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.

(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.

a b s t r a c tAlthough most work on prenatal stress has been conducted on mammalian species, birds provide useful alternative models since avian embryos develop outside the mother's body in a concealed environment, the egg, which is produced during a short time window of 4-14 days. This facilitates measurement of maternal substances provided for and manipulation of the embryo without interfering with the mother's physiology. We critically review prenatal corticosterone mediated effects in birds by reviewing both studies were females had elevated levels of plasma corticosterone during egg formation and studies applying corticosterone injections directly into the egg. A selected review of the mammalian literature is used as background. The results suggest that besides prenatal exposure to corticosterone itself, maternal corticosterone affects offspring's behaviour and physiology via alteration of other egg components. However, results are inconsistent, perhaps due to the interaction with variation in the post-natal environment, sex, age, developmental mode and details of treatment. The potential role of adaptive maternal programming has not been tested adequately and suggestions for future research are discussed.

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)).