Rupert Palme

Hormones as indicators of stress

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.

Measuring stress in wildlife: techniques for quantifying glucocorticoids

Stress responses play a key role in allowing animals to cope with change and challenge in the face of both environmental certainty and uncertainty. Measurement of glucocorticoid levels, key elements in the neuroendocrine stress axis, can give insight into an animal’s well-being and can aid understanding ecological and evolutionary processes as well as conservation and management issues. We give an overview of the four main biological samples that have been utilized [blood, saliva, excreta (feces and urine), and integumentary structures (hair and feathers)], their advantages and disadvantages for use with wildlife, and some of the background and pitfalls that users must consider in interpreting their results. The matrix of choice will depend on the nature of the study and of the species, on whether one is examining the impact of acute versus chronic stressors, and on the degree of invasiveness that is possible or desirable. In some cases, more than one matrix can be measured to achieve the same ends. All require a significant degree of expertise, sometimes in obtaining the sample and always in extracting and analyzing the glucocorticoid or its metabolites. Glucocorticoid measurement is proving to be a powerful integrator of environmental stressors and of an animal’s condition.

Stress hormones in mammals and birds. Comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples

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.

Measuring fecal steroids: guidelines for practical application.

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 animals 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.

Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals

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.

Antidepressants recruit new neurons to improve stress response regulation

Recent research suggests an involvement of hippocampal neurogenesis in behavioral effects of antidepressants. However, the precise mechanisms through which newborn granule neurons might influence the antidepressant response remain elusive. Here, we demonstrate that unpredictable chronic mild stress in mice not only reduces hippocampal neurogenesis, but also dampens the relationship between hippocampus and the main stress hormone system, the hypothalamo-pituitary-adrenal (HPA) axis. Moreover, this relationship is restored by treatment with the antidepressant fluoxetine, in a neurogenesis-dependent manner. Specifically, chronic stress severely impairs HPA axis activity, the ability of hippocampus to modulate downstream brain areas involved in the stress response, the sensitivity of the hippocampal granule cell network to novelty/glucocorticoid effects and the hippocampus-dependent negative feedback of the HPA axis. Remarkably, we revealed that, although ablation of hippocampal neurogenesis alone does not impair HPA axis activity, the ability of fluoxetine to restore hippocampal regulation of the HPA axis under chronic stress conditions, occurs only in the presence of an intact neurogenic niche. These findings provide a mechanistic framework for understanding how adult-generated new neurons influence the response to antidepressants. We suggest that newly generated neurons may facilitate stress integration and that, during chronic stress or depression, enhancing neurogenesis enables a dysfunctional hippocampus to restore the central control on stress response systems, then allowing recovery.

Excretion of infused 14C-steroid hormones via faeces and urine in domestic livestock

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.

Measurement of cortisol metabolites in faeces of ruminants.

Twenty-one metabolites were detected in faecal samples collected after infusion of (14C)cortisol into the jugular vein of sheep, using high-performance liquid chromatography/radiometric analysis plus mass spectrometry. One group of metabolites had molecular weights of between 302 and 308, and another group of 350, which indicates that the substances have a C19O3 or a C21O4 structure. Therefore, an enzyme immunoassay against 5β-androstane-3α-o1-11,17-dione-17-CMO:BSA was established. Faecal samples were collected from 10 cows immediately after transport and then during a course in which non-invasive diagnostic procedures were being taught (course 1). For comparison, faeces were sampled from another 5 cows that were being used for teaching invasive procedures (course 2). Six cows from a university farm served as controls. In the animals used in course 1, the highest concentrations of cortisol metabolites were measured immediately after transport to the university (median value: 2.2 μmol/kg faeces). During the first 5 days at the university, the concentrations decreased to 0.52 μmol/kg (median) and remained at this level during the rest of the course. The median concentration in the samples that were taken during course 2 (collected about 2 months after transport) was 0.48 μmol/kg. There was no significant difference in the excretion of cortisol metabolites between these cows and the controls. We conclude from these data that, using the enzyme immunoassay against 5β-androstane-3α-o1-11,17-dione-17-CMO, we were able to detect transport/novel environment stress but not the potential disturbance that cows experience during diagnostic procedures.

Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones

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.

Measurement of Corticosterone Metabolites in Birds' Droppings: An Analytical Approach

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.