Reducing stress during welfare inspection: validation of a non-intrusive version of the LayWel plumage scoring system for laying hens.

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.

Gestagens and glucocorticoids in chicken eggs.

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.

Stocking density effects on broiler welfare: identifying sensitive ranges for different indicators.

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)).

Corticosterone in chicken eggs.

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 4-7 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.

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

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.

Excretion of catecholamines in rats, mice and chicken.

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 (3)H-epinephrine or (3)H-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.

Measurement of corticosterone metabolites in chicken droppings.

(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.