Determination Of Corticosterone Levels Research Articles

Validation of an Alternative Feather Sampling Method to Measure Corticosterone.

Simple SummaryResearch projects on birds’ welfare or their stress physiology are often complemented by measurements of corticosterone level in feathers. Until now, the common standard for this method is to collect the feathers by plucking, a procedure which on living birds is presumed to be painful and to cause stress. Therefore, in most European countries an animal experiment application is required. The aim of this study was to validate an alternative, possibly less stressful sampling method: cutting the feathers close to the skin. The examined species were geese and ducks from a conventional poultry husbandry. There was no relevant difference between the two methods assessed according to statistical analysis. In conclusion, it is reasonable to assume that feather cutting could be established as an alternative sampling method for measuring corticosterone. Nevertheless, we recommend further research on other species to confirm these results.The most common feather sampling method for feather corticosterone measurement is by plucking the feathers from the bird’s skin. This procedure performed on living, restrained birds is qualified as an animal experiment according to German/European legislation, which has to be applied for from the competent authorities. The Directive 2010/63/EU requires the full implementation of the 3-R Principle of Russel and Burch in animal experiments, which means not only to replace the use of animals, but also to reduce the number of animals used and to refine procedures whenever possible. In response to this issue, the aim of this study was to validate an alternative, less invasive sampling method by cutting feathers close to the skin in comparison to the gold standard of plucking them. For this proof-of-principle study, a conventional poultry husbandry with trial groups of geese (Anser anser domesticus) and ducks (Anas sterilis) was selected. All birds were kept under the same living conditions to standardize the influencing factors regarding husbandry, and thus, their stress levels. Feather samples were collected between the shoulders from 46 geese and 51 ducks, both by cutting as well as by plucking, directly after slaughter for meat production. Feather corticosterone levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Results were compared using Bland–Altman plots and concordance correlation coefficients (CCC). It could be seen that concordance between corticosterone levels in cut and plucked feathers was rather poor: 0.38 for Anser, and 0.57 for Anas. However, comparing the mean corticosterone values in pg/mm of each species with their respective standard deviations, the differences between the methods were negligible. As the results showed that the differences between the individuals were markedly greater than the differences between the methods, the determination of corticosterone levels in cut feathers is valid compared to using plucked feathers. The validation tests of ELISA showed only acceptable repeatability and reliability. Hence, the results should be verified in further studies. In conclusion, it is recommended for future research to use cut instead of plucked feathers for corticosterone measurement.

An optimized method for the analysis of corticosterone in rat plasma by UV–HPLC

Animal models are useful in the study of stress disorders in that they offer the possibility of stimulating a human condition under controlled conditions in a simpler, more readily understandable system. Stress-related activation of the hypothalamic-pituitary-adrenal (HPA) axis characterized by an increase in plasma corticosterone (CT) levels in the rat is an important manifestation of the physiological stress response. Current available methods for the determination of peripheral corticosterone concentrations from trunk blood, is via a commercially available radio immunoassay (RIA) kit. The aim of this study was to optimize and validate a sensitive, specific and cost-effective high performance liquid chromatography (HPLC) method for the determination of CT levels in plasma of rats. A 500 µl plasma sample was extracted with 5 ml dichloromethane and analyzed by HPLC coupled to a diode array detector at 245 nm. The standard curve was linear over a concentration range of 10 - 500 ng/ml (r 2 =0.996). The percentage recovery was above 85%, the relative standard deviation was less than 7% and the limit of quantification was 10 ng/ml. Results from this method were compared with values obtained from a RIA method and the values were in close proximity of each other. We conclude that the current HPLC method that was optimized and validated is suitable for use in subsequent studies in rats.

Influence of Treatment with Intranasal Corticosteroids on the Nasal Mucosa, Weight, and Corticosteroid Concentration in Rats

The effect of intranasal corticosteroids on the nasal epithelium mucosa is an important parameter of treatment safety. This study was designed to examine whether treatment with topical corticosteroids in patients with allergic rhinitis causes atrophic nasal mucosal changes, when compared with systemic corticosteroids, in rats. Male Wistar rats were treated daily during 7 weeks with topical administration with 10 microliters of normal saline (control group), 10 microliters of mometasone furoate group, 10 microliters of triamcinolone acetonide (T group), and 8 mg/kg of daily subcutaneous injections of methylprednisolone sodium succinate (MP group). Body weight was evaluated weekly. At the end of the treatment, rats were killed by decapitation to collect blood for determination of corticosterone levels and nasal cavities were prepared for histological descriptive analyses. Treatment with T and MP decreased body weight. Plasma corticosterone concentration was significantly reduced by MP treatment and presented a clear tendency to decrease after T treatment. Histological changes observed in group T included ripples, cell vacuolization, increase in the number of nuclei, and decrease in the number of cilia in the epithelial cells. Growth and corticosterone concentration were impaired by T and MP at the same proportion, suggesting a role of this hormone in body gain. With the exception of T, intranasal or systemic treatment with the corticosteroids evaluated in this study did not affect nasal mucosa.

Baseline and stress-induced plasma corticosterone during long-distance migration in the bar-tailed godwit, Limosa lapponica.

The specific roles of corticosterone in promotion of avian migration remain unclear even though this glucocorticosteroid is elevated in many migrating bird species. In general, glucocorticosteroids promote metabolic homeostasis and may elicit effects on feeding and locomotion. Because the migratory stages of refueling and flight are characterized by distinct behaviors and physiology, the determination of corticosterone levels during each stage should help identify potential processes in which corticosterone is involved. We measured baseline levels of corticosterone in bar-tailed godwits (Limosa lapponica) during two distinct stages of migration: (1) immediately after arrival at a false stopover site just short of the Wadden Sea and (2) throughout the subsequent 4-wk refueling period on the Wadden Sea. Plasma corticosterone was higher in arriving than in refueling birds. In addition, corticosterone increased with size-corrected body mass during the refueling phase, suggesting that corticosterone rises as birds prepare to reinitiate flight. Therefore, elevated corticosterone appears associated with migratory flight and may participate in processes characterizing this stage. We also performed a capture stress protocol in all birds and found that corticosterone increased in both arriving and refueling godwits. Therefore, the normal course of migration may be typified by corticosterone concentrations that are lower than those associated with stressful and life-threatening episodes.

CIRCADIAN VARIATION IN THE SENSITIVITY OF THE PITUITARY–ADRENAL SYSTEM TO DEXAMETHASONE SUPPRESSION

Intact female rats were injected with 100 microgram dexamethasone/kg body weight or saline at 12.00, 24.00 or 04.00 h. Four hours later, i.e. 16.00, 04.00 or 08.00 h respectively, a double bleeding procedure was used to obtain blood samples from stressed and unstressed rats for subsequent fluorometric determination of corticosterone levels in the plasma. The difference between the level of corticosterone is stressed and unstressed rats (the stress increment) was determined and used as an index of the response of corticosterone in the plasma to stress. Increments in the levels of corticosterone in the plasma evoked by stress in rats injected at 12.00 h with dexamethasone and bled 4 h later were significantly (P less than 0.05) less than those in rats given dexamethasone at 24.00 or 04.00 and bled at 04.00 or 08.00 h respectively. In contrast, stress-induced increments in the level of corticosterone in the plasma of rats treated with saline did not vary with the time of day.

Acute effect of morphine administration on secretion of adrenal corticosterone in rats.

The purpose of this study is to determine the acute effect of morphine on ACTH secretion and to determine whether morphine has a direct action on the adrenal cortex by the adult female Wistar rat. ACTH was measured by the determination of corticosterone levels in adrenal glands, adrenal venous blood and circulatory blood. It should be emphasized that in this paper, mechanism of secretion of adrenal corticosterone by the acute effect (the early stage action) following morphine administration was described. Even in morphinization with five daily morphine (2mg/100g, B. W., i. p.) injections, the account of acute effect was determined immediately after the final injection of morphine administration. Morphine alone can not act as an inhibitor of ACTH release nor as an inhibitor of ACTH release due to histamine stress, and morphinization can not act to inhibit ACTH release or to block the effect of a histamine stress on the secretion of ACTH in the early stage action of the final morphine administration, as evidenced by corticosterone levels both in adrenal glands and in circulating blood of the intact rats. Morphine (2mg/100g, B. W., i. p.) alone caused stimulation of ACTH release as well as the final morphine injection of morphinization, and pretreatment of the rats with morphine (2mg/100g, B. W., i. p.) could not act to block the effect of a histamine (0.5mg/100g, B. W., i. p.) stress on the release of ACTH in the early stage action of morphine administration. A detailed account of the effect of morphine or morphinization with four daily morphine injections on corticosterone levels both in adrenal and in adrenal venous blood of the hypophysectomized-ACTH-treated rat has been reported. Morphine (2mg/100g, B. W., i. p.) alone or with pretreatment of pentobarbital (1mg/100g, B. W., i. p.) does not directly interfere with the response of the adrenal cortex to exogenous ACTH in the early stage action of morphine administration. Morphinization with four daily morphine (2mg/100g, B. W., i. p.) injections in the early stage action following the final morphine injection gave the same results.It seems likely that morphine does not directly act on adrenal corticoidogenesis in vivo. These results suggest in the early stage action of morphine administration that morphine acts to stimulate ACTH release at a higher level than the anterior pituitary and does not directly act on adrenal cortex, and that the acute effect of strong stimuli on the rate of ACTH release is not mediated by a decrease in the concentration of circulating adrenal corticosteroids.