Behavioural neurobiology
of feather pecking
Yvonne van Hierden
Beh
avioural n
eurob
iology of feather p
eckin
gYvonne van H
ierden 2003ISBN 90-6464-963-4
RIJKSUNIVERSITEIT GRONINGEN
Behavioural neurobiology of feather pecking
Proefschrift
ter verkrijging van het doctoraat in de
Wiskunde en Natuurwetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
vrijdag 10 oktober 2003
om 16:00 uur
door
Yvonne Margreet van Hierden
geboren op 11 december 1972
te Velp
Promotor: Prof. dr. J.M. Koolhaas
Co-promotor: Dr. S.M. Korte
Beoordelingscommissie: Prof. dr. L. Keeling
Prof. dr. B. Olivier
Prof. dr. T. Schuurman
ISBN: 90 6464 963 4
Paranimfen: Kathalijne Visser-Riedstra
Francesca Neijenhuis
Voor mijn ouders
The research described in this thesis was carried out at the Animal Welfare Group,
of the Animal Sciences Group, Lelystad, the Netherlands. The author was affiliated
at the Department of Animal Physiology, University of Groningen, as a member of
the Graduate School for Behavioral and Cognitive Neurosciences (BCN) of the
University of Groningen, the Netherlands. The research was funded by the Dutch
Organization for Scientific Research (NWO) as part of the research programme
(Grant ALW, PPWZ 805-46.051) and by the Dutch Ministry of Agriculture, Nature
Management and Fisheries. The birds were kindly provided by Hendrix Poultry
Breeders. The publication of this thesis was financially supported by the Animal
Sciences Group (Lelystad), University of Groningen, NWO and the Graduate
School for Behavioral and Cognitive Neurosciences
Cover design: Mechiel Korte and Yvonne van Hierden
Publisher: Ponsen & Looijen BV
© Y.M. van Hierden, Lelystad, 2003
All rights reserved. No part of this publication may be repoduced or transmitted in
any form or by any means, electronic or mechanical, including photocopying,
recording or any information storage or retrieval system, without written permission
from the author.
CONTENTS
Chapter page
1 General introduction 7
2 The development of feather pecking behaviour and targeting 27
of pecking in chicks from a high and low feather pecking line
of laying hens
3 Adrenocortical reactivity and central serotonin and dopamine 45
turnover in young chicks from a high and low feather pecking
line of laying hens
4 The control of feather pecking by serotonin 63
5 Chronic increase of dietary tryptophan decreases 85
feather pecking behaviour
6 Chicks from a high and low feather pecking line of laying 103
hens differ in apomorphine sensitivity
7 General Discussion (including summary of the results) 117
Samenvatting 137
References 145
Dankwoord 171
Curriculum Vitae 175
Chapter 1
General Introduction
8 Chapter 1
1. Introduction
Feather pecking is still one of the major problems in current housing
systems for laying hens, as it poses major welfare problems for the hens, and
constitutes an economical burden for the farmer. Hens peck and pull at the
feathers of conspecifics, causing damage to the plumage and loss of feathers. This
adversely affects the costs of egg production, since loss of feathers results in
considerable higher energetic needs and, consequently, higher feed requirements
of hens as a result of increased body heat loss (Leeson and Morrisson, 1978;
Tullet et al., 1980). Furthermore, feather pecking, which is painful in itself (Gentle
and Hunter, 1990), may result in severe damage to the integument of the birds,
including wounds of the skin. Wounded birds may become the victim of
cannibalism (Allen and Perry, 1975; Blokhuis et al., 2000; Hughes, 1982). This may
result in a loss up to 15% of the birds per production cycle (in alternative housing
systems) (Keeling et al., 1988). Apart from these serious economic losses, there is
a moral aspect to the problem as well. Clearly, feather pecking is detrimental to the
welfare of the birds.
Beak trimming, a common and effective precautionary measure practised
by poultry farmers to prevent serious feather damage and mortality, might be
associated with welfare problems. The procedure involves partial amputation of the
beak and behavioural studies indicate that hens may suffer from chronic pain,
presumably due to neuroma formation (Duncan et al., 1989; Gentle, 1986). Recent
studies showed that neuroma development can be prevented, when debeaking is
performed at an early age (1-10 days) (Gentle et al., 1997). Beak trimming
however, is without doubt painful in itself and has therefore been prohibited in
several European countries (e.g. Norway, Sweden, Switzerland). In the
Netherlands beak trimming will be legally banned in 2011.
A widespread introduction of loose-housing systems, specifically designed
with the aim to improve poultry welfare, is hampered due to the fact that large
outbreaks of feather pecking and cannibalism are more likely to occur in these
housing systems than in battery cages (Appleby and Hughes, 1991; Gunnarsson et
al., 1999). Most probably this is because the presence of a few feather peckers in a
General Introduction 9
free housing system has a much greater impact, simply because larger numbers of
potential victims are present (Allen and Perry, 1975). Thus, feather pecking
represents a serious problem that needs to be solved.
Because feather pecking in poultry practice reveals itself at group level, for
practical reasons alleged solutions are always applied on the whole flock. Most of
the measures taken involve adjusting management conditions, based on scientific
knowledge of environmental causal factors (see also section 3). However, despite
over 25 years of research and many efforts in practice to alleviate the problem of
feather pecking, an adequate solution to this problem has not been reached so far.
This strongly suggests that this type of abnormal behaviour cannot be completely
prevented by simply changing the environment of a group of birds. Research has
shown that only certain individuals in a flock initiate the feather pecking problem,
i.e. are the �real� feather peckers, whereas most birds receive pecks (Bessei, 1984;
Keeling, 1994; Wechsler et al., 1998). Furthermore, there is a growing body of
understanding that feather pecking results from a complex interaction between the
internal state of an animal and its environment (Blokhuis, 1989; Huber-Eicher and
Audigé, 1999; Hughes and Duncan, 1972; Leonard et al., 1995; Nicol et al., 2001;
Nørgaard-Nielsen et al., 1993). Feather pecking is generally considered an
abnormal behaviour, or even a behavioural pathology (Sanotra et al., 1995).
Following this latter interpretation, the aetiology of feather pecking may show
analogy to psychopathological disorders described in other species, like e.g.
(animal) stereotypies (Jenkins, 2001; Pitman, 1987; Pitman, 1989; Stein, 2000) and
self-mutilation disorders (Bordnick et al., 1994; Hartgraves and Randall, 1986;
Tiefenbacher et al., 2000; Weld et al., 1998). Despite a large body of scientific
knowledge on the causation of psychopathologies, surprisingly few efforts have
been made to approach feather pecking from the area of behavioural neuroscience
and psychiatry (Bilčík and Keeling, 2000; Kjaer et al., 2002).
Therefore, in this thesis feather pecking will be approached from the
assumption that it is a psychopathology, comparable to the ones mentioned above.
Current knowledge of the causal role of neurobiological systems in the
development of these behavioural disorders in both human and animal will be
applied to feather pecking. This implies shifting the focus of attention from purely
10 Chapter 1
studying the effects of environmental changes on a group of birds, towards
studying causal relationships between feather pecking and predisposing
behavioural or neuroendocrine factors within individual birds. Increased
understanding of the interaction between a birds environment and neuroendocrine
systems in relation to its propensity to perform feather pecking, may lead to new
insights and possible solutions to the feather pecking problem in the future.
2. Feather pecking: normal versus abnormal behaviour
Feather pecking in domestic fowl involves pecking at the feathers of
conspecifics. Its consequences for the recipient depend on the severity of pecking,
i.e. the force and velocity of the pecking movements (see for an overview, page 19;
Kjaer, 1999). The most gentle form of feather pecking is characterised by mild
(Keeling, 1995), often stereotypic pecks (Kjaer and Vestergaard, 1999), causing no
or very little damage to the feathers of the recipient birds. The more severe form of
feather pecking, characterised by feather pulling or even removal of feathers
(which in itself is painful), can result in serious damage to the feathers and
integument of the victims (Hughes and Duncan, 1972; Keeling, 1995). Severe
feather pecking can ultimately lead to cannibalistic acts, as wounded birds (i.e.
blood) are attractive for others to peck at (Blokhuis and Arkes, 1984; Savory and
Mann, 1997).
An important issue for debate among scientists is whether gentle and
severe feather pecking are related, i.e. depend on the same underlying
mechanisms (Kim-Madslien, 2000; Kjaer and Vestergaard, 1999; McAdie and
Keeling, 2002). There is some evidence indicating that gentle and severe feather
pecking may have a different origin and may be differentially affected by genetic
and environmental factors (Kjaer, 1999; Nicol et al., 1999). Furthermore, it appears
that gentle feather pecking at an early age does not always predict severe feather
pecking at a later age (Rodenburg et al., 2003). However, within a certain age
gentle and severe feather pecking are mostly positively correlated (Kim-Madslien,
2000; Rodenburg et al., 2003; Riedstra, 2003) suggesting that �both forms do
General Introduction 11
represent different extremes of the same behavioural continuum� (Kim-Madslien,
2000). Thus, the exact relationship between gentle and severe feather pecking is
still rather unclear.
It can be argued whether gentle feather pecking is of interest for poultry
welfare (McAdie and Keeling, 2002) as gentle feather pecking does not result in
feather damage. Gentle feather pecking in young chicks has even been regarded
part of normal social behaviour, representing social exploration (Riedstra and
Groothuis, 2002) or allopreening (Blokhuis, 1986; Harrison, 1965; Riedstra and
Groothuis, 2002; Vestergaard, 1994). However, high frequencies of gentle pecks
are often directed at the same spot on the body of another bird, giving gentle
feather pecking a very abnormal and stereotypic appearance (Kjaer and Sørensen,
1997; McAdie and Keeling, 2002). It has been recently suggested that �normal�
gentle feather pecking in young chicks may develop into stereotyped gentle feather
pecking and subsequently into severe feather pecking by either increased intensity
or increased severity of inter-bird pecking (McAdie and Keeling, 2002)
Neither this stereotyped form of gentle feather pecking nor severe feather
pecking has been reported to appear in the behavioural repertoire of hens living
under natural conditions and may therefore be considered abnormal behaviours
(Kim-Madslien, 2000; Kruijt, 1964; Vestergaard et al., 1993). As stereotypies are
generally a sign of maladaptation to the environment (Mason, 1991; Wiepkema and
Koolhaas, 1993), the occurrence of gentle feather pecking may be an early sign of
reduced welfare in birds.
3. Causation of feather pecking
Many years of research have revealed a wide range of factors influencing
the development or performance of (both gentle and severe) feather pecking
behaviour. Many of these factors affecting feather pecking are related to the
management of the birds (external factors). However, research has also revealed
numerous factors related to the nature of the birds (internal factors), including
12 Chapter 1
genetic predisposition, developmental stage, hormonal state, underlying
fearfulness, and social motivations.
3.1 External factors
Availability and quality of a floor substrate
One of the first and most important single causal factors associated with
the occurrence of feather pecking is the absence of (suitable) floor substrate
(Blokhuis and Arkes, 1984; Hughes and Duncan, 1972; Levy, 1938). Provision of
litter of a certain type or texture early in the development of the chick substantially
reduces feather pecking (Blokhuis and van der Haar, 1992; Huber-Eicher and
Wechsler, 1998). The two most influential current theories on the causation of
feather pecking assign an important role to this factor. It has been postulated that
feather pecking is a form of re- or misdirected pecking, related to the motivational
system of either feeding/foraging (Aerni et al., 2000; Blokhuis, 1989) or dustbathing
(Vestergaard, 1994). According to these theories, exposing chicks to litter early in
life would prevent them from perceiving feathers as a substrate for either foraging
or dustbathing. However, feather pecking is not fully eliminated by providing
suitable substrates (e.g. Huber-Eicher and Wechsler, 1998; Nicol et al., 2001),
suggesting the involvement of other causal factors.
Stocking density and group size
Feather pecking behaviour has been found to increase with group size
(Allen and Perry, 1975; Bilčík and Keeling, 1999; Keeling, 1994) and stocking
density (Allen and Perry, 1975; Appleby et al., 1988; Koelkebeck et al., 1987;
Simonsen et al., 1980) (Savory et al., 1999). Group size appears to be more
important than stocking density. However, since group size is often confounded
with stocking density, the role of the individual factors is difficult to distinguish
(Nicol et al., 1999; Savory et al., 1999).
General Introduction 13
Light intensity
The intensity of light influences the incidence of feather pecking. It is
generally agreed that increasing the brightness of the light increases the level of
feather pecking (Allen and Perry, 1975; Hughes and Black, 1974; Kjaer and
Vestergaard, 1999). Kjaer and Vestergaard (Kjaer and Vestergaard, 1999) found
that gentle and severe feather pecking are differently influenced by light intensity:
Birds reared at 30 lux showed less mild stereotypic pecking but more than twice as
much severe feather pecking as birds reared at 3 lux.
Diet and food form
Many of the earlier studies on feather pecking were based on the
hypothesis that it was related to deficiencies in certain nutritional components.
Outbreaks were ascribed to inadequate levels of e.g. calcium and protein
(aminoacids), dietary fibre, sodium chloride (for an overview see Hughes and
Duncan, 1972). Savory et al. (1999) reported of a suppression of feather pecking
damage with dietary supplementation with �higher� doses of the essential
aminoacid L-tryptophan.
Apart from the composition of the diet, dietary texture has also been found
to be an important factor. Several authors (for an overview see Hughes and
Duncan, 1972) report that pecking damage is more common when birds are fed on
pellets rather than on mash. In this respect, there might be an interaction between
diet and other environmental factors such as litter. For example, Savory and Mann
(1997) found that food form (mash or pellets) had no significant effect on feather
pecking in pullets kept in pens with litter-covered floors. Significant effects of
foraging material on feather pecking were found in studies in which the birds were
fed pellets (Huber-Eicher and Wechsler, 1997; Huber-Eicher and Wechsler, 1998).
Aerni et al. (2000) confirmed this interaction effect. High rates of feather pecking
and severe feather damage were only found in hens housed without access to
straw and fed food pellets.
14 Chapter 1
3.2 Internal factors
Genetic predisposition
Large variation in the performance of feather pecking exists between
strains of laying hens, even when they are kept in the same environment (e.g.
Bessei, 1986; Blokhuis and Beuving, 1993; Craig et al., 1975; Cuthbertson, 1980;
Kjaer, 1995). The use of breeding programmes in order to solve the feather
pecking problem requires knowledge on heritability of feather pecking and genetic
correlations with other traits (for instance production traits). Although some studies
indicate possibilities for such breeding programmes (Craig and Muir, 1996; Kjaer
and Sørensen, 1997; Muir, 1996), often the results are not consistent. For
instance, heritability estimates for feather pecking range from 0.04 � 0.56 (Bessei,
1984; Cuthbertson, 1980; Damme and Pirchner, 1984; Dickerson et al., 1961),
depending on age and method of recording (e.g. scoring of plumage condition,
direct observations).
Developmental stage
A number of studies indicate that feather pecking can already be observed
at a very early age (Hoffmeyer, 1969; Perry and Allen, 1976; Wennrich, 1975a).
Gentle feather pecking is performed by most members in groups of young chicks
(Kjaer and Sørensen, 1997; Wechsler et al., 1998). Severe feather pecking is
mostly observed at a later age (Huber-Eicher and Sebö, 2001), and is performed
by only a limited number of group members (Bessei, 1984; Keeling, 1994). Thus,
the intensity or severity of feather pecking seems to depend on age (Rodenburg
and Koene, 2003), with peaks appearing in different stages of development
(Blokhuis and Arkes, 1984; Hughes and Duncan, 1972). The intensity of feather
pecking also seems to change upon onset of lay and sexual maturity, possibly
under the influence of changes of gonadal hormones (Blokhuis and Arkes, 1984;
Hughes, 1973; Hughes and Duncan, 1972)
General Introduction 15
Furthermore, ontogenetic factors involving the interaction between
environmental conditions and birds at a young age have been reported to play an
important role in the development or occurrence of feather pecking later in life
(Blokhuis and van der Haar, 1992; Huber-Eicher and Wechsler, 1998; Johnsen et
al., 1998; Verbeek et al., 1994).
Hormonal state
Hughes (1973) tested the role of a range of gonadal hormones on feather
pecking, by using hormonal implants in hens at 12 weeks of age. Up to 18 weeks,
progesterone produced a moderate but significant increase in feather pecking.
Oestrogen and progesterone together resulted in a much greater increase. From
18 to 24 weeks the normal rise in feather pecking around the onset-of-lay was
prevented by testosterone. Hughes suggested that the increase in feather pecking
around the onset-of-lay is hormonally mediated, and can either be stimulated by
administering a combination of oestrogen and progesterone or be blocked by
giving testosterone.
Fearfulness
The role of fear in feather pecking behaviour is not clear. Some authors
have suggested that feather pecking is more likely to be initiated by fearful birds
e.g. (Johnsen et al., 1998; Vestergaard et al., 1993). However, most studies
indicate that fearfulness is a consequence of feather pecking, induced by feather
damage and pain, rather than a cause (Hansen and Braastad, 1994; Jones and
Hocking, 1999; Lee and Craig, 1991).
Social motivations
Although it appears that (severe) feather pecking is initially performed by a
restricted number of birds, feather pecking and particularly cannibalism can
escalate by spreading through a flock (McAdie and Keeling, 2000; Siren, 1963).
16 Chapter 1
The simplest mechanism is that birds are attracted to damaged feathers. Damage
to the integument or plumage has been found to facilitate and accelerate outbreaks
of feather pecking, leading to a domino effect (Allen and Perry, 1975; Freire et al.,
1999; McAdie and Keeling, 2000; Savory and Mann, 1997). Social transmission or
social learning have also been suggested to be involved in the spread of feather
pecking and cannibalism through a flock (Cloutier et al., 2002; McAdie and Keeling,
2002; Zeltner et al., 2000).
Recently, Riedstra and Groothuis (2002) proposed another mechanism
underlying the spread of gentle feather pecking in a flock. They argued that gentle
feather pecking plays an important functional role in the building (social
exploration) and maintenance of social relationships between chicks. Due to the
large group size in husbandry conditions, there might be an exponential increase in
the need to engage in and maintain such social relationships.
Jones (1995) and Blokhuis (2001) and their colleagues showed that birds
of a low feather pecking line displayed more social reinstatement behaviour than
birds of a high feather pecking line. They argued that if high feather pecking birds
are less socially motivated than low feather pecking birds, this might compromise
their ability to interact succesfully with their companions and to adapt to large social
groups. Social motivation (at 1, 17, 24 and 30 weeks of age) even predicted the
likelihood to develop gentle feather pecking (at 24 and 30 weeks of age) (Blokhuis
et al., 2001).
4. Summary and conclusion
From section 3 it is clear that research has succeeded in revealing many
factors influencing feather pecking. However, neither a single causal factor has
been identified so far, nor a combination of factors has resulted in an adequate
solution of the problem. It is generally agreed that feather pecking reflects
multifactorial processes (Hughes and Duncan, 1972), in which the interaction
between external (environmental) and internal (animal-based) factors affects its
occurrence.
General Introduction 17
As mentioned earlier, there is a growing acceptance that feather pecking is
a behavioural pathology (Sanotra et al., 1995; Zeltner et al., 2000). However, from
a neuroendocrine perspective, hardly any scientific evidence is available to provide
a foundation for this classification of feather pecking. To our knowledge, no
attempts were made to draw a parallel between feather pecking and behavioural
pathologies in other species, (e.g. stereotypies and obsessive compulsive
disorders). It is conceivable that neurobiological and neuroendocrine mechanisms
known to underlie such disorders may play a role in the development or
performance of feather pecking behaviour as well. Therefore, in this thesis, feather
pecking will be approached from a neurobiological angle (see also section 1).
5. A new approach to unravel mechanisms underlying feather pecking
5.1 Individual vulnerability
A wide range of studies, both in human and animals, demonstrated that
individuals can profoundly differ in their vulnerability for the development of a
behavioural pathology. The likelihood for an individual to develop dysfunctional
behaviour depends on a complex interaction between a genotypic (pre)disposition
and factors like ontogeny, adult life experiences and age. The outcome of this
interaction determines the capacity of an individual to cope with various
environmental demands.
Behavioural pathologies may develop when an individual fails to adapt to these
environmental demands (Koolhaas et al., 2001). Individual difference in
vulnerability for the development of behavioural pathology are accompanied by
clear differences in neuroendocrine reactivity and neurobiological makeup
(Koolhaas et al., 2001). For instance, the functioning of the hypothalamus-pituitary-
adrenal (HPA) axis, the serotonergic (5-HT) system and the dopaminergic (DA)
18 Chapter 1
system have been suggested to be divergent in those individuals that are prone to
the development of psychopathological disorders (see also box 1.2, 1.3 and 1.4).
As mentioned in section 3, large individual and strain differences have
been found for the performance of feather pecking as well. One of the first to
recognise that individual vulnerability for the development of feather pecking may
be a very useful tool in discovering underlying characteristics of birds, were
Blokhuis and Beutler (1992). They used two genetic lines of laying hens differing in
their propensity to feather peck. These so-called high (HFP) and low (LFP) feather
pecking lines of laying hens differed, at an adult age, in the level of feather pecking
damage (Blokhuis and Beutler, 1992) and feather pecking behaviour (Blokhuis and
Beuving, 1993). Apart from a marked difference in the level of feather pecking,
other authors showed that HFP and LFP birds also differ in several other
behavioural characteristics, such as fear and sociality (Johnsen and Vestergaard,
1996; Jones et al., 1995).
Korte and his colleagues (1997, 1999) were the first to investigate
physiological characteristics of adult birds of both lines. On the basis of the marked
behavioural differences between HFP and LFP birds in open-field behaviour found
by Jones et al. (1995), they anticipated differences between lines in the
behavioural and physiological reactivity to an acute stressor. In the experiments by
Korte, it was shown that in response to acute stress induced by manual restraint
(i.e. placing a bird on its side for 8 minutes), adult HFP birds displayed more
struggling behaviour, lower heart rate variability, higher plasma noradrenaline and
lower plasma corticosterone levels than LFP birds. Surprisingly, these behavioural
and physiological characteristics of HFP and LFP hens showed considerable
analogy to the characteristics of the proactive and reactive coping strategies,
respectively, previously found in rodents (Koolhaas et al., 2001). Korte (1997)
postulated that the propensity of a bird to perform feather pecking is related to its
coping strategy, i.e., the way it deals with environmental challenges, both
behaviourally and physiologically.
General Introduction 19
5.2 Coping strategies
Coping strategy is defined as the complex of individual behavioural,
physiological and neurobiological characteristics that determine how an individual
responds to environmental challenges (Koolhaas et al., 2001).
Coping strategies are characterised by noticeable differences in
behavioural response to environmental challenges. Generally, two extremes in
behavioural coping strategy are distinguished, originally based on differences in
the expression of territorial aggression (Benus et al., 1991b; Benus et al., 1987;
Koolhaas et al., 1997); i.e. the �active� fight/flight response, first introduced by
Canon (1915) and the �passive� conservation-withdrawal response originally
described by Engel and Schmale (1972).
Koolhaas and his colleagues (1997) introduced a new terminology for the
two coping strategies: �proactive� (previously labelled 'active') and reactive
(previously labelled 'passive') coping strategies. The basis of this new terminology
is the consistency in the way high and low aggressive mice behaviourally react in a
wide variety of environmental challenges, either proactively (�first do, then think�) or
reactively (�first think, then do�). A very fundamental difference seems to be the
degree in which behaviour is guided by environmental stimuli (Koolhaas et al.,
2001). Proactive animals act primarily on the basis of earlier experience and easily
forms routines, whereas the reactive copers are more guided by the information
actually present in their environment. These differences in behavioural control
mechanisms determine the adaptive character of the two coping strategies:
proactive animals are better adapted to stable, highly predictable environmental
conditions, whereas reactive copers may adapt well in variable and unpredictable
environmental conditions (Benus et al., 1990; Koolhaas et al., 1999).
Besides consistent behavioural differences between proactive and reactive
rodents, fundamental differences in the functioning of physiological and
neurobiological systems (see box 1.1) have been found, underlying the behavioural
differences (for a review on these differences see Koolhaas et al., 2001).
20 Chapter 1
BOX 1.1 Physiological and neuroendocrine characteristics of the proactive and reactive coping strategy in rodents
Cort = corticosterone, NA = Noradrenaline, A = Adrenaline, 5-HT = serotonin, DA = Dopamine
Proactive Reactive HPA-axis activity (Cort) Low Normal
HPA-axis reactivity (Cort) Low High
Neurosympathetic reactivity (NA) High Low
Adrenomedullary reactivity (A) High Medium
Parasympathetic reactivity Low High
Testosterone production High Low
5-HT1A receptor sensitivity High Low
Striatal DA receptor sensitivity High Low
(Koolhaas et al., 2001)
5.3 Coping strategy: a guide in feather pecking research
Research in both humans and animals has revealed a number of
neurobiological systems involved in the aetiology of psychopathological
behaviours. Especially the HPA-axis (box 1.2), the serotonergic (5-HT) system (box
1.3) and the dopaminergic (DA) system (box 1.4) have been implicated in several
behavioural pathologies, including depression, excessive aggressive behaviour,
(animal) stereotypies, and obsessive compulsive disorders (OCD) (see boxes for
references). Notably, these are also the neurobiological systems involved in the
differentiation between the proactive and reactive coping strategy in rodents (see
box 1.1).
As mentioned above, studies by Korte et al. (1997, 1999) suggest that the
neurobiological characteristics of adult birds of the HFP and LFP line are similar to
those of rodents displaying a proactive and reactive coping strategy, respectively.
Therefore, in this thesis, the concept of coping strategy will be used as a guide in
unravelling possible behavioural and neuroendocrine mechanisms underpinning
the development and performance of feather pecking.
General Introduction 21
BOX 1.2 Hypothalamus Pituitary Adrenal (HPA) axis In birds, as in mammals, high levels of glucocorticoids in the blood plasma can be indicative for a
response to acute stressors (Beuving and Vonder, 1978; Gross, 1990). In reaction to an acute stressor,
corticosterone, the main corticosteroid in the avian blood plasma, is released from the adrenal cortex, in
response to adrenocorticotropic Hormone (ACTH). ACTH is released from the pituitary under the
influence of corticotropin-releasing hormone (CRH), which in turn is produced by the hypothalamus.
Corticosterone exerts an inhibitory influence on the activity of the hypothalamus-pituitary-adrenal (HPA)-
axis (i.e. on CRH and ACTH release) and extra-hypothalamic structures through interaction with
corticosteroid receptors. The corticosteroid receptors in the brain consists of two distinct types of
receptors; the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Both intracellular
receptor types differ in primary structure, localisation and function (de Kloet et al., 1990). MRs bind
corticosteroids with a 10-fold higher affinity than GRs (de Kloet et al., 1998). MRs are highly
concentrated in the hippocampus and are mainly involved in the organisation of circadian-driven daily
activities and the regulation of basal activity of the HPA-axis. MRs determine the threshold or sensitivity
for stress-induced activation of the HPA system (de Kloet et al., 1991). GRs are more widely expressed
in the brain and are suggested to be involved (in conjunction with MRs) in corticosterone-mediated
feedback on stress-induced activation of the HPA axis.
In humans, functional abnormalities of the HPA-axis and disturbances in glucocorticoid regulation via
MRs and GRs, have been implicated in the aetiology of several behavioural disorders, such as
depression (Heuser, 1998; Holsboer and Barden, 1996; Reus and Wolkowitz, 2001; Seckl et al., 1990;
Steckler et al., 1999), anxiety (Korte, 1991; Korte et al., 1995) and aggression (Haller et al., 1998; Haller
et al., 2000).
6. Aim and outline of this thesis
The main scientific aim of this thesis is to identify behavioural,
neurobiological and neuroendocrine characteristics of laying hens, that may be
causally related to feather pecking behaviour. Birds of the HFP and LFP line will be
used as a model for high and low feather pecking, as was previously done by other
authors (Blokhuis and Beutler, 1992; Blokhuis and Beuving, 1993; Johnsen and
Vestergaard, 1996; Jones et al., 1995; Korte et al., 1997; Korte et al., 1999;
McAdie and Keeling, 2002). The extreme differences in behaviour and stress
physiology between birds of both lines may help to give a better insight in
22 Chapter 1
mechanisms underlying feather pecking behaviour. Neuroendocrine systems
associated with the differentiation of coping strategies, and known to be involved in
behavioural pathologies in other species are investigated, including the HPA axis,
the serotonergic and dopaminergic system.
In the present thesis it will be investigated whether:
1. the concept of coping strategy represents a useful framework for studying the
mechanisms underlying feather pecking. It will be investigated whether
differences in behaviour, physiology and neurobiology between birds of the
HFP and LFP line are consistent with current knowledge of the proactive and
reactive coping strategy, respectively.
2. the development and performance of feather pecking can be explained by
differences in neuroendocrinology (HPA-axis, 5-HT and DA) between the two
lines. In this thesis, emphasis will lie on investigating the (possible causal) role
of 5-HT in the development and performance of feather pecking behaviour.
As mentioned earlier, age was found to be an important factor in the
occurrence of feather pecking. Furthermore, different motivational systems have
been implicated in the development of feather pecking, i.e. feeding/foraging and
dustbathing (section 3). Adult HFP and LFP birds have been shown to differ in
feather pecking behaviour, however it is unclear at which developmental stage
HFP and LFP chicks start to show differences in feather pecking. Furthermore, it is
not known which motivational systems are involved in the development of feather
pecking in either line. Therefore, in chapter 2, a study is described investigating
the development of feather pecking and related behaviours during the first 8 weeks
of life of HFP and LFP chicks.
The clear differences between adult HFP and LFP birds in the behavioural
and physiological responsiveness were previously interpreted in terms of
differences in coping strategy (Korte et al. 1997; 1999). Furthermore, it was
postulated that these differences are causally related to the differences in feather
pecking between both lines (Korte et al. 1997; 1999). In Chapter 3 it is investigated
whether differences between lines in behavioural development (chapter 2) are
associated with physiological and neuroendocrine differences as well.
General Introduction 23
BOX 1.3 The serotonergic (5-HT) system
The neurotransmitter serotonin or 5-hydroxytryptamine (5-HT) is found throughout the central
nervous system (CNS). Within the CNS, the cell bodies of 5-HT neurons are clustered along the midline
(raphe nuclei) of the midbrain and the brain stem of both mammals and birds including chicken (Okado
et al., 1992). These neurons send ascending projections to most parts of the forebrain and descending
projections to lower brain stem regions and to the spinal cord. Serotonergic neurons in the brain
synthesise 5-HT from the amino acid L-tryptophan. The (rate limiting) enzyme, tryptophan-5-
hydroxylase, converts L-tryptophan into 5-hydroxytryptophan, which is then decarboxylated to 5-
hydroxytrypamine by 5-hydroxytryptophan decarboxylase (Blier and de Montigny, 1998). Dietary L-
tryptophan supplementation (Fernstrom, 1983; Harrison and D'Mello, 1986) or depletion (Klaassen et
al., 1999; Young and Leyton, 2002) can be used to increase or decrease 5-HT levels in the brain,
respectively.
5-HT is synthesised in nerve terminals and stored in vesicles. After release into the synaptic
cleft, 5-HT can bind to receptors of different subtypes. Via binding to these different receptors, 5-HT can
influence many parts of the brain involved in controlling a variety of physiologic functions, including
mood, behaviour, pain, appetite, endocrine secretion and cardiovascular function. After acting on pre-
and postsynaptic receptors, 5-HT is transported back into the nerve terminal via uptake carriers, where
it may be recycled in storage granules, or destroyed by a mitochondrial enzyme, monoamine oxidase
(MAO). The major degradation product, or main metabolite of 5-HT is 5-HIAA (Fuller, 1995).
5-HT neuron dysfunction appears to have an aetiological role in various behavioural
pathologies, since drugs that modify serotonin function are potentially useful in treating many
psychopathological disorders, including aggression (Chiavegatto et al., 2001; Lesch and Merschdorf,
2000; van der Vegt et al., 2001), depression (Blier and de Montigny, 1998; Lucki, 1998; Schreiber and
de Vry, 1993) and (animal) stereotypies (Ko�t'ál and Savory, 1995; Pitman, 1989; Schoenecker and
Heller, 2001), Obsessive Compulsive Disorder (OCD) (Blier and de Montigny, 1998; Luescher, 1998;
Pigott, 1996; Stein, 2000) . The role of 5-HT in aggression has been extensively studied. Low
concentrations of 5-HIAA in the cerebrospinal fluid have been consistently found as a trait characteristic
in highly violent, aggressive individuals (van der Vegt et al., 2001). In addition, many animal studies
show anti-aggressive effects of selective 5-HT1A and 5-HT1B receptor agonists (which lower 5-HT
release), supporting a role of the 5-HT system in aggression (Brown and Linnoila, 1990; de Boer et al.,
1999; de Boer et al., 2000; van der Vegt et al., 2001).
Treatment of psychiatric disorders like depression and OCD, involves administration of
tricyclic antidepressants or selective serotonin reuptake inhibitors (SSRIs), like for instance fluoxetine
(�Prozac�). Chronic administration of SSRIs, increases 5-HT neurotransmission, and alleviates the
symptoms of these disorders (Stein, 2000; Vaswani et al., 2003).
24 Chapter 1
As a first step, the development of adrenocortical (re)activity in HFP and
LFP chicks during the first 8 weeks of life was studied. Secondly, we studied DA
and 5-HT turnover in the brain of HFP and LFP chicks, at the age of 28 days.
The clear differences between adult HFP and LFP birds in the behavioural
and physiological responsiveness were previously interpreted in terms of
differences in coping strategy (Korte et al. 1997; 1999). Furthermore, it was
postulated that these differences are causally related to the differences in feather
pecking between both lines (Korte et al. 1997; 1999). In Chapter 3 it is investigated
whether differences between lines in behavioural development (chapter 2) are
associated with physiological and neuroendocrine differences as well. As a first
step, the development of adrenocortical (re)activity in HFP and LFP chicks during
the first 8 weeks of life was studied. Secondly, we studied DA and 5-HT turnover in
the brain of HFP and LFP chicks, at the age of 28 days.
The 5-HT and DA system have been implicated in the distinction between
coping strategy (Koolhaas et al., 2001) and in the aetiology of behavioural
pathologies (Brown and Linnoila, 1990; Ellison, 1994; Goodman et al., 1990;
McDougle et al., 1994; Stein, 2000). Therefore, we investigated the role of the 5-
HT system in the performance and development of feather pecking. The effect of a
decrease (chapter 4) and an increase (chapter 5) of 5-HT turnover in the brain of
LFP and HFP chicks on feather pecking behaviour are described.
Chapter 6, reports about a study, in which, as a first step of investigating a
possible role of the DA in feather pecking behaviour, we investigated the sensitivity
of the DA receptor system of HFP and LFP chicks. We examined the effect of
acute APO treatment on behavioural responses in an open field, to identify the
hypothesised difference in DA receptors sensitivity between chicks of both lines.
In chapter 7 an integrated discussion is given, by raising the main topics
and findings of this thesis.
General Introduction 25
BOX 1.4 The dopaminergic (DA) system The neurotransmitter dopamine (DA), together with adrenaline and noradrenaline, is called a
catecholamine. Dopamine is synthesised from tyrosine through the actions of two enzymes, tyrosine
hydroxylase and decarboxylase. Tyrosine hydroxylase, the rate-limiting enzyme in the process, converts
L-tyrosine to dihydroxy-L-phenylalanine, or L-DOPA. Then, the aromatic amino acid decarboxylase
immediately converts L-DOPA to DA. In some cells, DA is further converted to the neurotransmitter
noradrenaline or even further to adrenaline, but in many neurons DA itself serves as an active
neurotransmitter. Once DA is released from presynaptic DA containing vesicles in the terminals, it can
interact with postsynaptic DA receptors or different types of presynaptic autoreceptors that regulate
transmitter release, synthesis, or firing rate. DA receptors have been classified into two major families
(including many subtypes): D1 and D2 receptors. Interneuronally (i.e. after reuptake into the neuron) and
extraneuronally, DA is metabolised (e.g. by MAO) to the substances DOPAC and HVA, respectively
(Tzschentke, 2001).
Apomorphine (APO), a DA receptor agonist is frequently used to evaluate DA function or
activity in physiological processes or neuropsychiatric disorders (Lal, 1988), or predict individual
differences in sensitivity of DA receptors (Surmann and Havemann-Reinecke, 1995). In several animal
species, injection with APO induces stereotyped behaviour and increased locomotor activity (Berridge
and Aldridge, 2000; Bolhuis et al., 2000; Delius, 1988; Godoy et al., 2000; Surmann and Havemann-
Reinecke, 1995; Zarrindast and Amin, 1992).
Dopamine has been implicated in a variety of functions including motor control, cardiovascular
regulation, cognition, learning, endocrine regulation and emotion. Like the HPA-axis and 5-HT, DA
dysfunction has been implicated in the aetiology of psychopathological disorders, like schizophrenia
(Ellison, 1994) and OCD (Goodman et al., 1990; McDougle et al., 1994). For instance, excessive DA
neurotransmission in the forebrain is believed to underlie schizophrenia. Drugs blocking postsynaptic D2
receptors are effective in treating this disorder (Marcotte et al., 2001; Moore et al., 1999). In OCD,
augmentation of DA neurotransmission, either through administration of serotonin reuptake inhibitors,
via an interaction between 5-HT and DA (Stein, 2000) and/or haloperidol (van Ameringen et al., 1999),
is thought to alleviate the symptoms.
26 Chapter 1
Chapter 2
The development of feather pecking
behaviour and targeting of pecking in chicks from a high and low feather
pecking line of laying hens
Yvonne M. van Hierden1,2, S. Mechiel Korte1, E. Wim Ruesink1, Cornelis G. van Reenen1, Bas Engel1, Jaap M. Koolhaas2 and
Harry J. Blokhuis2
1Institute for Animal Science and Health (ID-Lelystad), P.O. Box 65, NL-8200
AB Lelystad, The Netherlands 2University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Applied Animal Behaviour Science 77:183-196, 2002
28 Chapter 2 Abstract
Large individual differences between adult laying hens in their propensity for
feather pecking are known to exist. However, not much research has been carried out into
the individual differences concerning the development of feather pecking behaviour. The
purpose of this study was to investigate whether contrasting levels of feather pecking,
observed among adult birds from two lines of laying hens, already occur at an early age.
Furthermore, an important question to be discussed was whether different behavioural
systems might be related to the occurrence of feather pecking. Therefore, this study
consisted of studying and comparing the behaviour of White Leghorn laying hens from a
high (HFP) and low feather pecking line (LFP) during the first 8 weeks of life. Chicks were
reared in litter-floor pens and were kept in groups of five animals per line (12 groups per
line).
HFP chicks showed significantly higher levels of gentle feather pecking (gentle FP)
than LFP chicks at the age of 14 and 28 days. Furthermore, HFP chicks spent significantly
more time preening than LFP chicks on days 14, 28 and 41. Duration of foraging behaviour
and feeding behaviour was significantly higher in the LFP line compared to the HFP line on
days 41 and 56 and days 28, 41 and 56, respectively. HFP chicks showed a significant
negative correlation between gentle FP and preening on days 3 (r = -0.49) and 41 (r = -
0.86). In the LFP line duration of feeding correlated negatively with gentle FP on day 3 (r = -
0.63). A principal component analysis (PCA) revealed that in the HFP line, gentle FP and
preening exhibited high and opposite loadings on the same component at all ages, whereas
feeding consistently loaded on the other component. This outcome contrasted with that of
the LFP line. In this line feeding predominantly loaded on the same principal component as
gentle FP, with loadings opposite to those of gentle FP, whereas preening showed the same
loadings as gentle FP, on days 3 and 41.
In conclusion, differences in feather pecking behaviour between HFP and LFP
chicks can already be observed at a very early age during development. Furthermore, our
results indicate that HFP and LFP chicks differ in the way pecking behaviour is targeted.
This difference could be related to the existence of a difference in underlying motivational
system controlling the development of feather pecking between the two lines.
Development of feather pecking 29
1. Introduction
The occurrence of feather pecking behaviour is, despite years of studying
this phenomenon, still rather unpredictable. Feather pecking, ranging from gentle
feather pecking (resembling a stereotypy; (Kjaer and Vestergaard, 1999) to pulling
and removing feathers of conspecifics, causes deterioration of the plumage,
injuries and ultimately leads to mortality (cannibalism). Hence, feather pecking
negatively affects poultry welfare (Blokhuis and Wiepkema, 1998).
Research has revealed numerous factors contributing to the development
of feather pecking (Hughes and Duncan, 1972). These include both animal related
(e.g. hormones, genetics) (Hughes, 1973; Kjaer, 1999) and environment related
factors, such as light intensity (Allen and Perry, 1975), diet (Hughes and Duncan,
1972), stocking density (Bilčík and Keeling, 2000; Kjaer and Vestergaard, 1999;
Simonsen et al., 1980) and availability and quality of floor substrate (Blokhuis,
1986; Huber-Eicher and Wechsler, 1998).
A number of studies indicate that feather pecking can already be observed
at a very early age (Hoffmeyer, 1969; Wennrich, 1975b) and it is suggested that a
relatively short sensitive period "for getting the right pecking experience" early in
life (Johnsen et al., 1998) is important in the development of this behaviour
(Johnsen et al., 1998; Vestergaard, 1994). In recent years, more interest has been
directed to the onset of feather pecking and the role of early life experience (i.e.
rearing conditions) in the causation and development of feather pecking in a group.
For instance, the provision of suitable litter during the rearing phase, is found to
substantially reduce feather pecking (e.g. Blokhuis and van der Haar, 1992; Huber-
Eicher and Wechsler, 1998).
It has been postulated that feather pecking is a form of re- or misdirected
pecking, related to the motivational system of either feeding and foraging (Blokhuis,
1989) or dustbathing (Vestergaard, 1994). According to these theories, exposing
chicks to litter early in life would prevent them from perceiving feathers as a
substrate for either foraging or dustbathing. However, feather pecking is not
eliminated by providing suitable substrates (e.g. Nicol et al., 2001) and, therefore
30 Chapter 2
behavioural systems other than dustbathing or feeding may also be linked to the
occurrence of feather pecking.
Large and consistent differences in feather pecking are observed between
breeds and lines (Bessei, 1986; Hughes and Duncan, 1972; Kjaer and Sørensen,
1997), as well as between individual birds within flocks of laying hens (Keeling,
1994). Blokhuis and Beutler (1992), reported two strains of White Leghorn layers
showing contrasting levels of feather pecking damage. Birds at the age of 24 and
30 weeks (Blokhuis et al., 2001) and 38 and 41 weeks (Blokhuis and Beuving,
1993) showed significantly higher levels of (gentle) feather pecking behaviour in
the so-called high feather pecking line (HFP) compared to the low feather pecking
line (LFP).
It is still unclear at which developmental stage LFP and HFP chicks start to
show differences in feather pecking. Furthermore, it remains unanswered which
motivational systems are involved in the development of feather pecking in either
line. It is essential to have a wide knowledge of these issues in order to further
unravel the underlying causation of feather pecking. Thus, this study was designed
to investigate the development of feather pecking and related behaviour of HFP
and LFP chicks during the first 8 weeks of life.
2. Methods
2.1 Birds and housing
In this study, 120 White Leghorn chicks from two strains were obtained
from a commercial supplier: 60 LFP and 60 HFP chicks. The two lines originate
from different breeding lines and the difference in feather pecking is a coincidental
result of a commercial selection program (Korte et al., 1997). All birds were female
and non-beak trimmed. Chicks arrived on the day of hatching and were individually
marked by a wingtag before housing. From the day of arrival chicks were kept in
groups of five animals per line (12 groups per line) and housed in pens (0.75
Development of feather pecking 31
m×1.0 m) with wood shavings. Visual contact between chicks in adjacent pens was
prevented by hardboard separations between the pens.
Pens were placed in a climate-controlled room. The environmental
temperature was lowered from 34 °C on day 1 to 18 °C at 8 weeks of age. On days
1 and 2 of age the light regime was alternately 4 h light and 4 h dark. From 3 days
to 8 weeks of age the light regime decreased from an 18 h light to a 10 h light
period.
All groups had access to three drinking cups and one square feeding
trough placed along one of the walls of the pen. Feeding regimes were those
recommended by suppliers of commercial layers, i.e. starter feed (mash) from 0 to
6 weeks; grower feed (mash) from 6 to 8 weeks. Water and a commercial feed
were provided ad libitum.
2.2 Behavioural measurements
The behaviour of the birds was studied at the age of 3, 14, 28, 41 and 56
days. On these days all pens were recorded on videotape between 13:00 and
17:00 h for a period of 30 min. At each age two focal birds were randomly chosen
from each pen and their behaviour was scored continuously for 30 min per bird
using The Observer® 3.0 software (Noldus, Wageningen, The Netherlands).
Duration and frequency of the behavioural elements scored are described in Table
1.
2.3 Statistical analysis
For behaviour, initially, averages of the observations for the two randomly
chosen birds per cage were analysed. First, separate analyses per age were
performed. HFP and LFP lines were compared with Wilcoxon's two-sample test
(Mann�Whitney test; Conover, 1980) applied to rank numbers of the data. Second,
all ages were compared pairwise. For each pair, the difference between the means
at the two ages was calculated for each cage. Wilcoxon's two-sample test was
applied on these differences to compare HFP and LFP lines. A significant result
32 Chapter 2
indicates an interaction between lines and ages, i.e. age effects for the two lines
differ. The test will show which line has the largest age effect. A non-significant
result was taken as an indication that a main effect model would apply. In the first
instance, as a follow-up, Wilcoxon's signed rank test (Wilcoxon's matched pairs
test; Conover, 1980) was applied to the differences for the HFP and LFP lines
separately.
This way it can be checked whether age effects within lines differ from 0. In
addition, parametric tests were performed per age with a generalised linear model,
with a logit link for fractions and a logarithmic link for counts, and a multiplicative
overdispersion parameter in the binomial and Poisson variance functions,
respectively. The parameters were estimated by maximum quasi-likelihood, the
overdispersion parameters were estimated from Pearson's chi-square statistic.
Tests were performed with the maximum quasi-likelihood ratio statistic. Details may
be found in McCullagh and Nelder (1989). Pairwise comparisons between ages
were made with a generalised linear mixed model, including random effects for
cages, according to methodology presented in Engel and Keen (1994) . Because
of the complicated correlation structure between observations, no analysis was
performed on all data with all ages in one model. The parametric and non-
parametric analyses basically produced the same results and only results from the
more simple rank tests will be discussed.
To study the relationship between pairs of variables individual observations
per animal were used. At each age, two different, but related, approaches were
followed. First, correlations were calculated between (Pearson) residuals saved
from separate analyses of the variables with fixed cage effects. These correlations
can be interpreted as pooled correlations within lines and cages. Second, one of
the variables was added as a covariate to the generalised linear mixed model for
the other variable. Tests where performed to see whether the coefficient of the
additional covariable significantly depended on the lines and differed from zero.
To visualise the correlation structures per line and age between the
behavioural parameters, separate principal component analysis (PCA) were
performed. Variables were expressed in terms of the first two principal components
in biplots. Each of these components is a linear combination of the variables gentle
Development of feather pecking 33
FP, foraging, feeding, preening, walking and resting. All statistical calculations were
done in Genstat® (1993, 1997). Differences or correlations were considered
significant if P < 0.05. Table 1. Ethogram showing the behavioural measurements (posture either standing or
sitting).
Behaviour Definition
Pecking frequencies
Gentle feather pecking (Gentle FP) Mild pecking at the feathers of conspecific, generally
performed in multiple bouts (every single peck is
counted as one occurrence)
Severe feather pecking Vigorous pecking/pulling/pinching at the feathers of
conspecific
Aggressive pecking Forceful and rapid singular pecks (mainly) at the head
of conspecific (or other parts of the facial region)
Comb pecking Pecking at the comb of a conspecific
Cage pecking Pecking at the walls of the cage
Other frequency
Ground scratching Bird, alternately, makes backward strokes with both
legs in the litter as part of foraging behaviour. (Every
stroke is recorded as one occurrence)
Duration
Feeding (FEED) Pecking at food in trough
Foraging (FORAG) Pecking at the litter and scratching (separately scored
as ground scratching) or moving with the head in a
lower position than the rump
Preening (PREEN) Preening behaviour as described by Kruijt (1964): e.g.
autopecking, nibbling, stroking, combing, head-
rubbing
Walking (WALK) Walking, running, jumping or flying (it may be
accompanied by wing-flapping)
Dustbathing Sitting and performing: vertical wing-shaking, body
shaking, litter pecking and/or scratching, bill raking,
side and head rubbing
Resting (REST) Sitting or standing inactive (no movement of the legs)
34 Chapter 2 Figure 1. Frequency or relative duration (percentage of the total observation time) of
behaviour of LFP and HFP chicks on days 3, 14, 28, 41 and 56 of age (levels expressed as
mean ± S.E.M.). ***P < 0.001,**P < 0.01,*P < 0.05,#0.05 < P < 0.08.
3 14 28 41 560
3
6
9
12
15
Freq
uenc
y/30
min
utes
Severe feather peckingB
#
3 14 28 41 560
10
20
30
4080
90
100
Rel
ativ
e du
ratio
n (%
)
ForagingC
****
3 14 28 41 560
10
20
30
4080
90
100
Rel
ativ
e du
ratio
n (%
)
PreeningE
***
#
****
#
3 14 28 41 560
3
6
9
12
15
Freq
uenc
y/30
min
utes
Gentle feather peckingLFP HFPA
***
3 14 28 41 560
10
20
30
4080
90
100
Rel
ativ
e du
ratio
n (%
)
WalkingF
**#
3 14 28 41 560
10
20
30
4080
90
100
Rel
ativ
e du
ratio
n (%
)
FeedingD
***
#*
Development of feather pecking 35
3. Results
3.1 Mean levels of behavioural elements
Significant age by line interactions (P < 0.05) were found for gentle FP,
severe feather pecking, foraging, feeding, preening and walking (Figure 1). Figure
1A shows that HFP chicks displayed significantly more gentle FP than LFP chicks,
at the age of 14 and 28 days. Levels of severe feather pecking (Figure 1B),
although quite low in either line, tended to be higher in HFP birds on 41 days of
age. Duration (i.e. percentage of total observation time) of foraging behaviour
(Figure 1C) was significantly higher in the LFP line compared to the HFP line on
days 41 and 56 of age.
Feeding behaviour (Figure 1D) was significantly higher in the LFP line
compared to the HFP line. The time spent feeding was higher on days 28, 41 and
56 of age. HFP chicks spent significantly more time preening ( Figure 1E) than LFP
chicks on days 14, 28 and 41 and tended to on days 3 and 56 of age. LFP spent
more time walking than HFP birds (Figure 1F), on days 28 (P < 0.01) and 41
(P = 0.07). LFP chicks showed significantly shorter duration of resting behaviour
than HFP chicks, on days 28 (28.18 ± 2.52% versus 46.86 ± 4.34%; P < 0.001), 41
(32.68 ± 1.81% versus 46.32 ± 3.24%; P<0.001) and 56 (34.64 ± 2.92% versus
48.12 ± 3.82%; P < 0.01) of age.
HFP birds pecked significantly (P < 0.05) more at the comb of a
conspecific than LFP birds, on 14 days (2.25 ± 1.06 versus 0.12 ± 0.09) and 56
days of age (5.87 ± 1.94 versus 2.08 ± 0.71). No significant line or age differences
were found for aggressive pecking (the overall level was 1.75 ± 0.211) and cage
pecking (overall 8.22 ± 0.97). No significant line or age effects were found for
ground scratching behaviour (overall 15.6 ± 1.95). Levels of dustbathing behaviour
were close to zero in either lines, and no significant line or age differences were
found.
36 Chapter 2
3.2 Correlations and PCA
To examine whether the observed differences in the development of
feather pecking behaviour between the two lines can be attributed to different
underlying motivational systems, the frequency of gentle FP was correlated with
the duration of several behavioural elements. In addition, using the same
behavioural elements, a PCA was performed to summarise the correlation matrix
and to further substantiate the possible existence of a common underlying factor.
Table 2 presents Pearson correlations between gentle FP and foraging, feeding,
preening, walking and resting. No correlations are shown for severe feather
pecking and dustbathing behaviour because of the very low levels for these
behavioural elements.
Foraging behaviour showed no clear correlation with gentle FP. Only on
day 14 a significant positive correlation was found in the LFP line. In the LFP line
duration of feeding correlated negatively with gentle FP on days 3 and 41 of age.
Resting behaviour correlated positively on days 41 and 56 in the LFP line and also
positively in the HFP line on days 3 and 41. In HFP chicks a significant negative
correlation was found between gentle FP and preening on days 3 and 41. Walking
correlated negatively on day 3 but positively on days 14 and 28 in this line.
The first two principal components explained 77, 67, 65 and 72% of the
variation for ages 3, 14, 28, 41 and 56, respectively for the LFP line and 78, 65, 64,
75 and 63% for the HFP line. In Figure 2, for each line and age, the five
behavioural parameters are expressed in terms of the first two components and
presented in a biplot. These plots are a visualisation of the correlation structure.
For instance, the high and opposite loadings for gentle FP and preening (PREEN)
for the HFP line were consistent with the moderate to high negative correlations
between these variables in Table 2. The fact that gentle FP hardly correlated with
feeding (FEED) for HFP chicks is consistent with the latter parameter always
loading on the other component (Figure 2F�J). In contrast to the HFP line, for LFP
chicks, FEED predominantly loaded on the same component as gentle FP, with
opposite loadings, while PREEN had similar loadings as gentle FP at 3 and 41
days of age (Figure 2A and D). Thus, at each age, the principal component with a
Development of feather pecking 37
high loading for gentle FP was differently associated with PREEN and FEED in
HFP and LFP chicks, respectively.
Table 2. Pearson correlations (r) between gentle feather pecking and foraging, feeding,
preening, walking and resting in LFP and HFP chicks on days 3, 14, 28, 41 and 56 of age. Gentle feather pecking
Age (days) LFP HFP
Foraging 3 0.15 -0.34
14 0.45* 0.26
28 0.16 0.15
41 0.02 0.34
56 0.18 -0.32
Feeding 3 -0.63*** -0.06
14 -0.24 -0.08
28 -0.28 -0.18
41 -0.35# 0.15
56 -0.22 -0.11
Preening 3 0.27 -0.49**
14 -0.01 -0.33
28 0.02 -0.16
41 0.16 -0.86***
56 0.14 -0.30
Walking 3 -0.26 -0.36#
14 0.26 0.59**
28 0.35# 0.44*
41 0.26 -0.32
56 -0.28 -0.12
Resting 3 0.23 0.50*
14 -0.26 -0.21
28 0.32 0.18
41 0.71*** 0.54**
56 0.42* 0.08 ***P < 0.001, **P < 0.01, *P < 0.05, #0.05 < P < 0.10
38
Cha
pter
2
Fi
gure
2.
Dis
tribu
tion
of b
ehav
iour
al p
aram
eter
s in
rel
atio
n to
the
firs
t tw
o co
mpo
nent
s of
a P
rinci
pal C
ompo
nent
Ana
lysi
s ac
hiev
ed f
rom
beha
viou
ral o
bser
vatio
ns o
f LFP
and
HFP
chi
cks
on d
ays
3, 1
4, 2
8, 4
1 an
d 56
of a
ge. E
ach
abbr
evia
tion
of a
beh
avio
ural
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Development of feather pecking 39
4. Discussion
4.1 The development of behaviour and targeting of pecking
In the present study, we investigated the development of feather pecking
and related behaviour in chicks of the LFP and HFP line. HFP chicks appear to
have a higher "drive" in performing feather directed behaviour than LFP chicks, as
shown by the higher levels of gentle FP and preening behaviour in HFP chicks at
several points in time during the first 8 weeks of development. LFP chicks
showed more interest in exploring and pecking the environment, i.e. were more
engaged in pecking feed and litter. Thus, the essential difference in pecking
behaviour between the two lines may not be a difference in the propensity to peck
per se, but in the way pecking is targeted.
Interesting in this regard is a study by Braastad (1990) , which shows that
targeting of pecking behaviour can be influenced during early development. In that
study, chicks were exposed to blue-dyed food during the first 6 days post-hatching
(i.e. the sensitive period for food imprinting according to Hess, 1964) , and then
provided with blue key-stimuli on the floor as adult birds. These hens pecked more
at the floor and showed significantly less preening and a better plumage (possibly
indicating less feather pecking) than other birds.
4.2 Gentle feather pecking and preening behaviour in HFP chicks
On the individual level preening behaviour was inversely related to gentle
FP in the HFP line but not in the LFP line. Preening behaviour appears to be
influenced by the same environmental factors as feather pecking (Aerni et al.,
2000; Blokhuis, 1986). Aerni et al. (2000) recorded preening significantly more
often in pens without straw than with straw and more often in hens fed on pellets
than in hens fed on mash. Savory and Mann (1997) found that in several strains of
laying hens, an increase in feather pecking on a group level coincided with an
increase in preening during development. They suggested that there may be an
element of allopreening in feather pecking and that increased attention towards a
40 Chapter 2
bird's own plumage may be associated with increased attention towards other
birds' plumage as well. Blokhuis (1986) suggested that a certain basal level of
pecking at conspecifics exists that is not controlled by the ground pecking system,
and that this feather pecking may therefore be considered exploratory behaviour or
allopreening (Harrison, 1965).
Roden and Wechsler (1998) observed that preening chicks sometimes
started to peck at the feathers of neighbouring birds, possibly not differentiating
between their own and the feathers of other birds. Unfortunately, their studies did
not provide information about a correlation between feather pecking and preening.
We hypothesise here that HFP chicks that spent less time pecking and
manipulating their own feathers (i.e. preening), may have redirected these pecks
towards the feathers of penmates.
4.3 Gentle feather pecking and feeding behaviour in LFP chicks
In the LFP line gentle FP was not related to preening. LFP chicks showed
higher levels of foraging and feeding behaviour. This finding seems in agreement
with the hypothesis of Blokhuis (1989) , that feather pecking is a form of re- or
misdirected pecking, under the control of the feeding system. However, in the LFP
line, on the individual level, gentle FP was inversely related to feeding but not to
foraging. An explanation could lie in the ethogram used in this study, in which the
scoring of feeding was restricted to the feeding trough and the scoring of foraging
and scratching was restricted to the litter. However, the feeding trough was large
enough for very young chicks to get into completely (and most of them did during
feeding), and we observed a lot of scratching in the food during feeding, a
behaviour associated with foraging. Therefore, it is likely that part of the behaviour
scored as feeding, did not actually involve feed intake, but was in fact foraging or
exploratory pecking behaviour, intended to gather information about the food and
not primarily to ingest it. Chicks do spend a considerable amount of time pecking at
their food without eating (Fujita, 1973).
The feeding system of chicks is not fully developed at the time of hatching
and during the first days of life pecking at food is not motivated by hunger as the
Development of feather pecking 41
presence of yolk sac reserves makes food ingestion totally unnecessary (Goodwin
and Hess, 1969). Most pecks made in that period of time are of an exploratory
nature (Vestergaard, 1994), serving no other immediate function than information
gathering.
Hence, we argue that young LFP chicks that spent less time pecking
exploratively at the food (scored as feeding) may have redirected these exploratory
pecks to the feathers of conspecifics.
4.4 Gentle feather pecking and severe feather pecking
The present observation of feather pecking behaviour from the age of 3
days post-hatching in both lines, agrees with findings of, e.g. (Hoffmeyer, 1969)
and (Wennrich, 1975b). HFP and LFP chicks only differed in gentle FP on days 14
and 28 of age. Previous studies (Blokhuis and Beuving, 1993) on adult birds from
the same experimental lines report consistent higher levels of gentle FP in the HFP
line compared to the LFP line, suggesting that the difference between chicks in the
present study is not merely reflecting a difference in developmental rate.
In the present experiment, the nature of the observed feather pecking was primarily
gentle. Levels of severe feather pecking were generally low, possibly due to
experimental conditions of low stocking density and availability of litter. A question
of particular relevance for practical husbandry is whether birds showing high levels
of gentle FP at an early age, may be more predisposed to becoming severe feather
peckers later on. Further research is needed in which individual chicks are
monitored from day 1 post-hatching to adulthood.
The associations between gentle FP and other behavioural elements
demonstrated in the present study may not be of any relevance to the development
of severe feather pecking. It has been suggested that gentle and severe feather
pecking originate from different motivational systems (Kjaer and Vestergaard,
1999). The motor pattern of gentle FP resembles that of stereotypic pecking (Kjaer
and Vestergaard, 1999) and is quite different from the motor pattern of severe
feather pecking. However, in this study, severe feather pecks were always
embedded within bouts of gentle FP (data not shown), providing support for the
42 Chapter 2
recent suggestion by Kim-Madslien (2000) that "gentle and severe feather pecking
represent different extremes of the same behavioural continuum", rather than two
separate behaviours. Unfortunately, in the present study levels of severe feather
pecking were too low to allow a reliable estimation of correlations between severe
feather pecking and other behavioural elements, as was done for gentle FP.
4.5 Summary and Conclusion
In summary, this study indicates that in HFP birds preening and gentle FP
are negatively associated, with chicks either performing a relatively high level of
preening together with a relatively low level of gentle FP, or vice versa. In LFP
chicks gentle FP is negatively associated to feeding. Results from the PCA
substantiate differences between lines concerning the way various behavioural
elements relate, in particular gentle FP, preening and feeding. The correlations of
feeding and preening with those principal components with high loadings for gentle
FP were profoundly different for the HFP and LFP line, respectively. From the
assumption that a principal component with a high loading for gentle FP
reflects an underlying factor related to the propensity to engage in (gentle) feather
pecking, we suggest that the motivational system controlling the performance of
(gentle) feather pecking may differ as to the genetical background (HFP versus
LFP).
Hence, we argue that young HFP chicks are more predisposed to direct
(exploratory) pecks at animate stimuli, whereas LFP chicks are more predisposed
to direct (exploratory) pecks at inanimate environmental stimuli. We hypothesise
that due to this difference, feather pecking, starting off as "normal" exploratory
pecking, may turn out to be controlled by different motivational systems in both
lines. Further research is necessary to test this hypothesis.
This supposition might be an explanation as to how feather pecking
develops in both lines. However, it does not account for the difference in the
frequency of gentle FP between the two lines. Korte (1997, 1999) showed that the
behavioural and physiological characteristics of adult HFP and LFP birds resemble
those of the so-called proactive and reactive coping strategy, respectively.
Development of feather pecking 43
Proactive individuals are more intrinsically driven and more prone to develop
behavioural routines, whereas reactive individuals react more to environmental
stimuli (Koolhaas et al., 1999). Feather pecking may well be an example of such a
routine-like behaviour. In future experiments, we will investigate whether
differences in behavioural, physiological and neurobiological (coping)
characteristics between chicks may account for the differences in the frequency of
feather pecking, not only in these experimental lines but also in commercial lines.
In conclusion, differences in feather pecking behaviour between HFP and
LFP chicks can already be observed at a very early age during development.
Furthermore, our results indicate that HFP and LFP chicks differ in the way pecking
behaviour is targeted. This difference could be related to the existence of a
difference in underlying motivational system controlling the development of feather
pecking between the two lines.
44 Chapter 2
Chapter 3
Adrenocortical reactivity and central serotonin and dopamine turnover in young
chicks from a high and low feather pecking line of laying hens
Yvonne M. van Hierden1,2, S. Mechiel Korte1, E. Wim Ruesink1, Cornelis G. van Reenen1, Bas Engel1, Gerdien A.H. Korte-
Bouws1, Jaap M. Koolhaas2 and Harry J. Blokhuis2
1Institute for Animal Science and Health (ID-Lelystad), P.O. Box 65, NL-8200
AB Lelystad, The Netherlands 2University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Physiology and Behaviour, 75: 653-659, 2002
46 Chapter 3
Abstract
Feather pecking in domestic fowl is a behavioural abnormality that consists of mild
or injurious pecking at feathers of conspecifics. Previously, it was shown that chicks from a
high feather pecking (HFP) and low feather pecking (LFP) line of laying hens already differ
in their propensity to feather peck at 14 and 28 days of age. As a first step in investigating a
possible relationship between the development of feather pecking and physiological and
neurobiological characteristics of laying hens, two subsequent experiments were carried out.
Firstly, we investigated the development of adrenocortical (re)activity in HFP and LFP chicks
during the first 8 weeks of life. Secondly, we studied dopamine (DA) and serotonin (5-HT)
turnover in the brain of 28-day-old HFP and LFP chicks. In both experiments, chicks were
exposed to manual restraint (placing the chicks on its side for 5 min).
Plasma corticosterone levels were lower (baseline on days 3 and 56; restraint-
induced on days 3, 14 and 28) in HFP chicks. Both brain DA and 5-HT turnover were lower
in the HFP chicks, as well. Possible consequences for the observed differences in (stress)
physiology and neurobiology between the two lines in relation to the feather pecking are
discussed.
Adrenocortical reactivity and central serotonin and dopamine turnover 47
1. Introduction
Feather pecking behaviour consists of mild pecking (gentle feather
pecking) or vigorous pulling at the feathers of conspecifics (severe feather
pecking). The latter can especially cause damage to the plumage and loss of
feathers, which increases susceptibility to further injury, like wounds of the skin. At
worst, injured birds may be pecked to death (i.e., cannibalism). Thus, feather
pecking behaviour negatively affects poultry welfare and is a serious problem in
poultry practice that needs to be solved (Blokhuis and Arkes, 1984).
Until now, no single causal factor has been identified that induces feather
pecking. There is general acceptance that the development of feather pecking
reflects multifactorial processes (Huber-Eicher and Audigé, 1999). Some
investigators have stressed the relevance of environmental factors (e.g., housing
conditions) (Blokhuis, 1989), while others have implicated animal-related factors
(e.g., genetics, hormones) (Kjaer et al., 2001) and animal�environment interactions
(e.g., ontogenetic factors) (Johnsen et al., 1998). Lately, studies of feather pecking
behaviour are broadening to include animal characteristics (Keeling and Jensen,
1995; Korte et al., 1997; Korte et al., 1999).
Previously, it has been shown that two strains of laying hens that differ in
their propensity to feather peck (Blokhuis and Beutler, 1992; Blokhuis and Beuving,
1993) also show differences in open-field reactions (Jones et al., 1995), social
motivation (Jones et al., 1995) and behavioural and physiological stress
responsivity (Korte et al., 1997; Korte et al., 1999). More specifically, it was shown
that in response to acute stress induced by manual restraint, adult birds of the high
feather pecking line (HFP) displayed more struggling behaviour, lower heart rate
variability, higher plasma noradrenaline and lower plasma corticosterone levels
than birds of the low feather pecking line (LFP).
The behavioural and physiological characteristics of birds of the HFP and
LFP line show considerable analogy to the characteristics of respectively the
proactive (active) and reactive (passive) coping strategy, known to exist in other
species like rodents (Benus et al., 1990) and pigs (Bolhuis et al., 2000; Ruis et al.,
2000). In mice, it has been shown that proactive copers are more intrinsically
48 Chapter 3
driven. This means that their behaviour is less guided by environmental stimuli but
more by internal mechanisms. They easily develop routines, a rather rigid form of
behaviour. In contrast, reactive copers are more flexible and react more to
environmental stimuli (Benus et al., 1990; Koolhaas et al., 1999). There is a
growing body of evidence that adopting a proactive coping strategy makes an
individual more vulnerable to develop behavioural abnormalities than a reactive
individual (see, for a review Koolhaas et al., 1999). A differential HPA axis
(re)activity between the two coping strategies (reflected in plasma corticosteroid
levels) is suggested to underlie this difference (Koolhaas et al., 1999). Due to their
lipophilic nature, corticosteroids may readily enter the brain to bind to specific
cytoplasmatic receptors (Korte, 2001). Consequently, corticosteroids may alter
neural transmission in the serotonergic (5-hydroxytryptamine, 5-HT) (Meijer and de
Kloet, 1998) and dopaminergic (DA) system (Lowry et al., 2001). Indirectly, it has
been shown that 5-HT as well as DA neurotransmission is altered in adult proactive
individuals compared to adult reactive individuals (Korte et al., 1996; Rots et al.,
1996). DA and 5-HT are known to be involved in the expression of (environmentally
induced) behavioural disorders (e.g., stereotypies, obsessive compulsive disorder)
in adult individuals of several species (Benus et al., 1990; Bolhuis et al., 2000;
Ko�t�ál and Savory, 1995). Stereotypies are generally defined as unvarying,
repetitive behaviour patterns that have no obvious goal or function (Mason, 1991).
It has been suggested that gentle feather pecking (usually performed in long bouts)
has stereotypic characteristics (Kjaer and Vestergaard, 1999), as its motor patterns
closely resemble drug-induced stereotypic pecking in chickens (Bilčík, 2000).
Severe feather pecking may have a less clear stereotypic nature. The number of
severe pecks per bout is rather low compared to gentle feather pecking, as its
performance often evokes a flight reaction of the peckee (Kjaer and Vestergaard,
1999). It can, however, be described as abnormal behaviour with repetitive c.q.
routine-like characteristics.
The available data suggest a possible causal role of DA and 5-HT
neurotransmission in the development of feather pecking, possibly modulated by
corticosteroids. Therefore, it could be hypothesised that the difference in the level
of feather pecking behaviour between birds of the HFP and LFP line reflects a
Adrenocortical reactivity and central serotonin and dopamine turnover 49
difference in sensitivity of the DA and 5-HT system in the brain, possibly through
interaction with corticosterone.
As mentioned earlier, adult HFP and LFP birds show a consistent
difference in feather pecking behaviour (Blokhuis and Beutler, 1992; Blokhuis and
Beuving, 1993). Recently, we (van Hierden et al., 2002b) showed that already at
an early age, HFP and LFP chicks show clear differences in feather pecking and
related behaviours. On days 14 and 28 (but not on days 41 and 56) posthatching,
HFP chicks showed significantly higher levels of feather pecking than LFP chicks
(van Hierden et al., 2002b). However, there is no knowledge on physiological and
neurobiological characteristics of HFP and LFP birds at a young age. The aim of
the present study is to investigate whether the differences in behavioural
development between the two lines go parallel with physiological and
neurobiological differences.
Therefore, as a first step in investigating the question of a possible
relationship between corticosteroids, 5-HT and DA turnover and the development
of feather pecking, two subsequent experiments were carried out. Firstly, we
investigated the development of adrenocortical (re)activity in HFP and LFP chicks
during the first 8 weeks of life. Secondly, we studied DA and 5-HT turnover in the
brain of HFP and LFP chicks on 28 days of age. In the present experiments,
feather pecking behaviour was not studied. In our indirect approach, we used the
manual restraint test, an acute stressor, as a model for coping with environmental
challenges.
2. Methods
2.1 Experiment 1. Adrenocortical (re)activity
Birds and housing
In this study, 480 White Leghorn chicks from two strains were used: 240 HFP
chicks and 240 LFP chicks (Korte et al., 1997; Korte et al., 1999). All birds were
50 Chapter 3
female and non-beak-trimmed. Chicks arrived on the day of hatching and were
housed in litter-floor pens (0.75×1.0 m) with four animals per line (60 pens per
line). The pens were placed in six identical climate-controlled rooms and the lines
were randomly assigned to the pens within the rooms. Visual contact between
chicks in adjacent pens was prevented by hardboard separations between the
pens. The environmental temperature was lowered from 34 °C on day 1 to 18 °C at
8 weeks of age. On days 1 and 2 of age, the light regime was alternately 4 h light
and 4 h dark. From 3 days to 8 weeks of age, the light regime decreased from an
18-h light to a 10-h light period. A standard vaccination program was applied during
rearing.
All groups had access to three drinking cups and one square feeding
trough placed along one of the walls of the pen. Water and a standard rearing feed
(mash) were provided ad libitum.
Manual restraint and blood sampling
On days 3, 14, 28, 41 and 56 of age, chicks were killed by rapid
decapitation. Trunk blood was collected and blood samples were analysed for
plasma corticosterone. Half of the birds (six pens per line per age) were
decapitated immediately (within 2 min) after removal from the pen; the other half
was manually restrained (i.e., placed on its side) for 5 min before decapitation.
Chicks from the same pen were removed, tested and decapitated simultaneously.
Treatments (line/age/restraint) were randomly assigned to the pens within the
rooms. Decapitation was always carried out between 9.00 and 12.00 h.
Corticosterone measurement
The blood samples were immediately transferred to chilled (0 °C) Lithium�
Heparin-coated centrifuge tubes. Blood was centrifuged for 10 min at 3000 rpm at
a temperature of 4 °C. Plasma samples for corticosterone analysis were stored at 4
°C in the presence of 0.1% (w/v) sodium azide. Corticosterone concentrations were
determined in unextracted, enzymatically pretreated plasma (DELFIA), as
Adrenocortical reactivity and central serotonin and dopamine turnover 51
described earlier (de Jong et al., 2001). The detection range of the corticosterone
assay was 0.2�44 ng/ml.
2.2 Experiment 2. DA and 5-HT turnover in the brain
Birds and housing
In this study, 15 LFP and 15 HFP chicks were used. All birds were female
and non-beak-trimmed. Chicks arrived on the day of hatching and were housed in
litter-floor pens (0.75×1.0 m) of four animals per line. The pens were placed in a
climate-controlled room. Chicks were reared under the same environmental and
management conditions as in Experiment 1.
Measurement of corticosterone levels and DA and 5-HT turnover
On 28 days of age the chicks were manually restrained for 5 min and
killed by rapid decapitation. Blood samples were collected and analysed for
corticosterone (see Section 2.1.3).
The brains were immediately frozen in a dry ice precooled tube containing
n-heptane and stored at - 70°C until the assays were performed. For the assay, a
brain was transversally cut, rostrally to the midbrain 5-HT neurons (Kuenzel and
Masson, 1988) (see figure 1). Thereafter, the rostral brain sections were used for
the measurement of serotonin (5-hydroxytryptamine; 5-HT) and dopamine (DA)
and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the DA
metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid
(HVA). Previously, it has been shown that 5-HT turnover is indicated by the 5-
HIAA/5-HT ratio (Korte-Bouws et al., 1996) and DA turnover by the
(DOPAC+HVA)/DA ratio (Thiffault et al., 2000).
In order to measure these neurotransmitters and their metabolites the
brain samples were homogenised in icewater in a 1000-µl solution containing 5
µM clorgyline, 5 µg/ml glutathione and 200 ng/ml N-ϖ-methylserotonin (internal
standard) with a MSE Soniprep 150 ultrasonic tissueprocessor (Beun de Ronde,
52 Chapter 3
NL). Thereafter, 50 µl 2 M HClO4 and 40 µl 2.5 M potassium acetate were added
to 200 µl of the homogenate. After 15 min the tissue samples were centrifuged for
15 min at 15000 x g (4°C). Thereafter, 30µl of the supernatant was diluted with
450 µl HPLC grade water.
Figure 1. Image of the chicken brain. The diagonal line represents the position at which the
brain was cut. The right (rostral) brain section was used for 5-HT and DA turnover
measurement. Cb=Cerebellum; CO= Chiasma opticum
Adrenocortical reactivity and central serotonin and dopamine turnover 53
The samples were injected onto a reverse-phase/ion-pair high
performance liquid chromatography (HPLC) setup with electrochemical detection
for the measurement of 5-HIAA, 5-HT, DA, DOPAC and HVA. The
chromatographic system consisted of X-Act degasing unit (Jour Research,
Sweden), a Perkin-Elmer series 410 HPLC pump (USA), a Perkin-Elmer ISS 101
autosampler (USA) with a 100-µ1 loop, the INTRO combined columnoven,
electrochemical detector (Antec Leyden, NL) and a column (150 mm x 4.6 mm
i.d.) packed with Hypersil ODS, 5 µm particle size (Alltech Associates, USA).
The mobile phase consisted of 0.051 M citric acid monohydrate, 0.051 M
Na2HPO4 - 2H20, 0.26 mM EDTA, 0.356 mM sodium octyl sulphonate, 0.265 mM
di-n-butylamine, 2.0 mM NaCl and 13% methanol. This buffer was filtered through
a 0.22-µm membrane filter (Schleicher & Schuell, Germany). Separation was done
at 25°C using a flow rate of 1 ml/min.
Detection of the 5-HT and 5-HIAA was performed using an electrochemical
detector (Antec, Leiden, Netherlands) with a glassy carbon working electrode set at
- 0.611 V versus an In Situ Ag/AgCI reference electrode. The data were recorded
with a chart recorder (Model BD112, Kipp and Zn., The Netherlands), and peak
heights of samples were compared with those of standards determined each day
for quantification. The limit of detection (signal/noise ratio 3: 1) was 9.5 fmol/100 µl.
2.3 Statistical analysis: Experiments 1 and 2
The data of experiment 1 were analysed with an analysis of variance
model with main effects and interactions for the factors line (HFP/LFP), restraint
stress (yes/no) and age. Data were checked for normal distribution and
homogeneity of variances. Preliminary analyses of the corticosterone data showed
that the variance increased with the mean. Corticosterone levels were log-
transformed prior to analysis (averages per pen were analysed). For
corticosterone, the log-transformation (in order to obtain normal distribution),
appropriate when the variance is proportional to the square of the mean, was not
satisfactory. The assumption that the variance was proportional to the mean fitted
54 Chapter 3
the data better and the analysis was performed accordingly. Statistical inference
was based on maximum quasi-likelihood. A multiplicative overdispersion parameter
in the variance was estimated from Pearson's 2 statistic. Significance tests were
based on the quasi-likelihood ratio statistic. Technical details may be found in
McCullagh and Nelder (1989).
In experiment 2, the variances for the two lines differed significantly for
some of the variables. Line means were compared with a t test for unequal
variances employing Satterthwaites approximation (Snedecor and Cochran, 1956).
Incidentally, no marked differences with results from the ordinary t test (based on
an equal variances assumption) were found. All statistical calculations were
performed with Genstat 5 (1993, 1997). P values below 0.05 were considered
significant.
3. Results
3.1 Development of adrenocortical (re)activity
Figure 2 shows the dynamics of baseline and restraint-induced
corticosterone levels for the LFP and HFP line during the first 8 weeks of life. No
significant interactions between Restraint, Line and Age were found. There were
significant effects of Restraint [F(1,106) = 678.6, P < 0.001], Line [F(1,106) = 32.2,
P < 0.001] and Age [F(4,106) = 92.2, P < 0.001] on corticosterone levels.
On 3 and 56 days of age, HFP chicks showed significantly lower baseline
corticosterone levels than LFP chicks. In the LFP line, baseline levels of
corticosterone decreased significantly from 14 to 28 days of age, maintaining the
same level on subsequent days. In the HFP chicks, baseline corticosterone levels
also decreased during aging (although less evident): day 14 was significantly
higher than days 41 and 56. After manual restraint, corticosterone levels were
lower in the HFP line compared to the LFP line on days 3, 14 and 28 of age.
Adrenocortical reactivity and central serotonin and dopamine turnover 55
Similar to baseline levels, stress-induced corticosterone levels also
decreased during aging. Corticosterone levels of LFP chicks declined significantly
from days 14 to 41 of age and remained constant. HFP chicks showed a similar
pattern, with corticosterone levels significantly declining from 14 to 41 days of age.
Figure 2. Baseline corticosterone levels (ng/ml) and corticosterone levels (ng/ml) after
manual restraint (5 min) in LFP and HFP chicks on days 3, 14, 28, 41 and 56. Levels are
expressed as means ± S.E.M. ***P < 0.001, **P < 0.01, *P < 0.05, #0.05 < P < 0.08.
3 14 28 41 56 3 14 28 41 56
Age (days)
0
10
20
30
40
Cor
ticos
tero
ne (n
g/m
l)
LFP HFP
*
#
**
***
***
Manual RestraintBaseline
56 Chapter 3 3.2 DA and 5-HT turnover
Table 1 shows plasma corticosterone levels and (turnover) levels of the
neurotransmitters DA and 5-HT in the brain of 28 days old LFP and HFP chicks,
that had been exposed to restraint stress. Corticosterone levels were significantly
lower in HFP chicks (approx. t=4.72, df=12.64) than in LFP chicks. The 5-HIAA/5-
HT ratio and the (DOPAC+HVA)/DA ratio are considered markers of the 5-HT and
DA turnover, respectively. Both 5-HT and DA turnover (Table 1) were significantly
lower in the HFP line compared to the LFP line (approx. t = 3.42, Df = 21.69 resp.
approx. t = 3.38, df = 17.82).
Table 1. Levels of corticosterone (ng/ml) and the neurotransmitters dopamine and serotonin
and their metabolites (ng/mg brain tissue). Levels expressed as mean ± S.E.M.
LFP (n=15) HFP (n=15)
[Corticosterone] 13.192 ± 1.651*** 5.579 ± 0.271
[5-HIAA/5-HT] 0.105 ± 0.006** 0.081 ± 0.004
[(DOPAC+HVA)/DA] 0.405 ± 0.029*** 0.300 ± 0.013
5-HT 1.705 ± 0.051 1.911 ± 0.059*
5-HIAA 0.176 ± 0.008 0.154 ± 0.009#
DA 0.453 ± 0.036 0.492 ± 0.027
DOPAC 0.087 ± 0.005* 0.072 ± 0.003
HVA 0.134 ± 0.007** 0.109 ± 0.004
***P < 0.001, **P < 0.01, *P < 0.05, #0.05 < P < 0.08
Adrenocortical reactivity and central serotonin and dopamine turnover 57
4. Discussion 4.1 Development of adrenocortical (re)activity
The main finding of this experiment is that young chicks of the HFP line are
characterised by lower adrenocortical (re)activity than LFP chicks. HFP chicks
showed lower baseline as well as restraint-induced levels of corticosterone
compared to LFP chicks on days 3 and 56, respectively, on days 3, 14 and 28 of
age. These findings are in agreement with previous findings (Korte et al., 1997) in
adult hens of these lines and strengthens the idea that the HFP and LFP line are
representatives of respectively the proactive and reactive coping style.
Corticosteroids are of crucial importance for the regulation of adaptive
behaviour, learning, memory and neural plasticity (Korte, 2001; Sandi and Rose,
1994). In several species, including birds, it has been shown that circulating
corticosteroids enter the brain, where they bind to intracellular mineralocorticoid
(MR) and glucocorticoid (GR) receptors, e.g., in the hippocampus and amygdala
(mammals) c.q. archistriatal complex (birds) (Korte, 2001; Kuenzel and Masson,
1988). A disturbed balance in MR/GR function is believed to alter responsiveness
to the environment, promote susceptibility to stress, alter behavioural adaptation
(Korte, 2001), and influence learning and memory processes (Sandi and Rose,
1994).
Chicks are precocial to ensure their survival, therefore, they need to learn
rapidly about the properties of their environment and retain this memory (Sandi and
Rose, 1994). Recently (van Hierden et al., 2002b), it was shown that LFP and HFP
chicks differ in the way they `experience' environmental stimuli and interact with it.
This was reflected in the different ways pecking behaviour was targeted in both
lines. LFP chicks showed more interest in exploring and pecking at nonanimate
environmental stimuli, i.e., are more engaged in pecking feed and litter. In contrast,
HFP chicks showed more interest in pecking at animate stimuli, i.e., showed higher
levels of feather pecking and preening (which also includes pecking at feathers). It
58 Chapter 3
was hypothesised that differences in learning processes may have lead to the
involvement of different underlying motivational systems (respectively, preening
and feeding behaviour) in the development of feather pecking in both lines.
In accordance with that hypothesis, we suggest here that the differences in
the development and performance of feather pecking between LFP and HFP
chicks are associated with (1) differences in behavioural and physiological (coping)
response to environmental stimuli and (2) differences in learning processes, during
early development. Furthermore, we hypothesise that (3) a different MR/GR
balance in the brain of LFP and HFP chicks may be underlying these differences.
In future experiments, it is necessary to further investigate whether
physiological and behavioural differences between LFP and HFP chicks arise from
differences in occupancy of MR and/or GR receptors (MR/GR balance).
4.2 DA and 5-HT turnover, coping and feather pecking
Previously, it has been suggested that proactive individuals, behaviourally
characterised by low behavioural inhibition, high routine formation, low cue
dependency and low flexibility, are more vulnerable for the development of
behavioural abnormalities than their reactive counterparts (Koolhaas et al., 1999).
There is accumulating evidence that this difference in vulnerability may be a
consequence of the differences in DA and 5-HT neurotransmission (e.g., turnover
levels, receptor expression levels and receptor sensitivity) between proactive and
reactive copers (Bolhuis et al., 2000; Korte et al., 1996).
For instance, in rodents and pigs (Benus et al., 1991a; Bolhuis et al.,
2000), the DA receptor agonist apomorphine produced a greater enhancement of
stereotyped behaviour in proactive coping individuals than in reactive coping
individuals. Furthermore, it was shown that proactive mice have lower 5-HT
neurotransmission (Korte et al., 1996) and (possibly), consequently, a more
sensitive (postsynaptic) 5-HT receptor system as compared to reactive mice (van
der Vegt et al., 2001). A difference in sensitivity of (postsynaptic) 5-HT receptors
are suggested to play a role in the differences in behavioural repertoire between
proactive and reactive individuals (Korte et al., 1996; van der Vegt et al., 2001).
Adrenocortical reactivity and central serotonin and dopamine turnover 59
The lower DA and 5-HT turnover in chicks of the HFP line as compared to
the LFP line found in the present study are in agreement with above findings in
adult pigs and rodents and support the assumption that the HFP and LFP lines are
representatives of the respectively proactive and reactive coping strategy.
Both DA and 5-HT have been shown to play a role in the expression of oral
stereotypies in fowl ( Ko�t�ál and Savory, 1995). Several DA receptor agonists,
e.g., CQP201-403 (Ferrari et al., 1993), apomorphine (Goodman et al., 1983) and
amphetamine (Goodman, 1981), induce stereotyped pecking responses in birds,
suggesting a possible involvement of the DA system in the development of
stereotypic gentle feather pecking. Bilčík (2000) investigated a possible
involvement of DA neurotransmission in the expression of feather pecking. His
findings were inconclusive as to whether DA plays a role in feather pecking. He did
not find a difference in DA sensitivity in young chicks, that were later (at an adult
age) identified as feather peckers and non-feather peckers. However, they did find
some minor differences in binding and densities of D1 and D2 dopamine receptor
subtypes in specific brain regions, between feather peckers and non-peckers.
Interestingly, increasing brain 5-HT levels by dietary supplementation with
L-tryptophan (precursor of 5-HT) suppressed feather pecking damage in growing
bantams (Savory et al., 1999). In line with these results, it did not come as a
surprise that LFP chicks were characterised by a higher 5-HT turnover. Self-
mutilating feather pecking disorder (FPD) in birds is a stereotypy that seems under
the control of 5-HT mechanisms. Clomipramine, a tricyclic antidepressive drug
inhibiting the reuptake of 5-HT and noradrenaline, was effective in alleviating
severe FPD in psittacine birds (parrots and parakeets) (Bordnick et al., 1994). In a
study of Blokhuis and his colleagues (1993), adult HFP and LFP hens, when
housed on battery cages, showed marked differences in the type of stereotypy
performed. Almost 60% of the observed HFP birds showed pecking at own
feathers, whereas only 6% of the observed LFP birds showed this kind of
stereotypy. It is tempting to hypothesise that the higher levels of self-mutilating
pecking, found in the experiment of Blokhuis and his colleagues (1993) and the
higher levels of feather pecking in HFP chicks found in our recent study (van
60 Chapter 3
Hierden et al., 2002b) may be associated with lower 5-HT turnover in the birds of
the HFP line compared to the LFP line.
In view of the above findings, we hypothesise that a lower DA and 5-HT
turnover in HFP chicks compared to LFP chicks predispose them to more easily
develop a stereotypy like (gentle) feather pecking. Further research is necessary to
investigate this possible relationship between DA and 5-HT neurotransmission and
the development of feather pecking in the HFP and LFP line.
4.3 Interaction of corticosteroids with DA and 5-HT pathways and feather pecking
Another possible way in which corticosterone may play a role in the
development of feather pecking is through interaction with DA and 5-HT pathways.
It is known that corticosteroids stimulate DA release in the brain and that a
corticosterone-induced increase in extracellular DA levels results in psychomotor
activation (Piazza and Le Moal, 1997). Furthermore, it was shown that
corticosteroids via GRs may play an important role in the sensitisation of the DA
system (Rivet et al., 1989). Interestingly, the development of divergence in DA
responsiveness in apomorphine susceptible and unsusceptible rat lines, that also
differ in coping strategy, is preceded by changes in pituitary�adrenal activity (Rots
et al., 1996).
Corticosteroids also stimulate 5-HT synthesis at the level of the raphe
nuclei, probably via glucocorticoid receptors, and this results in increased
extracellular 5-HT levels in limbic forebrain regions (Korte-Bouws et al., 1996).
Consequently, low corticosteroid levels in proactive individuals via these
mechanisms may play an important role in the increased vulnerability of these
individuals for the developing stereotypies (Korte, 2001).
4.4 Summary and Conclusion
In conclusion, young chicks of the HFP line are characterised by lower plasma
corticosterone levels, and both lower 5-HT and DA turnover as compared to LFP
Adrenocortical reactivity and central serotonin and dopamine turnover 61
chicks. To our knowledge this is the first time it has been shown that chicks, that
are known to differ in feather pecking, also differ in both stress physiology and
neurobiology.
Further research is needed to investigate whether a difference in binding of
corticosterone to corticosteroid receptors in the brain of HFP and LFP birds is
responsible for the differences in the development and performance of feather
pecking in both lines. Or, whether a difference in sensitivity of the DA system and
5-HT system, possibly under influence of corticosterone, may be the underlying
mechanism in the development of feather pecking.
62 Chapter 3
Chapter 4
Control of feather pecking by serotonin
Yvonne M. van Hierden1,2, S. Mechiel Korte1, Sietse F. de Boer2 and Jaap M. Koolhaas2
1 Animal Sciences Group, P.O. Box 65, NL-8200, AB Lelystad, The Netherlands
2University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
submitted for publication in Behavioural Neuroscience
64 Chapter 4
Abstract Feather pecking behaviour in laying hens may be considered a behavioural
pathology, comparable to human psychopathological disorders. Scientific knowledge on the
causation of such disorders strongly suggests involvement of the serotonergic (5-HT)
system in feather pecking. Previously, chicks from a high feather pecking (HFP) line were
found to display lower 5-HT turnover levels than chicks from a low feather pecking (LFP) line
(in response to acute stress).
The present study investigated whether low 5-HT neurotransmission is causally
underlying feather pecking. Firstly, S-15535, a somatodendritic 5-HT1A autoreceptor agonist,
demonstrated to be an excellent tool for reducing 5-HT turnover in the forebrain of LFP and
HFP chicks. Secondly, the most effective dose of S-15535 (4.0 mg S-15535/kg BW)
significantly increased severe feather pecking behaviour.
The results confirmed our postulation that the performance of feather pecking is
triggered by low 5-HT neurotransmission.
Control of feather pecking by serotonin 65
1. Introduction
There is considerable evidence available to support a pivotal role of the
serotonergic system in the development of behavioural pathologies. In contrast to
human or rodent studies, in the field of farm animal research considerable less
information is available on correlates between abnormal behaviour and the
functioning of the serotonergic system (Ko�t�ál and Savory, 1995). An example of
abnormal behavioural in farm animals, is feather pecking in laying hens. Feather
pecking involves the (stereotypic) pecking and (compulsive) pulling of each others
feathers, leading to injury and ultimately death (cannibalism). It is a multifactorial
problem (Hughes and Duncan, 1972), which due to complex interactions between
animal characteristics (e.g. genetic predisposition) and environmental factors (e.g.
housing) is hard to solve.
Recently (van Hierden et al., 2002a), we postulated that the central
serotonergic system is involved in the development and performance of feather
pecking behaviour. Chicks from a high feather pecking line of laying hens (HFP)
showed, in response to restraint stress, significantly lower serotonin (5-HT)
turnover levels in the forebrain than chicks from a low feather pecking line (LFP).
This suggests a difference in the functioning of the central 5-HT system between
the lines. A dysregulation of brain 5-HT transmission is implicated in various
impulsive states, i.e. disorders involving loss of impulse control, like aggression (de
Boer et al., 2000; van der Vegt et al., 2001) and obsessive compulsive disorders
(OCD) (Pigott, 1996; Stein, 2000). Feather pecking has clear compulsive
characteristics. Once birds start feather pecking, they tend to do so successively
and their pecking behaviour is difficult to discourage.
From the above it can be suggested that feather pecking is an impulsive
state caused by low serotonergic neurotransmission. This line of reasoning implies
that low 5-HT neurotransmission is causally involved in feather pecking. Therefore,
in the present study the hypothesis was tested that lowering 5-HT turnover in the
forebrain of laying hens increases their level of feather pecking.
One useful pharmacological research tool for transiently lowering 5-HT
turnover levels in the forebrain is activating the inhibitory somatodentritic 5-HT1A
66 Chapter 4
autoreceptor by administering a 5-HT1A receptor agonist. S-15535 is such a drug
that preferentially acts as an agonist on the somatodendritic 5-HT1A autoreceptors,
but has antagonistic (partial agonistic) properties on postsynaptic 5-HT1A receptors
(Millan et al., 1994; Millan et al., 1997; Millan et al., 1993). As a consequence of its
agonist action on the autoreceptor, S-15535 potently inhibits the firing of 5-HT
neurons and consequently reduces 5-HT release and turnover.
From the above, it is postulated that (1) S-15535 decreases 5-HT turnover
in the forebrain of LFP and HFP birds, which (2) results in higher levels of feather
pecking behaviour in both lines. To test these hypotheses, two experiments were
carried out.
Experiment 1 consisted of two identical ethopharmalogical
subexperiments. In these subexperiments the effect of different doses of S-15535
on the central 5-HT (and dopamine) turnover in the forebrains of HFP and LFP
chicks was investigated, respectively. In Experiment 2, the most effective dose of
S-15535 in lowering 5-HT turnover levels in the forebrain (in experiment 1) was
subsequently used in a study in which the effect of S-15535 injection on feather
pecking and related behaviour of LFP and HFP birds was investigated.
2. Methods
2.1 Experiment 1: Dose-response of S-15535
Birds and housing
In this study 60 HFP (experiment 1a) and 60 LFP (experiment 1b) White
Leghorn chicks were used (for line specifications see Korte et al., 1997).
Experiment 1a and 1b were carried out under identical experimental conditions (i.e.
housing, environment, experimental crew). Due to practical problems it was not
possible to combine both experiments.
All birds were female and non-beaktrimmed. Chicks arrived on the day of
hatching and were kept in groups of 5 animals per line (12 groups per line) and
Control of feather pecking by serotonin 67
housed in pens (0.75x1.0m) with woodshavings. The pens were placed in two
identical climate controlled rooms (6 pens per room). Individual pens were visually
isolated by hardboard partitions. The environmental temperature was lowered from
34°C on day one to 22°C at 5 weeks of age. On days 1 and 2 of age the chicks
received 24 hours of light. From 3 days to 5 weeks of age the light regime
decreased from an 18 h light to a 10 h light period.
All birds had access to three drinking cups and one square feeding trough
placed along one of the walls of the pen. Water and a commercial rearing feed
(mash) were provided ad libitum.
S-15535
S-15535-3 methanesulfonate (4-(benzodioxan-5-yl)1-(indan-2-
yl)piperazine, lot no E1798, molecular weight: 432.5) was provided by the Institut
de Recherches Internationales Servier, France. S-15535 was dissolved in sterile
distilled water (vehicle solution) and injections were given subcutaneously (s.c.) in
the neck, in a volume of 1 ml/kg body weight. Vehicle and solutions were at room
temperature when injected.
Manual restraint test
At 28 days of age, each chick per pen was injected s.c. with a different
dose of S-15535: 0, 0.4, 0.8, 4.0 or 8.0 mg S-15535/kg BW and placed back in the
homecage (12 chicks per line-treatment combination). After 30 minutes, chicks
were removed from the homecage and taken to adjacent test rooms. Chicks were
subjected to a manual restraint test, i.e. placed on the side for 5 minutes, and their
behaviour (latency time struggling, number of struggles, latency time vocalising and
number of vocalisations) was scored. Following the manual restraint test, chicks
were killed by rapid decapitation.
68 Chapter 4
Preparation and treatment of brain tissue
The brains were removed within one minute after decapitation and
immediately frozen in a dry ice precooled tube containing n-heptane and stored at
- 70°C until the assays were performed. For the assay three brain regions were
used: the hippocampus, the archistriatal complex (i.e. an amygdala like structure
in birds (Kuenzel and Masson, 1988) and remainder of the forebrain.
The brain sections were used for the measurement of serotonin (5-
hydroxytryptamine; 5-HT) and dopamine (DA) and the 5-HT metabolite 5-
hydroxyindoleacetic acid (5-HIAA) and the Dopamine (DA) metabolites 3,4-
dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Previously, it
has been shown that 5-HT turnover is indicated by the 5-HIAA/5-HT ratio (Korte-
Bouws et al., 1996) and DA turnover by the (DOPAC+HVA)/DA ratio (Thiffault et
al., 2000).
Analysis of monoamines by HPLC
In order to measure these neurotransmitters and their metabolites the
brain samples were homogenised in icewater in a 1000-µl solution containing 5
µM clorgyline, 5 µg/ml glutathione and 200 ng/ml N-ϖ-methylserotonin (internal
standard) with a MSE Soniprep 150 ultrasonic tissueprocessor (Beun de Ronde,
NL). Thereafter, 50 µl 2 M HClO4 and 40 µl 2.5 M potassium acetate were added
to 200 µl of the homogenate. After 15 min the tissue samples were centrifuged for
15 min at 15000 x g (4°C). Thereafter, 30µl of the supernatant was diluted with
450 µl HPLC grade water.
The samples were injected onto a reverse-phase/ion-pair high
performance liquid chromatography (HPLC) setup with electrochemical detection
for the measurement of 5-HIAA, 5-HT, DA, DOPAC and HVA. The
chromatographic system consisted of X-Act degasing unit (Jour Research,
Control of feather pecking by serotonin 69
Sweden), a Perkin-Elmer series 410 HPLC pump (USA), a Perkin-Elmer ISS 101
autosampler (USA) with a 100-µ1 loop, the INTRO combined columnoven,
electrochemical detector (Antec Leyden, NL) and a column (150 mm x 4.6 mm
i.d.) packed with Hypersil ODS, 5 µm particle size (Alltech Associates, USA).
The mobile phase consisted of 0.051 M citric acid monohydrate, 0.051 M
Na2HPO4 - 2H20, 0.26 mM EDTA, 0.356 mM sodium octyl sulphonate, 0.265 mM
di-n-butylamine, 2.0 mM NaCl and 13% methanol. This buffer was filtered through
a 0.22-µm membrane filter (Schleicher & Schuell, Germany). Separation was done
at 25°C using a flow rate of 1 ml/min.
Detection of the 5-HT and 5-HIAA was performed using an electrochemical
detector (Antec, Leiden, Netherlands) with a glassy carbon working electrode set at
- 0.611 V versus an In Situ Ag/AgCI reference electrode. The data were recorded
with a chart recorder (Model BD112, Kipp and Zn., The Netherlands), and peak
heights of samples were compared with those of standards determined each day
for quantification. The limit of detection (signal/noise ratio 3: 1) was 9.5 fmol/100 µl.
2.2 Experiment 2: S-15535 and feather pecking behaviour
Birds and housing
In this study 80 LFP and 80 HFP chicks were used. All birds were female
and non-beaktrimmed. Chicks arrived on the day of hatching and were kept in
groups of 8 animals per line and housed in pens (0.75 x 1.0m) with woodshavings.
The pens were placed in two identical climate controlled rooms. Chicks were
individually marked by colouring them with waterproof markers. Different patterns
of the colours blue, green, black and purple were applied on the back feathers of
the birds. Individual pens were visually isolated by hardboard partitions.
The environmental temperature was lowered from 34°C on day one to
18°C at 45 days of age onwards. On days 1 and 2 of age the chicks received 24
hours of light. From 3 days to 35 days onwards the light regime decreased from an
18 h light to a 10 h light period. All birds had access to three drinking cups and one
70 Chapter 4
square feeding trough placed along one of the walls of the pen. Water and a
commercial rearing feed (mash) were provided ad libitum.
Experimental design and behavioural measurements
The LFP and HFP chicks were raised in litter floor pens (with
woodshavings) until 35 days of age. The floor of the pen consisted of a slatted floor
on which cardboard was placed. A thick layer of woodshavings was applied onto
the cardboard. At 35 days of age the cardboard with the layer of woodshaving was
removed from the pens. At 50 days of age, the 8 chicks per pen were randomly
split into 2 groups of 4 animals, and redivided over 40 litter-floor pens (0.75 x 1.0
m) that were placed in the same two climate controlled rooms.
Pens were randomly assigned to either S-15535 or control treatment (10
pens per line-treatment combination). Two birds per cage were randomly chosen
and their behaviour after S-15535 or vehicle injection was studied either on 56
days (chick one) or 57 days of age (chick two). S-15535-chicks were injected
subcutaneously in the neck with 4.0 mg S-15535/kg BW, in a volume of 1 ml/kg
BW. Control-chicks were injected with distilled water, in a volume of 1 ml/kg BW.
Thirty minutes after injection the behaviour was recorded on videotape for
a period of 30 minutes. The behaviour of the birds was scored afterwards using
The Observer® 4.0 software (Noldus, Wageningen, The Netherlands). Duration
and frequency of the behavioural elements scored are described in Table 1.
2.3 Statistical analysis experiment 1 and 2
Experiment 1: Dose-response of S-15535
Levels of 5-HT and DA, and their metabolites in the different brain regions,
and behaviour during Manual Restraint were analysed with an analysis of variance
model, with main effects and interactions for experimental factors for Line (HFP or
Control of feather pecking by serotonin 71
LFP) and Treatment (5 levels). Pen was entered as a random effect in the analysis.
For the (post hoc) analyses of the levels of 5-HT and DA and their metabolites, a
common F-test was used.
The behavioural data during Manual Restraint were not normally
distributed. Therefore, these data were analysed according to techniques
described by Engel and Keen (1994) and Engel and Buist (1996), employing
Genstat 5 procedure IRREML (Keen and Engel, 1998). Latency time struggling and
latency time vocalising, were expressed as a percentage of the total duration of the
test (5 minutes). The Wald test (Rao, 1973) was used for estimation of Line x
Treatment, Line and Treatment effects. The Wald statistic (W) is an estimation of
the F-statistic in the IRREML procedure. Differences were considered significant if
P < 0.05. For all calculations GenStat® 6 (2002) was used.
Frequency of feather pecking was analysed with a log linear model with
main effects and interaction for Line and Treatment, i.e. log(m) is expressed as a
sum of main effects and interactions, where m is the expected mean. The model
comprised a multiplicative dispersion factor relative to the variance under a
Poisson distribution, i.e. the variance was assumed to be proportional to the mean
m.
Estimation of durations and frequencies was performed by maximum quasi-
likelihood. The dispersion parameter was estimated from Pearson�s chi-square
statistic. Significance tests were performed by referring the log quasi-likelihood
ratio to an F-distribution (with �residual degrees of freedom� corresponding to
Pearson�s chi-square statistic). Details may be found in McCullagh and Nelder
(1989). Significance tests for feather pecking as a binary variable were based on
the maximum likelihood ratio test. Differences were considered significant if P <
0.05. For all calculations GenStat® 6 (2002) was used.
72 Chapter 4
Table 1. Ethogram showing the behavioural measurements (posture either standing or
sitting).
Behaviour Definition
Frequencies
Gentle feather pecking Mild pecking at the feathers of conspecific,
generally performed in multiple bouts (single
pecks are counted as one occurrence)
Severe feather pecking Vigorous pecking/pulling/pinching at the
feathers of conspecific (single pecks are
counted as one occurrence)
Duration
Feeding Pecking at the litter and scratching (separately
scored as ground scratching) or moving with
the head in a lower position than the rump
Preening Preening behaviour as described by Kruijt
(1964): e.g. autopecking, nibbling, stroking,
combing, head-rubbing
Walking Walking, running, jumping or flying (it may be
accompanied by wing-flapping)
Resting Sitting or standing inactive (no movement of the
legs)
3. Results 3.1 Experiment 1: Dose-Response of S-15535
Figure 1 shows the levels of 5-HT and DA turnover for the different doses in
the different brain regions. No significant Line x Treatment interactions were found
for any of the variables.
Control of feather pecking by serotonin 73
For 5-HT turnover in the hippocampus (Figure 1A) a significant overall
Treatment effect [F(1,4) = 31.21, P < 0.001], but no significant overall Line effect
was found. Figure 1A shows that the two highest doses (4.0 and 8.0 mg)
significantly decreased 5-HT turnover, compared to the other doses of S-15535 (0,
0.4 and 0.8 mg).
A significant Treatment effect was found for 5-HT turnover in the
archistriatum [F(1,4) = 14.56, P = 0.006]. No significant Line effect was found. In
the HFP line (Figure 1B), 5-HT turnover levels in the archistriatum were lowest for
the 4.0 mg and 8.0 mg treatment compared to the control treatment.
For the remainder of the brain tissue a significant effect of Line [F(1,1) = 8.09, P =
0.004] and Treatment [F(1,4) = 28.37, P < 0.001] were found. Figure 1C shows
that LFP chicks have higher 5-HT turnover levels than HFP chicks and that a dose-
dependent decrease in 5-HT turnover is observed in both lines.
The dose-dependent decrease in 5-HT turnover in the different brain
regions, is the result of a decrease in 5-HIAA rather than 5-HT (data not shown).
For 5-HT no significant Treatment effects were found for the different brain regions.
For 5-HIAA significant Treatment effects were found for the hippocampus [F(1,4) =
23.85, P < 0.001], the archistriatum [F(1,4) = 8.81, P = 0.05] and the remainder of
the forebrain [F(1,4) = 26.73, P < 0.001]. S-15535 treatment dose-dependently
decreased the level of 5-HIAA in the forebrain (data not shown).
For DA turnover in the hippocampus, a significant Line [F(1,1) = 7.67, P =
0.006] and Treatment effect [F(1,4) = 13.74, P = 0.008] were found. For DA
turnover in the archistriatum trends for a Line [F (1,1) = 3.12, P = 0.08] and
Treatment effect [F(1,4) = 8.97, P < 0.06] were found. Overall HFP birds showed
higher DA levels in the hippocampus and archistriatum. For DA turnover in the
remainder of the forebrain a significant Treatment [F(1,4) = 16.68, P = 0.002], but
no significant Line effect was found. Figure 1D-1F show that the highest dose of S-
15535 significantly increases DA turnover levels in HFP line.
74
Cha
pter
4
Figu
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(afte
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Sig
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*** P
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, **P
< 0
.01,
* P <
0.0
5, # 0.
05 <
P <
0.1
0
00.
40.
84.
08.
0-
00.
40.
84.
08.
0
S-1
5535
(mg/
kg B
W)
0.00
0.04
0.08
0.12
5-HT turnover
Arc
hist
riatu
m
LFP
HFP
B
*#
00.
40.
84.
08.
0-
00.
40.
84.
08.
0S
-155
35 (m
g/kg
BW
)
0.00
0.04
0.08
0.12
5-HT turnover
Hip
poca
mpu
s
LFP
HFP
A
** *
***
***
00.
40.
84.
08.
0-
00.
40.
84.
08.
0
S-1
5535
(mg/
kg B
W)
0.00
0.04
0.08
0.12
5-HT turnover
Rem
aind
er o
f the
fore
brai
n
LFP
HFP
C
***
****
**
*** **
00.
40.
84.
08.
0-
00.
40.
84.
08.
0
S-1
5535
(mg/
kg B
W)
0.00
0.50
1.00
1.50
2.00
DA turnover
Arc
hist
riatu
m
LFP
HFP
E
#
00.
40.
84.
08.
0-
00.
40.
84.
08.
0
S-1
5535
(mg/
kg B
W)
0.00
0.50
1.00
1.50
2.00
DA turnover
Hip
poca
mpu
s
LFP
HFP
D*
00.
40.
84.
08.
0-
00.
40.
84.
08.
0
S-1
5535
(mg/
kg B
W)
0.00
0.50
1.00
1.50
2.00
DA turnover
Rem
aind
er o
f the
fore
brai
n
LFP
HFP
F
* *
Control of feather pecking by serotonin 75
The results of the behaviour during manual restraint are shown in Figure 2.
For the number of struggles, a significant Line [F (1,1) = 12.95, P < 0.001] and
Treatment [F (1,4) = 38.63, P < 0.001] effect were found. Figure 2A shows that in
both lines the two highest doses significantly increased the numbers of struggles
(Figure 2A). Overall HFP birds showed a higher number of struggles.
For the number of vocalisations (Figure 2B) a significant Treatment effect
[F(1,4) = 24.70, P < 0.001] but no significant Line effect was found. The number of
vocalisations dose-dependently increased in both lines. For the latency time
struggling (Figure 2C) a significant Treatment effect [F(1,4) = 38.26, P < 0.001] but
no Line effect was found. For the latency time vocalising (Figure 2D) significant
Line [F(1,1) = 9.00, P = 0.003] and Treatment effects [F(1,4) = 23.88, P < 0.001]
were found. Both 4.0 and 8.0 mg treatment significantly decreased both latency
times.
3.2 Experiment 2: Effect of S-15535 on feather pecking behaviour
For gentle feather pecking behaviour no significant Line x Treatment
interaction was found (Table 2). However, a significant overall Line effect [F(1,74) =
12.15, P < 0.001] and a trend for a overall Treatment effect [F(1,74) = 3.54, P =
0.06] were found (Table 2). Figure 3A shows that S-15535 treated HFP birds
tended (P = 0.08) to show higher levels of gentle feather pecking than control HFP
birds.
Similar to gentle feather pecking, also for severe feather pecking (Figure
3B) no significant Line x Treatment effect was found (Table 2). However, a
significant effect of Line [F (1,74) = 12.05, P < 0.001] and Treatment [F (1,74) =
5.67, P = 0.02] were found for this behaviour (Table 2). Figure 3B shows that S-
15535 treated HFP birds showed significantly (P = 0.05) more severe feather
pecking behaviour than control HFP birds.
76 Chapter 4
Figure 2. Number of struggles and vocalisations, and the latency to struggle and vocalise
(Mean ± S.E.M.) of LFP and HFP chicks (28 days of age), in response to different levels of
S-15535 (during a manual restraint test of 5 minutes). Significant effects (within lines,
compared to control treatment): ***P < 0.001, **P < 0.01, *P < 0.05, #0.05 < P < 0.10
0 0.4 0.8 4.0 8.0 - 0 0.4 0.8 4.0 8.0
S-15535 (mg/kg BW)
0
3
6
9
12
15
Num
ber o
f stru
ggle
s
Number of struggles
LFP HFP
A
***
***
**
#
0 0.4 0.8 4.0 8.0 - 0 0.4 0.8 4.0 8.0
S-15535 (mg/kg BW)
0
20
40
60
80
100
Late
ncy
time
stru
gglin
g (%
of t
otal
tim
e)
Latency time struggling
LFP HFPC
***
***
**
0 0.4 0.8 4.0 8.0 - 0 0.4 0.8 4.0 8.0
S-15535 (mg/kg BW)
0
20
40
60
80
100
Late
ncy
time
voca
lisin
g (%
of t
otal
tim
e)
Latency time vocalising
LFP HFP
D
* *
**
**
0 0.4 0.8 4.0 8.0 - 0 0.4 0.8 4.0 8.0
S-15535 (mg/kg BW)
0
25
50
75
100
Num
ber o
f voc
alis
atio
ns
Number of vocalisations
LFP HFP
B***
****
#
* *
#
Control of feather pecking by serotonin 77
For both foraging and feeding behaviour (Figure 3C and Figure 3B), trends
for a Line x Treatment interaction were found (resp. [F(1,74) = 3.72, P = 0.06] and
[F(1,74) = 2.81, P = 0.10]). For feeding also a trend for a Line effect was found
[F(1,74) = 2.90, P = 0.09]. Figure 3C shows that S-15535 treated HFP birds
showed significantly more foraging behaviour than control HFP birds. S-15535
treated LFP birds spent significantly more time feeding (Figure 3D) compared to S-
15535 treated HFP birds.
For preening behaviour no significant Line x Treatment, or Line effect were
found, however a significant Treatment effect was found [F(1,74) = 22.16, P <
0.001]. In both lines (Figure 3E), preening behaviour significantly decreased after
S-15535 treatment.
For duration of walking, a significant Line x Treatment effect [F (1,74) =
8.14, P = 0.01] and Treatment effect [F(1,74) = 10.73, P < 0.001] were found.
Figure 3F shows that LFP control birds tended to spent more time walking than
HFP control birds (P = 0.09). S-15535 treated HFP chicks showed significantly
more time walking compared to control HFP birds (P < 0.001).
For resting behaviour (data not shown) a significant Line effect [F(1,74) =
3.88, P < 0.001] was found. No interaction or Treatment effect was found. LFP
birds spent less time resting than HFP birds. Further results of the behavioural
elements are shown in Table 2.
78 Chapter 4
Figure 3. Frequency (Mean ± S.E.M) or relative duration (mean percentage of the total
observation time ± SEM) of the behaviour of LFP and HFP birds (56 or 57 days of age)
injected with either vehicle (control) or S-15535. Means without a common superscript differ
(P < 0 .10).
LFP HFP0
10
20
30
40
5075
100
125
150
Freq
uenc
y/30
min
utes
Severe feather peckingB
aaa
b
LFP HFP0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e du
ratio
n (%
)
ForagingC
a
babab
LFP HFP0
10
20
3080
90
100
Rel
ativ
e du
ratio
n (%
)
PreeningE
b
aa
b
LFP HFP0
25
50
75
100
125
150
Freq
uenc
y/30
min
utes
Gentle feather peckingControl S-15535A
aa
c
a
LFP HFP0
10
20
3080
90
100
Rel
ativ
e du
ratio
n (%
)
WalkingF
aa
c
b
LFP HFP0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e du
ratio
n (%
)
FeedingD
a
bab ab
Control of feather pecking by serotonin 79
4. Discussion
4.1 Experiment 1: Dose-response of S-15535 The results of the present experiment clearly demonstrate that S-15535 is
an excellent tool for reducing 5-HT turnover in the forebrain of LFP and HFP
chicks. This effect can be attributed to stimulation of the somatodendritic 5-HT1A
receptor, as was found for rodents (Gobert et al., 1995; Millan et al., 1997; Millan et
al., 1993). S-15535 treatment had a similar effect in reducing 5-HT turnover levels
in the forebrain of HFP and LFP chicks, suggesting a comparable number or
sensitivity of presynaptic 5-HT1A autoreceptors between the lines.
The most effective dose of S-15535, without affecting DA turnover, was 4.0
mg S-15535/kg BW. The highest dose (8.0 mg) enhanced DA turnover. This is
consisted with the fact that 5-HT1A receptors modulate the activity of serotonergic
as well as dopaminergic pathways (Lejeune et al., 1996; Lejeune and Millan,
1998). Indeed studies (Lejeune and Millan, 1998; Millan et al., 1997) showed that
S-15535 dose-dependently, but markedly, increased the firing rate of dopaminergic
neurons, and increased the DA release.
The behavioural response to manual restraint of HFP and LFP chicks, after
S-15535 injection, indicated that birds of both lines (although more pronounced in
the HFP line) became more proactive in their response (i.e. struggled more). This
is in agreement with earlier findings that HFP birds, characterised by lower levels of
5-HT metabolism in the forebrain (van Hierden et al., 2002a) compared to LFP
birds, display a proactive coping strategy (Korte et al., 1997; van Hierden et al.,
2002a) in response to manual restraint.
4.2 Experiment 2: Effect of S-15535 on feather pecking behaviour
The purpose of the present study was to assess the causal role of
serotonergic neurotransmission activity in the performance of feather pecking
behaviour in HFP and LFP chicks. The principal finding of this experiment was that
80 Chapter 4
acute S-15535 injection significantly increased the level of feather pecking
behaviour in HFP birds. This result confirms our hypothesis that low serotonergic
neurotransmission is causally involved in the performance of feather pecking
behaviour.
The increase in duration of foraging and walking of the HFP chicks, in
response to S-15535 injection, may be explained from the increase in severe
feather pecking. It is well known that birds that are being severely pecked will move
away from the feather pecker, which in turn will move to another bird to peck.
Normally, gentle feather pecking does not elicit a lot of activity in the pecker or
peckee, because this kind of pecking has no real adverse effects (pain or injury) for
the peckee. In the present study severe feather pecks were mostly embedded in
bouts of gentle feather pecking.
Previously (Korte et al., 1997, 1999), HFP and LFP birds have been found
to display a proactive and reactive coping strategy, respectively. Proactive rodents
are characterised by low serotonergic transmission and enhanced expression
(Korte et al., 1996; van Riel et al., 2002) and sensitivity of 5-HT1A receptors (van
der Vegt et al., 2001). The more pronounced behavioural response of HFP birds to
S-15535 treatment in both experiments may well be a consequence of a difference
in the sensitivity of 5-HT1A receptor system between both lines. The exact
mechanisms underlying the effect of a decrease in 5-HT turnover on feather
pecking, and the more pronounced behavioural response of HFP birds compared
to LFP birds, are still to be demonstrated. Several other 5-HT receptor
(ant)agonists should be applied in future studies In rodents, S-15535 has potent
anti-aggressive (anti-proactive) properties (de Boer et al., 2000; Millan et al., 1997;
van der Vegt et al., 2001). These effects are attributed to the complementary
agonistic effects of S-15535 on the 5-HT1A autoreceptors and antagonistic effects
on the post-synaptic 5-HT1A receptors. In our study, as in rodents, S-15535
treatment decreased the level of 5-HT neurotransmission, however it increased the
level of feather pecking and increased the levels of proactive behaviour during
manual restraint. This confirms ethological knowledge indicating that feather
pecking is not a form of aggressive behaviour (Bilčík and Keeling, 1999;
Hoffmeyer, 1969). Aggressive pecking in birds is displayed by one or two firm
Con
trol o
f fea
ther
pec
king
by
sero
toni
n
81
Tabl
e 2.
Effe
cts
of L
ine
and
Trea
tmen
t on
beha
viou
ral o
bser
vatio
ns o
f LFP
and
HFP
chi
cks
Li
ne
Tr
eatm
ent
P-va
lues
1
LF
P
H
FP
Con
trol
S-
1553
5
L
ine*
Trea
tmen
t Li
ne
Trea
tmen
t
Gen
tle F
P
4.
86 ±
5.1
4 6
8.70
± 1
9.34
1
8.20
± 1
0.2
55
.40
± 15
.3
ns
***
#
Sev
ere
FP
0.0
5 ±
0.28
1
4.29
± 4
.77
1.
74 ±
1.7
0
12.6
0 ±
4.46
ns
**
*
*
Fora
ging
(%)
31.0
6 ±
3.18
2
8.37
± 3
.09
26.
50 ±
3.0
8
32.
94 ±
3.2
0
#
ns
n
s
Feed
ing
(%)
33.6
0 ±
3.72
2
4.96
± 3
.41
28.
42 ±
3.5
9 3
0.14
± 3
.56
#
#
ns
Pre
enin
g (%
) 8.
10 ±
1.7
7
8.9
4 ±
1.85
1
4.39
± 2
.35
2
.65
± 1.
04
ns
ns
*
**
Wal
king
(%)
11.2
7 ±
1.48
1
3.31
± 1
.59
8.
71 ±
1.3
5 1
5.87
± 1
.71
**
ns
***
Res
ting
(%)
15.9
4 ±
2.46
2
3.37
± 2
.84
20.
92 ±
2.7
5 1
8.39
± 2
.56
ns
*
n
s
1 Sig
nific
ant e
ffect
: *** P
< 0
.001
, **P
< 0
.01,
* P <
0.0
5, # 0.
05 <
P <
0.1
0, n
s =
non
sign
ifica
nt
82 Chapter 4
pecks directed to the head of a conspecific, whereas feather pecking has a clear
repetitive structure of pecking and pulling feathers. This also implies that the
normal control of feather pecking involves different postsynaptic 5-HT receptors
than the control of aggression.
As feather pecking has compulsive rather than aggressive characteristics,
it might be considered an animal model for OCD. Low serotonergic
neurotransmission has been implicated in the pathogenesis of OCD as well.
Chronic administration of 5-HT reuptake inhibitors (SSRIs) exerts a therapeutic
effect in OCD patients (Bergqvist et al., 1999). The mechanism responsible for the
beneficial effects of SSRIs have been suggested to be due to a desensitisation of
the terminal 5-HT autoreceptor resulting in a chronically enhanced 5-HT
neurotransmission in certain regions of the brain (Blier and Bouchard, 1994). It has
also been suggested that prolonged exposure to SSRIs may lead to decreased
responsiveness of postsynaptic receptors (Peroutka and Snyder, 1981), suggesting
a basal hypersensitivity of the serotonergic system in OCD (Mundo et al., 1995).
This hypothesis seems to be confirmed by some studies (Erzegovesi et al., 2001;
Mundo et al., 1995; Pigott, 1996; Zohar and Judge, 1996) in which acute treatment
with 5-HT agonists lead to a worsening of obsessive/compulsive symptoms. This is
in agreement with our finding that acute 5-HT agonist treatment enhances feather
pecking behaviour.
Thus, the available data suggest that feather pecking behaviour may well
be a suitable animal model for OCD. In future studies it will be investigated whether
chronic enhancement of 5-HT neurotransmission in the chicken brain, will be
beneficial in decreasing feather pecking behaviour.
4.3 Summary and Conclusion
Results of the present study support our hypothesis that a difference in the
functioning of the serotonergic system is mediating the difference in the
performance of feather pecking between HFP and LFP birds. The data suggest
that feather pecking behaviour is an animal model for OCD, triggered by low
serotonergic neurotransmission.
Control of feather pecking by serotonin 83
In conclusion, differences in the sensitivity for the development of feather
pecking may well be associated with a difference in serotonergic function. In the
future, more pharmacological experiments studying the role of serotonergic or
other neurobiological systems (e.g. dopaminergic system) are necessary to reveal
the exact mechanisms underlying feather pecking behaviour.
84 Chapter 4
Chapter 5
Chronic increase of dietary L-Tryptophan decreases feather pecking behaviour
Yvonne M. van Hierden1,2, S. Mechiel Korte1 and Jaap M. Koolhaas2
1 Animal Sciences Group, P.O. Box 65, NL-8200, AB Lelystad, The Netherlands
2University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
submitted for publication in Applied Animal Behaviour Science
86 Chapter 5
Abstract Many studies show the involvement of the serotonergic (5-HT) system in the
performance of abnormal behaviour in both human and animals. Recently, we showed that
acute reduction of 5-HT turnover in the forebrain, increased gentle and severe feather
pecking behaviour in chicks from a high (HFP) and low feather pecking (LFP) line of laying
hens, suggesting that the performance of feather pecking behaviour involves low 5-HT
neurotransmission.
In the present study, we postulated that if low 5-HT is causally underlying feather
pecking, increasing 5-HT turnover in the forebrain will decrease the development and
performance of feather pecking. Augmentation of 5-HT neurotransmission in the brain was
induced by chronically increasing dietary levels of the essential amino acid L-tryptophan
(TRP) from which 5-HT is synthesised. From the age of 34 days, LFP and HFP chicks were
fed a diet containing 2 % TRP, whereas control birds of both lines were continuinly fed the
normal rearing feed (0.16 % TRP). From 35 days of age, litter was removed from the pens
(10 pens/line-treatment) and all chicks (10 chicks/pen) were on housed a slatted floor until
the end of the experiment. At 49 days of age, feather pecking behaviour was studied for 30
minutes. At 50 days of age baseline corticosterone, TRP and other Large Amino Acids
(LNAAs) were measured in the blood plasma of decapitated chicks (10 chicks per line-
treatment). Furthermore, plasma corticosterone and central 5-HT turnover levels in response
to manual restraint (5 min) were determined (10 chicks/line-treatment).
TRP treatment resulted in a significant overall decrease of the frequency of gentle
feather pecking (although more pronounced in the HFP line). For severe feather pecking a
similar but not significant pattern was found. Significant Line effects were found for gentle
and severe feather pecking. HFP birds showed significantly higher levels of gentle and
severe feather pecking behaviour than LFP birds. TRP treatment significantly increased the
TRP/LNAA ratio in the plasma of the chicks. Furthermore, TRP treatment overall increased
baseline and stress-induced levels of plasma corticosterone (although more pronounced in
the LFP line). TRP supplementation significantly increased 5-HT turnover in the
hippocampus and archistriatum and tended to do so in the remainder of the forebrain.
The results confirm our hypothesis that feather pecking behaviour is triggered by
low serotonergic neurotransmission, as increasing serotonergic tone, by increasing dietary
TRP, decreases feather pecking behaviour.
Dietary L-Tryptophan decreases feather pecking 87
1. Introduction
Feather pecking in laying hens, an abnormal form of allopecking, is
causing welfare problems in current poultry farming. Expressed in a more gentle,
stereotypic form of pecking, feather pecking is abnormal, but not very harmful. The
more severe, also rather compulsive form, feather pulling, ultimately results in
injuries, leading to cannibalism and death. The causation of feather pecking is
multifactorial (Hughes and Duncan, 1972), with interactions between animal
characteristics (e.g. genetic factors) and environmental factors that have been
proven hard to fathom.
Recently (van Hierden et al., 2003a), we found evidence for a causal role
of the serotonergic (5-HT) system in the performance of feather pecking behaviour.
In a study with high (HFP) and low feather pecking (LFP) chicks, acute reduction
of 5-HT turnover in the forebrain, increased gentle and severe feather pecking
behaviour, suggesting that the performance of feather pecking behaviour involves
low 5-HT neurotransmission in the forebrain. We postulated that if low 5-HT is
causally underlying feather pecking behaviour, increasing 5-HT turnover in the
forebrain should decrease the development and performance of feather pecking.
An effective tool for increasing 5-HT neurotransmission in the brain is
increasing dietary levels of the essential amino acid L-tryptophan (TRP) from which
5-HT is synthesised (Fernstrom, 1983). For access into the brain, TRP competes
with other large neutral amino acids (LNAA; i.e. tyrosine, phenylalanine, leucine,
isoleucine, valine), as all LNAAs depend on the same carrier for transport across
the blood-brain-barrier (Markus et al., 2000). An increase in the ratio of plasma
TRP to the sum of the other LNAAs (TRP/∑LNAAs), gives TRP an advantage in
the competition for access into the brain (Markus, 2000). TRP hydroxylase, the
rate-limiting enzyme on the pathway from TRP to serotonin is normally about half-
saturated with TRP (Young and Leyton, 2002). Thus, elevated dietary intake of
TRP, resulting in an increased plasma TRP/LNAA ratio, increases brain levels of
TRP and elevates rates of 5-HT synthesis and metabolism (Johnston et al., 1990;
Lepage et al., 2002).
88 Chapter 5
TRP has been given to both man and animal, in disorders where a low
level of serotonin has been suggested to be of etiological significance, for instance,
depression (Sandyk, 1992), aggression (Shea et al., 1990; Winberg et al., 2001)
and obsessive compulsive disorders (McDougle et al., 1999; Weld et al., 1998;
Young and Leyton, 2002). In rhesus monkeys, with a history of compulsive self-
injurious behaviour, 3 weeks of dietary TRP supplementation, significantly
increased 5-HIAA in the cerebrospinal fluid and significantly decreased the duration
of self-biting (Weld et al., 1998). In a study by Savory and his colleagues (1999),
dietary supplementation with TRP in growing bantams, resulted in a suppression of
pecking damage with the higher (22.6 g/kg diet) dose, compared to the control (2.6
g/kg diet) dose, at 4 and 6 weeks of age. This lower level of pecking damage is
very likely to be the result of a lower level of severe feather pecking behaviour. In
that experiment, bantams were reared on a wire mesh floor, a condition known to
increase feather pecking (Blokhuis, 1989). From the available data it can be postulated that increasing the dietary
TRP level in the feed of HFP and LFP birds, increases the level of central serotonin
turnover and attenuates the development of feather pecking. To test this
hypothesis, in the present study the effect of an increased dietary TRP level on the
performance of feather pecking in HFP and LFP chicks was investigated. The dose
of dietary TRP (1.6 vs 21 gram/kg diet) used in this study, was chosen on the basis
of results of studies from Savory et al. (1999), Rosebrough (1996) and Shea et al.
(1990).
2. Methods
2.1 Birds and housing
In this study 200 White Leghorn chicks were used: 100 LFP and 100 HFP
chicks (for line specifications see Korte et al. 1997). All birds were female and non-
beaktrimmed. Chicks arrived on the day of hatching and were kept in groups of 10
animals per line (20 groups per line) and housed in pens (0.75x1.0m) with
Dietary L-Tryptophan decreases feather pecking 89
woodshavings. The pens were placed in two identical climate controlled rooms.
Individual pens were visually isolated by hardboard partitions. Four of the 10 birds
per pen were individually marked with a waterproof marker.
The environmental temperature was lowered from 34°C on day one to
18°C at 45 days of age onwards. On days 1 and 2 of age the chicks received 24
hours of light. From 3 days to 35 days onwards the light regime decreased from an
18 h light to a 10 h light period.
All birds had access to three drinking cups and one square feeding trough
placed along one of the walls of the pen. Water and feed (mash) were provided ad
libitum.
2.2 Experimental design and treatment diets
From day 1 till day 34 of age, all chicks received a standard rearing feed,
containing 1.6 gram TRP per kg feed. From the age of 34 days, half of the HFP
and LFP birds, were fed a diet containing 2 % TRP (± 21 gram TRP per kg diet).
The other half of the birds were continuously fed the normal rearing feed (1.6
gram/kg diet). There were 10 pens per line-treatment combination.
The LFP and HFP chicks were raised in litter floor pens (with
woodshavings) until 35 days of age. The floor of the pen consisted of a slatted
floor, on which cardboard was placed. A thick layer of woodshavings was applied
onto the cardboard. At 35 days of age the cardboard with the layer of
woodshavings was removed from the pens.
2.3 Behavioural observations
At 49 days of age all pens were recorded on videotape for a period of 30
minutes. Afterwards, two of the four coloured birds per pen were randomly chosen
and their behaviour was scored continously for 30 minutes per bird using The
Observer® 4.0 software programme (Noldus, Wageningen, The Netherlands).
Frequency and duration of the behavioural elements scored are described in Table
1.
90 Chapter 5
Table 1. Ethogram showing the behavioural measurements (posture either standing or
sitting).
Behaviour Definition
Frequencies
Gentle feather pecking Mild pecking at the feathers of conspecific,
generally performed in multiple bouts (single
pecks are counted as one occurrence)
Severe feather pecking Vigorous pecking/pulling/pinching at the
feathers of conspecific (single pecks are
counted as one occurrence)
Duration
Feeding Pecking at the litter and scratching (separately
scored as ground scratching) or moving with
the head in a lower position than the rump
Preening Preening behaviour as described by Kruijt
(1964): e.g. autopecking, nibbling, stroking,
combing, head-rubbing
Walking Walking, running, jumping or flying (it may be
accompanied by wing-flapping)
Resting Sitting or standing inactive (no movement of the
legs)
2.4 Physiological and neurobiological measurements
The two observed coloured birds were used for physiological and
neurobiological measurements at the age of 50 days. One of the observed birds
per pen, was killed by rapid decapitation, upon removal from the pen, for baseline
corticosterone, TRP and LNAAs measurements in the blood plasma. The other
observed bird was removed from the pen, and manually restrained for 5 minutes
(i.e. placed on its side). Subsequently, chicks were decapitated and their blood and
Dietary L-Tryptophan decreases feather pecking 91
brains were collected, for corticosterone and serotonin turnover measurements,
respectively.
Trunk blood of both birds was collected in chilled (0 °C) Lithium-Heparin-
coated centrifuge tubes, for either baseline or stress-induced plasma
corticosterone measurements. Blood was centrifuged for 10 minutes at 3000 rpm
at a temperature of 4°C. Plasma samples for corticosterone analysis were stored at
4°C in the presence of 0.1% (w/v) sodium azide. Corticosterone concentrations
were determined in unextracted, enzymatically pretreated plasma (DELFIA), as
described earlier (de Jong et al., 2001). The detection range of the corticosterone
assay was 0.2 � 44 ng/ml.
Levels of TRP and Phenylanaline in the bloodplasma were analysed using
deproteinization with SSA (Sheep Serum Albumin). Analyses were performed with
RP-HPLC: Alltima C18 column, sodiumacetate/methanol as eluens, pH 6.0.
Detection with UV-detection at 207 nm. Plasma concentrations of the other LNAAs
were measured by high-performance liquid chromatography as described in de
Jonge and Breuer, 1994 (de Jonge and Breuer, 1994).
The brains of the restrained birds were removed within one minute after
decapitation and immediately frozen in a dry ice precooled tube containing n-
heptane and stored at - 70°C until the assays were performed. For the assay three
brain sections were used: the hippocampus, the archistriatal complex (i.e. an
amygdala like structure in birds (Kuenzel and Masson, 1988) and remainder of the
forebrain. The brain sections were used for the measurement of serotonin (5-
hydroxytryptamine; 5-HT) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-
HIAA). Previously, it has been shown that 5-HT turnover is indicated by the 5-
HIAA/5-HT ratio (Korte-Bouws et al., 1996).
The tissue samples were homogenised in 0.1 M perchloric acid and
subsequently centrifuged at 14000 rpm. 50 µl of the supernatant was injected onto
a reverse-phase/ion pair high performance liquid chromatography (HPLC) setup
with electrochemical detection for the measurement of 5-HT and 5-HIAA. The
chromatographic system consisted of an Pharmacia LKB HPLC pump 2158 (Kyoto,
Japan), a Gilson 231 Sample Injector, an Antec Decade potentiostate (Antec
Leyden, Leiden, The Netherlands) with a glassy carbon cell operated at +500 mV
92 Chapter 5
vs Ag/AgCl and a column (150mm x 4.6mm i.d.) packed with Supelcosil LC-18-DB,
3 µm particle size. The mobile phase consisted of sodium acetate (4.1 g/l), EDTA
(0.15 g/l), sodium octane-sulfonic acid (0.175 g/l), tetramethylammonium (0.15 g/l),
methanol (12 % v/v), pH 4.1. The flow rate was set at 1 ml/min.
2.5 Statistical analysis
Pens were considered to be the experimental units and the means of the
two observed animals per pen were analysed. Durations of behaviours (i.e.
percentages) were analysed with a logistic regression model with main effects and
interactions for experimental factors for Line (HFP or LFP) and Treatment (0 or 50
mg per kg bodyweight), i.e. log(P/100-P) was expressed as a sum of main effects
and interactions, where P is the expected percentage. The model comprised a
multiplicative dispersion factor relative to the binomial variance function, i.e. the
variance was assumed to be proportional to P*(100-P). Frequency of feather
pecking was analysed with a log linear model with main effects and interaction for
Line and Treatment, i.e. log(m) is expressed as a sum of main effects and
interactions, where m is the expected mean. The model comprised a multiplicative
dispersion factor relative to the variance under a Poisson distribution, i.e. the
variance was assumed to be proportional to the mean m.
Estimation of durations and frequencies was performed by maximum
quasi-likelihood. The dispersion parameter was estimated from Pearson�s chi-
square statistic. Significance tests were performed by referring the log quasi-
likelihood ratio to an F-distribution (with �residual degrees of freedom�
corresponding to Pearson�s chi-square statistic). Details may be found in
McCullagh and Nelder (1989) . Significance tests for feather pecking as a binary
variable were based on the maximum likelihood ratio test.
Corticosterone, 5-HIAA/5-HT ratio and TRP/LNAA data were analysed with
a mixed analysis of variance model with main effect and interactions for the factors
Line and Treatment. For the analyses a common F-test was used. Residuals were
checked for normal distribution and homogeneity of variances. Data showing
Dietary L-Tryptophan decreases feather pecking 93
heterogeneity, i.e. an increased variance with increasing mean, were
logarithmically transformed and reanalysed.
Differences were considered significant if P < 0.05. For all calculations
GenStat® 6 (2002) was used.
3. Results
3.1 Behavioural observations
Figure 1 shows the results of the behavioural observations at 7 weeks of
age.
For both gentle (Figure 1A) and severe feather pecking behaviour (Figure
1B) no significant Line x Treatment interaction was found (Table 2). For gentle
feather pecking, a significant overall Treatment effect [F(1,75) = 5.72, P = 0.02]
was found. For severe feather pecking no significant overall Treatment effect was
found [F(1,75) = 2.32, P = 0.13]. Figure 1A shows that TRP treated HFP birds
showed significantly (P = 0.03) less gentle feather pecking than control HFP birds.
For severe feather pecking a similar but not significant (P = 0.19) pattern was
found (Figure 1B). Significant overall Line effects were found for gentle [F(1,75) =
35.15, P < 0.001] and severe feather pecking [F(1,75) = 27.54, P < 0.001]. HFP
birds showed significantly higher levels of gentle and severe feather pecking
behaviour than LFP birds (table 2).
For duration of foraging behaviour no significant Line x Treatment
interaction was found. A trend for a Treatment effect [F(1, 75) = 3.15, P = 0.08] and
a significant effect of Line were found [F (1,75) = 9.93, P < 0.001]. LFP birds spent
more time foraging than HFP birds. Figure 1C shows that LFP control birds
significantly (P = 0.04) spent more time foraging than TRP treated LFP birds.
For feeding behaviour a significant Line x Treatment interaction was found
[F(1,74) = 5.40, P = 0.02]. Furthermore, significant effects of Line [F(1, 75) =
11.21, P < 0.001] and Treatment [F(1,75) = 30.20, P < 0.001] were found (Table 2).
Figure 1D shows that in both lines TRP treatment significantly increases the
duration of feeding behaviour, but more pronounced in the HFP line.
94 Chapter 5
Figure 1. Frequency or relative duration (percentage of the total observation time) of the
behaviour of control and TRP treated LFP and HFP chicks (49 days of age). Levels are
expressed as mean ± S.E.M. Means without a common superscript differ (P < 0.10).
LFP HFP0
10
20
30
40
50300
400
Freq
uenc
y/30
min
utes
Severe feather pecking
B
aa
b
b
LFP HFP0
10
20
3080
90
100
Rel
ativ
e du
ratio
n (%
)
ForagingC
ba a a
LFP HFP0
10
20
3080
90
100
Rel
ativ
e du
ratio
n (%
)
PreeningE
a
b
a
a
LFP HFP0
50
100
150
200
250
300
350
400
Freq
uenc
y/30
min
utes
Gentle feather peckingControl Trypt
a
A
aa
b
c
LFP HFP0
10
20
30
4080
90
100
Rel
ativ
e du
ratio
n (%
)
WalkingF
a a ab
LFP HFP0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e du
ratio
n (%
)
FeedingD
a
b
c
ab
Dietary L-Tryptophan decreases feather pecking 95
For preening behaviour no Line x Treatment effect was found, however a
significant Line effect [F(1,75) = 3.93, P = 0.05] and Treatment effect [F(1,75) =
9.09, P < 0.001] were found. Figure 1E shows that TRP treated HFP birds spent
significantly (P = 0.004) less time preening than control HFP birds.
For the duration of walking, a significant Line x Treatment effect was found
[F(1,74) = 4.73, P = 0.03]. No significant overall Line or Treatment effect were
found. Figure 1F shows that TRP treated HFP birds spent significantly (P = 0.02)
more time walking than control HFP birds.
For resting behaviour a significant Line x Treatment interaction [F(1,74) =
5.02, P = 0.03], Line [F(1,75) = 22.28, P < 0.001] and Treatment [F(1,75) = 22.88,
P < 0.001] effects were found. TRP treatment decreased the duration of resting in
both lines, but more pronounced in the HFP line (52.16 ± 0.04 vs 23.33 ± 0.03)
than in the LFP line (23.33 ± 0.03 vs 18.73 ± 0.03).
Further results of the behavioural elements are shown in Table 2.
3.2 Corticosterone, TRP/LNAA, and serotonin measurements For the TRP/LNAA ratio (Figure 2A) no significant Line x Treatment
interaction and Line effect were found. TRP treatment however significantly
increased [F(1,36) = 747.62, P < 0.001] the TRP/LNAA ratio in the plasma of LFP
and HFP chicks. Further results of the LNAAs are presented in Table 3.
For baseline corticosterone levels a trend for a Line x Treatment interaction
was found [F(1,35) = 3.43, P = 0.07]. A significant Treatment [F(1,36) = 3.92, P =
0.05] and Line effect [F(1,36) = 8.90, P = 0.005] were found, for baseline plasma
corticosterone levels (Table 3). Figure 2B shows that TRP treated LFP chicks
showed significantly higher baseline corticosterone levels compared to control LFP
birds (P = 0.01).
For corticosterone levels after manual restraint (Figure 2B), no significant
Line x Treatment interaction was found. Both Treatment [F(1,36) = 5.10, P = 0.03]
and Line [F(1,36) = 10.41, P = 0.003] effects were significant. Figure 2B shows that
also stress-induced corticosterone levels were significantly higher in the TRP
treated LFP chicks compared to control LFP birds (P = 0.01).
96 Chapter 5
Figure 2. Plasma levels of the TRP/LNAA ratio (Figure 2A), baseline and restraint-induced
corticosterone (Figure 2B), and forebrain levels of 5-HT turnover (5-HIAA/5-HT) after manual
restraint; LFP-C= LFP-control, LFP-T= LFP-tryptophan, HFP-C= HFP-control, HFP-T= HFP-
tryptophan. Levels expressed as mean ± S.E.M. Means without a common superscript differ
(P < 0.10).
0.00
0.25
0.50
0.75
1.00
Tryp
toph
an/L
NAA
ratio
LFP-C LFP-T HFP-C HFP-T
a
b
a
bA
Baseline Restraint0
1
2
3
4
5
6C
ortic
oste
rone
(ng/
ml)
ab
a
bB
a a
a
a
a
hippocampus archistriatum remainder0.00
0.25
0.50
0.75
1.00
5-H
IAA
/5-H
T
b
ab
C
ab
aa a a aab ab
Dietary L-Tryptophan decreases feather pecking 97
5-HT turnover levels (5-HIAA/5-HT) in the different brain regions are
presented in table 3 and Figure 2C. For non of the brain regions significant Line x
Treatment interactions or Line effects were found. TRP supplementation however
significantly increased 5-HT turnover in the hippocampus [F(1,35) = 3.85, P = 0.05]
and archistriatum [F(1,35) = 4.98, P = 0.03] and tended to do so in the remainder
of the forebrain [F(1,36) = 3.12, P = 0.09]. Figure 2C shows that TRP treatment in
LFP chicks significantly (P = 0.05) increased stress-induced 5-HT turnover levels in
the hippocampus compared to control LFP chicks. For the HFP birds, no significant
effect of TRP treatment on 5-HT turnover in the hippocampus was found. However,
TRP treatment significantly increased 5-HT turnover in the archistriatum (P = 0.01)
and the remainder of the forebrain (P = 0.05) in this line, whereas no significant
effects were found for the LFP line in these brain regions
4. Discussion
In the present study the effect of TRP supplementation on the development
of feather pecking behaviour was studied in HFP and LFP birds. The principal
finding was that a dose of 21 gram TRP/kg diet significantly reduced gentle feather
pecking behaviour and increased the duration of feeding behaviour.
4.1 Effect of TRP supplementation on physiological and neurobiological parameters
In both lines TRP treatment increased the TRP/LNAA ratio, which enabled
an increase in 5-HT turnover in the forebrain of the chicks. These results confirm a
study by Rosebrough (1996), that also showed TRP supplementation to be an
excellent tool for increasing 5-HT turnover in the forebrain of chickens.
In the LFP line 5-HT turnover was increased in the hippocampus, whereas
in the HFP line 5-HT turnover levels were increased in the archistriatum and the
remainder of the forebrain. We tentatively suggest that differences in TRP
98
Cha
pter
5
Tabl
e 2.
Effe
cts
of L
ine
and
Trea
tmen
t on
beha
viou
ral o
bser
vatio
ns o
f LFP
and
HFP
chi
cks
Line
Trea
tmen
t
P
-val
ues1
LFP
HFP
Con
trol
T
RP
L
ine*
Trea
tmen
t
L
ine
Trea
tmen
t
Gen
tle F
P
18.5
5 ±
9.05
1
84.1
8 ±
29.0
13
7.70
± 2
4.7
65
.00
± 17
.8
ns
***
*
Sev
ere
FP
2.0
8 ±
1.25
28.4
6 ±
4.29
1
6.18
± 3
.49
9
.36
± 2.
78
ns
***
ns
Fora
ging
(%)
5.5
8 ±
0.64
3.0
5 ±
0.49
5.03
± 0
.60
3
.60
± 0.
53
ns
***
***
Pre
enin
g (%
) 9
.61
± 1.
47
14
.16
± 1.
77
15.
38 ±
1.8
1
8.3
9 ±
1.43
ns
*
*
**
Wal
king
(%)
9.4
2 ±
0.82
7.9
6 ±
0.77
8.10
± 0
.76
9
.29
± 0.
82
*
ns
n
s
Res
ting
(%)
23.5
0 ±
2.08
37.9
3 ±
2.42
4
0.22
± 2
.42
21.
22 ±
2.0
8
*
***
***
1 Sig
nific
ant e
ffect
: *** P
<0.0
01, **
P<0
.01,
* P<0
.05,
# 0.05
<P<0
.10,
ns
= no
n si
gnifi
cant
Die
tary
L-T
rypt
opha
n de
crea
ses
feat
her p
ecki
ng
99
Tabl
e 3.
Effe
cts
of L
ine
and
Trea
tmen
t on
[TR
P/L
NA
A],
pla
sma
leve
ls o
f [C
ort-b
asel
ine]
and
[Cor
t-stre
ss] (
ng/m
l), a
nd b
rain
leve
ls o
f [5-
HIA
A] (
ng/m
g br
ain
tissu
e), [
5-H
T] a
nd [
5-H
IAA
]/[5-
HT]
afte
r man
ual r
estra
int;
hipp
o= h
ippo
cam
pus,
arc
h= a
rchi
stria
tum
, rem
=rem
aind
er o
f
fore
brai
n
L
ine
T
reat
men
t
P-va
lues
1
LFP
HFP
Con
trol
TRP
Li
ne*T
reat
men
t
L
ine
Trea
tmen
t
TRP
587.
10 ±
26.
2 59
6.50
± 2
6.4
121.
60 ±
11.
9 10
62.0
± 3
5.2
ns
ns
**
*
Tyro
sine
24
8.00
± 8
.05
203.
50 ±
7.3
0 21
6.75
± 7
.53
234
.75
± 7.
84
ns
***
ns
Phe
nyla
lani
ne
142.
18 ±
3.7
1 13
0.98
± 3
.56
150.
74 ±
3.8
2 1
22.4
2 ±
3.44
ns
*
**
*
Leuc
ine
30
7.78
± 9
.69
320.
08 ±
9.8
8 32
0.11
± 9
.88
307
.75
± 9.
69
ns
ns
ns
Isol
euci
ne
159.
58 ±
5.5
2
157.
75 ±
5.4
8 15
4.42
± 5
.43
162
.92
± 5.
57
ns
ns
ns
Val
ine
35
7.10
± 1
0.6
35
1.60
± 1
0.5
348.
90 ±
10.
5 3
59.8
0 ±
10.6
ns
ns
ns
5-H
IAA
hipp
o
0.
20 ±
0.0
4
0.2
0 ±
0.04
0.1
3 ±
0.04
0.2
7 ±
0.04
ns
ns
**
5-H
T hip
po
0.
92 ±
0.0
4
1.0
0 ±
0.05
0.8
1 ±
0.04
1.1
1 ±
0.04
ns
ns
***
5-H
IAA
arch
0.
11 ±
0.0
1
0.0
8 ±
0.01
0.0
6 ±
0.01
0.1
3 ±
0.01
**
*
**
*
5-H
T arc
h
0.
69 ±
0.0
7
0.6
0 ±
0.07
0.5
5 ±
0.07
0.7
4 ±
0.07
***
ns
*
5-H
IAA
rem
0.
10 ±
0.0
1
0.1
0 ±
0.01
0.0
8 ±
0.01
0.1
2 ±
0.01
ns
ns
***
5-H
T rem
0.
74 ±
0.0
2
0.6
8 ±
0.02
0.6
2 ±
0.02
0.7
9 ±
0.01
**
#
**
*
[TR
P/L
NA
A]
0
.49
± 0.
02
0
.51
± 0.
02
0
.10
± 0.
01
0
.89
± 0.
02
ns
ns
**
*
[Cor
t-bas
elin
e]
0
.68
± 0.
09
0
.45
± 0.
09
0
.42
± 0.
09
0
.70
± 0.
09
ns
**
*
[Cor
t-stre
ss]
3
.70
± 0.
42
1
.80
± 0.
37
2
.17
± 0.
39
3
.34
± 0.
37
ns
**
*
[5-H
IAA
]/[5-
HT]
hipp
o
0.
20 ±
0.0
3
0.1
9 ±
0.03
0.1
6 ±
0.02
0.2
4 ±
0.01
ns
ns
*
[5-H
IAA
]/[5-
HT]
arch
0.
17 ±
0.0
1
0.1
4 ±
0.02
0.1
3 ±
0.01
0.1
8 ±
0.01
ns
ns
*
[5-H
IAA
]/[5-
HT]
rem
0.
15 ±
0.0
1
0
.13
± 0.
02
0
.13
± 0.
01
0
.16
± 0.
01
ns
ns
#
Sig
nific
ant e
ffect
: *** P
<0.0
01, **
P<0
.01,
* P<0
.05,
# 0.05
<P<0
.10,
ns
= no
n si
gnifi
cant
100 Chapter 6
hydroxylase activity between both lines, in the different brain regions, are
responsible for the different levels of 5-HT turnover in response to TRP.
In the LFP line, an increased TRP/LNAA ratio, was accompanied by an increase in
both baseline and stress-induced plasma corticosterone levels, whereas in the
HFP line corticosterone levels were not affected.
The increased adrenocortical (re)activity in the LFP line in response to TRP
treatment, is in agreement with findings by Mench (1991) in broiler breeders.
Mench (1991) found that an elevation in corticosterone, was modulated by dietary
TRP. In mammals, 5-HT is known to be involved in the regulation of the HPA-axis
(Chaouloff, 2000). Release of ACTH from the pituitary and glucocorticosteroids
from the adrenal cortex is strongly and acutely stimulated by 5-HT via action
mediated by 5-HT1A receptors in the hypothalamus (Fuller, 1992). An increase of
corticosterone levels in the LFP, but not in the HFP line, is in agreement with
previous findings of a higher adrenocortical (re)activity in the LFP line (Korte et al.,
1997; van Hierden et al., 2002a).
4.2 Effect of TRP supplementation on feather pecking behaviour
The results of this experiment support our hypothesis that increasing 5-HT
neurotransmission in the forebrain of (HFP) chicks, by increasing dietary TRP,
attenuates the development of feather pecking behaviour. Our findings are in
agreement with earlier findings of Savory and his colleagues (1999), who found
TRP supplementation to decrease feather pecking damage in growing bantams at
4 and 6 weeks of age, suggesting a decrease in severe feather pecking. In our
study, TRP supplementation did not significantly decrease severe feather pecking.
However, severe feather pecks were mostly embedded in bouts of gentle feather
pecking, suggesting a common underlying motivation and neurobiological system
(Riedstra, 2003).
Savory and his co-workers (1999) suggested that sedative-like properties
of TRP were responsible for the decrease in feather pecking (damage). In our
study however, chronic TRP treatment did not result in sedation of the birds, as
Increase in dietary L-Tryptophan decreases feather pecking 101
HFP chicks even became more active. This difference might be explained from the
difference in experimental setup. In the study of Savory, from day one of age,
chicks were given supplemental TRP and were raised without access to litter.
However, in the present study, supplemental TRP was provided and litter was
removed, at a later developmental stage of the chicks. Possibly, a longer
administration of high dietary TRP levels, may lead to sedation of the birds. In
future studies such effects of chronic TRP supplementation should be investigated.
Supplementary TRP also affected other behavioural elements. In the HFP
line, an increase in both feeding and walking, was accompanied by a decrease in
preening behaviour. In the LFP line TRP treatment decreased foraging, and also
increased feeding behaviour. The increase in the duration of feeding behaviour in
both lines, might be explained from the role of the 5-HT system in the regulation of
hunger and satiation. However, most studies report of a decrease in feed intake in
response to TRP treatment (Lacy et al., 1986; Rosebrough, 1996). Six days of TRP
administration to commercial layer breeder hens at a level of 5 g/kg has been
reported to end the incidence of hysteria and stimulate feed consumption (Laycock
and Ball, 1990). However, in the present study, only the time spent with the head
in the feeding trough, and not the actual feed intake was not measured. We did not
observe an effect of TRP on bodyweight (data not shown), suggesting no effect on
feed intake. However, current results are hard to interpret and the effect of TRP on
(duration of) feeding should be investigated further.
Recently (van Hierden et al., 2003a), we suggested that feather pecking
behaviour might be a suitable animal model for obsessive compulsive disorder
(OCD), as feather pecking has clear compulsive characteristics. Once birds start
feather pecking, they tend to do so successively and their pecking behaviour is
difficult to discourage. Furthermore, impaired 5-HT neurotransmission is implicated
in obsessive compulsive disorders (for a review see Blier and de Montigny, 1998)
as well as feather pecking (van Hierden et al., 2003a). Chronic enhancement of 5-
HT neurotransmission via blocking of 5-HT reuptake with selective serotonin
reuptake inhibitors (SSRIs), alleviates OCD symptoms in most cases (Bergqvist et
al., 1999; Blier and Montigny de, 1999). In future studies it should be investigated
102 Chapter 6
whether chronic treatment with SSRIs decreases the performance of feather
pecking behaviour.
4.4 Summary and Conclusion
Results of the present study confirm our hypothesis that feather pecking
behaviour is triggered by low serotonergic neurotransmission, as increasing
serotonergic tone, by increasing dietary TRP, decreases feather pecking
behaviour.
In conclusion, differences in the sensitivity for the development of feather
pecking are associated with a difference in serotonergic function. In the future,
more pharmacological experiments, studying the role of serotonergic or other
neurobiological systems (e.g. dopaminergic system) are necessary to reveal the
exact mechanisms underlying feather pecking behaviour.
Chapter 6
Chicks from a high and low feather pecking line of laying hens differ in
apomorphine sensitivity
Yvonne M. van Hierden1,3, S. Mechiel Korte1, Ľubor Ko�t�ál2, Pavel Výboh2, Monika Sedlačková2, Marek Rajman2, Marian
Juráni2, Jaap M. Koolhaas3
1Animal Sciences Group, P.O. Box 65, NL-8200, AB Lelystad, The Netherlands 2Department of Endocrinology and Ethology, Institute of Animal
Biochemistry and Genetics, Slovak Academy of Sciences
900 28 Ivanka pri Dunaji, Slovakia 3University of Groningen, P.O. Box 14, 9750 AA Haren
The Netherlands
submitted for publication in Applied Animal Behaviour Science
104 Chapter 6
Abstract The dopaminergic (DA) system has been implicated in several behavioural
pathologies in both human and animal species. Previously, chicks from a high feather
pecking (HFP) line were found to display lower central DA activity than chicks from a low
feather pecking (LFP) line. HFP and LFP birds have been suggested to display a proactive
and reactive coping strategy, respectively. Proactive rodents show a larger behavioural
response to apomorphine (APO) than reactive copers, suggesting a more sensitive DA
(receptor) system in proactive individuals.
In the present study, it is investigated whether HFP chicks show a more enhanced
behavioural response to acute APO treatment than LFP birds in an open field. Furthermore,
it was determined whether behavioural variation between HFP and LFP birds, after APO
injection, is reflected by variation of D1 and D2 receptor densities in the brain.
APO treatment (0.5 mg/kg BW) significantly more enhanced locomotor activity (i.e.
total distance moved, mean and maximum velocity) in an open field in the HFP line
compared to the LFP line (during the first 15 minutes). There were no significant line
differences in D1 and D2 receptor densities in the brain.
The present study demonstrated that HFP birds have a higher sensitivity of the DA
(receptor) system in the brain compared to LFP birds. Thus, confirming our hypothesis that
HFP birds display a proactive and LFP birds a reactive coping strategy.
A possible relationship between the functioning of the DA system en feather
pecking is discussed.
Difference in APO sensitivity in HFP and LFP birds 105
1. Introduction
Feather pecking behaviour in laying strains of domestic fowl is a long-
standing welfare problem in the layer industry. It is characterised by rather
stereotypic pecking (Kjaer and Vestergaard, 1999) and compulsive pulling (McAdie
and Keeling, 2000) at feathers of conspecifics, ultimately leading to injury and
death. Despite years of research, its complex aetiology remains hard to fathom.
Feather pecking is usually performed by a limited number of individuals in a flock
(Keeling, 1994). Specific interaction between a genetic predisposition for the
development of feather pecking and environmental challenges is believed to
underlie this behavioural pathology.
Previously (van Hierden et al., 2002a), we reported that birds from a high
(HFP) and low feather pecking (LFP) line of laying hens displayed different
physiological and neurobiological response patterns when challenged. More
specifically, it was shown that in response to acute stress induced by manual
restraint, HFP chicks had lower plasma corticosterone levels and lower dopamine
(DA) and serotonin (5-HT) turnover levels in the forebrain than LFP chicks. The
results from the study supported earlier findings (Korte et al., 1997; Korte et al.,
1999) that the (physiological) characteristics of HFP and LFP birds show
considerable analogy to the characteristics of respectively the proactive and
reactive coping strategy, known to exist in other species like rodents and pigs.
From this study (van Hierden et al., 2002a) we also postulated that the difference in
feather pecking behaviour between both lines might be causally related to a
difference in functioning of the 5-HT and DA system.
Recently (van Hierden et al., 2003a), we found evidence for a causal role of the 5-
HT system in the development and performance of feather pecking. In the present
study a first step is taken by investigating a possible role of the DA system in
feather pecking behaviour.
It has been suggested that the neurobiological characteristics of �proactive�
individuals make them more vulnerable to develop (behavioural) pathologies than
�reactive� individuals (Cools et al., 1990; Degen et al., 2003; Koolhaas et al., 1999;
106 Chapter 6
Sontag et al., 2003; Teunis et al., 2002). A difference in the functioning or
sensitivity of the DA (receptor) system has been suggested to (partly) account for
this difference in vulnerability (Cools et al., 1993). Apomorphine (APO), a full
agonist of the dopaminergic D1 and D2 receptors, with similar intrinsic activity as
DA, is often used to predict individual differences in the sensitivity of the (receptor)
DA system (Lal, 1988; Surmann and Havemann-Reinecke, 1995). By stimulation of
the postsynaptic D1 and D2 receptors, APO (dose-dependently) induces an
increase of locomotor activity and stereotyped behaviour (Berridge and Aldridge,
2000), like stereotypic pecking in chickens (Osuide and Adejoh, 1973; Zarrindast
and Amin, 1992)Proactive copers show a larger behavioural response to injection
with APO than reactive copers (Benus et al., 1991a; Bolhuis et al., 2000),
suggesting a more sensitive DA (receptor) system in proactive individuals.
From the above we postulate that birds from the (proactive) HFP line have
a higher sensitivity of the DA (receptor) system, and will therefore show an
enhanced behavioural response to acute APO treatment compared to (reactive)
LFP birds. To test this hypothesis, the behaviour of LFP and HFP chicks in
response to an APO injection was studied using an open field. Another objective of
the present study was to determine whether behavioural variation (in an open field)
between HFP and LFP birds, after APO injection, is also reflected by variation of D1
and D2 receptor densities in the brain. Therefore, receptor binding capacities were
assessed by measuring specific binding of tritiated D1 and D2 receptor ligands in
different regions of the brain of control HFP and LFP chicks.
2. Methods
2.1 Birds and housing
In this study 96 White Leghorn chicks were used: 48 LFP and 48 HFP
chicks (for line specifications see Korte et al., 1997). All birds were female and
non-beaktrimmed. Chicks arrived on the day of hatching and were kept in groups of
Difference in APO sensitivity in HFP and LFP birds 107
4 animals per line (12 groups per line) and housed in pens (0.75x1.0m) with
woodshavings. The pens were placed in a climate controlled room. Individual pens
were visually isolated by hardboard partitions. Chicks were individually marked on
the back with waterproof markers (black, purple, blue and green) before housing.
The environmental temperature was gradually lowered from 34°C on day
one to 22°C at 5 weeks of age. On days 1 and 2 of age the chicks received 24
hours of light. From 3 days to 5 weeks of age the light regime gradually decreased
from an 18 h to a 10 h light period. All birds had access to three drinking cups and
one square feeding trough placed along one of the walls of the pen. Water and
commercial feed (mash) were provided ad libitum.
2.2 APO injection and open field test
Apomorphine hydrochloride (Sigma RBI, the Netherlands) was freshly
dissolved in distilled water (vehicle) every day. APO was injected into the breast
muscle at a dose of 0.5 mg/kg BW in a volume of 1 ml/kg BW. A pilot study showed
that this dose was the most effective in eliciting a change in behaviour of the chicks
(data not shown). This finding is in agreement with previous studies (Osuide and
Adejoh, 1973; Zarrindast and Amin, 1992). The control chicks were injected (i.m.)
with a volume of 1 ml distilled water/kg BW.
At either 29, 30 or 31 days of age each chick was individually tested in an
open field. A chick was captured individually, taken to the test room and injected
into the breast muscle with either APO or distilled water (control). In each pen two
APO and two control chicks were randomly chosen. The open field was situated in
a separate room, to ensure auditory isolation, and the ambient temperature and
humidity were maintained at a similar level to that of the home environment. The
open field consisted of a wooden box, measuring 1.5 x 1.5 x 1.5 m (LxWxH), with
white solid walls and woodshavings on the floor.
Immediately following APO or vehicle injection, the chick was placed in the
middle of the open field. The behaviour of the chicks was videotaped for 30
minutes using an overhead camera and scored afterwards using Ethovision® 2.1
software programme (Noldus, Wageningen, The Netherlands).
108 Chapter 6
2.3 Dopamine D1 and D2 receptor binding
For analysis of D1 and D2 receptor binding in LFP and HFP chicks, only the
control birds were used. At the age of 35 days the two control birds were captured
from their homecage and killed by rapid decapitation. Their brains were removed
immediately and dissected into 4 brain regions; telencephalic pallium, basal
telencephalon, diencephalon and the mesencephalon (for details see Ko�t�ál et al.,
1999). Dissected tissue samples were frozen in a mixture of isopentane and dry ice
and stored at -70°C. The frozen samples were transported on dry ice from
Lelystad (the Netherlands) to Ivanka pri Dunaji (Slovakia).
The densities of D1 and D2 receptors were determined according to the
method described by Ko�ťál et al. (1999). Each dissected brain region was
weighed individually and homogenised in cold 50 mmol/l Tris-HCl (1:10 w/v) buffer
(pH 7.8). The homogenate was diluted 1:4 with the same buffer and centrifuged at
48,000 g for 10 min at 4°C (Beckman L5- 65, Ty-65 rotor). The supernatant was
discarded, the pellet washed in the same buffer and recentrifuged as before. The
final pellet was resuspended in 4ml of 50 mmol/l Tris-HCl incubation buffer (pH
7.4), containing 1 mmol/l MgCl2, 2 mmol/l CaCl2, 120 mmol/l NaCl and 5 mmol/l
KCl and next diluted with the same buffer to give a concentration of 1 mg protein
per ml, based on estimated protein content (Lowry et al., 1951).
Membrane suspensions were incubated in duplicate for 30 min at 37°C.
For the estimation of specific binding to D1 receptors each plastic tube contained
300 µl of membrane suspension (1 mg protein/ml), 100 µl of 0.59 nmol/l [3H]SCH
23390 (75.5 Ci/mmol, DuPont NEN, USA) and 100 µl of incubation buffer (total
binding) or 100 µl of unlabelled SCH 23390 (1 µmol/l, RBI, USA; non-specific
binding). For the estimation of specific binding to D2 receptors each plastic tube
contained 300 µl of membrane suspension, 100 µl 0.05 nmol/l [3H]spiperone (23
Ci/mmol, Amersham, Great Britain) and 100 µl of incubation buffer (total binding) or
100 µl of (+)-butaclamol (1 µmol/l, RBI, USA; non-specific binding). Following
incubation, separation of free from bound ligand was achieved by centrifugation at
23,000 g for 6 min at 4°C. Supernatant was removed by aspiration, and the tips of
Difference in APO sensitivity in HFP and LFP birds 109
the tubes (containing unrinsed pellets) were cut off with a heated wire and placed
in scintillation vials. After addition of scintillant, the vials were shaken for 2 h,
allowed to equilibrate, and radioactivity (dpm) was counted (Beckman LS 6000SE,
USA).
2.4 Statistical analysis
For the analysis of the open field behaviour, the 30-minutes observation
period was divided into 6 periods of 5 minutes. Furthermore, the behaviour of the
birds was divided into the states �moving� and �not moving� (i.e. �velocity� = 0.5
cm/s in Ethovision) and statistical analysis was performed on these states
separately. For the �moving state� the mean and maximum velocity (cm/s), total
distance moved (cm) and the total time spent �moving� (% of time) were calculated.
The sample rate used was 3 samples/second.
Open field behaviour was analysed per time period with a mixed analysis
of variance model with main effect and interactions for the factors Age (day 29, 30
or 31), Open Field (1 or 2), Line (HFP/LFP) and Treatment (APO/Control). Pen was
entered as a random effect in the analysis. The behavioural data were not normally
distributed. Therefore, these data were analysed according to techniques
described by Engel and Keen (1994) and Engel and Buist (1996), employing
Genstat 5 procedure IRREML (Keen and Engel, 1998). The Wald test (Rao, 1973),
was used for estimation of Line x Treatment, Line and Treatment effects. No Age
or Open Field effects were found and therefore these factors were excluded from
the analysis.
For the analyses of the levels of the D1 and D2 receptors in the four brain
regions, components were estimated with Restricted Maximum Likelihood Model
(REML) procedure (Patterson and Thompson, 1971). The Wald test (Rao, 1973)
was used for estimation of the Line effect.
When used in the IRREML procedure, the Wald statistic (W) is an
estimation of the F-statistic, when used in the REML procedure, the Wald statistic
is equal to the F-statistic. Differences were considered significant if P < 0.05. For
all calculations GenStat® 6 (2002) was used.
110 Chapter 6
Figure 1. The behaviour of control and APO treated LFP and HFP chicks in an open field.
Levels expressed as mean ± S.E.M. LFP-C= LFP-Control, HFP-C=HFP-Control, LFP-A
=LFP-APO, HFP-A = HFP-APO
0-5 6-10 11-15 16-20 21-25 26-30Time period (min)
0
1000
2000
3000
4000
5000
Tota
l dis
tanc
e m
oved
(cm
)
Total distance movedLFP-C HFP-C LFP-A HFP-A
A
0-5 6-10 11-15 16-20 21-25 26-30Time period (min)
0
20
40
60
80
100
Tota
l dur
atio
n of
mov
ing
(%)
Total duration of movingD
0-5 6-10 11-15 16-20 21-25 26-30Time period (min)
0
5
10
15
20
Mea
n ve
loci
ty (c
m/s
)
Mean velocity
B
0-5 6-10 11-15 16-20 21-25 26-30Time period (min)
0
50
100
150
200
250
Max
imum
vel
ocity
(cm
/s)
Maximum velocityC
Difference in APO sensitivity in HFP and LFP birds 111
3. Results
3.1 Open field behaviour
Table 1 shows the Line x Treatment (L x T) interactions, Line and
Treatment effects, per time period, for the parameters measured in the open field.
No significant overall Line effects were found for open field behaviour. Significant
Line x Treatment interactions were only observed during the first 3 time periods.
Therefore, we restrict the discussion of the behavioural results of the LFP and HFP
birds presented in Figure 1 to these first 3 time periods.
For �total distance moved� and �mean velocity� (Figure 1A and 1B)
significant Line x Treatment effects were found. During the first 15 minutes, APO
treatment enhanced the �total distance moved� and �mean velocity�, in the HFP
birds significantly more than in the LFP birds. In fact no significant differences were
found between APO treated and control chicks of the LFP line during this period of
time.
The �maximum velocity� is shown in Figure 1C. In the first time period, a
significant Treatment effect was found. Both APO-treated HFP and LFP chicks
showed a significantly higher �maximum velocity� than control HFP and LFP chicks.
Furthermore, a significant Line x Treatment interaction was found for the first two
time period. APO treatment enhanced the �maximum velocity� in the HFP birds
significantly more than in the LFP birds.
For �total duration of moving� (Figure 1D), no significant Line x Treatment
effects were found. Except for time period 3, significant Treatment effects were
found. Control birds spent more time moving during time period 1 and 2, compared
to APO treated birds.
3.2 D1 and D2 receptor binding
There were no significant Line differences in the specific binding to
dopamine D1 and D2 receptors in the four brain regions studied (Figure 2).
112 Chapter 6
Figure 2. Brain densities of D1 and D2 receptors in control LFP and HFP chicks (35 days of
age)1= telencephalic pallium, 2=basal telencephalon, 3=diencephalon and 4=
mesencephalon (for details see Ko�t�ál et al., 1999)
4. Discussion
The purpose of the present study was to investigate whether HFP and LFP
birds differ in the sensitivity of the DA (receptor) system and whether differences in
D1 and D2 receptor densities in the brain are correlated with APO-induced
behavioural differences between the lines.
4.1 Behavioural effects of APO
APO treatment was effective in inducing a behavioural change in LFP and
HFP birds in an open field environment. The behaviour of the APO birds was
characterised by an increased locomotor activity, i.e. bouts of inactivity were
alternately followed by bouts of hyperactivity (i.e. running behaviour). This finding is
in agreement with other APO studies in chicks (Bilčík, 2000; Osuide and Adejoh,
1 2 3 4Brain region
0
10
20
30
40
50
60
spec
ific
bind
ing
(fmol
/mg
prot
ein)
LFP HFP D1
1 2 3 4Brain region
0
5
10
15
20
spec
ific
bind
ing
(fmol
/mg
prot
ein)
D2
Difference in APO sensitivity in HFP and LFP birds 113
1973) and rodents (Benus et al., 1991a; Surmann and Havemann-Reinecke,
1995). However, in our experiment no stereotypic pecking behaviour, like pecking
at the wall or pecking its own toes, was observed, as is often reported to occur with
APO treatment in chicks (Bilčík, 2000; Machlis, 1980; Osuide and Adejoh, 1973).
Possibly, the interaction between APO treatment and the novel environment, is
responsible for the absence of stereotypic behaviour in both lines. Harkin and his
colleagues (2000) showed that in mice, APO produced qualitatively different
responses under novel conditions when compared to those behaviours elicited in
the home cage. In their study APO-induced locomotion was more prominent in the
novel exploratory box than in the home cage. The absence of stereotypic pecking
may also be an age-related effect. Osuide and Adejoh (1973) showed that APO
induced more stereotypic pecking in very young chicks than in older birds.
The results of the present study clearly demonstrated a quantitative
difference in the behavioural response to acute APO treatment between HFP and
LFP birds in an open field. HFP chicks showed a higher increase in locomotor
activity in response to APO than birds of the LFP line. This is in agreement with the
previous findings that proactive rodents and proactive pigs show a higher
sensitivity to the effects of APO (Benus et al., 1991a; Bolhuis et al., 2000; Cools et
al., 1993) than their reactive counterparts. Thus, our data are consistent with the
concept of coping strategies, confirming our hypothesis that HFP birds display a
proactive and LFP birds a reactive coping strategy.
Recently (van Hierden et al., 2002a), we showed that HFP chicks display a
lower level of DA turnover compared to LFP birds. A less active DA system may be
accompanied by compensatory upregulation of DA receptors (Griffin and Ojeda,
1992). Therefore, we may have expected higher DA receptor densities in the HFP
line compared to the LFP line. However, the present study showed that the
different behavioural effects of APO in HFP and LFP birds, cannot be explained
from a difference in D1 and D2 receptor densities in both lines. This suggests that
other mechanisms account for the more enhanced behavioural effect of HFP
chicks to APO treatment compared to LFP chicks. Cools and his colleagues
(1990) also found that individuals with a functionally high activity of the DA system
show a weak response to APO and vice versa. They suggested that APO is poorly
114 Chapter 6
effective at high levels of fractional occupancy of the receptors by DA, but strongly
effective at low levels of fractional occupancy of the receptors by DA. It can be
speculated that, apart from a difference in receptor occupancy, differences in
functioning or sensitivity of intracellular signal transduction pathways may also
account for the more enhanced behavioural effect of HFP chicks to APO treatment
(Griffin and Ojeda, 1992). However, the exact mechanisms should be examined in
future studies.
4.3 Possible (causal) role of DA in feather pecking
Recently, we suggested that feather pecking might be a suitable animal
model for OCD (van Hierden et al., 2003a). Like the 5-HT system, dysfunction of
the DA system has been implicated in the pathophysiology of behavioural
disorders, like animal stereotypies (de Lanerolle, 1980; Nistico and Stephenson,
1979; Pitman, 1989; Schoenecker and Heller, 2001; Sharman, 1978) and
obsessive compulsive disorders (OCDs) (Jenkins, 2001; McDougle et al., 1994;
van Ameringen et al., 1999). Feather pecking has stereotypic as well as
compulsive characteristics. Once birds start feather pecking, they tend to do so
successively and it is almost impossible to discourage their feather pecking
behaviour. In two recent experiments we found evidence for a causal role of the 5-
HT system in the performance and development of feather pecking. An acute
decrease in 5-HT turnover in the forebrain increased feather pecking, while a
chronic increase in 5-HT turnover decreased feather pecking in HFP and LFP
chicks (van Hierden et al., 2003a; van Hierden et al., 2003b).
From the present study it can be postulated that sensitivity of the DA
system, is indicative for the development of feather pecking behaviour and that the
DA system may also play a (causal) role in the development and performance of
feather pecking behaviour. Bilčík (2000) found no evidence that differences in DA
sensitivity of young chicks (i.e. response to APO injection) can be used for
prediction of susceptibility to feather pecking. However, Kjaer et al. (2002) showed
that acute haloperidol treatment, significantly reduced feather pecking behaviour in
adult laying hens. Haloperidol, a drug with anti-OCD effects, is a D2 receptor
Difference in APO sensitivity in HFP and LFP birds 115
antagonist which, with acute administration, increases DA-turnover by blocking the
presynaptic DA autoreceptor (McElvain and Schenk, 1992). This suggests that low
DA neurotransmission might be involved in the performance of feather pecking
behaviour.
4.4 Summary and Conclusion
In the present study, a first step was taken in investigating a possible role
of the DA system in feather pecking behaviour. However, feather pecking
behaviour of the birds was not measured in this experiment. Therefore, in future
experiments direct effects of manipulation of the DA system on feather pecking
behaviour in HFP and LFP birds should be investigated, by applying DA
(ant)agonists like haloperidol. Furthermore, an extensive body of data supports the
existence of a functional interaction between central 5-HT and DA. For instance,
DA receptor stimulation, with receptor agonists, was found to increase 5-HT efflux
in several forebrain regions (Mendlin et al., 1998), suggesting a facilitatory DA
modulation of 5-HT neurotransmission. The interaction between the DA and the 5-
HT system, in relation to feather pecking behaviour should also be examined, in
pharmacological studies.
In summary, the present study demonstrated that HFP birds have a higher
sensitivity of the DA (receptor) system in the brain, compared to LFP birds, as
reflected by a more enhanced behavioural response to APO. This difference in
response cannot be explained from a difference in D1 and D2 receptor densities
between both lines. In the future, more pharmacological experiments, studying the
(interacting) role of the 5-HT, DA or other neurobiological systems, are necessary
to reveal the exact mechanisms underlying feather pecking behaviour.
116
C
hapt
er 6
Tabl
e 1.
Wal
d st
atis
tics
(W) a
nd P
-val
ues
(P) f
or o
pen
field
beh
avio
ur, p
er ti
me
perio
d (d
f =1)
. ns
= no
n-si
gnifi
cant
, if P
> 0
.05
Tim
eper
iod
Effe
ct
To
tal d
ista
nce
mov
ed
Mea
n ve
loci
ty
M
axim
um v
eloc
ity
Tota
l dur
atio
n of
m
ovin
g
1
L x
T
W =
4.2
0; P
= 0
.04
W
= 6
.55;
P =
0.0
1
W =
6.4
6; P
= 0
.01
ns
L
ns
ns
ns
ns
T
W =
8.1
0; P
= 0
.004
W
= 1
6.30
; P <
0.0
01
W =
59.
25; P
< 0
.001
W
= 1
5.28
; P <
0.0
01
2
L x
T
W =
7.3
7; P
= 0
.007
W
= 1
2.91
; P <
0.0
01
W =
6.7
2; P
= 0
.01
ns
L
ns
ns
ns
ns
T
ns
W =
6.0
5; P
= 0
.01
W
= 1
7.88
; P <
0.0
01
W =
18.
82; P
< 0
.001
3
L
x T
W
= 3
.61;
P =
0.0
5
W =
5.1
5; P
= 0
.02
ns
ns
L
ns
ns
ns
ns
T
W =
15.
45; P
< 0
.001
W
= 1
9.68
; P <
0.0
01
W =
16.
79; P
< 0
.001
ns
4
L
x T
ns
ns
ns
ns
L
ns
ns
ns
ns
T
W =
33.
67; P
< 0
.001
W
= 3
4.49
; P <
0.0
01
W =
21.
32; P
< 0
.001
W
= 6
.95;
P =
0.0
08
5
L x
T
ns
ns
ns
ns
L
ns
ns
ns
ns
T
W =
61.
81; P
< 0
.001
W
= 6
1.11
; P <
0.0
01
W =
36.
24; P
< 0
.001
W
= 1
1.50
; P <
0.0
01
6
L
x T
ns
ns
ns
ns
L
ns
ns
ns
ns
T
W =
38.
32 ;
P <
0.0
01
W =
37.
63; P
< 0
.001
W
= 4
.01;
P <
0.0
01
W =
8.3
2; P
< 0
.001
Chapter 7
General Discussion
118 Chapter 7
1. Summary of the results
The aim of this thesis was to identify individual behavioural and
neuroendocrine characteristics of laying hens in relation to feather pecking
behaviour. The starting-point of this thesis were the results of previous studies with
adult birds from a high (HFP) and low feather pecking (LFP) line of laying hens
(Blokhuis and Beutler, 1992; Blokhuis and Beuving, 1993; Johnsen and
Vestergaard, 1996; Jones et al., 1995; McAdie and Keeling, 2002). At an adult age,
HFP and LFP birds, exhibited differences in behavioural and physiological
characteristics related to the way birds of both lines cope with stressors (Korte et
al., 1997; Korte et al., 1999). Adult HFP birds and LFP birds were found to display
characteristics of the proactive and reactive coping strategy, respectively (Korte et
al., 1997; Korte et al., 1999). Thus, Korte and his colleagues (1997;1999)
postulated that the concept of coping strategy may represent a useful framework
to unravel the causation of feather pecking behaviour. Here, we also propose that
the application of the concept of coping strategy may substantiate the assumption
that feather pecking is a behavioural pathology, using a neurobiological approach.
These propositions provided the empirical basis for the experiments presented in
this thesis.
Adult HFP and LFP birds were previously found to display contrasting
levels of feather pecking and other behaviour (Blokhuis et al., 2001). However, no
data were available on the developmental stage at which the behaviour of these
lines starts diverging. Therefore, the first experiment of this thesis involved studying
the development of feather pecking and other behaviours in HFP and LFP chicks
during the first 8 weeks of life (chapter 2). Already at the age of 14 and 28 days
HFP chicks displayed higher levels of gentle feather pecking behaviour than LFP
chicks. Furthermore, at several ages, LFP chicks showed higher durations of
foraging and feeding behaviour, whereas HFP chicks spent more time preening. A
principal component analysis (PCA) was performed on the data (see box 7.1).
From the assumption that a principal component with a high loading for gentle
feather pecking reflects an underlying factor related to the propensity to engage in
(gentle) feather pecking, we suggested that the motivational system controlling the
General Discussion 119
performance of (gentle) feather pecking may differ between both lines. In the HFP
line, gentle feather pecking and preening exhibited high and opposite loadings on
the same component at all ages (i.e. were highly negatively correlated), whereas
feeding consistently loaded on the other component. Conversely, in the LFP line
feeding predominantly loaded on the same principal component as gentle feather
pecking, with loadings opposite to those of gentle feather pecking, whereas
preening showed the same loadings as gentle feather pecking, on days 3 and 41.
Chapter 3 describes a study investigating physiological and
neuroendocrine characteristics of young HFP and LFP chicks. Firstly, the
development of the adrenocortical (re)activity during the first 8 weeks of life was
studied. Secondly, we studied the levels of serotonin (5-HT) and dopamine (DA)
turnover in the brain of HFP and LFP chicks on 28 days of age. In these
experiments, levels of feather pecking behaviour were not measured. We used a
manual restraint test (i.e. placing a chick on its side for 5 minutes) as an
environmental challenge to study coping characteristics in HFP and LFP birds.
Plasma corticosterone levels were lower (baseline on days 3 and 56; restraint-
induced on day 3, 14, and 28) in HFP chicks compared to LFP chicks. These
findings agreed with previous findings (Korte et al., 1997) and strengthened the
idea that HFP and LFP birds resemble proactive and reactive copers, respectively.
Both brain 5-HT and DA turnover levels were lower in the HFP chicks compared to
LFP chicks, as well. Differences in the functioning of both the 5-HT and DA system
have also been implicated in the distinction between the proactive and reactive
coping strategy in rodents (Benus et al., 1991a; Korte et al., 1996; van der Vegt et
al., 2001) and pigs (Bolhuis et al., 2000). Furthermore, both systems, but
particularly the 5-HT system, have been found to play a pivotal role in the aetiology
of several behavioural disorders, such as animal stereotypies (Goodman et al.,
1983; Ko�t�ál and Savory, 1995; Nistico and Stephenson, 1979; Pitman, 1989;
Schoenecker and Heller, 2001) and obsessive compulsive disorders (OCD) (Blier
and de Montigny, 1998; Luescher, 1998; Pigott, 1996; Stein, 2000). This
information lead to our postulation that both neurobiological systems might play a
(causal) role in the development and performance of feather pecking.
120 Chapter 7
In chapter 4, a first study (consisting of two subexperiments) is described
aimed at investigating the causal role of the 5-HT system in the development and
performance of feather pecking behaviour. From chapter 3 we hypothesised that
low 5-HT turnover is causally related to the performance of feather pecking
behaviour. To test this hypothesis we used the pharmacological agent S-15535 (a
5-HT1A somatodendritic autoreceptor agonist). In rodents, S-15535 reduces 5-HT
synthesis and release in the forebrain (de Boer et al., 2000; Millan et al., 1997). In
a dose-response study (experiment 1), we found that also in (LFP and HFP)
chicks, S-15535 dose-depently reduces 5-HT turnover in the postsynaptic areas.
The most effective dose without affecting DA turnover was 4.0 mg S-15535/kg BW.
In experiment 2, we applied this dose to investigate its effect on feather pecking
behaviour in both lines. S-15535 significantly increased severe feather pecking and
tended to increase gentle feather pecking, confirming our hypothesis. The effect of
S-15535 treatment appeared more pronounced in the HFP line.
The findings in chapter 4 led to the postulation made in Chapter 5 that
increasing 5-HT neurotransmission in the brain would decrease feather pecking
behaviour. Increasing 5-HT neurotransmission in the brain can be achieved by
increasing the level of aminoacid L-Tryptophan (TRP), the precursor of 5-HT, in the
diet. Increasing the level of dietary TRP (from 1.6 to 21.0 gram/kg) in the feed for
14 days of LFP and HFP chicks resulted in a significant increase in 5-HT turnover
in the forebrain of the birds, and in a significant decrease of gentle feather pecking.
As with S-15535, the effect of manipulation of the 5-HT system with TRP
on feather pecking appeared to be more pronounced in the HFP line, suggesting a
difference in the sensitivity of the 5-HT receptor system in this line. This would
confirm previous studies in rodents, where a more sensitive 5-HT receptor system
in proactive animals has been found (van der Vegt et al., 2001). Apart from a
difference in the sensitivity of the 5-HT receptor system, proactive and reactive
individuals have been reported to differ in the sensitivity of the DA receptor system
(Benus et al., 1991a; Bolhuis et al., 2000). Enhanced sensitivities of 5-HT and DA
receptor systems have been suggested to underlie a higher vulnerability of
proactive animals for the development of behavioural abnormalities like
stereotypies (Koolhaas et al., 1999). Extrapolation of this idea to the present
General Discussion 121
findings in poultry would imply that HFP and LFP birds may also differ in the
sensitivity of the DA receptor system.
Therefore, in chapter 6 we reported of a study in which we investigated the
sensitivity of the DA receptor system in HFP and LFP chicks. HFP and LFP chicks
were injected with either distilled water, or the DA receptor agonist apomorphine
(APO). The behavioural response of the chicks to acute APO treatment was tested
in an open field. APO treatment induced a significantly greater enhancement of
locomotor activity (i.e. total distance moved, mean and maximum velocity) in the
HFP line compared to the LFP line. The differences in the behavioural response to
APO between the lines, could not be explained by differences in densities of the
DA receptors (D1 and D2) in the brain. Thus, in agreement with our hypothesis, this
study clearly indicated that birds from the HFP line have a higher sensitivity of the
DA receptor system than birds from the LFP line.
2. Coping strategy: a guideline for studying characteristics of LFP and HFP birds
2.1 Behavioural characteristics
Korte and his colleagues showed that the (stress-induced) behavioural and
physiological characteristics of adult HFP birds and LFP birds show great analogy
to the characteristics of the proactive and reactive coping strategy, respectively
(Korte et al., 1997; Korte et al., 1999). Hence, throughout this thesis the concept of
coping strategy has been a guideline in studying and understanding the behaviour
and neuroendocrinology of HFP and LFP birds.
A fundamental difference between proactive and reactive rodents is the
extent to which their behaviour is influenced by environmental cues. The behaviour
of proactive copers is more intrinsically driven (i.e. internally motivated) in
comparison with that of reactive copers. As a result proactive copers more easily
develop routine-like behavioural patterns, which are rigid and largely independent
122 Chapter 7
of actual external (inanimate) stimuli (Benus et al., 1990; Benus et al., 1987;
Verbeek et al., 1994). Reactive copers, on the other hand, are more flexible in their
behaviour. They are more sensitive to environmental stimuli and show a more
externally driven and directed behavioural orientation (Koolhaas et al., 2001).
In chapter 2, the (innate) difference in the targeting of pecking behaviour in
general and the difference in the level of feather pecking between HFP and LFP
chicks, on a group level, was explained from this difference in cue dependency (or
behavioural flexibility) between proactive and reactive individuals. HFP chicks
displayed higher levels of preening behaviour, whereas LFP birds displayed higher
levels of feeding and foraging behaviour. Preening in birds has been considered a
rather routine-like behaviour. The timing of preening is not critically dependent on
specific environmental events and preening behaviour is less triggered by external
cues (i.e. inanimate stimuli), than for instance feeding or foraging (Delius, 1988).
Feather pecking, because of its repetitive structure, may also be considered a
rather routine-like behaviour (Korte et al., 1997). Furthermore, it is well known that
once feather pecking has developed in a flock, it is hardly influenced by changing
environmental conditions. Hence, on a group level, the routine-like character of
feather pecking might explain the higher levels of (gentle) feather pecking in the
(proactive) HFP line compared to the (reactive) LFP line.
In chapter 2, the divergence in intrinsic (or extrinsic) regulation of
behaviour between HFP and LFP birds was also reflected at the individual level,
by consistent and differential loading patterns of behaviours of HFP and LFP
chicks, obtained in a multivariate principal component analyses (PCA, see box
7.1). Gentle feather pecking consistently exhibited high and opposite loadings on
the same component as preening and feeding in the HFP and LFP line,
respectively. This means that a high motivation for performing gentle feather
pecking was consistently associated with a low motivation to preen and a low
motivation to feed in the HFP and LFP line, respectively. Retrieval of principal
components with high (but opposite) loadings either for gentle feather pecking and
preening, or for gentle feather pecking and feeding suggests that both gentle
feather pecking and either preening (in HFP birds) or feeding (in LFP birds) share a
common underlying factor related to the propensity to engage in these behaviours.
General Discussion 123
It can be suggested that this common underlying factor represents the motivational
system controlling both behaviours. Previously, feather pecking has been
considered a form of redirected behaviour, related to the motivational system of
either feeding/foraging (Aerni et al., 2000; Blokhuis, 1989) or dustbathing
(Vestergaard, 1994). Here, we suggest that the type of motivational system
involved in the regulation of feather pecking depends on underlying characteristics
of the individual bird. In the LFP line, in accordance with the notion by Blokhuis
(1989) (Blokhuis, 1989) and Aerni (Aerni et al., 2000), gentle feather pecking may
be redirected feeding behaviour. In the HFP line, however, gentle feather pecking
may be considered a form of redirected preening. In chapter 2, severe feather
pecking was not included in a PCA since levels were too low during the first 8
weeks of development to allow reliable statistical analysis. In chapter 5,
presumably as a result of the removal of the litter and the higher age of the birds,
levels of both gentle and severe feather pecking were relatively high in the control
HFP and LFP birds, compared to those reported in chapter 2. It would be of
interest to know whether under these conditions the motivation underlying severe
feather pecking in both lines would be the same as gentle feather pecking
observed in chapter 2.
Figure 7.1 shows the results of a PCA of the behavioural data of the control
LFP and HFP birds of the tryptophan experiment described in chapter 5. In the
HFP line both severe and gentle feather pecking exhibit high and opposite loadings
on the same component as preening. In the LFP line both severe and gentle
feather pecking exhibit high and opposite loadings on the same component as
feeding. The biplots in figure 7.1 resemble the biplots in chapter 2. Furthermore,
the biplots show that gentle and severe feather pecking are highly positively
correlated. A principal component with high but equal loadings for gentle and
severe feather pecking also points to a common underlying motivation for these
behaviours. This strongly suggests that, within one age, the same motivational
system is underlying both gentle and severe feather pecking and that both
behaviours may reinforce each other. This suggestion is in agreement with
behavioural observations (data not shown) that bouts of severe pecks are mostly
124 Chapter 7
Figure 1. Distribution of behavioural parameters in relation to the first two components of a
Principal Component Analysis achieved from behavioural observations of LFP and HFP
chicks on 49 days of age (chapter 5). See also box 7.1.
-0.80 0.00 0.80
-0.80
0.00
0.80LFP
REST
FEEDWALK
GFP
FORAG
PREEN
SFP
-0.80 0.00 0.80
-0.80
0.00
0.80HFP
REST
FEED
WALK
GFP
FORAG
PREEN
SFP
General Discussion 125
imbedded in bouts of gentle feather pecks (Riedstra and Groothuis, 2002; van
Hierden et al., 2002b).
The consistency of loading patterns obtained with PCA across experiments
suggests that the motivational systems underlying the performance of feather
pecking in the HFP and LFP lines are trait characteristics, demonstrable under
different (environmental) conditions. The consistent finding of preening versus
feather pecking in the HFP line, and feeding versus feather pecking in the LFP line,
suggests that the development and/or performance of feather pecking may be less
extrinsically controlled in the HFP line than in the LFP line. The differential loading
patterns obtained with PCA in the present thesis support the existence of
differences between HFP and LFP birds in the extent to which their behaviour is
extrinsically (or intrinsically) controlled, and agree with an interpretation of
behavioural differences between both lines in terms of coping strategy. Thus, the
individual coping strategy of chicken might be an important determinant of the
motivational system underlying feather pecking.
BOX 7.1 Principal Component Analysis (PCA)
PCA was used to analyse and objectively summarise relationships between multiple variables (Jolliffe,
1986). Variables were scaled prior to PCA, i.e. PCA was performed on the Pearson correlation matrix.
Principal components produced by PCA are linear combinations of the original variables, and represent
condensed new variables reflecting independent characteristics underlying the correlation matrix. The
first component explains most of the variance (expressed in terms of first eigenvalue), the second
component explains most of the remaining variation, and so forth. The coefficients of the scaled
variables, the so-called loadings, indicate the importance of each of the original variables for the
principal components.Figure 7.1 shows the distribution of the variables included in the PCA in relation to
the first two principal components (with eigenvalues larger than 1). A biplots is a two-dimensional
visualisation of the correlation structure underlying the variables. Each of the components is a linear
combination of the variables gentle feather pecking (GFP), severe feather pecking (SFP), foraging
(FORAG), feeding (FEED), preening (PREEN), walking (WALK) and resting (REST). Variables loading
on the same component (X or Y axis), and placed opposite from each other are (highly) negatively
correlated. Variables loading on the same component and placed close to each other are (highly)
positively correlated.
126 Chapter 7
2.2 Physiological characteristics
The differences in HPA axis reactivity, 5-HT and DA neurotransmission in
young chicks of both lines (chapter 3) confirmed, at the neuroendocrine level, that
HFP birds display a proactive, and LFP birds display a reactive coping strategy
(Korte et al. 1997; 1999). Furthermore, in accordance with previous findings in
proactive and reactive rodents (Benus et al., 1991a; Korte et al., 1996; van der
Vegt et al., 2001) and pigs (Bolhuis et al., 2000), a more pronounced behavioural
response to manipulation of the 5-HT and DA (receptor) system was found in the
HFP line compared to the LFP line (chapter 4 and 5).
The neuroendocrine characteristics of HFP and LFP chicks support the
behavioural findings, in the assumption that feather pecking is associated with
fundamental traits of proactive and reactive copers, respectively (see box 7.2).
However, the relationship between putative neuroendocrine indicators of coping
strategy and the development of feather pecking, as presented in the current
thesis, is only of a correlational nature. So far, it remained an open question to
what extent the physiological differences between the HFP and the LFP line might
causally explain the difference in feather pecking as well.
3. Causal role of 5-HT in feather pecking
Application of the concept of coping strategy has helped us to reveal and
interpret behavioural and neuroendocrine characteristics of laying hens, differing in
the level of feather pecking. It led to hypotheses on the causality of these
characteristics in the development and performance of feather pecking (chapter 2
and 3).
Hence, it has brought us to investigate neuroendocrine systems (i.e. 5-HT
and DA) involved in the differentiation of the two lines in more detail. In this thesis,
emphasis was placed on the 5-HT system, as its significance in the aetiology of
several behavioural disorders in human and animal species has been clearly
identified. The difference in serotonergic tone (in response to a stressor) found
General Discussion 127
between LFP and HFP birds (chapter 3) suggested a role of 5-HT in the
development and performance of feather pecking. Chapter 4 and 5 revealed that,
at a group level, a negative relationship between the level of 5-HT
neurotransmission and feather pecking exists. Especially, the HFP line appeared
sensitive to manipulation of 5-HT neurotransmission, as a decrease (chapter 4) in
5-HT turnover levels resulted in more a pronounced increase of feather pecking,
and an increase (chapter 5) in 5-HT turnover levels resulted in a more pronounced
decrease of feather pecking behaviour in this line.
In chapter 5, both feather pecking and stress-induced 5-HT turnover levels
of individual LFP and HFP birds were measured. Figure 7.2 shows the relationship
between both measures. The figure clearly shows a negative correlation (rs = -
0.67; P < 0.001) between (the sum of gentle and severe) feather pecking and
stress-induced 5-HT levels both within and between the selection lines. It is of
interest to see that the few LFP animals that showed high levels of feather pecking
are characterised by a relatively low 5-HT turnover. This provides further support
for the idea that in both the LFP and HFP lines 5-HT neurotransmission plays a
causal role in feather pecking behaviour, not only on a group level but also on the
level of individual birds.
An important question to be raised is how the animal-related and
environmental factors (for an overview see chapter 1) known to affect feather
pecking, relate to the effects of low 5-HT neurotransmission on feather pecking. Or,
in other words, which factors trigger the �lowering� of 5-HT, resulting in the
development or performance of feather pecking, and what are the exact
mechanisms. So far, no scientific data in chickens can provide answers to this
question. However, research in the area of neuroscience and psychiatry has
provided a large body of scientific knowledge on the causation of
psychopathologies in humans, which might be of use in understanding
mechanisms underlying behavioural pathology in chicken
128 Chapter 7
BOX 7.2 Some physiological and neuroendocrine characteristics of the HFP and LFP line
Cort = corticosterone, NA = Noradrenaline, A = Adrenaline, 5-HT = serotonin, DA = Dopamine
HFP LFP
HPA-axis (re)activity (Cort)1,3 Low High
Neurosympathetic reactivity (NA)1 High Low
Parasympathetic reactivity2 Low High
5-HT1A receptor sensitivity4 High Low
Striatal DA receptor sensitivity5 High Low
Testosterone in eggs6 High Low
References
1.Korte et al. (1997)
2. Korte et al. (1999)
3. Chapter 3
4. Chapter 4
5. Chapter 6
6. Riedstra (2003)
General Discussion 129
Figure 2. Relationship between 5-HT turnover in the chicken brain and the frequency of total
feather pecking. Data were obtained in the experiment on the effect of dietary tryptophan on
5-HT turnover and feather pecking in HFP and LFP birds (Chapter 5). Average 5-HT
turnover levels were analysed with a generalized linear model (GLM) with a logarithmic link
function and variance proportional to the square of the mean. Inference was based on
maximum quasi-likelihood. Calculations were performed with GLM facilities in GenStat
(2002) specifying a gamma distribution. The model comprised the logarithm of the frequency
of total feather pecking (log(TFP)) as an explanatory variable in addition to main effects for
factors for dietary tryptophan (standard versus high) and lines (HFP versus LFP).
Significance tests showed that the coefficient of log(TFP) did not significantly depend on the
factor Diet and Line (P > 0.10), therefore a common coefficient b was assumed. The
estimated value for b was -0.16 (S.E = 0.03) and differed significantly (P < 0.001) from 0.
Hence, it could be concluded that 5-HT turnover was significantly and negatively associated
with the frequency of total feather pecking. The Spearman rank correlation between both
parameters was - 0.67 (P < 0.001).
0 1 2 3 4 5 6 7Frequency of total feather pecking per 30 min (Log [freq+1])
-2.40
-2.00
-1.60
-1.20
-0.805-
HT
turn
over
(Log
[mea
n 5-
HT]
)LFP HFP
130 Chapter 7
4. Feather pecking as a behavioural pathology
In this thesis a novel approach for the investigation of feather pecking
behaviour was introduced. We considered feather pecking a behavioural
pathology, and assumed that its aetiology is comparable to behavioural
pathologies found in humans and other species. However, defining feather pecking
as a behavioural pathology would only be valid if several criteria have been met.
The criteria for the most common behavioural pathologies are published in
�Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition (DSM-IV)
(First, M.B. (Editor), 1998). These criteria indicate that, for instance, characteristics
of the behaviour itself should be considered.
The most striking characteristic of feather pecking is its repetitive structure,
i.e. the stereotypic pecking at feathers and/or compulsive pulling of feathers. In
humans, a psychiatric disorder exists called �trichotillomania�, meaning compulsive
hair-pulling syndrome. According to the criteria for the most common mental
disorders, as published in �Diagnostic and Statistical Manual of Mental Disorders -
Fourth Edition (DSM-IV) (First, M.B. (Ed), 1998), trichotillomania is an OCD-like
disease classified as an �Impulse-Control Disorder�. An important criterion is the
inability to resist an impulse or psychological drive to act in a way harmful to
oneself (or others). The DSM IV criteria of Obsessive Compulsive Disorder (OCD)
further indicate that either obsessions or compulsions should be demonstrable,
which are severe enough to be time consuming or cause marked distress or
significant impairment. Obsessions are defined as recurrent and persistent ideas,
thoughts or impulses, that are experienced as intrusive and inappropriate and that
cause marked anxiety or distress. Compulsions are repetitive behaviours, aimed at
preventing or reducing anxiety or distress. By definition, compulsions are either
clearly excessive or are not connected in a realistic way with what they are
designed to neutralise or prevent (1998). We may conclude that feather pecking in
laying hens shares many behavioural characteristics of such a compulsive
disorder.
In neuroscientific research on behavioural pathologies in humans, the
involvement of the 5-HT system has been widely documented. The pathology of
General Discussion 131
behavioural pathologies appears attributable to aberrant 5-HT (and DA)
metabolism (Blier and de Montigny, 1998; Pigott, 1996; Stein, 2000). Augmenting
5-HT (and DA) neurotransmission through the use of tricyclic antidepressant or
serotonine selective reuptake inhibitors (SSRIs), has also potent anti-OCD effects.
For instance, the SSRI fluoxetine (Prozac), which increases 5-HT but also DA
availability, is successful in treating trichotillomania and other forms of OCD
(Luescher, 1998; Stein, 2000; van Ameringen et al., 1999). The finding (as
discussed earlier) that the 5-HT system is causally related to feather pecking, (at
least partly) justifies our assumption that feather pecking is a behavioural
pathology. The repetitive structure of feather pecking and the way it responds to
�pharmacological� treatment (chapter 4 and 5) suggests that feather pecking
resembles a form of OCD.
The exact causal factors or mechanisms behind the development of OCDs
in relation to 5-HT are still highly speculative (Franzblau et al., 1995; Joiner and
Sachs-Ericsson, 2001) and at this moment do not provide answers to the feather
pecking problem. However, the large degree of analogy on a behavioural and
neuroendocrine level between feather pecking and OCDs, does raise the question
whether feather pecking might be an appropriate animal model for studying OCDs,
like trichotillomania.
Several authors claimed to have found an animal model for OCD (Geyer
and Markou, 2000; Marcotte et al., 2001; Nurnberg et al., 1997; Pitman, 1989;
Sarter and Bruno, 2002; Sutanto and de Kloet, 1994). Bordnick and his colleagues
(1994) compared trichotillomania, to feather picking disorder in birds. Feather
picking in psittacine birds (parrots and parakeets), involves the pecking and
plucking of its own feathers (Iglauer and Rasim, 1993; Jenkins, 2001; Levine,
1984). With respect to analogous behaviour, proposed aetiologies, evoking cues,
response to behaviour therapy, and response to pharmacological treatments based
on serotonin-reuptake inhibitors, Bordnick suggested that feather picking disorder
has the potential to be a useful animal model of trichotillomania. Interestingly, it has
been recently suggested that the underlying aetiology of feather picking strongly
resembles feather pecking in laying hens (Meehan et al., 2003; Mench and
Keeling, 2001). Furthermore, HFP birds have been found to be more vulnerable for
132 Chapter 7
developing feather picking, compared to LFP birds (Blokhuis et al., 1993), thus
suggesting that feather pecking may be suitable for investigating an OCD like
trichotillomania.
However, feather pecking as animal model for OCD should meet the
validation criteria of animal models for human behavioural pathologies. Validation
criteria are general standards that are relevant to the evaluation of any model. The
basis of any animal model of a human disorder, is the assumption that there is
homology, or at least analogy, among the physiological and behavioural
characteristics of various species. Hence, extrapolations can be made from
animals to humans. The validity of a model refers to the extent to which a model is
useful for a given purpose. In neurobiological research, the purpose of an animal
model is the elucidation of the mechanisms underlying the human condition
(Overall, 2000). There is continuing scientific debate over the proper evaluation of
animal models (Geyer and Markou, 2000; Overall, 2000; Sarter and Bruno, 2002).
Authors do not seem to agree on the significance of the different aspects of validity
of a model, i.e., face validity, predictive validity and construct validity.
Currently, animal models are required to possess the following 3 types of validity
(Overall, 2000):
• Face validity, where the model is phenotypically similar and acts as a good
model of specific symptoms
• Predictive validity, where the model shows the same effect for drugs used in
treatment or induction of provocative states
• Construct validity, where the model either relies on or elucidates the same
basic underlying mechanism responsible for the condition in humans.
From the above, it can be argued that feather pecking as an animal model for
OCD may already have face validity and predictive validity. According to Geyer and
Markou (2000) demonstration of construct validity may be unnecessary, if an
animal model exhibits good face validity and solid predictive validity (e.g. by
predicting efficacy of therapeutic agents). However, Sarter and Bruno (2002)
argue that a model should have construct validity as well. True construct validity
General Discussion 133
implies that the �cause� of the behavioural response in the animal is sufficient to
provoke the same response in humans (Overall, 2000). Further investigation into
feather pecking as an animal model for OCD could lead to construct validity.
However, this may not be an easy task since it is more easy to study the complex
relationships between neurobiological and behavioural variables (the core focus of
construct validation) in chickens, than in humans. Especially, neurobiological
mechanisms, resulting from the animal model, may not be easily amenable to
study in humans.
5. Implications for future poultry research and - practice
5.1 Implications for genetic selection
During the past five decades, breeders of commercial strains of laying
hens, have stringently selected on traits that enhance the efficiency of egg
production. Selection emphasis changed periodically for different traits, some of
which included the size of eggs, number of egg produced, or age at first egg
(Jones et al., 2001). For many years primary breeders have been selecting for
early sexual maturity in commercial layers. Over time, age at first egg has steadily
decreased, whereas egg weight has increased (McMillan et al., 1990).
Potential dangers of overselection for a single performance trait, such as
alterations in health and behaviour, have been documented for a wide range of
species (see for an overview Rauw et al., 1998) and (Jones and Hocking, 1999)).
For laying hens, it was found that (individual) selection for early sexual maturity and
egg production was related to proneness to osteoporosis and increased
aggression (Craig et al., 1975). Kjaer (1999) suggested that selection for
production traits including enhanced feed conversion (resulting in smaller body
size) which has taken place in the commercial breeding programs, may have
contributed to the feather pecking problems in laying hens.
In a comparative study of different breeds of poultry, Schütz and Jensen
(2001) found that White leghorns selected for high feed efficiency were less active
134 Chapter 7
in fear tests, spent less time foraging or exploring the environment, and were less
involved in social interactions than the unselected red junglefowl or bantams.
These behavioural differences between breeds were interpreted as correlated
responses to selection for increased production. Subsequent molecular genetic
studies, employing specified pedigree of White leghorn and red jungle fowl parental
strains, provided support for this idea by revealing quantitative trait loci (QTL)
affecting both production-related variables and behavioural characteristics,
indicating linkage at the level of the genome (Schütz et al., 2002).
The HFP and LFP lines originate from different breeding lines, and are a
coincidental result of a commercial selection program. This selection program
included criteria such as number of eggs laid, eggshell quality, and mortality. The
LFP line was selected for these criteria starting in 1980 (for 15 generations) and
the HFP line starting in 1987 (for 8 generations); selection ended in 1995. In
comparison with LFP birds, HFP birds are characterised by e.g. lower age of first
egg, and higher egg weight (unpublished results; Riedstra, personal
communication). Furthermore, HFP birds have a higher egg production than their
LFP counterparts (personal communication Hendrix-Poultry).
From the above, it can be postulated that the differences in behavioural
and neuroendocrine characteristics are related to the differences in (selection on)
production traits between the lines. We may hypothesise that a differential
selection on production traits between the lines, via unintentional coselection on
neurobiological traits, has created differences in neuroendocrine states between
HFP and LFP birds, underlying differences in coping strategy and in feather
pecking. The notion that genetically unfavourable relationships may exist between
production traits and neurobiological characteristics in poultry may provide an
explanation for the fact that behavioural problems such as feather pecking, have
not been adequately solved to far, despite comprehensive efforts in terms of
adjusting housing and management of laying hens. We speculate that through
(over)selection on production traits, an �unbalanced� bird may be created which,
due to its neuroendocrine state, is not very adaptive to environmental changes and
hence very vulnerable for the development of feather pecking.
General Discussion 135
Furthermore, we propose that birds should be selected in the housing
system for which they are bred. Equally important to the traits of selection is the
environment of selection. Most commercial breeders use individual housing of hens
(Hunton, 1990). However, this selection procedure ignores the genotype x
environment interaction. For instance, cage-adapted hens may be able to produce
many eggs, also in alternative housing systems, but just few environmental
disturbances may result in outbreaks of feather pecking and cannibalism.
In conclusion, we suggest that future selection programs should not be
solely based on production traits, but should include a set of criteria related to the
(psycho)neuroendocrine state of birds. Results from this thesis may be a first step
in the development of new criteria or traits that can be used in breeding
programmes aimed at a more balanced genetic selection. However, more scientific
research is needed on the interactions between selection on production traits, the
neuroendocrine states of laying hens, and injurious behaviours including feather
pecking.
5.2 Implications for bird management
Apart from having implications for genetic selection, results reported in this
thesis may also provide clues for management of laying hens, in relation to feather
pecking behaviour. The neurobiological and neuroendocrine state (i.e. 5-HT
turnover levels) of a bird has not only been found to differ between the selection
lines (chapter 2), but has also been found to depend on management factors, such
as diet (chapter 6). Future research should focus on revealing the implications of
interactions between management factors (for instance light intensity, rearing
conditions, availability of suitable litter) and neurobiological and neuroendocrine
states for the development of feather pecking. This information should be
incorporated in future designs of housing systems for laying hens.
Finally, this thesis has provided information on coping strategies and
related personality and neuroendocrine traits of high and low feather pecking birds.
The question is how this kind of information on animal characteristics can help in
136 Chapter 7
solving the feather pecking problem? At this stage, only a comparison has been
drawn between birds of the HFP and LFP line. It should however be investigated
whether birds of other commercial lines of laying hens, differing in their propensity
to feather peck, also display similar behavioural and/or neurobiological features of
the proactive and reactive coping strategies. It should also be investigated whether
individual variation in feather pecking within lines is associated with individual
differences in coping characteristics. If future research confirms the suggested
(causal) relationship between feather pecking and coping strategy, then
behavioural and neurobiological knowledge (such as presented in the current
thesis) could be used for predicting the vulnerability of certain individuals or genetic
strains for the development of feather pecking under certain environmental
conditions. Finding the right match between management (e.g. housing) conditions
and characteristics (e.g. personality traits) of individual birds might be a useful
strategy in the introduction of free-housing systems and improving animal welfare.
For instance, following the concept of coping strategy, it would be predicted that
proactive (supposedly high feather pecking) birds would benefit from a stable,
highly predictable environment, whereas reactive (low feather pecking) birds would
also be able to adapt well to variable and unpredictable environmental conditions.
Samenvatting
138 Samenvatting
Achtergrond van het onderzoek
Verenpikken bij leghennen vormt een groot welzijnsprobleem in de huidige
(commerciële) leghennenhouderij. Hennen pikken en trekken aan elkaars veren,
en veroorzaken daarbij schade aan het verenpak en verwondingen aan de huid.
Gewonde kippen kunnen vervolgens het slachtoffer worden van kannibalisme.
Wetenschappelijk onderzoek naar verenpikken heeft in de afgelopen 25 jaar veel
informatie opgeleverd over mogelijke oorzaken van dit probleem. Het blijkt echter
dat niet één enkele factor, maar meerdere factoren een rol spelen bij het ontstaan
van verenpikken. Tot nu toe zijn oplossingen voor verenpikken voornamelijk
gezocht in het veranderen van de huisvesting en het management van groepen
dieren. Hiermee is echter nog geen definitieve oplossing van het verenpikprobleem
bereikt.
Uit onderzoek is ook gebleken dat er grote verschillen kunnen bestaan
tussen verschillende rassen en lijnen leghennen, in de mate waarin zij verenpikken
vertonen. Genetische aanleg voor verenpikken speelt dus ook een rol. Daarnaast
is bekend dat er ook tussen hennen (van eenzelfde ras of lijn), die onder dezelfde
omstandigheden worden gehouden, grote individuele verschillen kunnen bestaan
in de mate van verenpikken. Verenpikken blijkt dus te ontstaan uit een tot nu toe
onbegrepen samenspel van genetische aanleg en omgevingsfactoren.
Doel en uitgangspunten van dit proefschrift
In dit proefschrift is onderzocht hoe individuele verschillen in de mate van
verenpikken zouden kunnen worden veroorzaakt. Doel van het onderzoek was te
bestuderen welke eigenschappen (lees �verschillen in gedrag, fysiologie en
neurobiologie�) van leghennen, het niveau van verenpikken dat zij vertonen
Samenvatting 139
bepalen. Er is in dit proefschrift gekozen om verenpikken te benaderen als een
gedrags- of psychopathologie, zoals die bij mensen, maar ook andere diersoorten
voorkomen. Gekeken is naar die fysiologische en neurobiologische systemen van
leghennen, die (o.a.) bij mensen in verband worden gebracht met het voorkomen
van gedragsafwijkingen, zoals bijvoorbeeld obsessief compulsief gedrag (OCD).
Het niet goed functioneren van deze systemen zou wellicht ook verband kunnen
houden met het ontstaan of uitvoeren van verenpikken.
Aan de basis van dit proefschrift ligt onderzoek aan twee lijnen leghennen
die genetisch verschillen in de mate van verenpikken (Blokhuis and Beutler, 1992;
Blokhuis and Beuving, 1993; Johnsen and Vestergaard, 1996; Jones et al., 1995;
McAdie and Keeling, 2002). Deze hoog (HFP) en laag (LFP) verenpiklijnen,
verschillen niet alleen in de mate van verenpikken, maar ook in verschillende
gedrags- en fysiologische karakteristieken, die verband houden met de wijze
waarop deze dieren omgaan met veranderingen of stressoren in hun omgeving
(Korte et al., 1997; Korte et al., 1999). Op volwassen leeftijd vertonen HFP en LFP
hennen kenmerken van de zogenaamde proactieve en reactieve copingstrategie,
zoals bekend bij ratten en muizen (Koolhaas, 1997; 1999). Dit concept van
copingstrategieën is gebruikt bij het ontrafelen van de verschillen in verenpikken
tussen lijnen (/rassen) en individuen.
De resultaten
Zoals al aangegeven is bekend dat volwassen HFP en LFP hennen
verschillen in de mate van verenpikken. Er is echter nog niet bekend op welke
leeftijd de lijnen het verschil in verenpikken gaan vertonen. Daarom is in het eerste
experiment in dit proefschrift (hoofdstuk 2), de ontwikkeling van verenpikken en
ander gedrag in de HFP en LFP lijn, gedurende de eerste 8 levensweken,
onderzocht. Al op een leeftijd van 14 en 28 dagen vertoonden HFP kuikens meer
mild verenpikken dan LFP kuikens. Verder besteedden LFP kuikens op
verschillende leeftijden meer tijd aan foerageren en eten, terwijl HFP kuikens meer
tijd aan poetsen besteedden. Verder kwam uit het experiment naar voren dat de
140 Samenvatting
onderliggende motivatie om te gaan verenpikken lijkt te verschillen tussen beide
lijnen. In de HFP lijn zou het uitvoeren van verenpikken vanuit het motivationele
systeem van poetsen kunnen voortkomen, terwijl verenpikken in de LFP lijn
verband lijkt te houden met het motivationele systeem van eten. Hoofdstuk 3 beschrijft een studie waarin fysiologische en neuroendocrine
karakteristieken van HFP en LFP kuikens zijn onderzocht. Ten eerste is gekeken
naar de ontwikkeling van de hypofysebijnierschors (re)activiteit gedurende de
eerste 8 levensweken. Ten tweede zijn op een leeftijd van 28 dagen, de niveaus
van serotonine (5-HT) en dopamine (DA) turnover in de hersenen van HFP en LFP
kuikens bestudeerd. In deze experimenten werd geen verenpikken gemeten. Er
werd gebruik gemaakt van de zogenaamde �manual restraint test�, waarbij een
kuiken 5 minuten op haar zij wordt vastgehouden. De test wordt gebruikt om de
fysiologische en neuroendocrine respons van kuikens op een acute stressor te
meten. Aansluitend op de test werd via decapitatie het bloed en de hersenen van
kuikens verzameld.
Het gemiddelde bloedplasma niveau van het stresshormoon corticosteron
(dat wordt afgegeven door de bijnierschors) was lager in de HFP kuikens dan in de
LFP kuikens (basaal, d.w.z. zonder manual restraint, op dag 3 en 56; na manual
restraint op dag 3, 14, en 28). Dit resultaat bevestigde de resultaten in de
volwassen dieren van Korte en zijn collega�s (1997) en versterkte het idee dat HFP
en LFP dieren respectievelijk een proactieve en een reactieve copingstrategie
hebben. De hersenniveau�s van de 5-HT en DA turnover (na manual restraint)
waren lager in de HFP dan in de LFP kuikens.
Verschillen in de (re)activiteit van de hypofysebijnierschors en in het
functioneren van het serotonerge en dopaminerge systeem in de hersenen zijn ook
kenmerkend voor het verschil tussen de proactieve en reactieve copingstrategie.
Verder spelen deze systemen een belangrijke rol in de ontwikkeling van gedrags-
of psychopathologieën. Deze informatie leidde tot de veronderstelling in dit
proefschrift dat deze systemen weleens een rol zouden kunnen spelen bij de
ontwikkeling en uitvoering van verenpikken.
In hoofdstuk 4 wordt een studie beschreven (bestaande uit 2
experimenten), die als doel had te onderzoeken of het serotonerge systeem in het
Samenvatting 141
brein een oorzakelijke rol speelt bij de ontwikkeling en ontvoering van
verenpikgedrag. Uit hoofdstuk 3 volgde het idee dat HFP kuikens meer
verenpikken dan LFP kuikens, weleens veroorzaakt zou kunnen worden door de
lagere 5-HT turnover in de HFP lijn. Om deze hypothese te toetsen, werd in deze
studie het farmacon S-15535 gebruikt. S-15535 is een stof die door binding aan
een specifieke serotonine receptor in het brein, bij ratten en muizen, de aanmaak
en afgifte van 5-HT verlaagt.
Uit een dosis-respons experiment (experiment 1) kwam naar voren dat 4.0
mg S-15535/kg lichaamsgewicht, de meest specifieke en effectieve dosis is, om 5-
HT turnover in het brein van HFP en LFP kuikens te verlagen. In experiment 2
werd deze dosering toegepast, om het effect van verlaging van 5-HT op het
verenpikgedrag van HFP en LFP kuikens te bestuderen. S-15535 injectie (dus
verlaging van 5-HT) resulteerde in een significante toename van verentrekken en
een trend voor een verhoging van mild verenpikken.
De resultaten uit hoofdstuk 4, leidden tot de hypothese in hoofdstuk 5 dat
verhoging van 5-HT turnover in het brein verenpikken zal verlagen. Verhoging van
5-HT turnover kan worden bewerkstelligd door het gehalte van het aminozuur L-
Tryptofaan (TRP) in het dieet te verhogen. 5-HT wordt namelijk uit L-Tryptofaan
gemaakt. Een toename van TRP in het voer (van 1.6 naar 21.0 gram/kg) van LFP
en HFP kuikens, gedurende 14 dagen, resulteerde in een significante toename van
5-HT turnover in de hersenen en een significante afname van verenpikken.
Manipulatie van het 5-HT systeem in de hersenen, met S-15535 of TRP,
resulteerde in een grotere gedragsrespons in de proactieve HFP dan in de
reactieve LFP lijn. Deze resultaten zijn in overeenstemming met bevindingen in
proactieve ratten, wiens 5-HT systeem gevoeliger lijkt te zijn dan dat van reactieve
ratten (van der Vegt et al., 2001). Naast een verschil in de gevoeligheid van het 5-
HT systeem in het brein tussen proactieve en reactieve ratten, lijken deze
individueën ook te verschillen in de gevoeligheid van het DA systeem. Er zijn
aanwijzingen dat proactieve dieren gevoeliger zijn voor het ontwikkelen van
abnormaal gedrag door deze verschillen in de gevoeligheid van beide systemen.
Dit leidde tot de hypothese dat HFP dieren wellicht ook een gevoeliger DA
systeem zouden kunnen hebben dan LFP dieren.
142 Samenvatting
In hoofdstuk 6 wordt dan ook een experiment beschreven waarin
onderzocht is of er een verschil is in de gevoeligheid van het DA systeem tussen
HFP en LFP kuikens. Gevoeligheid van het DA systeem kan worden getest met de
stof apomorphine (APO). HFP kuikens werden injecteerd met APO of gedistilleerd
water (controle dieren). Direct na injectie werden de dieren individueel getest in
een zogenaamde �open field� test. De kuikens werden daarvoor losgelaten in de
�open field� (een vierkante bak van1,5 x 1,5 x 1,5 meter) en hun activiteit in deze
nieuwe omgevingwerd gescoord. Naast het observeren van het gedrag in de �open
field�, werden in de hersenen van de controle HFP en LFP dieren de concentraties
van DA receptoren bepaald (D1 en D2).
De gedragsrespons als reactie op APO injectie was groter in de HFP lijn
dan in de LFP lijn. HFP kuikens vertoonden een grotere toename van de activiteit
in de �open field� (d.w.z. een grotere afgelegde afstand en snelheid) dan LFP
kuikens. Dit betekent dat HFP dieren inderdaad een gevoeliger DA systeem
hebben dan LFP dieren. Er bleek echter geen verschil te zijn in de hoeveelheid D1
and D2 receptoren in de hersenen van HFP en LFP kuikens. Het verschil in
gevoeligheid van het DA systeem tussen beide lijnen kan dus niet verklaard
worden door een verschil in het aantal receptoren.
In hoofdstuk 7 wordt naar samenhang gezocht tussen de belangrijkste
resultaten uit dit proefschrift. Geconcludeerd kan worden dat het concept van
copingstrategieeën, een zeer bruikbaar kader was van waaruit de experimenten in
dit proefschrift zijn opgezet. De resultaten hebben daarnaast bevestigd dat HFP
dieren een proactieve en LFP dieren een reactieve copingstrategie hebben. Verder
zijn er aanwijzingen dat de verschillen in met name het 5-HT systeem tussen beide
lijnen het verschil in verenpikken zou kunnen verklaren. Daarnaast is er een
algemeen geldende (d.w.z. op beide lijnen van toepassing) negatieve correlatie
tussen 5-HT in het brein en de frequentie van verenpikken gevonden. Dat wil
zeggen dat kippen met een lager serotonine turnover in de hersenen na acute
stress, relatief meer verenpikken laten zien dan dieren met een hoger niveau van
serotonine. Deze rol van het 5-HT systeem zou een nieuwe oplossingsrichting voor
verenpikken kunnen betekenen.
Samenvatting 143
Tenslotte wordt in hoofdstuk 7 beargumenteerd dat vanwege de rol van 5-
HT en de compulsieve eigenschappen van het verenpikgedrag, verenpikken
weleens als model voor OCD bij de mens zou kunnen fungeren.
144 Samenvatting
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Dankwoord
Dankwoord 172
In de zomer van 1998 reageerde ik op een vacature waarin stond �wanneer
paarden uw wetenschappelijke interesse hebben�. Dat was het geval en ik mocht
op gesprek komen bij wat toen nog heette ID-DLO. Ik werd niet uitverkozen��en
dat was achteraf denk ik maar goed ook. Een van mijn paranimfen was de beste
kandidaat en dat is ook gebleken! Enkele maanden later kon ik weer reageren op
een advertentie van ID-DLO, waarin een OIO baan werd aangeboden. Dit maal
was het onderwerp �verenpikken bij leghennen� en nog wel de fysiologische kant
ervan. Aangezien ik me met beide zaken in mijn studie zeer intensief bezig had
gehouden, hoopte ik een kans te maken. Inderdaad mocht ik weer op gesprek
komen! Niet lang daarna kreeg ik jou, Mechiel, aan de lijn. Wat ik me van dat
gesprek kan herinneren is dat het van zeer Korte duur was, maar dat het goede
nieuws met een zeer groot enthousiasme door de telefoon heen schalde! Dat was
het begin van onze samenwerking, die gekenmerkt zou worden door een groot
persoonlijk leerproces voor zowel jou als mijzelf in die 4 jaar. Maar terugkijkend op
de afgelopen jaren, kan ik niet anders zeggen Mechiel, dan dat ik zeer blij ben dat
jij mijn begeleider bent geweest. Je enthousiasme, je kennis van zaken, je goede
zorgen en de vrijheid die jij mij ook heel bewust hebt gegeven om te groeien en om
beslissingen te nemen in dit project hebben geresulteerd in het boekje dat nu voor
ons ligt. Bedankt Mechiel!
Een ander persoon die in de eerste jaren van mijn OIO periode een zeer
belangrijke rol in mijn project speelde was Wim Ruesink. Wim, hoewel onze wegen
zo halverwege het project zijn gescheiden, ben je steeds geïnteresseerd gebleven
in het project. Dankzij jou weet ik hoe belangrijk het is om de praktische uitvoering
van een experiment vast goed door te denken van achter je bureau en niet pas in
de stal.
Jaap, je werd wat laat bij dit project betrokken. Toch twijfelde je niet en
wilde jij mijn promotor worden. Jouw heldere kijk op mijn onderzoek en je
enthousiasme gaven steeds weer nieuwe impulsen aan het project. Bedankt!
Kees (! !!!), jou wil ik toch nog even in het bijzonder bedanken! De eerste
jaren van mijn project waren voor mij vaak gevuld met onzekerheden en gebrek
aan kennis van zaken op een aantal gebieden (met name de statistiek of course).
Dankwoord 173
Het feit dat je steeds weer tijd vrij wilde maken voor mijn �vraagjes�, zijn een
belangrijke factor geweest voor het slagen van mijn project!
Experimenten kun je alleen goed uitvoeren als je mensen hebt die bereid
zijn om mee te denken, en flexibel mee te werken! Sander, Wouter, Albert, Alan,
Reinard, en anderen, bedankt, zonder jullie was het niet gelukt!
Voor goede gesprekken, gezelligheid en ondersteuning van mijn project
dank ik ook vele andere (ex)collega�s van GSM/Dierenwelzijn: Bonne, Dinand,
Gerdien, Godelieve, Hans, Henk, Hennie, Ina, Ingrid, Ingrid, Johan, Johanna,
Joop, Marc, Marko, Marjan, Maaike.
Rick, jou experiment heeft het boekje niet gehaald, maar desalniettemin
was het een geweldig leuk experiment en je was een zeer humoristische en goede
stagiaire!
Dit project stond niet op zichzelf. Bedankt, Bernd, Bas, Bart voor de
prettige samenwerking en Harry, Jan, Paul en Ton voor het opbouwende
commentaar en de leuke vergaderingen!
Bas en Willem, statistiek is nooit mijn sterkste punt geweest, dat van jullie
wel, en ik heb er veel van geleerd. Bedankt voor jullie hulp!
Naast collega�s zijn er nog tal van anderen die ik wil bedanken, mijn
vriend(inn)en bijvoorbeeld, Hester, Monique, Anite, Minie en Antoon, Antoinette,
Gert, Jelle en Karlijn. We hebben elkaar te weinig gezien in de afgelopen jaren,
toch hebben jullie steeds interesse getoond in mijn werk en de rest� Bedankt!
Netty en Chantal, bedankt voor de goede zorgen voor Imp en Bryndis.
Jullie bijdrage aan dit boekje was groter dan jullie weten!
Paranimfen, het was een makkelijke keuze! Francesca, door de
vriendschap en gastvrijheid van jou en Martin ging ik me snel thuisvoelen in �Swift�.
Bedankt ook voor alle liften naar stal� Kathalijne, ex-kamergenote, vriendin,
bijrijdster van Impje, bedankt voor de goede gesprekken en de gezelligheid! De
liefde voor paarden verbindt ons drieeën, maar onze vriendschap is zeker meer
dan alleen dat geworden. Bedankt dat jullie ja hebben gezegd tegen het
paranimfschap!
Martin en Bernd, mannen van de paranimfen, fijn dat ik ze effe een dagje
mag lenen en bovenal ook jullie bedankt voor de vriendschap!
Dankwoord 174
Groningen is niet alleen de stad waar ik promoveer, ondertussen is het ook
mijn tweede thuis geworden! Nel, Rob, Irith en Arvid, bedankt voor het interesse in
mijn werk en de fantastische ontvangst in jullie leven!
De mensen die ik de meeste dank verschuldigd ben zijn jullie, pap, mam
en Wouter. Jullie kennen mij het langst en zonder jullie steun, interesse en liefde
was dit boekje er zeker nooit gekomen! Bedankt voor alles!
Paarden zijn dan wel niet het begin geweest van mijn wetenschappelijke
carriere. Ze zijn wel het begin geweest van de rest van mijn leven�. Dankzij Impje
(jij ook bedankt jongen), heb ik nu een Vrindje! Arthur, lieve schat, we delen veel
meer dan onze liefde voor paarden (en andere beesten). Bedankt voor je rust, je
interesse in mijn werk, je geduld met mij, en bovenal je LIEFDE.
Curriculum Vitae
Curriculum Vitae 176
Yvonne van Hierden werd geboren op 11 december 1972 te Velp. Ze haalde in
1992 haar VWO diploma aan het Baudartius College te Zutphen. In datzelfde jaar
begon ze de studie Zoötechniek aan de Landbouwuniversiteit Wageningen. In
1997 studeerde zij af met als afstudeerrichtingen Ethologie en Fysiologie van Mens
en Dier. In januari van 1999 begon zij als OIO bij het instituut voor Dierhouderij en
Diergezondheid, bij het huidige cluster Dierenwelzijn. Haar onderzoeksproject
�physiological characteristics of feather pecking�, maakte deel uit van het
multidisciplinaire project �feather pecking in laying hens: a multidisciplinaire
approach�, waar in totaal 4 OIO�s aan werkten (Bas Rodenburg, Bart Buitenhuis en
Bernd Riedstra).
Vanaf 1 april 2003 is zij, voor een periode van 1 jaar, werkzaam als junior
onderzoeker bij de divisie Dier en Omgeving van de Animal Sciences Group
(Lelystad).
Behavioural neurobiology
of feather pecking
Yvonne van Hierden
Beh
avioural n
eurob
iology of feather p
eckin
gYvonne van H
ierden 2003ISBN 90-6464-963-4