Age matters: Differential effects of the exposure to elevated environmental temperatures on representative variables of the immune system in juvenile and adult female Japanese quail.

Several days under the influence of high environmental temperature negatively impact on some immune-related variables of poultry species. However, it is little known whether this exposure affects differentially the immune response on different life stages. This study was aimed at characterizing the immune responses of juvenile and adult female Japanese quail (Coturnix coturnix) when exposed to a cyclic chronic heat stressor. Independent sets of juvenile (24) and adult (21) female Japanese quail were bred under thermoneutral conditions or exposed to an increasing temperature from 24 °C to 34 °C, for 8 h along nine consecutive days (chronic heat stressor). The inflammatory response against a mitogen, the antibody production against a non-pathogenic antigen and the heterophil to lymphocyte (H/L) ratio were measured after the ninth day of chronic heat exposure. Adult females had a more robust inflammatory response compared to their juvenile counterpart. The inflammatory response, the production of antibodies, and the H/L ratio were suppressed by the chronic heat stressor in the juvenile females; while in their adult counterpart, the production of antibodies was not affected by the chronic heat stress. Under the exposure of high environmental temperatures, juvenile females would be more susceptible to potentially harmful pathogens that require a robust immune response, while their adult counterparts would be able to mount an optimal acquired immune response against pathogens requiring this type of response. Our findings suggest that while heat stress during breeding should be avoided in both juvenile and adult females, more precautions should be taken during early life stages.

Yolk corticosterone and progesterone levels in Greater Rhea (Rhea americana) eggs vary in a changing social environment.

Maternal hormones in avian egg yolks may signal and prepare offspring for the prevailing conditions. However, this adjustment requires some degree of flexibility in regulating yolk hormone deposition. The Greater Rhea (Rhea americana) has a particular mating system that combines mixed polygyny and polyandry, communal nesting, and exclusive paternal care of chicks. In this species, we previously found that yolk hormone deposition varies among eggs of different captive populations and could influence chicks' physiology and behavior. However, it is still unknown whether females can modify yolk hormone deposition in a changing social environment. Using a captive population of Greater Rheas, in this study, we quantified yolk hormone levels before and after a reduction in the number of females present in the population. We found that females deposited on average higher yolk corticosterone and lower yolk progesterone after the change in their social environment. Since corticosterone deposited into the yolk comes exclusively from the female's plasma, our results suggest that females had, on average, higher plasma corticosterone levels. The change in the number of females may increase the events of male-male competitions, courtships, and matings, leading to an increase of corticosterone in the females' plasma and then into their eggs. Since we previously found that higher yolk corticosterone and lower yolk progesterone were associated with the production of chicks that have an attenuated stress response, the present study results suggest that yolk hormone deposition is mediated by flexible mechanisms that could adjust development to the prevailing conditions.

Modulating offspring responses: concerted effects of stress and immunogenic challenge in the parental generation.

The perception, processing and response to environmental challenges involves the activation of the immuno-neuroendocrine (INE) interplay. Concerted environmental challenges might induce trade-off when resource allocation to one trait occurs at the expense of another, also producing potential transgenerational effects in the offspring. We evaluated whether concerted challenges, in the form of an immune inoculum against inactivated Salmonella enteritidis (immune challenge, ICH) and a chronic heat stress (CHS) exposure on adult Japanese quail, modulate the INE responses of the parental generation and their offspring. Adults were inoculated and later exposed to a CHS along nine consecutive days. For the last 5 days of the CHS, eggs were collected for incubation. Chicks were identified according to their parental treatments and remained undisturbed. Induced inflammatory response, heterophil/lymphocyte (H/L) ratio and specific humoral response against sheep red blood cells (SRBC) were evaluated in both generations. Regardless of the ICH, stressed adults showed a reduced inflammatory response and an elevated H/L ratio compared with controls. In offspring, the inflammatory response was elevated and the specific SRBC antibody titres were diminished in those chicks prenatally exposed to CHS, regardless of the ICH. No differences were found in the H/L ratio of the offspring. Together, our results suggest that CHS exposure influences the INE interplay of adult quail, establishing trade-offs within their immune system. Moreover, CHS not only affected parental INE responses but also modulated their offspring INE responses, probably affecting their potential to respond to future challenges. The adaptability of the developmental programming of offspring would depend on the environment encountered.

Effect of maternal environment on yolk immunoreactive corticosterone and its influence on adrenocortical and behavioral activity in chicks of Greater Rhea (Rhea americana).

Maternal corticosterone in avian eggs may modify offspring phenotype in order to increase survival in poor environments. In the Greater Rhea (Rhea americana), we previously found that yolk immunoreactive corticosterone is influenced by the quality of the maternal environment: eggs laid by females of the intensive rearing system (IRS), living in poor captive conditions, had higher yolk immunoreactive corticosterone than those produced by females of the semi-extensive rearing system (SRS), living in better conditions. Here, we evaluate if these different hormone levels are associated with the production of different phenotypes. We collected eggs from the IRS and SRS for hormonal quantification and artificial incubation. Then, half of the chicks selected from each environment were exposed to a capture and restraint protocol, and the rest remained undisturbed and were used as controls. In the IRS, we found that higher yolk immunoreactive corticosterone was associated with the production of chicks that had reduced hatchability, lower hatchling mass and higher baseline fecal glucocorticoid metabolites (FGM) than those produced by SRS females. Moreover, after capture and restraint, IRS chicks did not modify their FGM nor their behaviors compared to their controls, while SRS chicks increased their FGM and spent more time ambulating and less time pecking, compared to their controls. These results indicate that yolk immunoreactive corticosterone could modify offspring phenotype. Although future studies are needed to elucidate their implications for fitness, our results suggest that yolk corticosterone could be mediating an adaptive maternal effect that allows individuals to better cope with poor conditions.

Dynamics of thymol dietary supplementation in quail (Coturnix japonica): Linking bioavailability, effects on egg yolk total fatty acids and performance traits.

Phytogenic additives such as thymol are encountering growing interest in the poultry industry. However, there are still questions concerning dynamics of their bioavailability, biological action, optimal dosage and duration of supplementation needed to achieve meaningful effects, as well as persistence of induced changes after supplement withdrawal. We studied the link between the dynamics of free thymol concentration and the changes in fatty acids composition in quail egg yolk, both during a month-long chronic dietary supplementation and after 3 weeks of supplement withdrawal (post-supplementation). Fifty, 85 days-old, female quail of homogeneous body weights (251±1g) in egg-laying peak were used. To evaluate potential dose-dependent effects, three increasing doses 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6, respectively) and two controls were evaluated (n = 10). In parallel, we assessed the concomitant changes in free thymol excretion, potential liver histopathological changes, and birds´ performance traits. Egg yolk and droppings show a dose-dependent increase in THY concentration after 9 days of supplementation and a decrease after post-supplementation. In egg yolk, these changes were accompanied by reduced saturated fatty acid concentrations achieved by 28 days of supplementation in THY2 and 14 days of supplementation in THY4 and THY6. However, after post-supplementation the aforementioned effect disappeared in THY2 but not in THY4 and THY6. While THY2 failed to increase polyunsaturated fatty acids, THY4 and THY6 increased polyunsaturated fatty acids by day 14 of supplementation and remained increased after post-supplementation. Fatty acids changes induced by thymol are consistent with improved nutritional quality of eggs. No treatment effects were observed in liver histopathology and female performance. Findings suggest that both dose of thymol and duration of supplementation modulate thymol and fatty acids concentrations in egg yolk and thymol concentration in droppings. Furthermore, the persistence of those effects after post-supplementation period is also a dose-dependent phenomenon.

Dynamics of thymol dietary supplementation in quail (Coturnix japonica): Dataset on thymol bioavailability, egg yolk fatty acids profile and performance traits.

Dietary supplementation with thymol (2-isopropyl-5-methylphenol) has been proposed as a strategy to improve modern intensive poultry production. Moreover, its antioxidant properties and potential beneficial influence on lipid metabolism have fostered current research focusing on enhancing nutritional quality of meat and egg products. In general, studies have focused on the overall effects of dietary supplementation once the supplementation protocol has finished and using only one potential dose, without actually measuring bioactive compounds' concentration in the diet supplied or target tissues. Herein, we provide a unique dataset of the dynamics of thymol bioavailability and biological action, optimal dosage and duration of supplementation needed to achieve meaningful effects, as well as persistence of induced changes after chronic supplement withdrawal. Specifically, during a month-long supplementation period, 5 sampling points were evaluated separated by at least 1 week. Then, a last sampling point was studied after a 3-week withdrawal period. Three increasing doses of dietary thymol were used, and approximately 80 variables assessed. The measured variables were associated with free thymol concentration in feed, egg yolk and droppings, feed and egg yolk fatty acids profile (saturated, unsaturated, and polyunsaturated fatty acids), performance traits (body weight, feed intake, egg laying rate, egg physical characteristics), general welfare quality assessment (plumage state) and liver histopathology. The data can provide insights on the link between the dynamics of free thymol concentration and the changes in fatty acids profile in quail egg yolk, both during chronic thymol dietary supplementation and after supplement withdrawal. The comprehensive approach used herein for studying thymol supplementation outcome could help understanding the scope of its effects on a whole organism level.

State of the art knowledge in adrenocortical and behavioral responses to environmental challenges in a threatened South American ratite: Implications to in situ and ex-situ conservation.

The Greater Rhea (Rhea americana) is an endemic ratite to South America, whose wild populations have undergone a remarkable decrease due to habitat degradation and fragmentation by the expansion of the agricultural frontier, poaching and predation by dogs. Anthropogenic perturbations in wild environments, as well as the management in captivity, can generate different stress responses in this species, thus, the monitoring of adrenocortical and behavioral activities are considered primary assessment tools with both conservation and welfare implications. In this review we analyze and integrate the different measurements of glucocorticoids (in plasma, feces, and yolk) carried out in different captive and wild populations, taking into account the diverse predictable and unpredictable conditions to which the Greater Rhea responds in each of those environments. In addition, the translocation of this bird is presented as an application of stress physiology in field ecology for conservation purposes, in which we evaluated how this species responds when it is released into a novel environment. Our results indicate that this ratite has a striking high sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis compared to that of other bird species and shows a wide variety of adrenocortical responses depending on the environment in which it lives. This suggests that its HPA axis has a phenotypic plasticity that enables the rhea to cope with the environmental challenges. In this sense, we propose that one of the routes of this plasticity could be mediated by the maternal transfer of steroid hormones to the egg. Finally, we discuss the importance of integrating the monitoring of the adrenocortical response along with the environmental variables that define the life history of the species, in management and conservation programs ex-situ and in situ.

Stress Induced Polarization of Immune-Neuroendocrine Phenotypes in Gallus gallus.

Immune-neuroendocrine phenotypes (INPs) stand for population subgroups differing in immune-neuroendocrine interactions. While mammalian INPs have been characterized thoroughly in rats and humans, avian INPs were only recently described in Coturnix coturnix (quail). To assess the scope of this biological phenomenon, herein we characterized INPs in Gallus gallus (a domestic hen strain submitted to a very long history of strong selective breeding pressure) and evaluated whether a social chronic stress challenge modulates the individuals' interplay affecting the INP subsets and distribution. Evaluating plasmatic basal corticosterone, interferon-γ and interleukin-4 concentrations, innate/acquired leukocyte ratio, PHA-P skin-swelling and induced antibody responses, two opposite INP profiles were found: LEWIS-like (15% of the population) and FISCHER-like (16%) hens. After chronic stress, an increment of about 12% in each polarized INP frequency was found at expenses of a reduction in the number of birds with intermediate responses. Results show that polarized INPs are also a phenomenon occurring in hens. The observed inter-individual variation suggest that, even after a considerable selection process, the population is still well prepared to deal with a variety of immune-neuroendocrine challenges. Stress promoted disruptive effects, leading to a more balanced INPs distribution, which represents a new substrate for challenging situations.

Thymol feed supplementation in quail alters the percentages of nutritionally relevant egg yolk fatty acids: effects throughout incubation.

BACKGROUND: Polyunsaturated fatty acids (PUFA) are crucial components of egg yolk and particularly prone to oxidative damage, generating losses of nutrients for embryonic development and influencing the quality of eggs for human consumption. The aim of this study was to evaluate whether dietary thymol (a natural antioxidant) is related to changes in quail egg yolk total (T), triglyceride (TG) and phospholipid (PL) fatty acid composition at different stages of embryo development. Thus female Japanese quail (100 days of age) were assigned to one of two dietary treatments (12 individuals each): CON (basal diet) or THY (0.0016 mol thymol day-1 per bird). After 2 weeks of supplementation, eggs were incubated and samples were obtained at 0, 4 and 16 days of embryonic development. RESULTS: In 0 day THY eggs, α-linolenic acid and n-3 PUFA in T and TG, docosahexaenoic acid and PUFA in T and arachidonic acid in TG were increased, while saturated fatty acids (SFA) in T were reduced. From 4 days on, PUFA, n-3 PUFA and SFA from T and TG in THY eggs were found to be similar to those of CON eggs. The changes in PL throughout incubation were similar in both dietary treatments. CONCLUSION: Thymol would provide the embryo with PUFA for synthesis/deposition in membranes and/or assign to supply energy. Additionally, thymol supplementation would be advisable for the production of healthier table eggs. © 2017 Society of Chemical Industry.

The fractal organization of ultradian rhythms in avian behavior.

Living systems exhibit non-randomly organized biochemical, physiological, and behavioral processes that follow distinctive patterns. In particular, animal behavior displays both fractal dynamics and periodic rhythms yet the relationship between these two dynamic regimens remain unexplored. Herein we studied locomotor time series of visually isolated Japanese quails sampled every 0.5 s during 6.5 days (>106 data points). These high-resolution, week-long, time series enabled simultaneous evaluation of ultradian rhythms as well as fractal organization according to six different analytical methods that included Power Spectrum, Enright, Empirical Mode Decomposition, Wavelet, and Detrended Fluctuation analyses. Time series analyses showed that all birds exhibit circadian rhythms. Although interindividual differences were detected, animals presented ultradian behavioral rhythms of 12, 8, 6, 4.8, 4 h and/or lower and, irrespective of visual isolation, synchronization between these ultradian rhythms was observed. Moreover, all birds presented similar overall fractal dynamics (for scales ∼30 s to >4.4 h). This is the first demonstration that avian behavior presents fractal organization that predominates at shorter time scales and coexists with synchronized ultradian rhythms. This chronobiological pattern is advantageous for keeping the organism's endogenous rhythms in phase with internal and environmental periodicities, notably the feeding, light-dark and sleep-wake cycles.

High resolution, week-long, locomotion time series from Japanese quail in a home-box environment.

Temporal and spatial patterns of locomotion reflect both resting periods and the movement from one place to another to satisfy physiological and behavioural needs. Locomotion is studied in diverse areas of biology such as chronobiology and physiology, as well as in biomathematics. Herein, the locomotion of 24 visually-isolated Japanese quails in their home-box environment was recorded continuously over a 6.5 days at a 0.5 s sampling rate. Three time series are presented for each bird: (1) locomotor activity, (2) distance ambulated, and (3) zone of the box where the bird is located. These high resolution, week-long, time series consisting of 1.07×10(6) data points represent, to our knowledge, a unique data set in animal behavior, and are publically available on FigShare. The data obtained can be used for analyzing dynamic changes of daily or several day locomotion patterns, or for comparison with existing or future data sets or mathematical models across different taxa.

Influence of the rearing system on yolk corticosterone concentration in captive Greater Rheas (Rhea americana).

Many environmental conditions elevate plasma corticosterone in laying birds, leading to elevated hormone accumulation in the egg. We investigated whether maternal yolk corticosterone levels in Greater Rheas differ between fresh eggs collected from an intensive (IRS) and a semi-extensive (SRS) rearing system. After HPLC validation, yolk corticosterone was measured using a corticosterone (125) I radio-immunoassay kit. Results (mean ± SE) showed that eggs collected from the IRS exhibited a significantly higher corticosterone concentration than eggs from SRS (89.88 ± 8.93 vs. 45.41 ± 5.48 ng/g yolk, respectively). Our findings suggest that rearing conditions under an intensive scheme (e.g., small pens with bare ground, no direct foraging and handling) might be perceived as more stressful for Greater Rhea females than semi-extensive rearing conditions (e.g., low animal density distributed in extensive areas and direct foraging), which would result in the transfer of higher yolk corticosterone levels. A better understanding of environmental conditions and female traits that affect yolk corticosterone deposition provides a background for future studies concerning the roles of maternal corticosterone on offspring development. Zoo Biol. 35:246-250, 2016. © 2016 Wiley Periodicals, Inc.

Immune neuroendocrine phenotypes in Coturnix coturnix: do avian species show LEWIS/FISCHER-like profiles?

Immunoneuroendocrinology studies have identified conserved communicational paths in birds and mammals, e.g. the Hypothalamus-Pituitary-Adrenal axis with anti-inflammatory activity mediated by glucocorticoids. Immune neuroendocrine phenotypes (INPs) have been proposed for mammals implying the categorization of a population in subgroups underlying divergent immune-neuroendocrine interactions. These phenotypes were studied in the context of the LEWIS/FISCHER paradigm (rats expressing high or low pro-inflammatory profiles, respectively). Although avian species have some common immunological mechanisms with mammals, they have also evolved some distinct strategies and, until now, it has not been studied whether birds may also share with mammals similar INPs. Based on corticosterone levels we determined the existence of two divergent groups in Coturnix coturnix that also differed in other immune-neuroendocrine responses. Quail with lowest corticosterone showed higher lymphoproliferative and antibody responses, interferon-γ and interleukin-1ß mRNA expression levels and lower frequencies of leukocyte subpopulations distribution and interleukin-13 levels, than their higher corticosterone counterparts. Results suggest the existence of INPs in birds, comparable to mammalian LEWIS/FISCHER profiles, where basal corticosterone also underlies responses of comparable variables associated to the phenotypes. Concluding, INP may not be a mammalian distinct feature, leading to discuss whether these profiles represent a parallel phenomenon evolved in birds and mammals, or a common feature inherited from a reptilian ancestor millions of years ago.

Effect of postweaning handling strategies on welfare and productive traits in lambs.

Postweaning management strategies that include an element of social enrichment may reduce weaning stress and improve welfare and productive performance. We analyzed the effect of postweaning handling strategies on welfare and production traits in lambs. After weaning, 36 lambs were assigned to 3 experimental groups with 12 lambs each (control [C], fattening with gentle human female contact [H], and fattening with 2 adult ewes [E]). The average daily gain (ADG) was estimated. Blood samples were taken, and infrared thermography was used to estimate stress variables. There were significant differences among treatments (in favor of alternative strategies) regarding production and stress variables (cortisol, glucose, and creatine kinase). The results suggest that the lambs handled gently during the fattening were less reactive and better able to modulate their physiological stress. The E group adapted better to acute stress than the C group but was less efficient in modulating chronic stress. Both treatments showed higher slaughter live weights and better ADGs compared with the control. The use of social enrichment at weaning, especially to establish a positive human-nonhuman animal bond, alleviates lamb weaning stress and improves welfare and performance.

The effects of testosterone on immune function in quail selected for divergent plasma corticosterone response.

The immunocompetence handicap hypothesis (ICHH) suggests that the male sex hormone testosterone has a dual effect; it controls the development and expression of male sexually selected signals, and it suppresses the immune system. Therefore only high quality males are able to fully express secondary sexual traits because only they can tolerate the immunosuppressive qualities of testosterone. A modified version of the ICHH suggests that testosterone causes immunosuppression indirectly by increasing the stress hormone corticosterone (CORT). Lines of Japanese quail (Coturnix japonica) selected for divergent responses in levels of plasma CORT were used to test these hypotheses. Within each CORT response line (as well as in a control stock) we manipulated levels of testosterone in castrated quail by treatment with zero (sham), low or high testosterone implants, before testing the birds' humoral immunity and phytohaemagglutinin (PHA)-induced immune response, as well as body condition. The PHA-induced response was not significantly affected by CORT selected line, testosterone treatment or their interaction. There was, however, a significant effect of CORT line on humoral immunity in that the control birds exhibited the greatest antibody production, but there was no significant effect of testosterone manipulation on humoral immunity. The males in the sham implant treatment group had significantly greater mass than the males in the high testosterone group, suggesting a negative effect of high testosterone on general body condition. We discuss these results in the context of current hypotheses in the field of sexual selection.

Physiological stress in captive Greater rheas (Rhea americana): highly sensitive plasma corticosterone response to an ACTH challenge.

Up to the present no studies have been conducted either on baseline concentrations of adrenal hormones or on hormonal responses to stress in Greater rhea (Rhea americana) and most ratite species. The aims of this work were to assess the presence of corticosterone in plasma of Greater rhea, to validate a corticosterone (125)I-radioimmunoassay for determining corticosterone levels in plasma samples and to study the activation of the adrenal gland after an adrenocorticotrophic hormone (ACTH) challenge. Six captive Greater rhea juveniles of 10 months of age received an intravenous ACTH injection. Blood samples were taken at 0min (baseline pre-ACTH levels), and post-injection at 15, 30, 60min and at 24 and 48h. The high pressure liquid chromatography (HPLC) analysis of pooled plasma showed that corticosterone is the glucocorticoid found in the plasma of Greater rhea. Biochemical assays of standard validation (e.g., parallelism, exogenous corticosterone recovery) showed that measurements of corticosterone present in the plasma of the Greater rhea provided by commercial corticosterone (125)I-radioimmunoassay were accurate and precise. ACTH challenge induced a more than 40-fold increase in plasma corticosterone at 60min post-ACTH (from 4.0 to 166.5ng/ml, on average). The corticosterone response to ACTH in Greater rhea was higher than is usual in birds, an apparently typical characteristic of ratites.

Effects of the essential oils of Lippia turbinata and Lippia polystachya (Verbenaceae) on the temporal pattern of locomotion of the mosquito Culex quinquefasciatus (Diptera: Culicidae) larvae.

The essential oils (EO) of Lippia turbinata (TUR) and Lippia polystachya (POL) have shown lethal effects against mosquito larvae. The present work evaluated whether these EO at doses ranging from sublethal to lethal (20, 40 and 80 ppm) modify the temporal pattern of locomotion of Culex quinquefasciatus larvae. Larvae were individually placed in glass boxes, and their activity recorded at 0.3 s intervals during 40 min. Individuals treated with doses >40 ppm of either EO significantly decreased their ambulation speed and the percentage of total time ambulating compared to controls. TUR 80 ppm decreased their ambulation even sooner than POL 80 ppm, when compared to their respective controls. These findings are consistent with the neurotoxic effect against insects attributed to alpha-Thujone, a main component of both EO. A detrended fluctuation fractal analysis evaluating the complexity and organisation of the temporal pattern of locomotion showed fractal patterns in all animals. Both sublethal and lethal doses of TUR and POL increased the complexity of ambulation. Interestingly, for POL 20 ppm, an increase in complexity was observed, while no changes in general activity were detected, suggesting that fractal analysis may be more sensitive to detect behavioural changes than general activity evaluation.

Stress-induced decrement in the plasticity of the physical properties of chick brain membranes.

The molecular basis underlying the stress-induced increment in the density of central benzodiazepine receptor from chick forebrain, observed previously at 4 degrees C, was studied from a biophysical perspective. The thermal dependence of [3H]flunitrazepam binding to the central benzodiazepine receptor and the supramolecular organization were studied in forebrain membranes from chicks submitted to partial water immersion. The equilibrium dissociation constants increased with temperature in membrane from both control and stressed chicks. The heat capacity values in control samples (deltaC(p, CON)) were significantly less negative than deltaC(p STR). Changes in deltaH and deltaS between 4-37 degrees C were greater in stressed chicks compared to control; however, the binding was exothermic and driven by enthalpy in both conditions. At 4 degrees C, the receptor density (B(max)) was higher in stressed chicks compared to control. Such a difference was lost irreversibly upon temperature elevation, possibly due to the hysteresis between the heating and cooling behaviour of B(max, CON) and the constancy in B(max, STR). The fluorescence anisotropy of diphenylhexatriene was higher in control samples with respect to stressed chicks below 10 degrees C. A temperature-induced increment in protein intrinsic-fluorescence was observed only in control, and was quenched by acrylamide more easily at 4 degrees C than at 25 degrees C. A higher microviscosity at 4 degrees C in control favoured more external localizations of integral proteins; at higher temperatures, tryptophan residues moved to hydrophobic membrane-regions. Changes in the membrane-organization towards more fluid states favoured the accessibility of benzodiazepine to the central benzodiazepine receptor, expressed by the higher values of B(max) found in stressed samples at low temperatures with respect to control samples.