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Palme, R.; Rettenbacher, S.; Touma, C.; El-Bahr, S.M.; Mostl, E. |
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Title |
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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Year |
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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Pages |
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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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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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 @ |
Serial |
4083 |
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Touma, C.; Palme, R.; Sachser, N. |
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Title |
Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones |
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Journal Article |
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Year |
2004 |
Publication |
Hormones and Behavior |
Abbreviated Journal |
Horm Behav |
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Volume |
45 |
Issue |
1 |
Pages |
10-22 |
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Keywords |
Adrenal Cortex/drug effects; Adrenal Cortex Function Tests; Adrenocorticotropic Hormone/pharmacology; Analysis of Variance; Animals; Circadian Rhythm; Corticosterone/*analysis/metabolism; Dexamethasone/pharmacology; Feces/*chemistry; Female; Immunoenzyme Techniques/*methods; Male; Mice; Mice, Inbred C57BL; Models, Animal; Reproducibility of Results; Stress, Psychological/*metabolism |
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In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science. |
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Department of Behavioural Biology, University of Muenster, D-48149 Muenster, Germany. touma@uni-muenster.de |
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0018-506X |
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PMID:14733887 |
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Equine Behaviour @ team @ |
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4084 |
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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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Year |
2003 |
Publication |
General and Comparative Endocrinology |
Abbreviated Journal |
Gen Comp Endocrinol |
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Volume |
134 |
Issue |
2 |
Pages |
156-166 |
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Keywords |
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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Abstract |
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 |
Touma, C.; Sachser, N.; Mostl, E.; Palme, R. |
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Title |
Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice |
Type |
Journal Article |
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Year |
2003 |
Publication |
General and Comparative Endocrinology |
Abbreviated Journal |
Gen Comp Endocrinol |
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Volume |
130 |
Issue |
3 |
Pages |
267-278 |
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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 |
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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. |
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Department of Behavioral Biology, Institute of Neuro and Behavioral Biology, University of Muenster, Badestrasse 9, D-48149 Muenster, Germany. touma@uni-muenster.de |
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0016-6480 |
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PMID:12606269 |
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Equine Behaviour @ team @ |
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4086 |
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Author |
Griffin, B. |
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The use of fecal markers to facilitate sample collection in group-housed cats |
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Journal Article |
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2002 |
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Contemporary Topics in Laboratory Animal Science / American Association for Laboratory Animal Science |
Abbreviated Journal |
Contemp Top Lab Anim Sci |
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41 |
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2 |
Pages |
51-56 |
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Keywords |
Animals; Behavior, Animal; Biological Markers/*analysis; Cats/*physiology/psychology; Diet/veterinary; Feces/*chemistry; Food Coloring Agents/analysis; Housing, Animal; Individuality; Plastics/analysis; Specimen Handling/methods/*veterinary |
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The provision of proper social housing is a priority when designing an experiment using domestic cats as laboratory animals. When animals are group-housed, studies requiring analysis of stool samples from individual subjects pose difficulty in sample collection and identification. In this study, commercially available concentrated food colorings (known as bakers pastes) were used as fecal markers in group-housed cats. Cats readily consumed 0.5 ml of bakers paste food coloring once daily in canned cat food. Colorings served as fecal markers by imparting a distinct color to each cat s feces, allowing identification in the litter box. In addition, colored glitter (1/8 teaspoon in canned food) was fed to cats and found to be a reliable fecal marker. Long-term feeding of colorings and glitter was found to be safe and effective at yielding readily identifiable stools. |
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Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Alabama 36841, USA |
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1060-0558 |
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PMID:11958604 |
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Equine Behaviour @ team @ |
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4165 |
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Author |
Li, C.; Jiang, Z.; Tang, S.; Zeng, Y. |
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Title |
Influence of enclosure size and animal density on fecal cortisol concentration and aggression in Pere David's deer stags |
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Journal Article |
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Year |
2007 |
Publication |
General and Comparative Endocrinology |
Abbreviated Journal |
Gen Comp Endocrinol |
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Volume |
151 |
Issue |
2 |
Pages |
202-209 |
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Keywords |
*Aggression; Animals; *Deer; *Environment; Feces/*chemistry; Handling (Psychology); Housing, Animal; Hydrocortisone/*analysis; Male; Population Density |
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We investigated the impact of enclosure size and animal density on behavior and adrenocortical secretion in Pere David's deer in Dafeng Nature Reserve, China. From February 15 to April 16 in 2004, we conducted two experiments. First, we studied maintenance behavior and conflict behavior of Pere David's deer stags in a large enclosure (200 ha) with low animal density (0.66 deer/ha) and a small display pen (0.75 ha) with high animal density (25.33 deer/ha). The maintenance behavior we recorded included standing, locomotion, foraging and rest. During the behavioral observations, we collected fresh voided fecal samples from the stags periodically, and analyzed the fecal cortisol concentrations in those samples using radioimmunoassay technique. Second, we monitored the fecal cortisol concentrations of one group of stags (12 deer lived in an enclosure of 100 ha) before and after transferred into a small pen (0.5 ha). We found that in the first experiment: (1) there were significant differences in standing and rest whereas no significant differences of locomotion and foraging between the free-ranging group and the display group; (2) frequency of conflict behavior in the display group was significantly higher than those in the free-ranging group; and (3) fecal cortisol concentration of the display group (326.17+/-16.98 ng/g dry feces) was significantly higher than that of the free-ranging group (268.98+/-15.21 ng/g dry feces). In the second experiment, there was no significant difference of the fecal cortisol concentrations among sampling days, but the mean fecal cortisol concentration of the day after transferring (337.46+/-17.88 ng/g dry feces) was significantly higher than that of the day before transferring (248.44+/-7.99 ng/g dry feces). Comparison with published findings, our results indicated that enclosure size and animal density affect not only behaviors, but also adrenocortical secretion in Pere David's deer. Small living space with high animal density may impose physiological stress to captive Pere David's deer. Moreover, long-term physiological stress and increase of conflict behavior may subsequently affect survival and reproduction of the deer. |
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Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China |
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0016-6480 |
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PMID:17324429 |
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Equine Behaviour @ team @ |
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5475 |
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