
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PMID:16477600 |
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Equine Behaviour @ team @ |
Serial |
4078 |
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Palme, R.; Touma, C.; Arias,N.; Dominchin, M.F.; Lepschy, M. |
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Title |
Steroid extraction: Get the best out of faecal samples |
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2012 |
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Veterinary Medicine Austria |
Abbreviated Journal |
Vet. Med. Austria |
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100 |
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238-246 |
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Faecal steroid hormone metabolites are becoming increasingly popular as parameters for reproductive functions and stress. Theextraction of the steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition, so selection of a proper extraction procedure is essential. There have been some studies to address this complex but often neglected point. Radiolabelled
steroids (e.g. cortisol or progesterone) have frequently been added to faecal samples to estimate the efficiency of the extraction procedures used. However, native, unmetabolized steroids are normally not present in the faeces and therefore the results are artificial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites present in the given species. As a result, it is possible to evaluate different extraction methods for use with faecal samples. We present studies on sheep, horses, pigs, hares and dogs that utilized samples containing naturally metabolized, 14C-labelled steroids. |
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Review, faeces, extrac- tion, non-invasive hormone moni- toring, stress, reproduction. |
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Equine Behaviour @ team @ |
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6046 |
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Palme, R.; Touma, C.; Arias, N., Dominchin, M.N.; Lepschy, M. |
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Title |
Steroid extraction: Get the best out of faecal samples |
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Journal Article |
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2013 |
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Wiener Tierärztliche Wochenschriften |
Abbreviated Journal |
Wien Tierärztl Monat – Vet Med Austria |
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100 |
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238-246. |
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Review, faeces, extraction, non-invasive hormone monitoring, stress, reproduction. |
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Faecal steroid hormone metabolites are becoming increasingly popular as parameters for reproductive functions and stress. The extraction of the steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition, so selection of a proper extraction procedure is essential. There have been some studies to address this complex but often neglected point. Radiolabelled steroids (e.g. cortisol or progesterone) have frequently been added to faecal samples to estimate the efficiency of the extraction procedures used. However, native, unmetabolized steroids are normally not present in the faeces and therefore the results are artifi- cial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites present in the given species. As a result, it is possible to evaluate different extraction methods for use with faecal samples. We present studies on sheep, horses, pigs, hares and dogs that utilized samples containing naturally metabolized, 14C-labelled steroids. We recommend extracting faecal steroids by simply suspending the faeces in a high percentage of a primary alcohol (for glucocorticoid metabolites 80% aqueous methanol proved best suited for virtually all mammalian species tested so far). Not only does the procedure significantly increase the total amount of recovered radioactivity, it also increases the percentage of unconjugated metabolites, which are more likely to be recognized by the antibodies used in various immunoassays. The advantages of this extraction procedure are clear: it is very easy to use (no evaporation step is needed), it yields high recoveries and variation based on the extraction procedure is reduced to a minimum. |
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no |
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Equine Behaviour @ team @ |
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6520 |
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Author |
Palme, R. |
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Title |
Non-invasive measurement of glucocorticoids: Advances and problems |
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Journal Article |
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2019 |
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Physiology & Behavior |
Abbreviated Journal |
Physiol. Behav. |
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199 |
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229-243 |
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Glucocorticoids; Non-invasive; Faecal cortisol/corticosterone metabolites; Immunoassays; Physiological/biological validation |
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Glucocorticoids (GCs; i.e. cortisol/corticosterone) are a central component of the stress response and thus their measurement is frequently used to evaluate the impact of stressful situations. Their metabolites from faeces of various animal species are more and more taken as a non-invasive aid to assess GC release and thus adrenocortical activity. The current literature review includes an extensive collection (1327 papers) and evaluation (see also Supplementary Tables) of the literature on faecal cortisol/corticosterone metabolite (FCM) analysis published to date. It aims at giving reference for researchers interested in implementing FCM analysis into their study or seeking to improve such methods by providing background knowledge on GC metabolism and excretion, conveying insights into methodological issues and stating caveats of FCM analysis and by highlighting prerequisites for and some examples of a successful application of such methods. Collecting faecal samples and analysing FCMs may appear simple and straightforward, but researchers have to select and apply methods correctly. They also need to be aware of the many pitfalls and potentially confounding factors and, last but not least, have to carefully interpret results. Applied properly, measurement of FCMs is a powerful non-invasive tool in a variety of research areas, such as (stress) biology, ethology, ecology, animal conservation and welfare, but also biomedicine. |
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0031-9384 |
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Palme2019_attachment.pdf |
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Equine Behaviour @ team @ |
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6517 |
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Author |
Palme, R.; Moestl, E. |
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Measurement of cortisol metabolites in faeces of sheep as a parameter of cortisol concentration in blood |
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1997 |
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Zeitschrift für Säugetierkunde |
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J. Mammal. Biol. |
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62 |
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192-197 |
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glucocorticoids, metabolites, animal biology, sheep, immunoenzyme techniques, |
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Equine Behaviour @ team @ |
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6044 |
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Author |
Palme, R.; Rettenbacher, S.; Touma, C.; El-Bahr, S.M.; Mostl, E. |
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Stress hormones in mammals and birds: comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples |
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Journal Article |
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2005 |
Publication |
Annals of the New York Academy of Sciences |
Abbreviated Journal |
Ann N Y Acad Sci |
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Volume |
1040 |
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162-171 |
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Keywords |
Adrenal Glands/chemistry/metabolism; Animals; Birds; Catecholamines/analysis/chemistry/*metabolism; Feces/*chemistry; Glucocorticoids/analysis/chemistry/*metabolism; Hormones/analysis/metabolism; Mammals; Species Specificity; Stress/*metabolism |
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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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Institute of Biochemistry, Department of Natural Sciences, University of Veterinary Medicine, Vienna, Austria. rupert.palme@vu-wien.ac.at |
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0077-8923 |
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PMID:15891021 |
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Call Number |
Equine Behaviour @ team @ |
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4083 |
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Author |
Ganswindt, A.; Palme, R.; Heistermann, M.; Borragan, S.; Hodges, J.K. |
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Title |
Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth |
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Journal Article |
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2003 |
Publication |
General and Comparative Endocrinology |
Abbreviated Journal |
Gen Comp Endocrinol |
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134 |
Issue |
2 |
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156-166 |
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Adrenal Cortex/*metabolism/secretion; Adrenal Cortex Function Tests/methods/*veterinary; Adrenocorticotropic Hormone/physiology; Animals; Carbon Isotopes/diagnostic use; Chromatography, High Pressure Liquid/veterinary; Elephants/*metabolism/urine; Feces/*chemistry; Glucocorticoids/analysis/urine; Hydrocortisone/*analysis/diagnostic use/urine; Immunoenzyme Techniques/methods/veterinary; Male; Reproduction/physiology; Sexual Behavior, Animal/physiology; Stress, Psychological/diagnosis/*physiopathology; Testosterone/*analysis/diagnostic use/urine |
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Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species. |
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German Primate Centre, Department of Reproductive Biology, Kellnerweg 4, 37077 Gottingen, Germany. ganswindt@www.dpz.gdwg.de |
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0016-6480 |
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PMID:14511986 |
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Equine Behaviour @ team @ |
Serial |
4085 |
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Author |
Thiel, D.; Jenni-Eiermann, S.; Palme, R. |
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Measuring corticosterone metabolites in droppings of capercaillies (Tetrao urogallus) |
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Journal Article |
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Year |
2005 |
Publication |
Annals of the New York Academy of Sciences |
Abbreviated Journal |
Ann N Y Acad Sci |
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Volume |
1046 |
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96-108 |
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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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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. |
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Swiss Ornithological Institute, 6204 Sempach, Switzerland. dominik.thiel@vogelwarte.ch |
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0077-8923 |
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PMID:16055846 |
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Equine Behaviour @ team @ |
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4079 |
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Author |
Baltic, M.; Jenni-Eiermann, S.; Arlettaz, R.; Palme, R. |
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A noninvasive technique to evaluate human-generated stress in the black grouse |
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Journal Article |
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Year |
2005 |
Publication |
Annals of the New York Academy of Sciences |
Abbreviated Journal |
Ann N Y Acad Sci |
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Volume |
1046 |
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81-95 |
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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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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. |
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Zoological Institute, Division of Conservation Biology, Baltzerstrasse 6, CH-3012 Bern, Switzerland |
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0077-8923 |
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PMID:16055845 |
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no |
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Equine Behaviour @ team @ |
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4080 |
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Author |
Palme, R. |
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Title |
Measuring fecal steroids: guidelines for practical application |
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Journal Article |
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Year |
2005 |
Publication |
Annals of the New York Academy of Sciences |
Abbreviated Journal |
Ann N Y Acad Sci |
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1046 |
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75-80 |
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Animals; Feces/*chemistry; Immunoassay/methods; Reproducibility of Results; Specimen Handling/methods; Steroids/*analysis |
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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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Institute of Biochemistry, Department of Natural Sciences, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria. Rupert.Palme@vu-wien.ac.at |
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English |
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0077-8923 |
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PMID:16055844 |
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Equine Behaviour @ team @ |
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4081 |
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