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.     

Genetic differences in coping strategies in response to prolonged and repeated restraint in Japanese quail divergently selected for long or short tonic immobility

Exposure to fearful situations elicits behavioral and Hypothalamic–Pituitary–Adrenal (HPA) axis responses characteristic of the coping response of individual animals to counteract environmental challenges. The aim of this study was to investigate behavioral and corticotropic responses concomitantly following prolonged or repeated restraint stress by placing two genotypes of Japanese quail divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI) in a crush cage. In our study, STI quail exhibited higher corticosterone (CORT) levels than LTI quail in response to prolonged restraint. STI quail struggled sooner and much more than LTI quail, and struggling behavior in STI quail progressively decreased during the course of restraint whereas LTI quail displayed very little struggling behavior in the crush cage. LTI quail are thus more likely to adopt a passive behavior coping strategy upon exposure to threat whereas STI quail behave more as active copers. The corticosterone responses shown by LTI and STI quail under restraint stress suggest that adrenocortical correlates of coping behavior in these genotypes of quail may be different from the coping styles previously described in other species. Repeated restraint slightly decreased CORT responses to stress in all experimental groups, but more markedly in male STI quail, whereas adrenal sensitivity and maximum adrenal corticosterone response capacity did not change in any group. On the other hand, neither behavioral habituation nor sensitization processes occurred in the context of repeated restraint in female and male LTI quail and female STI quail, whereas the decreases observed in some behavioral responses were interpreted to be the result of a habituation process in male STI quail.     

Genetic differences in coping strategies in response to prolonged and repeated restraint in Japanese quail divergently selected for long or short tonic immobility

Exposure to fearful situations elicits behavioral and Hypothalamic–Pituitary–Adrenal (HPA) axis responses characteristic of the coping response of individual animals to counteract environmental challenges. The aim of this study was to investigate behavioral and corticotropic responses concomitantly following prolonged or repeated restraint stress by placing two genotypes of Japanese quail divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI) in a crush cage. In our study, STI quail exhibited higher corticosterone (CORT) levels than LTI quail in response to prolonged restraint. STI quail struggled sooner and much more than LTI quail, and struggling behavior in STI quail progressively decreased during the course of restraint whereas LTI quail displayed very little struggling behavior in the crush cage. LTI quail are thus more likely to adopt a passive behavior coping strategy upon exposure to threat whereas STI quail behave more as active copers. The corticosterone responses shown by LTI and STI quail under restraint stress suggest that adrenocortical correlates of coping behavior in these genotypes of quail may be different from the coping styles previously described in other species. Repeated restraint slightly decreased CORT responses to stress in all experimental groups, but more markedly in male STI quail, whereas adrenal sensitivity and maximum adrenal corticosterone response capacity did not change in any group. On the other hand, neither behavioral habituation nor sensitization processes occurred in the context of repeated restraint in female and male LTI quail and female STI quail, whereas the decreases observed in some behavioral responses were interpreted to be the result of a habituation process in male STI quail.     

Japanese quail selected for high plasma corticosterone response deposit high levels of corticosterone in their eggs

Poor habitat quality or body condition often correlates with high responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis rather than with elevated baseline levels of glucocorticoids. We hypothesized that, for egg-laying vertebrates, high responsiveness of the HPA axis would correspond to high concentrations of corticosterone in yolk. We tested the prediction that Japanese quail (Coturnix coturnix japonica) selected for high plasma corticosterone response to brief immobilization (HS quail) would lay eggs with higher yolk corticosterone concentrations than birds selected for low response (LS quail). Quail from both lines were left undisturbed, outside of the stressors associated with daily management, before a first round of egg collection. In a second experiment, quail of both lines were experimentally stressed during the week before egg collection. In both cases we found quail from the HS line to lay eggs with significantly higher yolk corticosterone concentrations than quail of the LS line. After exposure to added experimental stressors, the line difference was more pronounced (increasing from 62% to 96%). There was no line difference in concentrations of yolk testosterone. Our results suggest that (1) genetic differences underly differences in the transfer of maternal corticosterone to yolk and (2) females may be able to control deposition of corticosterone into yolk through a mechanism independent of baseline corticosterone titers.     

Characterization of CRF, AVT, and ACTH cDNA and pituitary-adrenal axis function in Japanese quail divergently selected for tonic immobility

Higher corticosterone (CORT) responses to acute stress have previously been reported in quail selected for short (STI) duration of tonic immobility (TI) than for long TI (LTI), although behavioral studies indicated that LTI quail were more fearful. To investigate adrenal and pituitary function in these quail lines and their possible involvement in the differences in hypothalamic-pituitary-adrenal (HPA) axis reactivity, we measured CORT responses to adrenocorticotropin (1-24 ACTH), corticotropin-releasing factor (CRF), and arginine vasotocin (AVT) after characterizing the nucleotide acid sequences of these peptides in quail. Although maximum adrenal responses, assessed by ACTH challenge, were higher in STI quail, adrenal sensitivity was comparable for the two genotypes. It is therefore unlikely that differences in HPA axis reactivity involved the adrenal level. AVT and ACTH induced comparable CORT responses in both genotypes, whereas those induced by CRF were much lower. AVT is thus more potent than CRF in quail, but the respective maximum pituitary capacity of both genotypes to secrete ACTH was similar, and it is doubtful that the AVT pathway is involved in the difference in HPA axis reactivity between genotypes. On the other hand, the higher CORT responses induced by CRF in STI quail suggest that CRF might be involved in the differences in HPA axis reactivity between LTI and STI genotypes.     

A genome scan with AFLP markers to detect fearfulness-related QTLs in Japanese quail

A quantitative trait loci (QTL) study was undertaken to identify genome regions involved in the control of fearfulness in Japanese quail (Coturnix japonica). An F2 cross was made between two quail lines divergently selected over 29 generations on duration of tonic immobility (DTI), a catatonic-like state of reduced responsiveness to a stressful stimulation. A total of 1065 animals were measured for the logarithm of DTI (LOGTI), the number of inductions (NI) necessary to induce the immobility reaction, open-field behaviour including locomotor activity (MOVE), latency before first movement (LAT), number of jumps (JUMP), dejections (DEJ) and shouts (SHOUT), corticosterone level after a contention stress (LOGCORT) and body weight at 2 weeks of age (BW2). A total of 310 animals were included in a genome scan using selective genotyping with 248 AFLP markers. A total of 21 suggestive or genome-wide significant QTL were observed. Two highly significant QTL were identified on linkage group 1 (GL1), one for LOGTI and one for NI. In the vicinity of the QTL for LOGTI, a nearly significant QTL for SHOUT and a suggestive QTL for LAT were also identified. On GL3, genome-wide significant QTL were observed for JUMP and DEJ as well as suggestive QTL for LOGTI, MOVE, SHOUT and LAT. A significant QTL for BW2 was observed on GL2 and a nearly significant one on GL1. These results may be useful in the understanding of fearfulness in quail and related species provided that fearfulness has the same genetic basis.     

The influence of stress treatment on femur cortical bone porosity and medullary bone status in Japanese quail selected for high and low blood corticosterone response to stress

1. Femurs taken from Japanese quail (Coturnix coturnix japonica) genetically selected for either exaggerated (HS; high-stress) or reduced (LS; low-stress) blood corticosterone (B) response to unfamiliar environments were examined to determine their cortical bone porosity (CBP; degree of osteoporosis) and medullary bone (MB) status following treatment with or without stress. 2. Femur CBP was elevated in both sexes of stressed, HS-line quail and was attributed directly to the presumably exaggerated blood B responses known to occur in this line as a consequence of the stress treatments used. 3. The increase in CBP in stressed, HS-line birds occurred independently of changes in the numbers of osteoclast cells (CLASTS) associated with cortical bone. 4. Stress treatment was associated with lower CLAST numbers/mm2 of MB and with increased MB accretion. 5. These stress-induced effects were considered to be the consequence of a reduced rate of lay found in stressed quail hens. 6. There was only marginal evidence for a direct effect of B in stimulating MB resorption.     

Ease of capture in lines of Japanese quail (Coturnix japonica) subjected to contrasting selection for fear or sociability

Japanese quail (Coturnix japonica) of lines, which have been subjected to contrasting selection for duration of the tonic immobility (TI) reaction or social reinstatement (SR) behaviour over many generations show corresponding differences in underlying fearfulness and sociality. As fearfulness and sociality are particularly influential traits in domesticated species, the finding that such traits respond to artificial selection may have important implications for poultry welfare and performance. However, it is not known if or how such selection has influenced human-animal interactions. The present experiment investigated the influence of fearfulness and SR behaviour on the ease with which birds could be caught and handled. Birds of lines selected for duration of the TI response or SR behaviour were reared in mixed line groups (LTI and STI or HSR and LSR) of 491 and 346 birds, respectively, until 6 weeks of age. When the birds were 2, 4, and 6 weeks of age, they were caught one by one and their individual capture ranks noted. In the group of birds selected for duration of the TI response, birds selected of the line selected for short duration of TI were caught before those selected for long duration of the response. In the group of birds selected for SR motivation, birds of the high line were caught before their low lines counterparts. Coefficients of concordance between capture ranks were significant and capture ranks did not differ significantly across ages. These results imply that selection for low levels of fear or high levels of sociality produces animals that are less disturbed by human interventions than animals selected for the opposite traits. The greater ease of capture of low fear line birds than high fear line birds may be explained by reduced fear of humans. The fact that the birds selected for high levels of SR behaviour are easier to catch than birds selected for low levels of sociality is less readily explicable. One hypothesis is that HSR line chicks tend to be more strongly imprinted on each other and the human caretaker. However, SR behaviour is highly species specific in both lines, existing evidence for line differences in social discrimination is limited and birds of the two lines show similar duration of the TI response. Despite this, whatever their underlying causation, these results demonstrate that genetic selection can be used to reduce negative reactions to human beings and may be of value in the improvement of both animal welfare and productivity.     

Aspects of “fear” in Japanese quail chicks (coturnix coturnix Japonica) genetically selected for different levels of locomotor activity

Domestic chicks selected for high and low locomotor activity in a novel environment have low and high levels of fearfulness, respectively. Using four commonly employed methods of estimating fear, namely emergence, open-field, response to a bell and tonic immobility tests, we found a similar effect in lines of Japanese quail selected for high and low locomotor activity, i.e. the active line appeared the least fearful. The present findings demonstrate that this effect is common to two gallinaceous species and two selection procedures.     

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.     

Adrenocortical function in mice selectively bred for different sensitivity to ethanol

The adrenocortical response to ethanol, histamine and electric shock was studied in the Short-Sleep (SS) and Long-Sleep (LS) mice. The plasma corticosterone levels measured at 60 min following ethanol injection were consistently higher in LS mice (males and females) than in SS mice. The corticosterone levels determined 60 min after histamine injection (50 mg/kg) or electric foot shock were not statistically different in these two lines of mice. The initial rise of the corticosteroid after ethanol injection was identical in SS and LS mice, whereas the costicosteroid response to mild stress of saline injection was different: SS mice showed higher levels of corticosterone than LS mice at 20 and 30 min after the injection. Corticosterone increment attributable to alcohol stress clearly seemed greater in LS than in SS mice, even though the adrenals of the SS mice were 20–30% heavier than those of LS. The possible significance of these endocrine differences with respect to sensitivity to ethanol and behavioral arousal level was discussed.     

Genetic selection on a behavioural fear trait is associated with changes in heart rate variability in quail

This study investigated whether genetic selection on a divergent behavioural trait of fearfulness (tonic immobility duration) was related to changes in the nervous control of the heart. Quail selected for either long or short tonic immobility (LTI or STI, respectively) duration was compared with an unselected control line (CTI). The autonomic control of the heart was assessed by heart rate variability analysis and pharmacological blockades. Quail were surgically fitted with a telemetric device. Heart rate before injection did not differ between the three lines. The vagal-sympathetic effect (VSE) at rest differed significantly from 1 in CTI and STI quail, suggesting that parasympathetic activity was dominant. In LTI quail, VSE did not differ from 1, suggesting a balance between parasympathetic and sympathetic activities. The intrinsic heart rate reached after the successive injections of propranolol and atropine did not differ between lines and was higher than the heart rate at rest in STI, which was in line with results of VSE at rest. After atropine injection, the sympathetic activity indicated by the low-frequency power was lower in CTI than in the two selected quail. After propranolol injection, the parasympathetic activity indicated by the root of the mean squares of successive differences and the high-frequency power was higher in STI than in CTI and LTI quail. Selection on tonic immobility duration thus appears to be associated with changes in the sympathovagal control of the heart, which may influence behavioural responses to stressful situations.     

Dietary restriction causes chronic elevation of corticosterone and enhances stress response in red-legged kittiwake chicks

Release of corticosterone in hungry kittiwake chicks facilitates begging and allows them to restore depleted energy reserves by increasing parental food provisioning. However, in order to avoid detrimental effects of chronic elevation of corticosterone, chicks might suppress adrenocortical activity in response to prolonged food shortages. In this study we examined temporal dynamics of corticosterone release in red-legged kittiwake (Rissa brevirostris) chicks exposed to prolonged restrictions in energy content and/or nutritional quality (low versus high lipid content) of their food. Starting at the age of 15 days, chicks were fed either high- or low-lipid fish at 40%, 65%, and 100% of ad libitum energy intake. Body mass measurements and baseline plasma samples were taken on a weekly basis after beginning of the treatment. After 3 weeks of treatment, chicks were exposed to a standardized acute handling and restraint stress protocol, where in addition to a baseline sample, three plasma samples were taken at intervals up to 50 min. We found that food-restricted chicks had lower body mass, chronically (during 2-3 weeks) elevated baseline and higher acute stress-induced levels of corticosterone compared to chicks fed ad libitum. Low lipid content of food further exacerbated these effects. An increase in baseline levels of corticosterone was observed within a week after energy requirements of food-restricted chicks exceeded their daily energy intake. A tendency for suppression of adrenocortical activity was observed in treatments fed low-lipid diets only at the end of the experiment. We suggest that nest-bound chicks, if food-stressed, might suffer deleterious effects of chronic elevation of corticosterone.     

Age and food deprivation affects expression of the glucocorticosteroid stress response in Magellanic penguin (Spheniscus magellanicus) chicks

We examined how the glucocortical stress response in free-living Magellanic penguin (Spheniscus magellanicus) chicks changes with age and whether adrenocortical function of chicks within a brood varies in relation to food provisioned by adults. Chicks showed little corticosterone response to capture stress shortly after hatching, an intermediate response around 45-d posthatch, and a robust stress response near fledging. However, in response to an adrenocorticotropic hormone (ACTH) challenge, hatchlings were capable of secreting corticosterone at adult-like levels. The larger sibling in broods of two showed a similar gradual stress-response development pattern. In contrast, by day 45, when differences in body condition were well established between siblings, the smaller, food-deprived chicks significantly increased baseline levels of corticosterone but showed normal stress-induced levels. Near fledging, baseline levels had returned to normal, but stress-induced levels were lower than expected. Similar to altricial species, normally developing semialtricial Magellanic penguin chicks do not express a robust corticosterone stress response until near fledging. Chronic stressors such as food deprivation cause corticosterone use to be up-regulated earlier than expected. However, in cases of extended chronic stress, down-regulation may ensue, thus avoiding the negative effects of chronically elevated levels of corticosterone.     

Sight of a Predator Induces a Corticosterone Stress Response and Generates Fear in an Amphibian

Amphibians, like other animals, generate corticosterone or cortisol glucocorticoid responses to stimuli perceived to be threatening. It is generally assumed that the corticosterone response of animals to capture and handling reflects the corticosterone response to stimuli such as the sight of a predator that are thought to be natural stressors. Fijian ground frogs ( Platymantisvitiana ) are preyed upon by the introduced cane toads ( Rhinellamarina ), and we used ground frogs to test the hypothesis that the sight of a predator will induce a corticosterone stress response in an amphibian. Urinary corticosterone metabolite concentrations increased in male ground frogs exposed to the sight of a toad for 1, 3 or 6 h, whereas corticosterone did not change in frogs exposed to another male ground frog, a ball, or when no stimulus was present in the test compartment. The frogs exposed to a toad initially moved towards the stimulus then moved away, whereas frogs exposed to another frog moved towards the test frog and remained closer to the frog than at the start of the test. Tonic immobility (TI) was measured as an index of fearfulness immediately after the test exposure of the frogs to a stimulus. The duration of TI was longer in frogs exposed to a toad than to another frog or to a ball. The results provide novel evidence that the sight of a predator can induce a corticosterone response and lead to increased fearfulness in amphibians. In addition, they show that endemic frogs can recognise an introduced predator as a threat.     

Pre- and post-hatching effects of corticosterone treatment on behavior of the domestic chick

We investigated the effect of 60 ìg of corticosterone administered to domestic chicks either before or after hatching on the behavioral response to isolation in a novel arena and performance in a task involving the simultaneous identification of food and detection of a predator (overhead silhouette of a hawk moving overhead). Following release into a novel arena, chicks treated with corticosterone at 18 days of incubation emitted more distress vocalizations. In contrast, no difference in the number of vocalizations was found between chicks treated with corticosterone at day 1 post-hatching and controls. Behavior in the home cages was generally similar across treatments, though chicks treated with corticosterone at 18 days of incubation slept more than control chicks. While searching for grain against a background of pebbles, chicks treated with corticosterone at embryonic day 18, but not chicks treated on day 1 post-hatching, took longer to detect the overhead image of a predator than did controls. Corticosterone treatment at both ages increased the rate of pecking at grains and pebbles. Our findings support work on other birds indicating that corticosterone treatment during incubation influences stress reactivity. The impairment in predator detection in chicks treated with corticosterone on day 18 of incubation appears to be caused by the known effects of corticosterone treatment at this age in preventing the development of lateralization of the thalamofugal visual projections. This further supports the hypothesis that brain lateralization provides an advantage in performing more than one task simultaneously.     

Fearfulness Affects Quail Maternal Care and Subsequent Offspring Development

Our study investigated relationships between a precocial bird’s fearfulness and maternal care, and the implication of maternal care as a vector for non-genomic transmission of fearfulness to chicks. We compared care given to chicks between two sets of female Japanese quail selected to present either high (LTI) or low fearfulness (STI). Chicks, from a broiler line, were adopted by these females following a sensitization procedure. Chicks’ fearfulness after separation from their mother was assessed by well-established procedures. LTIs took longer to present maternal responses, pecked chicks more during the first days post-hatch, presented impaired maternal vocal behaviour and were globally less active than STI females. Chicks mothered by LTIs presented more fearful reactions than did chicks mothered by STIs, supporting the hypothesis of a non-genetic maternal transmission of fearfulness. We suggest that the longer latencies required by LTIs to become maternal are a consequence of their greater fear of chicks, and that their lower general and vocal activity could be components of a heightened antipredatory strategy. We discuss the transmission of maternal fearfulness to fostered chicks, taking into account the possible implication of several well-known mechanisms underlying maternal effects.     

Effects of social isolation and crowding upon adrenocortical reactivity and behavior in the rat

The effects of social isolation and crowding on adrenocortical function and upon behavioral responsiveness to electric shock have been studied in male and female rats. All female experimental groups showed higher corticosterone levels and heavier adrenals than their male counterparts. The major effect of housing condition concerned the corticosterone response to stress, while basal hormone concentration was not modified. Socially housed rats showed a more intense adrenocortical response and also a greater behavioral reactivity to electric shock than the isolates.     

Baseline and stress-induced levels of corticosterone during different life cycle substages in a shorebird on the high arctic breeding grounds

After a migratory flight of several thousand kilometers to their high arctic breeding grounds, red knots (Calidris canutus islandica, Scolopacidae) showed high baseline concentrations of plasma corticosterone (58 ng/mL). Such high baseline corticosterone levels may be conditional for the right behavioral and metabolic adjustments to environmental and social stresses that shorebirds experience on arrival in an unpredictable tundra breeding environment. Despite the high baseline levels of corticosterone, red knots still showed a marked stress response during the postarrival period, with corticosterone concentrations increasing significantly during a 60-min period of confinement. Baseline levels of corticosterone declined as the breeding season progressed. Red knots with brood patches, that is, birds that had completed egg laying and commenced incubation, had a reduced adrenocortical response to the stress of confinement compared with red knots with no, or with half-developed, brood patches. This is consistent with the idea that birds breeding in extreme environments with short breeding seasons may exhibit a decreased adrenocortical response to stressful events to prevent high corticosterone concentrations from inducing interruptions of reproductive behavior.     

Comparison of plasma ACTH and corticosterone levels after embryo bursectomy

Plasma levels of both adrenocorticotropic hormone (ACTH) and corticosterone (B) were determined in embryos (day 15 of incubation), chicks (day 3 after hatch) and young chickens (8 weeks). Experimental animals were bursectomized at 80 hr of incubation, i.e., before any anlage of the bursa of Fabricius could develop. Bursectomized (BFX) animals were compared to sham-operated controls (T), in basal, resting condition and 7 (ACTH) or 14 min (B) after ether stress was delivered for 30 sec. Basal B and ACTH levels seemed not to be significantly modified in BFX embryos, chicks and chickens. Hypophysial and adrenocortical response to stress appeared more precociously in BFX embryos (day 15 of incubation) than in intact ones (day 19). The non stress-responsive period that was observed for one week after hatch of T birds did not appear in 3-day-old BFX chicks whose both B and ACTH stress-induced levels were as high as in intact adults. In contrast, adrenocortical and pituitary corticotropic responses to stress were markedly impaired (by 50%) in adult BFX chickens as compared to intact controls.