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Author Palme, R. doi  openurl
  Title Measuring fecal steroids: guidelines for practical application Type Journal Article
  Year 2005 Publication Annals of the New York Academy of Sciences Abbreviated Journal Ann N Y Acad Sci  
  Volume 1046 Issue Pages 75-80  
  Keywords Animals; Feces/*chemistry; Immunoassay/methods; Reproducibility of Results; Specimen Handling/methods; Steroids/*analysis  
  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.  
  Address Institute of Biochemistry, Department of Natural Sciences, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria. Rupert.Palme@vu-wien.ac.at  
  Corporate Author Thesis  
  Publisher Place of Publication Editor  
  Language English Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0077-8923 ISBN Medium  
  Area Expedition Conference  
  Notes PMID:16055844 Approved no  
  Call Number Equine Behaviour @ team @ Serial 4081  
Permanent link to this record
 

 
Author Touma, C.; Palme, R. doi  openurl
  Title Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation Type Journal Article
  Year 2005 Publication Annals of the New York Academy of Sciences Abbreviated Journal Ann N Y Acad Sci  
  Volume 1046 Issue Pages 54-74  
  Keywords Animals; Birds/*metabolism; Circadian Rhythm; Feces/*chemistry; Glucocorticoids/*analysis; Mammals/*metabolism; Reproducibility of Results; Seasons; Sex Factors  
  Abstract In recent years, the noninvasive monitoring of steroid hormone metabolites in feces of mammals and droppings of birds has become an increasingly popular technique. It offers several advantages and has been applied to a variety of species under various settings. However, using this technique to reliably assess an animal's adrenocortical activity is not that simple and straightforward to apply. Because clear differences regarding the metabolism and excretion of glucocorticoid metabolites (GCMs) exist, a careful validation for each species and sex investigated is obligatory. In this review, general analytical issues regarding sample storage, extraction procedures, and immunoassays are briefly discussed, but the main focus lies on experiments and recommendations addressing the validation of fecal GCM measurements in mammals and birds. The crucial importance of scrutinizing the physiological and biological validity of fecal GCM analyses in a given species is stressed. In particular, the relevance of the technique to detect biologically meaningful alterations in adrenocortical activity must be shown. Furthermore, significant effects of the animals' sex, the time of day, season, and different life history stages are discussed, bringing about the necessity to seriously consider possible sex differences as well as diurnal and seasonal variations. Thus, comprehensive information on the animals' biology and stress physiology should be carefully taken into account. Together with an extensive physiological and biological validation, this will ensure that the measurement of fecal GCMs can be used as a powerful tool to assess adrenocortical activity in diverse investigations on laboratory, companion, farm, zoo, and wild animals.  
  Address Max Planck Institute of Psychiatry, Department of Behavioral Neuroendocrinology, Kraepelinstrasse 2-10, D-80804 Munich, Germany. touma@mpipsykl.mpg.de  
  Corporate Author Thesis  
  Publisher Place of Publication Editor  
  Language English Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0077-8923 ISBN Medium  
  Area Expedition Conference  
  Notes PMID:16055843 Approved no  
  Call Number Equine Behaviour @ team @ Serial 4073  
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Author Palme, R.; Rettenbacher, S.; Touma, C.; El-Bahr, S.M.; Mostl, E. doi  openurl
  Title Stress hormones in mammals and birds: comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples Type Journal Article
  Year 2005 Publication Annals of the New York Academy of Sciences Abbreviated Journal Ann N Y Acad Sci  
  Volume 1040 Issue Pages 162-171  
  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  
  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.  
  Address Institute of Biochemistry, Department of Natural Sciences, University of Veterinary Medicine, Vienna, Austria. rupert.palme@vu-wien.ac.at  
  Corporate Author Thesis  
  Publisher Place of Publication Editor  
  Language English Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0077-8923 ISBN Medium  
  Area Expedition Conference  
  Notes PMID:15891021 Approved no  
  Call Number Equine Behaviour @ team @ Serial 4083  
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Author Christensen, J.W.; Ahrendt, L.P.; Lintrup, R.; Gaillard, C.; Palme, R.; Malmkvist, J. doi  openurl
  Title Does learning performance in horses relate to fearfulness, baseline stress hormone, and social rank? Type Abstract
  Year 2012 Publication Applied Animal Behaviour Science Abbreviated Journal App Anim Behav Sci  
  Volume 140 Issue 1 Pages 44-52  
  Keywords Horse; Learning; Fearfulness; Stress; Reinforcement; Social rank  
  Abstract The ability of horses to learn and remember new tasks is fundamentally important for their use by humans. Fearfulness may, however, interfere with learning, because stimuli in the environment can overshadow signals from the rider or handler. In addition, prolonged high levels of stress hormones can affect neurons within the hippocampus; a brain region central to learning and memory. In a series of experiments, we aimed to investigate the link between performance in two learning tests, the baseline level of stress hormones, measured as faecal cortisol metabolites (FCM), fearfulness, and social rank. Twenty-five geldings (2 or 3 years old) pastured in one group were included in the study. The learning tests were performed by professional trainers and included a number of predefined stages during which the horses were gradually trained to perform exercises, using either negative (NR) or positive reinforcement (PR). Each of the learning tests lasted 3 days; 7min/horse/day. The NR test was repeated in a novel environment. Performance, measured as final stage in the training programme, and heart rate (HR) were recorded. Faeces were collected on four separate days where the horses had been undisturbed at pasture for 48h. Social rank was determined through observations of social interactions during feeding. The fear test was a novel object test during which behaviour and HR were recorded. Performance in the NR and PR learning tests did not correlate. In the NR test, there was a significant, negative correlation between performance and HR in the novel environment (rS=-0.66, P<0.001, i.e. nervous horses had reduced performance), whereas there was no such correlation in the home environment (both NR and PR). Behavioural reactions in the fear test correlated significantly with performance in the NR test in the novel environment (e.g. object alertness and final stage: rS=-0.43, P=0.04), suggesting that performance under unfamiliar, stressful conditions may be predicted by behavioural responses in a fear test. There was a negative correlation between social rank and baseline stress hormones (rS=-0.43, P=0.04), i.e. high rank corresponded to low FCM concentrations, whereas neither rank nor FCM correlated with fearfulness or learning performance. We conclude that performance under stressful conditions is affected by activation of the sympathetic nervous system during training and related to behavioural responses in a standardised fear test. Learning performance in the home environment, however, appears unrelated to fearfulness, social rank and baseline FCM levels.  
  Address  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0168-1591 ISBN Medium  
  Area Expedition Conference  
  Notes Approved no  
  Call Number Equine Behaviour @ team @ S0168-1591(12)00168-2 Serial 5769  
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Author Touma, C.; Sachser, N.; Mostl, E.; Palme, R. openurl 
  Title Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice Type Journal Article
  Year 2003 Publication General and Comparative Endocrinology Abbreviated Journal Gen Comp Endocrinol  
  Volume 130 Issue 3 Pages 267-278  
  Keywords Animals; Chromatography, High Pressure Liquid; Circadian Rhythm/*physiology; Corticosterone/*metabolism/urine; Feces/*chemistry; Female; Immunoenzyme Techniques; Kinetics; Male; Mice; Mice, Inbred C57BL; Reference Values; Sex Factors; Stress/metabolism; Time Factors; Tritium  
  Abstract Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.  
  Address Department of Behavioral Biology, Institute of Neuro and Behavioral Biology, University of Muenster, Badestrasse 9, D-48149 Muenster, Germany. touma@uni-muenster.de  
  Corporate Author Thesis  
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  Language English Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0016-6480 ISBN Medium  
  Area Expedition Conference  
  Notes PMID:12606269 Approved no  
  Call Number Equine Behaviour @ team @ Serial 4086  
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Author Palme, R. pdf  isbn
openurl 
  Title Non-invasive monitoring of stress hormones for welfare assessment in domestic and wild equids Type Conference Article
  Year 2015 Publication Proceedings of the 3. International Equine Science Meeting Abbreviated Journal Proc. 3. Int. Equine. Sci. Mtg  
  Volume Issue Pages  
  Keywords  
  Abstract Stress responses play an important role in allowing animals to cope with challenges. Glucocorticoids, key elements in the neuroendocrine stress axis, are traditionally measured as a parameter for welfare assessment. As blood sample collection itself disturbs an animal, non-invasive or minimal invasive methods have gained importance for assessing stress. In horses saliva and faeces are most frequently used. Faecal samples offer the advantage that they can be collected easily and stress-free. In faecal samples circulating hormone levels are integrated over a certain period of time. As a consequence faecal glucocorticoid metabolites represent the cumulative secretion and they are less affected by short episodic fluctuations of hormone secretion.

However, in order to gain reliable information about an animal’s adrenocortical activity, certain criteria have to be met: Depending whether the impact of acute or chronic stressors is assessed, frequent sampling might be necessary whereas in other cases, single samples will suffice. Background knowledge regarding the metabolism and excretion of glucocorticoids is essential and a careful validation is obligatory. In addition, this presentation will address analytical issues regarding sample storage, extraction procedures, and immunoassays and various examples of a successful application in equids will be given. Applied properly, non-invasive techniques to monitor stress hormones are a useful tool for animal welfare assessment.
 
  Address  
  Corporate Author Palme, R. Thesis  
  Publisher Xenophon Publishing Place of Publication Wald Editor Krueger, K.  
  Language Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title Proc. 3. Int. Equine. Sci. Mtg  
  Series Volume in prep Series Issue Edition  
  ISSN ISBN 978-3-95625-000-2 Medium  
  Area Expedition Conference  
  Notes Id - Approved no  
  Call Number Equine Behaviour @ team @ Serial 5795  
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Author Palme, R.; Moestl, E. openurl 
  Title Measurement of cortisol metabolites in faeces of sheep as a parameter of cortisol concentration in blood Type Journal Article
  Year 1997 Publication Zeitschrift für Säugetierkunde Abbreviated Journal J. Mammal. Biol.  
  Volume 62 Issue Pages 192-197  
  Keywords glucocorticoids, metabolites, animal biology, sheep, immunoenzyme techniques,  
  Abstract  
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  Call Number Equine Behaviour @ team @ Serial 6044  
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Author Palme, R.; Touma, C.; Arias,N.; Dominchin, M.F.; Lepschy, M. openurl 
  Title Steroid extraction: Get the best out of faecal samples Type Journal Article
  Year 2012 Publication Veterinary Medicine Austria Abbreviated Journal Vet. Med. Austria  
  Volume 100 Issue Pages 238-246  
  Keywords  
  Abstract 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.
 
  Address Review, faeces, extrac- tion, non-invasive hormone moni- toring, stress, reproduction.  
  Corporate Author Thesis  
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  Series Editor Series Title Abbreviated Series Title  
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  Area Expedition Conference  
  Notes Approved no  
  Call Number Equine Behaviour @ team @ Serial 6046  
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Author Palme, R.; Touma, C.; Arias, N., Dominchin, M.N.; Lepschy, M. openurl 
  Title Steroid extraction: Get the best out of faecal samples Type Journal Article
  Year 2013 Publication Wiener Tierärztliche Wochenschriften Abbreviated Journal Wien Tierärztl Monat – Vet Med Austria  
  Volume 100 Issue Pages 238-246.  
  Keywords Review, faeces, extraction, non-invasive hormone monitoring, stress, reproduction.  
  Abstract 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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  Notes Approved no  
  Call Number Equine Behaviour @ team @ Serial 6520  
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Author Merl, S.; Scherzer, S.; Palme, R.; Möstl, E. url  doi
openurl 
  Title Pain causes increased concentrations of glucocorticoid metabolites in horse feces Type Journal Article
  Year 2000 Publication Journal of Equine Veterinary Science Abbreviated Journal J Equine Vet Sci  
  Volume 20 Issue 9 Pages 586-590  
  Keywords  
  Abstract The concentration of 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites, was measured using enzyme immunoassay in fecal samples of horses experiencing painful episodes. One group of horses consisted of 10 stallions castrated (samples were collected daily for 10 days); the other group was made up of 29 horses which were brought to an animal hospital because of signs of colic (samples were collected twice daily for six days). Before castration, median concentrations of 10.5 nmol/kg feces were measured. On days 1 and 2 after castration, median 11,17-DOA values increased up to 26.2 and 50.0 nmol/kg feces, respectively, and decreased thereafter to levels lower than at the beginning of the sampling period. High variations were measured between individual cases of colic. In animals with colic, all horses excreted more than 33 nmol 11,17-DOA/kg feces for various periods. The highest concentration measured was 885 nmol/kg feces. One animal out of the 29 colic horses did not show any clinical signs of pain upon arrival in the hospital. The 11,17-DOA values were below 17 nmol/kg feces in all those samples. From this data we conclude, that the concentration of 11,17-DOA in feces is a parameter for painful situations that have occurred one or two days earlier.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0737-0806 ISBN Medium  
  Area Expedition Conference  
  Notes Approved no  
  Call Number Equine Behaviour @ team @ Serial 6047  
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