In view of the consequences of glucocorticoid (GC) production, inter-individual comparisons of the hormonal response to challenges are of interest. GC excretion is widely assessed non-invasively via measurements of immunoreactive glucocorticoid metabolites (CM) in excreta by means of enzyme immunoassays (EIAs). A few methodological issues need to be considered to ensure valid results. We examined whether and how patterns (mixture) of excreted CM change during development (early and late nestling stage, adults) in blue tits and compared two EIAs. Assay 1 had been successfully validated for a number of bird species. Assay 2 used an antibody against the same antigen but raised in a different individual. The difference could affect antibody cross-reactivity and therefore the suitability of assay 2 had to be validated. We compared the results of the two assays in samples of adult blue tits (handling stress experiment), chicken and quail (ACTH challenges). The CM patterns of 3-4 days old blue tit nestlings differed markedly from those of 11-12 days old nestlings and adults. Sex differences in CM patterns were found in all age classes. While assay 2 cannot be recommended for measuring stress responses in adult blue tits, it was suitable for use in chicken and quail. Our results show that a change in antibody charge may have a remarkable impact on antibody cross-reactivities with CM and may affect the biological sensitivity of an assay to measure hormonal stress responses. Furthermore, the suitability of an assay is highly species-dependent.

The welfare and productivity of South American camelids may be affected by stressful events. The purpose of this study was to validate a non-invasive method for stress monitoring using faecal samples and to apply it to evaluate a stressful event, such as confinement. For physiological validation, nine alpacas (Vicugna pacos) and six llamas (Lama glama) were subjected to pharmacological stimulation of their adrenal cortex. Serial faecal samples were collected during 48 h before and after stimulation. During confinement, faecal samples from six llamas were collected twice per day during six consecutive days. Faeces belonging to 18 vicuñas (Vicugna vicugna) were collected before and one day after their capture for confinement (Chacu). Faecal cortisol metabolites (FCM) were extracted from each sample

It was recently found that high concentrations of chicken yolk gestagens and gestagen metabolites hamper corticosterone quantification via immunoassays. However, the situation in chicken albumen is still unresolved. In addition, the ratio of steroid hormone in the yolk of wild birds might differ. To investigate these matters, corticosterone and gestagens were measured in individual fractions of high-performance liquid-chromatographic separations of chicken albumen and yolk of red jungle fowl. Similarly, yolk extracts of hens with corticosterone-releasing implants or placebos were analysed to assess the impact of elevated plasma corticosterone concentrations on authentic yolk corticosterone levels. We also compared the results of a previously used corticosterone enzyme immunoassay (EIA) to those from a commercial radioimmunoassay (RIA) kit. The analytical validations of chicken albumen, bankiva yolk and yolks from hens with or without artificially elevated plasma corticosterone levels indicated that the main share of the immunoreactivity measured via corticosterone immunoassays was caused by substances other than authentic corticosterone. In albumen, the concentration of authentic corticosterone was below the detection limit. Analysis of bankiva yolk revealed three major gestagen peaks with concentrations of up to 2000 ng per fraction and a corticosterone peak of about 0.8 ng per fraction. Both corticosterone assays found a slightly higher corticosterone peak in a corticosterone-implanted hen's yolk (EIA: 0.7 ng; RIA: 0.5 ng per fraction) compared to the sham-treated female (EIA: 0.5 ng; RIA: 0.2 ng per fraction) but both antibodies also bound to several other substances, presumably gestagens. Although a certain amount of circulating corticosterone might pass into the yolk, direct quantification of corticosterone in non-homogenized avian egg samples via immunoassays is not advisable.

1. The objectives of the present study were to validate a reduced, non-intrusive version (RLS) of the LayWel plumage scoring system in domestic laying hens with reference to complete, intrusive scoring (CLS) and to investigate the effect of these two scoring methods on corticosterone metabolite concentrations. 2. A total of 312 medium-heavy laying hens from 4 commercial hybrids kept in 24 floor pens were scored by two experienced teams. Another 150 hens from two hybrids kept in 6 pens were used for estimating scoring treatment effects on corticosterone metabolites in droppings. 3. Plumage scores were in general higher using the RLS method compared to the CLS method. The agreement between teams for plumage scores (CLS) were on a high (total score) to an excellent (single body part except breast and cloaca) level. 4. Birds subjected to CLS tended to have higher concentrations of corticosterone metabolites in droppings 2 h after scoring compared with birds in the control treatment (not scored). Birds subjected to RLS had intermediate concentrations. 5. It was concluded that a reduced version of the LayWel scoring system is a valid and reliable scoring method which tends to induce less stress to the subjects than the original procedure.

Faecal cortisol metabolite concentrations are useful indicators of stress in domestic and game animals. Their excretion by trained or raced horses was used as a suitable indicator of stress in the present study. The reference range of concentrations of faecal 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites, for racing standardbreds was calculated from data of 18 healthy regularly trained and raced trotters. The 95 % confidence interval was 14-31 nmol/kg faeces. Some unfit horses showed values out of this reference range, e.g. 2 of 4 horses with recurrent exertional rhabdomyolysis both had 11,17-DOA concentrations of 50 nmol/kg.In a further trial, the relation between faecal 11,17-DOA levels of 5 treadmill-trained trotters and exercise was investigated. Median 11, 17-DOA levels gradually increased from 39 nmol/kg at the start of the training program to a maximum of 146 nmol/kg prior to the 3(rd) training session. The 11,17-DOA levels of one rather nervous horse even reached 1,478 nmol/kg, prior to its 3(rd) training session. The day after the 1(st),2(nd), 3(rd) and 4(th) training session, median 11,17-DOA levels were 33, 55, 65 and 115 nmol/kg, respectively. The median 11, 17-DOA levels prior to 4 standard exercise test (SET) were 26, 43, 216 and 43 nmol/kg, respectively. The median post SET 11, 17-DOA levels were 63, 281, 197 and 81 nmol/kg.Plasma cortisol levels increased after exercise, but at 18 hrs it appeared that the circadian pattern was restored. The type of exercise and the time in relation to the exercise had significant effects on plasma cortisol concentrations. Plasma cortisol concentrations prior to SETs were higher than those prior to training.Increased baseline 11,17-DOA levels prior to exercise indicated increased hypothalamic-pituitary-adrenal axis activity during the entire trial. Hence, all training of horses and performing SETs indicates stress. However, when horses became accustomed to the exercise they excreted lower levels of faecal cortisol metabolites, suggesting successful adaptation to the stress of intensive exercise.

Although stocking density is perceived as a topic of major importance, no consensus has been reached on what density would allow for good welfare. In the present study, the welfare of 4 replicates of birds stocked at 8, 19, 29, 40, 45, 51, 61, and 72 broilers per pen (or 6, 15, 23, 33, 35, 41, 47, and 56 kg actually achieved BW/m(2)) was studied using 6 welfare indicators. Density did not affect bursa weight, mortality, or concentrations of corticosterone metabolites in droppings but did influence leg health (P = 0.015) and footpad and hock dermatitis (P < 0.001) and tended to influence fearfulness (P = 0.078). However, not every increase in density or group size, or both, led to poorer welfare for the affected indicators: leg health and fearfulness showed unexpected peaks at intermediate densities. Furthermore, the indicators were influenced at different densities: leg strength showed a steep decrease from 6 to 23 kg/m(2), hock dermatitis rose from 35 to 56 kg/m(2), and footpad dermatitis and fearfulness were only significantly higher at the highest density of 56 kg/m(2). No threshold stocking density above which all aspects of welfare were suddenly altered was found in this study. Instead, different aspects of welfare were influenced at different densities or group sizes, or both. Thus, evaluating the effects of stocking density on welfare as a whole would require either identification of acceptable levels for each separate indicator or a weighting of the indicators in an integrated welfare score. A tentative attempt to such an integration, made using equal weights for all parameters, showed a decrease in welfare as density increased (P < 0.001). The lowest 2 densities (6 and 15 kg/m(2)) scored better than most middle densities (23, 33, 35, and 47 kg/m(2)), whereas all densities scored better than the highest density (56 kg/m(2)).

Avian eggs contain a variety of steroid hormones, which have been attributed as a tool for maternal phenotypic engineering. The majority of studies focuses on androgens, but also significant amounts of progesterone as well as other steroid hormones have been measured. The question if corticosterone is also present in eggs of chickens is currently under debate. The only analytical validation performed so far has failed to demonstrate corticosterone in the yolk of chickens, suggesting that antibodies for corticosterone measurement cross-react with other steroids present in the yolk. In order to investigate this assumption and to characterise potential cross-reacting hormones in more detail, we performed high-performance liquid chromatographic (HPLC) analyses of chicken yolk extracts and determined the concentration of immunoreactive corticosterone, progesterone and cortisol.The progesterone antibody revealed several immunoreactive substances, including progesterone, pregnenolone and two substances with lower polarity. The corticosterone enzyme immunoassay detected immunoreactive substances at exactly the same elution positions as the progesterone assay and a very small peak at the elution position of corticosterone. Immunoreactive cortisol was not found. In addition, inner and outer regions of the yolk sphere were analysed separately via HPLC. We found different concentrations of immunoreactive substances between the inner and outer yolk regions, probably reflecting the steroidogenic activity of the follicle cells during oocyte growth. We conclude that in homogenised yolk extracts without previous clean-up, the measured corticosterone concentrations may actually reflect those of progesterone and its precursors, most probably being 5 alpha- and 5 beta-pregnanes and pregnenolone. (C) 2009 Elsevier Inc. All rights reserved.

Abstract The hormone content of a birds’ egg may reflect the environmental conditions of the bird. In this study we measured hormone concentrations of eggs from hens living under different housing conditions. Eggs from 16 floor-housed and 16 singly caged hens were analysed for androstenedione and estradiol. The concentrations of these hormones were highest in the yolk of eggs from floor-housed hens (P≤0.05). The concentration of estradiol in the albumen of eggs was highest for caged birds (P≤0.0001). In caged hens, the concentrations of both hormones varied significantly over days in the egg yolk, but not in the albumen. As the concentration of androstenedione and estradiol in the yolk of chicken eggs is environmentally dependent, these hormones may provide a mechanism by which the hen signals the state of the environment to her progeny.

Stress assessment favours methods, which do not interfere with an animal’s endocrine status. To develop such non-invasive methods, detailed knowledge about the excretion of hormone metabolites in the faeces and urine is necessary. Our study was therefore designed to generate basic information about catecholamine excretion in rats, mice and chickens. After administration of 3H-epinephrine or 3H-norepinephrine to male and female rats, mice and chickens, all voided excreta were collected for 4 weeks, 3 weeks or for 10 days, respectively. Peak concentrations of radioactivity appeared in one of the first urinary samples of mice and rats and in the first droppings in chickens 0.2–7.2 h after injection. In rats, between 77.3 and 95.6% of the recovered catecholamine metabolites were found in the urine, while in mice, a mean of 76.3% were excreted in the urine. Peak concentrations in the faeces were found 7.4 h post injection in mice, and after about 16.4 h in rats (means). Our study provides valuable data about the route and the profile of catecholamine excretion in three frequently used species of laboratory animals. This represents the first step in the development of a reliable, non-invasive quantification of epinephrine and norepinephrine to monitor sympatho-adrenomedullary activity, although promising results for the development of a non-invasive method were found only for the chicken.

The critical evaluation and reduction of stressful processes involved in farm animal husbandry, transport and slaughter represent serious issues in animal welfare discussion. Elevated concentrations of adrenocortical hormones are potential indicators of various forms of stress, however, the negative effects of the blood sampling procedure pose serious limitations to their measurement in the plasma. For non-invasive stress-assessment, profound knowledge about the metabolism of stress hormones is required. The present study aimed to monitor the distribution of corticosterone in the body of chickens, and to investigate if and to which extent, corticosterone is metabolised to other substances. Therefore, radiolabelled (H-3 -) corticosterone was administered intravenously to 24 broilers. Each 6 broilers were killed 1, 2, 4 or 8 h after hormone injection and radioactivity was measured in blood, tissue and faecal samples. Within 8 hours, the main part of the radiolabelled steroids was voided via the droppings. Only trace amounts were found in tissue samples at any time. Reversed-phase high-performance liquid-chromatographic (RP-HPLC) separations were performed on samples of the bile. They revealed that 1 hour after injection, H-3-corticosterone was completely metabolised into more polar substances. Furthermore, the results showed that the faecal corticosterone metabolites (CM) were already present in the bile and that a cortisone enzyme immunoassay (EIA) was more suited for the measurement of immunoreactive CM in the chicken than a corticosterone EIA. The findings from this study underline the suitability of recently developed non-invasive methods, which by evaluating stressful conditions can help to improve animal welfare.

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.

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.

Abstract: Birds are discussed as models for prenatal stress. In this study, several experiments were conducted to gain basic knowledge of if, how, and when maternal adrenocortical activity is reflected by corticosterone concentrations in the egg. Radiolabeled corticosterone was administered to 10 laying hens to investigate the uptake into as well as the distribution within the eggs. The yolk was dissected in concentric layers and analyzed. Less than 1% of the administered radioactivity entered the egg but was, however, not evenly distributed. On the day after injection, highest radioactivity (Bq/g) was detected in the albumen and the outmost layer, whereas concentration peaked –7 days later in the inner layers. In two other experiments, increased plasma levels of corticosterone were induced by injection of adrenocorticotropic hormone (ACTH) or feeding of corticosterone. Again, yolk disks were cut in layers and analyzed with a corticosterone enzyme immunoassay. No effect of the ACTH administration was detected, whereas feeding of corticosterone resulted in increased immunoreactive corticosterone concentrations in the yolk. Straight‐phase high‐performance liquid chromatographic (HPLC) separations were also performed to characterize immunoreactive steroids in the yolk. Two close‐eluting peaks at the approximate elution position of corticosterone could be observed after the feeding experiment, whereas in untreated control eggs they were absent. It was concluded that transfer from plasma to egg is low for corticosterone and that further investigations concerning the transport mechanisms and the exact nature of yolk steroids are necessary.

1. A non-invasive technique for stress assessment is needed. Therefore, an enzyme immunoassay (EIA) for measurement of glucocorticoid metabolites in chicken droppings was established and validated. 2. Radiolabelled corticosterone was administered intravenously to detect the time course of excreted metabolites. The metabolites were then characterised by chemical and immunological methods to find a suitable antibody. 3. Reversed-phase high-performance liquid chromatography (RP-HPLC) separations of the peak concentration samples revealed that corticosterone was extensively metabolised, mainly to more polar substances. 4. HPLC fractions were tested in several EIAs for glucocorticoid metabolites, where the highest quantities were detected by a newly established cortisone assay, measuring metabolites with a 3,11-dione structure. 5. The biological relevance of this cortisone EIA was confirmed by stimulation of adrenocortical activity by adrenocorticotropic hormone (ACTH). 6. With this newly developed EIA it should be possible to measure adrenocortical activity non-invasively in chickens and other galliformes, thus providing a tool for a variety of research fields, such as poultry production, ethology and behavioural ecology.