Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail

Studies in our laboratory have focused on endocrine, neuroendocrine, and behavioral components of reproduction in the Japanese quail. These studies considered various stages in the life cycle, including embryonic development, sexual maturation, adult reproductive function, and aging. A major focus of our research has been the role of neuroendocrine systems that appear to synchronize both endocrine and behavioral responses. These studies provide the basis for our more recent research on the impact of endocrine disrupting chemicals (EDCs) on reproductive function in the Japanese quail. These endocrine active chemicals include pesticides, herbicides, industrial products, and plant phytoestrogens. Many of these chemicals appear to mimic vertebrate steroids, often by interacting with steroid receptors. However, most EDCs have relatively weak biological activity compared to native steroid hormones. Therefore, it becomes important to understand the mode and mechanism of action of classes of these chemicals and sensitive stages in the life history of various species. Precocial birds, such as the Japanese quail, are likely to be sensitive to EDC effects during embryonic development, because sexual differentiation occurs during this period. Accordingly, adult quail may be less impacted by EDC exposure. Because there are a great many data available on normal development and reproductive function in this species, the Japanese quail provides an excellent model for examining the effects of EDCs. Thus, we have begun studies using a Japanese quail model system to study the effects of EDCs on reproductive endocrine and behavioral responses. In this review, we have two goals: first, to provide a summary of reproductive development and sexual differentiation in intact Japanese quail embryos, including ontogenetic patterns in steroid hormones in the embryonic and maturing quail. Second, we discuss some recent data from experiments in our laboratory in which EDCs have been tested in Japanese quail. The Japanese quail provides an excellent avian model for testing EDCs because this species has well-characterized reproductive endocrine and behavioral responses. Considerable research has been conducted in quail in which the effects of embryonic steroid exposure have been studied relative to reproductive behavior. Moreover, developmental processes have been studied extensively and include investigations of the reproductive axis, thyroid system, and stress and immune responses. We have conducted a number of studies, which have considered long-term neuroendocrine consequences as well as behavioral responses to steroids. Some of these studies have specifically tested the effects of embryonic steroid exposure on later reproductive function in a multigenerational context. A multigenerational exposure provides a basis for understanding potential exposure scenarios in the field. In addition, potential routes of exposure to EDCs for avian species are being considered, as well as differential effects due to stage of the life cycle at exposure to an EDC. The studies in our laboratory have used both diet and egg injection as modes of exposure for Japanese quail. In this way, birds were exposed to a specific dose of an EDC at a selected stage in development by injection. Alternatively, dietary exposure appears to be a primary route of exposure; therefore experimental exposure through the diet mimics potential field situations. Thus, experiments should consider a number of aspects of exposure when attempting to replicate field exposures to EDCs.     

Behavioural and endocrine fear responses in Japanese quail upon presentation of a novel object in the home cage

Most tests used to study fear in birds involve transferring them to a novel environment, which constitutes a bias in studies aiming at identifying the neural correlates of a specific fear-inducing situation. In order to investigate fear in birds with minimum interference by humans, behavioural and endocrine responses to the presentation of a novel object in the home cage were investigated in two lines of Japanese quail divergently selected for long or short duration of tonic immobility, a behavioural index of fear. Presentation of the novel object induced typical fear responses (avoidance of the object, increased pacing and increased plasma corticosterone levels) that were similar in the two lines of quail. Presentation of a novel object in the home cage thus appears to be a suitable stimulus to induce fear reactions in quail, with minimum interference from other motivational systems. The fact that quail of both lines reacted similarly in this test, while they are known to differ greatly in their behavioural responses to other fear-inducing tests, illustrates the multidimensional nature of fear.     

Effects of early embryonic exposure to genistein on male copulatory behavior and vasotocin system of Japanese quail

Genistein is a phytoestrogen, particularly abundant in soybeans that can bind estrogen receptors and sex hormone binding proteins, exerting both estrogenic and antiestrogenic activity. In this study we used the Japanese quail embryo as a test end-point to investigate the effects of early embryonic exposure to genistein on male copulatory behavior and on vasotocin parvocellular system. Both differentiate by the organizational effects of estradiol during development and may therefore represent an optimal model to study the effects of xenoestrogens. We injected two doses of genistein (100 and 1000 microg) into the yolk of 3-day-old Japanese quail eggs. Other eggs were treated with either 25 microg of estradiol benzoate or sesame oil as positive and negative controls. At the age of 6 weeks, behavioral tests revealed a significant decrease of all aspects of copulatory behavior (in comparison to the control group) in estradiol-treated birds. In contrast, genistein-treated animals demonstrated various degrees of decrease in the mean frequencies of some aspects of the sexual behavior. The computerized analysis of vasotocin innervation in medial preoptic, stria terminalis and lateral septum nuclei revealed a statistically significant decreased immunoreactivity in treated animals compared to control ones. These results demonstrate that genistein, similarly to estradiol, has an organizational effect on quail parvocellular vasotocin system and on copulatory behavior. In conclusion, present results confirm, in this avian model, that embryonic exposure to phytoestrogens may have life-long effects on sexual differentiation of brain structures and behaviors.     

Selection for tonic immobility duration does not affect the response to novelty in quail

Genetic selection on a single fear test, the tonic immobility test, seems to result in selection on fearfulness, i.e. the propensity to exhibit fear responses, whatever the fear tests used. However, the conception of fear as a single variable has been challenged by the recognition that fear is multidimensional. This study was designed to test whether genetic selection on a classic index of fear in birds, tonic immobility duration, is accompanied by changes in the response to a single dimension of fear – novelty.Two lines of quail divergently selected for long (LTI) or short (STI) duration of tonic immobility were exposed to a novel object in their home cage. Quail of both lines showed typical fear reactions in response to novelty but there was no difference between lines. We conclude that genetic selection for tonic immobility duration does not affect all dimensions of fear, notably not novelty. Further studies are needed to investigate the dimensions of fear on which the two lines of quail could have been selected.     

Effect of a cranium-directed daily illumination cycle on the laying rhythm in Japanese quail

This study tests the effect on the laying rhythm of a light cycle reaching directly the encephala via a diode in Japanese quail maintained in constant darkness. In DD, all the birds expressed their free-running laying rhythm (period close to 25 h). When the diode is switched on 14 h per 24 h cycle, females showed the same organization as in LD with the same laying time. Thus, a photoperiodic cycle, where light was only perceived through the skull of the female quail, could synchronize its laying rhythm. This device with a LED is an interesting alternative solution to eye-patching or blinding of birds.     

Selection of social traits in juvenile Japanese quail affects adults’ behaviour

The inability of animals to cope with their social environment in husbandry systems could be a major source of problems for both animal welfare management and economy. In this context, previous studies selected lines of domestic Japanese quail diverging for their level of social reinstatement, hypothesizing that quail with high levels of social motivation would adapt better to crowded social situations than quail with low levels of social motivation. However, these selections were based only on social motivation evaluated in young birds. As behavioural traits can vary with age, this study analysed the persistence of selected social traits into adulthood. With this in mind, we used several behavioural tests to estimate social motivation of adult females from selected lines for high social reinstatement (HSR, n = 24) and for low social reinstatement (LSR, n = 24). The tests were: open-field tests, emergence tests, separation tests, runway tests and confrontation tests. As birds’ behaviour in these tests can be influenced by the emotive characteristics of the subjects, we measured the emotional reactivity of quail not only during these tests but also in specific emotive tests: tonic immobility tests. Our results showed that the social motivation characteristics of birds persist in adulthood. Adult HSR females still presented a higher social motivation than adult LRS females: their response to social isolation was stronger and their motivation to reach conspecifics and to stay close to them was higher. However, the behavioural expression of social motivation appeared to be modified during development. Whereas vocal behaviour reflected the social motivation of chicks, in isolated contexts, this characteristic was not observed in adult quail.     

Japanese Quail’s Genetic Background Modulates Effects of Chronic Stress on Emotional Reactivity but Not Spatial Learning

Chronic stress is known to enhance mammals’ emotional reactivity and alters several of their cognitive functions, especially spatial learning. Few studies have investigated such effects in birds. We investigated the impact of a two-week stress on Japanese quail’s emotional reactivity and spatial learning. Quail is an avian model widely used in laboratory studies and for extrapolation of data to other poultry species. As sensitivity to chronic stress can be modulated by intrinsic factors, we tested juvenile female Japanese quail from three lines, two of them divergently selected on tonic immobility duration, an indicator of general fearfulness. The different emotional reactivity levels of quail belonging to these lines can be revealed by a large variety of tests. Half of the birds were submitted to repeated unpredictable aversive events for two weeks, whereas the other half were left undisturbed. After this procedure, two tests (open field and emergence tests) evaluated the emotional reactivity of treated and control quails. They were then trained in a T-maze for seven days and their spatial learning was tested. The chronic stress protocol had an impact on resting, preening and foraging in the home cage. As predicted, the emotional reactivity of treated quails, especially those selected for long tonic immobility duration, was higher. Our spatial learning data showed that the treatment enhanced acquisition but not memorization. However, intrinsic fearfulness did not seem to interact with the treatment in this test. According to an inverted U-shaped relationship between stress and cognition, chronic stress can improve the adaptability of birds to a stressful environment. We discussed the mechanisms possibly implied in the increase of emotional reactivity and spatial abilities.     

Difference of social motivation in quail selected for divergent circadian activity

Endogenous rhythms are adaptive responses to predictable changes of the environment, like the day/night cycle. Some researches demonstrated that social cycles can influence the circadian rhythm, while no study investigated the effect of endogenous rhythmicity on the sociability in Vertebrates. This study investigated whether differences in the functioning of the circadian system was associated with social motivation in Japanese quail (Coturnix c. japonica).We compared quail from a line expressing a robust circadian rhythm of feeding activity (R) to quail from a line expressing circadian arrhythmicity of feeding activity (A) under constant darkness. Classic behavioral tests evaluated social motivation of these birds.When socially isolated, the motivation of R quail to re-establish contact with conspecifics appeared stronger than that of A quail. When in the presence of conspecifics, R quail faced a stressful situation (change of environment) more calmly than did A quail. Thus, variation of circadian behavioral rhythmicity is associated with variation in social motivation in Japanese quail. Rhythmic animals appeared to respond more appropriately to environmental challenges than arrhythmic animals.     

Polymorphism of dopamine receptor D4 exon I corresponding region in chicken

In stockbreeding, there are indications that behavioral traits of livestock have an effect on breeding and production. If the variation in individual behavior is related to that in neurotransmitter-related genes such as in humans, it would be possible to breed pedigrees composed of individuals having behavioral traits that are useful to production and breeding using selection based on genotypes. In this study, we investigated the exon I region of dopamine receptor D4 (DRD4), in which variation is related to psychiatric disorder in humans, in major poultry species namely Japanese quail (Coturnix japonica), chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and helmeted guinea fowl (Numida meleagris). Furthermore, we investigated Japanese cormorant (Phalacrocorax capillatus) and Japanese jungle crow (Corvus macrorhynchos) as an out-group. In these species of birds, the repeat of proline was identified in the region corresponding to the human polymorphic region. The repeat number was 9 in Japanese quail, ring-necked pheasant and Japanese cormorant; 12 in helmeted guinea fowl; and 3 in Japanese jungle crow. However, no polymorphism was found in these species. In contrast, polymorphism was observed in chicken and two alleles with 8 and 9 repeats were identified. Although 9 repeats (allele 9) were predominant in most chicken breeds, Black Minorca had only 8 repeats (allele 8). Intra-breed polymorphism was found in 6 out of 12 breeds, and two alleles (alleles 8 and 9) were detected in these breeds. This polymorphism, which is the first to be reported on a neurotransmitter-related gene in birds, would contribute significant information for elucidation of differences in behavioral traits in chicken breeds.     

Distribution and metabolism of selenite and selenomethionine in the Japanese quail

Compared to the many studies on the physiological and toxicological effects of selenium (Se) in mammals, avian Se metabolism is still an unexplored topic. Some birds are useful as poultry for human nutrition. Moreover, birds belong to higher trophic levels in the biosphere and thus may play an important role in Se circulation in the ecosystem in the same way as mammals do. In this study, we analyzed the distribution and metabolism of Se in an experimental bird, the Japanese quail, which was fed drinking water containing sodium selenite or selenomethionine (SeMet). The highest concentration of Se was detected in the pancreas, followed by down feathers, liver, and kidneys. SeMet was more efficiently incorporated into the quail than selenite. The specific and preferable distribution of Se to the high molecular weight fraction in the serum of the quail was observed only in the SeMet-ingestion group. As in mammals, selenosugar and trimethylselenonium were the major metabolites in quail excreta. Three unknown Se metabolites were detected by HPLC-ICP-MS. Although part of the metabolic pathway of Se in the Japanese quail fed selenite and SeMet was the same as that observed in mammals, the bird also showed certain avian-specific metabolic process for Se.     

Are farm-reared quails for game restocking really common quails (Coturnix coturnix)?: a genetic approach

The common quail (Coturnix coturnix) is a popular game species for which restocking with farm-reared individuals is a common practice. In some areas, the number of released quails greatly surpasses the number of wild breeding common quail. However, common quail are difficult to raise in captivity and this casts suspicion about a possible hybrid origin of the farmed individuals from crosses with domestic Japanese quail (C. japonica). In this study we used a panel of autosomal microsatellite markers to characterize the genetic origin of quails reared for hunting purposes in game farms in Spain and of quails from an experimental game farm which was founded with hybrids that have been systematically backcrossed with wild common quails. The genotypes of these quail were compared to those of wild common quail and domestic strains of Japanese quail. Our results show that more than 85% of the game farm birds were not common quail but had domestic Japanese quail ancestry. In the experimental farm a larger proportion of individuals could not be clearly separated from pure common quails. We conclude that the majority of quail sold for restocking purposes were not common quail. Genetic monitoring of individuals raised for restocking is indispensable as the massive release of farm-reared hybrids could represent a severe threat for the long term survival of the native species.     

Mucuna pruriens seed powder feeding influences reproductive conditions and development in Japanese quail Coturnix coturnix japonica

This study was designed to test whether Mucuna pruriens, a natural source of l-dihydroxyphenylalanine (l-DOPA, a dopamine precursor) feeding, can influence development and reproductive conditions in the high food value bird, Japanese quail, Coturnix coturnix japonica. Experiments were performed in both male and female Japanese quail. One-week-old quail chicks were divided into three groups of 36 birds each. Group I was provided with normal diet and served as control. Group II was provided with food mixed with l-DOPA (50 mg/15 g food) and Group III was provided with food mixed with M. pruriens seed powder (480 g/kg food). At the age of 3 weeks (when birds were sexually distinguished) Group I was divided into two sub-groups IA (male) and IB (female) of six birds each. Similarly, Groups II and III were sub-divided into IIA (male), IIB (female) and IIIA (male), IIIB (female), respectively, of six birds each. Observations were made up to the age of 5 weeks. Male experimental groups (IIA and IIIA) showed significantly increased testicular activity, cloacal gland volume, body weight (BW), plasma testosterone and LH level in comparison to control (IA). Similarly female experimental groups (IIB and IIIB) showed significantly greater weight of reproductive organs (uterus, ovary, oviduct and ovarian follicle), BW, egg weight and size and number of follicles. On the other hand, plasma prolactin level was significantly low in comparison to control (IB). Results suggest that M. pruriens is a rich natural source of l-DOPA and the development and reproduction in Japanese quail might be associated with the dopaminergic system of the brain.     

Effects of 2 G hypergravity exposure on Bobwhite (Colinus virginianus) and Japanese quail (Coturnix coturnix japonica)

We compared reproductive fitness and early postnatal growth of Bobwhite (Colinus virginianus) and Japanese (Coturnix coturnix japonica) quail incubated and hatched during 2 G centrifugation. Fertilized Bobwhite and Japanese quail eggs were placed in portable incubators on the 8-ft International Space Station Test Bed (ISSTB) Centrifuge at NASA Ames Research Center. The quail eggs were incubated throughout hatching and reared until Postnatal day (P)4 at either 1.0, 1.2 or 2.0 G. Two days before hatching, candling revealed significantly greater numbers of viable Bobwhite than Japanese quail eggs at all g-loads. Bobwhite quail exhibited significantly better hatching success at all g-loads than did Japanese quail. Bobwhite hatchlings were sensitive to gravitational loading as evidenced by reduced postnatal body mass and length of 2 G hatchlings relative to 1 G control hatchlings. In contrast, mass and length of Japanese quail hatchlings were unaffected by 1.2 or 2 G exposure. Together, our findings provide evidence for superior viability and hatching success in Bobwhite quail relative to Japanese quail, coupled with greater sensitivity of postnatal body growth and development to 2 G loading. Bobwhite quail may be better suited than Japanese quail for scientific studies on space biology platforms.     

Experimental Chlamydia psittaci Infection of Japanese Quail

Japanese quail were used for the infection model of avian chlamydiosis. One-day-old Japanese quail were highly susceptible to lethal infection by a Chlamydia psittaci strain of budgerigar origin upon inoculation via the air sac route with 10^4.1 FFU of the organism, showing an acute and lethal course with chlamydial propagation. In contrast, 7-day-old quail developed resistance to the infection as shown by the lack of lethal effect with the same dose. The resistance of 7-day-old birds was abolished by immunosuppressive treatment with cyclophosphamide. Upon inoculation with a sublethal dose of 10^2.1 FFU, latent infection was established in 1-day-old birds with a minimum number of the organism. The latent infection in the birds was converted to the lethal form by treatment with cyclophosphamide along with chlamydial propagation and suppression of antibody production.     

Immediate early gene expression associated with induction of brooding behavior in Japanese quail

Certain species can be induced to foster infant or neonatal animals through the process of sensitization. We induced brooding behavior in adult Japanese quail through repeated exposure to foster chicks across five 20-min trials. Brooding behavior was characterized by a bird allowing chicks to approach and remain underneath its wings while assuming a distinctive stationary crouching posture, preening, and feather fluffing. Birds who did not show brooding behavior actively avoided chicks. Among the birds that brooded chicks, females brooded chicks for longer durations compared to males. Brooding females continued a regular daily egg laying pattern; males showed no significant changes in testosterone levels after exposure to chicks. In a second experiment, we measured expression of two immediate early gene (IEG) protein products, ZENK and Fos, to identify the brain regions activated or inhibited by brooding behavior in females. ZENK and Fos expression in brooding or sensitized females (SF) were compared with expression in nonmaternal females with chicks (NMF) and with females without chicks and with blocks as control objects (BL). There was a reduced density of ZENK-like immunoreactive (ZENK-lir) cells in the medial preoptic nucleus (POM) in NMF birds. In SF birds, the density of Fos-like immunoreactive (Fos-lir) cells was elevated in the bed nucleus stria terminalis, medial portion (BSTm), and ectostriatum (E). These experiments begin to define the neural circuitry underlying brooding behavior in Japanese quail, and establish a model for future studies of the neural mechanisms of avian parental behavior.     

Melatonin does not link the eyes to the rest of the circadian system in quail: a neural pathway is involved

Blinding by enucleation has a dramatic effect on the circadian activity rhythm of Japanese quail. The activity patterns of enucleated birds held under 24-hr light-dark cycles are disrupted, although entrainment can persist in many birds. In constant darkness (DD), blinded birds are rendered arrhythmic. These results demonstrate that the eyes are a major component of the circadian system, and that insofar as enucleation produces arrhythmicity in DD, the eyes' role is not merely a photosensory one. The eyes of quail can synthesize and secrete the hormone melatonin, which has been implicated as a blood-borne messenger relaying timing information between elements of the circadian system in some avian species. However, the way in which the eyes communicate with the rest of the circadian system in quail appears to be neural, since (1) optic nerve section produces the same effects as blinding by enucleation on the circadian activity rhythm, and (2) eyes subjected to optic nerve section retain their ability to synthesize and secrete melatonin.     

Acute oral toxicity of sodium cyanide in birds

Sensitivities of six avian species, black vulture (Coragyps atratus), American kestrel (Falco sparverius), Japanese quail (Coturnix japonica), domestic chicken (Gallus domesticus), eastern screech-owl (Otus asio), and European starling (Sturnus vulgaris), to acute poisoning by sodium cyanide (NaCN) were compared by single dose LD50's. Three species, domestic chickens, black vultures, and turkey vultures (Cathartes aura), were dosed with NaCN to determine cyanide residues in those that died and also in survivors, in addition to postmortem fate. Three flesh-eating species (black vulture, American kestrel, and eastern screech-owl; LD50's 4.0-8.6 mg/kg) were more sensitive to NaCN than three species (Japanese quail, domestic chicken, and European starling; LD50's 9.4-21 mg/kg) that fed predominantly on plant material. Elevated concentrations of cyanide were found in the blood of birds that died of cyanide poisoning; however, concentrations in birds that died overlapped those in survivors. Blood was superior to liver as the tissue of choice for detecting cyanide exposure. No gross pathological changes related to dosing were observed at necropsy.     

Evidence for introgressive hybridization of wild common quail (Coturnix coturnix) by domesticated Japanese quail (Coturnix japonica) in France

Many cases of introgressive hybridization have been reported among birds, particularly following introduction to the natural environment of individuals belonging to non-native similar taxa. This appears to be the case for common quail (Coturnix coturnix) in France where wild populations artificially come into contact with domesticated Japanese quail (Coturnix japonica) raised for meat and egg production but sometimes released for hunting purposes. In order to highlight the possible existence of gene flows between both taxa, a comparison of nuclear (25 microsatellite loci) and mitochondrial (sequencing and RFLP) DNA polymorphisms was performed on 375 common quails (from France, Spain and Morocco) and 140 Japanese quails (from France and Japan). Genetic diversity was assessed, and analyses (Factorial Correspondence Analysis, Bayesian admixture) of molecular polymorphisms revealed clear differentiation between the two taxa, making it possible to detect for hybrids among quails sampled in the wild. Eight birds expected to be common quail were found to be two pure Japanese quail, one probable backcross to C. japonica, three F1/F2 hybrids, and two probable backcrosses to Coturnix coturnix. These results show that Japanese quails were released and suggest that the two taxa hybridize in the wild. They confirm the urgent need for preventing the release of pure Japanese or hybrid quails to preserve the genetic integrity of C. coturnix. The tools developed for this study should be useful for accurate monitoring of wild quail populations within the framework of avifauna management programs.     

Effect of long-term simulated microgravity on some sexual traits of male Japanese quail

The aim of this study was to investigate the effects of long-term stay of Japanese quail in simulated microgravity on some sexual features of males. As a model for simulating microgravity exposure of birds in laboratory conditions was used hypodynamia. The mean left testis weight in males reared under hypodynamia from day 3 up to 180 days of age was lower than that of control (P<0.05), but the right testis weight differences between both groups were not significant. Also the area, volume and foam production of cloacal gland was not significantly decreased in hypodynamia birds at the end of experiment. By contrast, the plasma testosterone concentration of males living in hypodynamia was reduced about 50 % at 90 and 180 days of age in comparison to control (P<0.05) suggesting some negative effects on sexual development. Our results demonstrated that male quail kept under simulated microgravity conditions were sexually competent although their sexual efficiency was reduced especially in terms of plasma testosterone.