Chronic treatment with glucocorticoids does not affect egg quality but increases cortisol deposition into egg albumen and elicits changes to the heterophil to lymphocyte ratio in a sex-dependent manner.

During chronic stress, there is an initial increase in glucocorticoid (GC) levels, but they then return to low, albeit not baseline, levels. Recent studies have renewed interest in cortisol in that it may also have important roles in the stress response. The purpose of our study was to test the hypothesis that chronic treatment with low levels of either corticosterone or cortisol would alter HLR and immune organ morphometrics. Further, we wanted to determine if chronic treatment with either GC would elicit an increase in cortisol levels in egg albumen. To test our hypotheses, we implanted silastic capsules that contained corticosterone, cortisol, or empty capsules as controls (N = 5/sex/treatment). Blood serum, smears, body weights, and egg quality data were collected. Ducks were then euthanized and body weight, weights of spleens, livers, and the number of active follicles were recorded. Albumen GC levels were assessed using mass spectrometry. Data were analyzed using a 2- or 3-way ANOVA as appropriate and post-hoc with Fishers PLSD. No treatment elicited differences in egg quality measures or body weight compared to controls. Corticosterone treatment did elicit an increase in serum corticosterone (p < 0.05), but not cortisol, levels compared to controls in both sexes. Both cortisol and corticosterone treatments increased (p < 0.05) serum levels of cortisol compared to controls. Relative spleen weights were higher (p < 0.05) in hens following corticosterone but not cortisol treatment. No other organs showed any differences among the treatment groups. Both GCs elicited an increase (p < 0.001) in HLR in hens at all time-points over the 2-week treatment period compared to controls. Cortisol, not corticosterone, only elicited an increase in HLR for drakes (p < 0.05) compared to controls but only at day 1 after implants. Chronic treatment with cortisol, but not corticosterone, elicited an increase (p < 0.01) in egg albumen cortisol levels compared to other groups. Corticosterone was not detected in any albumen samples. Our results suggest that glucocorticoids elicit differential effects and although corticosterone has been stated to be the predominant GC in avian species, cortisol may provide critical information to further understand bird welfare.

Research Note: Sex difference in changes in heterophil to lymphocyte ratios in response to acute exposure of both corticosterone and cortisol in the Pekin duck.

Poultry scientists have utilized both direct and indirect measures of stress hormones for monitoring the state of avian welfare. For decades, it has been assumed that the mammalian and avian hypothalamic pituitary adrenal (HPA) function similarly to one another. However, there are considerable differences between the 2. Further, it has been assumed that the predominate glucocorticoid (GC) in birds was corticosterone, but recent studies have suggested that both corticosterone and cortisol are secreted. GC release is associated with an increase in blood heterophils due to increased migration from the lymph nodes and a decrease in lymphocytes due to marginalization. Both actions account for an increase in heterophil to lymphocyte ratios (HLR). The goal of this project was to determine the effect of each GC on HLR over time. To achieve this, we intramuscularly injected 2.0 mg/kg of corticosterone or cortisol, a lower dose cortisol treatment (0.5 mg/kg), or safflower oil as vehicle control. Blood was collected prior to intramuscular (IM) injections and blood collected 3 more times at every hour. Blood smears were also collected to assess HLR at the same four time points. HLR assays were completed by avian pathologists from an independent lab who were unaware of the treatments. Data were analyzed by 3-way repeated measures ANOVA with a P < 0.05 considered significant. We found significant sex (P < 0.001) x treatment (P < 0.001) x time (P < 0.001) effects with significant interactions (P = 0.0055). In hens, both GC resulted in significant increase in HLR at 1 h after injection compared to controls. In drakes, however, both GC showed a significant increase in HLR but not until 2 h after injection. The low dose cortisol had no significant effect on HLR in either sex. These data suggest that sex differences need to be considered when assessing duck welfare, and that cortisol may play a role in the HPA axis in ducks.

Sex differences in serum glucocorticoid levels and heterophil:lymphocyte ratios in adult pekin ducks (Anas platyrhynchos domesticus).

It is becoming more common for poultry scientists to utilize direct and indirect measures of stress hormones to monitor bird welfare. However, it has been clear that our understanding of the avian hypothalamic-pituitaryadrenal axis (HPA) is insufficient as evidenced by the many conflicting reports regarding stress responses, such as transportation stress, in poultry. It has long been assumed that the poultry HPA functions similarly to that of mammals, but now we know that there are considerable differences in the avian HPA compared to mammals. Synthesis and release of glucocorticoids (GC) are stimulated by adrenocorticotropin hormone (ACTH); GC are synthesized from a common pathway that begins with cholesterol and pregnenolone. The synthesis of one of the glucocorticoids does not depend upon the synthesis of the other. The purpose of our study was to test the hypothesis that ACTH will stimulate both corticosterone and cortisol release in ducks. To test this hypothesis, we injected artificial ACTH (cosyntropin; 0.0625 mg/kg, 0.031 mg/kg, or 0.016 mg/kg or saline as control) intramuscularly into adult drakes and hens (N = 10/sex/dose). Both glucocorticoids (GC) were assayed in serum using previously verified ELISAs. Blood smears were also assessed for heterophil to lymphocyte ratios (HLR). Data were analyzed by repeated measures 3-way ANOVA with Fishers PLSD as an ad hoc test. We observed that both GC were secreted in significantly (p = 0.0002) different patterns in a dose-dependent manner compared to controls, and that there was a significant (p = 0.0001) sex difference in both GC compared to saline controls. Further, we observed that all doses of ACTH elicited a significant (p = 0.004) sex difference in the HLR response compared to controls, but no dose-dependent effects were noted. Our data suggest that ducks, at least, may utilize more than just corticosterone to maintain physiological homeostasis in response to stress. Further, the time course of the stressor to release GC and subsequent HLR response may be dependent upon sex. More detailed analyses of the HPA are necessary in all avian species to better understand stress responses as we utilize biological bases for welfare assessments and stress responses.