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The dopaminergic system and aggression in laying hens
The dopaminergic system and aggression in laying hens R. L. Dennis*† and H. W. Cheng*1 *Livestock Behavior Research Unit, USDA-ARS, West Lafayette, IN 47907; and †Purdue University, Department of Animal Science, West Lafayette, IN 47907 trols (n = 12). In experiment 2, the dominant birds from a separate flock were treated with D1 antagonist, D2 antagonist, or saline controls (n = 12). Treatmentassociated changes in aggressive behaviors and central neurotransmitters were measured. Aggression was increased in all strains in response to D1 agonism but increased only in the less aggressive HGPS birds with D2 agonism. Aggression was decreased and hypothalamic serotonin and epinephrine were increased in birds from all strains treated with D2 receptor antagonist. The D1 receptor antagonism elicited different behavioral and neurotransmitter responses based on the aggressive phenotype of the genetic strains. Aggressive strains DXL and LGPS but not the HGPS strain decreased aggressiveness following antagonism of the D1 receptor. The data show evidence for distinct neurotransmitter regulation of aggression in high and low aggressive strains of hens through different receptor systems. These chicken lines could provide new animal models for the biomedical investigation of the genetic basis of aggression.
Key words: dopamine, laying hen, aggression 2011 Poultry Science 90:2440–2448 doi:10.3382/ps.2011-01513
INTRODUCTION Maintenance of birds at high stocking density allows producers to meet an increasing demand for eggs at the lowest, most competitive price in the market. Birds maintained in modern production systems can exhibit a high degree of aggression and feather pecking. Beak trimming is a common practice used to reduce damage from pecking. However, it is also a source of both pain for the birds and controversy with activists and consumers concerned with bird welfare (Hughes and Gentle, 1995). It has been suggested that the best solution for these problems is to breed animals that exhibit minimal aggression and cannibalism (Cheng and Muir, ©2011 Poultry Science Association Inc. Received March 29, 2011. Accepted July 30, 2011. 1 Corresponding author: [email protected]
2007). Several studies have identified gene variants that may predispose individuals to aggressive or feather pecking behaviors (Flisikowski et al., 2009; Biscarini et al., 2010). However, the cellular mechanisms of such behaviors in birds are still unclear and biomarkers for selection of such animals are severely lacking. Behavioral and physiological homeostasis of animals is controlled by neuronal activities in the central nervous system. The hypothalamus is an essential region for the mediation of behavioral response to stimuli. It is critical in the neural mediation of the endocrine system (Kruk et al., 1998), and hypothalamic regulation of aggressive behavior has been shown in many species (Roberts and Kiess, 1964; Roeling et al., 1994). Electrical stimulation of distinct regions within the hypothalamus has been shown to elicit aggressive behavior in mammals (rats: Roeling et al., 1994; cats: Gregg and Siegel, 2001; Hassanain et al., 2005). Neurotransmission within the hypothalamus, especially via dopamine
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ABSTRACT The dopaminergic system is involved in the regulation of aggression in many species, especially via dopamine (DA) D1 and D2 receptor pathways. To investigate heritable differences in this regulation, 2 high aggressive strains [Dekalb XL (DXL) and low group egg productivity and survivability (LGPS)] and one low aggressive strain (low group egg productivity and survivability; HGPS) of laying hens were used in the study. The HGPS and LGPS lines were diversely selected using group selection for high and low group production and survivability. The DXL line is a commercial line selected through individual selection based on egg production. Heritable differences in aggressive propensity between the strains have been previously assessed. The birds were pair housed within the same strain and labeled as dominant or subordinate based on behavioral observation. For both experiments 1 and 2, behavioral analysis was performed on all 3 strains whereas neurotransmitter analysis was performed only on the most aggressive (DXL) and least aggressive (HGPS) strains. In experiment 1, the subordinate birds were treated with D1 agonist, D2 agonist, or saline con-
AGGRESSION AND DOPAMINE
This study was designed to test our hypothesis that selection-induced changes of the dopaminergic systems play an important role in controlling animal behaviors, and that D1 and D2 receptors are involve to differing degrees in regulating processes of aggression in birds described previously as exhibiting high and low aggressiveness. The objectives of the present study were to investigate the genetic differences in neuroendocrine and behavioral change (specifically aggressive and other impulsive behaviors such as feather pecking) of high and low aggressive strains of birds to selective D1 and D2 receptor agonists and antagonists.
MATERIALS AND METHODS Genetic Strains and Experimental Birds Hens from DXL and LGPS strains have been shown to display high levels of aggressive behaviors compared with hens of the HGPS strain (Cheng and Muir, 2007). Birds were not beak trimmed, and 12 hens were kept in each cage. The birds were housed under high light intensity, which allowed aggressive behavior to be displayed more frequently. Group productivity was based on average rate of lay per cage, and longevity was based on average days of survival up to 72 wk of age. Birds were removed at the first sign of injury to either the head and neck (aggression initiated) or the cloaca and other areas (feather pecking initiated) and were counted as mortalities. Birds were chosen or rejected for future breeding based on cage production and longevity statistics (for a full review of selection process see Cheng and Muir, 2007). The eleventh selected generation of birds from the HGPS and LGPS as well as DXL strains was used in this study. The DXL (or Dekalb XL) strain is a commercial strain individually selected based on production criteria by industry poultry breeders. This colony has been maintained without further active selection since 1987 at Purdue University. Both DXL and LGPS hens display more aggressive behaviors compared with HGPS birds (Cheng and Muir, 2007). Early behavioral analysis showed HGPS hens displayed less agonistic behaviors but similar levels of both gentle and severe feather pecking compared with DXL [Craig and Muir, 1996a,b; strains are referred to as S (HGPS) and X (DXL) in select cited references]. In the study, bird care guidelines were in strict accordance with the rules and regulations set by Craig et al. (1999). Experimental protocols were approved by the institutional Animal Care and Use Committee at Purdue University (protocol no. 00–008–06).
Experimental Design All birds were housed in grower cages within the same strain from 1 d of age. At 19 wk of age, the birds were transferred from the grower facility to the layer facility and randomly paired within the same strain in novel
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(DA) and serotonin (5-HT), has been shown to mediate different types of aggressive responsiveness (Nelson and Chiavegatto, 2001; Nelson and Trainor, 2007). Increased DA concentrations have been shown to be related to increased aggression in many species (chicken: Cheng et al., 2003; mice: Nikulina and Kapralova, 1992; rats: Ferrari et al., 2003). Aggression can be a reinforcing behavior, especially through mesocorticolimbic DA via the D1 and D2 receptors (Miczek et al., 2002; Couppis and Kennedy, 2008). Agonism of the D1 receptor has previously been shown to decrease aggressiveness in mice without altering locomotor abilities and differently from the D2 receptor mediation (Tidey and Miczek, 1992). Investigation of high and low aggressive strains of rats found that agonism of the D2 receptor increased stimulation-induced aggression in low aggressive strains (Nikulina and Kapralova, 1992). Excitation of D2 receptor pathways may mediate the aggression differently in high and low aggressive strains. These findings of the interactions of neurotransmitters and aggressiveness in mammals should give us clues regarding the functions of neurotransmitters in controlling bird aggression because central neuronal morphofunctions are similar between mammals and birds (Davidson et al., 2000; Tramontin and Brenowitz, 2000). To examine the cellular mechanism of genetic basis of stress responses and aggression, 2 genetically selected lines of White Leghorn chickens were developed at Purdue University, West Lafayette, Indiana (Craig and Muir, 1996a,b; Muir, 1996; Muir and Craig, 1998; Cheng et al., 2001a,b). These lines were diversely selected based on high (HGPS) or low (LGPS) group egg productivity and survivability, resulting in part from high or low aggression and cannibalism, in colony cages (Craig and Muir, 1996a,b; Craig et al., 1999). Previous studies have found that the HGPS hens (referred to in select references as KGB) were less aggressive and had an improved rate of lay, survival, and feather score as well as reduced cannibalism and flightiness compared with hens from the Dekalb XL (DXL) a commercial line (individually selected) and the reverse-selected (group selected) LGPS line (Craig and Muir, 1996a,b; Cheng et al., 2001a; Cheng and Muir, 2007). Compared with hens from the DXL line, HGPS hens also had better and faster adaptation to various stressors such as social interaction, handling, cold, and heat (Hester et al., 1996a,b,c). In addition, HGPS hens displayed greater cell-mediated immunity and a higher ratio of CD4+:CD8+ T cells, whereas LGPS hens exhibited eosinophilia and heterophilia and had a greater heterophil:lymphocyte ratio in single-hen cages (Cheng et al., 2001b). Eosinophilia and heterophil:lymphocyte ratio have been used as stress indicators in animals, including chickens (Gross and Siegel, 1983; Maxwell, 1993; Woolaston et al., 1996; Hohenhaus et al., 1998). Collectively, genetic selection has resulted in lines with significantly different phenotypes, each of which has unique characteristics in physical indices, behavior, and resistance to stressors.
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cm2/bird.
Behavioral Observations In experiment 1, pairs were observed live in their home cages for frequency of aggressive behaviors and feather pecking. Each pair was observed for 20 min/d for 3 out of the 5 d before drug administration and 3 out of the 5 d during drug administration to the least aggressive hen in each cage (the drugs were administered for all 5 d). Time used for behavioral observations was based on previous studies on aggression in the present strains (Dennis et al., 2006). Days of observation were assigned in a randomized block design over the 5-d period. This was done so that all observations could be made by the same observer during the lights-on hours. Live observations were taken by the same observer to eliminate between-observer variability. Observations were performed one cage at a time in a randomized complete block design in time and location within the room (3 observations/cage were taken over 5 consecutive days between 0600 and 2000 h; injections were delivered immediately before the observation period). In experiment 2, pairs were video recorded for 3 d before injection and d 1 through 5 of injection to the most aggressive hen in each cage. Observations were taken for
1 h/d. Recorded behaviors were used for observations in experiment 2 to obtain more information than was possible using live observations with a single observer. This change was made in an attempt to improve the power of behavioral analysis.
Aggressive Behaviors Frequency of aggressive behaviors was analyzed as the number of incidents of aggressive pecking and threats delivered to conspecifics over a 20-min period. Aggression toward cage mate and hens in neighboring cages were both counted. Aggressive pecking was defined as forceful downward pecks directed at the head or neck of other birds. A threat was defined as one bird standing with its neck erect and hackle feathers raised (maybe only slightly) in front of another bird. Feather pecking was defined as one bird pecking at feathers of another bird; feather pecking could be gentle (nibbling or gentle pecking in which feathers are not removed or pulled) or severe (vigorous pecking to feathers in which feathers are often pulled, broken, or removed). Severe pecking in the present trials was so rare that both types of feather pecking were analyzed together.
HPLC Assay: Central 5-HT, Epinephrine, Norepinephrine, and DA, and Their Metabolites In behavioral analysis of control hens, DXL and HGPS hens were found to be the most distinct. The DXL strain was the most aggressive and HGPS was the least aggressive. Therefore, neurotransmitter analysis was performed only on hens from these 2 strains. The brains of the hens (DXL and HGPS only) were removed immediately following cervical dislocation; the dorsal raphe nucleus and the hypothalamus were dissected using a stereotaxic atlas (Kuenzel and Masson, 1988; Puelles et al., 2007) and were maintained at −80°C until they were prepared for assay by HPLC. The hypothalamus was selected because of its regulatory control over aggression and other pecking behaviors that are, in large part, mediated by DA and 5-HT, which intervening processes arise from the raphe nucleus. Central 5-HT, epinephrine (EP), norepinephrine (NE), DA and their metabolites, 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid, and homovanillic acid (HVA) were measured in duplicate from brain samples. Samples were acidified in duplicate using a 10:1 dilution of 0.2 M perchloric acid and 3% ascorbic acid. Samples were centrifuged and diluted with 50% mobile phase before being filtered through a 0.2-µg nylon filter. The mobile phase flow rate was 1.2 mL/min, and the concentration of neurotransmitters and metabolites was calculated from a reference curve made using standards; peaks were removed if they deviated from the range set by the standard curve of 0.1 to 300 ng/mL. Hens measuring outside of this range were
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cages allowing 1,000 Behavioral observations were taken live in experiment 1 and by video recordings in experiment 2. Hens were marked with blue or green livestock marker for individual identification. The light schedule was 16L:8D, and feed and water were available ad libitum throughout the study. Birds were visually inspected daily and any birds with injuries or appearing ill were removed from the study. Experiment 1. At 21 wk of age, the least aggressive bird (determined through behavior observations before administering treatment as described below) from each pair received a daily intraperitoneal injection of one of the following chemicals for 5 consecutive days: D1 agonist SKF-38393 (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich, St Louis, MO; n = 12/strain), D2 agonist quinpirole (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich; n = 12/strain), or saline vehicle only (control; n = 12/ strain). Body weight was taken at the beginning of the study to determine proper dosage. Experiment 2. Using a separate set of birds at 21 wk of age, the most aggressive individual from each pair received a daily intraperitoneal injection of the D1 antagonist SCH-23390 (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich; n = 12/strain), D2 antagonist raclopride (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich; n = 12/strain), or saline vehicle only (control; n = 12/ strain) for 5 consecutive days. Body weight was taken at the beginning of the study to determine proper dosage. Based on observations taken during experiment 1, body weight was taken again at just before sampling to determine weight gain or loss during the 5-d trial. Agonists and antagonists were selected based on selectivity, availability, and ability to cross the blood brain barrier.
AGGRESSION AND DOPAMINE
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removed from all analyses, including behavior. Concentrations were obtained as nanograms per milliliter.
Statistics Behavior data were analyzed using a mixed model repeated measure ANOVA for differences over time of days of treatment. Log transformation was used in ANOVA of HPLC analysis data. The appropriate transformations (log and square root) were used in analysis of behavioral data as needed. The statistical model used was
+ (day; d)k + (animal; a)l(ij) + (Gd)ik + (Td)jk + (GTd)ijk + (ad)ijkl. Original (untransformed) least squares means and SEM were reported for all groups. Contrasts were used to determine significance using the Bonferroni adjustment to maintain an experimental α of 0.05 (0.10 was considered a trend). Data were analyzed using PROC MIXED of SAS 8.2 software (SAS Institute, Cary, NC). Main effects included genetic strains, agonist or antagonist treatment, and observation day as the repeated measure for behavior data. All interactions between main effects were considered and were removed if the P-value was greater than 0.25.
RESULTS Behavior Experiment 1. Agonist of the D1 receptor, SKF38393, increased aggressive behavior in all tested birds from the 3 strains, whereas D2 agonist, quinpirole, increased aggressive behavior in HGPS birds only [P < 0.05; Figure 1; F(4,78.5) = 2.5]. The D1 agonist increased feather pecking in DXL and HGPS birds but not LGPS birds (P < 0.05; Figure 2). The D2 receptor agonism did not alter feather pecking behavior in any of the strains used (P > 0.05; Figure 2). Experiment 2. Dopamine D1 receptor antagonism decreased aggression only in the high aggressive strains LGPS and DXL [P < 0.05; Figure 3; F(4,69) = 4.89]. The D2 antagonism decreased aggression in all strains tested (P < 0.05; Figure 3). A main effect of treatment showed a tendency for a reduction in feather pecking following D1 and D2 receptor antagonists across all strains [P < 0.10; Figure 4; F(2,71.2) = 2.42].
Figure 1. Effects of dopamine D1 and D2 agonists on aggressive behaviors of subordinate birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean aggressive behaviors (±SEM). Asterisk denotes significant difference (P < 0.05) from control mean.
F(2,32) = 1.56 and F(2,29) = 1.34; EP, F(2,32) = 0.45 and F(2,29) = 0.01; NE, F(2,32) = 0.16 and F(2,29) = 0.08). In addition, D1 and D2 agonists did not affect the concentrations of dopaminergic and serotonergic transmitter expression and turnover in the hypothalamus and raphe nucleus in all the tested birds from all the 3 examined strains [P > 0.05; Table 1; 5-HT turnover, F(2,32) = 0.28 and F(2,29) = 1.55; DA turnover, F(2,32) = 0.80 and F(2,29) = 0.77]. In experiment 1 using subordinate birds, we noted a strain effect in the analysis of hypothalamic 5-HIAA, with HGPS hens having consistently lower concentrations (P < 0.05). Experiment 2. In the hypothalamus, EP concentrations were increased in both DXL and HGPS strains following antagonism of D2 receptor [P < 0.05; Table 2; F(2,40) = 4.06]. In DXL hens, hypothalamic 5-HT concentrations were increased following antagonism of
Physiology Experiment 1. No change was found in the concentrations of 5-HT, DA, and catecholamines (EP and NE) in the hypothalamus and raphe nucleus of the tested birds following both D1 and D2 agonists [P > 0.05; Table1; 5-HT, F(2,32) = 0.78 and F(2,29) = 0.45; DA,
Figure 2. Effects of dopamine D1 and D2 agonists on feather pecking of subordinate birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean feather pecking (±SEM). Asterisk denotes significant difference (P < 0.05) from control mean.
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xijkl = (genetic strain; G)i + (treatment; T)j + GTij
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D1 and D2 receptors [P < 0.05; Table 2; F(2,38) = 5.28]. In HGPS hens, hypothalamic 5-HT concentrations were increased following antagonism of the D2 receptors only [P < 0.05; Table 2; F(2,38) = 5.28]. In the raphe nucleus, no effect of treatment was found on 5-HT levels [P > 0.05; Table 2; F(2,38) = 0.50]; however, the metabolite 5-HIAA concentrations were increased in HGPS hens following D1 antagonism [P < 0.05; Table 2; F(2,40) = 3.80]. In experiment 2 us-
ing dominant hens, we noted strain effects. The HGPS hens had consistently higher raphe nucleic HVA concentrations and hypothalamic HVA and 3,4-dihydroxyphenylacetic acid concentrations compared with DXL hens [P < 0.05; Table 2; F(2,40) = 6.39 and F(2,40) = 4.58 and 4.79]. Body weight gain was determined in experiment 2. The DXL and LGPS hens treated with D1 antagonist gained significantly more weight over the 5-d period than saline controls (P < 0.05). Saline-treated DXL, LGPS, and HGPS hens gained 33 ± 12, 30 ± 12, and 41 ± 13 g, respectively. The D1 antagonist DXL, LGPS,
Table 1. Central serotonin and catecholamine concentrations (ng/mL) of subordinate hens1 treated with dopamine D1 agonist, D2 agonist, or saline control2 Control Item Raphe nucleus Norepinephrine Epinephrine Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid Dopamine turnover Serotonin 5-Hydroxyindoleacetic acid Serotonin turnover Hypothalamus Norepinephrine Epinephrine Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid Dopamine turnover Serotonin 5-Hydroxyindoleacetic acid3 Serotonin turnover 1DXL
DXL
D1 agonist HGPS
DXL
D2 agonist HGPS
DXL
60 9.4 2.2 85 4.7 83 51 18 4.8
± ± ± ± ± ± ± ± ±
15.6 2.10 0.57 36.8 2.02 59.8 17.5 3.0 2.53
68 6.8 1.5 104 3.8 176 25 12 3.0
± ± ± ± ± ± ± ± ±
18.4 2.49 0.67 43.5 2.39 70.7 26.7 3.5 3.87
64 11.7 2.7 33 5.7 47 51 10 0.4
± ± ± ± ± ± ± ± ±
18.4 2.49 0.67 43.5 2.39 70.7 20.7 3.9 3.35
54 9.6 2.4 92 4.2 130 56 18 6.4
± ± ± ± ± ± ± ± ±
15.6 2.10 0.57 36.8 2.02 59.8 17.5 3.0 2.53
57 8.9 2.1 62 2.8 119 52 17 5.9
± ± ± ± ± ± ± ± ±
15.6 2.10 0.57 36.8 2.02 59.8 17.5 3.0 2.53
54 7.2 3.4 40 1.9 64 65 21 2.1
± ± ± ± ± ± ± ± ±
16.8 2.27 0.61 39.7 2.18 64.6 20.1 3.2 3.00
100 19 7.1 99 15.5 67 51 20.6 2.8
± ± ± ± ± ± ± ± ±
28.6 7.20 2.04 43.0 5.97 31.4 20.5 2.87 0.97
114 31 5.6 149 12.5 119 40 19.1 3.2
± ± ± ± ± ± ± ± ±
33.0 8.31 2.35 49.7 6.89 36.9 22.2 3.31 1.05
72 22 6.2 72 6.4 70 77 19.7 1.9
± ± ± ± ± ± ± ± ±
46.7 11.76 3.33 70.3 9.75 51.3 31.4 4.68 1.49
127 32 6.1 134 14.9 70 62 14.4 1.6
± ± ± ± ± ± ± ± ±
30.5 7.70 2.18 46.0 6.4 33.6 20.5 3.06 0.97
122 26 4.0 95 9.6 73 47 17.5 2.3
± ± ± ± ± ± ± ± ±
28.6 7.20 2.04 43.0 5.97 31.4 19.2 2.87 0.91
112 29 9.0 44 4.8 22 89 16.7 0.9
± ± ± ± ± ± ± ± ±
33.0 8.31 2.35 49.7 6.89 36.3 24.3 3.31 1.15
= DeKalb XL, high aggressive strain; HGPS = high group egg productivity and survivability, low aggressive strain. as least squares means ± SEM. 3Main effect of strain (P < 0.05). 2Reported
HGPS
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Figure 3. Effects of dopamine D1 and D2 antagonists on aggressive behavior of dominant birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean aggressive behavior (±SEM). Asterisk indicates significant difference (P < 0.05) from control mean.
Figure 4. Effects of dopamine D1 and D2 antagonists on feather pecking of dominant birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean feather pecking (±SEM).
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AGGRESSION AND DOPAMINE Table 2. Central serotonin and catecholamine concentrations (ng/mL) of dominant antagonist, or saline control2 Control Item
DXL 33 5.1 1.7 1.1 0.3
± ± ± ± ±
7.9 0.87ab 0.46 0.26 0.10
D1 antagonist HGPS
21 6.4 2.5 2.0 0.4
treated with dopamine D1 antagonist, D2
± ± ± ± ±
9.6 1.07b 0.56 0.32 0.12
DXL 41 6.0 2.6 1.6 0.4
± ± ± ± ±
7.9 0.87b 0.46 0.26 0.11
D2 antagonist
HGPS 44 5.7 2.5 1.7 0.4
± ± ± ± ±
8.9 0.99ab 0.52 0.29 0.11
DXL 37 4.1 2.4 0.9 0.4
± ± ± ± ±
8.3 0.92a 0.49 0.28 0.11
HGPS 34 5.8 2.2 1.7 0.4
± ± ± ± ±
8.9 0.99ab 0.52 0.29 0.11
1.3 ± 0.37 71 ± 17.2 9 ± 2.9
1.4 ± 0.45 89 ± 18.6 9 ± 3.6
0.8 ± 0.39 74 ± 16.1 12 ± 2.9
0.9 ± 0.41 71 ± 18.6 18 ± 3.3
0.6 ± 0.39 68 ± 16.1 13 ± 3.1
0.6 ± 0.39 100 ± 20.3 13 ± 3.3
85 ± 69.8
141 ± 75.4
36 ± 65.3
122 ± 75.4
13 ± 65.3
1 ± 82.6
66 17.3 5.1 2.7 0.47
± ± ± ± ±
19.8 4.69a 1.40 0.44 0.094a
63 12.7 6.0 3.0 0.62
± ± ± ± ±
24.2 5.74a 1.72 0.53 0.116ab
77 15.3 6.7 2.9 0.59
± ± ± ± ±
19.8 4.69a 1.40 0.44 0.094ab
66 15.8 7.2 3.6 0.69
± ± ± ± ±
22.4 5.31a 1.59 0.49 0.107b
84 27.9 7.0 3.3 0.65
± ± ± ± ±
21.0 4.97b 1.49 0.46 0.100b
95 28.1 9.0 3.0 0.89
± ± ± ± ±
22.4 5.31b 1.59 0.49 0.107b
1.1 ± 1.46 42 ± 14.1a 6.7 ± 2.54
1.0 ± 0.79 50 ± 17.2a 8.8 ± 3.11
3.8 ± 1.46 95 ± 16.0b 9.8 ± 2.54
0.8 ± 1.66 76 ± 16.0ab 9.3 ± 2.88
0.6 ± 1.55 93 ± 14.9b 10.5 ± 2.69
0.6 ± 1.66 95 ± 16.0b 5.6 ± 2.88
44 ± 17.0
25 ± 20.8
1 ± 19.3
30 ± 19.3
17 ± 18.0
1 ± 19.3
a,bMeans
with different superscripts are significantly different (P < 0.05). 1DXL = DeKalb XL, high aggressive strain; HGPS = high group egg productivity and survivability, low aggressive strain. 2Reported as least squares means ± SEM. 3Rows indicate treatment differences (P < 0.05). 4Rows indicate main effect of strain (P < 0.05).
and HGPS hens gained 59 ± 12, 61 ± 13, and 28 ± 13 g, respectively. The D2 antagonist DXL, LGPS, and HGPS hens gained 45 ± 12, 22 ± 13, and 15 ± 13 g, respectively, and showed no significant difference from control (P > 0.05).
DISCUSSION Dopaminergic involvement in aggression mediation has been shown in many studies (Dennis et al., 2006; Nelson and Trainor, 2007; Couppis and Kennedy, 2008; Seo et al., 2008). Hyperactivity in the dopaminergic system is associated with increased aggression in animals and humans (Netter and Rammsayer, 1991; Friedel, 2004). In addition, locomotor and stationary forms of social behaviors can both be altered by pharmaceutical manipulation of the D1 receptor (Gendreau et al., 1997). The D1 agonism elicited different social and emotional behavioral responses in mice bred for high and low activity level, suggesting a link in D1 control over locomotor behaviors and social response (Gendreau et al., 1997). In the present strains, agonism of the D1 receptor increased aggression in all strains observed. Although overall activity levels were not investigated in this study, it is possible that an increase in activity in all birds treated with D1 agonist resulted in an increase in aggressive social encounters. However, we would also expect to see an increase in other active behaviors such as feather pecking, which was not
increased in all strains. Agonism of D1 receptor pathways can also cause a disturbance of memory, as seen in avoidance tasks and resistance to amnesia in some mice (Dubrovina, 2006). Alterations to social memory can also affect performance of social behaviors such as aggression (Marino et al., 2005) separately from feather pecking behaviors. Strain-based effectiveness of D1 antagonism in regulating aggression may be directly related to D1 receptor density or indirectly related through sensitivity of its downstream effects, including increased central serotonin response in DXL birds. The paraventricular nucleus of the hypothalamus is a critical region involved in the food reward system; stimulation of this region with neuropeptide Y increases weight gain and feeding behavior (Stanley et al., 1989). Increased weight gain in the DXL and LGPS birds suggests a potential role of the paraventricular nucleus or other feed intake-regulating regions of the hypothalamus. Increased pecking in domestic hens has been suggested to be related to misdirected foraging behaviors (Huber-Eicher and Wechsler, 1997). Increased 5-HIAA concentration was also evident in the raphe nucleus of the HGPS birds treated with D1 antagonist. Reduced 5-HIAA concentrations in human cerebrospinal fluid have been shown to be associated with a history of aggressive and suicidal behaviors (Brown et al., 1982). Increased 5-HIAA in these birds suggests an increase in 5-HT turnover in response to dopaminergic manipulation. This reduction
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Raphe nucleus Norepinephrine Epinephrine3 Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid Dopamine turnover Serotonin 5-Hydroxyindoleacetic acid 5-Serotonin turnover Hypothalamus Norepinephrine Epinephrine3 Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid3,4 Dopamine turnover Serotonin3 5-Hydroxyindoleacetic acid 5-Serotonin turnover
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Increased central concentrations of 5-HT are most frequently associated with reduced levels of aggression (Nelson and Chiavegatto, 2001). Serotonin is one of the primary moderators of aggressive behavior. Previous and ongoing research in these birds has also identified heritable differences in the response to pharmaceutical alterations of the 5-HT system (Dennis et al., 2008). Reduced hypothalamic EP responsiveness is seen during acute and chronic stress (Roth et al., 1982). Increased hypothalamic EP coinciding with reduced aggressiveness may be suggestive of a less stressful social environment. Increased hypothalamic EP response is also seen with increased 5-HT levels following 5-HT agonism in rats (Hemrick-Luecke and Fuller, 1995). Epinephrine response can also indicate activation of a reward center (Kalra et al., 1988). Increased hypothalamic secretions of EP into the portal system may help modulate anterior pituitary regulation of stress coping (Gibbs, 1985). Alterations to the reward system via the D2 receptor is the most shared pathway for controlling aggressiveness between the present strains. In conclusion, our findings suggest the importance of genotype or epigenotype in understanding the mechanisms regulating these behaviors and in their pharmaceutical manipulation. Our results also suggest the potential for using indicators such as different response to pharmaceutical receptor agonism for more efficiently selecting for low aggressive behaviors.
ACKNOWLEDGMENTS The authors thank the technicians at the Livestock Behavior Research Unit of the USDA (West Lafayette, IN), as well as Fred Haan and the staff of the Purdue University Poultry Facility (West Lafayette, IN) for their outstanding assistance. We also thank Paul Collodi, Robert Meisel, and William Muir for their consultation. The work was supported by a grant of USDANRI #2032.
REFERENCES Biscarini, F., H. Bovenhuis, J. van der Poel, T. B. Rodenburg, A. P. Jungerius, and J. A. M. van Arendonk. 2010. Across-line SNP association study for direct and associative effects on feather damage in laying hens. Behav. Genet. 40:715–727. Brown, G. L., M. H. Ebert, P. F. Goyer, D. C. Jimerson, W. J. Klein, W. E. Bunney, and F. K. Goodwin. 1982. Aggression, suicide, and serotonin: Relationships to CSF amine metabolites. Am. J. Psychiatry 139:741–746. Calogero, A. E., G. Bagdy, and R. D’Agata. 1998. Mechanisms of stress on reproduction: Evidence for a complex intra-hypothalamic circuit. Ann. N. Y. Acad. Sci. 851:364–370. Chen, T. J., K. Blum, D. Mathews, L. Fisher, N. Schnautz, E. Braverman, J. Schoolfield, B. Downs, and D. Comings. 2005. Are dopaminergic genes involved in a predisposition to pathological aggression? Hypothesizing the importance of “super normal controls” in psychiatricgenetic research of complex behavioral disorders. Med. Hypotheses 65:703–707. Cheng, H. W., G. Dillworth, P. Singleton, Y. Chen, and W. M. Muir. 2001a. Effect of genetic selection for productivity and longevity
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in 5-HT was accompanied by a reduction in feather pecking but not by alterations to hypothalamic 5-HT or aggressive changes. These data suggests that mediation of feather pecking in HGPS birds is distinct from that mediating aggressiveness. Feather pecking behaviors can be altered by manipulation of the dopaminergic system (Kjaer et al., 2004). The D1 agonism resulted in increases in stereotypic feather pecking in DXL and HGPS birds but not in LGPS birds. Such maladaptive stereotypic behaviors are often associated with anxiety-related behaviors. D1 agonism has been shown to interact with both the central serotonergic and adrenal functions, and heritable factors can influence both the type and extent of response. Treatment-associated feather pecking increases seen in DXL and HGPS birds may be more closely related to heritable similarities in these strains that may be correlated to high productivity. Both DXL and HGPS hens share a phenotype for high production parameters, and previous studies have suggested a link between stereotypic behaviors and high production-related stressors. Increased demand on the hen to produce could alter the dopaminergic system through interactions with reproductive systems (Calogero et al., 1998) Agonism of the D2 receptor showed no effect on aggressiveness in either of the high aggression strains, increasing aggression only in HGPS birds. Similar to our findings, an investigation of high and low aggressive strains of rats found that agonism of the D2 receptor increased stimulation-induced aggression in only the low aggressive strains (Nikulina and Kapralova, 1992). Excitation of D2 receptor pathways may mediate aggression differently in high and low aggressive strains. Analysis of the 2 isoforms of the D2 receptor, D2L (long) and D2S (short), evidenced that D2L plays a vital role in mediating offensive aggressiveness (Vukhac et al., 2001). Human studies have also identified a specific isoform of the dopamine transporter gene (DAT1) that is related to increased aggressiveness (Chen et al., 2005). Strain differences in relative isoform expression could greatly affect their response to receptor excitation. The functions of D2 receptor and its subtypes in mediating behavior in the present strains will be examined in further investigations. Antagonism of the D2 receptors can prevent a behavior, such as aggression, from being perceived as a reinforcing or rewarding behavior through the D2 pathway (Couppis and Kennedy, 2008). In this way all strains may have reduced their frequency of aggressive and feather pecking behaviors because of the lack of positive reinforcement. A reduction in D2 receptor density, as is frequently seen in aggressive individuals, may reduce the effectiveness of D2 agonism, resulting in an increase in aggression only in the less aggressive HGPS strain. Antagonism of the D2 receptor also increased hypothalamic EP and 5-HT concentrations in all strains.
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Herpfer, I., and K. Lieb. 2005. Substance P receptor antagonists in psychiatry: Rationale for development and therapeutic potential. CNS Drugs 19:275–293. Hester, P. Y., W. M. Muir, and J. V. Craig. 1996a. Group selection for adaptation to multiple-hen cages: Humoral immune response. Poult. Sci. 75:1315–1320. Hester, P. Y., W. M. Muir, J. V. Craig, and J. L. Albright. 1996b. Group selection for adaptation to multiple-hen cages: Hematology and adrenal function. Poult. Sci. 75:1295–1307. Hester, P. Y., W. M. Muir, J. V. Craig, and J. L. Albright. 1996c. Group selection for adaptation to multiple-hen cages: Production traits during heat and cold exposures. Poult. Sci. 75:1308– 1314. Hohenhaus, M. A., M. J. Josey, C. Dobson, and P. M. Outteridge. 1998. The eosinophil leucocyte, a phenotypic marker of resistance to nematode parasites, is associated with calm behaviour in sheep. Immuno. Cell Biol. 76:153–158. Huber-Eicher, B., and B. Wechsler. 1997. Feather pecking in domestic chicks: Its relation to dustbathing and foraging. Anim. Behav. 54:757–768. Hughes, B. O., and M. J. Gentle. 1995. Beak trimming of poultry: Its implications for welfare. World’s Poult. Sci. J. 51:51–61. Kalra, S. P., J. T. Clark, A. Sahu, M. G. Dube, and P. S. Kaira. 1988. Control of feeding and sexual behaviors by neuropeptide Y: Physiological implications. Synapse 2:254–257. Kjaer, J. B., B. M. Hjarvard, K. H. Jensen, J. Hansen-Moller, and O. N. Larsen. 2004. Effects of haloperidol, a dopamine D2 receptor antagonist, on feather pecking behaviour in laying hens. Appl. Anim. Behav. Sci. 86:77–91. Kruk, M. R., K. G. C. Westphal, A. M. M. Van Erp, J. van Asperen, B. J. Cave, E. Slater, J. de Koning, and J. Haller. 1998. The hypothalamus: Cross-roads of endocrine and behavioural regulation in grooming and aggression. Neurosci. Biobehav. Rev. 23:163–177. Kuenzel, W. J., and M. Masson. 1988. A Stereotaxic Atlas of the Brain of the Chick (Gallus domesticus). Johns Hopkins University Press, Baltimore, MD. Marino, M. D., B. N. Bourdelat-Parks, L. C. Liles, and D. Weinshenker. 2005. Genetic reduction of noradrenergic function alters social memory and reduces aggression in mice. Behav. Brain Res. 161:197–203. Maxwell, M. H. 1993. Avian blood leucocyte responses to stress. World’s Poult. Sci. J. 49:34–43. Miczek, K. A., E. W. Fish, J. F. de Bold, and R. M. M. de Almeida. 2002. Social and neural determinants of aggressive behaviour: Pharmacotherapeutic targets at serotonin, dopamine and gamma-aminobutyric acid systems. Psychopharmaco. 163:434–458. Muir, W. M. 1996. Group selection for adaptation to multiplehen cages: Selection program and direct responses. Poult. Sci. 75:447–458. Muir, W. M., and J. V. Craig. 1998. Improving animal well-being through genetic selection. Poult. Sci. 77:1781–1788. Nelson, R. J., and S. Chiavegatto. 2001. Molecular basis of aggression. Trends Neurosci. 24:713–719. Nelson, R. J., and B. C. Trainor. 2007. Neural mechanisms of aggression. Nat. Rev. Neurosci. 8:536–546. Netter, P., and T. Rammsayer. 1991. Reactivity to dopaminergic drugs and aggression-related personality traits. Pers. Individ. Dif. 12:1009–1017. Nikulina, E. M., and N. S. Kapralova. 1992. Role of dopamine receptors in the regulation of aggression in mice: Relationship to genotype. Neurosci. Behav. Physiol. 22:364–369. Puelles, L., M. Martinez-de-la-Torre, G. Paxinos, C. Watson, and S. Martinez. 2007. The chick brain in stereotaxic coordinates. An atlas featuring neuromeric subdivisions and mammalian homologies. Elsevier. New York, NY. Roberts, W. W., and H. O. Kiess. 1964. Motivational properties of hypothalamic aggression in cats. J Comp. Physiol. Psychol. 58:187–193. Roeling, T. A., J. G. Veening, M. R. Kruk, J. P. Peters, M. E. Vermelis, and R. Nieuwenhuys. 1994. Efferent connections of the hypothalamic “aggression area” in the rat. Neuroscience 59:1001–1024.
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on blood concentrations of serotonin, catecholamine and corticosterone of chickens. Poult. Sci. 80:1278–1285. Cheng, H. W., S. D. Eicher, Y. Chen, P. Singleton, and W. M. Muir. 2001b. Effect of genetic selection for group productivity and longevity on immunological and hematological parameters of chickens. Poult. Sci. 80:1079–1086. Cheng, H. W., and W. M. Muir. 2007. Mechanisms of aggression and production in chickens: Genetic variations in the functions of serotonin, catecholamine, and corticosterone. World’s Poult. Sci. J. 63:233–254. Cheng, H. W., P. Singleton, and W. M. Muir. 2003. Social stress differentially regulates neuroendocrine responses in laying hens: I. Genetic basis of dopamine responses under three different social conditions. Psychoneuroendocrinology 28:597–611. Couppis, M. H., and C. H. Kennedy. 2008. The rewarding effect of aggression is reduced by nucleus accumbens dopamine receptor antagonism in mice. Psychopharmacology (Berl.) 197:449–456. Craig, J. V., W. F. Dean, G. B. Havenstein, K. K. Kruger, K. E. Nestor, G. H. Purchase, P. B. Siegel, and G. L. van Wicklen. 1999. Guidelines for poultry husbandry. Pages 55–56 in Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Federation of Animal Science Societies, Champaign, IL. Craig, J. V., and W. M. Muir. 1996a. Group selection for adaptation to multiple-hen cages: Behavioral responses. Poult. Sci. 75:1145–1155. Craig, J. V., and W. M. Muir. 1996b. Group selection for adaptation to multiple hen cages: Beak-related mortality, feathering, and body weight responses. Poult. Sci. 75:294–302. Davidson, R. J., D. C. Jackson, and N. H. Kalin. 2000. Emotion, plasticity, context, and regulation: Perspectives from affective neuroscience. Psychol. Bull. 126:890–909. Dennis, R. L., Z. Q. Chen, and H. W. Cheng. 2008. Serotonergic mediation of aggression in high and low aggressive chicken strains. Poult. Sci. 87:612–620. Dennis, R. L., W. M. Muir, and H. W. Cheng. 2006. Effects of raclopride on aggression and stress in diversely selected chicken lines. Behav. Brain Res. 175:104–111. Dubrovina, N. I. 2006. Effects of activation of D1 dopamine receptors on extinction of a conditioned passive avoidance reflex and amnesia in aggressive and submissive mice. Neurosci. Behav. Physiol. 36:679–684. Ferrari, P. F., A. M. van Erp, W. Tornatzky, and K. A. Miczek. 2003. Accumbal dopamine and serotonin in anticipation of the next aggressive episode in rats. Eur. J. Neurosci. 17:371–378. Flisikowski, K., H. Schwarzenbacher, M. Wysocki, S. Weigend, R. Preisinger, J. B. Kjaer, and R. Fries. 2009. Variation in neighbouring genes of the dopaminergic and serotonergic systems affects feather pecking behaviour of laying hens. Anim. Genet. 40:192–199. Friedel, R. O. 2004. Dopamine dysfunction in borderline personality disorder: A hypothesis. Neuropsychopharmacology 29:1029– 1039. Gendreau, P. L., J. M. Petitto, J. L. Gariepy, and M. H. Lewis. 1997. D1 dopamine receptor mediation of social and nonsocial emotional reactivity in mice: Effects of housing and strain difference in motor activity. Behav. Neurosci. 111:424–434. Gibbs, D. M. 1985. Hypothalamic epinephrine is released into hypophysial portal blood during stress. Brain Res. 335:360–364. Gregg, T. R., and A. Siegel. 2001. Brain structures and neurotransmitters regulating aggression in cats: Implications for human aggression. Prog. Neuropsychopharmacol. Biol. Psychiatry 25:91– 140. Gross, W. B., and H. W. Siegel. 1983. Evaluation of the heterophil/ lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27:972–979. Hassanain, M., S. Bhatt, S. Zalcman, and A. Siegel. 2005. Potentiating role of interleukin-1 beta (IL-1 beta) and IL-1 beta type 1 receptors in the medial hypothalamus in defensive rage behavior in the cat. Brain Res. 1048:1–11. Hemrick-Luecke, S. K., and R. W. Fuller. 1995. Decreased hypothalamic epinephrine concentration by quipazine and other serotonin agonists in rats. Biochem. Pharmacol. 49:323–327.
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Roth, K. A., I. M. Mefford, and J. D. Barchas. 1982. Epinephrine, norepinephrine, dopamine and serotonin: Differential effects of acute and chronic stress on regional brain amines. Brain Res. 239:417–424. Seo, D., C. J. Patrick, and P. J. Kennealy. 2008. Role of serotonin and dopamine system interactions in the neurobiology of impulsive aggression and its comorbidity with other clinical disorders. Aggress. Violent. Behav. 13:383–395. Stanley, B. G., K. C. Anderson, M. H. Grayson, and S. F. Leibowitz. 1989. Repeated hypothalamic stimulation with neuropeptide Y increases daily carbohydrate and fat intake and body weight gain in female rats. Physiol. Behav. 46:173–177.
Tidey, J. W., and K. A. Miczek. 1992. Morphine withdrawal aggression: Modification with D1 and D2 receptor agonists. Psychopharmacology (Berl.) 108:177–184. Tramontin, A. D., and E. A. Brenowitz. 2000. Seasonal plasticity in the adult brain. Trends Neurosci. 23:251–258. Vukhac, K. L., E. B. Sankoorikal, and Y. Wang. 2001. Dopamine D2L receptor- and age-related reduction in offensive aggression. Neuroreport 12:1035–1038. Woolaston, R. R., P. Manueli, S. J. Eady, I. A. Barger, L. F. Le Jambre, D. J. D. Banks, and R. G. Windon. 1996. The value of circulating eosinophil count as a selection criterion for resistance of sheep to trichostrongyle parasites. Int. J. Parasitol. 26:123–126.
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Key words: dopamine, laying hen, aggression 2011 Poultry Science 90:2440–2448 doi:10.3382/ps.2011-01513
INTRODUCTION Maintenance of birds at high stocking density allows producers to meet an increasing demand for eggs at the lowest, most competitive price in the market. Birds maintained in modern production systems can exhibit a high degree of aggression and feather pecking. Beak trimming is a common practice used to reduce damage from pecking. However, it is also a source of both pain for the birds and controversy with activists and consumers concerned with bird welfare (Hughes and Gentle, 1995). It has been suggested that the best solution for these problems is to breed animals that exhibit minimal aggression and cannibalism (Cheng and Muir, ©2011 Poultry Science Association Inc. Received March 29, 2011. Accepted July 30, 2011. 1 Corresponding author: [email protected]
2007). Several studies have identified gene variants that may predispose individuals to aggressive or feather pecking behaviors (Flisikowski et al., 2009; Biscarini et al., 2010). However, the cellular mechanisms of such behaviors in birds are still unclear and biomarkers for selection of such animals are severely lacking. Behavioral and physiological homeostasis of animals is controlled by neuronal activities in the central nervous system. The hypothalamus is an essential region for the mediation of behavioral response to stimuli. It is critical in the neural mediation of the endocrine system (Kruk et al., 1998), and hypothalamic regulation of aggressive behavior has been shown in many species (Roberts and Kiess, 1964; Roeling et al., 1994). Electrical stimulation of distinct regions within the hypothalamus has been shown to elicit aggressive behavior in mammals (rats: Roeling et al., 1994; cats: Gregg and Siegel, 2001; Hassanain et al., 2005). Neurotransmission within the hypothalamus, especially via dopamine
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ABSTRACT The dopaminergic system is involved in the regulation of aggression in many species, especially via dopamine (DA) D1 and D2 receptor pathways. To investigate heritable differences in this regulation, 2 high aggressive strains [Dekalb XL (DXL) and low group egg productivity and survivability (LGPS)] and one low aggressive strain (low group egg productivity and survivability; HGPS) of laying hens were used in the study. The HGPS and LGPS lines were diversely selected using group selection for high and low group production and survivability. The DXL line is a commercial line selected through individual selection based on egg production. Heritable differences in aggressive propensity between the strains have been previously assessed. The birds were pair housed within the same strain and labeled as dominant or subordinate based on behavioral observation. For both experiments 1 and 2, behavioral analysis was performed on all 3 strains whereas neurotransmitter analysis was performed only on the most aggressive (DXL) and least aggressive (HGPS) strains. In experiment 1, the subordinate birds were treated with D1 agonist, D2 agonist, or saline con-
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This study was designed to test our hypothesis that selection-induced changes of the dopaminergic systems play an important role in controlling animal behaviors, and that D1 and D2 receptors are involve to differing degrees in regulating processes of aggression in birds described previously as exhibiting high and low aggressiveness. The objectives of the present study were to investigate the genetic differences in neuroendocrine and behavioral change (specifically aggressive and other impulsive behaviors such as feather pecking) of high and low aggressive strains of birds to selective D1 and D2 receptor agonists and antagonists.
MATERIALS AND METHODS Genetic Strains and Experimental Birds Hens from DXL and LGPS strains have been shown to display high levels of aggressive behaviors compared with hens of the HGPS strain (Cheng and Muir, 2007). Birds were not beak trimmed, and 12 hens were kept in each cage. The birds were housed under high light intensity, which allowed aggressive behavior to be displayed more frequently. Group productivity was based on average rate of lay per cage, and longevity was based on average days of survival up to 72 wk of age. Birds were removed at the first sign of injury to either the head and neck (aggression initiated) or the cloaca and other areas (feather pecking initiated) and were counted as mortalities. Birds were chosen or rejected for future breeding based on cage production and longevity statistics (for a full review of selection process see Cheng and Muir, 2007). The eleventh selected generation of birds from the HGPS and LGPS as well as DXL strains was used in this study. The DXL (or Dekalb XL) strain is a commercial strain individually selected based on production criteria by industry poultry breeders. This colony has been maintained without further active selection since 1987 at Purdue University. Both DXL and LGPS hens display more aggressive behaviors compared with HGPS birds (Cheng and Muir, 2007). Early behavioral analysis showed HGPS hens displayed less agonistic behaviors but similar levels of both gentle and severe feather pecking compared with DXL [Craig and Muir, 1996a,b; strains are referred to as S (HGPS) and X (DXL) in select cited references]. In the study, bird care guidelines were in strict accordance with the rules and regulations set by Craig et al. (1999). Experimental protocols were approved by the institutional Animal Care and Use Committee at Purdue University (protocol no. 00–008–06).
Experimental Design All birds were housed in grower cages within the same strain from 1 d of age. At 19 wk of age, the birds were transferred from the grower facility to the layer facility and randomly paired within the same strain in novel
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(DA) and serotonin (5-HT), has been shown to mediate different types of aggressive responsiveness (Nelson and Chiavegatto, 2001; Nelson and Trainor, 2007). Increased DA concentrations have been shown to be related to increased aggression in many species (chicken: Cheng et al., 2003; mice: Nikulina and Kapralova, 1992; rats: Ferrari et al., 2003). Aggression can be a reinforcing behavior, especially through mesocorticolimbic DA via the D1 and D2 receptors (Miczek et al., 2002; Couppis and Kennedy, 2008). Agonism of the D1 receptor has previously been shown to decrease aggressiveness in mice without altering locomotor abilities and differently from the D2 receptor mediation (Tidey and Miczek, 1992). Investigation of high and low aggressive strains of rats found that agonism of the D2 receptor increased stimulation-induced aggression in low aggressive strains (Nikulina and Kapralova, 1992). Excitation of D2 receptor pathways may mediate the aggression differently in high and low aggressive strains. These findings of the interactions of neurotransmitters and aggressiveness in mammals should give us clues regarding the functions of neurotransmitters in controlling bird aggression because central neuronal morphofunctions are similar between mammals and birds (Davidson et al., 2000; Tramontin and Brenowitz, 2000). To examine the cellular mechanism of genetic basis of stress responses and aggression, 2 genetically selected lines of White Leghorn chickens were developed at Purdue University, West Lafayette, Indiana (Craig and Muir, 1996a,b; Muir, 1996; Muir and Craig, 1998; Cheng et al., 2001a,b). These lines were diversely selected based on high (HGPS) or low (LGPS) group egg productivity and survivability, resulting in part from high or low aggression and cannibalism, in colony cages (Craig and Muir, 1996a,b; Craig et al., 1999). Previous studies have found that the HGPS hens (referred to in select references as KGB) were less aggressive and had an improved rate of lay, survival, and feather score as well as reduced cannibalism and flightiness compared with hens from the Dekalb XL (DXL) a commercial line (individually selected) and the reverse-selected (group selected) LGPS line (Craig and Muir, 1996a,b; Cheng et al., 2001a; Cheng and Muir, 2007). Compared with hens from the DXL line, HGPS hens also had better and faster adaptation to various stressors such as social interaction, handling, cold, and heat (Hester et al., 1996a,b,c). In addition, HGPS hens displayed greater cell-mediated immunity and a higher ratio of CD4+:CD8+ T cells, whereas LGPS hens exhibited eosinophilia and heterophilia and had a greater heterophil:lymphocyte ratio in single-hen cages (Cheng et al., 2001b). Eosinophilia and heterophil:lymphocyte ratio have been used as stress indicators in animals, including chickens (Gross and Siegel, 1983; Maxwell, 1993; Woolaston et al., 1996; Hohenhaus et al., 1998). Collectively, genetic selection has resulted in lines with significantly different phenotypes, each of which has unique characteristics in physical indices, behavior, and resistance to stressors.
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cm2/bird.
Behavioral Observations In experiment 1, pairs were observed live in their home cages for frequency of aggressive behaviors and feather pecking. Each pair was observed for 20 min/d for 3 out of the 5 d before drug administration and 3 out of the 5 d during drug administration to the least aggressive hen in each cage (the drugs were administered for all 5 d). Time used for behavioral observations was based on previous studies on aggression in the present strains (Dennis et al., 2006). Days of observation were assigned in a randomized block design over the 5-d period. This was done so that all observations could be made by the same observer during the lights-on hours. Live observations were taken by the same observer to eliminate between-observer variability. Observations were performed one cage at a time in a randomized complete block design in time and location within the room (3 observations/cage were taken over 5 consecutive days between 0600 and 2000 h; injections were delivered immediately before the observation period). In experiment 2, pairs were video recorded for 3 d before injection and d 1 through 5 of injection to the most aggressive hen in each cage. Observations were taken for
1 h/d. Recorded behaviors were used for observations in experiment 2 to obtain more information than was possible using live observations with a single observer. This change was made in an attempt to improve the power of behavioral analysis.
Aggressive Behaviors Frequency of aggressive behaviors was analyzed as the number of incidents of aggressive pecking and threats delivered to conspecifics over a 20-min period. Aggression toward cage mate and hens in neighboring cages were both counted. Aggressive pecking was defined as forceful downward pecks directed at the head or neck of other birds. A threat was defined as one bird standing with its neck erect and hackle feathers raised (maybe only slightly) in front of another bird. Feather pecking was defined as one bird pecking at feathers of another bird; feather pecking could be gentle (nibbling or gentle pecking in which feathers are not removed or pulled) or severe (vigorous pecking to feathers in which feathers are often pulled, broken, or removed). Severe pecking in the present trials was so rare that both types of feather pecking were analyzed together.
HPLC Assay: Central 5-HT, Epinephrine, Norepinephrine, and DA, and Their Metabolites In behavioral analysis of control hens, DXL and HGPS hens were found to be the most distinct. The DXL strain was the most aggressive and HGPS was the least aggressive. Therefore, neurotransmitter analysis was performed only on hens from these 2 strains. The brains of the hens (DXL and HGPS only) were removed immediately following cervical dislocation; the dorsal raphe nucleus and the hypothalamus were dissected using a stereotaxic atlas (Kuenzel and Masson, 1988; Puelles et al., 2007) and were maintained at −80°C until they were prepared for assay by HPLC. The hypothalamus was selected because of its regulatory control over aggression and other pecking behaviors that are, in large part, mediated by DA and 5-HT, which intervening processes arise from the raphe nucleus. Central 5-HT, epinephrine (EP), norepinephrine (NE), DA and their metabolites, 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid, and homovanillic acid (HVA) were measured in duplicate from brain samples. Samples were acidified in duplicate using a 10:1 dilution of 0.2 M perchloric acid and 3% ascorbic acid. Samples were centrifuged and diluted with 50% mobile phase before being filtered through a 0.2-µg nylon filter. The mobile phase flow rate was 1.2 mL/min, and the concentration of neurotransmitters and metabolites was calculated from a reference curve made using standards; peaks were removed if they deviated from the range set by the standard curve of 0.1 to 300 ng/mL. Hens measuring outside of this range were
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cages allowing 1,000 Behavioral observations were taken live in experiment 1 and by video recordings in experiment 2. Hens were marked with blue or green livestock marker for individual identification. The light schedule was 16L:8D, and feed and water were available ad libitum throughout the study. Birds were visually inspected daily and any birds with injuries or appearing ill were removed from the study. Experiment 1. At 21 wk of age, the least aggressive bird (determined through behavior observations before administering treatment as described below) from each pair received a daily intraperitoneal injection of one of the following chemicals for 5 consecutive days: D1 agonist SKF-38393 (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich, St Louis, MO; n = 12/strain), D2 agonist quinpirole (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich; n = 12/strain), or saline vehicle only (control; n = 12/ strain). Body weight was taken at the beginning of the study to determine proper dosage. Experiment 2. Using a separate set of birds at 21 wk of age, the most aggressive individual from each pair received a daily intraperitoneal injection of the D1 antagonist SCH-23390 (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich; n = 12/strain), D2 antagonist raclopride (0.5 mg/kg in 1 mL of saline; Sigma-Aldrich; n = 12/strain), or saline vehicle only (control; n = 12/ strain) for 5 consecutive days. Body weight was taken at the beginning of the study to determine proper dosage. Based on observations taken during experiment 1, body weight was taken again at just before sampling to determine weight gain or loss during the 5-d trial. Agonists and antagonists were selected based on selectivity, availability, and ability to cross the blood brain barrier.
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removed from all analyses, including behavior. Concentrations were obtained as nanograms per milliliter.
Statistics Behavior data were analyzed using a mixed model repeated measure ANOVA for differences over time of days of treatment. Log transformation was used in ANOVA of HPLC analysis data. The appropriate transformations (log and square root) were used in analysis of behavioral data as needed. The statistical model used was
+ (day; d)k + (animal; a)l(ij) + (Gd)ik + (Td)jk + (GTd)ijk + (ad)ijkl. Original (untransformed) least squares means and SEM were reported for all groups. Contrasts were used to determine significance using the Bonferroni adjustment to maintain an experimental α of 0.05 (0.10 was considered a trend). Data were analyzed using PROC MIXED of SAS 8.2 software (SAS Institute, Cary, NC). Main effects included genetic strains, agonist or antagonist treatment, and observation day as the repeated measure for behavior data. All interactions between main effects were considered and were removed if the P-value was greater than 0.25.
RESULTS Behavior Experiment 1. Agonist of the D1 receptor, SKF38393, increased aggressive behavior in all tested birds from the 3 strains, whereas D2 agonist, quinpirole, increased aggressive behavior in HGPS birds only [P < 0.05; Figure 1; F(4,78.5) = 2.5]. The D1 agonist increased feather pecking in DXL and HGPS birds but not LGPS birds (P < 0.05; Figure 2). The D2 receptor agonism did not alter feather pecking behavior in any of the strains used (P > 0.05; Figure 2). Experiment 2. Dopamine D1 receptor antagonism decreased aggression only in the high aggressive strains LGPS and DXL [P < 0.05; Figure 3; F(4,69) = 4.89]. The D2 antagonism decreased aggression in all strains tested (P < 0.05; Figure 3). A main effect of treatment showed a tendency for a reduction in feather pecking following D1 and D2 receptor antagonists across all strains [P < 0.10; Figure 4; F(2,71.2) = 2.42].
Figure 1. Effects of dopamine D1 and D2 agonists on aggressive behaviors of subordinate birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean aggressive behaviors (±SEM). Asterisk denotes significant difference (P < 0.05) from control mean.
F(2,32) = 1.56 and F(2,29) = 1.34; EP, F(2,32) = 0.45 and F(2,29) = 0.01; NE, F(2,32) = 0.16 and F(2,29) = 0.08). In addition, D1 and D2 agonists did not affect the concentrations of dopaminergic and serotonergic transmitter expression and turnover in the hypothalamus and raphe nucleus in all the tested birds from all the 3 examined strains [P > 0.05; Table 1; 5-HT turnover, F(2,32) = 0.28 and F(2,29) = 1.55; DA turnover, F(2,32) = 0.80 and F(2,29) = 0.77]. In experiment 1 using subordinate birds, we noted a strain effect in the analysis of hypothalamic 5-HIAA, with HGPS hens having consistently lower concentrations (P < 0.05). Experiment 2. In the hypothalamus, EP concentrations were increased in both DXL and HGPS strains following antagonism of D2 receptor [P < 0.05; Table 2; F(2,40) = 4.06]. In DXL hens, hypothalamic 5-HT concentrations were increased following antagonism of
Physiology Experiment 1. No change was found in the concentrations of 5-HT, DA, and catecholamines (EP and NE) in the hypothalamus and raphe nucleus of the tested birds following both D1 and D2 agonists [P > 0.05; Table1; 5-HT, F(2,32) = 0.78 and F(2,29) = 0.45; DA,
Figure 2. Effects of dopamine D1 and D2 agonists on feather pecking of subordinate birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean feather pecking (±SEM). Asterisk denotes significant difference (P < 0.05) from control mean.
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xijkl = (genetic strain; G)i + (treatment; T)j + GTij
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D1 and D2 receptors [P < 0.05; Table 2; F(2,38) = 5.28]. In HGPS hens, hypothalamic 5-HT concentrations were increased following antagonism of the D2 receptors only [P < 0.05; Table 2; F(2,38) = 5.28]. In the raphe nucleus, no effect of treatment was found on 5-HT levels [P > 0.05; Table 2; F(2,38) = 0.50]; however, the metabolite 5-HIAA concentrations were increased in HGPS hens following D1 antagonism [P < 0.05; Table 2; F(2,40) = 3.80]. In experiment 2 us-
ing dominant hens, we noted strain effects. The HGPS hens had consistently higher raphe nucleic HVA concentrations and hypothalamic HVA and 3,4-dihydroxyphenylacetic acid concentrations compared with DXL hens [P < 0.05; Table 2; F(2,40) = 6.39 and F(2,40) = 4.58 and 4.79]. Body weight gain was determined in experiment 2. The DXL and LGPS hens treated with D1 antagonist gained significantly more weight over the 5-d period than saline controls (P < 0.05). Saline-treated DXL, LGPS, and HGPS hens gained 33 ± 12, 30 ± 12, and 41 ± 13 g, respectively. The D1 antagonist DXL, LGPS,
Table 1. Central serotonin and catecholamine concentrations (ng/mL) of subordinate hens1 treated with dopamine D1 agonist, D2 agonist, or saline control2 Control Item Raphe nucleus Norepinephrine Epinephrine Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid Dopamine turnover Serotonin 5-Hydroxyindoleacetic acid Serotonin turnover Hypothalamus Norepinephrine Epinephrine Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid Dopamine turnover Serotonin 5-Hydroxyindoleacetic acid3 Serotonin turnover 1DXL
DXL
D1 agonist HGPS
DXL
D2 agonist HGPS
DXL
60 9.4 2.2 85 4.7 83 51 18 4.8
± ± ± ± ± ± ± ± ±
15.6 2.10 0.57 36.8 2.02 59.8 17.5 3.0 2.53
68 6.8 1.5 104 3.8 176 25 12 3.0
± ± ± ± ± ± ± ± ±
18.4 2.49 0.67 43.5 2.39 70.7 26.7 3.5 3.87
64 11.7 2.7 33 5.7 47 51 10 0.4
± ± ± ± ± ± ± ± ±
18.4 2.49 0.67 43.5 2.39 70.7 20.7 3.9 3.35
54 9.6 2.4 92 4.2 130 56 18 6.4
± ± ± ± ± ± ± ± ±
15.6 2.10 0.57 36.8 2.02 59.8 17.5 3.0 2.53
57 8.9 2.1 62 2.8 119 52 17 5.9
± ± ± ± ± ± ± ± ±
15.6 2.10 0.57 36.8 2.02 59.8 17.5 3.0 2.53
54 7.2 3.4 40 1.9 64 65 21 2.1
± ± ± ± ± ± ± ± ±
16.8 2.27 0.61 39.7 2.18 64.6 20.1 3.2 3.00
100 19 7.1 99 15.5 67 51 20.6 2.8
± ± ± ± ± ± ± ± ±
28.6 7.20 2.04 43.0 5.97 31.4 20.5 2.87 0.97
114 31 5.6 149 12.5 119 40 19.1 3.2
± ± ± ± ± ± ± ± ±
33.0 8.31 2.35 49.7 6.89 36.9 22.2 3.31 1.05
72 22 6.2 72 6.4 70 77 19.7 1.9
± ± ± ± ± ± ± ± ±
46.7 11.76 3.33 70.3 9.75 51.3 31.4 4.68 1.49
127 32 6.1 134 14.9 70 62 14.4 1.6
± ± ± ± ± ± ± ± ±
30.5 7.70 2.18 46.0 6.4 33.6 20.5 3.06 0.97
122 26 4.0 95 9.6 73 47 17.5 2.3
± ± ± ± ± ± ± ± ±
28.6 7.20 2.04 43.0 5.97 31.4 19.2 2.87 0.91
112 29 9.0 44 4.8 22 89 16.7 0.9
± ± ± ± ± ± ± ± ±
33.0 8.31 2.35 49.7 6.89 36.3 24.3 3.31 1.15
= DeKalb XL, high aggressive strain; HGPS = high group egg productivity and survivability, low aggressive strain. as least squares means ± SEM. 3Main effect of strain (P < 0.05). 2Reported
HGPS
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Figure 3. Effects of dopamine D1 and D2 antagonists on aggressive behavior of dominant birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean aggressive behavior (±SEM). Asterisk indicates significant difference (P < 0.05) from control mean.
Figure 4. Effects of dopamine D1 and D2 antagonists on feather pecking of dominant birds of Dekalb XL (DXL), low group egg productivity and survivability (LGPS), and high group egg productivity and survivability (HGPS) strains. Data are presented as least squares mean feather pecking (±SEM).
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AGGRESSION AND DOPAMINE Table 2. Central serotonin and catecholamine concentrations (ng/mL) of dominant antagonist, or saline control2 Control Item
DXL 33 5.1 1.7 1.1 0.3
± ± ± ± ±
7.9 0.87ab 0.46 0.26 0.10
D1 antagonist HGPS
21 6.4 2.5 2.0 0.4
treated with dopamine D1 antagonist, D2
± ± ± ± ±
9.6 1.07b 0.56 0.32 0.12
DXL 41 6.0 2.6 1.6 0.4
± ± ± ± ±
7.9 0.87b 0.46 0.26 0.11
D2 antagonist
HGPS 44 5.7 2.5 1.7 0.4
± ± ± ± ±
8.9 0.99ab 0.52 0.29 0.11
DXL 37 4.1 2.4 0.9 0.4
± ± ± ± ±
8.3 0.92a 0.49 0.28 0.11
HGPS 34 5.8 2.2 1.7 0.4
± ± ± ± ±
8.9 0.99ab 0.52 0.29 0.11
1.3 ± 0.37 71 ± 17.2 9 ± 2.9
1.4 ± 0.45 89 ± 18.6 9 ± 3.6
0.8 ± 0.39 74 ± 16.1 12 ± 2.9
0.9 ± 0.41 71 ± 18.6 18 ± 3.3
0.6 ± 0.39 68 ± 16.1 13 ± 3.1
0.6 ± 0.39 100 ± 20.3 13 ± 3.3
85 ± 69.8
141 ± 75.4
36 ± 65.3
122 ± 75.4
13 ± 65.3
1 ± 82.6
66 17.3 5.1 2.7 0.47
± ± ± ± ±
19.8 4.69a 1.40 0.44 0.094a
63 12.7 6.0 3.0 0.62
± ± ± ± ±
24.2 5.74a 1.72 0.53 0.116ab
77 15.3 6.7 2.9 0.59
± ± ± ± ±
19.8 4.69a 1.40 0.44 0.094ab
66 15.8 7.2 3.6 0.69
± ± ± ± ±
22.4 5.31a 1.59 0.49 0.107b
84 27.9 7.0 3.3 0.65
± ± ± ± ±
21.0 4.97b 1.49 0.46 0.100b
95 28.1 9.0 3.0 0.89
± ± ± ± ±
22.4 5.31b 1.59 0.49 0.107b
1.1 ± 1.46 42 ± 14.1a 6.7 ± 2.54
1.0 ± 0.79 50 ± 17.2a 8.8 ± 3.11
3.8 ± 1.46 95 ± 16.0b 9.8 ± 2.54
0.8 ± 1.66 76 ± 16.0ab 9.3 ± 2.88
0.6 ± 1.55 93 ± 14.9b 10.5 ± 2.69
0.6 ± 1.66 95 ± 16.0b 5.6 ± 2.88
44 ± 17.0
25 ± 20.8
1 ± 19.3
30 ± 19.3
17 ± 18.0
1 ± 19.3
a,bMeans
with different superscripts are significantly different (P < 0.05). 1DXL = DeKalb XL, high aggressive strain; HGPS = high group egg productivity and survivability, low aggressive strain. 2Reported as least squares means ± SEM. 3Rows indicate treatment differences (P < 0.05). 4Rows indicate main effect of strain (P < 0.05).
and HGPS hens gained 59 ± 12, 61 ± 13, and 28 ± 13 g, respectively. The D2 antagonist DXL, LGPS, and HGPS hens gained 45 ± 12, 22 ± 13, and 15 ± 13 g, respectively, and showed no significant difference from control (P > 0.05).
DISCUSSION Dopaminergic involvement in aggression mediation has been shown in many studies (Dennis et al., 2006; Nelson and Trainor, 2007; Couppis and Kennedy, 2008; Seo et al., 2008). Hyperactivity in the dopaminergic system is associated with increased aggression in animals and humans (Netter and Rammsayer, 1991; Friedel, 2004). In addition, locomotor and stationary forms of social behaviors can both be altered by pharmaceutical manipulation of the D1 receptor (Gendreau et al., 1997). The D1 agonism elicited different social and emotional behavioral responses in mice bred for high and low activity level, suggesting a link in D1 control over locomotor behaviors and social response (Gendreau et al., 1997). In the present strains, agonism of the D1 receptor increased aggression in all strains observed. Although overall activity levels were not investigated in this study, it is possible that an increase in activity in all birds treated with D1 agonist resulted in an increase in aggressive social encounters. However, we would also expect to see an increase in other active behaviors such as feather pecking, which was not
increased in all strains. Agonism of D1 receptor pathways can also cause a disturbance of memory, as seen in avoidance tasks and resistance to amnesia in some mice (Dubrovina, 2006). Alterations to social memory can also affect performance of social behaviors such as aggression (Marino et al., 2005) separately from feather pecking behaviors. Strain-based effectiveness of D1 antagonism in regulating aggression may be directly related to D1 receptor density or indirectly related through sensitivity of its downstream effects, including increased central serotonin response in DXL birds. The paraventricular nucleus of the hypothalamus is a critical region involved in the food reward system; stimulation of this region with neuropeptide Y increases weight gain and feeding behavior (Stanley et al., 1989). Increased weight gain in the DXL and LGPS birds suggests a potential role of the paraventricular nucleus or other feed intake-regulating regions of the hypothalamus. Increased pecking in domestic hens has been suggested to be related to misdirected foraging behaviors (Huber-Eicher and Wechsler, 1997). Increased 5-HIAA concentration was also evident in the raphe nucleus of the HGPS birds treated with D1 antagonist. Reduced 5-HIAA concentrations in human cerebrospinal fluid have been shown to be associated with a history of aggressive and suicidal behaviors (Brown et al., 1982). Increased 5-HIAA in these birds suggests an increase in 5-HT turnover in response to dopaminergic manipulation. This reduction
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Raphe nucleus Norepinephrine Epinephrine3 Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid Dopamine turnover Serotonin 5-Hydroxyindoleacetic acid 5-Serotonin turnover Hypothalamus Norepinephrine Epinephrine3 Dopamine Homovanillic acid 3,4-Dihydroxyphenylacetic acid3,4 Dopamine turnover Serotonin3 5-Hydroxyindoleacetic acid 5-Serotonin turnover
hens1
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Increased central concentrations of 5-HT are most frequently associated with reduced levels of aggression (Nelson and Chiavegatto, 2001). Serotonin is one of the primary moderators of aggressive behavior. Previous and ongoing research in these birds has also identified heritable differences in the response to pharmaceutical alterations of the 5-HT system (Dennis et al., 2008). Reduced hypothalamic EP responsiveness is seen during acute and chronic stress (Roth et al., 1982). Increased hypothalamic EP coinciding with reduced aggressiveness may be suggestive of a less stressful social environment. Increased hypothalamic EP response is also seen with increased 5-HT levels following 5-HT agonism in rats (Hemrick-Luecke and Fuller, 1995). Epinephrine response can also indicate activation of a reward center (Kalra et al., 1988). Increased hypothalamic secretions of EP into the portal system may help modulate anterior pituitary regulation of stress coping (Gibbs, 1985). Alterations to the reward system via the D2 receptor is the most shared pathway for controlling aggressiveness between the present strains. In conclusion, our findings suggest the importance of genotype or epigenotype in understanding the mechanisms regulating these behaviors and in their pharmaceutical manipulation. Our results also suggest the potential for using indicators such as different response to pharmaceutical receptor agonism for more efficiently selecting for low aggressive behaviors.
ACKNOWLEDGMENTS The authors thank the technicians at the Livestock Behavior Research Unit of the USDA (West Lafayette, IN), as well as Fred Haan and the staff of the Purdue University Poultry Facility (West Lafayette, IN) for their outstanding assistance. We also thank Paul Collodi, Robert Meisel, and William Muir for their consultation. The work was supported by a grant of USDANRI #2032.
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in 5-HT was accompanied by a reduction in feather pecking but not by alterations to hypothalamic 5-HT or aggressive changes. These data suggests that mediation of feather pecking in HGPS birds is distinct from that mediating aggressiveness. Feather pecking behaviors can be altered by manipulation of the dopaminergic system (Kjaer et al., 2004). The D1 agonism resulted in increases in stereotypic feather pecking in DXL and HGPS birds but not in LGPS birds. Such maladaptive stereotypic behaviors are often associated with anxiety-related behaviors. D1 agonism has been shown to interact with both the central serotonergic and adrenal functions, and heritable factors can influence both the type and extent of response. Treatment-associated feather pecking increases seen in DXL and HGPS birds may be more closely related to heritable similarities in these strains that may be correlated to high productivity. Both DXL and HGPS hens share a phenotype for high production parameters, and previous studies have suggested a link between stereotypic behaviors and high production-related stressors. Increased demand on the hen to produce could alter the dopaminergic system through interactions with reproductive systems (Calogero et al., 1998) Agonism of the D2 receptor showed no effect on aggressiveness in either of the high aggression strains, increasing aggression only in HGPS birds. Similar to our findings, an investigation of high and low aggressive strains of rats found that agonism of the D2 receptor increased stimulation-induced aggression in only the low aggressive strains (Nikulina and Kapralova, 1992). Excitation of D2 receptor pathways may mediate aggression differently in high and low aggressive strains. Analysis of the 2 isoforms of the D2 receptor, D2L (long) and D2S (short), evidenced that D2L plays a vital role in mediating offensive aggressiveness (Vukhac et al., 2001). Human studies have also identified a specific isoform of the dopamine transporter gene (DAT1) that is related to increased aggressiveness (Chen et al., 2005). Strain differences in relative isoform expression could greatly affect their response to receptor excitation. The functions of D2 receptor and its subtypes in mediating behavior in the present strains will be examined in further investigations. Antagonism of the D2 receptors can prevent a behavior, such as aggression, from being perceived as a reinforcing or rewarding behavior through the D2 pathway (Couppis and Kennedy, 2008). In this way all strains may have reduced their frequency of aggressive and feather pecking behaviors because of the lack of positive reinforcement. A reduction in D2 receptor density, as is frequently seen in aggressive individuals, may reduce the effectiveness of D2 agonism, resulting in an increase in aggression only in the less aggressive HGPS strain. Antagonism of the D2 receptor also increased hypothalamic EP and 5-HT concentrations in all strains.
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