behavioural neurobiology of feather pecking · 2016. 3. 5. · feather pecking, characterised by...

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


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).


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)


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.


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.


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.


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.


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.


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


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


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

***

#*


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


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

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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


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.


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


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


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


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.


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


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).


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).


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


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


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,


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


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).


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


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


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.


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

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-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

* *


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***

****

#

* *

#


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


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.


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.


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


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


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).


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


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


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


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.


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


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


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


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.


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


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


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


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).


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


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


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


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.


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


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.


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


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


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)


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


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


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.


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

behavioural neurobiology of feather pecking · 2016. 3. 5. · feather pecking, characterised by...

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