Preening cups increase apparent wet preening behaviors, but have no impact on other behaviors, body condition, growth, or body morphometrics of grow-out Pekin ducks.

Preening cups may be a form of open water that would allow ducks to express preening behaviors. We set out to test the hypothesis that preening cups would not have detrimental effects on ducks or their environment. Control pens (N = 6, 65 ducks/pen) had nipple lines while experimental pens (N = 6, 65 ducks/pen) had the same nipple line plus one preening cup (PC). Body weights of 30 ducks per pen, and body condition scores on 50 ducks per pen were recorded weekly. On d 18 and 43, 5 ducks per pen were euthanized and their spleens, Bursas, liver, and uropygial glands were weighed. Behavior data were collected using scan sampling with video being recorded for 72 continuous hours at 4 different ages: 25 d, 30 d, 36 d, and 40 d. Body morphometrics were analyzed by 2-way ANOVA with repeated measures. Body condition scoring was analyzed by Pearson's chi-square. The GLIMMIX procedure (SAS 9.4) was used for behavioral analyses to examine treatment differences in the proportion of ducks performing dry preening, wet preening, eating, drinking, standing, and laying down. Feather pecking, feather picking, preening conspecifics (also known as allopreening), dunking head, and drinking from preening cup were analyzed using PROC LOGISTIC with the Firth bias correction for quasi-complete separation and odds ratios were calculated. More PC ducks housed with PC performed wet preening compared to control ducks (25 d: F1,26 = 6.90, P = 0.0143; 30, 36, and 40 d; F1,78 = 24.53, P < 0.0001). Ducks in the PC group were also more likely to lay down compared to controls (25 d: F1,33 = 4.95, P = 0.0330). No differences were observed for any other behavior, body condition score, body weight or morphometrics at any age. Although ducks in the preening cup group showed an increase in wet preening, our data suggest that open water is not necessary to maintain feather condition or uropygial gland size.

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.

Chronic heat stress part 1: Decrease in egg quality, increase in cortisol levels in egg albumen, and reduction in fertility of breeder pekin ducks.

Global warming poses detrimental effects on poultry production leading to substantial economic losses. The goal of our experiment was to test the hypothesis that heat stress (HS) would alter welfare and egg quality (EQ) of breeder ducks. Furthermore, we wanted to test if HS would increase cortisol levels in egg albumen. Adult Pekin ducks were randomly assigned to two different rooms at 85% lay with 60 hens and 20 drakes per room. Baseline data including body weight, body condition scores (BCS), and egg production/quality were collected the week preceding heat treatment. Ducks were subjected to cyclic HS of 35°C for 10h/day and 29.5°C for the remaining 14h/day for 3 weeks while the control room was maintained at 22°C. Eggs were collected daily and analyzed weekly for quality assessment, and for albumen glucocorticoid (GCs) levels using mass spectrometry. One week before the exposure to HS, 10 hens and 5 drakes were euthanized and the same number again after 3 weeks and birds necropsied. Data analyses were done by 1- or 2-way ANOVA as appropriate with a Tukey-Kramer post hoc test. BCS were analyzed using a chi-squared test. A p ≤ 0.05 was considered significant. Circulating levels of corticosterone were significantly (p < 0.01) elevated at week 1 only in the HS hens. The circulating levels of cortisol increased significantly at week 1 and 2 (p < 0.05), and week 3 (p < 0.01) in the hens and at weeks 2 and 3 only (p < 0.05) in the drakes. Feather quality scores (p < 0.01), feather cleanliness scores (p < 0.001) and footpad quality scores (p < 0.05) increased significantly in the HS group. HS elicited a significant (p < 0.001) decrease in egg production at weeks 1 and 3. Hens in the HS group showed significantly decreased BW (p < 0.001) and number of follicles (p < 0.05). Shell weight decreased significantly at week 1 only (p < 0.05) compared to controls. Yolk weight decreased significantly at week 3 (p < 0.01) compared to controls. HS elicited a significant increase in albumen cortisol levels at week 1 (p < 0.05) and week 3 (p < 0.05). Thus, cortisol may provide critical information to further understand and to improve 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.

Sex differences in glucocorticoid responses to shipping stress in Pekin ducks.

Some concerns have been raised recently about the assay of corticosterone vs. cortisol in poultry species. Thus, we tested the hypothesis that ducks secrete both glucocorticoids. First, we validated two commercially ELISA kits for the two glucocorticoids by first charcoal stripping duck serum in order to remove all steroid hormones. We ran serial dilutions of spiked, charcoal-stripped serum on kits of opposite glucocorticoid as well as a serial dilution using the respective ELISA buffer of the opposite assay kit. We found that the glucocorticoid standard curve in duck serum matched the respective curve in that kit's own buffer. However, when the opposite hormone was run in each kit in both duck serum or ELISA buffer, a near zero slope was obtained. Second, we further validated the presence of both glucocorticoids using mass spectrometry. Third, we tested the hypothesis that exogenous ACTH would stimulate the release of both corticosterone and cortisol. And, fourth, we tested the hypothesis that each glucocorticoid would have different serum levels in response to shipping stress. To test this hypothesis, we collected serum from 10 drakes and 10 hens from 2 flocks (N = 20 per time point per sex): 24 h prior to shipping, at shipping as ducks were walked off the truck, 24 h after shipping, and 1 wk after shipping. Data were analyzed by 2-way repeated measures ANOVA. Surprisingly, we also observed a sex difference in both glucocorticoid levels in that hens showed higher (P < 0.01) serum levels than did drakes at all-time points in response to either ACTH or transportation. Finally, no differences were observed in either glucocorticoid levels associated with shipping in either sex. The fact that both glucocorticoids are released in measurable amounts lends to the possibility that they may be differentially regulated, or at least there is a sex difference in the neural pathways associated with glucocorticoid release in ducks. Although corticosterone is the likely predominate glucocorticoid in ducks, serious attention should be given to the role of cortisol in poultry. Further consideration of sex, age, and timing of blood collection to stressor needs to be considered when assessing glucocorticoid levels in any avian species.