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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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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 @ |
Serial |
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 |
Type |
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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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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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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Schwarzenberger, F.; Mostl, E.; Palme, R.; Bamberg, E. |
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Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals |
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Journal Article |
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1996 |
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Animal Reproduction Science |
Abbreviated Journal |
Animal Reproduction: Research and Practice |
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42 |
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1-4 |
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515-526 |
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Faecal steroids; Non-invasive monitoring; Oestrogens; Progesterone metabolites; Reproductive hormones |
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Non-invasive faecal oestrogen and progesterone metabolite evaluations are well established approaches for monitoring reproductive function in a variety of mammalian species. The route of excretion of steroid hormone metabolites varies considerably among species, and also between steroids within the same species. Steroid concentrations in faeces exhibit a similar pattern to those in plasma, but have a lag time, which depending upon the species, can be from 12 h to more than 2 days. Faecal steroid metabolites in mammals are mainly unconjugated compounds. Faecal oestrogens consist predominantly of oestrone and/or oestradiol-17α or -17β. Therefore, specific oestrogen antibodies or antibodies against total oestrogens can be used for their determination. Progesterone is metabolised to several 5α- or 5β-reduced pregnanediones and hydroxylated pregnanes prior to its faecal excretion. Therefore, relevant antibodies for their determination show considerable cross-reactivities with several pregnane metabolites, whereas specific progesterone antibodies are less suitable. Faecal oestrogen evaluations have been used as reliable indicators of pregnancy in several ungulate and some primate species. They have also been used to determine the preovulatory period in carnivores, corpus luteum activity in New World primates, and to diagnose cryptorchidism in horses. Faecal progesterone metabolite analysis has been successfully used for monitoring corpus luteum function and pregnancy, abortion, seasonality and treatment therapies in an ever expanding list of species. |
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refbase @ user @ |
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327 |
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Mostl, E.; Palme, R. |
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Title |
Hormones as indicators of stress |
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Journal Article |
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2002 |
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Domestic Animal Endocrinology |
Abbreviated Journal |
Fourth International Conference on Farm Animal Endocrinology |
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23 |
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1-2 |
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67-74 |
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Animal welfare is of increasing importance and absence of chronic stress is one of its prerequisites. During stress, various endocrine responses are involved to improve the fitness of the individual. The front-line hormones to overcome stressful situations are the glucocorticoids and catecholamines. These hormones are determined as a parameter of adrenal activity and thus of disturbance. The concentration of glucocorticoids (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantage that they can be easily collected and this procedure is feedback free. Recently, enzyme immunoassays (EIA) have been developed and successfully tested, to enable the measurement of groups of cortisol metabolites in animal feces. The determination of these metabolites in fecal samples is a practical method to monitor glucocorticoid production. |
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Equine Behaviour @ team @ |
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4067 |
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Palme, R.; Fischer, P.; Schildorfer, H.; Ismail, M.N. |
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Excretion of infused 14C-steroid hormones via faeces and urine in domestic livestock |
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Journal Article |
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1996 |
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Animal Reproduction Science |
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43 |
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1 |
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43-63 |
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Sheep--endocrinology; Pig--endocrinology; Pony; 14C-steroids; Faeces; Urine; Blood |
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The aim of this comparative study was to gain more information about the excretion of steroid hormones in farm animals. This should help to establish or improve non-invasive steroid monitoring procedures, especially in zoo and wildlife animals. Over a period of 4 h the 14C-steroid hormones (3.7 MBq) progesterone (three females), testosterone (three males), cortisol and oestrone (two males, two females) were infused intravenously in sheep, ponies and pigs. Faeces were collected immediately after defecation. Urine was sampled via a permanent catheter in females and after spontaneous urination in males. A total of 88 +/- 10% (mean +/- SD) of the administered radioactivity was recovered. Considerable interspecies differences were measured both in the amounts of steroid metabolites excreted via faeces or urine and the time course of excretion. Progesterone and oestrone in ewes, and progesterone in mares were excreted mainly in the faeces (over 75%). The primary route of excretion of all other 14C-steroids was via the urine but to a different extent. In general, sheep showed the highest degree of faecal excretion and pigs the least. The highest radioactivity in urine (per mmol creatinine) was observed during the infusion or in one of the next two samples thereafter, whereas in faeces it was measured about 12 h (sheep), 24 h (ponies) or 48 h (pigs) after the end of the infusion. Thereafter the radioactivity declined and reached background levels within 2-3 weeks. In faeces, steroid metabolites were present mainly in an unconjugated form, but in blood and urine as conjugates. Mean retention time of faecal radioactivity suggested that the passage rate of digesta (duodenum to rectum) played an important role in the time course of the excretion of steroids. The information derived from this investigation could improve the precision of sampling as well as the extraction of steroids from the faeces. Furthermore, the study demonstrates that it should be possible to establish methods for measuring faecal androgen and cortisol metabolites for assessing male reproductive endocrinology and stress in animals. |
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Equine Behaviour @ team @ |
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4069 |
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Möstl, E.; Palme, R. |
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Hormones as indicators of stress |
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Journal Article |
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2002 |
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Domestic Animal Endocrinology |
Abbreviated Journal |
Domest. Anim. Endocrinol. |
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23 |
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1–2 |
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67-74 |
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Animal welfare is of increasing importance and absence of chronic stress is one of its prerequisites. During stress, various endocrine responses are involved to improve the fitness of the individual. The front-line hormones to overcome stressful situations are the glucocorticoids and catecholamines. These hormones are determined as a parameter of adrenal activity and thus of disturbance. The concentration of glucocorticoids (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantage that they can be easily collected and this procedure is feedback free. Recently, enzyme immunoassays (EIA) have been developed and successfully tested, to enable the measurement of groups of cortisol metabolites in animal feces. The determination of these metabolites in fecal samples is a practical method to monitor glucocorticoid production. |
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0739-7240 |
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Equine Behaviour @ team @ |
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5930 |
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Christensen, J.W.; Ahrendt, L.P.; Lintrup, R.; Gaillard, C.; Palme, R.; Malmkvist, J. |
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Does learning performance in horses relate to fearfulness, baseline stress hormone, and social rank? |
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Abstract |
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2012 |
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Applied Animal Behaviour Science |
Abbreviated Journal |
App Anim Behav Sci |
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140 |
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1 |
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44-52 |
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Horse; Learning; Fearfulness; Stress; Reinforcement; Social rank |
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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. |
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0168-1591 |
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Equine Behaviour @ team @ S0168-1591(12)00168-2 |
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5769 |
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Author |
Palme, R. |
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Monitoring stress hormone metabolites as a useful, non-invasive tool for welfare assessment in farm animals |
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Journal Article |
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2012 |
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Animal Welfare |
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21 |
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3 |
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331-337 |
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animal welfare, corticosterone, cortisol, faeces, farm animals, stress |
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A multitude of endocrine mechanisms are involved in coping with challenges. Glucocorticoids, secreted by the adrenal glands, are in the front line of the battle to overcome stressful situations. They are usually measured in plasma samples as parameters of adrenal activity and thus of disturbance. Unfortunately, collecting blood samples itself can disturb an animal. Thus, non-invasive methods for the determination of glucocorticoids or their metabolites have become increasingly popular. The pros and cons of various non-invasive sample materials (saliva, excreta, milk, hair/feathers and eggs) for glucocorticoid determination are given. Above all, faecal samples offer the
advantage that they can be collected easily. In faecal samples, circulating hormone levels are integrated over a certain period of time and represent the cumulative secretion of hormones. Thus, the levels are less affected by short fluctuations or the pulse-like nature of hormone secretion. However, using this technique to assess an animal’s adrenocortical activity is not especially simple. Whether frequent sampling is necessary or single samples will suffice depends upon the study’s aim (whether one is examining the impact of acute or chronic stressors). Background knowledge of the metabolism and excretion of cortisol/corticosterone metabolites is required and a careful validation for each species and sex investigated is obligatory. The present review also addresses analytical issues regarding sample storage, extraction procedures and immunoassays and includes a comprehensive list of published studies (up to 2011) describing the use of such methods in farmed animals. Applied properly, non-invasive techniques to monitor glucocorticoid metabolites in faecal samples of various species are a useful tool for welfare assessment, especially as they are easily applied at farm or group level. |
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0962-7286 |
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Equine Behaviour @ team @ |
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5793 |
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Author |
Palme, R. |
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Title |
Non-invasive monitoring of stress hormones for welfare assessment in domestic and wild equids |
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Conference Article |
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Year |
2015 |
Publication |
Proceedings of the 3. International Equine Science Meeting |
Abbreviated Journal |
Proc. 3. Int. Equine. Sci. Mtg |
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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. |
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Corporate Author |
Palme, R. |
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Publisher |
Xenophon Publishing |
Place of Publication |
Wald |
Editor |
Krueger, K. |
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Proc. 3. Int. Equine. Sci. Mtg |
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Series Volume |
in prep |
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ISBN |
978-3-95625-000-2 |
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Id - |
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no |
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Call Number |
Equine Behaviour @ team @ |
Serial |
5795 |
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