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Author |
Touma, C.; Palme, R. |
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Title |
Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation |
Type |
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 |
Issue |
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Pages |
54-74 |
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Keywords  |
Animals; Birds/*metabolism; Circadian Rhythm; Feces/*chemistry; Glucocorticoids/*analysis; Mammals/*metabolism; Reproducibility of Results; Seasons; Sex Factors |
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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. |
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Max Planck Institute of Psychiatry, Department of Behavioral Neuroendocrinology, Kraepelinstrasse 2-10, D-80804 Munich, Germany. touma@mpipsykl.mpg.de |
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0077-8923 |
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PMID:16055843 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
4073 |
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Author |
Mostl, E.; Rettenbacher, S.; Palme, R. |
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Title |
Measurement of corticosterone metabolites in birds' droppings: an analytical approach |
Type |
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 |
Issue |
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Pages |
17-34 |
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Keywords |
Animals; Birds/*metabolism; Corticosterone/*analysis/metabolism; Feces/*chemistry; Gas Chromatography-Mass Spectrometry; Immunoassay; Molecular Structure; Reproducibility of Results; Sensitivity and Specificity |
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Abstract |
Fecal steroid analyses are becoming increasingly popular among both field and laboratory scientists. The benefits associated with sampling procedures that do not require restraint, anesthesia, and blood collection include less risk to subject and investigator, as well as the potential to obtain endocrine profiles that are not influenced by the sampling procedure itself. In the feces, a species-specific pattern of metabolites is present, because glucocorticoids are extensively metabolized. Therefore, selection of adequate extraction procedures and immunoassays for measuring the relevant metabolites is a serious issue. In this review, emphasis is placed on the establishment and analytical validation of methods to measure glucocorticoid metabolites for a noninvasive evaluation of adrenocortical activity in droppings of birds. |
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Institute of Biochemistry, Department of Natural Sciences, University of Veterinary Medicine, Vienna, Veterinarplatz 1, A-1210 Vienna, Austria. erich.moestl@vu-wien.ac.at |
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0077-8923 |
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PMID:16055841 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
4082 |
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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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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. |
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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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Call Number |
Equine Behaviour @ team @ |
Serial |
4086 |
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Author |
Traversa, D.; Giangaspero, A.; Iorio, R.; Otranto, D.; Paoletti, B.; Gasser, R.B. |
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Title |
Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces |
Type |
Journal Article |
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Year |
2004 |
Publication |
Parasitology |
Abbreviated Journal |
Parasitology |
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Volume |
129 |
Issue |
Pt 6 |
Pages |
733-739 |
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Keywords |
Animals; DNA, Helminth/*analysis; DNA, Ribosomal Spacer/*chemistry; Feces/*chemistry; Female; Horse Diseases/*diagnosis/parasitology; Horses; Male; Polymerase Chain Reaction/*methods; Species Specificity; Spirurida Infections/diagnosis/*veterinary; Spiruroidea/*genetics |
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Abstract |
Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae. |
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Department of Biomedical Comparative Sciences, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy. traversa@unite.it |
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0031-1820 |
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Notes |
PMID:15648696 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
2631 |
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Author |
Schwarzenberger, F.; Mostl, E.; Bamberg, E.; Pammer, J.; Schmehlik, O. |
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Title |
Concentrations of progestagens and oestrogens in the faeces of pregnant Lipizzan, trotter and thoroughbred mares |
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Journal Article |
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Year |
1991 |
Publication |
Journal of reproduction and fertility. Supplement |
Abbreviated Journal |
J Reprod Fertil Suppl |
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Volume |
44 |
Issue |
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Pages |
489-499 |
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Keywords |
Animals; Estrogens/*analysis; Feces/*chemistry; Female; Gestational Age; Horses/*metabolism; Immunoenzyme Techniques; Labor, Obstetric; Pregnancy; Pregnancy, Animal/*metabolism; Pregnenes/analysis; Progestins/*analysis |
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Faecal samples were collected at weekly intervals from pregnant Lipizzan mares during Weeks 7-16 following mating and from Lipizzan, Trotter and Thoroughbred mares during the last 3 months of gestation. After parturition, samples were taken daily from the Thoroughbred mares for another 6 days. Non-pregnant mares served as controls. The concentrations of unconjugated oestrogens (Eg), 20 alpha-OH-progestagens (20 alpha-G) and 20 beta-OH-progestagens (20 beta-G) were measured by enzyme immunoassay. In the faeces of Lipizzan mares, immunoreactive progestagens were significantly (P less than 0.01) elevated above the levels in non-pregnant mares by Week 11, and Eg by Week 13 of pregnancy onwards. During the last 3 months of gestation, concentrations of Eg were significantly higher in Trotter mares than in Lipizzan and Thoroughbred mares. Concentrations of 20 alpha-G and 20 beta-G increased to maximal values in the last month of gestation. There was no significant difference among the 3 breeds with respect to 20 alpha-G but, during the 10 weeks before parturition, concentrations of 20 beta-G in the Lipizzan mares were significantly lower (P less than 0.05) than those in the Thoroughbred mares. They were also significantly lower than those of the Trotter mares during the last 4 weeks of gestation. After parturition, the concentrations of Eg and progestagens had declined to baseline values by Days 3 and 4 respectively. From these results we conclude that high concentrations of progestagens with 20 alpha- and 20 beta-hydroxyl groups are present in the faeces of pregnant mares, especially during the last month of gestation. |
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Institut fur Biochemie, Veterinary Medical University, Vienna, Austria |
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0449-3087 |
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PMID:1795293 |
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Call Number |
refbase @ user @ |
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322 |
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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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Volume |
1046 |
Issue |
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Pages |
75-80 |
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Keywords |
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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0077-8923 |
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PMID:16055844 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
4081 |
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