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M, E., östl,., Messmann, S., Bagu, E., Robia, C., & Palme, R. (1999). Measurement of Glucocorticoid Metabolite Concentrations in Faeces of Domestic Livestock. J. Vet. Med. A, 46(10), 621–631.
Abstract: After 14C-labelled cortisol infusion in ponies and pigs, faecal samples were collected. Extraction of 0.5 g faeces with 5 ml 80–90 % methanol yielded the highest radioactivity in the supernatant. Most of the metabolites were ether soluble. After high performance liquid chromatography (HPLC), the presence of immunoreactive metabolites was demonstrated by measuring each HPLC fraction using enzyme immunoassays for cortisol, corticosterone and 11-oxoaetiocholanolone. Only the assay for 11-oxoaetiocholanolone revealed peaks with co-eluting radioactivity. For biological validation of the test system, adrenocorticotrophic hormone (ACTH) and dexamethasone were injected intravenously successively in both species (n = 6). Cortisol concentration in blood and the 11-oxoaetiocholanolone immunoreactive substances in faeces were determined. In horse faeces, basal values of 2.3–35.2 nmol/kg were measured. After ACTH administration, an increase (more than 200 % above basal values) of these metabolites was seen about 1 day after ACTH administration. After dexamethasone injection the levels decreased, reaching minimum concentrations 2 days after administration. In pigs, an increase in these metabolites was measured in only three animals after ACTH; dexamethasone did not cause a decrease. The stability of the samples after defecation was tested by storing samples from cows, horses and pigs at room temperature. It was shown that there was a significant increase in the concentration of measured cortisol metabolites in bovine, equine and porcine faeces after storage for 1 h, 4 h and 24 h, respectively. In frozen samples this effect was diminished after thawing samples at 40°C; thawing the samples at 95°C prevented an increase in immunoreactive substances.
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Touma, C., Palme, R., & Sachser, N. (2004). Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones. Horm Behav, 45(1), 10–22.
Abstract: 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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Palme, R., Fischer, P., Schildorfer, H., & Ismail, M. N. (1996). Excretion of infused 14C-steroid hormones via faeces and urine in domestic livestock. Animal Reproduction Science, 43(1), 43–63.
Abstract: 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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Schwarzenberger, F., Mostl, E., Palme, R., & Bamberg, E. (1996). Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Animal Reproduction: Research and Practice, 42(1-4), 515–526.
Abstract: 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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Mostl, E., & Palme, R. (2002). Hormones as indicators of stress. Fourth International Conference on Farm Animal Endocrinology, 23(1-2), 67–74.
Abstract: 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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Möstl, E., & Palme, R. (2002). Hormones as indicators of stress. Domest. Anim. Endocrinol., 23(1–2), 67–74.
Abstract: 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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Palme, R. (2012). Monitoring stress hormone metabolites as a useful, non-invasive tool for welfare assessment in farm animals. Animal Welfare, 21(3), 331–337.
Abstract: 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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Merl, S., Scherzer, S., Palme, R., & Möstl, E. (2000). Pain causes increased concentrations of glucocorticoid metabolites in horse feces. J Equine Vet Sci, 20(9), 586–590.
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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Pawluski, J., Jego, P., Henry, S., Bruchet, A., Palme, R., Coste, C., et al. (2017). Low plasma cortisol and fecal cortisol metabolite measures as indicators of compromised welfare in domestic horses (Equus caballus). Plos One, 12(9), e0182257.
Abstract: The hypothalamic-pituitary-adrenal (HPA) axis response to chronic stress is far from straight forward, particularly with regards to animal welfare. There are reports of no effect as well as both decreases and increases in cortisol after chronic stressors. Therefore, the first aim of the present study was to determine how measures of compromised welfare, such as chronic pain and haematological anomalies, related to cortisol levels in domestic horses (Equus caballus). Domestic horses are an informative model to investigate the impact of chronic stress (due to environment, pain, work, housing conditions...) on the HPA axis. The second aim was to determine whether levels of fecal cortisol metabolites (FCM) may be used as an indicator of welfare measures. The present study used fifty-nine horses (44 geldings and 15 mares), from three riding centres in Brittany, France. The primary findings show that horses whose welfare was clearly compromised (as indicated by an unusual ears backward position, presence of vertebral problems or haematological anomalies, e.g. anaemia) also had lower levels of both FCM and plasma cortisol. This work extends our previous findings showing that withdrawn postures, indicators of depressive-like behavior in horses, are associated with lower plasma cortisol levels. We also found that evening plasma cortisol levels positively correlated with FCM levels in horses. Future research aims to determine the extent to which factors of influence on welfare, such as living conditions (e.g. single stalls versus group housing in pasture or paddocks), early life factors, and human interaction, act as mediators of cortisol levels in horses.
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Krueger, K., Marr, I., Dobler, A., & Palme, R. (2019). Preservation of fecal glucocorticoid metabolites and immunoglobulin A through silica gel drying for field studies in horses. conphys, 7(1).
Abstract: Non-invasive methods enable stress evaluation through measuring fecal glucocorticoid metabolites (FGMs), and immunoglobulin A (IgA) in the feces avoiding stressful blood drawing or stressful restraining of animals in the field. However, FGMs and IgA are mostly analysed in freshly frozen samples, which is difficult when fresh samples cannot be frozen immediately or frozen samples cannot be stored or transported. Good results were also derived from air-dried fecal samples, which are hampered by unstable air humidity in the field. These difficulties may be overcome, when drying of samples could be induced with colorless silica gel (SiO2) granules in a secure set-up, such as an air tight tube. We determined the speed of drying 1.5 g of a fresh fecal sample from six horses on air and on silica gel. Furthermore, FGMs and IgA were analysed in differently stored subsamples from 12 horses: in frozen fecal samples, in air- or silica gel-dried samples stored for 1 day and for 7 days, and in wet fecal samples kept in a tube at room temperature for 7 days. FGM levels remained stable in feces dried on air or on silica gel for 7 days, whereas IgA quantities showed a significant loss. Under field conditions, when freezing or transporting the frozen samples is not possible and humidity hampers air drying, drying samples on silica gel in air tight tubes appears to be very helpful and reliable for analysing FGMs.
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