Does Environmental Enrichment Reduce Stress? An Integrated Measure of Corticosterone from Feathers Provides a Novel Perspective

Abstract

Enrichment is widely used as tool for managing fearfulness, undesirable behaviors, and stress in captive animals, and for studying exploration and personality. Inconsistencies in previous studies of physiological and behavioral responses to enrichment led us to hypothesize that enrichment and its removal are stressful environmental changes to which the hormone corticosterone and fearfulness, activity, and exploration behaviors ought to be sensitive. We conducted two experiments with a captive population of wild-caught Clark's nutcrackers (Nucifraga columbiana) to assess responses to short- (10-d) and long-term (3-mo) enrichment, their removal, and the influence of novelty, within the same animal. Variation in an integrated measure of corticosterone from feathers, combined with video recordings of behaviors, suggests that how individuals perceive enrichment and its removal depends on the duration of exposure. Short- and long-term enrichment elicited different physiological responses, with the former acting as a stressor and birds exhibiting acclimation to the latter. Non-novel enrichment evoked the strongest corticosterone responses of all the treatments, suggesting that the second exposure to the same objects acted as a physiological cue, and that acclimation was overridden by negative past experience. Birds showed weak behavioral responses that were not related to corticosterone. By demonstrating that an integrated measure of glucocorticoid physiology varies significantly with changes to enrichment in the absence of agonistic interactions, our study sheds light on potential mechanisms driving physiological and behavioral responses to environmental change.

About the Authors:

Graham D. Fairhurst

* E-mail: [email protected]

Affiliation: Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Matthew D. Frey

Affiliation: Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

James F. Reichert

Affiliation: Department of Psychology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Izabela Szelest

Affiliation: Department of Psychology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Debbie M. Kelly

Current address: Department of Psychology, University of Manitoba, Winnipeg, Manitoba, Canada

Affiliation: Department of Psychology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Gary R. Bortolotti

Affiliation: Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Introduction

Enrichment is the modification of a captive animal's environment with the goals of increasing environmental complexity [1] and improving biological functioning [2]. The majority of enrichment research has focused on combating fearfulness and harmful abnormal and stereotypic behaviors arising in captive production (i.e., farm), laboratory, and companion animals ([3]; see also [4]) because animal welfare is both economically and ethically important [1], [5]–[7]. Behavioral ecologists have indirectly studied enrichment in different contexts, and more frequently use non-domesticated animals as models. Investigations of, for example, exploration behavior [8], [9] information acquisition [10], dominance [11], and personality [12], [13] can involve de facto enrichment and provide data comparable to studies of other captive animals. Enrichment has numerous behavioral effects (for reviews see [6], [14], [15]), but has been shown to reduce fear responses [1], [13], [16], increase movement and activity [17]–[19], and induce changes in exploration behavior [8], [10], [11], [20], [21].

Studies assessing physiological responses to enrichment frequently measure levels of glucocorticoid (GC) hormones like corticosterone (CORT) or cortisol because they vary with exposure to environmental perturbations [22]–[25]. Prolonged activation of the HPA axis and sustained elevated levels of GCs have detrimental effects on health and reproduction [26], [27] and GC levels can correlate with fitness components [25], [28]–[30]. However, while some studies have reported that enrichment can lower GC levels in the blood [31], [32], others have reported no effect [19], [20], [33], [34], or even increases [21], [35], [36]. Such studies have been inconsistent in their procedures for measuring GC physiology and there is still a lack of consensus and understanding of the effects of enrichment on GCs [37], [38]. Furthermore, studies addressing simultaneous behavioral and physiological responses to enrichment have found that animals may react behaviorally in measurable ways yet exhibit no measurable GC response. For example, garden warblers (Sylvia borin) exposed to a toy exhibited active exploration [20] and great tits (Parus major) exposed to a box showed increased activity [19], yet neither showed a change in circulating CORT levels. Likewise, steers (Bos primigenius) given a drum can [39] and calves given toys [40] significantly increased active behaviors, yet showed no change in cortisol levels. How abnormal behaviors relate to stressors is unclear. For example, Dybkjaer [41] reported that belly-nosing behavior in pigs is an indicator of stressful rearing conditions, but when Gardner and colleagues [42] manipulated pig density as a means of lowering stress, they did not detect a change in that behavior. Furthermore, Le Maho and colleagues [43] found that although domestic geese appeared calm and exhibited no behavioral signs of stress during a routine procedure to which they had been adjusted, several-fold increases in CORT levels were detected following the procedure. This collective evidence suggests that behavior and stress physiology are context dependent and may operate independently of each other.

From the perspective of the animal, enrichment constitutes an unpredictable environmental change. Thus, an animal's response to enrichment may not be caused by the enrichment objects per se, but rather by the associated change. Although some behavioral responses to enrichment, such as exploration and play, can be attributed to the objects themselves, physiological responses may more likely be caused by the unpredictable nature of the change in environment. Vertebrates are well known to respond physiologically to such change by releasing GCs as part of the “stress response” [23], [24], [28].

While the vast majority of research has addressed the effects of enrichment, relatively little is known about how animals respond to a change from an enriched to a more impoverished environment. This is an important knowledge gap because it is an animal's response to removal of enrichment objects that would shed light on the importance of associated environmental change. A barren environment can affect behavior [44] and physiology [45], and sparse evidence suggests that removal of enrichment can have negative physiological [31], [39] and psychological [46] effects. However, studies experimentally testing the relationship between behavioral and GC responses to enrichment and its removal within the same animal are rare, especially for non-domesticated birds [3].

It is important to determine how well a change in behavior correlates with measures of physiological stress [47], especially if behavioral responses to enrichment are to be integrated effectively into measures of emotional state and, subsequently, well-being and quality of life of captive animals (see [7], [46], [48]). Enrichment is used as a stress-reduction technique ([49] and references therein; [50]) and stress is believed to mediate the relationship between problem behaviors and well-being [51]–[53]. However, inconsistencies in the literature make it necessary to clarify how and when the HPA axis responds to enrichment. Better understanding the relationships between enrichment, behavior, and stress will help refine techniques for assessing the outcomes of enrichment procedures [53], which will benefit a broad spectrum of research.

Although the lack of consensus regarding enrichment and GC levels may be partly context dependent (e.g., different enrichment protocols; [37]), all previous studies measuring GC levels have utilized blood or, less frequently, fecal [54]–[56] or salivary [57] sampling. These techniques have known limitations and biases ([58], [59]; and see [60]) and provide measures of GC physiology over short time periods (i.e., minutes or hours). Thus, our understanding of how enrichment affects stress physiology would benefit from a long-term perspective on GC secretion. Here we use a technique to track stress physiology of birds through changes in CORT found in feathers. Feather CORT integrates the intensity and frequency of the physiological response because values incorporate the amplitude and duration of all CORT secretion, including response to stressors, during the period of feather growth [60], [61]. Therefore, feather CORT does not rely solely on baseline or stress-induced values, but instead integrates the two into a biologically-relevant measure of total CORT secretion (sensu [24]).

We conducted two experiments to help clarify the relationships between enrichment and its removal, GC physiology, and behavior. Although domestic chickens (Gallus gallus) are the typical avian model for enrichment research, we wanted the results of our study to also be applicable to behavioral ecologists, so we used a captive population of wild-caught Clark's nutcrackers (Nucifraga columbiana). During experiment 1 we exposed nutcrackers to short-term (10-d) enrichment to test the hypothesis that enrichment attenuates stress physiology. If this were true, nutcracker feather CORT should be significantly reduced following short-term enrichment. Alternatively, if short-term enrichment does not affect nutcracker stress physiology, or if nutcrackers perceive enrichment as a stressor, we predict no effect or an increase in feather CORT, respectively. In experiment 2 we exposed nutcrackers to enrichment objects continuously for three months, then removed the objects. This design allowed us to replicate experiment 1, using both short- and long-term enrichment, and also test the hypothesis that the change of environment associated with enrichment and its removal is perceived as a stressor. If the environmental change were a stressor, feather CORT should increase immediately following both addition and removal of enrichment objects. Additionally, we were interested in how well behavioral measures can be used as a proxy for physiological responses to enrichment, so in experiment 2 we examined the relationships between feather CORT and fearfulness, activity, and exploration behaviors.

Materials and Methods

Ethics Statement

All aspects of this research complied fully with the rules and regulations governing the use and care of animals in research at the University of Saskatchewan, and were conducted under approval #20040088 from the Animal Research Ethics Board, University of Saskatchewan.

Housing and daily routine

During 2000–2002, 41 wild nutcrackers were caught in Colorado, USA, so all birds had been in captivity for at least 4 years prior to our first experiment in 2007. All birds were housed individually at the Western College of Veterinary Medicine, University of Saskatchewan, Canada, in a single windowless colony room in standard metal pet bird cages constructed from thin (∼3 mm) metal bars with a removable metal floor tray (1 m×0.75 m×1 m). All cages had a wooden perch and separate wood and metal swing. All birds were checked regularly by veterinarians and were deemed in good health before we began our experiments. Prior to experiments, all birds had experienced the same daily cleaning and feeding routine that we continued for the duration of the experiments: morning weighing, feeding, and water changing; afternoon water changing; weekly cage changes; and additional twice weekly cage bottom cleaning. Nutcrackers were fed a 95% ad libitum diet of turkey starter, parrot pellets, sunflower seeds, peanuts, pine nuts, mealworms, and vitamin supplement, as well as water and grit ad libitum. Food and grit was provided in plastic food cups snapped into the cage walls, and water was provided in circular plastic bowls. Cages were arranged on moveable racks that could accommodate three cages above and three cages below. Light was maintained at 12 h light:12 h dark. None of the birds in our experiments had previously received any form of cage enrichment other than their perch and swing.

Experiment 1

Beginning in October 2007, 16 randomly selected nutcrackers (8 male, 8 female) were moved from the colony room into a similar windowless experimental room and assigned randomly to one of two walls that faced each other. After 2 weeks, a plastic curtain was installed that divided the experimental room in half: eight birds on one side of the divider were visually isolated from eight birds on the other side of the divider. Birds were allowed to adjust to the divider for an additional 2 weeks (Fig. 1).

[Figure omitted. See PDF.]

Figure 1. Experimental timelines with diagrams illustrating how feather sections reflect periods of the experiments.

Feathers from experiment 1 were cut into three sections corresponding to periods prior to pre-enrichment (A), short-term enrichment (B), and removal of enrichment (C). The first feather from experiment 2 was cut into two sections corresponding to pre-enrichment (A) and short-term enrichment (B), and the second feather from experiment 2 was cut into three sections corresponding to long-term enrichment (C), removal of enrichment (D), and non-novel enrichment (E). See text for descriptions of time periods. Note: illustrations not to scale.

https://doi.org/10.1371/journal.pone.0017663.g001

We then pulled the right secondary feather #1 (adjacent to primary #1) from each nutcracker to induce new feather growth. All feathers pulled were fully grown and dead. Subsequent feather growth was measured every 5 days for the remainder of the experiment. After the first 10 days of feather growth enrichment objects to which the birds were naïve were installed in the cages of birds on one side of the divider only, thus separating birds into an enriched experimental group (n = 8) and a non-enriched control group (n = 8). Enrichment objects comprised three plastic bird toys (balls: 195 mm×45 mm, rings: 190 mm×50 mm, and a mirror lantern: 140 mm×35 mm) and one wooden chew toy (250 mm×55 mm) that all hung inside the cages, plus an artificial pine garland (300 mm×300 mm) installed on the outside of the upper left back corner of the cage. All objects were added at the same time. After 10 days of enrichment, objects were removed and the regrowing feathers were allowed to grow for an additional 10 days, at which point they were pulled out (Fig. 1).

We chose to group all experimental birds together on one side of the room rather than randomly assign treatments to birds. Random assignment would have resulted in experimental birds being neighbors with non-experimental control birds and to reduce obvious bias we would have been forced to visually isolate neighbors. We chose to not do this for two reasons. First, it would have been difficult to keep control birds from seeing enrichment objects during cage changes. Second, and more importantly, little is known about nutcracker sociality, but it is suggested that they are moderately social birds [62]. Birds in our experiments had always been maintained in a colony setting, so rather than confound our data by subjecting birds to a potentially stressful social situation, we chose to group birds by treatment, thus allowing individuals within a treatment group to see each other.

Experiment 2

Beginning in October 2008, 23 nutcrackers (12 m, 11 f) that had not previously received cage enrichment were selected randomly, moved into the experimental room, and assigned randomly to one of two walls that faced each other. The birds' daily routine was maintained throughout the experiment and was the same as that described above. We repeated the adjustment procedure as in experiment 1, except that the divider separated nutcrackers into groups of 11 and 12. We then pulled a feather from each bird to induce new feather growth (Fig. 1). All feathers pulled were fully grown and dead. We pulled the right secondary feather #1 from 17 of the 23 birds, but six birds were missing or growing that feather so we selected the next morphologically similar feather in sequence: right secondary feather #2 (4 birds) or #3 (1 bird), or right primary feather #1 (1 bird).

Regrowing feathers were measured every 5 days until feathers were approximately half-grown (mean ± SD  = 61.74±8.95 mm). Then, as in experiment 1, enrichment objects to which the birds were naïve were installed in the cages of birds on one side of the divider only, forming an enriched group (n = 11) and a non-enriched control group (n = 12). All objects were added at the same time. The induced feathers were allowed to complete their growth and were subsequently pulled 31 days later to ensure that feathers were fully grown (Fig. 1).

Once birds had been exposed to enrichment objects for 67 days, we pulled a second feather from each individual to induce new feather growth (Fig. 1). All feathers pulled were fully grown and dead. We pulled left secondary #1 from 17 birds, but three birds were missing or growing that feather. For those individuals, we selected the next morphologically similar feather in sequence: left secondary feather #4 (1 bird), or right secondary feather #1 (2 birds) or #2 (1 bird). Feathers were allowed to grow for 25 days, at which point the enrichment objects were removed from cages. After 11 days the enrichment objects were re-installed into the same cages as before and feathers were allowed to grow for a final 10 days before being collected for analysis. We were only able to sample second feathers from 21 birds because two birds died and, in a third, an induced feather did not regrow.

Feather sections

We cut all feathers using growth measurements as guides such that cut sections corresponded to the different time periods of the experiment (Fig. 1). Feathers from experiment 1 were cut into three sections: the distal section was grown prior to enrichment (Fig. 1, top, A: “pre-enrichment”), the middle section was grown while enrichment objects were present in the cages (Fig. 1, top, B: “short-term enrichment”), and the proximal section was grown after objects were removed (Fig. 1, top, C: “removal of enrichment”). The first feathers from experiment 2 were cut into two sections: the distal section was grown prior to enrichment (Fig. 1, bottom, A: “pre-enrichment”) and the proximal section was grown while enrichment objects were present in the cages (Fig. 1, bottom, B: “short-term enrichment”). The second feathers from experiment 2 were cut into three sections: the distal section was grown while enrichment objects were still present in the cages (Fig. 1, bottom, C: “long-term enrichment”), the middle section was grown after objects were removed (Fig. 1, bottom, D: “removal of enrichment”), and the proximal section was grown when objects were re-installed (Fig. 1, bottom, E: “non-novel enrichment”).

Behavior

We recorded nutcracker behavior during experiment 2 using a small digital video camera that was able to record all birds on one side of the room simultaneously (Table 1). The camcorder was mounted on a small tripod that stood on a utility cart that was used for daily feeding and was therefore familiar to the birds. We used the digital timestamp on the recordings for calculating elapsed time.

[Figure omitted. See PDF.]

Table 1. Observation schedule for nutcracker behaviors during Experiment 2.

https://doi.org/10.1371/journal.pone.0017663.t001

We measured latency to feed (LTF) for each nutcracker as the time taken by a bird to approach its food dish after being reintroduced into the cage. Previous studies have used latency to feed as a measure of fearfulness [13], [63]–[65]. Our nutcrackers normally feed readily in the presence of caretakers, and even jump on food cups and begin to feed before the cups are fully snapped into place on the cage. Thus, we knew a priori that the birds should not be afraid of caretakers or food during feeding. We made 5-minute video recordings of both experimental and control nutcrackers during normal feeding time (∼11:00 hrs) on three separate occasions as they were released into fresh cages that already had food in cups. The first recording was made immediately following the installation of enrichment objects into experimental cages. This recording captured the initial behavioral responses of all birds at the time when experimental individuals were first exposed to enrichment objects. The second and third recordings were made when birds had received 2 months of continuous exposure to objects (“long-term enrichment”), and immediately following removal of enrichment objects 26 days later (“removal of enrichment”).

Birds are known to express behaviors ranging from freezing to active investigation when exposed to novelty [64]. We therefore measured two behaviors likely to vary with exposure to novel objects: activity level (AL), quantified by counts of all hops around the cage and positional changes (i.e., turning around 180° but remaining in the same place when perched); and exploration (EXP), quantified by counting the number of pecks at any object within the cage or at any part of the cage itself. Recordings of AL and EXP lasted 5 minutes, as in previous work (e.g., [31]), and were made several hours after afternoon water changes (at ∼1700 hrs) and in the absence of caretakers to ensure that these behaviors were not affected by human presence. Observations of AL and EXP were made during four periods of our experiment: 2 weeks prior to enrichment (“pre-enrichment”), immediately following the installation of enrichment objects (“short-term enrichment”), after birds had 2 months of continuous exposure to objects (“long-term enrichment”), and immediately following removal of enrichment objects 26 days later (“removal of enrichment”).

Feather CORT assays

We extracted CORT from feathers in three separate extractions using a methanol-based technique following [60]. We measured the lengths of all feathers, and then cut, removed, and discarded the calamus. The remaining feather sample was cut with scissors into very small pieces (<5 mm²) and 10 mL of methanol (HPLC grade, VWR International, Mississauga, Ontario, Canada) was added. We placed samples in a sonicating water bath at room temperature for 30 min, then incubated them at 50°C overnight in a water bath. We separated the methanol from the feather material by vacuum filtration, and the methanol extract was placed in a 50°C water bath and allowed to evaporate in a fume hood. Extracts were later reconstituted in a small volume of phosphate buffer system (PBS; 0.05 M, pH 7.6) and frozen at −20°C until analyzed by radioimmunoassay (RIA). We assessed the efficiency of each of the three methanol extractions by including feather samples spiked with a small amount (approximately 5000 CPM) of ³H-corticosterone in each extraction (see Appendix S1 in [60] for more details). On average, greater than 95% of the radioactivity was recoverable in the reconstituted samples.

Feather CORT levels were determined by RIA as in previous studies [60], [61], [66]–[68]. Measurements were performed on reconstituted methanol extracts and were duplicated. Samples were measured in four assays with an intra-assay coefficient of variation of 7.4%, an inter-assay coefficient of variation of 14.1%, and mean (± SD) limit of detection (ED80) of 12.9±2.2 pg CORT/assay tube. Data values are expressed as pg CORT per mm of feather, which gives a valid estimate of CORT per unit time of feather growth (see [60], [61], [69] for validation). CORT assays were performed at the University of Saskatchewan, Canada.

Statistical analyses

To determine if feather sections used for CORT analyses differed in length between enriched and non-enriched controls we combined all data from both experiments and used a mixed model (PROC MIXED; SAS v. 9.1). We used length of feather section as the response variable, treatment as the explanatory variable, and experiment (i.e., 1 or 2) as a random factor. We included a repeated statement to account for multiple measurements taken from the same individual over time [70].

We used mixed modeling (PROC MIXED) to compare CORT values from feather sections grown during the periods of our experiments. For experiment 1 we compared pre-, short-term, and removal of enrichment. For experiment 2 we compared pre-, short-term, and long-term enrichment; removal of enrichment; and non-novel enrichment. We modeled feather CORT as the response variable, time period and treatment (i.e., experimental or control) as fixed factors, and included a time period × treatment interaction term. We used a repeated statement to account for multiple measurements taken from the same individual over time. Two feathers were collected from each bird in experiment 2, so our model included a random factor to account for possible variation between feathers.

To determine the influence of enrichment on behavior, and to address the relationship between behavior and CORT, we used mixed models (PROC MIXED and PROC GLIMMIX; SAS v. 9.1). We modeled behaviors individually, using behavioral data (LTF or AL) as the response variable, feather CORT as a covariate, and time period, treatment (i.e., control or experimental), and a time period × treatment interaction term as fixed factors. LTF data were fitted to models using a normal error distribution and an identity link function in PROC MIXED. AL data were counts and were therefore fitted to models using a negative binomial error distribution and a log link function in PROC GLIMMIX. We used random statements to account for variation between feathers, and used repeated (PROC MIXED) or random (PROC GLIMMIX) statements to account for the multiple measurements taken from the same individual over time.

EXP data were counts, but were zero-inflated and we were not able to get models to converge using PROC MIXED or PROC GLIMMIX. Instead, we used a zero-inflated Poisson model (PROC GENMOD) to address the relationship between EXP and CORT. We used counts of pecks as the response variable, CORT as a covariate, and time period and treatment (i.e., enriched or control) as explanatory variables. Although this approach had the advantage of accounting for the high incidences of zeros in our data, it did not allow us to include an interaction term or random or repeated statements. However, considering how few counts of pecks were actually recorded throughout the experiment, we do not believe the absence of interaction and random terms affected our results significantly.

Results

Lengths of feather sections used for CORT analyses did not differ between control and experimental groups in any time period of either experiment (F_1,142 = 0.09, p = 0.77).

Experiment 1

Overall, mean CORT values differed significantly between time periods (F_2,22 = 13.76, p<0.001; Fig. 2), but not between treatment and control birds (F_1,12 = 3.96, p = 0.07), and the time period × treatment interaction was not significant in our model (F_2,22 = 2.76, p = 0.09). However, post hoc comparisons revealed that CORT levels increased significantly from pre-enrichment to short-term enrichment in experimental birds (t_1,22 = −4.66, p<0.001), but not in controls (t_1,22 = −1.57, p = 0.13). CORT values from the enrichment removal period did not differ significantly from pre- enrichment values for either group (experimental: t_1,22 = −1.47, p = 0.16; control: t_1,22 = −1.92, p = 0.07).

[Figure omitted. See PDF.]

Figure 2. Mean (± SE) nutcracker feather CORT values (pg/mm) from experiment 1.

Filled circles: experimental birds; open circles: non-enriched controls. See text for explanation of time periods.

https://doi.org/10.1371/journal.pone.0017663.g002

Experiment 2

Similar to experiment 1, mean CORT values differed significantly between time periods (F_4,93 = 16.48; p<0.0001; Fig. 3), but not between treatment and control (F_1,93 = 0.07; p = 0.79), and there was no significant interaction between treatment and time period (F_4,93 = 1.92, p = 0.20). CORT increased significantly between pre-enrichment and short-term enrichment periods for both experimental (t_1,93 = −3.82, p = 0.0002) and control birds (t_1,93 = −4.71, p<0.0001). Long-term enrichment CORT values in controls were similar to pre-enrichment values (t_1,93 = 0.03, p = 0.98), but in experimental birds long-term enrichment CORT values were significantly lower than pre-enrichment values (t_1,93 = 3.16, p = 0.002). CORT levels from feather sections grown after removal of enrichment objects increased significantly from long-term enrichment levels in experimental birds (t_1,93 = −2.63, p<0.01) but not in controls (t_1,93 = −1.86, p = 0.07). CORT levels increased significantly following non-novel enrichment for both control and experimental birds and were significantly higher than both pre-enrichment (t_1,93 = −5.15, p<0.0001) and enrichment levels (t_1,93 = −4.49, p<0.0001).

[Figure omitted. See PDF.]

Figure 3. Mean (± SE) nutcracker feather CORT values (pg/mm) from experiment 2.

Filled circles: experimental birds; open circles: non-enriched controls. (A) shows the first four time periods of the experiment; (B) shows the fifth time period (non-novel enrichment) and includes short-term (novel) enrichment for comparison. Note different scales on y-axes. See text for explanation of time periods.

https://doi.org/10.1371/journal.pone.0017663.g003

Behavior

There was a significant interaction between the effects of treatment and time period on LTF (F_2,52 = 4.30; p<0.02). During short-term enrichment, LTF was significantly greater in experimental birds than in controls (t_1,52 = −2.58; p = 0.01; Fig. 4), but this effect disappeared during long-term enrichment when LTF in experimental birds was reduced (t_1,52 = 3.89; p = 0.0003) to levels seen in controls. CORT was not significantly related to LTF in either group (F_1,52 = 1.33; p = 0.25).

[Figure omitted. See PDF.]

Figure 4. Mean (± SE) latency (sec) to feed (LTF) of nutcrackers during experiment 2.

Filled circles: experimental birds; open circles: non-enriched controls. See text for explanation of time periods.

https://doi.org/10.1371/journal.pone.0017663.g004

There was no significant interaction between treatment and time period (F_1,73 = 0.11; p = 0.96) in our model of AL, so we interpreted the main effects directly. AL did not differ between time periods (F_1,73 = 0.88; p = 0.45; Fig. 5) or between treatments (F_1,73 = 0.72; p = 0.40), and was not significantly related to CORT (F_1,73 = 0.50; p = 0.48).

[Figure omitted. See PDF.]

Figure 5. Mean (± SE) counts of nutcracker hops and positional changes (activity level, AL) during experiment 2.

Filled circles: experimental birds; open circles: non-enriched controls. See text for explanation of time periods.

https://doi.org/10.1371/journal.pone.0017663.g005

Our model of EXP revealed that, overall, experimental birds were more likely to show pecking behavior than control birds (Wald chi-square = 5.5; p<0.01; Fig. 6) and that pecking was significantly more likely during the short-term enrichment than in other periods (Wald chi-square = 13.33; p<0.001). EXP was not related to CORT (Wald chi-square = 0.26; p = 0.61) for either group.

[Figure omitted. See PDF.]

Figure 6. Mean (± SE) counts of nutcracker pecks (exploration, EXP) during experiment 2.

Filled circles: experimental birds; open circles: non-enriched controls. See text for explanation of time periods.

https://doi.org/10.1371/journal.pone.0017663.g006

Discussion

Did enrichment attenuate stress physiology in captive nutcrackers? The integrated measure of CORT increased significantly from pre-enrichment levels following short-term exposure to enrichment objects in two separate experiments, indicating that stress physiology was likely enhanced, not reduced, following the manipulation. However, individuals exposed to long-term enrichment (i.e., the final 25 days of a 92-d enrichment period) expressed CORT levels that were significantly lower than pre-enrichment values, suggesting a physiological benefit. These results led us to hypothesize that nutcrackers perceived initial enrichment as a stressful change of environment, and we predicted that the change of environment associated with removal of enrichment would likewise produce elevated CORT levels. Contrary to our predictions, CORT levels in experiment 1 decreased following removal of enrichment and returned to pre-enrichment levels. However, in experiment 2, CORT levels increased significantly to levels seen following short-term enrichment, in accordance with our predictions.

How do we explain these seemingly opposite responses to removal of enrichment in our two experiments? The main difference between experiments 1 and 2 was length of exposure to enrichment objects prior to removal. The longer exposure likely allowed birds in experiment 2 to habituate to the objects such that the objects became part of their normal environment, similar to acclimation seen in previous studies ([71], [72] and see [24]). The fact that CORT levels during long-term enrichment (the final 25 days of a 92-d enrichment) were not significantly higher than those prior to enrichment provide evidence that birds had indeed adjusted to the presence of objects. Thus, removal of the objects in experiment 2 constituted a change of environment to which birds responded by elevating CORT. This was not the case in experiment 1 where the shorter 10-day exposure period did not allow for the same length of adjustment. Objects were therefore not recognized as part of the environment and instead were perceived as a stressor. Thus, the removal of the objects was seen as a removal of a stressor, to which birds responded by decreasing CORT secretion, albeit non-significantly, to pre-enrichment levels.

Were the birds physiologically stressed by short-term enrichment? Although none of the birds showed any sign of illness or discomfort during either of our experiments, two birds died during, and one shortly after, experiment 2. Interestingly, all three fatalities were in the experimental group, and those individuals had three of the four highest CORT values for the short-term enrichment period. The provisioning of enrichment must be done carefully and in the context of the species and environment. It is possible that the installation of four objects inside the cage was perceived as an over-enrichment by nutcrackers, and the birds reacted with sustained CORT responses. Sustained elevated CORT secretion can suppress immune response [23], [73], so elevated CORT levels may have been a contributing factor in the deaths. However, CORT values during short-term enrichment, when responses would be expected to be most robust, were not the highest seen in the experiment, so we doubt that birds were over-enriched. Thus, we cannot conclude that enrichment per se caused stress sufficient enough to result in these deaths.

Behaviorally, only LTF and EXP behavior were influenced by the addition of enrichment objects, but these responses were not seen two months later, nor were they seen upon removal of the objects. By contrast, feather CORT increased significantly in periods following both the addition and removal of enrichment objects. Importantly, a lack of relationship between feather CORT levels and concurrently measured behaviors support the idea that stress-related behavior and GC physiology may act independently of each other within an individual, and highlights a physiological response that was not detectable through the behaviors we measured. Despite evoking a physiological response to enrichment, is possible that the objects were not engaging enough to elicit anything other than transient behavioral responses. This is in contrast to previous studies that found behavioural, but not physiological, responses to enrichment [19], [20], [39], [40], [43]. These results suggest that both context and type of enrichment are important determinants of responses to enrichment.

Control birds showed similar feather CORT levels to experimental birds in all but one period of experiment 2, despite not being able to see enrichment objects. Although we made every effort to ensure that control birds were treated identically to experimental birds other than not receiving enrichment objects, we cannot rule out the possibility that some factor common to both groups influenced our results. Controls were only visually, and not aurally, isolated from their experimental counterparts. Nutcrackers are semi-social corvids that possess complex vocal communication systems [62]. Nutcrackers in both treatment groups were noisy throughout the experiment, so it is possible that vocalizations from experimental birds were changed or interrupted by the presence of enrichment objects, and the perception of these altered vocalizations promoted a release of CORT by control birds. Public information can affect CORT levels in birds [74], so it is possible that vocalizations work in a similar way, alerting individuals. This intriguing possibility requires further research.

Our results challenge the notion that all types of enrichment are immediately beneficial for captive animals and indicate that initial reactions to enrichment need not be positive or, in the case of some behaviors, may even be absent. Furthermore, a lack of relationship between feather CORT and the behaviors we measured suggests that relying solely upon behavioral measures of stress to assess captive animal well-being can be misleading, an assertion supported by other studies [19], [20], [39], [40], [43]. We are not suggesting that enrichment is harmful to captive animals. In fact, our results indicate that long-term enrichment generally reduced activity of the HPA axis and therefore may provide some physiological relief for captive animals experiencing otherwise stressful conditions. This result was not seen in control birds, so the effect was likely caused by exposure to enrichment objects. Furthermore, the fearfulness we observed in response to short-term enrichment was not detected following long-term enrichment, suggesting that this effect was temporary.

Likewise, although we did not detect a behavioral response to removal of enrichment objects, we are not suggesting that impoverishment has no behavioral consequences. On the contrary, numerous studies have documented deleterious behavioral effects of impoverished environments (e.g., [39], [40]). However, in our study we were only concerned with behavioral responses occurring very soon after experimental manipulations; as we did not detect any change in the behaviors we measured following removal of enrichment objects, we conclude that nutcrackers did not respond behaviorally to removal of enrichment.

Previous work has suggested that novelty is an important property of enrichment to which animals respond [13], [16], [65], but results have been mixed [75]. Our experimental design allowed us to assess the effect of novelty on nutcracker GC secretion because birds in experiment 2 were re-enriched with objects to which they had previously been exposed. Had novelty been a factor influencing GC responses in the short-term enrichment period in experiment 2, feather CORT values during the non-novel enrichment period (i.e., second exposure to the objects) should have been lower than or equal to levels following initial exposure. To the contrary, we found feather CORT values to be significantly higher when birds were no longer naïve to the enrichment objects. This indicates that nutcrackers mounted a stronger physiological response the second time they were exposed to the same objects, a result previously seen only in mammals [75]. The non-novel enrichment period in our study was only 10 days, compared to 25 days for the short-term enrichment period, so it is plausible that the shorter growth period resulted in a higher average CORT. However, the feather sections in experiment 1 were also grown over 10 days, yet their CORT values were half that of the non-novel enrichment values in experiment 2 and comparable to the values from longer feather sections grown during experiment 2. Thus, it appears that the length of feather growth period is not responsible for the high CORT values seen during the non-novel enrichment period. As an alternative explanation, the experimental birds may have developed a negative association between the enrichment objects and the activities of our experiment. Although all nutcrackers in our experiment had been handled daily for several years prior to our experiment, the extra handling required for measuring feather lengths and collecting feathers may have been perceived as stressors by the birds. The enrichment objects may thus have acted as a cue and upon seeing the enrichment objects a second time birds responded more strongly because they had a negative prior experience.

Conclusions

Enrichment can undoubtedly alter both physiological and behavioral functioning in animals [32], [35], [38], [45], [76], [77]. By demonstrating that an integrated measure of GC physiology varies significantly with changes to enrichment in the absence of agonistic interactions, our study sheds light on potential mechanisms driving those physiological and behavioral effects. Our work adds an avian perspective to studies addressing GC responses to novelty, and suggests that when a non-novel stimulus acts as a cue, acclimation may be overridden by negative past experience. Importantly, our findings suggest that how animals perceive enrichment and its removal depends on the duration of exposure: shorter-term enrichment may be experienced as a stressor, but longer-term enrichment allows for acclimation and therefore subsequent removal of enrichment constitutes a change to the environment. Future research should work to identify the factors that affect the rate at which individuals transition between these two psycho-physiological states. Studying such factors in captive and free-living animals will improve our understanding of how and why animals adapt to environmental change.

Acknowledgments

We thank S. Cabezas, M. Vögeli, G. Treen, and two anonymous referees for thoughtful comments on the manuscript; P. Mason, T. Friesen, and M. Weiss for helping take care of the nutcracker colony; and T. Marchant for the use of her lab and her valuable insights into physiology.

Author Contributions

Conceived and designed the experiments: GDF GRB. Performed the experiments: GDF MDF JFR IS. Analyzed the data: GDF GRB MDF. Contributed reagents/materials/analysis tools: GRB DMK. Wrote the paper: GDF GRB.

Citation: Fairhurst GD, Frey MD, Reichert JF, Szelest I, Kelly DM, Bortolotti GR (2011) Does Environmental Enrichment Reduce Stress? An Integrated Measure of Corticosterone from Feathers Provides a Novel Perspective. PLoS ONE 6(3): e17663. https://doi.org/10.1371/journal.pone.0017663

References

1. Jones RB (1996) Fear and adaptability in poultry: Insights, implications and imperatives. Worlds Poultry Science Journal 52: 131–174.RB Jones1996Fear and adaptability in poultry: Insights, implications and imperatives.Worlds Poultry Science Journal52131174

2. Newberry RC (1995) Environmental enrichment: increasing the biological relevance of captive environments. Appl Anim Behav Sci 44: 229–243.RC Newberry1995Environmental enrichment: increasing the biological relevance of captive environments.Appl Anim Behav Sci44229243

3. de Azevedo CS, Cipreste CF, Young RJ (2007) Environmental enrichment: A GAP analysis. Appl Anim Behav Sci 102: 329–343.CS de AzevedoCF CipresteRJ Young2007Environmental enrichment: A GAP analysis.Appl Anim Behav Sci102329343

4. Morgan KN, Tromborg CT (2007) Sources of stress in captivity. Appl Anim Behav Sci 102: 262–302.KN MorganCT Tromborg2007Sources of stress in captivity.Appl Anim Behav Sci102262302

5. Leone EH, Estevez I (2008) Economic and welfare benefits of environmental enrichment for broiler breeders. Poult Sci 87: 14–21.EH LeoneI. Estevez2008Economic and welfare benefits of environmental enrichment for broiler breeders.Poult Sci871421

6. Mason G, Clubb R, Latham N, Vickery S (2007) Why and how should we use environmental enrichment to tackle stereotypic behaviour? Appl Anim Behav Sci 102: 163–188.G. MasonR. ClubbN. LathamS. Vickery2007Why and how should we use environmental enrichment to tackle stereotypic behaviour?Appl Anim Behav Sci102163188

7. Mendl M, Burman OHP, Parker RMA, Paul ES (2009) Cognitive bias as an indicator of animal emotion and welfare: Emerging evidence and underlying mechanisms. Appl Anim Behav Sci 118: 161–181.M. MendlOHP BurmanRMA ParkerES Paul2009Cognitive bias as an indicator of animal emotion and welfare: Emerging evidence and underlying mechanisms.Appl Anim Behav Sci118161181

8. Mettke-Hofmann C (2007) Object exploration of garden and sardinian warblers peaks in spring. Ethology 113: 174–182.C. Mettke-Hofmann2007Object exploration of garden and sardinian warblers peaks in spring.Ethology113174182

9. Sabbatini G, Stammati M, Tavares MCH, Visalberghi E (2007) Response toward novel stimuli in a group of tufted capuchins (Cebus libidinosus) in Brasilia National Park, Brazil. Am J Primatol 69: 457–470.G. SabbatiniM. StammatiMCH TavaresE. Visalberghi2007Response toward novel stimuli in a group of tufted capuchins (Cebus libidinosus) in Brasilia National Park, Brazil.Am J Primatol69457470

10. Mettke-Hofmann C, Wink M, Winkler H, Leisler B (2005) Exploration of environmental changes relates to lifestyle. Behav Ecol 16: 247–254.C. Mettke-HofmannM. WinkH. WinklerB. Leisler2005Exploration of environmental changes relates to lifestyle.Behav Ecol16247254

11. Fox RA, Ladage LD, Roth TC, Pravosudov VV (2009) Behavioural profile predicts dominance status in mountain chickadees, Poecile gambeli. Anim Behav 77: 1441–1448.RA FoxLD LadageTC RothVV Pravosudov2009Behavioural profile predicts dominance status in mountain chickadees, Poecile gambeli.Anim Behav7714411448

12. McDougall PT, Reale D, Sol D, Reader SM (2006) Wildlife conservation and animal temperament: causes and consequences of evolutionary change for captive, reintroduced, and wild populations. Anim Conserv 9: 39–48.PT McDougallD. RealeD. SolSM Reader2006Wildlife conservation and animal temperament: causes and consequences of evolutionary change for captive, reintroduced, and wild populations.Anim Conserv93948

13. Fox RA, Millam JR (2007) Novelty and individual differences influence neophobia in orange-winged Amazon parrots (Amazona amazonica). Appl Anim Behav Sci 104: 107–115.RA FoxJR Millam2007Novelty and individual differences influence neophobia in orange-winged Amazon parrots (Amazona amazonica).Appl Anim Behav Sci104107115

14. Balcombe JP (2006) Laboratory environments and rodents' behavioural needs: a review. Laboratory Animals 40: 217–235.JP Balcombe2006Laboratory environments and rodents' behavioural needs: a review.Laboratory Animals40217235

15. Lutz CK, Novak MA (2005) Environmental enrichment for nonhuman primates: Theory and application. ILAR J 46: 178–191.CK LutzMA Novak2005Environmental enrichment for nonhuman primates: Theory and application.ILAR J46178191

16. Meehan CL, Mench JA (2002) Environmental enrichment affects the fear and exploratory responses to novelty of young Amazon parrots. Appl Anim Behav Sci 79: 75–88.CL MeehanJA Mench2002Environmental enrichment affects the fear and exploratory responses to novelty of young Amazon parrots.Appl Anim Behav Sci797588

17. deMonte M, LePape G (1997) Behavioural effects of cage enrichment in single-caged adult cats. Anim Welf 6: 53–66.M. deMonteG. LePape1997Behavioural effects of cage enrichment in single-caged adult cats.Anim Welf65366

18. Kells A, Dawkins MS, Borja MC (2001) The effect of a ‘freedom food’ enrichment on the behaviour of broilers on commercial farms. Anim Welf 10: 347–356.A. KellsMS DawkinsMC Borja2001The effect of a ‘freedom food’ enrichment on the behaviour of broilers on commercial farms.Anim Welf10347356

19. Cockrem JF, Silverin B (2002) Sight of a predator can stimulate a corticosterone response in the great tit (Parus major). Gen Comp Endocrinol 125: 248–255.JF CockremB. Silverin2002Sight of a predator can stimulate a corticosterone response in the great tit (Parus major).Gen Comp Endocrinol125248255

20. Mettke-Hofmann C, Rowe KC, Hayden TJ, Canoine V (2006) Effects of experience and object complexity on exploration in garden warblers (Sylvia borin). Journal of Zoology 268: 405–413.C. Mettke-HofmannKC RoweTJ HaydenV. Canoine2006Effects of experience and object complexity on exploration in garden warblers (Sylvia borin).Journal of Zoology268405413

21. Cummings D, Brown JL, Rodden MD, Songsasen N (2007) Behavioral and physiologic responses to environmental enrichment in the maned wolf (Chrysocyon brachyurus). Zoo Biol 26: 331–343.D. CummingsJL BrownMD RoddenN. Songsasen2007Behavioral and physiologic responses to environmental enrichment in the maned wolf (Chrysocyon brachyurus).Zoo Biol26331343

22. Wingfield JC, Ramenofsky M (1999) Hormones and the behavioral ecology of stress. In: Balm PHM, editor. Stress Physiology in Animals. Sheffield: Sheffield Academic Press. pp. 1–51.JC WingfieldM. Ramenofsky1999Hormones and the behavioral ecology of stress.PHM BalmStress Physiology in AnimalsSheffieldSheffield Academic Press151

23. Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21: 55–89.RM SapolskyLM RomeroAU Munck2000How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions.Endocr Rev215589

24. Romero LM (2004) Physiological stress in ecology: lessons from biomedical research. Trends Ecol Evol 19: 249–255.LM Romero2004Physiological stress in ecology: lessons from biomedical research.Trends Ecol Evol19249255

25. Bonier F, Martin PR, Moore IT, Wingfield JC (2009) Do baseline glucocorticoids predict fitness? Trends Ecol Evol 24: 634–642.F. BonierPR MartinIT MooreJC Wingfield2009Do baseline glucocorticoids predict fitness?Trends Ecol Evol24634642

26. Dobson H, Smith RF (1995) Stress and reproduction in farm animals. J Reprod Fertil 451–461.H. DobsonRF Smith1995Stress and reproduction in farm animals.J Reprod Fertil451461

27. Charmandari E (2005) Endocrinology of the stress response. Annu Rev Physiol 67: 259–284.E. Charmandari2005Endocrinology of the stress response.Annu Rev Physiol67259284

28. Wingfield JC, Kitaysky AS (2002) Endocrine responses to unpredictable environmental events: Stress or anti-stress hormones? Integr Comp Biol 42: 600–609.JC WingfieldAS Kitaysky2002Endocrine responses to unpredictable environmental events: Stress or anti-stress hormones?Integr Comp Biol42600609

29. Blas J, Bortolotti GR, Tella JL, Baos R, Marchant TA (2007) Stress response during development predicts fitness in a wild, long lived vertebrate. Proc Natl Acad Sci U S A 104: 8880–8884.J. BlasGR BortolottiJL TellaR. BaosTA Marchant2007Stress response during development predicts fitness in a wild, long lived vertebrate.Proc Natl Acad Sci U S A10488808884

30. Breuner CW, Patterson SH, Hahn TP (2008) In search of relationships between the acute adrenocortical response and fitness. Gen Comp Endocrinol 157: 288–295.CW BreunerSH PattersonTP Hahn2008In search of relationships between the acute adrenocortical response and fitness.Gen Comp Endocrinol157288295

31. Boinski S, Swing SP, Gross TS, Davis JK (1999) Environmental enrichment of brown capuchins (Cebus apella): Behavioral and plasma and fecal cortisol measures of effectiveness. Am J Primatol 48: 49–68.S. BoinskiSP SwingTS GrossJK Davis1999Environmental enrichment of brown capuchins (Cebus apella): Behavioral and plasma and fecal cortisol measures of effectiveness.Am J Primatol484968

32. Pohle K, Cheng HW (2009) Comparative effects of furnished and battery cages on egg production and physiological parameters in White Leghorn hens. Poult Sci 88: 2042–2051.K. PohleHW Cheng2009Comparative effects of furnished and battery cages on egg production and physiological parameters in White Leghorn hens.Poult Sci8820422051

33. Barnett JL, Tauson R, Downing JA, Janardhana V, Lowenthal JW, et al. (2009) The effects of a perch, dust bath, and nest box, either alone or in combination as used in furnished cages, on the welfare of laying hens. Poult Sci 88: 456–470.JL BarnettR. TausonJA DowningV. JanardhanaJW Lowenthal2009The effects of a perch, dust bath, and nest box, either alone or in combination as used in furnished cages, on the welfare of laying hens.Poult Sci88456470

34. van de Weerd HA, Day JEL (2009) A review of environmental enrichment for pigs housed in intensive housing systems. Appl Anim Behav Sci 116: 1–20.HA van de WeerdJEL Day2009A review of environmental enrichment for pigs housed in intensive housing systems.Appl Anim Behav Sci116120

35. Marashi V, Barnekow A, Ossendorf E, Sachser N (2003) Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice. Horm Behav 43: 281–292.V. MarashiA. BarnekowE. OssendorfN. Sachser2003Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice.Horm Behav43281292

36. Moncek F, Duncko R, Johansson BB, Jezova D (2004) Effect of environmental enrichment on stress related systems in rats. J Neuroendocrinol 16: 423–431.F. MoncekR. DunckoBB JohanssonD. Jezova2004Effect of environmental enrichment on stress related systems in rats.J Neuroendocrinol16423431

37. Fox C, Merali Z, Harrison C (2006) Therapeutic and protective effect of environmental enrichment against psychogenic and neurogenic stress. Behav Brain Res 175: 1–8.C. FoxZ. MeraliC. Harrison2006Therapeutic and protective effect of environmental enrichment against psychogenic and neurogenic stress.Behav Brain Res17518

38. Pena Y, Prunell M, Rotllant D, Armario A, Escorihuela RM (2009) Enduring effects of environmental enrichment from weaning to adulthood on pituitary-adrenal function, pre-pulse inhibition and learning in male and female rats. Psychoneuroendocrinology 34: 1390–1404.Y. PenaM. PrunellD. RotllantA. ArmarioRM Escorihuela2009Enduring effects of environmental enrichment from weaning to adulthood on pituitary-adrenal function, pre-pulse inhibition and learning in male and female rats.Psychoneuroendocrinology3413901404

39. Ishiwata T, Uetake K, Abe N, Eguchi Y, Tanaka T (2006) Effects of an environmental enrichment using a drum can on behavioral, physiological and productive characteristics in fattening beef cattle. Anim Sci J 77: 352–362.T. IshiwataK. UetakeN. AbeY. EguchiT. Tanaka2006Effects of an environmental enrichment using a drum can on behavioral, physiological and productive characteristics in fattening beef cattle.Anim Sci J77352362

40. Veissier I, Chazal P, Pradel P, LeNeindre P (1997) Providing social contacts and objects for nibbling moderates reactivity and oral behaviors in veal calves. J Anim Sci 75: 356–365.I. VeissierP. ChazalP. PradelP. LeNeindre1997Providing social contacts and objects for nibbling moderates reactivity and oral behaviors in veal calves.J Anim Sci75356365

41. Dybkjaer L (1992) The identification of behavioural indicators of stress in early weaned piglets. Appl Anim Behav Sci 35: 135–147.L. Dybkjaer1992The identification of behavioural indicators of stress in early weaned piglets.Appl Anim Behav Sci35135147

42. Gardner JM, Duncan IJH, Widowski TM (2001) Effects of social “stressors” on belly-nosing behaviour in early-weaned piglets: is belly-nosing an indicator of stress? Appl Anim Behav Sci 74: 135–152.JM GardnerIJH DuncanTM Widowski2001Effects of social “stressors” on belly-nosing behaviour in early-weaned piglets: is belly-nosing an indicator of stress?Appl Anim Behav Sci74135152

43. Le Maho Y, Karmann H, Briot D, Handrich Y, Robin JP, et al. (1992) Stress in birds due to routine handling and a technique to avoid it. Am J Physiol 263: R775–R781.Y. Le MahoH. KarmannD. BriotY. HandrichJP Robin1992Stress in birds due to routine handling and a technique to avoid it.Am J Physiol263R775R781

44. Bolhuis JE, Schouten WGP, Schrama JW, Wiegant VM (2005) Behavioural development of pigs with different coping characteristics in barren and substrate-enriched housing conditions. Appl Anim Behav Sci 93: 213–228.JE BolhuisWGP SchoutenJW SchramaVM Wiegant2005Behavioural development of pigs with different coping characteristics in barren and substrate-enriched housing conditions.Appl Anim Behav Sci93213228

45. Pohle K, Cheng HW (2009) Furnished cage system and hen well-being: Comparative effects of furnished cages and battery cages on behavioral exhibitions in White Leghorn chickens. Poult Sci 88: 1559–1564.K. PohleHW Cheng2009Furnished cage system and hen well-being: Comparative effects of furnished cages and battery cages on behavioral exhibitions in White Leghorn chickens.Poult Sci8815591564

46. Bateson M, Matheson SM (2007) Performance on a categorisation task suggests that removal of environmental enrichment induces ‘pessimism’ in captive European starlings (Sturnus vulgaris). Anim Welf 16: 33–36.M. BatesonSM Matheson2007Performance on a categorisation task suggests that removal of environmental enrichment induces ‘pessimism’ in captive European starlings (Sturnus vulgaris).Anim Welf163336

47. Rushen J (2000) Some issues in the interpretation of behavioural responses to stress. In: Moberg GP, Mench JA, editors. The Biology of Animal Stress - Basic principles and implications for animal welfare. New York: CAGI Publishing. pp. 23–42.J. Rushen2000Some issues in the interpretation of behavioural responses to stress.GP MobergJA MenchThe Biology of Animal Stress - Basic principles and implications for animal welfareNew YorkCAGI Publishing2342

48. Paul ES, Harding EJ, Mendl M (2005) Measuring emotional processes in animals: the utility of a cognitive approach. Neurosci Biobehav Rev 29: 469–491.ES PaulEJ HardingM. Mendl2005Measuring emotional processes in animals: the utility of a cognitive approach.Neurosci Biobehav Rev29469491

49. Young RJ (2003) Environmental enrichment for captive animals. Oxford: Blackwell Science. RJ Young2003Environmental enrichment for captive animals.OxfordBlackwell Science228 p

50. Carlstead K, Shepherdson D (2000) Alleviating stress in zoo animals with environmental enrichment. In: Moberg GP, Mench JA, editors. The Biology of Animal Stress - Basic principles and implications for animal welfare. New York: CABI Publishing. pp. 337–354.K. CarlsteadD. Shepherdson2000Alleviating stress in zoo animals with environmental enrichment.GP MobergJA MenchThe Biology of Animal Stress - Basic principles and implications for animal welfareNew YorkCABI Publishing337354

51. Wolfe TL (2000) Understanding the role of stress in animal welfare: practical considerations. In: Moberg GP, Mench JA, editors. The Biology of Animal Stress - Basic principles and implications for animal welfare. New York: CABI Publishing. pp. 355–368.TL Wolfe2000Understanding the role of stress in animal welfare: practical considerationsGP MobergJA MenchThe Biology of Animal Stress - Basic principles and implications for animal welfareNew YorkCABI Publishing355368

52. Hemsworth PH (2003) Human-animal interactions in livestock production. Appl Anim Behav Sci 81: 185–198.PH Hemsworth2003Human-animal interactions in livestock production.Appl Anim Behav Sci81185198

53. Boissy A, Manteuffel G, Jensen MB, Moe RO, Spruijt B, et al. (2007) Assessment of positive emotions in animals to improve their welfare. Physiol Behav 92: 375–397.A. BoissyG. ManteuffelMB JensenRO MoeB. Spruijt2007Assessment of positive emotions in animals to improve their welfare.Physiol Behav92375397

54. Wasser SK, Hunt KE (2005) Noninvasive measures of reproductive function and disturbance in the barred owl, great horned owl, and northern spotted owl. Bird Hormones and Bird Migrations: Analyzing Hormones in Droppings and Egg Yolks and Assessing Adaptations in Long-Distance Migration 109–137.SK WasserKE Hunt2005Noninvasive measures of reproductive function and disturbance in the barred owl, great horned owl, and northern spotted owl.Bird Hormones and Bird Migrations: Analyzing Hormones in Droppings and Egg Yolks and Assessing Adaptations in Long-Distance Migration109137

55. Staley AM, Blanco JM, Dufty AM, Wildt DE, Monfort SL (2007) Fecal steroid monitoring for assessing gonadal and adrenal activity in the golden eagle and peregrine falcon. J Comp Physiol B Biochem Syst Environ Physiol 177: 609–622.AM StaleyJM BlancoAM DuftyDE WildtSL Monfort2007Fecal steroid monitoring for assessing gonadal and adrenal activity in the golden eagle and peregrine falcon.J Comp Physiol B Biochem Syst Environ Physiol177609622

56. Wasser SK, Hunt KE, Brown JL, Cooper K, Crockett CM, et al. (2000) A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. Gen Comp Endocrinol 120: 260–275.SK WasserKE HuntJL BrownK. CooperCM Crockett2000A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species.Gen Comp Endocrinol120260275

57. Menargues A, Urios V, Mauri M (2008) Welfare assessment of captive Asian elephants (Elephas maximus) and Indian rhinoceros (Rhinoceros unicornis) using salivary cortisol measurement. Anim Welf 17: 305–312.A. MenarguesV. UriosM. Mauri2008Welfare assessment of captive Asian elephants (Elephas maximus) and Indian rhinoceros (Rhinoceros unicornis) using salivary cortisol measurement.Anim Welf17305312

58. Romero LM, Reed JM (2005) Collecting baseline corticosterone samples in the field: is under 3 min good enough? Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology 140: 73–79.LM RomeroJM Reed2005Collecting baseline corticosterone samples in the field: is under 3 min good enough?Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology1407379

59. Carlsson HE, Lyberg K, Royo F, Hau J (2007) Quantification of stress sensitive markers in single fecal samples do not accurately predict excretion of these in the pig. Res Vet Sci 82: 423–428.HE CarlssonK. LybergF. RoyoJ. Hau2007Quantification of stress sensitive markers in single fecal samples do not accurately predict excretion of these in the pig.Res Vet Sci82423428

60. Bortolotti GR, Marchant TA, Blas J, German T (2008) Corticosterone in feathers is a long-term, integrated measure of avian stress physiology. Funct Ecol 22: 494–500.GR BortolottiTA MarchantJ. BlasT. German2008Corticosterone in feathers is a long-term, integrated measure of avian stress physiology.Funct Ecol22494500

61. Bortolotti GR, Marchant T, Blas J, Cabezas S (2009) Tracking stress: localisation, deposition and stability of corticosterone in feathers. J Exp Biol 212: 1477–1482.GR BortolottiT. MarchantJ. BlasS. Cabezas2009Tracking stress: localisation, deposition and stability of corticosterone in feathers.J Exp Biol21214771482

62. Tomback DF (1998) Clark's nutcracker. Birds N Am 331: 1–24.DF Tomback1998Clark's nutcracker.Birds N Am331124

63. Greenberg R, Mettke-Hofmann C (2001) Ecological aspects of neophobia and neophilia in birds. In: Nolan V Jr, Thompson CF, editors. Curr Ornithol. New York: Kluwer Academic/Plenum. pp. 119–178.R. GreenbergC. Mettke-Hofmann2001Ecological aspects of neophobia and neophilia in birds.V. Nolan JrCF ThompsonCurr OrnitholNew YorkKluwer Academic/Plenum119178

64. Mettke-Hofmann C, Winkler H, Leisler B (2002) The significance of ecological factors for exploration and neophobia in parrots. Ethology 108: 249–272.C. Mettke-HofmannH. WinklerB. Leisler2002The significance of ecological factors for exploration and neophobia in parrots.Ethology108249272

65. Fox RA, Millam JR (2004) The effect of early environment on neophobia in orange-winged Amazon parrots (Amazona amazonica). Appl Anim Behav Sci 89: 117–129.RA FoxJR Millam2004The effect of early environment on neophobia in orange-winged Amazon parrots (Amazona amazonica).Appl Anim Behav Sci89117129

66. Wayland M, Gilchrist HG, Marchant T, Keating J, Smits JE (2002) Immune function, stress response, and body condition in arctic-breeding common eiders in relation to cadmium, mercury, and selenium concentrations. Environ Res 90: 47–60.M. WaylandHG GilchristT. MarchantJ. KeatingJE Smits2002Immune function, stress response, and body condition in arctic-breeding common eiders in relation to cadmium, mercury, and selenium concentrations.Environ Res904760

67. Harms NJ, Fairhurst GD, Bortolotti GR, Smits JEG (2010) Variation in immune function, body condition, and feather corticosterone in nestling Tree Swallows (Tachycineta bicolor) on reclaimed wetlands in the Athabasca oil sands, Alberta, Canada. Environ Pollut 158: 841–848.NJ HarmsGD FairhurstGR BortolottiJEG Smits2010Variation in immune function, body condition, and feather corticosterone in nestling Tree Swallows (Tachycineta bicolor) on reclaimed wetlands in the Athabasca oil sands, Alberta, Canada.Environ Pollut158841848

68. Bortolotti GR, Mougeot F, Martinez-Padilla J, Webster LMI, Piertney SB (2009) Physiological Stress Mediates the Honesty of Social Signals. PLoS ONE 4: GR BortolottiF. MougeotJ. Martinez-PadillaLMI WebsterSB Piertney2009Physiological Stress Mediates the Honesty of Social Signals.PLoS ONE4

69. Bortolotti GR (2010) Flaws and pitfalls in the chemical analysis of feathers: bad news-good news for avian chemoecology and toxicology. Ecol Appl 20: 1766–1774.GR Bortolotti2010Flaws and pitfalls in the chemical analysis of feathers: bad news-good news for avian chemoecology and toxicology.Ecol Appl2017661774

70. Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O (2006) SAS for Mixed Models, Second Edition. Cary, NC: SAS Institute, Inc. RC LittellGA MillikenWW StroupRD WolfingerO. Schabenberger2006SAS for Mixed Models, Second EditionCary, NCSAS Institute, Inc

71. Dobrakovova M, Kvetnansky R, Oprsalova Z, Jezova D (1993) Specificity of the effect of repeated handling on sympathetic-adrenomedullary and pituitary-adrenocortical activity in rats. Psychoneuroendocrinology 18: 163–174.M. DobrakovovaR. KvetnanskyZ. OprsalovaD. Jezova1993Specificity of the effect of repeated handling on sympathetic-adrenomedullary and pituitary-adrenocortical activity in rats.Psychoneuroendocrinology18163174

72. Bosson CO, Palme R, Boonstra R (2009) Assessment of the Stress Response in Columbian Ground Squirrels: Laboratory and Field Validation of an Enzyme Immunoassay for Fecal Cortisol Metabolites. Physiol Biochem Zool 82: 291–301.CO BossonR. PalmeR. Boonstra2009Assessment of the Stress Response in Columbian Ground Squirrels: Laboratory and Field Validation of an Enzyme Immunoassay for Fecal Cortisol Metabolites.Physiol Biochem Zool82291301

73. Saino N, Suffritti C, Martinelli R, Rubolini D, Moller AP (2003) Immune response covaries with corticosterone plasma levels under experimentally stressful conditions in nestling barn swallows (Hirundo rustica). Behav Ecol 14: 318–325.N. SainoC. SuffrittiR. MartinelliD. RuboliniAP Moller2003Immune response covaries with corticosterone plasma levels under experimentally stressful conditions in nestling barn swallows (Hirundo rustica).Behav Ecol14318325

74. Cornelius JM, Breuner CW, Hahn TP (2010) Under a neighbour's influence: public information affects stress hormones and behaviour of a songbird. Proc R Soc B 277: 2399–2404.JM CorneliusCW BreunerTP Hahn2010Under a neighbour's influence: public information affects stress hormones and behaviour of a songbird.Proc R Soc B27723992404

75. Gagliano H, Fuentes S, Nadal R, Armario A (2008) Previous exposure to immobilisation and repeated exposure to a novel environment demonstrate a marked dissociation between behavioral and pituitary-adrenal responses. Behav Brain Res 187: 239–245.H. GaglianoS. FuentesR. NadalA. Armario2008Previous exposure to immobilisation and repeated exposure to a novel environment demonstrate a marked dissociation between behavioral and pituitary-adrenal responses.Behav Brain Res187239245

76. Renner MJ, Rosenzweig MR (1987) Enriched and Impoverished Environments - Effects on Brain and Behavior. New York: Springer-Verlag. MJ RennerMR Rosenzweig1987Enriched and Impoverished Environments - Effects on Brain and Behavior.New YorkSpringer-Verlag

77. Morley-Fletcher S, Rea M, Maccari S, Laviola G (2003) Environmental enrichment during adolescence reverses the effects of prenatal stress on play behaviour and HPA axis reactivity in rats. Eur J Neurosci 18: 3367–3374.S. Morley-FletcherM. ReaS. MaccariG. Laviola2003Environmental enrichment during adolescence reverses the effects of prenatal stress on play behaviour and HPA axis reactivity in rats.Eur J Neurosci1833673374