HPLC MS-MS Analysis Shows Measurement of Corticosterone in Egg Albumen Is Not a Valid Indicator of Chicken Welfare

SIMPLE SUMMARY: Growing interest in the welfare of farmed animals, particularly those in restrictive housing, has stimulated attempts to define simple measures of welfare. One such claimed measure involves the analysis of so-called stress hormones. In chickens, the main ‘stress hormone’ is corticosterone. In Australia, reviews of legislation relating to the welfare of chickens housed in cages have relied heavily on non-invasive measures of corticosterone, in particular those using egg white (albumen). All of those measures have used antibodies to quantify the corticosterone. Recently, doubts have been raised about the specificity of these measurement techniques. In this study, we demonstrate that high-resolution chromatographic separation of extracted egg albumen, followed by mass spectrometry, reveals that corticosterone is barely detectable in chicken egg albumen. Previous work using immunoassays reported levels of 0.5 to 20 ng/g. We have found egg albumen corticosterone concentrations of about 50 pg/g. We conclude there is so little corticosterone in egg albumen that it is not routinely usable as an indicator of chicken welfare. We have also found significant amounts of other steroids (progesterone, cortisol) in chicken egg white, which may have contributed to the levels reported in the antibody studies. ABSTRACT: Assessment of animal welfare can include analysis of physiological parameters, as well as behavior and health. Levels of adrenocortical hormones such as cortisol (and corticosterone in chickens) have been relied on as indicators of stress. Elevations in those hormones have been said to be correlated with poor welfare, while levels in the normal range have been interpreted to mean that animals are in a good state of welfare. Procuring blood samples from animals for hormone measures can in itself be stressful and cause increases in the target hormones. To overcome this problem, indirect measures of cortisol and corticosterone have been developed. In chickens, corticosterone levels in egg albumen are said to be a useful indirect measure, and have been used in several recent studies as indicators of chicken welfare. All of the measures of chicken egg albumen corticosterone in welfare studies have used immunoassays, and have reported values ranging from about 0.5 to over 20 ng/g. Using these measures, egg albumen from chickens housed in conventional cages or free ranging has been said to have indistinguishable corticosterone levels. This has been used to support the conclusion that chickens kept in conventional cages are not experiencing stress and are in a good state of welfare. In this study, we have used high-pressure liquid chromatography (HPLC) coupled with mass spectrometry (MS) to measure corticosterone in egg albumen. We found levels of corticosterone (median level about 50 pg/g) in egg albumen which were just above the limit of detection. By contrast, we found significant levels of progesterone and cortisol, hormones which would be expected to cross react with anti-corticosterone antibodies, and which therefore might explain the high reported levels of corticosterone using immunoassay. We conclude that because corticosterone levels in egg albumen are negligible, they cannot be used as an indicator of chicken welfare.