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Appearance Matters: Artificial Marking Alters Aggression and Stress
Environment, Well-Being, and Behavior Appearance Matters: Artificial Marking Alters Aggression and Stress R. L. Dennis,*1 R. C. Newberry,† H.-W. Cheng,‡ and I. Estevez*2 *Department of Animal and Avian Sciences, University of Maryland, College Park 20742; †Center for the Study of Animal Well-Being, Department of Animal Sciences and Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman 99164-6520; and ‡Livestock Behavior Research Unit, USDA-Agricultural Research Service and Animal Sciences Department, Purdue University, West Lafayette, IN 47907 a group were artificially marked, the marked birds received more aggression and had lesser body mass than the unmarked individuals within the same group. Furthermore, in groups in which only a small proportion of the individuals were marked, we report altered plasma epinephrine and dopamine levels in marked individuals. These effects of marking were imperceptible when all birds in a group were marked. This finding has important implications for animal research because, when only a subset of group members is artificially marked and used for data collection, the results obtained may not be representative of the population.
Key words: aggression, stress, social behavior, dominance, identification 2008 Poultry Science 87:1939–1946 doi:10.3382/ps.2007-00311
INTRODUCTION Artificial marking is a clear manipulation of the physical appearance of individual animals that can affect behavior and reproductive success. For example, color and symmetry of leg bands has been shown to influence mate choice and mate guarding behavior of wild song birds (Burley, 1988; Johnsen et al., 1997, 2000) and radio-tagging of fledgling Louisiana Waterthrushes resulted in their removal from the nest and abandonment by adults (Mattsson et al., 2006). Physical alterations to the feathers and comb of domestic fowl (Gallus gallus domesticus) have also been found to modify behavior and responses from conspecifics, including aggressive interactions (Guhl, 1953; Guhl and Ortman, 1953; Marks et al., 1960; Siegel and Hurst, 1962). Markings can potentially alter multiple behavioral and physiological systems in animals. Here we use ag©2008 Poultry Science Association Inc. Received July 25, 2007. Accepted April 22, 2008. 1 Current address: Livestock Behavior Research Unit, USDA-Agricultural Research Service and Animal Sciences Department, Purdue University, West Lafayette, IN 47907. 2 Corresponding author: [email protected]
gression in the domestic fowl as a model for examining effects of markings because aggression and dominance hierarchies have been well studied in chickens with and without the use of different artificial marking systems. In groups of domestic fowl with an established dominance hierarchy, most aggressive interactions are directed toward subordinates (Guhl, 1953; Candland et al., 1969; Queiroz and Cromberg, 2006). The formation of dominance hierarchies and the propensity to show aggression toward unfamiliar birds, is probably related to individual recognition (Bradshaw, 1991; Lindberg and Nicol, 1996). If so, chickens housed in large groups may be unable to form dominance relationships with all other birds in the group because they may be unable to recognize every other bird individually and, thus, establish a pair-wise relationship with each bird (McBride, 1964). This may explain why aggressive displays are relatively rare in large groups of domestic fowl (D’Eath and Keeling, 2003). On the other hand, aggression and dominance have been linked to factors such as body mass, comb size, and previous experience of winning or losing (Guhl, 1953; Cloutier and Newberry, 2000, 2002), and theoretical models demonstrate that dominance hierarchies can emerge in large groups of individuals varying in phenotype in the absence of individual recognition (e.g., Dugatkin and Earley,
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ABSTRACT Artificial marking of animals for identification is frequently employed by researchers in the behavioral, biomedical, agricultural, and environmental sciences. The impact of artificial marking on experimental results is rarely explicitly considered despite evidence demonstrating that changes in phenotypic appearance can modify animal behavior and reproductive success. Here we present evidence that artificial marking of individuals within a social group has frequencydependent effects on the behavior and physiology of domestic fowl (Gallus gallus domesticus). We demonstrate that when only 20 or 50% of individuals within
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MATERIALS AND METHODS Birds Twelve hundred and sixty male 1-d-old domestic fowl of a rapidly growing (meat-type) white-feathered strain were randomly assigned to 1 of 21 groups of 10 birds and 21 groups of 50 birds for a density of 2.22 and 11.11 birds/m2, respectively. In each group, 20, 50, or 100% of the birds were marked by placing a mark on the back of the head using a permanent black marker and applying a pair of identification tags, yielding 7 replicate groups for each percentage marked within GS. Identification tags (Cornetto et al., 2002) were made of white paper disks (3 cm diameter), with a 2-digit black number printed on both sides that was unique to each bird. Tags were laminated to maintain their integrity and affixed to either side of the neck at one day of age with plastic filaments injected under the skin using the Swiftack system (Heartland Animal Health Inc., Fair Play, MO). Unmarked birds had no black mark or identification tags. A block design was used, with 7 complete blocks located in 2 rooms of one animal facility at the University of Maryland. Food and water were available ad libitum. A low density feed was used to slow growth and minimize chances of mobility problems. An artificial lighting program of 14L:10D was followed. Animal care was provided in adherence with Institutional Animal Care and Use Committee guidelines. Instances of mor-
tality were low and were corrected for in the statistical analysis.
Behavioral Observations Direct behavioral observations were made by the same observer for 10 min, 5 times in every 2-wk period from 3 to 10 wk of age. For all threats, aggressive pecks directed toward the head, and nonaggressive pecks directed toward identification tags observed in each of the groups, the observer recorded the giver and receiver and whether they were marked or unmarked, following the behavioral definitions of Estevez et al. (2003) and Cornetto et al. (2002). Data were collected using The Observer 3.0 software (Noldus Information Technology, Wageningen, the Netherlands).
Physical and Physiological Measures Data were obtained on body mass to the nearest gram (BM) at 2, 5, and 11 wk of age, fecal corticosterone (CORT) at 5 wk of age, and plasma epinephrine (EP), norepinephrine (NE), and dopamine (DA) concentrations at 11 wk of age. Data were collected from a random sample of 3 marked and 3 unmarked birds from each group, except the 20% marked groups of GS 10, from which 2 marked birds and 4 unmarked birds were sampled. To minimize bias and disturbance of the birds, birds to be sampled for blood, feces, and BM were gently picked up and removed from the pen by a single handler who walked very slowly in the pens. To control for time of day, birds were sampled by experimental block, with the order of sampling pens within block balanced across blocks. Birds sampled within each pen were selected in random order. For CORT determination, birds were placed individually in a large cage where a fecal sample was collected immediately following excretion, placed in a plastic bag, and maintained at −80°C until extraction of CORT using 90% ethanol. Each sampled bird was returned to its home pen immediately after collection. All fecal samples were collected between 1000 and 1500 h in a single day. Following extraction (Dehnhard et al., 2003), CORT concentrations were analyzed using a commercial 125I CORT radioimmunoassay kit from MP Biomedicals (Montreal, CA) as described previously by Cheng et al. (2001). Concentrations of CORT were calculated from a reference curve that ranged from 0.1 to 4.0 ng/mL, and the correlation coefficient was 0.995. Blood samples were collected from the brachial vein into heparinized tubes at 11 wk of age following a manual restraint of at least 45 s, and the restraint time (i.e., time from removal from the pen to completion of blood collection) was documented for use in the statistical analysis of catecholamine concentrations. Samples were centrifuged for 15 min at 3,000 rpm, and plasma was removed and maintained at −80°C until extraction and HPLC analysis of plasma catecholamines using the Coulochem II electrochemical detector from ESA
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2004). Pagel and Dawkins (1997) suggested that, in large groups of domestic fowl, status signals may take the place of dominance relationships based on individual recognition, proposing that identifiable marks, or status badges, may enable recognition of the social status of the bird, if not the individual itself. The observation that runts in large groups appear to be regularly dominated by other birds suggests that some form of dominance hierarchy based on phenotype is operating in these groups. In previous research on group size (GS) and aggressive behavior in domestic fowl, artificially marked focal birds delivered fewer, and received more, aggressive acts with increasing GS (Estevez et al., 2003). This result may have been an artifact of marking a constant number of focal birds within each group because the proportion of marked to unmarked birds declined with increasing GS. This possibility led us to hypothesize that application of artificial marks to individuals within a group results in frequency-dependent discrepancies in the behavior and physiology of marked vs. unmarked individuals. Here, we compare the effects of artificial marking in groups in which 20, 50, or 100% of the individuals were marked in both small and large GS (10 and 50 birds per group, respectively) on the aggression received and given by marked birds, and the stress parameters of marked and unmarked birds.
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RESULTS Aggression Received
Figure 1. Artificial marking of a proportion of the flock alters aggression received (least squares mean ± SEM per bird in 10 min; letters show differences at P < 0.05). a) Aggressive pecks received (marking treatment; F4,111 = 16.83, P < 0.0001, n = 7); b) aggressive threats received (marking treatment; F4,39 = 5.25, P = 0.0018, n = 7); and c) pecks at identification tags received (marking treatment by age period; F12,147 = 9.81, P < 0.0001, n = 7). A,BDifferences between age periods (3 to 4, 5 to 6, 7 to 8, and 9 to 10 wk) within treatment group; a,bdifferences between treatment groups within age period.
Biosciences (Chelmsford, MA) as described previously (Cheng et al., 2001). The concentrations of DA, EP, and NE were calculated from a reference curve that ranged from 1.0 to 100 pg/mL, and the correlation coefficient was 0.9998.
Statistical Analysis Behavioral data were analyzed in a mixed-model ANOVA with block as a random effect and age (four 2-wk periods from 3 to 10 wk of age) as a repeated measure that was fit to the appropriate covariance matrix
Marked birds in both 20 and 50% pens were found to receive significantly more pecks than their unmarked pen mates (Figure 1a) and the marked birds in the positive control group (100% marked group). No significant interactions were found between treatment and group size for pecks received. Similarly, a significant treatment effect was found for threats received (Figure 1b), with marked birds in 20% pens receiving significantly more threats than their unmarked pen mates or the birds in the 100% pens, but not significantly more than marked birds in 50% pens. Frequency of threats received also tended to be greater for marked than unmarked birds in 50% pens. Marked birds received significantly more pecks at their identification tags in 20 and 50% pens compared with the birds in 100% pens during period 1 (3 to 4 wk of age) but not at later ages (Figure 1c). Pecks at tags in 20 and 50% pens were significantly more frequent during period 1 compared with the later periods; however, this difference across periods was not seen in 100% pens (Figure 1c).
Aggression Given Not only did marked birds receive more aggression in groups containing both marked and unmarked birds but they also delivered less aggression than their unmarked flock mates. Specifically, in age period 4 (9 to 10 wk of age) and GS 50, marked birds delivered significantly fewer aggressive pecks than unmarked flock mates (ANOVA: marking treatment by GS by age period, F12,125 = 2.47, P = 0.006; Pairwise comparisons: t = 3.24, P = 0.013 for 20 vs. 100% marked groups, and t
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for each behavior. Variance partitioning was used to correct for heterogeneity of variance and log-transformation was used when appropriate to attain normality. There were 5 marking treatments (marked birds from 20, 50, and 100% marked pens, and unmarked birds from 20 and 50% marked pens). Behaviors both received and given by marked and unmarked individuals were compared across block, marking treatment, GS, and age period. Per bird measures were analyzed to correct for differences in GS due to mortalities. Least square means were determined and contrasts were used for means comparisons, using the Sidak adjustment to maintain an experimental α of 0.05. Body mass and CORT were analyzed using a blocked factorial ANOVA. Catecholamines were analyzed using a blocked factorial analysis of covariance (ANCOVA) with nested analysis; restraint time was the covariate. Because EP and NE have been reported to increase logarithmically with the time taken to sample blood (Matt et al., 1997), using restraint time as a covariate allowed for unbiased comparisons between treatment groups.
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Table 1. Artificial marking alters aggression given Proportion of flock marked Aggressive pecks, GS 10 20% 50% 100% Aggressive pecks, GS 50 20% 50% 100% Aggressive threats 20% 50%
50% 100%
Period 1
Period 2
Period 3
Period 4
Marked Unmarked Marked Unmarked Marked
0.00 ± 0.01A,a 0.04 ± 0.02A,a 0.05 ± 0.02A,a 0.11 ± 0.02B,a 0.03 ± 0.02A,a
0.06 ± 0.02A,a 0.01 ± 0.02A,a 0.03 ± 0.02A,a 0.06 ± 0.02AB,a 0.04 ± 0.02A,a
0.07 ± 0.02A,a 0.02 ± 0.02A,a 0.01 ± 0.02A,a 0.03 ± 0.02A,a 0.01 ± 0.02A,a
0.00 ± 0.02A,a 0.03 ± 0.02A,a 0.01 ± 0.02A,a 0.07 ± 0.02AB,a 0.05 ± 0.02A,a
Marked Unmarked Marked Unmarked Marked
0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a
0.01 ± 0.01A,a 0.03 ± 0.01A,a 0.01 ± 0.01A,a 0.03 ± 0.01A,a 0.02 ± 0.01A,a
0.02 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.05 ± 0.01AB,a 0.03 ± 0.01A,a
0.05 ± 0.01A,a 0.09 ± 0.01B,b* 0.04 ± 0.01A,a 0.07 ± 0.01B,b* 0.04 ± 0.01A,a
Marked Unmarked Marked Unmarked Marked
0.02 ± 0.02A,a 0.05 ± 0.02A,a 0.04 ± 0.02A,a 0.14 ± 0.02B,b* 0.07 ± 0.02A,a
0.04 ± 0.01A,a 0.07 ± 0.01A,ab 0.07 ± 0.01A,ab 0.12 ± 0.01B,b* 0.04 ± 0.01A,a
0.05 ± 0.02A,a 0.06 ± 0.02A,a 0.03 ± 0.02A,a 0.09 ± 0.02AB,a 0.03 ± 0.02A,a
0.05 ± 0.01A,a 0.05 ± 0.01A,a 0.03 ± 0.01A,a 0.07 ± 0.01A,a 0.06 ± 0.01A,a
Marked Unmarked Marked Unmarked Marked
0.005 ± 0.003A,a 0.015 ± 0.003B,a 0.035 ± 0.003B,b* 0.027 ± 0.003B,ab 0.016 ± 0.003B,a
0.000 ± 0.003A,a 0.001 ± 0.003A,a 0.008 ± 0.003A,a 0.005 ± 0.003A,a 0.005 ± 0.003AB,a
0.000 ± 0.003A,a 0.000 ± 0.003A,a 0.003 ± 0.003A,a 0.000 ± 0.003A,a 0.000 ± 0.003A,a
0.000 ± 0.003A,a 0.000 ± 0.003A,a 0.001 ± 0.003A,a 0.001 ± 0.003A,a 0.001 ± 0.003A,a
A,B Differences (P < 0.05) between age periods (3 to 4, 5 to 6, 7 to 8, and 9 to 10 wk) within treatment, with significantly more frequent events in boldface. a,b Differences between marking treatments within age period, with significantly more frequent events marked with an asterisk (*). 1 Values indicate least squares means ± SEM of number of acts given per bird in 10 min (n = 7) by marking treatment, group size (GS), and age period.
= 2.97, P = 0.028 for 50 vs. 100% marked groups; Table 1). In age period 1 (3 to 4 wk of age), marked birds in 50% marked groups of GS 10 and 50 also delivered fewer threats than their unmarked counterparts (marking treatment by age, F12,77 = 2.07, P = 0.029; Table 1). No significant differences were detected in giving pecks at tags.
Body Mass Main effect contrasts revealed a significant effect of marking on BM (least square means and SEM) in both 20 and 50% pen treatments and at both GS. Marked birds were found to have significantly lesser BM than unmarked penmates at 2 wk (245 ± 7.1 vs. 259 ± 7.5 g; F1,54 = 7.16, P = 0.010) and 5 wk (1,681 ± 44.2 vs. 1,773 ± 43.1 g; F1,54 = 9.82, P = 0.003) of age. Body mass at 11 wk was not significantly affected by marking (marked, 4,999 ± 69.0 g; unmarked, 5,020 ± 62.9 g; F1,52 = 0.02, P = 0.888). There was no significant main effect of percentage marked per group on BM at any age, but there was a significant interaction between percentage marked and presence or absence of marking on the bird at 5 wk of age. Least square means and SEM were as follows: 1,754 ± 28.2, 1,675 ± 28.2, 1,806 ± 28.2, 1,695 ± 33.4, and 1,742 ± 26.4 g for 5-wk-old birds from 100% pens, marked in 50% pens, unmarked in 50% pens, marked in 20% pens, and unmarked in 20% pens, respectively, where unmarked birds in 50% pens were significantly
heavier than their marked pen mates (t = 3.30, P = 0.003) and, although not significant, marked birds in 100% pens tended to be heavier than marked birds in 50% pens (t = 2.00, P = 0.058).
Catecholamines and CORT A significant treatment effect was found in plasma EP concentration. Marked birds in 20% pens had significantly lesser EP concentrations compared with their unmarked counterparts and the positive control birds in 100% marked groups (Figure 2a). A treatment by GS interaction of plasma DA concentration was found to be significant. Marked birds in 20% pens of GS 50 had significantly greater DA concentrations than their unmarked counterparts (Figure 2b). No significant differences were found between treatments in plasma NE concentration (F4,50 = 2.00, P = 0.108; least square mean ± SEM, 3.77 ± 0.67 pg/mL) or fecal CORT metabolite (F4,28.3 = 1.10, P = 0.377; least square mean ± SEM, 2.35 ng/mL ± 0.30) concentration.
DISCUSSION We have considered 4 possible mechanisms to explain the increase in aggression toward marked birds: 1) application of novel marks, 2) xenophobia based on phenotypic dissimilarity, 3) marks perceived as signals of low or high status, and 4) social challenge to conspicuous individuals.
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100% Pecks at tags 20%
Birds within flock
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Xenophobia Based on Phenotypic Dissimilarity Kin recognition theory suggests that cooperation and reduced aggression may be controlled by perceived relatedness (Keller, 1997), which in some species appears to be based on degree of phenotypic similarity (Hamilton, 1964a,b; Jaisson, 1991). Although phenotypic matching could be accomplished by comparing one’s own appearance with that of others, mirror studies suggest that fowl do not recognize their own image (Panksepp et al., 1980). A more likely mechanism for phenotypic matching would be growing up with others of the same phenotype, producing a preference for the familiar phenotype. All birds were unmarked for a few hours after hatch, which might have provided sufficient time to develop a preference for unmarked birds. However, this would not explain the relatively low aggression in the 100% marked groups. After marking in the 20 and 50% marked groups, both phenotypes were kept
Figure 2. Effects of artificial marking of 20, 50, or 100% of the flock on mean plasma catecholamine concentrations (±SEM) following manual restraint. Letters show differences at P < 0.05. a) Plasma epinephrine (EP) concentration (marking treatment, F4,121 = 3.43, P = 0.0108, n = 7); b) plasma dopamine (DA) concentration [group size (GS) by marking treatment, F4,181 = 6.08, P < 0.0001, n = 7]. A,B Differences between GS within marking treatment; a,bdifferences between treatment groups within GS.
together, thereby providing no opportunity for birds to develop exclusive familiarity with members of their own phenotype. If phenotypic preference was based on greater familiarity with the most common phenotype in the population, it would not explain why marked birds received more aggression than unmarked birds in 50% marked groups where both phenotypes occurred at equal frequency. Therefore, xenophobia based on phenotypic dissimilarity provides an implausible explanation for our findings.
Marks Perceived as Signals of Low or High Status Artificial marks could inadvertently signal low competitive ability similar to the way that dull or soiled plumage signals reduced fitness associated with parasitic infection (Hamilton and Zuk, 1982), injury, or pathogenic disease. Likewise, a small or artificially dubbed comb signals low status and attracts aggression (Marks et al., 1960; Siegel and Hurst, 1962; Cloutier and Newberry, 2000). In our study, the artificial marks could have had a similar effect, especially the
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Novelty can elicit fear and aggression (Marin et al., 2001), and handling, usually necessary for application of marks, can alter stress and behavior (Gariepy et al., 2002; Queiroz and Cromberg, 2006). Pecks at identification tags occurred primarily during period 1 and then declined to a low level, suggesting that these pecks were primarily associated with novelty of the tags during the initial period after their application and represented exploratory behavior rather than aggression. If elevated aggression toward marked birds was primarily driven by the novelty of artificially applied marks, or changes in behavior resulting from handling received during marking, we would have expected the level of threats and aggressive pecks directed toward the heads of marked birds to decline with age as observed for pecks at tags. In contrast, aggressive pecks remained elevated over time in a frequency-dependent manner according to the proportion of marked birds in the group. Moreover, the greater frequency of aggressive pecks and threats received by marked birds in 20 and 50% marked groups compared with 100% marked groups throughout the study provides evidence that aggression was not triggered simply by the application of novel marks but was, rather, influenced by the frequency of the marked phenotype within the population. Because aggressive behavior is generally directed toward the head region, and behavior specifically directed toward the identification tags was shortlasting, it is plausible that these effects of marking on aggression were attributable solely to the presence of the black mark on the back of the head. However, because we marked birds with both a black mark and identification tags, we cannot conclude from this study whether one of these forms of marking alone or both forms in combination were responsible for the effects on aggression.
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Social Challenge to Conspicuous Individuals Instinctive deference to individuals bearing conspicuous marks would enable rapid invasion by cheaters bearing such marks without accompanying fighting ability, leading to the idea that there are costs associated with status badge bearing that only honest signalers can withstand (Rohwer, 1982; Senar, 1998). If social challenge is one such cost (Tibbetts and Dale, 2004), birds bearing conspicuous artificial marks should attract elevated aggression relative to unmarked birds in the same flock and the fewer conspicuously marked birds in a population, the more challenges they should receive. Our results are consistent with this prediction. The lowest level of aggression was received by marked birds in the 100% marked flocks, where the marks were not conspicuous because they were held by all birds. Although we did not find a significant difference in aggression received by marked birds in the 20 and 50% marked groups, the latter did tend to receive levels of aggression intermediate between those in 20 and 100% marked groups. The question remains as to why aggression toward marked birds would continue over time in a randomly marked population with a theoretically equal chance of winning and losing challenges. Estevez et al. (2007)
emphasize the importance of an individual’s ability to recoup the cost of aggressive encounters in their decision to engage in these contests as an important factor in multiple different aggression models. If there is a social cost associated with badge holding (Rohwer and Rohwer, 1978; Cloutier and Newberry, 2002; Tibbetts and Dale, 2004) and, on average, badge holders were not of greater competitive ability than nonbadge holders and thus able to overcome this cost, one would expect that, on average, the condition of marked birds would deteriorate relative to that of unmarked birds, thereby increasing their probability of losing encounters (Cloutier and Newberry, 2000). Given that previous experience of losing is an important determinant of the outcome of future social encounters (Hsu and Wolf, 1999; Cloutier and Newberry, 2000; Beacham, 2003), losing encounters would further cement low status, explaining why marked birds gave less aggression than they received. In accordance with the proposal that marking and consequent increased aggressive challenge would reduce body condition (or fitness), we found that marked birds had lower BM than unmarked birds in both 20 and 50% marked groups, differences suggestive of stress (Nicol et al., 1999; Keeling et al., 2003). Lower BM in the marked birds may have been related to energy costs incurred in an attempt to avoid aggression, lower food intake due to monopolization of food resources by the more aggressive unmarked birds, or stress associated with low social status (Senar et al., 2000). Differences in BM were attenuated by 11 wk of age, probably because growth slows as birds approach their mature size allowing smaller birds to catch up in BM. Marking affected catecholaminergic reactivity at 11 wk of age. Marked birds in the 20% marked groups had a depressed EP response and unaltered NE response, suggestive of reduced stress-coping ability and a suppressed fight-or-flight response compared with their unmarked counterparts or birds in 100% marked groups (Figure 2a). As stress hormones, both EP and NE participate in several physiological and behavioral processes (Kvetnansky and Mikulaj, 1970; Dillon et al., 1992; Dobrakovova et al., 1993; Matt et al., 1997). Whereas acute stress is associated with elevated plasma EP (Matt et al., 1997; Wortsman, 2002), prolonged chronic or chronic intermittent stress regimens have been observed to lower EP concentrations without altering NE concentrations in rats (Kvetnansky and Mikulaj, 1970; Dobrakovova et al., 1993) and humans (Matthews et al., 2001; Grant et al., 2003). Sensitivity of the EP system has been shown following pharmacological and genetic manipulation of aggressiveness in laying hens without altering NE (Dennis et al., 2006). Our results are consistent with previous findings indicating a dissociated stress response between the functions of adrenal medullary (indicated by EP) and sympathetic neural (indicated by NE) activity. Suppression of sympathetic adrenal responsiveness is consistent
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black mark on the back of the head, because of its proximity to the socially significant comb. However, our birds were sexually immature, and signals of status are usually not evident at such a young age but become prominent at sexual maturity. Even if the marks were initially perceived as signaling low status, why would marked birds in the 20 and 50% marked groups continue to attract elevated aggression over time? We applied marks to randomly selected birds that, on average, should have been equally likely to win or lose fights. Therefore, we might have expected that birds would learn that marks were not reliable signals of low status and would no longer direct aggression differentially toward marked birds. Conversely, the marks may have been perceived as badges of status. Badges of status may be used as honest signals of dominance in large groups of domestic fowl in which establishment of dominance through fighting would be costly and inefficient (Pagel and Dawkins, 1997). In birds, conspicuously pigmented feather patches may provide a particularly potent signal of fighting ability, enabling discrimination of dominant individuals without risking injury and wasting energy engaging in fights (Senar, 1998; Senar and Camerino, 1998). If the artificial marks applied to our birds were perceived by onlookers as representing honest badges of status, we might have expected that birds bearing them would have received less aggression than unmarked birds in the same group; in fact, we found the reverse.
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Conclusion We conclude that application of artificial identification marks to only a proportion of the individuals in a group can alter aggressive behavior, BM, and endocrine parameters in the domestic fowl. Thus, when artificial marking is required for this species, both the number and proportion of birds marked within a study population should be taken into consideration in experimental design and interpretation of results. Further research will be needed to elucidate the social impact of form, color, and body location of different types of artificial marking. The domestic fowl is highly dependent on both vision and pecking with the beak for sensory information, and the extent to which marking affects species less dependent on these sensory modalities is as yet unknown. Nevertheless, our findings sound a cautionary note for animal researchers because, when only a subset of group members is artificially marked and used for data collection, the results obtained may not be representative of the population.
ACKNOWLEDGMENTS We thank L. Douglass (University of Maryland, College Park) for statistical analysis, the graduate students and laboratory staff of I. Estevez and H.-W. Cheng for technical assistance, and the farm crew at University of Maryland’s Upper Marlboro facility for animal care. This research was funded by a competitive grant from the Maryland Agriculture Experiment Station to I. Estevez.
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with the reduction in aggressiveness of the marked birds. We also observed increased plasma DA concentration in marked birds of 20% groups at GS 50 (Figure 2b), suggestive of increased levels of chronic social stress (Cheng et al., 2001) as previously seen in chickens subjected to the chronic social stress of high stocking density (10 birds per cage) compared with siblings maintained at 2 birds per cage (Cheng et al., 2001). These results support our assertion that effects of artificial marking were stronger, to the point of producing endocrinological changes, the lower the proportion of marked individuals in a group. Although consistent with the hypothesis that, by 11 wk of age, marked birds in the 20% marked groups were experiencing chronic stress as a consequence of receiving repeated aggression, a definitive explanation cannot be given because we did not collect blood at earlier ages. Whereas acute social stress would be expected to increase CORT (Carere et al., 2003), marking had no effect on fecal CORT metabolite concentrations in our study, possibly due to dampening of CORT responsiveness in birds exposed to chronic social stress as a consequence of being marked. Consistent with this interpretation, Hester et al. (1996) found that chronic heat stress in chickens resulted in no significant effect on circulating CORT concentrations. Fecal CORT metabolite concentrations have been previously validated for domestic fowl and have been shown to exhibit a delay of approximately 4 h compared with plasma CORT levels (Dehnhard et al., 2003). Circadian fluctuation of CORT is unlikely to provide a plausible explanation for our results because fecal samples were collected by experimental block and time was accounted for in the statistical model. Furthermore, because fecal sampling provides an average estimate of circulating CORT levels over time, it is relatively insensitive to precise sampling time (Möstl and Palme, 2002).
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Dennis et al. statement responses of domestic chicks to cagemates and strangers. Appl. Anim. Behav. Sci. 71:57–66. Marks, H. L., P. B. Siegel, and C. Y. Kramer. 1960. Effects of comb and wattle removal on the social organization of mixed flocks of chickens. Anim. Behav. 8:192–196. Matt, K. S., M. C. Moore, R. Knapp, and I. T. Moore. 1997. Sympathetic mediation of stress and aggressive competition: Plasma catecholamines in free-living male tree lizards. Physiol. Behav. 61:639–647. Matthews, K. A., B. B. Gump, and J. F. Owens. 2001. Chronic stress influences cardiovascular and neuroendocrine responses during acute stress and recovery, especially in men. Health Psychol. 20:403–410. Mattsson, B. J., J. M. Meyers, and R. J. Cooper. 2006. Detrimental impact of radiotransmitters on juvenile Louisiana Waterthrushes. J. Field Ornithol. 77:173–177. McBride, G. 1964. Social discrimination + subflock structure in domestic fowl. Anim. Behav. 12:264–267. Möstl, E., and R. Palme. 2002. Hormones as indicators of stress. Domest. Anim. Endocrinol. 23:67–74. Nicol, C. J., N. G. Gregory, T. G. Knowles, I. D. Parkman, and L. J. Wilkins. 1999. Differential effects of increased stocking density, mediated by increased flock size, on feather pecking and aggression in laying hens. Appl. Anim. Behav. Sci. 65:137–152. Pagel, M., and M. S. Dawkins. 1997. Peck orders and group size in laying hens: Future contracts for non-aggression. Behav. Processes 40:13–25. Panksepp, J., N. J. Bean, P. Bishop, T. Vilberg, and T. L. Sahley. 1980. Opioid blockage and social comfort in chicks. Pharmacol. Biochem. Behav. 13:673–683. Queiroz, S. A., and V. U. Cromberg. 2006. Aggressive behavior in the genus Gallus sp. Braz. J. Poult. Sci. 8:1–14. Rohwer, S., and F. C. Rohwer. 1978. Status signalling in Harris sparrows – Experimental deceptions achieved. Anim. Behav. 26:1012–1022. Rohwer, S. A. 1982. The evolution of reliable and unreliable badges of fighting ability. Am. Zool. 22:531–546. Senar, J. C. 1998. Plumage coloration as a signal of social status. Pages 1669–1686 in Proc. 22nd Int. Ornithological Congr., Durban, South Africa. N. J. Adams and R. H. Slotow, ed. BirdLife South Africa, Johannesburg. Senar, J. C., and M. Camerino. 1998. Status signalling and the ability to recognize dominants: An experiment with siskins (Carduelis spinus). Proc. R. Soc. Lond. B. Biol. Sci. 265:1515–1520. Senar, J. C., V. Polo, F. Uribe, and M. Gomendio. 2000. Status signalling, metabolic rate and body mass in the siskin: The cost of being subordinate. Anim. Behav. 59:103–110. Siegel, H. S., and D. C. Hurst. 1962. Social interaction among females in dubbed and undubbed flocks. Poult. Sci. 41:141–145. Tibbetts, E. A., and J. A. Dale. 2004. A socially enforced signal of quality in a paper wasp. Nature 432:218–222. Wortsman, J. 2002. Role of epinephrine in acute stress. Endocrinol. Metab. Clin. North Am. 31:79–106.
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Estevez, I., I. L. Andersen, and E. Naevdal. 2007. Group size, density and social dynamics in farm animals. Appl. Anim. Behav. Sci. 103:185–204. Estevez, I., L. J. Keeling, and R. C. Newberry. 2003. Decreasing aggression with increasing group size in young domestic fowl. Appl. Anim. Behav. Sci. 84:213–218. Gariepy, J. L., R. M. Rodriguiz, and B. C. Jones. 2002. Handling, genetic and housing effects on the mouse stress system, dopamine function and behavior. Pharmacol. Biochem. Behav. 73:7–17. Grant, I., C. L. McKibbin, M. J. Taylor, P. Mills, J. Dimsdale, M. Ziegler, and T. Patterson. 2003. In-home respite intervention reduces plasma epinephrine in stressed Alzheimer caregivers. Am. J. Germ. Psych. 11:62–72. Guhl, A. M. 1953. Social behavior of the domestic fowl. Technical Bull. 73:1–43. Kansas Agricultural Experimental Station, Manhattan. Guhl, A. M., and L. L. Ortman. 1953. Visual patterns in the recognition of individuals among chickens. Condor 55:287–298. Hamilton, W. D. 1964a. The genetic evolution of social behavior. I. J. Theor. Biol. 7:1–17. Hamilton, W. D. 1964b. The genetic evolution of social behavior. II. J. Theor. Biol. 7:17–52. Hamilton, W. D., and M. Zuk. 1982. Heritable true fitness and bright birds a role for parasites? Science 218:384–387. Hester, P. Y., W. M. Muir, and J. V. Craig. 1996. Group selection for adaptation to multiple-hen cages: Humoral immune response. Poult. Sci. 75:1315–1320. Hsu, Y., and L. L. Wolf. 1999. The winner and loser effect: Integrating multiple experiences. Anim. Behav. 57:903– 910. Jaisson, P. 1991. Kinship and fellowship in ants and social wasps. Pages 60–93 in Kin Recognition. P. Hepper, ed. Cambridge University Press, Cambridge, UK. Johnsen, A., P. Fiske, T. Amundsen, J. T. Lifjeld, and P. A. Rohde. 2000. Color bands, mate choice and paternity in the bluethroat. Anim. Behav. 59:111–119. Johnsen, A., J. T. Lifjeld, and P. A. Rohde. 1997. Colored leg bands affect male mate-guarding behavior in the blue throat. Anim. Behav. 54:121–130. Keeling, L. J., I. Estevez, R. C. Newberry, and M. G. Correia. 2003. Production-related traits of layers reared in different sized flocks: The concept of problematic intermediate group sizes. Poult. Sci. 82:1393–1396. Keller, L. 1997. Indiscriminate altruism: Unduly nice parents and siblings. Trends Ecol. Evol. 12:99–103. Kvetnansky, R., and L. Mikulaj. 1970. Adrenal and urinary catecholamines in rats during adaptation to repeated immobilization stress. Endocrinology 87:738–743. Lindberg, A. C., and C. J. Nicol. 1996. Effects of social and environmental familiarity on group preferences and spacing behavior in laying hens. Appl. Anim. Behav. Sci. 49:109–123. Marin, R. H., P. Freytes, D. Guzman, and R. B. Jones. 2001. Effects of an acute stressor on fear and on the social rein-
Key words: aggression, stress, social behavior, dominance, identification 2008 Poultry Science 87:1939–1946 doi:10.3382/ps.2007-00311
INTRODUCTION Artificial marking is a clear manipulation of the physical appearance of individual animals that can affect behavior and reproductive success. For example, color and symmetry of leg bands has been shown to influence mate choice and mate guarding behavior of wild song birds (Burley, 1988; Johnsen et al., 1997, 2000) and radio-tagging of fledgling Louisiana Waterthrushes resulted in their removal from the nest and abandonment by adults (Mattsson et al., 2006). Physical alterations to the feathers and comb of domestic fowl (Gallus gallus domesticus) have also been found to modify behavior and responses from conspecifics, including aggressive interactions (Guhl, 1953; Guhl and Ortman, 1953; Marks et al., 1960; Siegel and Hurst, 1962). Markings can potentially alter multiple behavioral and physiological systems in animals. Here we use ag©2008 Poultry Science Association Inc. Received July 25, 2007. Accepted April 22, 2008. 1 Current address: Livestock Behavior Research Unit, USDA-Agricultural Research Service and Animal Sciences Department, Purdue University, West Lafayette, IN 47907. 2 Corresponding author: [email protected]
gression in the domestic fowl as a model for examining effects of markings because aggression and dominance hierarchies have been well studied in chickens with and without the use of different artificial marking systems. In groups of domestic fowl with an established dominance hierarchy, most aggressive interactions are directed toward subordinates (Guhl, 1953; Candland et al., 1969; Queiroz and Cromberg, 2006). The formation of dominance hierarchies and the propensity to show aggression toward unfamiliar birds, is probably related to individual recognition (Bradshaw, 1991; Lindberg and Nicol, 1996). If so, chickens housed in large groups may be unable to form dominance relationships with all other birds in the group because they may be unable to recognize every other bird individually and, thus, establish a pair-wise relationship with each bird (McBride, 1964). This may explain why aggressive displays are relatively rare in large groups of domestic fowl (D’Eath and Keeling, 2003). On the other hand, aggression and dominance have been linked to factors such as body mass, comb size, and previous experience of winning or losing (Guhl, 1953; Cloutier and Newberry, 2000, 2002), and theoretical models demonstrate that dominance hierarchies can emerge in large groups of individuals varying in phenotype in the absence of individual recognition (e.g., Dugatkin and Earley,
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ABSTRACT Artificial marking of animals for identification is frequently employed by researchers in the behavioral, biomedical, agricultural, and environmental sciences. The impact of artificial marking on experimental results is rarely explicitly considered despite evidence demonstrating that changes in phenotypic appearance can modify animal behavior and reproductive success. Here we present evidence that artificial marking of individuals within a social group has frequencydependent effects on the behavior and physiology of domestic fowl (Gallus gallus domesticus). We demonstrate that when only 20 or 50% of individuals within
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MATERIALS AND METHODS Birds Twelve hundred and sixty male 1-d-old domestic fowl of a rapidly growing (meat-type) white-feathered strain were randomly assigned to 1 of 21 groups of 10 birds and 21 groups of 50 birds for a density of 2.22 and 11.11 birds/m2, respectively. In each group, 20, 50, or 100% of the birds were marked by placing a mark on the back of the head using a permanent black marker and applying a pair of identification tags, yielding 7 replicate groups for each percentage marked within GS. Identification tags (Cornetto et al., 2002) were made of white paper disks (3 cm diameter), with a 2-digit black number printed on both sides that was unique to each bird. Tags were laminated to maintain their integrity and affixed to either side of the neck at one day of age with plastic filaments injected under the skin using the Swiftack system (Heartland Animal Health Inc., Fair Play, MO). Unmarked birds had no black mark or identification tags. A block design was used, with 7 complete blocks located in 2 rooms of one animal facility at the University of Maryland. Food and water were available ad libitum. A low density feed was used to slow growth and minimize chances of mobility problems. An artificial lighting program of 14L:10D was followed. Animal care was provided in adherence with Institutional Animal Care and Use Committee guidelines. Instances of mor-
tality were low and were corrected for in the statistical analysis.
Behavioral Observations Direct behavioral observations were made by the same observer for 10 min, 5 times in every 2-wk period from 3 to 10 wk of age. For all threats, aggressive pecks directed toward the head, and nonaggressive pecks directed toward identification tags observed in each of the groups, the observer recorded the giver and receiver and whether they were marked or unmarked, following the behavioral definitions of Estevez et al. (2003) and Cornetto et al. (2002). Data were collected using The Observer 3.0 software (Noldus Information Technology, Wageningen, the Netherlands).
Physical and Physiological Measures Data were obtained on body mass to the nearest gram (BM) at 2, 5, and 11 wk of age, fecal corticosterone (CORT) at 5 wk of age, and plasma epinephrine (EP), norepinephrine (NE), and dopamine (DA) concentrations at 11 wk of age. Data were collected from a random sample of 3 marked and 3 unmarked birds from each group, except the 20% marked groups of GS 10, from which 2 marked birds and 4 unmarked birds were sampled. To minimize bias and disturbance of the birds, birds to be sampled for blood, feces, and BM were gently picked up and removed from the pen by a single handler who walked very slowly in the pens. To control for time of day, birds were sampled by experimental block, with the order of sampling pens within block balanced across blocks. Birds sampled within each pen were selected in random order. For CORT determination, birds were placed individually in a large cage where a fecal sample was collected immediately following excretion, placed in a plastic bag, and maintained at −80°C until extraction of CORT using 90% ethanol. Each sampled bird was returned to its home pen immediately after collection. All fecal samples were collected between 1000 and 1500 h in a single day. Following extraction (Dehnhard et al., 2003), CORT concentrations were analyzed using a commercial 125I CORT radioimmunoassay kit from MP Biomedicals (Montreal, CA) as described previously by Cheng et al. (2001). Concentrations of CORT were calculated from a reference curve that ranged from 0.1 to 4.0 ng/mL, and the correlation coefficient was 0.995. Blood samples were collected from the brachial vein into heparinized tubes at 11 wk of age following a manual restraint of at least 45 s, and the restraint time (i.e., time from removal from the pen to completion of blood collection) was documented for use in the statistical analysis of catecholamine concentrations. Samples were centrifuged for 15 min at 3,000 rpm, and plasma was removed and maintained at −80°C until extraction and HPLC analysis of plasma catecholamines using the Coulochem II electrochemical detector from ESA
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2004). Pagel and Dawkins (1997) suggested that, in large groups of domestic fowl, status signals may take the place of dominance relationships based on individual recognition, proposing that identifiable marks, or status badges, may enable recognition of the social status of the bird, if not the individual itself. The observation that runts in large groups appear to be regularly dominated by other birds suggests that some form of dominance hierarchy based on phenotype is operating in these groups. In previous research on group size (GS) and aggressive behavior in domestic fowl, artificially marked focal birds delivered fewer, and received more, aggressive acts with increasing GS (Estevez et al., 2003). This result may have been an artifact of marking a constant number of focal birds within each group because the proportion of marked to unmarked birds declined with increasing GS. This possibility led us to hypothesize that application of artificial marks to individuals within a group results in frequency-dependent discrepancies in the behavior and physiology of marked vs. unmarked individuals. Here, we compare the effects of artificial marking in groups in which 20, 50, or 100% of the individuals were marked in both small and large GS (10 and 50 birds per group, respectively) on the aggression received and given by marked birds, and the stress parameters of marked and unmarked birds.
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RESULTS Aggression Received
Figure 1. Artificial marking of a proportion of the flock alters aggression received (least squares mean ± SEM per bird in 10 min; letters show differences at P < 0.05). a) Aggressive pecks received (marking treatment; F4,111 = 16.83, P < 0.0001, n = 7); b) aggressive threats received (marking treatment; F4,39 = 5.25, P = 0.0018, n = 7); and c) pecks at identification tags received (marking treatment by age period; F12,147 = 9.81, P < 0.0001, n = 7). A,BDifferences between age periods (3 to 4, 5 to 6, 7 to 8, and 9 to 10 wk) within treatment group; a,bdifferences between treatment groups within age period.
Biosciences (Chelmsford, MA) as described previously (Cheng et al., 2001). The concentrations of DA, EP, and NE were calculated from a reference curve that ranged from 1.0 to 100 pg/mL, and the correlation coefficient was 0.9998.
Statistical Analysis Behavioral data were analyzed in a mixed-model ANOVA with block as a random effect and age (four 2-wk periods from 3 to 10 wk of age) as a repeated measure that was fit to the appropriate covariance matrix
Marked birds in both 20 and 50% pens were found to receive significantly more pecks than their unmarked pen mates (Figure 1a) and the marked birds in the positive control group (100% marked group). No significant interactions were found between treatment and group size for pecks received. Similarly, a significant treatment effect was found for threats received (Figure 1b), with marked birds in 20% pens receiving significantly more threats than their unmarked pen mates or the birds in the 100% pens, but not significantly more than marked birds in 50% pens. Frequency of threats received also tended to be greater for marked than unmarked birds in 50% pens. Marked birds received significantly more pecks at their identification tags in 20 and 50% pens compared with the birds in 100% pens during period 1 (3 to 4 wk of age) but not at later ages (Figure 1c). Pecks at tags in 20 and 50% pens were significantly more frequent during period 1 compared with the later periods; however, this difference across periods was not seen in 100% pens (Figure 1c).
Aggression Given Not only did marked birds receive more aggression in groups containing both marked and unmarked birds but they also delivered less aggression than their unmarked flock mates. Specifically, in age period 4 (9 to 10 wk of age) and GS 50, marked birds delivered significantly fewer aggressive pecks than unmarked flock mates (ANOVA: marking treatment by GS by age period, F12,125 = 2.47, P = 0.006; Pairwise comparisons: t = 3.24, P = 0.013 for 20 vs. 100% marked groups, and t
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for each behavior. Variance partitioning was used to correct for heterogeneity of variance and log-transformation was used when appropriate to attain normality. There were 5 marking treatments (marked birds from 20, 50, and 100% marked pens, and unmarked birds from 20 and 50% marked pens). Behaviors both received and given by marked and unmarked individuals were compared across block, marking treatment, GS, and age period. Per bird measures were analyzed to correct for differences in GS due to mortalities. Least square means were determined and contrasts were used for means comparisons, using the Sidak adjustment to maintain an experimental α of 0.05. Body mass and CORT were analyzed using a blocked factorial ANOVA. Catecholamines were analyzed using a blocked factorial analysis of covariance (ANCOVA) with nested analysis; restraint time was the covariate. Because EP and NE have been reported to increase logarithmically with the time taken to sample blood (Matt et al., 1997), using restraint time as a covariate allowed for unbiased comparisons between treatment groups.
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Table 1. Artificial marking alters aggression given Proportion of flock marked Aggressive pecks, GS 10 20% 50% 100% Aggressive pecks, GS 50 20% 50% 100% Aggressive threats 20% 50%
50% 100%
Period 1
Period 2
Period 3
Period 4
Marked Unmarked Marked Unmarked Marked
0.00 ± 0.01A,a 0.04 ± 0.02A,a 0.05 ± 0.02A,a 0.11 ± 0.02B,a 0.03 ± 0.02A,a
0.06 ± 0.02A,a 0.01 ± 0.02A,a 0.03 ± 0.02A,a 0.06 ± 0.02AB,a 0.04 ± 0.02A,a
0.07 ± 0.02A,a 0.02 ± 0.02A,a 0.01 ± 0.02A,a 0.03 ± 0.02A,a 0.01 ± 0.02A,a
0.00 ± 0.02A,a 0.03 ± 0.02A,a 0.01 ± 0.02A,a 0.07 ± 0.02AB,a 0.05 ± 0.02A,a
Marked Unmarked Marked Unmarked Marked
0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a
0.01 ± 0.01A,a 0.03 ± 0.01A,a 0.01 ± 0.01A,a 0.03 ± 0.01A,a 0.02 ± 0.01A,a
0.02 ± 0.01A,a 0.04 ± 0.01A,a 0.04 ± 0.01A,a 0.05 ± 0.01AB,a 0.03 ± 0.01A,a
0.05 ± 0.01A,a 0.09 ± 0.01B,b* 0.04 ± 0.01A,a 0.07 ± 0.01B,b* 0.04 ± 0.01A,a
Marked Unmarked Marked Unmarked Marked
0.02 ± 0.02A,a 0.05 ± 0.02A,a 0.04 ± 0.02A,a 0.14 ± 0.02B,b* 0.07 ± 0.02A,a
0.04 ± 0.01A,a 0.07 ± 0.01A,ab 0.07 ± 0.01A,ab 0.12 ± 0.01B,b* 0.04 ± 0.01A,a
0.05 ± 0.02A,a 0.06 ± 0.02A,a 0.03 ± 0.02A,a 0.09 ± 0.02AB,a 0.03 ± 0.02A,a
0.05 ± 0.01A,a 0.05 ± 0.01A,a 0.03 ± 0.01A,a 0.07 ± 0.01A,a 0.06 ± 0.01A,a
Marked Unmarked Marked Unmarked Marked
0.005 ± 0.003A,a 0.015 ± 0.003B,a 0.035 ± 0.003B,b* 0.027 ± 0.003B,ab 0.016 ± 0.003B,a
0.000 ± 0.003A,a 0.001 ± 0.003A,a 0.008 ± 0.003A,a 0.005 ± 0.003A,a 0.005 ± 0.003AB,a
0.000 ± 0.003A,a 0.000 ± 0.003A,a 0.003 ± 0.003A,a 0.000 ± 0.003A,a 0.000 ± 0.003A,a
0.000 ± 0.003A,a 0.000 ± 0.003A,a 0.001 ± 0.003A,a 0.001 ± 0.003A,a 0.001 ± 0.003A,a
A,B Differences (P < 0.05) between age periods (3 to 4, 5 to 6, 7 to 8, and 9 to 10 wk) within treatment, with significantly more frequent events in boldface. a,b Differences between marking treatments within age period, with significantly more frequent events marked with an asterisk (*). 1 Values indicate least squares means ± SEM of number of acts given per bird in 10 min (n = 7) by marking treatment, group size (GS), and age period.
= 2.97, P = 0.028 for 50 vs. 100% marked groups; Table 1). In age period 1 (3 to 4 wk of age), marked birds in 50% marked groups of GS 10 and 50 also delivered fewer threats than their unmarked counterparts (marking treatment by age, F12,77 = 2.07, P = 0.029; Table 1). No significant differences were detected in giving pecks at tags.
Body Mass Main effect contrasts revealed a significant effect of marking on BM (least square means and SEM) in both 20 and 50% pen treatments and at both GS. Marked birds were found to have significantly lesser BM than unmarked penmates at 2 wk (245 ± 7.1 vs. 259 ± 7.5 g; F1,54 = 7.16, P = 0.010) and 5 wk (1,681 ± 44.2 vs. 1,773 ± 43.1 g; F1,54 = 9.82, P = 0.003) of age. Body mass at 11 wk was not significantly affected by marking (marked, 4,999 ± 69.0 g; unmarked, 5,020 ± 62.9 g; F1,52 = 0.02, P = 0.888). There was no significant main effect of percentage marked per group on BM at any age, but there was a significant interaction between percentage marked and presence or absence of marking on the bird at 5 wk of age. Least square means and SEM were as follows: 1,754 ± 28.2, 1,675 ± 28.2, 1,806 ± 28.2, 1,695 ± 33.4, and 1,742 ± 26.4 g for 5-wk-old birds from 100% pens, marked in 50% pens, unmarked in 50% pens, marked in 20% pens, and unmarked in 20% pens, respectively, where unmarked birds in 50% pens were significantly
heavier than their marked pen mates (t = 3.30, P = 0.003) and, although not significant, marked birds in 100% pens tended to be heavier than marked birds in 50% pens (t = 2.00, P = 0.058).
Catecholamines and CORT A significant treatment effect was found in plasma EP concentration. Marked birds in 20% pens had significantly lesser EP concentrations compared with their unmarked counterparts and the positive control birds in 100% marked groups (Figure 2a). A treatment by GS interaction of plasma DA concentration was found to be significant. Marked birds in 20% pens of GS 50 had significantly greater DA concentrations than their unmarked counterparts (Figure 2b). No significant differences were found between treatments in plasma NE concentration (F4,50 = 2.00, P = 0.108; least square mean ± SEM, 3.77 ± 0.67 pg/mL) or fecal CORT metabolite (F4,28.3 = 1.10, P = 0.377; least square mean ± SEM, 2.35 ng/mL ± 0.30) concentration.
DISCUSSION We have considered 4 possible mechanisms to explain the increase in aggression toward marked birds: 1) application of novel marks, 2) xenophobia based on phenotypic dissimilarity, 3) marks perceived as signals of low or high status, and 4) social challenge to conspicuous individuals.
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100% Pecks at tags 20%
Birds within flock
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Xenophobia Based on Phenotypic Dissimilarity Kin recognition theory suggests that cooperation and reduced aggression may be controlled by perceived relatedness (Keller, 1997), which in some species appears to be based on degree of phenotypic similarity (Hamilton, 1964a,b; Jaisson, 1991). Although phenotypic matching could be accomplished by comparing one’s own appearance with that of others, mirror studies suggest that fowl do not recognize their own image (Panksepp et al., 1980). A more likely mechanism for phenotypic matching would be growing up with others of the same phenotype, producing a preference for the familiar phenotype. All birds were unmarked for a few hours after hatch, which might have provided sufficient time to develop a preference for unmarked birds. However, this would not explain the relatively low aggression in the 100% marked groups. After marking in the 20 and 50% marked groups, both phenotypes were kept
Figure 2. Effects of artificial marking of 20, 50, or 100% of the flock on mean plasma catecholamine concentrations (±SEM) following manual restraint. Letters show differences at P < 0.05. a) Plasma epinephrine (EP) concentration (marking treatment, F4,121 = 3.43, P = 0.0108, n = 7); b) plasma dopamine (DA) concentration [group size (GS) by marking treatment, F4,181 = 6.08, P < 0.0001, n = 7]. A,B Differences between GS within marking treatment; a,bdifferences between treatment groups within GS.
together, thereby providing no opportunity for birds to develop exclusive familiarity with members of their own phenotype. If phenotypic preference was based on greater familiarity with the most common phenotype in the population, it would not explain why marked birds received more aggression than unmarked birds in 50% marked groups where both phenotypes occurred at equal frequency. Therefore, xenophobia based on phenotypic dissimilarity provides an implausible explanation for our findings.
Marks Perceived as Signals of Low or High Status Artificial marks could inadvertently signal low competitive ability similar to the way that dull or soiled plumage signals reduced fitness associated with parasitic infection (Hamilton and Zuk, 1982), injury, or pathogenic disease. Likewise, a small or artificially dubbed comb signals low status and attracts aggression (Marks et al., 1960; Siegel and Hurst, 1962; Cloutier and Newberry, 2000). In our study, the artificial marks could have had a similar effect, especially the
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Novelty can elicit fear and aggression (Marin et al., 2001), and handling, usually necessary for application of marks, can alter stress and behavior (Gariepy et al., 2002; Queiroz and Cromberg, 2006). Pecks at identification tags occurred primarily during period 1 and then declined to a low level, suggesting that these pecks were primarily associated with novelty of the tags during the initial period after their application and represented exploratory behavior rather than aggression. If elevated aggression toward marked birds was primarily driven by the novelty of artificially applied marks, or changes in behavior resulting from handling received during marking, we would have expected the level of threats and aggressive pecks directed toward the heads of marked birds to decline with age as observed for pecks at tags. In contrast, aggressive pecks remained elevated over time in a frequency-dependent manner according to the proportion of marked birds in the group. Moreover, the greater frequency of aggressive pecks and threats received by marked birds in 20 and 50% marked groups compared with 100% marked groups throughout the study provides evidence that aggression was not triggered simply by the application of novel marks but was, rather, influenced by the frequency of the marked phenotype within the population. Because aggressive behavior is generally directed toward the head region, and behavior specifically directed toward the identification tags was shortlasting, it is plausible that these effects of marking on aggression were attributable solely to the presence of the black mark on the back of the head. However, because we marked birds with both a black mark and identification tags, we cannot conclude from this study whether one of these forms of marking alone or both forms in combination were responsible for the effects on aggression.
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Social Challenge to Conspicuous Individuals Instinctive deference to individuals bearing conspicuous marks would enable rapid invasion by cheaters bearing such marks without accompanying fighting ability, leading to the idea that there are costs associated with status badge bearing that only honest signalers can withstand (Rohwer, 1982; Senar, 1998). If social challenge is one such cost (Tibbetts and Dale, 2004), birds bearing conspicuous artificial marks should attract elevated aggression relative to unmarked birds in the same flock and the fewer conspicuously marked birds in a population, the more challenges they should receive. Our results are consistent with this prediction. The lowest level of aggression was received by marked birds in the 100% marked flocks, where the marks were not conspicuous because they were held by all birds. Although we did not find a significant difference in aggression received by marked birds in the 20 and 50% marked groups, the latter did tend to receive levels of aggression intermediate between those in 20 and 100% marked groups. The question remains as to why aggression toward marked birds would continue over time in a randomly marked population with a theoretically equal chance of winning and losing challenges. Estevez et al. (2007)
emphasize the importance of an individual’s ability to recoup the cost of aggressive encounters in their decision to engage in these contests as an important factor in multiple different aggression models. If there is a social cost associated with badge holding (Rohwer and Rohwer, 1978; Cloutier and Newberry, 2002; Tibbetts and Dale, 2004) and, on average, badge holders were not of greater competitive ability than nonbadge holders and thus able to overcome this cost, one would expect that, on average, the condition of marked birds would deteriorate relative to that of unmarked birds, thereby increasing their probability of losing encounters (Cloutier and Newberry, 2000). Given that previous experience of losing is an important determinant of the outcome of future social encounters (Hsu and Wolf, 1999; Cloutier and Newberry, 2000; Beacham, 2003), losing encounters would further cement low status, explaining why marked birds gave less aggression than they received. In accordance with the proposal that marking and consequent increased aggressive challenge would reduce body condition (or fitness), we found that marked birds had lower BM than unmarked birds in both 20 and 50% marked groups, differences suggestive of stress (Nicol et al., 1999; Keeling et al., 2003). Lower BM in the marked birds may have been related to energy costs incurred in an attempt to avoid aggression, lower food intake due to monopolization of food resources by the more aggressive unmarked birds, or stress associated with low social status (Senar et al., 2000). Differences in BM were attenuated by 11 wk of age, probably because growth slows as birds approach their mature size allowing smaller birds to catch up in BM. Marking affected catecholaminergic reactivity at 11 wk of age. Marked birds in the 20% marked groups had a depressed EP response and unaltered NE response, suggestive of reduced stress-coping ability and a suppressed fight-or-flight response compared with their unmarked counterparts or birds in 100% marked groups (Figure 2a). As stress hormones, both EP and NE participate in several physiological and behavioral processes (Kvetnansky and Mikulaj, 1970; Dillon et al., 1992; Dobrakovova et al., 1993; Matt et al., 1997). Whereas acute stress is associated with elevated plasma EP (Matt et al., 1997; Wortsman, 2002), prolonged chronic or chronic intermittent stress regimens have been observed to lower EP concentrations without altering NE concentrations in rats (Kvetnansky and Mikulaj, 1970; Dobrakovova et al., 1993) and humans (Matthews et al., 2001; Grant et al., 2003). Sensitivity of the EP system has been shown following pharmacological and genetic manipulation of aggressiveness in laying hens without altering NE (Dennis et al., 2006). Our results are consistent with previous findings indicating a dissociated stress response between the functions of adrenal medullary (indicated by EP) and sympathetic neural (indicated by NE) activity. Suppression of sympathetic adrenal responsiveness is consistent
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black mark on the back of the head, because of its proximity to the socially significant comb. However, our birds were sexually immature, and signals of status are usually not evident at such a young age but become prominent at sexual maturity. Even if the marks were initially perceived as signaling low status, why would marked birds in the 20 and 50% marked groups continue to attract elevated aggression over time? We applied marks to randomly selected birds that, on average, should have been equally likely to win or lose fights. Therefore, we might have expected that birds would learn that marks were not reliable signals of low status and would no longer direct aggression differentially toward marked birds. Conversely, the marks may have been perceived as badges of status. Badges of status may be used as honest signals of dominance in large groups of domestic fowl in which establishment of dominance through fighting would be costly and inefficient (Pagel and Dawkins, 1997). In birds, conspicuously pigmented feather patches may provide a particularly potent signal of fighting ability, enabling discrimination of dominant individuals without risking injury and wasting energy engaging in fights (Senar, 1998; Senar and Camerino, 1998). If the artificial marks applied to our birds were perceived by onlookers as representing honest badges of status, we might have expected that birds bearing them would have received less aggression than unmarked birds in the same group; in fact, we found the reverse.
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Conclusion We conclude that application of artificial identification marks to only a proportion of the individuals in a group can alter aggressive behavior, BM, and endocrine parameters in the domestic fowl. Thus, when artificial marking is required for this species, both the number and proportion of birds marked within a study population should be taken into consideration in experimental design and interpretation of results. Further research will be needed to elucidate the social impact of form, color, and body location of different types of artificial marking. The domestic fowl is highly dependent on both vision and pecking with the beak for sensory information, and the extent to which marking affects species less dependent on these sensory modalities is as yet unknown. Nevertheless, our findings sound a cautionary note for animal researchers because, when only a subset of group members is artificially marked and used for data collection, the results obtained may not be representative of the population.
ACKNOWLEDGMENTS We thank L. Douglass (University of Maryland, College Park) for statistical analysis, the graduate students and laboratory staff of I. Estevez and H.-W. Cheng for technical assistance, and the farm crew at University of Maryland’s Upper Marlboro facility for animal care. This research was funded by a competitive grant from the Maryland Agriculture Experiment Station to I. Estevez.
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