Chronic stress and environmental enrichment as opposite factors affecting the immune response in Japanese quail (Coturnix coturnix japonica)

Abstract

Procedures in the commercial production of animals involve stressful situations which lessen the animal's welfare. This study on Japanese quail evaluated whether an environmental enrichment manipulation can affect avian immune responses and if combined with a chronic stressor exposure can help to counteract the negative effects of stress on the immune system. Potential gender effects were also considered. After hatch, half of the birds were housed in non-enriched boxes and half were housed in environmentally enriched boxes. From day 33 to 42 of age, all birds within half of the non-enriched and enriched boxes remained undisturbed while the other half were daily exposed to a 15 min restraint stressor (chronic stressor). The inflammatory response (lymphoproliferation after phytohemagglutinin-p), percentage of lymphocytes, heterophil/lymphocyte (H/L) ratio and primary antibody response against sheep red blood cells were assessed. The chronic stressor application and the enrichment procedure, respectively, either increased or reduced the four immunological parameters evaluated and always in opposite directions. Males consistently showed lower antibody titres than females and presented the highest H/L ratio in response to the stressor when reared in the non-enriched environment. The findings indicate that submitting these animals to an enriched environment can be effectively used to improve their immune response and to reduce the detrimental effects of a stressor exposure.

Keywords::

• Antibody response
• chronic stress
• environmental enrichment
• heterophil/lymphocyte ratio
• immune response
• Japanese quail

Introduction

The commercial production of animals implies capture, handling and other procedures that may involve stressful situations which lessen the animals' welfare (Schulz et al. 2000; Marco et al. 2006; Dickens et al. 2009, 2010). According to Kuenzel and Jurkevich (2010), stress in birds, as in mammals, comprises a variety of conditions or forces that affect an organism. These stressors may be external to the body and disturb homoeostasis, producing a state of stress (Siegel 1995). Two physiological stress response strategies were proposed (de Kloet and Derijk 2004). One involves an immediate response that organises the behavioural, sympathetic and hypothalamo-pituitary–adrenal (HPA) response to a stressor. The other is a slower response that facilitates behavioural adaptation, promotes recovery and re-establishes homoeostasis. The stress response has a large number of documented physical and physiological manifestations. In poultry, negative consequences of stress for reproductive function involve diminished egg production, fertility and hatchability among others (Marin et al. 2002; Marin and Satterlee 2004; Leone and Estévez 2008); altered social interaction with conspecifics (Jones 1996; Marin et al. 2001; Guzman and Marin 2008); and a strong incapability to adequately use resources (Jones 1996; Mormede et al. 2007). Stress may also differentially affect animals according to their gender. Several studies indicate that avian males seem more susceptible to stressors than their female counterparts (Huff et al. 1999; Wideman and French 2000; Marin et al. 2002), resulting in higher levels of glucocorticoids (GCs) released in to the bloodstream after stressor exposure (Marin et al. 2002).

One of the systems most affected by the increased activation of the HPA axis is the immune system (Glick 1984; Dohms and Metz 1991; Fair et al. 1999; Shini et al. 2008a,b; Bauer et al. 2009). The consequences of a sustained stress response on the immune system were widely reported. For example, nestling collared doves respond to within-brood competition by elevating corticosterone secretion and down-regulating cell-mediated immunoresponsiveness (Eraud et al. 2008); inoculation of chickens with corticosterone using pharmacological doses results in a rapid lymphoid depletion in the thymus, bursa and spleen (Dohms and Metz 1991) and antibody production decreases in laying hens treated with adrenocorticotrophic hormone (Mumma et al. 2006).

A great effort is directed to ameliorating the negative effects of stressor exposure which are generally unavoidable during animal rearing, i.e. at times of routine maintenance chores, animal capture, crating, weighing or transport. Among the proposed solutions, environmental enrichment (EE) is a technique that may lead to improvements in animal welfare. It is reported that EE reduces disturbance and aggression among animals (Cornetto et al. 2002), fear and stress responses (Nicol 1992; Reed et al. 1993; Grigor et al. 1995; Bizeray et al. 2002), attenuates abnormal behaviours (Huber-Eicher and Wechsler 1998; Meehan et al. 2003) and decreases levels of anxiety (Miller and Mench 2005; Fox et al. 2006). EE consists of the addition of different physical elements that modify the space where animals live (Leone and Estévez 2008). The main purpose of this addition is to provide the animals with new behavioural opportunities that could lead to improvements in their biological functioning (Newberry 1995). According to the type of interaction that an animal can have with the new modified environment, the EE is considered to fall into four general categories: foraging opportunities, structural complexity, sensory stimulation/novelty and social companionship (Miller and Mench 2005).

EE has been linked to a modulation of immunity in rats, but shows alterations that were not all consistent (Marashi et al. 2002). In poultry, little is known about the effects of EE on the immune system. Huff et al. (2003) documented that enriching the brooding environment during the first 2 weeks after hatch may be detrimental to turkeys with a fast T-maze solving response. However, the EE elements provided were replaced every day by ones that were novel to the birds. Therefore, the negative results reported could be related to the enrichment procedure that may also be considered as chronic stressful stimulation (Jones 1996; Huff et al. 2003; Richard et al. 2008).

In the present study, groups of newly hatched Japanese quail were kept in regular unstimulated rearing conditions while the other groups were exposed to an EE, where different sets of elements were presented to the birds and changed on a weekly base. This procedure was proposed as a compromise between reducing the stressor component of the novel elements and minimising potential boredom induced by the habituation to those elements. Half of the groups either remained undisturbed or were exposed to a chronic repeated stressor by brief physical restraint before their cellular and humoral immune responses were assessed. This enabled us to address three main questions. First, can EE affect the immune responses of the birds? Second, does the chronic stressor exposure applied show an immuno-depressor effect on quail? If so, can the EE help reduce the negative effects of the chronic stressor exposure? Finally, considering the gender differences in stress responses mentioned above, we also assessed whether males and females differ in their immune responses to the EE and/or the chronic stressor application.

Material and methods

Animals and husbandry

Japanese quail (Coturnix coturnix japonica) were used in the present study because they are recognised as a useful laboratory model for avian studies and for extrapolation of data to chickens and other commercially important poultry species (Mills and Faure 1992; Jones 1996), as well as being an important agricultural species in several countries (Baumgartner 1994; Faure et al. 2003). One hundred and eighty mixed-sex Japanese quail hatchlings were randomly housed in 12 white wooden boxes (15 quail each) (see treatment procedure below) measuring 90 × 45 × 60 cm (length × width × height), and remained in the same pen until the end of the study (day 49 of age). Each box had one feeder and 8 automatic nipple drinkers. A wiremesh floor (1 cm grid) was raised to 5 cm to allow the passage of excreta, and a lid prevented the birds from escaping. Brooding temperature was 37.5°C during the first week of life, with a weekly decline of 3.0°C until room temperature (24–27°C) was achieved. A quail starter diet (28% CP; 2800 Kcal ME/kg) and water were provided ad libitum. At 28 days of age, the birds were sexed by plumage colouration and wing banded for later individual identification. Coincident with banding, birds were given a laying ration (21% protein, 2750 Kcal ME/kg) and water continued ad libitum. Quail were subjected to a daily cycle of 14-h light (300–320 lx): 10-h dark during the study. Lights were turned on at 06:00 h and turned off at 22:00 h.

Treatment procedure

After hatch, half of the birds were housed in six boxes with a feeder and water nipples as above (non-enriched boxes; control) while the other half were housed in the remaining six boxes that were provided with a set of EE elements (enriched boxes). Three different sets of enrichment elements were used during the study, consisting of hanging bottle caps, hanging coloured wool and Velcro cylinders (foraging enrichment), different wooden platforms (structural enrichment) or a combination of hanging bottle caps and wooden platforms (mixed enrichment) (Figure 1A–D). All of these were applied to each of the six enriched boxes using a weekly rotation procedure that allowed all birds within the enrichment treatment to be sequentially exposed to all three enrichment sets.

Figure 1.  Overhead view of boxes showing the four rearing environments used during the study. (A) non-enriched environment, (B) structural enrichment:

= different wooden platforms, (C) foraging enrichment: ○ = bottle caps ▪ = Velcro cylinders

= coloured wool, and (D) mixed structural and foraging enrichment.

During a 10-day period, from day 33 to 42 of age, all birds in three non-enriched and in three Enriched boxes remained undisturbed (not-stressed, control) while the birds within the other boxes were daily and individually captured and immediately restrained in wooden restraint baskets for 15 min (stressed). Birds were restrained between 10:00 and 11:00 h. The baskets used served as crush cage devices and were similar to those used by Satterlee and Johnson (1988) for the genetic selection of their low or high quail stress lines. The restraint consisted in preventing all but slight movements of the quail's head and legs, and those associated with respiration. Following each daily stress session, the birds were immediately returned to their home boxes.

Thus, there were four treatment combinations: non-enriched/not-stressed; non-enriched/stressed; enriched/not-stressed; enriched/stressed and there were three independent replications for each of them. Within each of the group combinations, also the gender (male or female) was considered in the data analysis (see below). Six birds per box (three females and three males) were used for this study and their cellular and humoral immune responses were evaluated, providing a final number of 72 animals. The remaining nine quail per box were maintained under the exact same conditions as the others and used for a later behavioural study.

Immunological assays

Four parameters were evaluated in order to assess the condition of the immune system of each of the 72 birds: (a) lymphoproliferative responses to phytohemagglutinin-p (PHA-P); (b) percentage of lymphocytes; (c) heterophil/lymphocyte (H/L) ratio, both analysed in blood smears, and (d) primary antibody response against sheep red blood cells (SRBC) using a microagglutination assay.

To determine the cell-mediated immunity, the responses to PHA-P injection (a lectin from Phaseolus vulgaris (Sigma Chemical, St Louis, MO, USA)) was measured in the wing web of each bird following the methods described elsewhere (Stadecker et al. 1977; Smits et al. 1999; Roberts et al. 2009). Briefly, at 43 days of age (24 h after finishing the chronic stress treatment) a 0.075 ml intradermal injection of a solution of PHA-P in phosphate saline buffer (1000 μg/ml) was given in the wing web of each bird, at 2 mm from the brachial vein. The dermal swelling response was measured as the percentage increase in wing web thickness at the injection site 24 h post-PHA-P injections, and was calculated using the following formula: percentage of inflammation = (inflammation previous 24 h/inflammation post 24 h) × 100. Measurements were recorded to the nearest 0.01 mm using a mechanical micrometer. Following Smits et al. (1999) only one wing was injected, without a control vehicle injection in the other wing, because the inflammation resulting from the injection of phosphate buffered saline alone disappears gradually 24 h later, thus the inflammation measured is a consequence of the migration and recruitment processes due to the PHA-P injection. On the same day, the brachial vein of the opposite wing (left wing) was punctured in order to obtain one blood drop for smears (see below). Then, the birds were intraperitoneally injected with 0.75 ml of a 10% SRBC suspension in order to induce a humoral immune response (see below).

Leukocyte counts were obtained by analysing blood smears stained with May Grünwald Giemsa. Smears were done one and seven days after finishing the chronic stress treatment (days 43 and 49 of age, respectively). Differential counts of 100 white cells per blood smear were made. The percentage of lymphocytes data relates the number of lymphocytes before and one week after finishing the stressor treatment and was calculated using the following formula: lymphocyte (%) = (number of lymphocytes on day 49/number of lymphocytes on day 43) × 100. This variable was analysed in order to assess the evolution of this subpopulation of white cells after the stimulation with SRBC and concomitant with finishing the stress treatment. The H/L ratio of day 43 and the percentage of lymphocytes were compared within each bird sample.

To evaluate the induced humoral immune response, antibody titres were measured in blood samples one week after (49 days of age) the administration of the SRBC suspension. The blood samples were centrifuged at 2500g for 15 min and the serum obtained was then stored at − 20°C until further primary antibody response analysis. The antibody response was assessed with a microagglutination assay (Sever 1961; Smits and Williams 1999). Briefly, 20 μl of complement-inactivated (through heating to 56°C) plasma was serially diluted in 20 μl of phosphate buffered saline (PBS) (1:2, 1:4, 1:8, up to 1:512). Next, 20 μl of a 2% suspension of SRBC in PBS was added to all wells. Microplates were incubated at 40°C for 1 h and hemagglutination of the test plasma samples was compared to the blanks (PBS only) and negative controls (wells with no SRBC suspension). Antibody titres were reported as the Log₂ of the highest dilution yielding significant agglutination.

All experiments were carried out in accordance with the National Institute of Health Guide for the care and use of laboratory animals (NIH Publications No. 80 23, revised 1978).

Statistical analysis

Data within each treatment combination replicate were averaged before statistical analysis. Analyses were performed using a three-way ANOVA which examined the main effects of EE treatment (non-enriched, enriched boxes), chronic stress treatment (not-stressed, stressed birds), gender (male, female) and their interactions. Assumptions of the ANOVA were verified. H/L ratio data were subjected to a square root transformation before analysis to fit the ANOVA assumptions. Transformations were not required for inflammatory response, percentage of lymphocytes and antibody titre data. Post-hoc treatment group comparisons were conducted using the Fisher least significant difference (LSD) test. A p-value of < 0.05 was considered to represent significant differences.

Results

Inflammatory response (cutaneous basophil hypersensitivity test)

The effects of EE and stress treatment on the inflammatory response of male and female quail are given in Figure 2. The ANOVA revealed a significant main effect of the EE treatment (F(1,16) = 11,42; p < 0.01) and stress treatment (F(1,16) = 5.61; p = 0.03). No significant differences between male and female responses to the injection of PHA-P (F(1,16) = 0.03; p = 0.88) were detected. The post-hoc analysis was then performed combining male and female data. The test analysis showed that the inflammatory response was greater in the birds reared in the enriched environment and in the birds that were not-stressed compared with their respective non-enriched and stressed counterparts. Quail stressed in a non-enriched environment was the treatment combination that showed the lowest inflammatory response. The other group combinations (indicated with letter “a” in Figure 2) showed higher values (similar among these groups) of inflammatory response.

Figure 2.  Percentage of change in the wing web thickness 24 h post injection of PHA-P in Japanese quail submitted to a chronic stress treatment and reared under an enriched or non-enriched environment. ^a,bDifferent letters indicate significant (p < 0.05; Fisher LSD test) differences between groups. Filled columns represent treatment means and lines the SE of the mean (number of birds per group = 18).

Percentage of lymphocytes

The effects of EE and stress treatment on the percentage of peripheral blood lymphocytes one week after finishing the chronic stressor application in male and female quail are given in Figure 3. The ANOVA revealed a significant main effect of the EE treatment (F(1,16) = 39.59; p < 0.001) and stress treatment (F(1,16) = 31.69; p < 0.001). No significant differences between males and females (F(1,16) = 2.79; p = 0.10) were detected in this variable. Thus, post-hoc analysis was performed combining male and female data. The test showed that the percentage of lymphocytes was higher in the animals reared in the enriched environment and in the birds that were not-stressed compared with their respective treatment counterparts (i.e., birds reared in a non-enriched environment and birds that were stressed, respectively). Quail reared in an enriched environment that were not-stressed and stressed birds reared in a non-enriched environment showed, respectively, the higher and lower percentage of lymphocytes. Not-stressed birds reared in a non-enriched environment and stressed birds reared in an enriched environment showed similar and intermediate values (Figure 3).

Figure 3.  Percentage of peripheral blood lymphocytes one week after finishing a chronic restraint stressor application in Japanese quail reared under an enriched or non-enriched environment. ^a-cDifferent letters indicate significant (p < 0.05; Fisher LSD test) differences between groups. Filled columns represent treatment means and the lines the SE of the mean (number of birds per group = 18).

H/L ratio

The effects of EE and stress treatment on the H/L ratio of male and female quail are given in Figure 4. The ANOVA revealed a significant main effect of the EE treatment (F(1,16) = 34.49; p < 0.001) and stress treatment (F(1,16) = 41.51; p < 0.001). A significant interaction among EE, stress and gender was also observed (F(1,16) = 4.34; p = 0.05). Post-hoc analysis revealed that not-stressed birds reared in an enriched environment and stressed birds reared in a non-enriched environment showed, respectively, the lower and the higher H/L ratio. The not-stressed birds reared in a non-enriched environment and the stressed birds reared in an enriched environment showed similar and intermediate values (Figure 4). Moreover, the stressed males reared in the non-enriched environment showed a significantly (p < 0.05) even higher H/L ratio than their female counterparts.

Figure 4.  H/L ratio 24 h after finishing a chronic restraint stressor application in Japanese quail reared under an enriched or non-enriched environment. ^a-cDifferent letters indicate significant (p < 0.05; Fisher LSD test) differences between groups. Filled columns represent treatment means and the lines the SE of the mean (number of birds per group = 18).

SRBC antibody titre

The analysis of the humoral component of immunity revealed a significant main effect of the three factors studied on the antibody titre measured: EE (F(1,16) = 9.77; p < 0.01), stress (F(1,16) = 10.50; p = 0.01) and of gender (F(1,16) = 5.63; p = 0.03). Post-hoc analysis showed that the humoral response was higher in the animals reared in the enriched environment and in the not-stressed birds compared with their respective non-enriched and stressed counterparts. Moreover, regardless of EE or stress treatment, females showed higher antibody responses than males (Figure 5).

Figure 5.  Serum antibody titres one week after the injection of SRBC in Japanese quail submitted to a chronic restraint stress treatment and reared under an enriched or non-enriched environment. Particular significant (p < 0.05; Fisher LSD test) differences between groups are indicated in the figure. Filled columns represent treatment means and the lines the SE of the mean (number of birds per group = 18).

Discussion

The present study evaluated the effects of rearing female and male Japanese quail in an enriched environment and/or the exposure to a repeated (chronic) stressor on their immune response. Previous studies have shown that an EE procedure may alter components of immunity in rats (Marashi et al. 2002). In a turkey population presenting a fast T-maze solving response, EE may be considered potentially stressful with detrimental results on immunity (Huff et al. 2003). Our findings support the contention that submitting birds to EE can effectively improve the immune response and may even reduce the detrimental effects of a chronic stressor exposure. This modulation also shows some features that depend on the component of immunity studied (see below).

It was established that the stressor used in this study significantly increases circulating corticosterone levels in Japanese quail, submitted to either an acute or repeated (chronic) exposure (Satterlee and Johnson 1988; Jones et al. 2000). GCs have, among others, two well-known effects: anti-inflammatory action and retardation of the migration and cellular recruitment mechanisms (Dhabhar and McEwen 1997; Mumma et al. 2006). The analysis of the PHA-P response showed that the mechanisms involved in the inflammatory response were affected by both the EE and the stress procedure. Quail exposed to the chronic stressor and reared in a non-enriched environment showed the lower response to the PHA-P injection. Interestingly, the birds reared in an enriched environment showed higher responses to the PHA-P injection regardless of having been exposed or not to the chronic stressor. The results support the hypothesis that EE can be considered beneficial for the immunity of stressed animals.

Dhabhar et al. (1995, 1996) indicated that the induction of the adrenocorticotropic hormone (ACTH)/GC axis generally reduces the lymphocyte number (predominantly B and natural killer cells). The quail reared in the enriched environment that were not-stressed showed little variation in their percentage of lymphocytes one week after finishing the chronic stressor application. By contrast, stressed quail reared in a non-enriched environment showed the greatest fall in this population. Our findings suggest that the EE exposure may have provided a compensatory effect on the immunosuppressant action of stress.

The H/L ratio is a haematological variable that provides information about chronic stress status in poultry (Gross and Siegel 1983; Siegel 1995). Induction of GC secretion not only reduces the lymphocytes number but also increases the number of neutrophils (equivalent to heterophils in birds) (Dhabhar et al. 1995, 1996). In the present study, stressed quail reared in a non-enriched environment were the group with the higher H/L ratio, with not-stressed birds in an enriched environment showing the lower values of this parameter. This may be due to a higher and chronic activation of the HPA axis. This would be consistent with the results of the inflammatory response presented. Regardless of the mechanism underlying this phenomenon, it is clear that the immunosuppressive effects of the stressor are reduced by the enriching of the rearing environment. The finding that the males reared in the non-enriched environment that were submitted to the chronic stress treatment showed even a higher H/L ratio than their female counterparts is consistent with the reported differences in stress susceptibility between male and female poultry (Jones 1987; Marin et al. 2002).

The measurement of SRBC antibody titres provides information about the humoral immune response. Overall, this parameter was lower in birds submitted to the chronic stressor. Also, males showed lower values than females. These results confirm the reported immunosuppressive effect of stress (Dohms and Metz 1991; Fair et al. 1999; Mumma et al. 2006) and also agree with the contention that males are more susceptible to stressors than females (Huff et al. 1999; Wideman and French 2000; Marin et al. 2002). Our results also showed that the EE for hatchlings increased the bird's antibody production, indicating that this procedure helps to improve the humoral response of the confined birds.

The mechanism underlying the improvement in the immune responses of the stressed birds that were reared in an enriched environment may be related to an improved ability (probably learned) to deal with stressful situations (i.e. new enriching objects every week). This statement is supported by the findings for animals showing lowered fear and/or stress responses in an enriched environment (Nicol 1992; Reed et al. 1993; Grigor et al. 1995; Bizeray et al. 2002) than in a non-enriched rearing environment. Thus, it is conceivable that the more experienced animals (reared in the enriched environment) are better suited to cope or to habituate to the stressful components of the subsequent daily exposures to the restraint stressor (that includes not only a partial immobilisation but also the capturing by the experimenter and temporary housing in a new environment). Indeed, stressed birds in the enriched environment showed a reduced H/L ratio compared to the stressed birds in the non-enriched environment. During the study, some dust and waste could accumulate on the enrichment elements as a consequence of their use. Thus, we cannot rule out that birds in the enriched environment could have been exposed to a different microbiological environment in comparison to the control birds, which may consequently have altered their immune system. However, regardless of the mechanism involved in the resulting immunological changes, it is clear that birds reared in the enriched environment showed significantly different immune response compared to their control counterparts.

Norris and Evans (2000) pointed out that for immune function to be viewed in a life history context, investment in immune activity must trade-off with life history components because maintenance of the immune system requires resources (Klasing 1998). Thus, it is conceivable that the observed immune alteration of the EE may be also based on the potential optimisation of the energy assigned to different components during the life history of the animals.

Immune responses can be characterised mainly by a cellular and an inflammatory component (pro-inflammatory responses) or by a humoral and poorly cellular component (anti-inflammatory responses). Given that the alteration of immunity in this study changed the cellular and humoral responses, we may conclude that the effects of enriching the environment are not selective towards one component of immunity.

Given that birds can show a non-specific stress response to a wide variety of stressors (e.g. cold, crating, feed and water deprivation, manual restraint, water immersion and social tension) (Satterlee and Johnson 1988; Jones et al. 1994; Jones 1996; Marin and Arce 1996; Marin and Jones 1999), the improved immune response to the repeated restraint stressor shown herein might also be expected under other stressful situations during growth and, therefore, have important implications for their welfare.

Acknowledgements

This research was supported by grants from Fondo para Investigación Científica y Tecnológica (FONCYT) (Project 34157), Secretaría de Ciencia y Técnica, Universidad Nacional de Córdoba (SECyT, UNC) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Argentina. FNN holds a research fellowship from the latter institution. RHM is a Career Member of CONICET, Argentina. The authors wish to thank Dr R.B. Jones for his comments during the planning of this study and Dario C. Arbelo for his technical assistance.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.