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Daily and photoperiod variations of hypothalamic-pituitary-adrenal axis responsiveness in Japanese quail selected for short or long tonic immobility
Daily and Photoperiod Variations of Hypothalamic-Pituitary-Adrenal Axis Responsiveness in Japanese Quail Selected for Short or Long Tonic Immobility D. Hazard, M. Couty, J. M. Faure, and D. Gue´mene´1 Institut National de la Recherche Agronomique, Station de Recherches Avicoles, Centre de Tours-Nouzilly 37380 Nouzilly, France Higher hypothalamic-pituitary-adrenal (HPA) axis responsiveness to restraint in a crush cage was also measured in female quail reared under the long photoperiod, and similar responses were measured under both photoperiods in males. This result suggests that the effects of photoperiod length involve both local and more central mechanisms in the control of HPA axis responsiveness according to sex. On the other hand, we showed that the genetic selection program for TI responses induced greater increases in the B level following restraint in STI quail than in LTI quail of both sexes under both photoperiods, but the B adrenal response capacity was similar for both lines and sexes. Although further investigations on both lines regarding adrenal sensitivity are necessary before being able to conclude definitively, our findings strongly suggest that the differences observed in HPA axis responsiveness to restraint between lines are probably not due to differences in adrenal function itself but may involve upstream structures of the HPA axis.
(Key words: tonic immobility, corticosterone, photoperiod, Japanese quail) 2005 Poultry Science 84:1920–1925
INTRODUCTION To explore the possibility of reducing fearfulness and the expression of undesirable behavioral responses having deleterious consequences in domestic birds, a genetic selection program for behavioral traits related to welfare has been developed in birds (Mills and Faure, 1991). Thus, to explore this approach, 2 lines of Japanese quail have been divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI; Mills and Faure, 1991). TI is an unlearned catatonic state that is a reliable indicator of underlying fearfulness (Jones, 1986), and these LTI and STI lines thus constitute an appropriate model for the
2005 Poultry Science Association, Inc. Received for publication June 6, 2005. Accepted August 20, 2005. 1 To whom correspondence should be addressed: guemene@tours. inra.fr.
study of the putative relationship between fearfulness and hypothalamic-pituitary-adrenal (HPA) axis responsiveness. Successful adaptation to frightening or stressful stimuli requires not only the ability to perceive and respond to a stimulus but also the ability to control stress responses appropriately. As a response to a stressor, HPA axis activity leads to the secretion of glucocorticoids that promotes changes in central and peripheral mechanisms, which in turn allows birds to cope with harmful stimuli and maintain homeostasis (Siegel, 1971). Corticosterone (B) is the primary adrenal steroid found in the plasma of birds subjected to stress (Siegel, 1980; Harvey and Hall, 1990), and its measurement is the method most widely used to investigate changes in the activity of the HPA axis in birds. To characterize HPA axis responsiveness, it is important to determine intrinsic and environmental factors that might affect B levels. Abbreviation Key: ACTH = adrenocorticotropic hormone; B = corticosterone; HPA = hypothalamic-pituitary-adrenal; LTI = long tonic immobility; STI = short tonic immobility; TI = tonic immobility.
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ABSTRACT The aims of this study were to investigate the existence of a circadian rhythm of basal corticosterone (B) plasma concentrations in male and female Japanese quail lines divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI) and the possible effects of photoperiod length on corticotropic axis reactivity. Significant peaks in B levels were observed throughout the day in 3 out of the 4 groups used in our experiments. However, B levels remained very low for all groups (<5.0 ng/mL) and there was no consensus between groups. We therefore have no evidence from our results that basal B levels follow a circadian rhythm in adult STI and LTI quail held under a long photoperiod (16L:8D). We also showed that rearing under a long photoperiod (16L:8D) was associated with higher basal B levels and higher B adrenal response capacity to 1-24 adrenocorticotropic hormone (ACTH) injection in the STI and LTI lines compared with a shorter period (8L:16D).
HPA AXIS RESPONSIVENESS IN JAPANESE QUAIL
MATERIALS AND METHODS Birds and Management Japanese quail (Coturnix japonica) from the 36th generation of a selection program for LTI and STI responses were used in this study (Mills and Faure, 1991). Quail were identified by wing-banding on the day of hatching. All quail were reared collectively in battery cages, and food and water were provided ad libitum. The caretaker checked the quail daily and refilled the feeders whenever necessary in the morning (from 0830 h), but food was not refilled on the day of the experiment.
Experimental Procedures Experiment 1: Circadian Rhythm. A total of approximately 400 quail were exposed to continuous light until the age of 21 d and then were exposed to a long photope-
riod (16L:8D, light on at 0600 h, light off at 2200 h). Groups of 6 or 7 quail of the same line and the same sex were randomly placed in the rearing battery cages 1 wk before experimentation. Six or 7 quail of both lines and sexes were quickly sampled at regular time intervals [0600 (prior to lights on), 0700, 0900, 1100, 1300, 1500, 1700, 1900, 2100, 2300, 0100, 0300, and 0500 h] at the age of 44 d. During the scotoperiod, quail were captured in complete darkness. After capture, quail were transferred to a different room and decapitated, and all the blood sampling procedures lasted less than 30 s. Sampling of all birds from a single point was performed equally before and after the exact time point, in less than 10 min in total, except at the 0600 h sampling point, when all quail were bled prior to lights on. Experiment 2: Effects of Photoperiod. A total of approximately 200 quail were exposed to continuous lighting until the age of 21 d and then to 8L:16D (light on 0830 h) or 16L:8D (light on 0600 h) rhythms thereafter. Groups of 4 quail of the same line and sex were randomly placed in the rearing cages 1 wk before experimentation. An average of 7 quail were used for each experimental group, and quail were bled by decapitation at the age of 43 or 44 d between 0830 and 1230 h. Quail were bled after an interval of 10 min following physical or pharmacological treatments. We investigated HPA axis responsiveness by subjecting the quail to a physical treatment that consisted of a restraint in a crush cage, which has been shown to induce increases in B levels (Beuving and Vonder, 1986; Jones et al., 1994). After capture in the home cage, quail were transferred individually to a test room and then placed in a wooden box measuring 15 × 5 × 10 cm (length × width × height). The B adrenal response capacity has been addressed by pharmacological challenges consisting of the injection of 1-24 adrenocorticotropic hormone (ACTH; Immediate Synacthen, Novartis, France; 1 mg = 100 IU) at 100 g/kg of BW diluted in physiological serum (0.9% NaCl wt/vol) in the pectoralis major muscle. The 1-24 ACTH is very effective in stimulating the production of B in birds (Beuving and Vonder, 1986; Koelkebeck et al., 1986; Gue´mene´ et al., 2001). After capture in the home cage and transfer to the test room, quail were weighed individually prior to injection to determine the volume of 1-24 ACTH to be injected. They were placed back in the home cages for the required period immediately after injection. Two additional groups of quail were included in the study to assess basal B concentrations and the effects of capturing, weighing, and injecting. They were bled either immediately after capture in their home cage or 10 min after being captured, weighed, and injected with a representative volume of the vehicle (0.9% NaCl wt/vol, 400 to 700 L). Blood Sample Collection and B Assay. Blood was collected from each quail directly after decapitation in a tube containing EDTA (2 mg/mL of blood) and temporarily stored on ice. After centrifugation at 2,000 g for 15 min at 4°C, plasma samples were separated and stored
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Circadian rhythms in plasma B levels have been reported in various classes and species of animals including birds (Perkoff et al., 1959; Boissin et al., 1969; Joseph and Meier, 1973; Weitzman, 1976; Beuving and Vonder, 1977; Rich and Romero, 2001), and they might also be one important source of variability in B levels. However, there is no consensus in the literature concerning the circadian pattern of variations in plasma B levels. A peak occurring near the onset of the photoperiod and a low point early in the scotoperiod have been reported in adult domestic laying hens (Johnson and van Tienhoven, 1981). Other authors have reported the opposite, with a major peak occurring during the scotoperiod and low levels during the photoperiod (Beuving and Vonder, 1977; Etches, 1979). In quail, basal B concentrations have also previously been shown to fluctuate with circadian rhythm (Boissin et al., 1969). Although lower average basal B levels were measured during the dark compared with the light period, B concentrations increased throughout the dark period and the highest levels were reported by the end of the dark period (Boissin et al., 1969). Variations in plasma B levels are also affected by other external factors such as the length of the photoperiod, but again there is controversy concerning the nature of such effects. Continuous light or very long photoperiods have been reported to be associated with elevated basal B levels in some studies (Wilson and Cunningham, 1981; Lauber et al., 1987; Rich and Romero, 2001), whereas plasma B levels are reported to be similar in birds held under long and short photoperiods (Joseph and Meier, 1973; Deviche et al., 2001; Rich and Romero, 2001). The existence of circadian rhythms in plasma B levels has not been previously investigated in the lines of Japanese quail selected for LTI or STI response. Thus, the first aim of this study was to determine whether these lines show such rhythms or not. The second aim was to assess the effects of short (8L) and long (16L) photoperiods on their basal B levels, HPA axis responsiveness, and B adrenal response capacity.
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the day (time effect, P < 0.0001) but at different times according to line (time/line interaction, P = 0.009) and sex (time/sex interaction, P = 0.09). Maximum basal B levels were measured at 1900 h in female LTI quail, at 0300 h in female STI quail, and at 0600 h in male quail of both lines. Mean B concentrations for the day were similar in males (LTI, 2.4 ± 0.1 ng/mL; STI, 2.9 ± 0.2 ng/mL) and in females (LTI, 1.5 ± 0.2 ng/mL; STI, 1.5 ± 0.2 ng/mL) of both lines (line effect, P = 0.12), but B levels were higher (sex effect, P < 0.0001) in males than in females (sex effect: LTI, P = 0.0002; STI, P < 0.0001).
Effects of Photoperiod
at −20°C until measurement of B levels using a specific radio immunoassay (Etches, 1976). Statistical Analysis. The B values were subjected to a multifactorial ANOVA to assess the effects of line, sex, and treatment factors and their respective interactions using the Statview IV program (Abacus Concept Inc., Berkeley, CA). Whenever ANOVA reached significance (P < 0.05), post hoc tests were performed using the Fisher’s protected least significant difference test. The B values are expressed as means ± SE, and the level of significance is P < 0.05 unless otherwise stated.
RESULTS Circadian Rhythm The results indicated that maximum plasma B concentrations for sexes and lines remained lower than 5 ng/ mL (Figure 1). Occasional peaks were found throughout
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Figure 1. Daily variations of basal corticosterone (B) levels (ng/mL of plasma) in long tonic immobility (LTI) and short tonic immobility (STI) quail held under a long photoperiod (16L:8D). Samples bled during the scotoperiod and the photoperiod are represented by dark circles and open circles, respectively. The scotoperiod from 2200 to 0600 h is shown by the horizontal shaded bars (n = 6 per group on average; means ± SE).
Basal plasma B levels at 6 wk of age varied with length of photoperiod (P < 0.0001) for line and sex, but neither the line effect nor the sex effect was significant (Figure 2A). Quail subjected to a long photoperiod (16L:8D rhythm) showed 2.5-fold higher basal B levels (3.0 ± 0.3 ng/mL) than those exposed to a short photoperiod (8L:16D rhythm; 1.2 ± 0.2 ng/mL). Comparison of restraint-induced B responses to basal B levels showed that restraint in the crush cage for 10 min induced increases in B levels (P < 0.0001) in all experimental groups, with the exception of male LTI quail subjected to a short photoperiod (Figure 2B). In females, B levels following restraint were 2.8-fold higher in LTI quail and 2.2-fold higher in STI quail (photoperiod effect, P = 0.001) subjected to a long photoperiod (LTI, 11.9 ± 2.9 ng/mL; STI, 24.0 ± 3.6 ng/mL) than in those exposed to a short photoperiod (LTI, 4.3 ± 0.9 ng/mL; STI, 11.0 ± 3.6 ng/mL). In males of a specific line, plasma B levels following restraint did not differ significantly under either photoperiod (4.6 ± 2.5 ng/mL vs. 6.9 ± 1.4 ng/mL in the LTI line and 12.1 ± 1.8 ng/mL vs. 13.1 ± 2.2 ng/ mL in the STI line). On the other hand, STI quail of both sexes had higher B levels following restraint than LTI quail under both photoperiods (line effect, P < 0.0001). The B levels were 2.6- and 2.0-fold higher in STI and LTI quail following 10 min restraint under a short photoperiod (8L:16D) and a long photoperiod (16L:8D), respectively. Capture, immobilization, weighing, and a single i.m. injection of a saline solution induced significant increases in B levels during a 10-min interval in females of both lines subjected to a short photoperiod and in STI females subjected to a long photoperiod but not in the other groups (Figure 2C). The B increases in response to saline injection reached a maximum of 7.3 ± 1.4 ng/mL in female STI quail exposed to a long photoperiod. Injection of 1-24 ACTH at a dose of 100 g/kg of BW led to significant and similar increases in B concentrations in both lines and sexes (Figure 2D). Photoperiod length had a significant effect on B levels in response to 1-24 ACTH injection, with higher (P < 0.0001) B levels in quail subjected to a long photoperiod (16L:8D; 27.0 ± 1.7 ng/ mL) than in those subjected to a short photoperiod (8L:16D; 15.2 ± 0.9 ng/mL).
HPA AXIS RESPONSIVENESS IN JAPANESE QUAIL
DISCUSSION The aims of this study were to investigate the existence of a circadian rhythm of B levels in male and female Japanese quail lines divergently selected for LTI or STI and the possible effect of the length of photoperiod on corticotropic axis reactivity. Although B levels remained at very low levels throughout the day for all groups (<5.0 ng/mL) in our study, significant peaks were also observed in 3 of the 4 groups of mature quail used in our experiments. The existence of a circadian rhythm for B levels has previously been reported in different species of birds (Perkoff et al., 1959; Joseph and Meier, 1973; Weitzman, 1976; Beuving and Vonder, 1977; Rich and Romero, 2001), including adult male quail exposed to a natural photoperiod that was similar in length to the one used in this study (Boissin et al., 1969). Boissin et al. (1969) hypothesized that darkness triggered the daily rise in plasma B concentrations in quail because the increase in B levels occurred during the dark period, reaching a peak by the end of this period. However, in the present study, peaks were found at different sampling times during the
photoperiod and the scotoperiod according to line and sex. We cannot exclude the possibility that these discrepancies could be due to the difference in genotype used because differences between genotypes within the same species have previously been reported (Beuving and Vonder, 1977; Etches, 1979; Johnson and van Tienhoven, 1981). However, in our study, peaks appeared to result from the fact that higher B levels were measured for 1 or 2 out of 6 or 7 quail at the specific sampling times. Because different quail were used to measure B levels throughout the day in our study, we cannot state whether these high levels resulted from experimental artifact or had a physiological significance. It could be argued that a serial sampling approach would have been better to assess circadian rhythms, but repeated sampling has previously been reported to induce increases in B levels in birds (Beuving and Vonder, 1977; Harvey et al., 1980; Johnson, 1981; Beuving and Vonder, 1986). Moreover, the sampling method can have an effect on the results of assessment of basal B levels in quail (Hazard et al., 2004), as previously reported in other species (Eskeland and Blom, 1979; Harvey et al., 1980; Johnson, 1981). Thus, by minimizing the interval to sampling after capture, it appears to us that sampling after decapitation provided a more accurate measurement of basal B levels under our experimental conditions (compared with intracardiac, jugular, and wing vein puncture) and could be used for basal B level measurement and to assess the effects of very mild stress on B responses (D. Hazard, unpublished data). In the first experiment, most of the quail exposed to a long photoperiod (16L:8D) were already sexually mature, and the existence of a preovulatory B peak has been previously documented in hens (Etches, 1979). Consequently, it is possible that the erratic rises measured in females corresponded to preovulatory peaks. Nor can we exclude the possibility that peaks were due to other environmental factors, such as the occurrence of social interactions or other activities. However, peaks occurred only in very few birds, whereas for the factors proposed we would have expected more females to be affected or the majority of birds from a group at a specific time. Thus, we have no explanation for why we observed these erratic peaks, but our feeling is that these variations do not follow any consistent circadian rhythms. Therefore, although we cannot fully exclude the possibility, we have no evidence from our results that basal B levels follow a circadian rhythm in adult STI and LTI quail held under a long photoperiod (16L:8D). We noticed that basal B levels remained quite stable for all groups between 0800 and 1700 h, a period during which it is thus possible to investigate corticotropic axis reactivity further. From the results of the second experiment, we observed that quail reared under a long photoperiod (16 h) had higher basal B levels than those reared under a short photoperiod (8 h). This result is consistent with previous reports showing that basal B levels were higher in chickens reared under a long photoperiod than under a short one (Wilson and Cunningham, 1981; Lauber et al., 1987). However, basal B concentrations have also previously
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Figure 2. Corticosterone (B) concentrations (ng/mL of plasma) in long tonic immobility (LTI) and short tonic immobility (STI) quail subjected to long (16L:8D) and short (8L:16D) photoperiods. A) Basal B levels. B) B levels following 10 min of restraint in a crush cage. C) B levels 10 min after i.m. injection of a representative volume of the vehicle (NaCl 9%). D) B levels 10 min after i.m. injection of 1-24 adrenocorticotropic hormone (immediate synacthen) at 100 g/kg of BW (n = 7 per group on average; means ± SE). *Means are significantly different from those of the corresponding controls (P < 0.05).
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tained in male quail from an early generation of the same lines (Remignon et al., 1998). The fact that similar effects of 1-24 ACTH injection are measured in both lines suggests that the B response to restraint is independent of the adrenal B response capacity and in such a case may involve an upstream structure of the HPA axis. Such differences in HPA axis responsiveness between lines may depend on the nature of the stimulus applied and on an overall difference in its perception. This hypothesis is strongly supported by previous findings showing that exposure to a putatively milder stressor, consisting of the introduction of a novel object, elicited an increase in plasma B levels of LTI but not of STI quails (Launay, 1993). On the other hand, responses measured for both lines following 1-24 ACTH injection were of much greater amplitude than those measured following restraint. For this reason the results of the 1-24 ACTH injection may be more representative of a pharmacological than of a physiological effect. Under such a hypothesis, it cannot be excluded that the differences in response to restraint observed may have originated from a difference in sensitivity at the adrenal level. It will therefore be necessary to investigate adrenal sensitivity further before being able to reach conclusions about underlying mechanisms involved in the apparent difference in HPA axis responsiveness between LTI and STI quail lines. Whatever the underlying mechanisms, the apparently conflicting findings (i.e., higher B responses to restraint associated with shorter TI responses) in our lines and the reverse situation in others could be viewed as evidence that TI reactions and adrenocortical responses are not mutually coselected. This hypothesis is further supported by the fact that no relationship between B concentration after restraint and TI was found in a second generation cross (F2) between the LTI and STI lines (Mignon-Grasteau et al., 2003). In conclusion, we did not observe any consistent circadian B rhythm in sexually mature quail of STI or LTI lines held under a long photoperiod (16L:8D). We showed that rearing under a long photoperiod was associated with higher basal B levels and higher adrenal B responsiveness compared with a shorter period (8L:16D). Higher B levels were also measured following restraint in female quail reared under the long photoperiod, but they were similar in males. Therefore, further investigations regarding the respective effects, depending on sex, of photoperiod length and acquisition of sexual maturity are required. Nevertheless, these results suggest the involvement of both local and more central mechanisms in the control of HPA axis responsiveness. Interestingly, we have shown that the genetic selection program conducted on TI responses affected HPA axis responsiveness to strong acute stress, with higher B responses to restraint measured under both photoperiods for STI line compared with LTI line. Further studies will have to be undertaken regarding the respective adrenal sensitivity of lines and the effects of differing types of stress to be able to conclude definitively upon the causes of the observed differences in HPA axis responsiveness between lines.
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been reported to be similar under short and long photoperiods in house sparrows (Rich and Romero, 2001), common pigeons (Joseph and Meier, 1973), and dark-eyed Juncos (Deviche et al., 2001). In our study, it is impossible to distinguish between the possibility of a direct effect of photoperiod length and the possibility of an indirect effect resulting from acquisition of sexual maturity. Indeed, in the second experiment, quail at 6 wk of age exposed to a long photoperiod were already sexually mature, but those exposed to a short period were not (unpublished data). Rearing under a long photoperiod also was associated with higher B responses to restraint in female quail of both lines but not in males. Whether photoperiod directly or indirectly affects HPA axis responsiveness differently according to sex but regardless of line is questionable. Different hypotheses can be proposed to provide a valid explanation of the mechanisms underlying the present findings. The differences could involve changes in adrenal gland function, which is thought to be one of the major sites of the HPA axis at which B responses are modulated (Harvey and Hall, 1990). To investigate this hypothesis, adrenal function was investigated by pharmacological challenge, consisting of injection with 100 g/kg of BW of 1-24 ACTH. Interestingly, this challenge led to significantly higher B levels in quail subjected to 16L:8D than those subjected to 8L:16D rhythms. Adrenal responsiveness was thus clearly greater in the sexually mature quail held under a long photoperiod. However, although the B levels reached after challenge with 1-24 ACTH were similar between lines and sexes, the differences in B responses to restraint according to photoperiod were only observed for the female groups. Taken together, these results suggest that the higher B levels induced in response to restraint in females exposed to the long photoperiod could be due to this change in their B adrenal response capacity. However, other mechanisms must be involved at least in males since their responses to restraint were similar under the 2 photoperiods tested, but their respective adrenal response capacity was not. It more likely involves upstream structures of the HPA axis, probably in interaction with the acquisition of sexual maturity. Significant line effect was found in the response to restraint; higher responses were observed for the STI line under both of the photoperiods used. The divergent lines used in the present study have been selected on the basis of differential TI responses, a behavior that is positively correlated with fear (Mills and Faure, 1991). We would therefore have expected that the LTI line would have been more responsive to physical challenge (i.e., the opposite result from the one observed). Indeed, the use of a reverse strategy consisting of selecting quails on physiological responses (i.e., on high or low B responses to a restraint; Satterlee and Johnson, 1988), showed that the high stress line exhibited the highest B response and the longest TI responses (Jones et al., 1992). Although our results were unexpected, these differences in B response to restraint between lines are consistent with previous results ob-
HPA AXIS RESPONSIVENESS IN JAPANESE QUAIL
ACKNOWLEDGMENTS We thank A. D. Mills, who managed the selection program with J. M. Faure and supplied quail used in this study. We thank A. D. Mills and D. Raine for their valuable contributions to improving the quality of the manuscript. The authors also thank all those who contributed to this study, especially J.-M. Hervouet and J.-M. Brigand for expert technical assistance. D. Hazard was supported by grants from Institut National de la Recherche Agronomique and the Conseil Re´gional de la Re´gion Centre for completion of a Ph.D.
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Beuving, G., and G. M. Vonder. 1977. Daily rhythm of corticosterone in laying hens and the influence of egg laying. J. Reprod. Fertil. 51:169–173. Beuving, G., and G. M. Vonder. 1986. Comparison of the adrenal sensitivity to ACTH of laying hens with immobilization and plasma baseline levels of corticosterone. Gen. Comp. Endocrinol. 62:353–358. Boissin, J., I. Assenmacher, and J. D. Bayle. 1969. Circadian rhythm of adrenal cortex function, general activity and internal temperature in quails. Bull. Biol. Fr. Belg. 103:305–312. Deviche, P., C. Breuner, and M. Orchinik. 2001. Testosterone, corticosterone, and photoperiod interact to regulate plasma levels of binding globulin and free steroid hormone in darkeyed Juncos, Junco hyemalis. Gen. Comp. Endocrinol. 122:67–77. Eskeland, B., and A. K. Blom. 1979. Plasma corticosteroid levels in laying hens. Effect of two different blood sampling techniques and of rough handling of the animals. Acta Vet. Scand. 20:270–275. Etches, R. J. 1976. A radioimmunoassay for corticosterone and its application to the measurement of stress in poultry. Steroids 28:763–773. Etches, R. J. 1979. Plasma concentrations of progesterone and corticosterone during the ovulation cycle of the hen (Gallus domesticus). Poult. Sci. 58:211–216. Gue´mene´, D., G. Guy, J. Noirault, M. Garreau-Mills, P. Gouraud, and J. M. Faure. 2001. Force feeding procedure and physiological indicators of stress in male mule ducks. Br. Poult. Sci. 42:650–657. Harvey, S., and T. R. Hall. 1990. Hormones and stress in birds: Activation of the hypothalamo-pituitary-adrenal axis. Prog. Comp. Endocrinol. 342:453–460. Harvey, S., B. J. Merry, and J. G. Phillips. 1980. Influence of stress on the secretion of corticosterone in the duck (Anas platyrhynchos). J. Endocrinol. 87:161–171. Hazard, D., J. M. Faure, and D. Gue´mene´. 2004. Effects of sampling sites, photoperiod, and age on adrenal responsiveness in Japanese quail. Page 75 in Proc. 8th Int. Symp. Avian Endocrinol. Scottsdale, AZ. Johnson, A. L. 1981. Comparison of three serial blood sampling techniques on plasma hormone concentrations in the laying hen. Poult. Sci. 60:2322–2327. Johnson, A. L., and A. van Tienhoven. 1981. Plasma concentrations and corticosterone relative to photoperiod, oviposition,
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(Key words: tonic immobility, corticosterone, photoperiod, Japanese quail) 2005 Poultry Science 84:1920–1925
INTRODUCTION To explore the possibility of reducing fearfulness and the expression of undesirable behavioral responses having deleterious consequences in domestic birds, a genetic selection program for behavioral traits related to welfare has been developed in birds (Mills and Faure, 1991). Thus, to explore this approach, 2 lines of Japanese quail have been divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI; Mills and Faure, 1991). TI is an unlearned catatonic state that is a reliable indicator of underlying fearfulness (Jones, 1986), and these LTI and STI lines thus constitute an appropriate model for the
2005 Poultry Science Association, Inc. Received for publication June 6, 2005. Accepted August 20, 2005. 1 To whom correspondence should be addressed: guemene@tours. inra.fr.
study of the putative relationship between fearfulness and hypothalamic-pituitary-adrenal (HPA) axis responsiveness. Successful adaptation to frightening or stressful stimuli requires not only the ability to perceive and respond to a stimulus but also the ability to control stress responses appropriately. As a response to a stressor, HPA axis activity leads to the secretion of glucocorticoids that promotes changes in central and peripheral mechanisms, which in turn allows birds to cope with harmful stimuli and maintain homeostasis (Siegel, 1971). Corticosterone (B) is the primary adrenal steroid found in the plasma of birds subjected to stress (Siegel, 1980; Harvey and Hall, 1990), and its measurement is the method most widely used to investigate changes in the activity of the HPA axis in birds. To characterize HPA axis responsiveness, it is important to determine intrinsic and environmental factors that might affect B levels. Abbreviation Key: ACTH = adrenocorticotropic hormone; B = corticosterone; HPA = hypothalamic-pituitary-adrenal; LTI = long tonic immobility; STI = short tonic immobility; TI = tonic immobility.
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ABSTRACT The aims of this study were to investigate the existence of a circadian rhythm of basal corticosterone (B) plasma concentrations in male and female Japanese quail lines divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI) and the possible effects of photoperiod length on corticotropic axis reactivity. Significant peaks in B levels were observed throughout the day in 3 out of the 4 groups used in our experiments. However, B levels remained very low for all groups (<5.0 ng/mL) and there was no consensus between groups. We therefore have no evidence from our results that basal B levels follow a circadian rhythm in adult STI and LTI quail held under a long photoperiod (16L:8D). We also showed that rearing under a long photoperiod (16L:8D) was associated with higher basal B levels and higher B adrenal response capacity to 1-24 adrenocorticotropic hormone (ACTH) injection in the STI and LTI lines compared with a shorter period (8L:16D).
HPA AXIS RESPONSIVENESS IN JAPANESE QUAIL
MATERIALS AND METHODS Birds and Management Japanese quail (Coturnix japonica) from the 36th generation of a selection program for LTI and STI responses were used in this study (Mills and Faure, 1991). Quail were identified by wing-banding on the day of hatching. All quail were reared collectively in battery cages, and food and water were provided ad libitum. The caretaker checked the quail daily and refilled the feeders whenever necessary in the morning (from 0830 h), but food was not refilled on the day of the experiment.
Experimental Procedures Experiment 1: Circadian Rhythm. A total of approximately 400 quail were exposed to continuous light until the age of 21 d and then were exposed to a long photope-
riod (16L:8D, light on at 0600 h, light off at 2200 h). Groups of 6 or 7 quail of the same line and the same sex were randomly placed in the rearing battery cages 1 wk before experimentation. Six or 7 quail of both lines and sexes were quickly sampled at regular time intervals [0600 (prior to lights on), 0700, 0900, 1100, 1300, 1500, 1700, 1900, 2100, 2300, 0100, 0300, and 0500 h] at the age of 44 d. During the scotoperiod, quail were captured in complete darkness. After capture, quail were transferred to a different room and decapitated, and all the blood sampling procedures lasted less than 30 s. Sampling of all birds from a single point was performed equally before and after the exact time point, in less than 10 min in total, except at the 0600 h sampling point, when all quail were bled prior to lights on. Experiment 2: Effects of Photoperiod. A total of approximately 200 quail were exposed to continuous lighting until the age of 21 d and then to 8L:16D (light on 0830 h) or 16L:8D (light on 0600 h) rhythms thereafter. Groups of 4 quail of the same line and sex were randomly placed in the rearing cages 1 wk before experimentation. An average of 7 quail were used for each experimental group, and quail were bled by decapitation at the age of 43 or 44 d between 0830 and 1230 h. Quail were bled after an interval of 10 min following physical or pharmacological treatments. We investigated HPA axis responsiveness by subjecting the quail to a physical treatment that consisted of a restraint in a crush cage, which has been shown to induce increases in B levels (Beuving and Vonder, 1986; Jones et al., 1994). After capture in the home cage, quail were transferred individually to a test room and then placed in a wooden box measuring 15 × 5 × 10 cm (length × width × height). The B adrenal response capacity has been addressed by pharmacological challenges consisting of the injection of 1-24 adrenocorticotropic hormone (ACTH; Immediate Synacthen, Novartis, France; 1 mg = 100 IU) at 100 g/kg of BW diluted in physiological serum (0.9% NaCl wt/vol) in the pectoralis major muscle. The 1-24 ACTH is very effective in stimulating the production of B in birds (Beuving and Vonder, 1986; Koelkebeck et al., 1986; Gue´mene´ et al., 2001). After capture in the home cage and transfer to the test room, quail were weighed individually prior to injection to determine the volume of 1-24 ACTH to be injected. They were placed back in the home cages for the required period immediately after injection. Two additional groups of quail were included in the study to assess basal B concentrations and the effects of capturing, weighing, and injecting. They were bled either immediately after capture in their home cage or 10 min after being captured, weighed, and injected with a representative volume of the vehicle (0.9% NaCl wt/vol, 400 to 700 L). Blood Sample Collection and B Assay. Blood was collected from each quail directly after decapitation in a tube containing EDTA (2 mg/mL of blood) and temporarily stored on ice. After centrifugation at 2,000 g for 15 min at 4°C, plasma samples were separated and stored
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Circadian rhythms in plasma B levels have been reported in various classes and species of animals including birds (Perkoff et al., 1959; Boissin et al., 1969; Joseph and Meier, 1973; Weitzman, 1976; Beuving and Vonder, 1977; Rich and Romero, 2001), and they might also be one important source of variability in B levels. However, there is no consensus in the literature concerning the circadian pattern of variations in plasma B levels. A peak occurring near the onset of the photoperiod and a low point early in the scotoperiod have been reported in adult domestic laying hens (Johnson and van Tienhoven, 1981). Other authors have reported the opposite, with a major peak occurring during the scotoperiod and low levels during the photoperiod (Beuving and Vonder, 1977; Etches, 1979). In quail, basal B concentrations have also previously been shown to fluctuate with circadian rhythm (Boissin et al., 1969). Although lower average basal B levels were measured during the dark compared with the light period, B concentrations increased throughout the dark period and the highest levels were reported by the end of the dark period (Boissin et al., 1969). Variations in plasma B levels are also affected by other external factors such as the length of the photoperiod, but again there is controversy concerning the nature of such effects. Continuous light or very long photoperiods have been reported to be associated with elevated basal B levels in some studies (Wilson and Cunningham, 1981; Lauber et al., 1987; Rich and Romero, 2001), whereas plasma B levels are reported to be similar in birds held under long and short photoperiods (Joseph and Meier, 1973; Deviche et al., 2001; Rich and Romero, 2001). The existence of circadian rhythms in plasma B levels has not been previously investigated in the lines of Japanese quail selected for LTI or STI response. Thus, the first aim of this study was to determine whether these lines show such rhythms or not. The second aim was to assess the effects of short (8L) and long (16L) photoperiods on their basal B levels, HPA axis responsiveness, and B adrenal response capacity.
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the day (time effect, P < 0.0001) but at different times according to line (time/line interaction, P = 0.009) and sex (time/sex interaction, P = 0.09). Maximum basal B levels were measured at 1900 h in female LTI quail, at 0300 h in female STI quail, and at 0600 h in male quail of both lines. Mean B concentrations for the day were similar in males (LTI, 2.4 ± 0.1 ng/mL; STI, 2.9 ± 0.2 ng/mL) and in females (LTI, 1.5 ± 0.2 ng/mL; STI, 1.5 ± 0.2 ng/mL) of both lines (line effect, P = 0.12), but B levels were higher (sex effect, P < 0.0001) in males than in females (sex effect: LTI, P = 0.0002; STI, P < 0.0001).
Effects of Photoperiod
at −20°C until measurement of B levels using a specific radio immunoassay (Etches, 1976). Statistical Analysis. The B values were subjected to a multifactorial ANOVA to assess the effects of line, sex, and treatment factors and their respective interactions using the Statview IV program (Abacus Concept Inc., Berkeley, CA). Whenever ANOVA reached significance (P < 0.05), post hoc tests were performed using the Fisher’s protected least significant difference test. The B values are expressed as means ± SE, and the level of significance is P < 0.05 unless otherwise stated.
RESULTS Circadian Rhythm The results indicated that maximum plasma B concentrations for sexes and lines remained lower than 5 ng/ mL (Figure 1). Occasional peaks were found throughout
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Figure 1. Daily variations of basal corticosterone (B) levels (ng/mL of plasma) in long tonic immobility (LTI) and short tonic immobility (STI) quail held under a long photoperiod (16L:8D). Samples bled during the scotoperiod and the photoperiod are represented by dark circles and open circles, respectively. The scotoperiod from 2200 to 0600 h is shown by the horizontal shaded bars (n = 6 per group on average; means ± SE).
Basal plasma B levels at 6 wk of age varied with length of photoperiod (P < 0.0001) for line and sex, but neither the line effect nor the sex effect was significant (Figure 2A). Quail subjected to a long photoperiod (16L:8D rhythm) showed 2.5-fold higher basal B levels (3.0 ± 0.3 ng/mL) than those exposed to a short photoperiod (8L:16D rhythm; 1.2 ± 0.2 ng/mL). Comparison of restraint-induced B responses to basal B levels showed that restraint in the crush cage for 10 min induced increases in B levels (P < 0.0001) in all experimental groups, with the exception of male LTI quail subjected to a short photoperiod (Figure 2B). In females, B levels following restraint were 2.8-fold higher in LTI quail and 2.2-fold higher in STI quail (photoperiod effect, P = 0.001) subjected to a long photoperiod (LTI, 11.9 ± 2.9 ng/mL; STI, 24.0 ± 3.6 ng/mL) than in those exposed to a short photoperiod (LTI, 4.3 ± 0.9 ng/mL; STI, 11.0 ± 3.6 ng/mL). In males of a specific line, plasma B levels following restraint did not differ significantly under either photoperiod (4.6 ± 2.5 ng/mL vs. 6.9 ± 1.4 ng/mL in the LTI line and 12.1 ± 1.8 ng/mL vs. 13.1 ± 2.2 ng/ mL in the STI line). On the other hand, STI quail of both sexes had higher B levels following restraint than LTI quail under both photoperiods (line effect, P < 0.0001). The B levels were 2.6- and 2.0-fold higher in STI and LTI quail following 10 min restraint under a short photoperiod (8L:16D) and a long photoperiod (16L:8D), respectively. Capture, immobilization, weighing, and a single i.m. injection of a saline solution induced significant increases in B levels during a 10-min interval in females of both lines subjected to a short photoperiod and in STI females subjected to a long photoperiod but not in the other groups (Figure 2C). The B increases in response to saline injection reached a maximum of 7.3 ± 1.4 ng/mL in female STI quail exposed to a long photoperiod. Injection of 1-24 ACTH at a dose of 100 g/kg of BW led to significant and similar increases in B concentrations in both lines and sexes (Figure 2D). Photoperiod length had a significant effect on B levels in response to 1-24 ACTH injection, with higher (P < 0.0001) B levels in quail subjected to a long photoperiod (16L:8D; 27.0 ± 1.7 ng/ mL) than in those subjected to a short photoperiod (8L:16D; 15.2 ± 0.9 ng/mL).
HPA AXIS RESPONSIVENESS IN JAPANESE QUAIL
DISCUSSION The aims of this study were to investigate the existence of a circadian rhythm of B levels in male and female Japanese quail lines divergently selected for LTI or STI and the possible effect of the length of photoperiod on corticotropic axis reactivity. Although B levels remained at very low levels throughout the day for all groups (<5.0 ng/mL) in our study, significant peaks were also observed in 3 of the 4 groups of mature quail used in our experiments. The existence of a circadian rhythm for B levels has previously been reported in different species of birds (Perkoff et al., 1959; Joseph and Meier, 1973; Weitzman, 1976; Beuving and Vonder, 1977; Rich and Romero, 2001), including adult male quail exposed to a natural photoperiod that was similar in length to the one used in this study (Boissin et al., 1969). Boissin et al. (1969) hypothesized that darkness triggered the daily rise in plasma B concentrations in quail because the increase in B levels occurred during the dark period, reaching a peak by the end of this period. However, in the present study, peaks were found at different sampling times during the
photoperiod and the scotoperiod according to line and sex. We cannot exclude the possibility that these discrepancies could be due to the difference in genotype used because differences between genotypes within the same species have previously been reported (Beuving and Vonder, 1977; Etches, 1979; Johnson and van Tienhoven, 1981). However, in our study, peaks appeared to result from the fact that higher B levels were measured for 1 or 2 out of 6 or 7 quail at the specific sampling times. Because different quail were used to measure B levels throughout the day in our study, we cannot state whether these high levels resulted from experimental artifact or had a physiological significance. It could be argued that a serial sampling approach would have been better to assess circadian rhythms, but repeated sampling has previously been reported to induce increases in B levels in birds (Beuving and Vonder, 1977; Harvey et al., 1980; Johnson, 1981; Beuving and Vonder, 1986). Moreover, the sampling method can have an effect on the results of assessment of basal B levels in quail (Hazard et al., 2004), as previously reported in other species (Eskeland and Blom, 1979; Harvey et al., 1980; Johnson, 1981). Thus, by minimizing the interval to sampling after capture, it appears to us that sampling after decapitation provided a more accurate measurement of basal B levels under our experimental conditions (compared with intracardiac, jugular, and wing vein puncture) and could be used for basal B level measurement and to assess the effects of very mild stress on B responses (D. Hazard, unpublished data). In the first experiment, most of the quail exposed to a long photoperiod (16L:8D) were already sexually mature, and the existence of a preovulatory B peak has been previously documented in hens (Etches, 1979). Consequently, it is possible that the erratic rises measured in females corresponded to preovulatory peaks. Nor can we exclude the possibility that peaks were due to other environmental factors, such as the occurrence of social interactions or other activities. However, peaks occurred only in very few birds, whereas for the factors proposed we would have expected more females to be affected or the majority of birds from a group at a specific time. Thus, we have no explanation for why we observed these erratic peaks, but our feeling is that these variations do not follow any consistent circadian rhythms. Therefore, although we cannot fully exclude the possibility, we have no evidence from our results that basal B levels follow a circadian rhythm in adult STI and LTI quail held under a long photoperiod (16L:8D). We noticed that basal B levels remained quite stable for all groups between 0800 and 1700 h, a period during which it is thus possible to investigate corticotropic axis reactivity further. From the results of the second experiment, we observed that quail reared under a long photoperiod (16 h) had higher basal B levels than those reared under a short photoperiod (8 h). This result is consistent with previous reports showing that basal B levels were higher in chickens reared under a long photoperiod than under a short one (Wilson and Cunningham, 1981; Lauber et al., 1987). However, basal B concentrations have also previously
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Figure 2. Corticosterone (B) concentrations (ng/mL of plasma) in long tonic immobility (LTI) and short tonic immobility (STI) quail subjected to long (16L:8D) and short (8L:16D) photoperiods. A) Basal B levels. B) B levels following 10 min of restraint in a crush cage. C) B levels 10 min after i.m. injection of a representative volume of the vehicle (NaCl 9%). D) B levels 10 min after i.m. injection of 1-24 adrenocorticotropic hormone (immediate synacthen) at 100 g/kg of BW (n = 7 per group on average; means ± SE). *Means are significantly different from those of the corresponding controls (P < 0.05).
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tained in male quail from an early generation of the same lines (Remignon et al., 1998). The fact that similar effects of 1-24 ACTH injection are measured in both lines suggests that the B response to restraint is independent of the adrenal B response capacity and in such a case may involve an upstream structure of the HPA axis. Such differences in HPA axis responsiveness between lines may depend on the nature of the stimulus applied and on an overall difference in its perception. This hypothesis is strongly supported by previous findings showing that exposure to a putatively milder stressor, consisting of the introduction of a novel object, elicited an increase in plasma B levels of LTI but not of STI quails (Launay, 1993). On the other hand, responses measured for both lines following 1-24 ACTH injection were of much greater amplitude than those measured following restraint. For this reason the results of the 1-24 ACTH injection may be more representative of a pharmacological than of a physiological effect. Under such a hypothesis, it cannot be excluded that the differences in response to restraint observed may have originated from a difference in sensitivity at the adrenal level. It will therefore be necessary to investigate adrenal sensitivity further before being able to reach conclusions about underlying mechanisms involved in the apparent difference in HPA axis responsiveness between LTI and STI quail lines. Whatever the underlying mechanisms, the apparently conflicting findings (i.e., higher B responses to restraint associated with shorter TI responses) in our lines and the reverse situation in others could be viewed as evidence that TI reactions and adrenocortical responses are not mutually coselected. This hypothesis is further supported by the fact that no relationship between B concentration after restraint and TI was found in a second generation cross (F2) between the LTI and STI lines (Mignon-Grasteau et al., 2003). In conclusion, we did not observe any consistent circadian B rhythm in sexually mature quail of STI or LTI lines held under a long photoperiod (16L:8D). We showed that rearing under a long photoperiod was associated with higher basal B levels and higher adrenal B responsiveness compared with a shorter period (8L:16D). Higher B levels were also measured following restraint in female quail reared under the long photoperiod, but they were similar in males. Therefore, further investigations regarding the respective effects, depending on sex, of photoperiod length and acquisition of sexual maturity are required. Nevertheless, these results suggest the involvement of both local and more central mechanisms in the control of HPA axis responsiveness. Interestingly, we have shown that the genetic selection program conducted on TI responses affected HPA axis responsiveness to strong acute stress, with higher B responses to restraint measured under both photoperiods for STI line compared with LTI line. Further studies will have to be undertaken regarding the respective adrenal sensitivity of lines and the effects of differing types of stress to be able to conclude definitively upon the causes of the observed differences in HPA axis responsiveness between lines.
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been reported to be similar under short and long photoperiods in house sparrows (Rich and Romero, 2001), common pigeons (Joseph and Meier, 1973), and dark-eyed Juncos (Deviche et al., 2001). In our study, it is impossible to distinguish between the possibility of a direct effect of photoperiod length and the possibility of an indirect effect resulting from acquisition of sexual maturity. Indeed, in the second experiment, quail at 6 wk of age exposed to a long photoperiod were already sexually mature, but those exposed to a short period were not (unpublished data). Rearing under a long photoperiod also was associated with higher B responses to restraint in female quail of both lines but not in males. Whether photoperiod directly or indirectly affects HPA axis responsiveness differently according to sex but regardless of line is questionable. Different hypotheses can be proposed to provide a valid explanation of the mechanisms underlying the present findings. The differences could involve changes in adrenal gland function, which is thought to be one of the major sites of the HPA axis at which B responses are modulated (Harvey and Hall, 1990). To investigate this hypothesis, adrenal function was investigated by pharmacological challenge, consisting of injection with 100 g/kg of BW of 1-24 ACTH. Interestingly, this challenge led to significantly higher B levels in quail subjected to 16L:8D than those subjected to 8L:16D rhythms. Adrenal responsiveness was thus clearly greater in the sexually mature quail held under a long photoperiod. However, although the B levels reached after challenge with 1-24 ACTH were similar between lines and sexes, the differences in B responses to restraint according to photoperiod were only observed for the female groups. Taken together, these results suggest that the higher B levels induced in response to restraint in females exposed to the long photoperiod could be due to this change in their B adrenal response capacity. However, other mechanisms must be involved at least in males since their responses to restraint were similar under the 2 photoperiods tested, but their respective adrenal response capacity was not. It more likely involves upstream structures of the HPA axis, probably in interaction with the acquisition of sexual maturity. Significant line effect was found in the response to restraint; higher responses were observed for the STI line under both of the photoperiods used. The divergent lines used in the present study have been selected on the basis of differential TI responses, a behavior that is positively correlated with fear (Mills and Faure, 1991). We would therefore have expected that the LTI line would have been more responsive to physical challenge (i.e., the opposite result from the one observed). Indeed, the use of a reverse strategy consisting of selecting quails on physiological responses (i.e., on high or low B responses to a restraint; Satterlee and Johnson, 1988), showed that the high stress line exhibited the highest B response and the longest TI responses (Jones et al., 1992). Although our results were unexpected, these differences in B response to restraint between lines are consistent with previous results ob-
HPA AXIS RESPONSIVENESS IN JAPANESE QUAIL
ACKNOWLEDGMENTS We thank A. D. Mills, who managed the selection program with J. M. Faure and supplied quail used in this study. We thank A. D. Mills and D. Raine for their valuable contributions to improving the quality of the manuscript. The authors also thank all those who contributed to this study, especially J.-M. Hervouet and J.-M. Brigand for expert technical assistance. D. Hazard was supported by grants from Institut National de la Recherche Agronomique and the Conseil Re´gional de la Re´gion Centre for completion of a Ph.D.
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