Department of Physics, Chemistry and Biology
Master Thesis
Domestication related genotype on ADRA2C
— a determinant of fear response in chickens?
Amir Fallahshahroudi
LiTH-IFM- Ex-12/2625-SE
Supervisor: Per Jensen, Linköping University Examiner: Dominic Wright, Linköping University
Department of Physics, Chemistry and Biology Linköpings universitet
Rapporttyp Report category Examensarbete D-uppsats Språk/Language Engelska/English Titel/Title:
Domestication related genotype on ADRA2C — a determinant of fear response in chickens?
Författare/Author:
Amir Fallahsharoudi Sammanfattning/Abstract:
White Leghorn (WL) breed is homozygous for an allele on the α2C-AR gene while the Red Junglefowl (RJF) is mostly heterozygous for that. The gene is also hypermethylated in WL. The gene codes for the α2C -adrenergic receptor, which plays various roles including regulation of
neurotransmitter release from adrenergic neurons in the central nervous system and sympathetic nerves The aim of this study was to investigate the effects of the variation of α2C-AR gene on the chickens’ behaviour. Various behavioural tests mostly associated with fear and stress was conducted in progeny of an F9 generation of the advanced intercross line (AIL) between RJF and WL, selected for homozygosity of the alternative alleles on the α2C-AR gene. In the emergence test, the stress effect on both poking and total exit latency was significantly higher in WL genotype females in comparison to RJF genotype females (effect on head poking time: WL 70.62 ± 18.603 versus RJF 15.63 ± 29.069; effect on total exit time WL -72.14 ± 20.582 RJF 5.64 ± 30.140). In the aerial predator test RJF genotype birds showed significantly more agitated behaviours after the predator exposure in comparison to WL genotype birds (RJF 27.65 ± 0.700 versus WL 25.09 ± 0.915)Although we found differences in behaviour of individuals with WL genotype, more research is needed to find out how the variation on the ADRA2C gene has played a role in domestication of layer chicken.
ISBN
LITH-IFM-A-EX—12\2625—SE
__________________________________________________ ISRN
__________________________________________________ Serietitel och serienummer ISSN
Title of series, numbering
Handledare/Supervisor Per Jensen
Ort/Location: Linköping
Nyckelord/Keyword: ADRA2C, domestication, genetic, chicken, fearfulness
Datum/Date
2012-05-25
URL för elektronisk version
Institutionen för fysik, kemi och biologi
Department of Physics, Chemistry and Biology
Avdelningen för biologi
Content
1 Abstract ... 2
2 Introduction ... 2
3 Material & methods ... 8
3.1 Birds ... 8
3.2 Genotyping ... 8
3.3 Housing ... 8
3.4 Morphological measurements ... 9
3.5 Behaviour tests ... 9
3.5.1 Open field test ... 9
3.5.2 Learning test ... 10
3.5.3 Emergence test ... 11
3.5.4 Tonic immobility ... 11
3.5.5 Aerial Predator Test ... 12
3.6 Statistical analysis ... 13 4 Results ... 14 4.1 Morphological measurements ... 14 4.1.1 Weight ... 14 4.1.2 Metatarsus length ... 15 4.2 Behaviour test: ... 16
4.2.1 Open field test ... 16
4.2.2 Memory test ... 16
4.2.4 Tonic immobility test ... 17
4.2.5 Aerial predator test ... 17
4.3 PCA ... 18
5 Discussion ... 20
5.1 Conclusions ... 23
6 References ... 23
1 Abstract
White Leghorn (WL) breed is homozygous for an allele on the α2C-AR
gene while the Red Junglefowl (RJF) is mostly heterozygous for that. The gene is also hypermethylated in WL. The gene codes for the α2C
-adrenergic receptor, which plays various roles including regulation of neurotransmitter release from adrenergic neurons in the central nervous system and sympathetic nerves The aim of this study was to investigate the effects of the variation of α2C-AR gene on the chickens’ behaviour.
Various behavioural tests mostly associated with fear and stress was conducted in progeny of an F9 generation of the advanced intercross line (AIL) between RJF and WL, selected for homozygosity of the alternative alleles on the α2C-AR gene. In the emergence test, the stress effect on both
poking and total exit latency was significantly higher in WL genotype females in comparison to RJF genotype females (effect on head poking time: WL -70.62 ± 18.603 versus RJF 15.63 ± 29.069; effect on total exit time WL -72.14 ± 20.582 RJF 5.64 ± 30.140). In the aerial predator test RJF genotype birds showed significantly more agitated behaviours after the predator exposure in comparison to WL genotype birds (RJF 27.65 ± 0.700 versus WL 25.09 ± 0.915). Although we found differences in behaviour of individuals with WL genotype, more research is needed to find out how the variation on the ADRA2C gene has played a role in domestication of layer chicken.
2 Introduction
The high speed evolutionary process in which animals are adapted to live close to humans is called domestication. During domestication, the
pattern of selection pressures changes, i.e. there is a relaxation of natural selection pressures such as starvation or predation, and there is a selection pressure for the human-desired traits such as tameness and high
production (Price 1999). Darwin appreciated the extensive variations within domesticated species and used them as an example of his theories throughout his books; ―The origin of species‖ and ―Variation under domestication‖. At least since then, biologists have been interested in understanding the process of domestication (Wiener & Wilkinson 2011). Domestication has led to various morphological, physiological and
behavioural changes, which includes changes in growth pattern and body size, changes in reproductive cycles and endocrine responses, increased sociability, and decreased anti-predator responses and fear (Jensen 2006, Kuenzl et al. 1999) The chicken is a suitable model animal to study domestication for various reasons. The Red Junglefowl (Gallus gallus), is the main ancestor of domesticated chicken and can be found both wild in South-East Asia and in many zoos all over the world (Eriksson et al. 2008). As with dogs, chicken show a greater breed variability in
comparison to most of other domesticated animals because different chicken breeds have been selected for particular purposes such as
appearance, aggression, egg production, and fast growth. The possibility to cross the domestic breeds with the wild ancestor and acquire fertile offspring, a short generation time, quick embryonic development, and the fact that they are easy to keep in small areas makes the chicken an
exceptional model for genetic studies (Jensen 2006).
The behaviour of an individual animal can be affected by both the
environment and the genes. The genes can alter the behaviour in various ways, for example if a mutation occurs in a coding region of a receptor gene, it may cause a structural change in the receptor that may prevent it from binding with the signaling molecule. So the individuals which carry the mutant allele may react to a certain stimuli in a different way (Wiren et al. 2011). Thanks to the recent advances in genetic and statistical technologies, the genetic changes that have happened during
domestication can now be characterized. Linkage mapping is a powerful approach for gene mapping of domesticated species. The parts of the genome which are related with certain traits such as quantitative trait loci (QTL) can be recognized with this approach. For instance, association between PMEL17 gene which codes for plumage colour and various behavioural traits have been shown in a cross between the ancestral red junglefowl and White Leghorn (Nätt et al. 2007, Karlsson et al. 2010). The individuals who were homozygous for the RJF allele (i\i) were black and the birds that were homozygous for WL allele (I/I) were white. i/i birds were more vocal, aggressive, social, explorative, and they were less active in a test that measures fear from humans in comparison to I/I individuals. The authors suggested that WL breed have been selected for the white plumage colour and therefore have been co-selected for the
associated behavioural traits but on the other hand selection for behavioural traits may also lead to associated morphological changes such as one experiment involving silver fox (Belyaev 1979). During the experiment the foxes were selected and bred according to their tameness. After a few generations, the tame foxes developed some domestic dog like behaviours such as tail wagging and licking. Besides, some new morphological trait such as curly tails, floppy ears and shortened snouts which are common in domestic dogs were also developed in some of the foxes (Belyaev 1979).
Another approach to characterize the genetics behind domestication is to search for genomic regions with extended homozygosity. The basis of this method is the ―hitch hiking theory (Smith & Haigh 1974). According to the theory when a favorable mutation arises and gets strongly selected, the associated neutral alleles get accidental advantage and hence, the neutral polymorphisms would decrease. A common method to find these genomic regions is to use single-nucleotide polymorphism (SNP) data. When a single nucleotide in the genome is different between members of a species, a SNP occurs. The reduction in the variation of SNPs in some regions of genome can be linked with a recently fixed beneficial mutation and is known as selective sweep (Nielsen 2005). For instance, Parker et al. (2009) revealed that on chromosome 18, in a 24 kb region of reduced heterozygosity, an expressed retrogene encoding fibroblast growth factor 4 (IGF4) is present in chondrodysplastic dog breeds and is responsible for the short leg trait. Sutter et al. (2007) suggested that a single IGF1 allele is the main factor of small size in dogs. A region of reduced
heterozygosity is also present near IGF1 in small size dog breeds. To study genotype –phenotype correlation, an advanced intercross line (AIL) between WL and ancestral RJF was made by pairing an originally wild caught population of RJF with a Scandinavian population of WL (Shultz et al. 2002). Each generation of crossing present more genetic
recombination and hence, the genome of later generations of AIL birds will have a more random mixture of the WL and the RJF genomes. To study the effects of a particular gene on behaviour or morphological traits, it is possible to select parent birds on the basis of their genotype on a specific locus to gain offspring with desired genotypes at the specific locus (selected advanced intercross line; SAIL) whereas the other part of their genome is a random mixture of WL and RJF genome (Wiren et al. 2011).
Rubin et al. (2010) sequenced the DNA pools of 8 domesticated breeds of chicken and searched for regions with high degree of SNP fixation. The genetic differentiation was measured between breeds with pairwise
fixation index (FST) and the validity of SNPs was confirmed. Allele
counts were used at recognized SNP position to identify signs of selection in sliding 40-kb windows. For each window pooled heterozygosity (HP
values) was calculated and transformed into Z scores. A threshold of ZHP
≤ -6 was applied for putative selective sweeps. In layer comparison, 6 genes existed within 20 kb of windows with ZHP ≤ -6 , including one
gene candidate close to a window with no genes overlapping ±20 kb, located at the locus encoding α2C- adrenoceptor (α2C-AR) on chromosome
4 of layer breeds, in other words, most of the layer breeds are
homozygous for a variant allele on the mentioned gene. Nätt et al. (2012) studied gene expression and methylation profiles in
thalamus/hypothalamus of WL and RJF and suggested the probability of selection of genotypes which affect epigenetic states during
domestication. In part of the study Nätt et al. (2012) considered the probability of accumulation of epigenetic differences in selective sweeps and hence, compared their data with Rubin et al. (2010) and showed that
α2C-AR gene lies among the sweeps which are also hyper-methylated in
WL in comparison to RJF. It is worth noting that DNA methylation is commonly considered to cause stable gene silencing (Cedar et al. 2009, Raynal et al. 2012).
Adrenoceptors (ARs) are a part of G protein-coupled receptors (GPCRs). GPCRs are a big family of integral membrane proteins and are
responsible for several physiological responses to hormones,
neurotransmitters, odorants, taste and light (Lefkowitz et al. 2000) Nine different subtypes of adrenoceptor have been characterized; three α1, three α2, and three β receptors. The adrenoceptors are the interface between catecholamines (epinephrine and norepinephrine) and a broad range of target cells throughout the body, and hence, are responsible for mediating the biological functions of the sympathetic nervous system (SNS). The SNS contains three parts: sympathetic neurons in central nervous system, peripheral sympathetic neurons, and the adrenal medulla. The main neurotransmitter of sympathetic neurons is norepinephrine, while epinephrine is secreted from chromaphin cells of adrenal medulla and reaches its target cells via blood stream. Norepinephrine also can inhibit the neurotransmitter release by stimulating presynaptic
adrenoceptors (reviewed by Brede et al. 2003). α2-ARs include three subtypes (α2A, α2B and α2C) and are involved in a variety of physiological
functions. They mediate the effects of norepinephrine on CNS and participate in regulating the release of norepinephrine, serotonin (5-HT) and dopamine (DA) (Ruffolo et al. 1988). Considering that specific α2AR subtype-selective drugs had not been available until very recently
(Jnoff et al. 2012), genetically engineered mice had been used to study the physiological roles of various AR subtypes (Philipp et al. 2004). Hein et al. (1999) studied the release of neurotransmitters in mice with genetically disrupted AR subtypes. The authors showed that in the heart and in the central noradrenergic neurons, both α2A and α2C subtypes are
involved in presynaptic regulation of neurotransmitter release from sympathetic nerves. It was shown that α2A-ARs modulate
neurotransmission when the nerve activity is high, whereas, α2C-ARs
inhibit neurotransmitter release at lower stimulation frequencies. The study revealed that the mice lacking both α2A- and α2C-ARs had higher
plasma norepinephrine concentrations and had a hypertrophic heart at four months of age which shows the physiological importance of both subtypes. Brede et al (2003) suggested that α2A-ARs modulate the release
of catecholamines from sympathetic nerves, while α2C-ARs control
epinephrine release from the adrenal glands. Using mice with genetically altered AR expression, Sallinen et al. (1999) studied the participation of
α2C-ARs in physiological and behavioural responses to stress. The α2C-AR
knockout mice (α2C-KO) were more active in the forced swimming test,
while α2C-overexpressed mice showed increased immobility in the test.
The authors suggested that α2C-AR overexpression may promote
behavioural despair. Additionally, α2C-KO mice had a significantly lower
corticosterone concentration level after repeated stress in comparison to both the control and the α2C-overexpressing mice. Moreover, the study
showed that α2C-AR expression can also affect dopamine and serotonin
balance in the brain and α2C-KO seemed to cause stress protective effect.
Sallinen et al. (1998) studied the involvement of α2C-ARs in
isolation-induced aggression and prepulse inhibition (PPI) of the startle reflex in genetically engineered mice. The lack of α2C-AR expression led to
decreased attack latency in the isolation-aggression test, enhanced startle reactivity and reduced PPI. Björklund et al. (1998) investigated the behavioural functions of α2C-ARs in executing spatial and non-spatial
scape strategies in mice and showed that α2C-AR-overexpressing mice
developed impaired scape strategies in the water maze tests. The α2C
-overexpressing subjects could find neither the visible nor the hidden platform as precisely as the wild type, and spent more time swimming near the wall of the pool.
Gibbs (2008) reviewed the roles of norepinephrine and various ARs in memory acquisition and consolidation and suggested that α2-ARs are involved in memory consolidation in chicken. Tachibana et al. (2009) investigated the involvement of α2-ARs on feeding behaviour in layer-type chicks and showed that α2-ARs are associated with the stimulation
of feeding in layer-type chicks. The results supported the findings of a previous study conducted in broiler-type chicks (Bungo et al. 1999). Cheng et al. (2006) investigated the effect of clonidine (α2-AR agonist) on chicken immune function and suggested that sympathetic nervous system participates in chicken immune system by regulating activations of α2-ARs. Finally, α-ARs are involved in the ovulatory process at the ovarian level in chicken (Moudgal et al. 1981).
The anatomical distribution of α2-ARs in the chicken CNS was studied by Díez-alarcia et al. (2006). The data demonstrated that in the chicken brain α2A-AR was the dominant subtype, whereas the expression of α2B and α2C was limited to the telencephalon. Presence of α2-ARs in hyperpallia
and tectum opticum which are associated with avian visual pathways supported the findings suggesting the involvement of α2-AR system in acquiring and processing of visual information. Applying a method called functional autoradiographic binding; Díez-alarcia et al. (2009) studied the activation of α2-ARs after presenting epinephrine as agonist in chicken brain. The highest correlation between the functional autoradiography and anatomical distribution of α2-ARs was observed in the bed nucleus of the stria terminalis (BSt) and the nucleus supraopticus. BSt is
associated with reactions to both acute and chronic stress (Choi et al. 2008) and fear reactions (Fendt et al. 2005) in mammals, whereas,
nucleus supraopticus is associated with social recognition and emotional behaviours (Yoshida et al. 2009).
Fear can be defined as the reaction of an individual to a potentially dangerous situation. Fear response can increase the chance of an
individual to escape from the danger and hence increase the fitness of an individual in its’ natural environment by adjusting the behaviour and physiology. But in captivity, fearfulness may decrease the adaptability of an individual to the captive environment by increasing its’ stress level (Dwyer et al. 2004).We can guess that during early domestication the selection pressure favored the individuals which were less fearful toward humans. Various studies have suggested that selection for low fearfulness has been a critical part of the early domestication. For example Campler & Jensen (2009) assessed the fearfulness of the WL and their ancestor RJF according to four various fear tests and showed that the RJF was more fearful.
The alteration of α2C-AR expression affects physiological and behavioural
responses to stress in mice (Sallinen et al. 1999). The stress causes delay in onset of lay and decreased egg production (Shini et al. 2009) and the WL breed has been selected for commercial egg production and high feed conversion efficiency (West 1988).We can assume that the genotypes
which helped the birds to cope with the commercial environment (e.g. 4-5 individuals in each battery cage) and hence, led to production of more eggs were favored during layer selection. We hypothesized that the individuals with different genotypes on their α2C-AR gene would behave
differently in fear related behavioural tests. We predicted the WL genotype birds may react to the stress less intensively in comparison to the RJF genotype birds. Various behavioural tests mostly associated with fear and stress, were conducted in F10 generation SAIL birds, selected for homozygosity of alternative alleles on the α2C-AR gene. The
behavioural tests consisted of the open field, the hole in the box, the tonic immobility (TI), the aerial predator and the learning tests. Simultaneously bone elongation rate and growth rate were measured. To measure the stress level of the birds, blood smears were prepared and the
heterophil/lymphocyte ratio was evaluated.
3 Material & methods 3.1 Birds
The chickens used in this experiment were the intercross (N = 145) obtained from the F9 generation of the RJF×WL intercross. The parental RJF population had been acquired from a Swedish zoo and the parental WL had been obtained from a Scandinavian population which was bred for crossbreeding and selection experiment (described in detail by Schutz 2002). All the F9 AIL birds had been genotyped for an allele on α2C-AR
gene. Three different genotypes were recognized among them: birds homozygous for either the RJF or WL allele, and a third group heterozygous for both. 28 heterozygous AIL birds (14 males and 14 females) were selected and paired to produce the F10 SAIL generation. It was expected that F10 generation would contain 25% RJF homozygous, 25% WL homozygous and 50% heterozygous individuals. 41 RJF, 44 WL and 76 heterozygous genotype birds were recognized and studied.
3.2 Genotyping
The DNA was extracted from the blood using DNeasy extraction kit from Qiagen according the instruction of the manufacturer. The birds were genotyped using molecular Taqman method according the instruction. 41 RJF genotype, 76 heterozygous and 44 WL genotype birds were
recognized and studied.
3.3 Housing
The experimental birds were raised in two batches with a 28 days interval. The same F9 AIL parents paired to obtain the F10 SAIL
generation in the batch 2. The birds in batch 1 and batch 2 were treated as similar as possible, i.e. the rearing condition, vaccination program, blood sampling dates and behavioural tests dates were similar in both batches. All birds were hatched, marked and raised at the hatching facility at Linköping University. All birds were raised in mix sex and genotype groups of 85 (batch 1) and 60 (batch 2) in a 1-2 m2 (depending on their age) pen. The birds were provided with woodshavings on the floor, the perches and ad libitum food and water. At 7 weeks of age, the bird were transferred to a research facility at Vreta (10 km north of Linköping) in the same mixed groups and were kept in 6-8 m2 pens provided with wood shavings on the floor, perches, nests and ad libitum food and water. At 18 weeks of age male and female birds were separated and kept in different rooms.
3.4 Morphological measurements
To measure the growth rate the birds were weighed with a digital scale weekly from the hatching date until they were 7 weeks old and on day 88. At the same time, the length of the metatarsus was also measured using a digital caliper and bone elongation rate was assessed for each bird.
3.5 Behaviour tests
All behavioural tests were conducted blind, i.e. the observer was not aware of genotype of the individuals until the last behavioural test was conducted.
3.5.1 Open field test
At 4 weeks of age, an open field test was conducted. The test is well validated in chicken and shows fear but also social dependency (Gallup et al. 1980). The birds were moved to the experiment room and left there for 3 hours for habituation. After turning off the light, birds were captured gently, carried in a carton box and introduced into one specific corner of a 110 × 75 cm arena, made of wooden board, with a light brown floor. The arenas were covered with a wire mesh lid. Four birds were tested in four separate arenas simultaneously. After introducing all birds into arenas, the observer sat behind a wall, the lights were turned on and the movements of the birds in the arenas were recorded by a computer
connected to four ceiling-mounted video cameras for 600 s. After testing all birds, Ethovision software v3.1 was used to score the movements of the birds in the arenas. The arena had been virtually divided into 9 zones with two horizontal and two vertical virtual lines. The latency of exiting the releasing zone, the total movement, the velocity and the spent time in each zone were scored.
3.5.2 Learning test
At 5 weeks of age a learning test was performed. The test was based on associative learning and colour discrimination. The test consisted of four 110×75 cm arenas. In each arena, two plastic cups (yellow and blue) were attached to the floor in the corners opposite to the releasing site. In each arena one cup contained meal worms and the other one did not contain any reward, but to decrease the chance that birds see the mealworms from the distance, both cups contained small amount of wood shavings.
Considering that two colours were used and there were two corners (left and right) in front of the releasing site, four arrangements were possible: Yellow cup containing meal worm (Left/right) and blue cup containing worm (left/right) (Fig 1). Each arena had a unique arrangement and each chicken was randomly assigned to one of the arenas before the test. During the first trial, the chickens were put into the assigned arena and left alone to find the meal worms by themselves. If a chicken could not find the meal worms without help in 10 minutes, the observer would intervene and throw a few mealworms in front of the correct cup trying to lead the chicken toward the cup. A trained social companion was
presented when the experimental bird did not eat the mealworms alone at all. The second trial was similar to the first one but the observer did not intervene and a few birds which were reluctant to go for the mealworms in 10 minutes were excluded from the test. The latency to look into the correct cup, and whether the first cup visited was the one that bird had found mealworms inside during the first day were recorded. The third trial on the third day was conducted exactly like the second trial. To investigate if the chickens went to the cup they learned or if they could see, smell or hear the worms, a probe test was conducted on the fourth day. The probe trial was similar to previous trials but both cups were empty. The fifth trial was similar to the second trial. To find out if the chickens learned the colour or if they learned the side of correct cups, during the 6th trial the sides of the cups were swapped but the same colour as usual held the worms. To find out the duration that bird
remember the colours the test was repeated 12 and 42 days after the first trial.
Figure 1.The circles show the position and the colour of the cups, * represents the reward and the triangle shows the release site.
3.5.3 Emergence test
At 7 weeks of age, the emergence test was conducted. The test shows the willingness of the animal to enter a novel and bright area from a dark and sheltered one. It is a test of fear and is based on the hypothesis that fearful animals stay longer in the sheltered area before emerging into a novel and bright one (Jones et al. 1987). The dark area consisted of a carton box (32 × 23 × 25 cm) with two openings; one on the top and the other adjacent to the floor. The opening on the top was covered by a wooden lid and the other opening was closed with a guillotine trapdoor. The box was placed in a well-lighted 110 × 75 cm arena covered with wood shavings. Each bird was tested two times in two consecutive days. On the first day, each individual was put in the box from the top opening which was covered immediately afterward. After 30 seconds of habituation, the guillotine door was lifted using a rope and pulley allowing the bird to exit the box. Two scores were measured; the latency to poke the head out of the box and the latency to go out of the box completely. The maximum score of 360 s was given to the birds that did not exit the box after 6 min. On the second day, the same procedure was repeated, but before the test, each bird was restrained in a net for 3 min. The observer was hidden behind a wall and used a camera and a monitor to watch the birds.
3.5.4 Tonic immobility
The TI response has been considered as a valid measure of the fear level in chicken in various studies (Forkman et al. 2007). At 7 weeks of age, chickens were caught randomly with minimum disturbance and moved to
* *
the adjacent room where the experiment was conducted. To induce TI, each bird was placed on its back in a V-shaped wooden cradle and a gentle pressure was applied on its chest for 10 sec. If the bird stayed still for 10 sec after the pressure was removed, the induction attempt was considered successful and a stopwatch was started. If the bird righted itself within 10 sec, the induction was considered unsuccessful and it was repeated immediately (five times maximum). After successful TI
inductions, the number of attempts to induce TI, the latency of first head movement and the latency of righting were recorded. The maximum score of 600 sec was given to the birds that did not right in 10 minutes.
3.5.5 Aerial Predator Test
At 14 weeks of age an aerial predator test was conducted. The test measures the reactions of the subjects to a simulated aerial predator (Håkansson & Jensen 2008). The birds were caught one by one and
placed in a 150 × 50 × 50 cm test arena covered with wire mesh. The test comprised three parts, namely, pre-exposure period, exposure, and post exposure period. After 2 minutes of habituation, the subject’s behaviour was recorded for 5 minutes (pre-exposure) using instantaneous
sampling with 10s intervals. The observer recorded the subject’s
behaviour on the instant of each sample point. The ratio of behaviours was calculated by dividing the number of a preformed behaviour with the total number of sample points. Eight types of behaviours were measured:
Exploring: Walking or standing with head close to the ground (below
back), eyes focusing on the ground items.
Ground pecking: Pecks at items (visible or not) on the ground. Sitting: Sitting (legs bent, body touches the ground).
Preen: Uses beak to trim and arrange feathers. Vocalizing: All vocalizations.
Standing alert: Standing with head up attending to the environment
(legs erect).
Walking alert: Two or more steps with posture with head up.
Escape: Attempt to escape out from the test arena by jumping or
making fly attempts towards the roof.
Afterwards, a wooden hawk model (wingspan of 62 cm, length 31 cm) was presented over the arena. A steep rope was attached above the arena and the model which was attached to a pulley, slid above the arena (120 cm average distance from the lid) with a presentation time of around 2 seconds. The model was out of the bird’s sight before and after the predator exposure. The immediate intensity of the bird’s reaction to the simulated predator was scored according to a five level scale (0= No reaction, 1=Lift head once, 2= Lift head once and look around for more than 3 seconds, 3= Attempts to run or fly away and 4= Intense reaction, jump high)
Instantly after the exposure of the model predator, the second 5 minutes of behavioural observation was conducted (post-exposure) similar to the pre-exposure period.
3.6 Statistical analysis
All statistical analyses were made in SPSS Statistics v.20 (IBM Inc. 2011). To assess the consistency of the birds in different fear related tests the correlations between the results of the tests were measured using two-tailed spearman correlation test. The data were tested for normality and if the result were sufficiently normally distributed General Linear Models (GLM) were used to compare between and within sample effects of genotype, batch and sex on the different behavioral variables (significant level of P < 0.05). If significant family effects were detected the variable was excluded from the test otherwise sex × genotype interaction was used in the model to analyze the data. All deviations from the mean are given as ±1 SEM. To reduce the number of variables while retaining as much information from the original variables as possible a principal component analysis (PCA) was conducted. The number of retained factors for
additional analysis was measured with consideration of scree plot and the total explained variance. Each bird was given a score for the identified factors and after testing for normality GLM was performed on these scores.
4 Results
4.1 Morphological measurements 4.1.1 Weight
There were no significant differences in mean weight between the genotypes at any age. However, at day 88 WL genotype males were around 10% heavier than RJF genotype males (WL 742.62 34.339 versus RJF 673.67 18.660, P=0.06) (figure 2&3).
Figure 2.Weight at different ages (±1 SEM).
0 100 200 300 400 500 600 700
Day 0 Day 8 Day 15 Day 22 Day 29 Day 36 Day 43 Day 50 Day 88
Gr am (G) RJF Hz WL
Figure 3.Weight at day 88 in males (±1 SEM).
4.1.2 Metatarsus length
No significant difference was found in metatarsus length between genotypes at any age (Figure 4).
Figure 4.Metatarsus length at different ages (±1 SEM).
580 600 620 640 660 680 700 720 740 760 780 800 RJF Hz WL G ram (g) 0 10 20 30 40 50 60 70 80
Day 0 Day 8 Day15 Day 22 Day 29 Day 36 Day 43 Day 50 Day 88
M ili m e te r ( m m ) RJF Hz WL
16
4.2 Behaviour test: 4.2.1 Open field test
In the open field test no significant differences were found in neither of measured parameters (latency of exiting the release zone, total movement and time spent in each zone) between genotypes.
4.2.2 Memory test
In the memory test on the average, RJF genotype males made
significantly more correct choices in comparison to WL genotype males (RJF 96% ± 1.97% versus WL 82% ± 4.92%, p < 0.05), but females of both genotypes performed almost equally (figure 5).
Figure 5. Mean percentage of total correct choices in the learning test in males (±1 SEM). *= P < 0.05
4.2.3 Emergence test
In the emergence test, on the first day, WL genotype females took significantly more time before poke their head out of the box in comparison to RJF females (RJF 134.30 ±25.622 versus WL 71.72 ± 16.624). On the second day when birds were kept in a net for three
0% 20% 40% 60% 80% 100% 120% RJF Hz WL M e an p e rc e n tage * [ T y p e a q u o t e f r o m t h e d o
minutes before the test, the stress effect on both poking and total exit latency was significantly higher in WL genotype females in comparison to RJF genotype females (effect on head poking time: WL -70.62 ± 18.603 versus RJF 15.63 ± 29.069; effect on total exit time WL -72.14 ± 20.582 RJF 5.64 ± 30.140) (figure 6).
Figure 6. Average time (s) spent in the box before head
emergence and the alteration of average head emergence time after stress treatment in the emergence test in females (±1 SEM). *= P 0.05
4.2.4 Tonic immobility test
No significant differences were found in measured parameters related to the TI test (induction attempts, head movement latency and righting latency) between genotypes.
4.2.5 Aerial predator test
In the aerial predator test RJF genotype birds showed significantly more agitated behaviours in comparison to WL genotype birds (post exposure: RJF 27.65 ± 0.700 versus WL 25.09 ± 0.915) (figure 7). -150 -100 -50 0 50 100 150 200
Head emergence (Non stress) Head emergence (Stressed) Ti m e ( s) RJF Hz WL Head emergence Stress effect * * *
Figure 7. Mean numbers of relax and agitated behaviours before and after presentation of hawk model in the aerial predator test (±1 SEM). *= P < 0.05
4.3 PCA
66% of variance was explained by 4 components (component 1=29%, 2= 14%, 3= 12% and 4=10%) (Table 2)
Table 2. PCA components.
Component
1 2 3 4
Head emergence .909
Head emergence stress
effect -.858
Total emergence .854
Total emergence stress
effect -.828
Aerial predator Intensity
level .718 .324
Memory trial 8 .550 .481
Memory trial 2 .505 .449
TI Head movement -.429 .423 .342
TI iduction attempts -.676
Open field latency of exiting the release site
.670 -.311
Aerial predator Explore 2 -.487 .506
0.00 5.00 10.00 15.00 20.00 25.00 30.00 Relax behaviour (pre-exposure) Agitated behaviour (pre-exposure) Relax behaviour (post-exposure) Agitated behaviour (post-exposure) M e an n u m b e r o f o b ser vation s RJF Hz WL *
Figure 8. Mean PCA scores in females (±1 SEM). *= P < 0.05
Figure 9. Mean PCA scores in males (±1 SEM). *= P < 0.05 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40 0.60 0.80 PCA sco re Component 1 Component 2 RJF Hz WL -1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 2.00 PCA sco re Component 2 Component 3 RJF Hz WL * * *
5 Discussion
No significant differences were detected in weight or metatarsus length of the birds with different genotype, however it is worth noting that the WL genotype males were around 10% heavier (P=0.06) than RJF males at day 88. In the open field and TI tests we did not find significant differences between RJF and WL genotypes. RJF genotype males solved the learning test with significantly higher precision in comparison to WL genotype males. At the first trial of the emergence test, WL genotype females took significantly more time before poking out their head, at the second trial when birds were restrained in a net for 3 minutes before the test, WL genotype females were affected by the treatment significantly more in comparison to the RJF females. The results from the TI test were not significantly different in WL and RJF genotype birds. In the aerial
predator test, the RJF genotype birds showed significantly higher number of agitated behaviours and less relax behaviour in comparison to the WL genotype birds.
The conducted behavioural tests are among the most validated
behavioural tests of measuring fear in chicken (Forkman et al. 2007). General fear and social dependency of the birds are the major
determinants of the behaviours in the open field test (Gallup et al. 1980) but some behaviours such as latency of first movement seem to be more influenced by fear rather than the social motivation of the birds (Forkman et al. 2007). Buitenhuis et al. (2004) identified QTLs which are involved in open field response in layer hens but the exact QTL was different in young birds and adults. The emergence test is based on Light/Dark test in mice and shows the hesitancy of the subjects to enter the light component of the test (Cheng et al. 1994). Evidence do not suggest that light is
aversive for chicken (Forkman et al. 2007) but considering that the junglefowl has evolved to hide underbrush to avoid predation (Forkman et al. 2007), it makes sense that fearful individuals hesitate more before exiting from a hiding. It is not clear whether the birds perceived the box as a shelter and considered the light component potentially dangerous or not, but the test was significantly correlated with other validated fear tests which suggests the validity of the emergence test in chicken. TI is a
classic test of fear in chicken but the notion behind it is not clear, some studies have suggested that the rationale behind TI is an anti-predatory response called ―death feigning‖ (Forkman et al. 2007). For example Thompson et al. (1981) showed that in quail TI reduced the chance of predation by cats. Schütz et al. (2004) found 3 different QTLs on chromosome 1that were involved in duration of TI; one QTL on
chromosome 4 was involved in behaviour related to the novel object test, and one QTL on chromosome 11 was associated with behaviour in a
restraint test. Fear does not seem to be a unitary concept, and hence different tests may measure different aspects of fear i.e. fear types (Forkman et al. 2007).
In the Aerial predator test and the emergence tests where the effect of stress on the behaviour was assessed, the RJF genotype birds behaved significantly different from the WL genotype birds. In the emergence test, latency of head poking and total exit were significantly altered in WL genotype birds while, they were not changed significantly in RJF
genotype. At first look, it sounds like WL genotype is more susceptible to stress but we should also consider that the habituation effect is also
involved in tendency of birds to exit the box. I could not find any study showing the effect of habituation on the result of the test, but my
assumption is that birds would stay shorter in the box on the second day because the outside area is not novel for them anymore. Based on this assumption and the fact that fearful birds would emerge from the box later (Forkman et al. 2007), it can be assumed that WL genotype birds were more resistant to the stressor because they emerged from the box after the stress faster than the first day but the RJF genotype birds stayed in the box almost as long as they stayed there on the first day. In the aerial predator test, the RJF genotype birds showed significantly more agitated behaviour after the exposure of predator. The results of the aerial predator test support the findings of the emergence test where WL
genotype birds showed attenuated fear reaction to the stressor. Sallinen et al. (1999) showed that in mice, the expression of α2C-ARs is linked with
increased corticosterone level and α2C-KO mice had lower corticosterone
reaction to both acute and chronic stress which suggests that down-regulation of the gene may lead to stress protective effects. Male RJF genotype birds solved the learning test with significantly lower number of mistakes. In mice it has been shown that alteration of α2C-ARs expression
can lead to change in brain dopamine level (Sallinen et al. 2007) which has been shown to play a role in reward seeking behaviours.
We need to answer some fundamental questions before judging the results of the current experiment. The SNP that the SAIL birds were genotyped for is a synonymous SNP, i.e. the same amino acids are coded regardless of the SNP and hence, the receptor structure and function will not be affected by the genotype. The SNP lies in a 40kb sweep (Rubin et al. 2010) and α2C-AR gene occupies around 5kb of the region, the rest of
the region should be sequenced using more markers to find probable change in cis or trans regulatory regions around the gene that might had been the target of selection, in human for example many signature of positive selection has been found in promoters of genes related to the
nervous system (Haygood et al. 2007). DNA methylation is commonly considered to cause permanent gene silencing (Cedar et al. 2009, Raynal et al. 2012). Nätt et al. (2012) showed that α2C-AR gene is hyper
methylated in WL in comparison to the RJF and it was shown that 3 out 4 studied genes kept their methylation pattern after 8 generations of
intercrossing but the methylation pattern of α2C-AR gene in the AIL birds
has not been studied yet. An additional step of deciphering the reasons behind the presence of the sweep can be studying the methylation pattern in the SAIL birds. Studying cytosine methylation, especially in so called CpG islands in the promoter region of α2C-AR gene might yield
interesting results.
The α2C-ARs are expressed in adrenal glands and control release of
epinephrine in mice (Brede et al. 2003). The expression studies of α2C
-ARs in the brain of both mice and human have shown that a significant portion of the a2-adrenoceptors in striatum is of the a2C subtype
(Fagerholm et al. 2008). Another step that can explain significant behavioural differences found at this study could be investigating the expression of α2C-AR gene in the striatum and adrenal glands. One
challenge facing this method is that α2-ARs are expressed in different regions of chickens’ brain in different proportions (Díez-alarcia et al. 2006) and maybe different genotypes alter the gene expression pattern in some specific brain regions. A specific α2C-AR subtype-selective agonist
became available very recently (Jnoff et al. 2012). If the recently discovered α2C-AR subtype-selective agonist affects physiology and
behaviour of SAIL birds of various genotypes differently, it can provide more unbiased knowledge on the probable adoptive value of different genotypes in different environments.
A big challenge for population genetics–based signatures is determining whether a signature is a result of selection or is a result of the effects of population demographic background, such as bottlenecks (periods of decreased population size) and divided populations (Sabeti et al.2006). WL breed had been originated from a small base population (Muir et al. 2008). This founder effect caused an important population bottle neck and as a result, some DNA regions with reduced genetic diversity can be expected. Rubin et al. (2010) sequenced the genome of 3 WL strains; therefore generalization on selection history of layer breeds based
exclusively on this experiment is uncertain. It will be interesting to know whether α2C-AR gene lies in a selective sweeps in other layer breeds or
5.1 Conclusions
In the aerial predator test and the emergence tests where the effect of stress on the behaviour was assessed, the RJF genotype birds behaved significantly different from the WL genotype birds. To reach fixation, the newly arising beneficial alleles should have substantial adaptive
advantages. Although we found differences in behaviour of individuals with WL genotype, more research is needed to find out how the variation on the ADRA2C gene, might have played a role in domestication of layer chicken.
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