4 the Role of the Kynurenine Pathway in the Development of Feather Pecking in Domestic Chicken Lines

Tryptophan Metabolism and Feather Pecking in Laying Hens

Patrick Birkl

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Doctor in Philosophy in Animal Biosciences

Guelph, Ontario, Canada

TRYPTOPHAN METABOLISM AND FEATHER PECKING IN LAYING HENS

Patrick Birkl Advisor:

University of Guelph, 2020 Dr. Alexandra Harlander

Feather pecking (FP) in laying hens is one of the greatest welfare issues in the husbandry of chickens kept for egg-laying. FP may result in severe feather damage or cannibalism, which can increase flock mortality. FP behaviour, mostly described as gentle or severe, is affected by multiple internal and external factors, but the biological mechanisms that regulate the development and continuation of FP are poorly understood. While tryptophan (TRP) metabolism has been hypothesized to impact FP, nutritional manipulation of plasma TRP using acute tryptophan depletion (ATD) for studying changes in pecking behaviour in a social and non-social environment has not been explored, to the best of our knowledge. Further, the influence of the social environment on the plasma TRP metabolism, along the kynurenine (KYN) pathway and

FP, has not been considered. To manipulate TRP metabolism, we developed a nutritional, non-invasive ATD treatment for laying hens. The effect of ATD on gentle and severe FP in a social environment, as well as on pecking in an operant chamber, was evaluated. Disrupting social structures was utilized to observe any alterations in gentle and severe FP, and blood TRP metabolites along the KYN pathway. The results of this thesis indicate that ATD in laying hens reduces plasma TRP levels and increases pecking activity in a social (gentle FP) and non-social (operant pecking) environment.

Furthermore, disrupting social structures has been found to lower KYN/TRP ratios and increase the likelihood of FP (gentle and severe merged). These findings indicate that even acute TRP metabolism-manipulation may impact pecking behaviour, and that the

KYN pathway of TRP metabolism could help broaden our understanding of the biological mechanisms behind FP. iv

ACKNOWLEDGEMENTS First, I would like to thank my supervisor Dr. Alexandra Harlander for her continuous support during my PhD program and for enabling me to not only conduct experiments and write a thesis, but to attend numerous conferences and publish my work throughout my program. I would also like to thank my advisory committee members Dr. Lee Niel, Dr. Paul Forsythe, Dr. Joergen Kjaer and Dr. Georgia Mason for their constructive comments and criticism. My sincere gratitude goes to the numerous graduate, undergraduate, summer and visiting students without whom this work could not have been done. In alphabetical order, I would like to thank Dr. Aitor Arrazola, Angeli Li, Dr. Bishwo Pokharel, Dr. Brittany Lostracco, Claire Mindus, Claire Toinon, Florian Daufin, Jacky Chow, Julia Krumma, Dr. Michael Ross and Peter McBride who have all helped and contributed tremendously to this project. Also, I would like to thank the Arkell Poultry Research Station staff for taking care of the birds. I would also like to thank Dr. Dietmar Fuchs and his lab in Innsbruck for analyzing our plasma samples and providing expertise for the interpretation and discussion of our data. I also acknowledge the funding sources of Natural Sciences and Engineering Research Council of Canada (NSERC) – Discovery Grant (#400983) (chapter 2,3,4), Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) - #27267 (chapter 2, 3); #2015-2384 (chapter 4); and Egg Farmers of Canada (chapter 4), who contributed funding to Dr. Alexandra Harlander and provided funding for my scholarship and for the experimental setups. Also, I want to express my sincere gratitude for the support I received from Dr. Teresa Crease and Dr. Benjamin Bradshaw at the final stage of completing this thesis. Finally, I would like to thank my wife Katharina for her continuous support and never failing to make me look ahead instead of back.

TABLE OF CONTENTS

ABSTRACT ...... ii

Acknowledgements ...... iv

Table of Contents ...... v

List of Tables ...... ix

List of Figures ...... x

1 Literature Review ...... 1

1.1 Feather pecking in laying hens: A roadmap to pecking behaviour ...... 1

1.2 Causes of SFP: Early references and scientific study of severe feather pecking 4

1.3 Understanding severe feather pecking: The ethological view ...... 6

1.3.1 Severe FP and redirected foraging behaviour ...... 6

1.3.2 The influence of stress...... 7

1.3.3 Feather pecking phenotypes: Active, recipient and neutral ...... 11

1.4 Understanding severe feather pecking: The neurobiological view ...... 12

1.4.1 Introduction ...... 12

1.4.2 Neurobiology of severe feather pecking: The monoamines dopamine and serotonin ...... 12

1.4.3 Aromatic amino acid metabolism: Tryptophan ...... 15

1.4.4 Aromatic amino acid metabolism: Phenylalanine ...... 16

1.5 Acute tryptophan depletion: a tool to study serotonergic synthesis in humans and rodents ...... 17

1.6 Acute tryptophan depletion effects on affective states and behaviour ...... 18

1.7 The role of genetics in feather pecking ...... 20

1.7.1 Experimental selection to study severe feather pecking behaviour ...... 20

1.7.2 Target genes involved in feather pecking behaviour ...... 21

1.8 The role of nutrition in severe feather pecking ...... 22

1.8.1 Tryptophan, protein metabolism and protein-digestion ...... 26

1.9 Specific objectives ...... 28

1.9.1 Objective 1: Validation of Acute Tryptophan Depletion (ATD) in laying hens to reduce tryptophan in plasma and to test whether ATD causes an increase in FP in a social group or increased pecking in an operant chamber ...... 29

1.9.2 Objective 2: The effects of a social disruption stress treatment on TRP metabolism and FP behaviour in laying hens ...... 30

1.10 References ...... 31

2 Acute tryptophan depletion: the first method validation in an avian species (Gallus gallus domesticus) ...... 50

2.1 Introduction ...... 50

2.3 Materials and Methods ...... 52

2.3.1 Animals and Housing ...... 52

2.3.2 Acute Tryptophan Depletion Mixture and Sample Collection ...... 52

2.3.3 Amino Acid Analysis ...... 53

2.3.4 Statistical Analysis ...... 54

2.4 Results and Discussion ...... 54

2.2 References ...... 57

2.3 Tables and Figures ...... 59

3 Effects of acute tryptophan depletion on pecking behaviour in laying hens ...... 61

3.1 Introduction ...... 62

3.2 Materials and methods ...... 63

3.2.1 Animals and housing ...... 63

3.2.2 Operant chamber test equipment ...... 64

3.2.3 Acute tryptophan depletion and balanced control capsule ...... 64

vii

3.2.4 Tryptophan–deficient amino acid mixtures to groups of laying hens – feather pecking...... 65

3.2.5 Tryptophan–deficient amino acid mixtures to laying hens in an operant chamber – operant pecking ...... 66

3.2.6 Statistical analysis ...... 67

3.3 Results ...... 68

3.3.1 Effects of ATD/BC on feather-pecking behaviour in group housing ...... 68

3.3.2 Effects of ATD/BC on performance in the operant chamber ...... 68

3.4 Discussion ...... 69

3.5 References ...... 74

3.6 Figures ...... 77

4 The role of the kynurenine pathway in the development of feather pecking in domestic chicken lines ...... 81

4.1 Introduction ...... 82

4.2 Materials and methods ...... 84

4.2.1 Animals and housing ...... 84

4.2.2 Social disruption stress treatment ...... 84

4.2.3 Behavioural observations ...... 85

4.2.4 Body weight ...... 85

4.2.5 Blood measurements ...... 86

4.2.6 Statistical analysis ...... 87

4.3 Results ...... 87

4.3.1 Effects of social disruption and genetics on FP behaviour and body weight 87

4.3.2 Effects of social disruption and genetics on biomarkers of immune activation ...... 89

4.4 Discussion ...... 89

viii

4.5 References ...... 96

4.6 Tables ...... 101

4.7 Figures ...... 107

5 General Discussion ...... 109

5.1 Introduction ...... 109

5.2 Chapter 2) and 3): ATD increases FP and operant pecking ...... 110

5.3 Chapter 4) Social disruption increases FP ...... 114

5.4 General considerations and limitations ...... 118

5.5 Conclusion ...... 120

5.6 References ...... 122

Appendix ...... 127

LIST OF TABLES

Table 1 Plasma tryptophan (TRP) concentration [nmol/ml], TRP/large neutral amino acids (LNAA) and TRP/aromatic amino acids (AAA) ratios over time in 10 individuals (Ind) receiving either a balanced control (n = 5) or an acute tryptophan depletion (ATD) treatment (n=5)……………………………………………………………………………………...……..59

Table 2 Concentrations [nmol/ml] of amino acids during ATD and control treatment (C) for 10 individuals (HFP) over 6 hours post-treatment: Tryptophan (TRP), Phenylalanine (PHE), Tyrosine (TYR), Glycine (GLN), Gultamic acid (GLU), Valine (VAL), Leucine (LEU) and Isoleucine (ILEU)…………………………………………………………………………………………...60

Table 3 Descriptive statistics: Mean ± Standard Error of the Mean (SEM) for biological markers before (PRE) and after (POST) social disruption treatment in disrupted and undisrupted groups……………………………………………………………………………...………….101

Table 4 Descriptive statistics: Mean ± Standard Error of the Mean (SEM) for biological markers before (PRE) and after (POST) social disruption treatment in high (H), low (L) and unselected control (UC) lines in disrupted and undisrupted groups………………………………...…………………………………..……………..…….102

Table 5 Mean concentrations [nmol/ml] of amino acids and Standard Deviation (SD) before and after social disruption: Tyrosine (TYR), Phenylalanine (PHE), Tryptophan (TRP), Glycine (GLN), Gultamic acid (GLU), Valine (VAL), Leucine (LEU) and Isoleucine (ILEU). Line; 5= unselected control (UC), 51=low feather pecking (LFP), 52= high feather pecking (HFP). Baseline values (week 16) and values after treatment (week 17) are presented…………………………………………………………………….105

Table 6 Analyzed nutrient composition of diet fed to FP and non-FP birds…….……..127

LIST OF FIGURES

Figure 1 Concentration of plasma levels of tryptophan (nmol/l) at the time points T0-T6 after the intake of a balanced control (C) and an acute tryptophan depletion (ATD) mixture. The data are represented as the mean values ± Standard Deviation (SD)……………………………………………………………………………………………..60

Figure 2 Schematic representation of experimental timeline for the acute tryptophan depletion (ATD)/balanced control (BC) group housing and operant chamber testing schedule………………………………………………………………………….…………….78

Figure 3 Schematic of Operant Chamber set-up. A) The red key is illuminated, and a key peck will result in food reward after a 5-second delay. The food trough is in a closed position. B) The food trough is opened after a 5-second delay and the hen can access the food reward. The red key is not illuminated. Key pecks at the un-illuminated key do not result in food reward………………………………………………………………………79

Figure 4 Timeline of 5-second delay schedule of reward………………………………….80

Figure 5 Key peck/minute (mean ± SE) during the delay across all three phenotypes after ATD and UC treatment; Feather peckers (P), recipients (R) and bystanders (B)………...81

Figure 6 FP-bouts/hour (mean ± SD) after treatment between Disrupted and Undisrupted birds……………………………………………………………………………………………108

Figure 7 FP-bouts/hour (mean ± SD) after treatment between Disrupted and Undisrupted birds……………………………………………………………………………………………108

Figure 8 Body-weight gain in [g] (mean ± SD) of disrupted and undisrupted groups, three weeks post-mixing……………………………………………………………………………109

1 Literature Review

1.1 Feather pecking in laying hens: A roadmap to pecking behaviour

A variety of bird-to-bird pecking behaviours in domesticated chickens (Gallus gallus domesticus) and other species of birds exist, some of which fall under the umbrella term of injurious pecking (IP) (van Staaveren and Harlander, 2019). IP consists of pecks that are directed at feathers or exposed skin of conspecifics. For laying hens, IP can be divided into several descriptive categories, which were first introduced by Savory (1995) as tissue pecking (TP), aggressive pecking (AP) and feather pecking (FP).

Pecking directed at exposed skin or tissue is defined here as TP (van Staaveren and Harlander, 2019), often also referred to as cannibalistic pecking behaviour. Different forms of TP directed at certain body parts, such as toes (toe pecking) or the cloacal region (vent pecking), are typically considered separately from IP (Savory, 1995). IP may develop from FP via further pecking at denuded areas (Rodenburg et al., 2008), or independently thereof, such as vent-pecking (Savory 1995). Birds could be drawn to peck at injured areas of skin where feathers have been removed via FP (Cloutier et al., 2000). This would, however, not explain why TP occurs in birds with undamaged feather-cover (Gunnarsson et al., 1999; Pötzsch et al., 2001; Newberry, 2004).

AP behaviour in laying hens is considered crucial in establishing and maintaining social hierarchies (Savory, 1995). AP consists of forceful pecks, mostly aimed towards a bird’s head, comb or neck-area (Guhl, 1968). A connection between damaging levels of FP and AP has been suggested in the literature (Bilcik and Keeling, 1993; Bennewitz et al. 2014). However, these behaviours do not necessarily occur simultaneously in the same flock (e.g., Daigle et al., 2015; Birkl et al., 2017a). While a correlation of different forms of IP (e.g., AP and FP) may not mean that these behaviours are caused by a common underlying mechanism, various forms of IP may, however, could be triggered by similar risk factors (Lambton et al., 2015). Therefore, when investigating forms of IP within a research- or industry-platform, it is crucial to communicate as accurately as possible which form of IP is being studied.

FP, a heterogeneous behaviour that includes pecks that are directed at the feathers of conspecifics and is mostly sub-divided into gentle (GFP) and severe (SFP) FP (Lambton et al., 2010). The terminology introduced here will be used throughout this thesis, referring to FP as an umbrella term for GFP and SFP if not otherwise specified. GFP consists of gentle pecks at the tips and edges of feathers causing little to no damage to feathers (McAdie and Keeling, 2002). It has been suggested to further distinguish forms of GFP into exploratory pecking (low frequency), pecks directed at feathers (stereotyped and high frequency) and pecks that are not directed at feathers but rather at particles on feathers and, as such, do not fall under the term FP (Rodenburg et al., 2004). It has also been suggested that the exploratory form of GFP (in young chicks) plays a role in establishing and maintaining social hierarchies (Riedstra and Groothuis, 2002). GFP might be more similar to allopreening or exploratory pecking in other birds (Riedstra and Groothuis, 2002; Rodenburg et al., 2004).

The second category of FP, SFP, however, is described as forceful pecking at feathers, often resulting in the removal of the feather or fragments thereof, which leads to significant damage to the feather cover (Savory, 1995). Due to the potential damage inflicted by SFP, and the high prevalence thereof, this type of FP represents the most relevant form of IP for egg-production systems (Lambton et al., 2010). Research showed that damaged feathers attract more SFP than GFP, and a subsequent outbreak of cannibalism (or TP) was observed (McAdie and Keeling, 2000). This would indicate that birds performing SFP are more likely to develop TP. Some forms of SFP are accompanied by consumption of feathers (McKeegan and Savory, 1999; Harlander- Matauschek and Bessei, 2005; Ramadan and von Borell, 2008; Harlander-Matauschek and Häusler, 2009). Caution should be taken when using the term SFP, since some sub-categories of SFP involve the consumption of feathers, while others may not (van Staaveren and Harlander, 2019).

The relationship between gentle and severe forms of FP is unclear. Riedstra and Groothuis (2004) described that when SFP appeared in young chicks, it was initially embedded in GFP bouts and that differences in SFP-expression between genetic lines

of laying hens were reflected by early development of GFP. However, GFP has also been described as an unreliable predictor for SFP later in life (Rodenburg et al., 2004). Yet even SFP appears to be inconsistent over time (Daigle et al., 2015). Research using fixed action pattern morphology indicates that SFP and GFP stem from different behaviour systems (Dixon et al., 2008). While SFP resembles pecking associated with foraging behaviour, GFP patterns appear to be different from all other pecking patterns (Dixon et al., 2008). In summary, the relationship between GFP and SFP remains unclear. Ultimately, this controversial relationship between SFP and GFP may come as a result of the descriptive nature of these terms, without clear limits separating them. Also, GFP and SFP are mostly used as umbrella-terms without further discrimination, which complicates the attempt at an integrated understanding of a possible relationship between forms of SFP and GFP (van Staaveren and Harlander, 2019).

In commercial egg-production, SFP by laying hens is mostly noticed and discussed due to deterioration of feather-cover and can develop in all types of housing situations; cage systems (i.e., conventional and enriched cages) and non-cage systems such as barn, free-range and organic systems (Pötzsch et al., 2001). In non-cage housing systems, where large flocks of birds are kept in one enclosure, each SFP bird can: 1) inflict feather damage on a large number of individuals and 2) factors such as social learning (McAdie and Keeling, 2002; Zeltner et al., 2000) can contribute to the spread of SFP from one invididual to another. Hence, alleviating the issue of SFP in non-cage housing systems is of importance both from an animal-welfare and a production perspective since the victim of SFP might suffer from feather damage or potential skin damage due to TP (Cloutier et al., 2000), and production may be adversely affected due to increased mortality rates (Kjaer and Sorensen, 2002). Also, the deterioration of feather cover due to SFP often results in increased feed-consumption, as the birds require more energy to maintain their body temperature when the insulating effect of the plumage is compromised (Glatz, 2001).

Therefore, to counteract SFP (and TP) in practice, the most widely used method is beak-trimming, where a part of the bird’s beak is removed (Jendral and Robinson, 2004). While beak-trimming can reduce the damage of SFP within a flock (Struwe et al.,

1992), the treatment causes both acute and chronic pain to the birds (Duncan et al., 1989; Hughes and Gentle, 1995; Kuenzel, 2007), and the mutilation of the bird’s primary tactile organ is both troubling from an ethical and public-perspective point of view (Glatz, 2005). Public pressure has thus led to a ban of beak trimming in some countries such as Norway, Finland, and Sweden. Beak trimming is also discouraged in Austria, where 95% of flocks are non-beak-trimmed, due to voluntary welfare standards across food retail chains (Fiks-van Nikerk and DeJong, 2007). Beak-trimming may ameliorate the symptoms of IP but does not tackle the underlying causes. Hence, this practice fails to solve the issues of SFP (Dixon, 2008).

For tackling the issue of SFP in practice, it is critical to understand underlying mechanisms that contribute to the development of SFP. Early reports of IP date back over 100 years and already speculate about multiple risk factors affecting this behaviour. These reports shall be briefly introduced next (section 1.2), before moving on to a review of recent scientific investigations regarding the causes and development of SFP, which are discussed from an ethological and a neurobiological point of view in section 1.3 and section 1.4.

1.2 Causes of SFP: Early references and scientific study of severe feather pecking

In contrast to self-directed feather picking in psittacine birds (Jenkins, 2001), SFP in laying hens is almost always directed at other birds and, therefore, takes place in a social context. Early references did not distinguish between subcategories of IP, hence SFP will be used as an umbrella term to discuss these references. SFP has been a known issue for over a century (e.g., Oettel, 1873), and hints towards potential reasons for this issue have been suggested as early as in the 19th century. In 1895 the author Liebeskind, in his book on poultry husbandry, gives a more differentiated view on the issue and describes IP to occur most likely in a “crowded and barren environment” due to the resulting “boredom” of the birds (Liebeskind, 1895). In this book, the author also identified nutritional (e.g., fiber and protein) and genetic risk-factors (breed), which were assumed to contribute to the likelihood of a bird engaging in this “bad habit” of SFP.

When scientific investigations began to focus on SFP behaviour in laying hens between 1950 and 1985, the term feather-pulling or eating was still used frequently in the scientific literature for this phenomenon, adopted from earlier observational and anecdotal reports (e.g., Marsboon and Sierens, 1962; Willimon and Morgan, 1953). In the seventies of the last century, feather pulling/eating was replaced by the word FP (e.g., Allen and Perry, 1975; Hughes and Duncan, 1972). Hughes (1981) stated that an extensive literature on FP causes exists, falling into four groups: dietary composition, environment, hormonal influences, and “psychic” factors. One could also describe these factors as bird-related and environment-related (social or physical environment). In addition to these four categories, Hoffmeyer (1969) explained the motivation of FP in ethological terms as the expression of a “pecking drive”, which will be directed at feathers if there is no other appropriate substrate available in the environment. More recent research found that birds performing SFP consumed feathers and introduced the term “feather eating” once more in the scientific literature (McKeegan and Savory, 1999, 2001; Harlander-Matauschek and Bessei, 2005). Today, nutrition (Kjaer and Bessei, 2013, Stenfeldt et al., 2007; van Krimpen, 2008), genetics (Bennewitz et al., 2014; Huges and Duncan, 1972; Kjaer et al., 2001), management (Kjaer and Vestergaard, 1999; Rodenburg et al., 2008) and environmental factors (El-Lethey et al., 2000; Bestman and Wagenaar, 2003) are still understood to play a key role in the development of SFP-behaviour (Blokhuis and Wiepkema, 1998; Lambton et al., 2010; Rodenburg et al., 2013). Yet, the relative relevance of, and interplay between these external (environment-related) and internal (bird-related) risk-factors is poorly understood.

Generally, the risk factors contributing to the development of IP (in this thesis, the focus will lie on SFP, the most relevant and severe form of IP) are approached via two views: the ethological view and the neurobiological view. While the first view focuses on the role of the physical and social environment in the development of behaviour, the latter focuses on possible underlying neurobiological mechanisms (bird-related) due to priming factors, such as inappropriate rearing conditions (Mills, 2003).

The following sections 1.3 and 1.4 shall introduce both views in detail before identifying the gaps of knowledge, which lead to the work presented in this thesis.

1.3 Understanding severe feather pecking: The ethological view

1.3.1 Severe FP and redirected foraging behaviour

The ethological view explains SFP as the inability of the birds to express highly motivated natural behaviour in a physically and socially deprived environment. This may lead to the development and manifestation of behaviours to cope with an unnatural environment (Blokhuis, 1986; Duncan, 1998; Wennrich, 1975; Wood-Gush and Vestergaard, 1989) such as SFP. SFP behaviour has been postulated to replace or compensate for activities such as foraging behaviour (Huber-Eicher and Wechsler, 1998) or dust-bathing (Vestergaard et al., 1993). There exists less evidence to support the notion that SFP is derived from frustrated dust-bathing behaviour.

The interplay of internal and external stimuli describe the basis for motivation, which results in the expression of a certain behaviour (Mason and Bateson, 2009). In a naturalistic environment, even in the presence of feed, the red jungle fowl has been reported to spend over 60% of their active time with foraging-related pecking behaviour (ground-pecking) (Dawkins, 1989). This shows that foraging behaviour in chickens is highly motivated. Foraging behaviour consists of an appetitive or exploratory phase, where food is searched, followed by a consummatory phase, where food is ingested (Keeling, 2002).

Mitigation of both phases - the appetitive (food searching) and the exploratory (food ingestion) phase - have been linked to an increased risk of SFP development. For example, lack of opportunity to search for food (absence of foraging substrate) has been linked to an increased risk to develop SFP (Blokhuis, 1986; Decina, 2018), as have feed restriction or reducing the time spent consuming feed by providing a pelleted diet (Savory and Mann, 1997; Van Krimpen et al., 2005).

It is hence generally assumed that SFP is influenced by the motivational system of foraging and feeding (Blokhuis, 1986; Dixon et al., 2008; Hoffmeyer, 1969; Huber-

Eicher and Wechsler, 1997, 1998; Wennrich, 1975). Assuming most behaviour problems have their origins in normal, functional mechanisms (natural behaviour), it is important to understand the processes involved in the regulation of such behaviours (Mills, 1993).

The fact that the ingestion of feathers affects feed passage time in the gut (Harlander- Matauschek et al., 2006) indicates that this behaviour may serve a purpose (or physiological function). Consuming feathers may express a bird´s attempt to supplement a deficient diet, which shall be discussed in more detail in section 1.4.2. If such an attempt was successful, this would mean the animal overcomes a challenge in their environment by adapting its behaviour in response to a challenge (Mills 2003).

The idea that SFP arises due to lacking opportunity to forage, or because individuals might be facing nutritional deficits, is an intriguing one but there exists a plethora of research challenging this view. For instance, early life experiences, such as the absence of foraging substrate (El-Lethey et al. 2000; Huber-Eicher and Wechsler, 1998) or motherless rearing (Rodenburg et al., 2004) can increase the risk of developing SFP. Even later in life, the presence of litter (Aerni et al., 2001) as well as access to a range/pasture (Bestman and Wagenaar, 2003), significantly reduces but fails to terminate SFP entirely. This indicates that the opportunity to forage in a natural environment is not sufficient to prevent SFP (e.g. Petek et al., 2015). Hence, there are likely other mechanisms at play that prime an individual to perform SFP. The view that some or all sub-categories (with or without consumption of feathers) of SFP originate solely from a lack of opportunity to forage, and could possibly be reverted by providing such an opportunity, hence appears to be false. In particular, there exists a large body of literature discussing inheritable priming risk-factors that may play an important role in the development of SFP. These risk factors include the response to stress, which shall be discussed next.

1.3.2 The influence of stress

The terms “stress” and “distress” are often used interchangeably, which may result in confusion. Yet, according to Moberg (2000) “stress and distress are dissociable

concepts, distinguished by an animal’s ability or inability to cope or adapt to changes in its immediate environment and experience” and that “stress responses are normal reactions to environmental or internal perturbations and can be considered adaptive in nature. Distress occurs when stress is severe, prolonged or both”. While an integrated review of stress-related terminology is beyond the scope of this thesis, some basic concepts shall be discussed briefly before reviewing work on the role of physical and social stress in the development of SFP in laying hens.

Stress is associated with a state of arousal and the induction of a physiological stress response, namely activation of the autonomic, neuroendocrine, metabolic, and immune system. Activation of these systems may, over time, harm the individual and are hence terminated when the state of stress is overcome (McEwen, 2012). Activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to intense or sustained stress (distress) may result in permanent and irreversible changes to the CNS or circuits thereof (Ishiwata et al., 2005; Guilliams and Edwards, 2010; Silverman and Sternberg, 2012). Functional or morphological changes due to HPA-activation have been shown to affect the limbic system (hippocampus, amygdala, hypothalamus and prefrontal cortex) of mammals (Ziabreva et al., 2003a, b), the cortical-basal ganglia circuitry (Whitehouse and Lewis, 2015), and similar changes might be expected to the analogous structures in the avian brain (Reiner, 2005; Güntürkün, 2005). Alterations in these regions of the brain can also impact affective states such as anxiety, fearfulness or mood as well as motor control or motivation (Langen et al., 2011; McEwen, 2012). The extent to which an individual is affected by stress depends on the adversity, duration and the developmental stage during which the individual is exposed to stress (Mason and Rushen, 2006). Whether, and to what extent, an individual is affected by stress during a vulnerable developmental stage will depend on how stress-resilient a certain individual may be (Riboni and Belzung, 2017). More resilient animals are known to be more capable of terminating a stress-response via reorganization of neurobiological pathways to better cope with challenges, while less resilient animals are incapable of eliciting such changes and may, therefore, develop abnormal behaviour (Maier and Watkins, 2010; Koolhaas et al., 2011; Riboni and Belzung, 2017).

Work has been carried out investigating the effects of a variety of stressors, mostly suggesting a positive relationship between exposure to stress and SFP. Experiments with the same genetic lines as were used for the experiments in this thesis showed that these lines might differ in their stress-response to manual restraint (acute physical stress) in that high FP birds showed higher heart rates, lower heart rate variability and higher levels of stress-induced corticosterone compared to low FP birds (Kjaer and Guémené 2009, Kjaer and Jørgensen, 2011; Kops et al., 2017). However, other studies have found that deterioration of feather cover was related to lower stress-induced corticosterone levels (Jensen et al., 2005), yet no such differences were found between birds performing SFP and non-SFP birds (Jensen et al., 2005; Daigle et al., 2015; van der Eijk et al. 2019). In summary, while (acute physical) stress seems to be relevant in the development of SFP, the direction and exact impact is still unclear.

However, SFP behaviour might also be fostered by frustration as a result of more chronic stressors, such as a non-species-specific environment, not only in terms of an inadequate physical environment (e.g., lacking opportunity to forage) but also in terms of an inadequate social environment (e.g., maternal deprivation, inadequate group-size or social disruption). Severe reoccurring or sustained stress during vulnerable developmental stages (e.g. prenatal or postnatal) can cause abnormal social behaviour (Sandi and Haller, 2015) or repetitive motor actions (Lewis and Kim, 2009). A disruption of social bonds, or a lack thereof, as via maternal deprivation (Martin, 2002), social stress (Sandi and Haller, 2015) or a barren physical environment (Mills, 2003), are stressors that can result in distress and foster permanent and irreversible changes of the CNS. Since laying hens (in non-cage systems) are reared in large social groups of several thousand birds, the establishment of species-specific social bonds (pecking order) is made nearly impossible (Rodenburg and Koene, 2007). The lack of such bonds or stable hierarchies together with the re-grouping of birds from rearing to laying facilities may constitute a severe chronic stressor (Cheng et al., 2003; Patzke et al., 2009).

It has been reported that laying hens housed in large groups of several thousand birds express increased levels of SFP (D´Eath et al., 2003). In commercial egg-production

systems, as hens reach their sexual maturity and commence egg laying, they are usually transferred from a rearing barn to a layer barn, disrupting existing social structures (Bestman et al., 2009). Introducing laying hens to unfamiliar conspecifics is stressful for them (Grigor et al., 1995; Lindberg and Nicol, 1996). Studies in humans and mammals have found that social stress during vulnerable stages in the development of an individual – such as the adolescence period – can have a severe and long-lasting impact on psychological wellbeing (Amat et al., 2005; Sterlemann et al., 2010).

In studies investigating the effects of long-term exposure to social stress, animals have shown increased hypothalamic-pituitary-adrenal (HPA) axis-activity and high mortality rates (Albeck et al., 1997). A number of validated models and experimental procedures exist to induce social stress in rodents (Henry and Stephens, 1972; Klein et al., 1992; Schmidt et al., 2007) and other animals, such as pigs (de Groot et al., 2001; Jarvis et al., 2006; Tuscherer et al., 1998; Koopmans et al., 2005), beef cows (Mench et al., 1990) and others (Proudfoot and Habing, 2015), based on the principle of disrupting a social group and thereby providing an unstable social environment. While there are a handful of studies investigating social stress in laying hens, these studies have been conducted in a cage-environment (Cheng et al., 2002; 2003; Cheng and Muir, 2004) and it is very difficult to draw any conclusion as to whether and how this kind of social stress treatment affected SFP behaviour without direct behavioural observations. However, there is a need to investigate the effects of social stress on SFP in a floor- based housing environment and in adolescent birds, since social disruption in this vulnerable stage of the birds´ life might represent a risk factor. Hence, chapter 4) will investigate the effect of social stress on SFP. From the previous sections it becomes clear that SFP arises in response to challenges, which the individual laying hen faces in a commercial production system. The review of these challenges is not yet concluded and challenges such as nutrition of the birds will be adressed in section 1.4. First, however, some consideration shall be given to different phenotypes involved in SFP. Since SFP behaviour takes place in a social setting where one bird delivers pecks while another one receives them, this situation produces at least three distinct phenotypes which shall be introduced next.

1.3.3 Feather pecking phenotypes: Active, recipient and neutral

The three distinct phenotypes in SFP are active peckers, recipients of pecks, and, if birds neither peck others nor receive pecks, individuals who are neither (neutrals) (Daigle et al., 2015). It has been suggested that these phenotypes differ from one another in more than just their behaviour, but also in terms of serotonin turnover rates in the dorsal thalamus at a young age after an acute stressor (which may indicate differences in impulse-control), with active peckers showing higher turnover rates than recipients or neutrals (Kops et al., 2013). In a study comparing gene expression in the brain (hypothalamic RNA) between active FP birds, recipients and neutrals, Brunberg et al., (2011) found differences between RNA-transcripts (linked to the immune system) of active FP birds and the other two phenotypes, while recipients and control birds appeared similar. These findings are important when studying FP, as not just the active pecker needs to be in focus, but also the recipient of FP, while neutral birds may serve as a control group. This manifestation of behaviour, and irreversibility thereof, presents a challenge to the view that SFP may be adaptive, redirected foraging behaviour. SFP is known to occur even in systems with access to a range, where birds can perform their species-specific foraging behaviour (e.g. Bestman and Wagenaar, 2014; Rodenburg et al., 2013; Nicol et al., 2013). Furthermore, outbreaks of SFP cannot be alleviated by providing opportunities to forage later (Lambton et al., 2015), demonstrating that SFP might not be reversible. Irreversibility or manifestation of an abnormal repetitive behaviour would suggest an underlying dysfunction. If a disruption of a neurobiological mechanism is identified to cause SFP, it may be viewed as “malfunctional behaviour” as defined by Mills, (2003). Whereas the lack of a disruption of neurobiological systems would mean SFP may represent a “maladaptive behaviour” (Mills, 2003). While a detailed discussion of whether SFP constitutes either a maladaptive or malfunctional behaviour is beyond the scope of this thesis, section 1.4 shall introduce the neurobiological view and review relevant literature that associates SFP with underlying neurobiological mechanisms. Therefore, there might be underlying physiological changes such as monoaminergic system and amino acid precursors which could be responsible for SFP. This will be outlines in next section.

1.4 Understanding severe feather pecking: The neurobiological view

1.4.1 Introduction

SFP has been introduced to develop as a sign of frustration due to the inability to perform foraging behaviour (section 1.3). However, this fails to explain how SFP manifests despite the possibility to forage. Hence, it seems that other mechanisms are at play, which result in SFP to manifest independently of improved physical environment. In this section, associations between physiological mechanisms and SFP shall be discussed. The term physiology covers a broad spectrum of disciplines, such as immunology, neurology, endocrinology, and others. This thesis, however, will focus on the role that aromatic amino acid metabolism and activation of the immune system might play in FP behaviour in laying hens. Particularly, it will focus on the metabolism of the two essential amino acids Tryptophan (TRP) and Phenylalanine (PHE). Both amino acids play an important role as precursors of the monoamines serotonin (5-HT) and dopamine (DA), respectively. Both precursors compete with one another for transport across the blood-brain barrier (BBB), making their central availability for monoamine- synthesis interdependent (Fernstrom and Wurtmann, 1972). Also, both amino acid pathways contain rate-limiting enzymes that are sensitive to stress and play a role in immune-activation (Strasser et al., 2016). As will be discussed in the following sections, 5-HT and DA have been identified to play a role in FP behaviour (section 1.4.2, 1.4.3, 1.4.4.and 1.4.6). The three effects: 1) competitive effects for central uptake, 2) role as precursors of 5-HT and DA and 3) enzymes sensitive to stress, make TRP and PHE metabolism prime targets for investigating underlying mechanisms of FP. In the following sections, the role of the neurotransmitters derived from these essential amino acids in the development of FP shall be discussed before moving on to amino acid metabolism (1.4.3 and 1.4.4).

1.4.2 Neurobiology of severe feather pecking: The monoamines dopamine and serotonin

Previous work has shown associations between SFP and two main neurotransmitters dopamine (DA) and serotonin (5-HT) involved in the development of various behavioural

processes (De Haas and van der Eijk, 2018). The neurotransmitter DA derives from the amino acid Tyrosine (TYR) via L-3,4-dihydroxyphenylalanine (L-DOPA), by tyrosine- hydroxylase (Kanehisa et al., 2017). TYR is either sourced through dietary intake or derived from PHE via phenylalanine-hydroxylase (Fernstrom and Fernstrom, 2007). Both TYR and PHE, for central dopamine bio-synthesis, are transported across the BBB via active transport through the heterodimeric membrane transport protein LAT1, which preferentially transports branched chain (Valine, Leucine, Isoleucine) and aromatic amino acids (PHE, TYR, TRP), with TYR being the precursor of DA.

Research on birds performing FP indicates that differences exist in DA turnover rates between birds divergently selected for high versus low FP activity (van Hierden et al., 2002). Yet Kops et al. (2013) found no link between FP phenotypes and altered central DA turnover rates, which the authors attributed to higher brain D1 and D2-receptor density or sensitivity. In a study using haloperidol (a DA-receptor antagonist) FP but not AP was reduced in treated birds (Kjaer et al., 2004). The exact role that the dopaminergic system plays in the development of AP and FP in laying hens remains unclear. However, these studies all indicate that DA plays an important role in the development of FP.

The neurotransmitter 5-HT is derived from the essential amino acid TRP by tryptophan- hydroxylase (TPH), which metabolizes TRP to 5-Hydroxytryptophan, which in turn is converted to 5-HT by aromatic-L- amino-acid decarboxylase (Fitzpatrick,1999).

Investigations on commercial birds differing in FP performance by van Hierden et al. (2002) found lower brain 5-HT turnover rates after manual restraint in high FP birds, suggesting a difference in the physiological response to this stress-treatment. While knowledge on 5-HT receptor-distribution in the avian brain is sill limited to date (Herold et al., 2012), immunohistochemical studies on the pigeon brain revealed that serotonin fibers and terminals are widely distributed in the avian brain. They are prominent in the telencephalon, diencephalon, mesencephalon and e.g. in the cranial nerve (Krebs et al.,

1991; Challet et al., 1996). The most widespread 5-HT receptor type (5-HT1A), which plays a role in functions such as learning and reinforcement, feeding behaviour and

locomotor activity (Zilles et al., 2000), was found to be similarly distributed in the avian brain as it is in homologous structures of the mammalian brain (Herold et al., 2012). The density of 5-HT1A -receptors was found to be highest in the nucleus pretectalis, followed by the tectum and the telencephalic nidopallium, the nidopallium and the hyperpallium.

The lowest 5-HT1A -density was found in the hippocampal formation, the amygdaloid complex the basal ganglia and the thalamic nuclei (Herold et al., 2012). A study using

S-15535, a somatodendritic 5-HT1A autoreceptor agonist showed that FP can be triggered by low 5-HT neurotransmission (van Hierden et a., 2004), showing possible involvement of this receptor in FP.

In chickens, various brain regions were studied in relation to 5-HT/DA and FP. Some work focused on the rostral section (“brain traversally cut rostrally to the midbrain 5-HT neurons”, rostral section was used for measurements). The 5-HT metabolite 5- hydroxyindoleacetic acid (5-HIAA)/5-HT ratio and the 3,4-dihydroxyphenylacetic acid (DOPAC) + homovanillic acid (HVA)/DA were investigated (van Hierden et al., 2002) and found to be lower in high feather pecking (HFP) birds compared to birds of the low feather pecking (LFP) line. Another study investigated 5-HT/DA turnover in four brain areas belonging to the basal ganglia-thalamopallial circuit: the dorsal thalamus, the medial striatum, the arcopallium and the hippocampus (Kops et al., 2013). Differences in 5-HT turnover between FP-phenotypes (severe FP) and non-peckers or victims were only found in the dorsal thalamus, GFP was recorded but not analyzed. No differences were found in DA turnover or between peckers and non-peckers in other brain regions. The authors concluded that serotonergic neurotransmission in the dorsal thalamus and striatum might depend on differences in behavioural phenotype, such as SFP, victims or neutrals (Kops et al., 2013). Another study investigated monoamine turnover in young and adolescent birds (Kops et al., 2017). The birds used in this study were of the same genetic lines as the birds which were used in this thesis. The authors studied seven brain-regions in relation to FP behaviour: the medial striatum, the thalamus, the hippocampus, the caudolateral nidopallium, the caudocentral nidopallium, the arcopallium and the amygdala. Results showed that HFP and LFP birds differed mostly in serotonergic activity in the dorsal thalamus, medial striatum, amygdala, caudolateral nidopallium and arcopallium. Differences in dopaminergic activity were found to a lesser

extent. However, Kops et al., (2017) found a negative relationship between low monoamine-turnover rates and high occurrence of SFP from an early age onward. While the authors specifically investigated SFP, they did report that allo-pecking in general was observed more often in HFP birds (Kops et al., 2017). In summary, while low central 5-HT activity in young chicks seems to be related to FP, there is a lack of information on the role of 5-HT later in life.

Considering the potential role of these neurotransmitters in the development of SFP, it is important to understand the pathway of how they are metabolized, which is explained in more detail in the following sections 1.4.3 and 1.4.4.

1.4.3 Aromatic amino acid metabolism: Tryptophan

The major metabolic pathway of TRP, the kynurenine pathway (Fig. 1.), consumes around 99% of TRP (Stone and Darlington, 2002) via TRP breakdown in the liver, leaving only a small proportion of TRP for 5-HT synthesis (Tyce, 1990). Central 5-HT production is, however, dependent on TRP crossing the BBB via the large amino acid transporter-1 (LAT1), since 5-HT in the periphery cannot cross the BBB. TRP competes with other branched chain amino acids for transport at the LAT1 transporter. Central TRP availability for 5-HT synthesis relies on peripheral availability of TRP. Increased activity of tryptophan hydroxylase in the periphery, sending more TRP down the kynurenine pathway (Fig.1.), may, therefore, play a role in reducing TRP availability for central 5-HT synthesis. In the first metabolic step of TRP towards kynurenine (KYN), the indole ring is opened by an oxidative reaction, which is catalysed by TDO and IDO- 1/IDO-2. While TDO is active almost exclusively in the liver, IDO is found in the periphery (macrophages), as well as in the central nervous system. Measured in the periphery, KYN to TRP ratios reflect IDO activity (Wiedner et al., 1999). IDO activity is increased in the course of inflammatory responses by T-helper-cell cytokines such as interferon and likely plays a role in immune-regulation and communication between the immune and the nervous system (Amirkhani et al., 2002; Bell et al., 2001; Grohman et al., 2003; Maneglier et al., 2004). While changes in the activity of IDO have been shown to be indicative of a broad spectrum of diseases ranging from renal diseases (Pawlak et

al., 2009), cancer (Suzuki et al., 2010) or Parkinson’s disease (Wiedner et al., 2001), the kynurenine pathway also lies at the centre of psychiatric pathologies, such as depression (Laugeray et al., 2010; Miura et al., 2009; Wichers et al., 2005). In summary, the kynurenine pathway is both stress-activated and the major metabolic branch of TRP, influencing the availability of TRP for 5-HT production, which likely plays an important role in the development of FP (1.4.6). Hence, TRP metabolism and the kynurenine pathway appear to be a promising mechanism to study in the context of FP behaviour. Yet, no study has investigated KYN to TRP ratios in relation to FP and it is still unclear whether IDO activity would be sensitive to social stress.

1.4.4 Aromatic amino acid metabolism: Phenylalanine

The conversion of PHE to TYR is the first metabolic step in the dopaminergic pathway. Phenylalanine-hydroxylase (PAH) requires molecular oxygen, iron and the co-factor tetrahydrobiopterin (BH4). PAH activity is measured by calculating PHE to TYR ratios. In the most common inborn error of amino acid metabolism in infants, phenylketonuria, a mutation in the PAH gene results in low expression of the gene and, hence, in low activity of the enzyme, resulting in high, even toxic concentrations of PHE from dietary intake. Aberrant behaviour and psychiatric symptoms can also become apparent during the development of children (Blau et al., 2010). Whereas this severe medical condition is directly linked to PAH activity, PAH is also involved in a number of other pathologies and research has focused on the role of PAH activity during infection and inflammatory response (Beisel, 1975; Wannemacher, 1975; Wannemacher et al., 1976; Zangerle et al., 2010). PHE/TYR reduction has also been shown to affect mood in healthy human subjects (Leyton et al., 2000) and influence addictive behaviour (Venugopalan et al., 2010). Yet the responsiveness of PHE / TYR ratios to the inflammatory and physical stress challenges (Strasser et al., 2016) make the PHE / TYR ratio an interesting target when investigating stress-responses.

1.5 Acute tryptophan depletion: a tool to study serotonergic synthesis in humans and rodents

Acute tryptophan depletion (ATD) is a nutritional tool to reduce blood TRP (Young, 2013). The principle underlying ATD is that dietary administration of an amino acid mixture lacking TRP enhances protein synthesis, which utilizes TRP in the blood system and tissue. This leads to a rapid decrease of TRP in the bloodstream (Moja et al., 1984; Young et al., 1985). Also, TRP competes at the blood-brain-barrier (BBB) with other large neutral amino acids (LNAA) for active transport across the BBB, and these competitive effects reduce TRP- intake into the brain when LNAA levels increase (Fernstrom and Wurtmann, 1979). This procedure was chosen to manipulate the central serotonergic system over feeding animals with lower levels of TRP in their diet, since feeding a diet with amino acid imbalance would result in a rapid decrease in food-intake (Young, 2013). When first employed as a method to investigate psychiatric disorders related to the serotonergic system, it was still a theoretical assumption that ATD actually decreases central 5-HT synthesis in humans (Ellenbogen et al., 1999). However, as early as 1974, Biggio et al. (1974) showed a drastic central decrease in brain TRP and 5-HT in rats. More recent studies have also confirmed the theory that ATD manipulates brain 5-HT levels (Hood et al., 2005), where a decrease in brain 5-HT blunts neurotransmission, which is assumed to be the underlying mechanism for behavioural effects. Alternative mechanisms, other than a reduction of central 5-HT synthesis, have also been suggested to be involved in causing behavioural responses to ATD, such as brain nitric oxide, decreased brain-derived neurotrophic factor and decreased kynurenine pathway metabolites (van Donkelaar et al., 2010). Although there is a need for positive control treatments, such as the acute PHE and TYR depletion, to exclude that any behavioural effect may be caused by alterations of these amino acid concentrations, ATD is still acknowledged as a valid tool in studying serotonergic synthesis by central TRP depletion (Young, 2013). Whereas the amino acid mixture used for ATD was, in the case of rats, based on minimal nutritional recommendations to allow growth (Biggio et al., 1974), recipes for humans were based on the amino acid composition of either breast milk or a 500g steak (Young et al., 1985). Instead of

administrating the amino acid mixture pure, which sometimes involved side effects, such as nausea, another method of ATD was developed, where the amino acids would be administered in peptide-form via a gelatine-based protein almost completely lacking TRP (Blokland et al., 2004). For birds, no ATD mixture has yet been developed or tested, yet any amino acid mixture intended for the use to deplete plasma levels of TRP would have to be based on species-specific nutrient requirements. In this thesis, ATD was studied to reduce TRP in birds (with the aim of manipulation 5-HT pathway and SFP). The ATD recipe was based on the laying hen nutritional guidelines by Leeson and Summers (2009).

1.6 Acute tryptophan depletion effects on affective states and behaviour

A number of psychiatric conditions in humans, such as depression, are thought to be influenced by underlying alterations to the serotonergic system (Meltzer, 1989). Hence, ATD has been used on a variety of healthy or psychiatric populations to study behavioural effects (Mendelsohn et al., 2009). ATD has been shown to alter mood (affective state) in volunteers with a family history of mood-disorders of underlying 5-HT dysfunction (Benkelfat et al., 1994; Klaassen et al., 1999), suggesting that these individuals may be particularly vulnerable to alterations in the serotonergic system. Studies with healthy subjects have described a lowering of mood post treatment (Young et al., 1985; Smith et al., 1987), yet subsequent studies have failed to replicate these results for healthy volunteers (Benkelfat et a., 1994; Knott et al., 1999). This discrepancy can possibly be explained by the baseline mood status of the subjects; studies which report a mood-altering response to ATD used subjects with depression scores indicative of an underlying mood disorder, while studies in which no effect of ATD was reported used subjects without diagnosed mood-disorders (Bell et al., 2001). ATD has also been reported to result in significant impairment of cognitive functioning in subjects suffering from dementia (Alzheimer’s disease) (Porter et al., 2000). ATD has also been reported to affect cognition by impairing the formation of long-term memory (Schmitt et al., 2000).

There is some evidence that ATD triggers an increase in the expression of schizophrenic symptoms in subjects suffering from schizophrenia (Sharma et al., 1997). For bipolar disorders, a role for 5-HT in mania has been hypothesized (Yatham et al., 1999), yet no effects of ATD have been reported for bipolar disorders (Cassidy et al., 1998). In obsessive compulsive disorders (OCD), selective serotonin reuptake inhibitors (SSRI’s) have been successfully used to treat symptoms of this disorder and it was hence hypothesized that ATD would result in a worsening of the symptoms, yet studies subjecting OCD patients to ATD have not confirmed this (Bar et al., 1994; Smeraldi et al., 1997). OCD patients express a lack of behavioural inhibition, which results in negative self-destructive behaviour (such as hair-pulling; trichotillomania) or negative social behaviour towards others (Coles et al., 2006; Strauss et al., 1988). These behaviours resemble some negative social behaviours that can be observed in animals, such as FP or barbering in mice, which has been proposed as a model for trichotillomania (Kurien et al., 2005).

Numerous studies have investigated the effects of ATD in rats. Some studies report that ATD significantly impairs object recognition (Olivier et al., 2008), while not causing depressive-like behaviour or anxiety-related behaviour (Lieben et al., 2004). Another study has, however, found anxiogenic and depression-like effects but no cognitive impairment in rats treated with ATD (Blokland et al., 2002). In another experiment, the effects of ATD on serial reversal learning was tested but no effect of ATD on the performance of the rats was reported (van der Plasse, 2008).

In conclusion, ATD has been widely used in research in rodents and humans to study the involvement of 5-HT in behavioural disorders. In particular, increased motor- disinhibition could be demonstrated in a number of rodent and human studies (see 1.6). It is hypothesized that such an effect of increased of motor-disinhibition in laying hens would result in increased pecking performance, both in an operant conditioning paradigm and in a group-housing situation.

1.7 The role of genetics in feather pecking

Breed, and therefore genetics, have long been identified as factors that contribute to the likelihood of a bird developing FP. Among poultry keepers of the late 19th century, crested chicken breeds such as “the polish chicken” were assumed to be at highest risk to develop FP behaviour. Poultry keepers observed that feed particles got stuck in the bird’s head-plumage and pecking of conspecifics was directed towards the head and face area of flock mates, which often resulted in severe damage of the decorative head- crest (Liebeskind, 1895).

1.7.1 Experimental selection to study severe feather pecking behaviour

In a selection experiment, Kjaer et al. (2001) divergently selected birds from a pure-bred line of White Leghorns based on their propensity to express FP behaviour. Within two generations, birds from the high FP line distinguished themselves from the low FP line by the number of FP bouts (Kjaer et al., 2001). Later, FP was proposed as a model for hyperactivity disorder due to a correlation between high FP and general activity (Kjaer, 2009). Yet a recent study was unable to show a correlation between FP and general activity (Kjaer, 2017). However, recent work shows that these lines differ in their performance in behavioural tests, such as a social test (reaction to unfamiliar birds) or manual restraint test, where low FP birds appear to perceive these challenges as less stressful than high FP birds (Kjaer and Jørgensen, 2011), meaning these lines may differ in their stress-respone (see 1.3.2). Current studies have also found significant differences between the two lines concerning their behavioural responses in standardized tests aimed at investigating fear-related responses, such as novel object tests and open field tests. In these tests, high FP phenotype and genotype showed a more active response to the behavioural tests, suggesting lower levels of fearfulness (van der Eijk et al., 2018a). Differences between FP-behaviour (FP-phenotype did not always correspond with genotype), even within the same genetic line, were described and call for caution as the pheno-genotype relationship is complex and not well understood (van der Eijk et al., 2018b). These results emphasize the importance of

investigating phenotypic information, even within lines, since these do not always correspond with behaviour predicted from genotype information.

1.7.2 Target genes involved in feather pecking behaviour

In section 1.3 the ethological link between FP and foraging/feeding behaviour has been discussed. It is noteworthy that such a link may also exist in terms of genetics, as shown by a study investigating genetic relationships between feather eating, FP and general motor activity. This study found that FP is, in part, affected by feather eating and by general motor activity (Lutz et al., 2016). Target genes have also been identified that are directly or indirectly associated with the absorption of nutrients from the intestine and mechanisms of the immune system (Brunberg et al., 2011; Parmentier et al., 2009). Other authors have also proposed a genetic link between the immune system and FP (Biscarini et al. 2010; Hughes and Buitenhuis 2010).

Genes involved in the regulation of the monoaminergic systems have also been in the focus of research trying to find key variables in the development of FP; Flisikowski et al. (2009) studied genetic variation associated with FP in laying hens and identified a gene which regulates the transcription of the 5-HT 1A receptor gene as a candidate gene, potentially underlying FP. Regulatory genes in the transcription of the 5-HT 1A receptor- gene have also been studied for their relevance in FP behaviour across a variety of genotypes (Wysocki et al., 2010; Wysocki et al., 2013). One candidate gene of interest in this study was the Tryptophan hydroxylase 1 (TPH1), which encodes the rate limiting enzyme in the synthesis of 5-HT. The PRKG1 gene, which encodes cGMP-dependent protein kinase type 1, was also suggested as a candidate gene for FP (Wysocki et al., 2007). Although Wysocki (2007) did not identify significant differences between gene expression of the selected genes between commercial lines. The authors suggested that the use of selected high and low FP lines may yield different results.

Other candidate genes have been associated with FP behaviour that are involved in plumage colour expression (PMEL17) by Keeling et al. (2004), who also found a highly significant quantitative trait locus (QTL) for feather damage, coinciding with the locus for white plumage colour, with white birds being significantly less susceptible to feather

damage than pigmented animals. This observation has been confirmed by Bright (2007), who also found that pigmented laying hens suffered from more feather damage than white birds. Nätt et al. (2007) also observed that between two PMEL17 homozygous groups (white vs wild-type plumage), the white type showed less FP behaviour than the wild-type. Certainly, while these genes may be relevant in the development of FP, there exist all-white leghorn lines selected for high FP activity, in which case plumage colour is irrelevant and, hence, the PMEL-gene cannot be the sole driver causing FP. Furthermore, while there have been no reports of FP in wild or feral fowl kept in a natural habitat (McBride 1969), it is noteworthy that even the most ancient genetic makeup of Gallus gallus, the red jungle fowl, express FP when confronted with a stressful environment (Vestergaard et al., 1993).

1.8 The role of nutrition in severe feather pecking

When reviewing literature on the role of nutrition per se in the development of FP, it is apparent that various aspects of nutrition have been associated with the development of FP (for a general overview see van Krimpen et al., 2005). A number of minerals and trace elements have also been associated with the development of FP, and some studies could show that there are components able to suppress FP behaviour effectively. For example, adding sodium-bicarbonate to drinking water has been reported to treat outbreaks of cannibalism (FP and cannibalism are not clearly distinguished in this publicatin) effectively (Swarbrick and Parsons, 1992), yet a deprivation of sodium-carbonite by Hughes and Wood-Gush (1973) did not lead to FP outbreaks. Higher protein content of feedstuff was also reported to counteract FP (Gerum and Kirchgessner, 1978; Ambrosen and Petersen, 1997). The source of protein (animal vs. plant based) also seems to play a role, with contradictory findings (McKeegan et al., 2001; Pfirter and Walser, 1998; Hadorn et al., 1998) as to whether a plant-based or animal-derived source of protein reduces or increases the likelihood to develop FP.

Another key component of poultry nutrition - of great relevance when it comes to the development of FP - is dietary fiber. Fiber has been shown to play a role in the development of FP (Wahlström et al., 1998; Hartini et al., (2002); van Krimpen et al.,

2009). A study by Esmail (1997) showed that an increase in crude fiber was negatively correlated with incidences of FP and mortality, while positively correlated to plumage condition. Furthermore, studies have found that the insoluble fraction of fiber helps prevent FP in laying hens (Aernie al., 2000; El Lethey et al., 2000; Hetland and Choct, 2003). While there is evidence that provision of feathers in the diet reduces SFP (Kriegeis et al., 2012), there exists evidence to the contrary too, reporting that loose, available feathers were positively correlated with FP (Ramadan and Borell 2008). The underlying mechanisms for fiber to reduce FP are not fully understood and it has been postulated that increased fiber-intake may lead to increased satiety levels or more time spent feeding (van Krimpen et al., 2005). It has also been reported that increased intake of insoluble fiber could increase gut-viscosity and gut-fill (Hartini et al., 2002). The ingestion of feathers may substitute the ingestion of fiber in that both have comparable effects on the digestive tract. By consuming feathers, feed-passage time is significantly increased (Harlander-Matauschek et al., 2006), an effect that is also achieved by the insoluble fraction of fiber (Kalmendal and Bessei, 2012). Furthermore, it has also been shown that birds selected for high FP consume more feathers than birds selected against FP or unselected control birds (Harlander-Matauschek et al., 2007). The assumption that birds eat feathers to compensate for the lack of structure-components in their feed is supported by the work of Hetland et al., (2005). Kalmental and Bessei, (2012) investigated the preference of birds selected for or against FP and found that birds selected for FP consumed a larger proportion of dietary fiber than did birds selected against FP.

While it appears well-established that feathers may serve as a partial substitute for insoluble fiber, ingestion of feathers could provide additional nutritional benefits to the birds besides mechanical effects in the digestive tract. In animal nutrition, feathers are hardly utilized as feed-stuff for their poor digestibility and low availability of essential amino acids such as methionine, lysine and tryptophan (Dalev et al., 1997; Latshaw et al., 1994; Papadopoulos et al., 1985). Conventional processing of feathers to obtain a more suitable feed-ingredient (increase digestibility) involve hydrothermal degradation. However, this process comes at the cost of further reducing essential amino acids such as lysine, methionine and tryptophan (Latshaw et al., 1994; Wang and Parsons 1997). It

has therefore been suggested to use biotechnological processes to break down the protein keratin into utilizable nutrients using bacteria or fungi (Onifade et al., 1998) - a process that also takes place in the gut of animals. As even in laboratory conditions the amounts and composition of amino acids gained from enzymatic breakdown of feathers via keratinolytic proteases vary greatly (Onifade et al., 1998), it is unclear to what extent the ingestion of feathers offers an additional source of nutrients (amino acids) to FP birds. Accumulating evidence, however, does indicate that birds who perform FP differ in the composition of their gut-microbiome (Birkl et al., 2018; van der Eijk et al., 2019) and metabolites produced thereof (Meyer et al., 2013). It remains unclear whether or to what extend this alteration in microbiome metabolism presents a cause or consequence of consuming feathers.

In terms of amino acids, studies have found that decreased lysine induces FP (Conson and Peterson, 1986), while increased arginine was found to stop FP behaviour (Siren, 1963). However, another study could not confirm any effect of arginine on FP behaviour (Madsen, 1966). Increased methionine in the diet was found to counteract FP (Hughes and Duncan, 1972), yet earlier studies failed to find any effect of methionine on FP behaviour (Creek and Dandy, 1957 loc. cit. in; Marshboon and Sierens, 1962). Dänner and Bessei in 2000, however, found that increased methionine improved feather-cover of birds. Yet it must be mentioned that methionine + cysteine levels were manipulated in this study and the effect can therefore not solely be attributed to methionine levels but to methionine + cysteine levels.

As previously mentioned, TRP metabolism plays a role in 5-HT synthesis and, therefore, possibly SFP development (see section 1.4). However, TRP levels in poultry nutrition have traditionally not been of particular interest, as it is assumed that the “minimum (amount of TRP) requirement necessary for tissue growth” is provided by standard laying hen diets (Summers and Leeson, 2009). This assumption may mean that a standard layer diet undersupplies birds with TRP who may have an individually increased need for TRP due to altered TRP metabolism (Russo et al., 2009). Recent work has already shown that at peak reproductive stress, birds may require higher levels of TRP than supplied by a standard layer ration (Khattak and Helmbrecht, 2018).

Existing requirements vary from 0.16 to 0.23% TRP in diet for laying hens (kept in cages) (NRC 1994; Harms and Russel, 1999, 2000; Peganova et al., 2003). While the work of Khattak and Helmbrecht, (2018) has shown that the requirement of TRP for laying hens, kept in colony cages, at peak egg-production is 0.22%, no such updated data exists for birds kept in floor-based housing systems. To the best of our knowledge, there have been no attempts to evaluate changes in TRP requirements caused by an increase in egg-laying. Similarly, possible differences in TRP requirements between genetics with high output in egg-mass (e.g. commercial hybrids) and genetics with a lower output in egg-mass (e.g. heritage breeds) have not been investigated.

TRP is the most well-studied amino acid in human and rodent research for its involvement in modulating central 5-HT synthesis and, hence, its influence on behaviours (Markus, 2008; Richard et al., 2009). TRP availability for central 5-HT synthesis is not solely dependent on absolute TRP concentration in plasma. Central TRP availability is determined by its concentration in plasma in relation to other large neutral amino acid concentrations, which depend on concentration-dependent transport across the blood-brain-barrier by a shared transport protein; large amino acid transporter 1 (LAT1) (Wurtman et al., 2003; Fernstrom, 2013).

TRP concentration in plasma is primarily dependent on dietary intake and the rate of protein synthesis (Richard et al., 2009). However, there also exist metabolic processes influencing inevitable TRP-loss, such as the activity of enzymes along the kynurenine pathway; tryptophan 2,3 –dioxyngenase (TDO) and indoleamine 2,3-dioxygenase-1/-2 (IDO-1/IDO-2) are activated by glucocorticoids and pro-inflammatory cytokines and are sensitive to stress (Ellen et al., 2014; Le Floch et al., 2011).

TRP supplementation of commercial diets has been shown to increase growth and reduce aggressive behaviour in broiler-type chickens (Rosebrough, 1996; Shea et al., 1990, Shea-Moore et al., 1996), and reduce occurrence of gentle FP in bantam chickens (Savory et al., 1999; Savory, 1998). Dietary supplementation of TRP in layers has also been shown to reduce the occurrence of gentle FP in experimental lines (van Hierden et al., 2004), while this effect could not be confirmed for commercial birds (Dewart, 2014). Yet, caution is advised when comparing these studies of TRP-

supplementation, since not only were different types of birds (e.g. bantam vs commercial hybrids) used, but also different concentrations of TRP-supplementation (Dewart, (2014): 0.31% vs van Hierden et al., (2004): 2% and Savory, (1998): 1% and 2%). Also, birds between studies were of different ages (chicks vs sexually mature layers). The role of amino acids in plasma, their involvement in neurobiological pathways, stress response and development of FP in laying hens has been looked at previoulsy in laying hens. It was found that lower TRP availability (expressed by TRP/ aromatic amino acid ratio) was positively linked to an early onset of injurious pecking behaviour, mostly affecting aggressive pecking (Birkl et al., 2017).

PHE, an essential amino acid in poultry nutrition, can be either utilized for protein synthesis or metabolized by phenylalanine hydroxylase (PAH) to tyrosine (TYR), the precursor of thyroid hormones and catecholamines such as DA. At the beginning of the dopaminergic pathway, the ratio of PHE/TYR is a meausre of PAH activity in the liver. In humans this measure is commonly used to monitor the immune status of patients during infection and as an indicator of tetrahydrobiotperin deficiency (Neurauter et al., 2008; Mangge et al., 2013).

1.8.1 Tryptophan, protein metabolism and protein-digestion

L-Tryptophan (TRP) is an essential amino acid for monogastric animals as it cannot be synthesized by these organisms but needs to be sourced via dietary intake (Huether et al., 2012). Although the ultimate source of TRP is dietary, the pool of free TRP in plasma is influenced by two mechanisms: dietary intake and intracellular protein- degradation. Diet provides approx. 1/3 of TRP and protein-degradation 2/3 (Yao et al., 2011) per day. Despite the fact that microbes in the large intestine may ferment undigested food and synthesize TRP, this source of TRP is likely insignificant for the host due to the small amount being produced and the limited absorption of TRP by colonocytes (Huether et al., 2012). In laying hens, a significant amount of protein and, hence, amino acids are partitioned towards daily reproduction (egg-production) and amino acid requirements need to be able to sustain the daily output of protein via egg- laying (Russel and Harms, 1999). Recent work showed that commercially recommended TRP requirements of laying hens may be underestimated during peak

reproduction (Khattak and Helmbrecht, 2018). Also, with respect to TRP levels in feed towards other large neutral amino acids (Peganova et al., 2003), studies in pigs showed that the TRP requirement is influenced by the supply of large neutral amino acids (Boomgaardt and Baker, 1973; Henry et al., 1992; Peisker et al., 1998). Peganova et al., (2018) suggest that deficient dietary TRP and competitive effects of TRP towards other large neutral amino acids may lead to an imbalanced influx of amino acids across the blood-brain barrier into the central nervous system. Competitive effects of amino acids and the experimental study of reduced TRP-influx into the central nervous system are further discussed in 1.7 and 1.8, 2.1 and 3.1.

Amino acids are derived from breaking down dietary protein into di- and tri-peptides or free amino acids before they can be absorbed into enterocytes in the mucosa of the small intestine (Webb et al., 1992). In poultry, microbial fermentation can take place in the crop, but the main process of digestion starts with hydrolysis of protein by pepsin and hydrochloric acid of the proventriculus an gizzard. In the small intestine, dietary protein is then further digested by proteases of the pancreas, such as trypsin and chymotrypsin. The carboxyl end of phenylalanine, tyrosine, tryptophan, valine and leucine are cleaved by chymotrypsin, while trypsin cleaves arginine and lysine. (Riviere and Tempst, 2001). Then, at the brush border membrane of the small intestinal mucosa, peptidases further digest protein before absorption (Leeson and Summers, 2009). The duration from dietary intake to a postprandial increase of plasma amino acids is influenced by four factors: the speed of the digesta along the gastro-intestinal tract, the hydrolysis rate of protein, the rate of absorption of amino acids and peptides in the small intestine, and amino acid metabolism of the small intestinal mucosa. Depending on the protein-source, digestion of dietary protein may take place faster or slower, which is measured by examining the post-prandial increase in amino acids (Boirie et al., 1997). For the experiments presented in this thesis, a pure amino acid mixture was used, aimed at lowering plasma TRP levels, as is the standard procedure for Acute Tryptophan Depletion studies (Young, 2013). Since it was unclear how long it would take for a post-prandial change in plasma amino acid levels, due to a pure amino acid mixture, post-prandial amino acid levels were measured over the course of several hours (chapter 2). Administering a meal lacking TRP (ATD) depletes plasma levels of

TRP by activating hepatic protein-synthesis, which removes extracellular TRP due to incorporation of TRP into protein mainly in the liver after ingesting a meal (Harper and Benevenga, 1970). Since the depletion of plasma levels of TRP can be blocked by the protein-inhibitor cycloheximide, protein synthesis is the main mechanism depleting plasma levels of TRP and ultimately decreasing 5-HT in the brain (van Donkelaar et al., 2011). There are studies that have investigated the time between meal-intake after fasting and post-prandial changes in somatotrophic and thyrotrophic hormones in plasma (Buise et al., 2002). An experiment in two strains of mice showed, however, that an ATD mixture reaches maximal depletion of plasma and hippocampal TRP levels after 100 minutes. Hippocampal levels 5-HT metabolite 5-HIAA reach a minimum after 200 minutes (Biskup et al., 2012). Another study on mice reports central effects (35- 60% depletion of TRP and 5-HIAA and 20-34% depletion of 5-HT) after 2.5 hours (Sanchez et al., 2015). A rough idea of the time intervals one might expect for chickens could be provided by comparing the duration of digestion (gastrointestinal transit time, GIT) of mice and chickens. One study reports that feed (a pill of oatmeal, barium sulfide and water) passed through the small intestine of fasted layers within 2 hours (Hillerman et al., 1953); 3 hours and 40 minutes were reported by the same authors for total GIT. In comparison, GIT in fasted mice is approximately 3 hours (Myagmarjalbuu et al., 2013). Ultimately, however, only the measurement of central effects of ATD on TRP and 5-HT will provide clear information of the magnitude and time frame of ATD in laying hens (see section 5.4). However, the first step will be to develop a dietary protocol for ATD in laying hens and measure whether plasma-depletion of TRP in the periphery can be achieved by such a protocol.

1.9 Specific objectives

The main goal of this thesis was to investigate effects of a nutritional (acute depletion of TRP) and a social stress (social disruption) treatment on neurobiological characteristics that may be causally related to the development of SFP. For this purpose, genetic lines selected for high or low levels of SFP were used as a model.

In Chapter 2), based on findings from human and rodent-studies (1.5) and knowledge about the involvement of 5-HT in SFP (1.4), we tested whether a mixture of amino acids lacking 5-HT-precursor TRP could reduce plasma levels of TRP. We also determined the time it would take for such a dietary mixture of pure amino acids to affect plasma levels of TRP and TRP ratios towards LNAA and AAA.

In Chapter 3) we then tested whether ATD would affect SFP behaviour. We predicted that after ATD-treatment HFP birds would engage in more SFP and increase their pecking activity in an operant conditioning paradigm, while non-FP birds would show no such response.

Finally, in Chapter 4) we investigated whether social disruption would result in an increase in SFP behaviour, upregulation of IDO and TDO (TRP/KYN metabolism) and increased PAH activtiy (PHE/TYR metabolism). We also tested whether HFP birds were most responsive to social mixing with respect to these immune-parameters.

To test the above formulated hypothesis, this thesis is composed of two main objectives:

Chapter 2) describes the development and test of a method of ATD in laying hens; a non-invasive, non-pharmacological means to reduce plasma levels of TRP.

Chapter 3) reports on a test for an increase in FP following ATD in a group-housing situation and a test for an increase in unrewarded pecking in an operant conditioning chamber.

1.9.2 Objective 2: The effects of a social disruption stress treatment on TRP metabolism and FP behaviour in laying hens

Chapter 4) evaluates the influence of social stress on FP behaviour and TRP metabolism of laying hens divergently selected for FP.

Chapter 5) provides a synthesis of the thesis and discusses major findings, shortcomings and directions for future research.

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2 Acute tryptophan depletion: the first method validation in an avian species (Gallus gallus domesticus) 1

Abstract

Acute tryptophan depletion (ATD) is a valuable non-invasive nutritional tool in human and rodent research to study the effects of reduced tryptophan availability on behaviour, mostly in the context of behavioural disorders linked to the serotonergic system. The serotonergic system is thought to be involved in the development of feather pecking behaviour of laying hens, one of the most relevant welfare and production issues in modern intensive egg-production systems. ATD temporarily compromises the influx of tryptophan (TRP) across the blood brain barrier (BBB), which reduces central availability of TRP, the substrate for serotonin (5-HT) synthesis. However, ATD has never before been developed and evaluated in birds. In this work, we administered an ATD-amino acid mixture and a control mixture containing TRP to 10 laying hens. Blood samples were taken every full hour and plasma levels of TRP were measured. We hereby report that ATD in laying hens effectively reduces plasma levels of TRP to 50% of the baseline concentration, 4 hours after administration. Furthermore, ATD was found to reduce the ratio of TRP towards aromatic amino acids (AAA) by 60% and the ratio of TRP towards large neutral amino acids (LNAA) by 70%, three hours after administration. Further studies are needed to determine the effects of peripheral depletion on brain TRP and 5-HT levels in birds. However, our study showed for the first time in an avian species that ATD causes the lowering of both plasma TRP and the ratio in plasma of TRP towards other AAA or LNAA.

2.1 Introduction

Feather pecking (FP) is one of the most serious welfare and production issues in modern intensive egg-producing systems (Lambton et al., 2010), since FP behaviour

1 An edited version of this chapter has been published in Poultry Science 96(9), pp.3021-3025, with the following authors: Birkl, P., Kjaer, J.B., Szkotnicki, W., Forsythe, P. and Harlander-Matauschek, A.

and resulting cannibalism can lead to high rates of mortality (Kjaer, 1996). Studies investigating underlying mechanisms of FP behaviour point towards a serotonergic involvement in the development of FP (van Hierden et al., 2002; 2004). In humans and rodents, one way of investigating serotonergic involvement in behavioural disorders is to acutely deplete individuals of the serotonin (5-HT) precursor tryptophan (TRP) via oral administration of an acute tryptophan depletion (ATD) amino acid mixture lacking TRP (Dingerkus et al. 2012). TRP competes with other large neutral amino acids (LNAA) and particularly with aromatic amino acids (AAA) for active, concentration-dependent transport across the blood brain barrier (BBB), via the large amino acid transporter-1 (Christensen and Arbor, 1990). As ATD reduces plasma levels of TRP, yet increases levels of LNAAs, the TRP availability for central 5-HT synthesis is temporarily reduced (Young, 2013). The dietary method of ATD reduces brain 5-HT in mammals to around 40-60% of baseline levels (Lieben et al., 2004; Olivier et al., 2008). Pharmacological inhibition of the TRP hydroxylase (Sjoeerdsma et al., 1970), via parachlorphenylalanine (PCPA) can decrease brain 5-HT to below 5% (e.g. Meek and Neff, 1972). However, this might be less representative of a physiological state. Also, pharmacological 5-HT depletion by PCPA has been reported to involve side effects, such as insomnia (Mouret et al., 1968), toxicity (Young, 2013), and loss of appetite after treatment (Panksepp and Nance, 1974), in rats and humans. Similar side effects can be assumed in birds and might bias avian behaviour involving an appetitive motivational component, such as FP.

Yet, to utilize ATD in avian research, the following three key questions need to be answered: Firstly, does ATD cause a substantial lowering of plasma TRP and the ratio in plasma of TRP towards other LNAA (TRP/ΣLNAA)? Secondly, if ATD is lowering plasma TRP and/or ratios, is there a decline in brain TRP and 5-HT? Thirdly, what implications does such brain decline have on avian FP behaviour?

To address the first question, we composed an amino acid mixture lacking TRP, which was orally administered to laying hens. We then measured the plasma concentrations of TRP and other LNAA, allowing us to test whether such a mixture can decrease TRP levels and the ratios of TRP towards LNAA (TRP/ΣLNAA) and AAA (TRP/ΣAAA) effectively in laying hens.

2.3 Materials and Methods

2.3.1 Animals and Housing

For this experiment, we used 10 non-beak trimmed female laying hens (White Leghorn) of a genetic line selected for high levels of feather pecking behaviour (Kjaer et al., 2001). Birds were housed in 10 groups of 16 individuals per group under conventional management conditions at the research facility of the University of Guelph, Canada. Daily care and handling of birds was in correspondence with the procedures postulated in the Animal User Protocol No. 3206, which was approved by the animal care committee of the University of Guelph. Water and feed was provided ad libitum. We used an Arkell Research Station layer mash diet with the following nutritional specifications; crude protein (min): 18%, crude fat (min): 5.5%, crude fiber (max): 2%, calcium (actual): 4.24%, phosphorus (actual): 0.68%, sodium (actual): 0.18%, vitamin A (min) 16,500 IU/kg, vitamin D (min) 4,130 IU/kg, vitamin E (min): 60 IU/kg. Analytical feed specifications are provided in the Appendix (Table 6). Each pen was equipped with 12 cm of feeder space per hen, 8 nipple drinkers, wood shavings as litter substrate, 18 cm of elevated perch-space per hen and 3 nest boxes. Visual contact between groups was prevented by placing opaque PVC boards between the pens. Commercial lamps were used for the lighting schedule recommended by the LSL- Lohmann management guide for birds at 22 weeks or older; of 16 h light: 8 h dark periods at 15 lux light intensity. Average daily room temperature was 20° C. At the time of the experiment the birds were 40 weeks of age and bodyweights of birds ranged from 1912 g to 2123 g, with a mean weight of 1991.4 g ± 92 (M ± SD) for the five birds receiving the control treatment, and 1987.6 g ± 73.9 for the five birds receiving the ATD treatment. The birds received no feed during the time of the experiment but had free access to water.

2.3.2 Acute Tryptophan Depletion Mixture and Sample Collection

The amino acid mixture used for ATD in laying hens with an average body weight of 2 kg was composed of the following 12 amino acids: Arginine (0.145 g), Cysteine (0.081 g), Glycine (0.208 g), Histidine (0.035 g), Isoleucine (0.135 g), Leucine (0.17 g), Lysine (0.145 g), Methionine (0.062 g), Phenylalanine (0.098 g), Threonine (0.098 g), Tyrosine

(0.08 g), Tryptophan (0.033 g for the balanced control treatment) and Valine (0.145 g). The total amount of 1.435 g was homogenized and aliquoted into two 1 g gelatine capsules. Aliquots were rounded to two decimal places. The amounts are based on minimum daily requirements for white leghorn type laying hens, as stated in the Nutritional Requirements for Poultry 1994, by the National Research Council, divided by four to account for the duration of the experiment (6 hours).

At 5:45 am, 15 minutes before the beginning of dawn in the light program and therefore prior to feed intake, 10 birds (feather pecking phenotype and genotype) were taken out of their home pens and two gelatine capsules were orally administered to each bird, containing either the ATD (n = 5) or the control (C) treatment (n = 5). Palpation of the crop was performed to ensure swallowing of the capsules and to confirm the crops were empty prior to administration of the capsules. The hens were then transferred into individual pens where they were provided with access to water. Blood samples were collected at the following time points after administering the ATD/C treatment: 0 hours, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours post-treatment. Sampling times were chosen according to the duration of digestion in poultry (Leeson and Summers 2001). 1.0 ml of blood per bird per sample was collected from the wing or metatarsal vein using 21 Gauge-needle winged infusion kits, into 2 ml EDTA (3.6 mg) coated tubes. Tubes were immediately placed on ice until samples were centrifuged at 2000 x g for 15 minutes at 4°C to obtain plasma, which was then transferred into 1.5 ml micro centrifuge tubes and stored at -80°C until amino acid analysis.

2.3.3 Amino Acid Analysis

The Amino Acid Analysis was performed on a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system. Aliquots of the plasma samples were transferred into a 6 by 55 mm glass culture tube and dried under vacuum using a centrifugal evaporator. After drying, the samples were treated with a re-drying solution consisting of methanol: water: triethylamine (2:2:1), vortex-mixed and dried under vacuum for 15 minutes. Then the samples were derivatized for 20 minutes at room temperature with a derivatizing solution made up of methanol: water: triethylamine: phenylisothiocyanate (PITC)

(7:1:1:1). After 20 minutes, the sample is dried under vacuum for 15 minutes. Then the derivatized sample was again washed with the re-drying solution, vortex-mixed and dried under vacuum for 15 minutes. The derivatized sample was dissolved in a given amount of sample diluent (pH 7.40) and an aliquot injected into the column, running on a modified PICO-TAG gradient. Column temperature was at 48°C. The derivatized amino acids were detected at 254 nm. The Waters Acquity UPLC system employed consisted of a Binary Solvent Manager, a Sample Manager, a TUV Detector and a Waters Acquity UPLC BEH C18 column (2.1 X 100 mm). Data was collected, stored and processed using Waters Empower 3 Chromatography software. Drying was done using a Tomy CC-181 Centrifugal Concentrator with a Sargent-Welch Model 8821 Vacuum pump. The analysis was performed at the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre (SPARC) Biocentre at the SickKids Hospital, Toronto.

2.3.4 Statistical Analysis

We used the Proc Mixed procedure in SAS version 9.4 (SAS Institute Inc., Cary, NC, USA, 2012) to compare ATD to the control treatment. In this model tryptophan concentration was the response variable. Treatment was treated as a fixed effect, and the interaction of time (linear, quadratic, and cubic) with treatment was also fitted. Compound symmetry structure was used (TYPE=CS) with individual as the repeated measure subject. The interaction of treatment by time (linear, quadratic, and cubic) was tested for significance. Degrees of freedom were calculated using a between and within method approach (DDFM=BW).

2.4 Results and Discussion

Oral administration of the ATD mixture decreased plasma TRP levels by 50%, TRP/ΣAAA ratios by 60% and TRP/ΣLNAA ratios by 73% (Table 1). The results of the entire amino-acid assay are presented in Table 2 of this thesis. A Proc Mixed procedure in SAS revealed that the fitted curves for control and ATD treatment differ significantly from one another (the test of fixed effects revealed significant linear F1,2=15.5, P <

0.0003, quadratic F1,2=8.21, P < 0.0009 and cubic effects F1,2=6.95, P < 0.0024).

Tryptophan levels reached a minimum at 4 hours after administration of the ATD mixture (Table 1, Figure 1), whereas TRP/ΣAAA and TRP/ΣLNAA ratios were lowest at 3 hours post treatment. This discrepancy can be explained by the peak of AAA (tyrosine and phenylalanine) and LNAA (Tyrosine, Phenylalanine, Valine, Leucine and Isoleucine; data presented in supplementary material) occurring at 3 hours post administration of the control and ATD amino acid mixtures. This rise in LNAA and AAA levels after the ATD and control treatment resulted in decreased TRP/ΣLNAA ratios despite an increase in TRP concentrations in the control treatment (Figure 1). The reduction in plasma TRP levels achieved in this study (-50%) is lower than what studies in humans (Young et al., 1985) or in rats had previously reported (-75%) (Gessa et al., 1974). There are a number of possible reasons why the amino acid mixture used in laying hens did not deplete plasma levels of TRP to the extent measured in rodent and human studies. Chickens, unlike rodents or primates, possess a crop where an entire or partial meal can be stored. Although the initial ingestion of the capsules was controlled by palpating the crop of the chickens after ingesting the capsule, it can be assumed that a part of the amino acid mixture was regurgitated into the crop, delaying the passage time of the mixture from the gizzard into the duodenum, while also reducing the initial amount of mixture that reaches the duodenum. Such a process could also explain how the maximum depletion of TRP occurred at different points in time for some individuals (Table 1). However, as there are no digestive processes taking place in the crop (Leeson and Summers, 2001; Svihus, 2014), the achieved decrease of TRP levels may also relate to properties of the amino acid mixture itself. ATD mixtures for humans are based on the amino acid composition of a 500 g steak (Young et al., 1985) or on the amino acid composition of breast milk (Young et al., 1987). Our recipe was based on the daily amino acid requirements for laying hens (National Research Council, 1994), calculated for the length of the experiment (6 hours). As there is little information on daily requirements for non-essential amino acids in laying hens (e.g. cysteine), we derived the concentration of these amino acids for the ATD recipe by calculating the relative amounts of these amino acids towards TRP from an ATD recipe for humans (Daly et al., 2014). For cysteine, Daly et al. (2014) used 2.7 g of cysteine towards 2.3 g of TRP. As our recipe contained 0.033 g TRP (derived from daily nutritional

requirements, calculated for 6 hours), we used 0.462 g of cysteine. This results in a 1.4 : 1 ratio of cysteine towards TRP in both recipes for humans and chickens. However, our histidine: TRP, tyrosine : TRP and leucine : TRP ratios are based on minimum nutritional requirements for laying hens (National Research Council, 1994). Therefore, we used 0.035 g of histidine towards 0.033 g of TRP, 0.080 g of tyrosine and 0.170 g of leucine, resulting in a histidine: TRP ratio of 1 : 1 , compared to 1.4 : 1 in Daley et al.’s recipe, a tyrosine : TRP ratio of 2.5 : 1, compared to a 3 : 1 ratio, and a leucine : TRP ratio of approximately 5 : 1, compared to 6 : 1 ratio in the human study. These smaller relative amounts of amino acids towards TRP could be responsible for why the observed depletion of TRP did not exceed a 50% reduction of plasma TRP levels 3 hours after ATD intake. Reduced availability of essential amino acids, such as histidine or leucine, might limit protein synthesis and therefore prevent further depletion of plasma TRP (Harper and Benevenga, 1970). While our study was able to demonstrate that ATD might be a useful non-pharmacological tool to lower plasma TRP levels in the periphery, further studies are needed to determine the effects of a peripheral depletion on central TRP and 5-HT levels in birds. Also, further fine-tuning is required to determine the ideal ATD recipe for birds. However, this present study demonstrates that ATD in laying hens effectively reduces plasma TRP levels. ATD may therefore be considered as a promising, non-invasive, nutritional tool to investigate serotonergic involvement in a spectrum of atypical behaviours for which a serotonergic involvement is assumed, such as FP in laying hens (van Hierden et al., 2002) or compulsive feather picking in parrots (Grindlinger, 1993).

Acknowledgement:

This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC DG Grant No. 0531194). We thank the Evonik AG, especially Dr. Ariane Helmbrecht, for providing the amino acids free of charge.

2.2 References

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A. Blokland. (2008). Acute tryptophan depletion dose dependently impairs object memory in serotonin transporter knockout rats. Psychopharmacology (Berl.), 200, 243-254.

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Svihus, B. (2014). Function of the digestive system. Journal of Applied Poultry Research 23, 306–314. van Hierden, Y. M., S. F. de Boer, J. M. Koolhaas, and S. M. Korte. (2004). The control of feather pecking by serotonin. Behavioral Neuroscience 118, 575–583. van Hierden, Y. M., S. M. Korte, E. W. Ruesink, C. G. van Reenen, B. Engel, G. A. Korte-Bouws, J. M. Koolhaas, and H. J. Blokhuis. (2002). Adrenocortical reactivity and central serotonin and dopamine turnover in young chicks from a high and low feather-pecking line of laying hens. Physiology & Behavior 75, 653–659.Kjaer, J. (1996).

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2.3 Tables and Figures

Time Treatment Ind. 1 Ind. 2 Ind. 3 Ind. 4 Ind. 5 Average deviation from baseline [h]

0 49.5 72.6 46.7 61.9 56.9 TRP TRP/LNAA TRP/AAA

2 50.9 89.8 105.3 92.5 64.1 23.0% -37.3% 8.8%

3 27.0 68.4 50.6 65.5 57.8 -3.6% -38.7% -0.9% Control 4 44.4 61.8 41.9 49.9 54.1 -7.1% -32.8% 6.8%

5 46.1 63.5 42.4 46.1 54.4 -7.0% -28.1% 6.5%

6 41.2 62.9 43.1 42.8 49.9 -9.5% -26.8% -1.0%

0 54.2 48.3 62.9 59.4 56.2 TRP TRP/LNAA TRP/AAA

2 32.9 39.7 57.5 38.9 33.6 -15.7% -61.2% -54.0%

3 25.0 33.6 36.4 39.2 29.3 -23.5% -73.1% -60.1% ATD 4 25.8 34.4 27.4 38.6 37.4 -49.7% -65.0% -51.0%

5 38.0 40.4 30.6 42.5 41.3 -48.5% -54.1% -55.6%

6 36.8 45.8 33.2 36.7 45.0 -48.3% -47.5% -31.7%

Treatment Timepoint [h] TRP [nmol/ml] PHE [nmol/ml] TYR [nmol/ml] Gln [nmol/ml] Glu [nmol/ml] Val [nmol/ml] Leu [nmol/ml] Ileu [nmol/ml] c 0 58 120 226 715 196 366 316 188 c 2 81 170 275 546 175 1213 490 568 c 3 54 121 206 473 142 862 308 365 c 4 50 108 176 583 151 744 285 274 c 5 50 112 173 639 128 671 294 237 c 6 48 115 176 657 119 596 287 213 Treatment Timepoint [h] TRP [nmol/ml] PHE [nmol/ml] TYR [nmol/ml] Gln [nmol/ml] Glu [nmol/ml] Val [nmol/ml] Leu [nmol/ml] Ileu [nmol/ml] t 0 56 116 153 734 196 260 239 119 t 2 41 174 247 625 169 982 294 328 t 3 33 146 246 639 147 944 287 295 t 4 33 115 205 674 134 747 217 192 t 5 39 113 182 780 134 643 223 165 t 6 39 110 166 821 114 549 218 143

3 Effects of acute tryptophan depletion on pecking behaviour in laying hens2

Abstract

Pecking at the feather cover of other birds (FP) is one of the most important welfare problems in domestic birds, possibly linked to underlying changes in the serotonergic system. Acute tryptophan depletion (ATD) is a widely used method for studying serotonergic function in mammals, and a recipe to deplete peripheral tryptophan levels has been recently validated in birds (chapter 2). However, no studies to date have assessed the impacts of a tryptophan-deficient amino acid mixture on bird social behaviour, including feather pecking, or motor performance. In this work, 160 White Leghorn laying hens were kept in 10 groups of 16 birds. Each group included birds from two genetic lines, divergently selected to perform high (HFP; 4 birds per group) or low (LFP; 3 birds per group) levels of FP, and from an unselected control line (UC; 9 birds per group). Each group was assessed for feather pecking following exposure to ATD and balanced control (BC) feeding treatments; treatment order was counterbalanced across groups. The effect of ATD/BC on motor response (pecking) was also investigated using a 5-second delayed reward task in an operant chamber. Responses were assessed in 10 phenotypic feather peckers, 10 recipients of feather pecking and 10 bystanders (who neither performed nor received feather pecks). ATD given to groups of birds induced gentle feather pecking in all genotypes. Following ATD, phenotypic feather peckers performed more poorly during the delayed reward task, as seen by their higher number of non-rewarded key and non-key pecks towards an illuminated pecking key, in the operant chamber. In conclusion, ATD impacted the hens’ pecking behaviour by increasing the number of gentle feather pecks at conspecifics. Furthermore, feather peckers were more likely to peck while waiting for a reward after ATD.

2 An edited version of this manuscript has been published in Frontiers in Veterinary Sciences, with the following authors: Birkl, P., Chow, J., McBride, P., Kjaer, J.B., Kunze, W., Forsythe, P. and Harlander- Matauschek, A.

3.1 Introduction

Birds kept for egg-laying engage in a variety of social interactions. However, some behaviours can become problematic, such as feather pecking (FP) (Mench and Blatchford, 2014), such as severe feather pecking (SFP).

Ethologists consider SFP to be an expression of frustration arising from a lack of adequate opportunity to perform foraging behaviour (Wennrich, 1975). However, SFP is also known to occur even when birds are provided with outdoor access to accommodate foraging (Lambton et al., 2010). Additionally, the biological basis of motor-behaviour may include the modulatory role of 5-HT, which is involved in various forms of motor activity (Harmer et al., 2009; Langen et al., 2011; Soubrie et al., 1986). Similarly, it has been suggested that the avian 5-HT system (de Haas and van der Eijk, 2018) and the precursor tryptophan (TRP) are intimately linked to FP. Furthermore, genetic factors (Kjaer et al., 2001), such as candidate genes linked to the 5-HT system (Biscarini et al., 2010), lower forebrain 5-HT turnover levels (van Hierden et al., 2002; 2004), and nutritional factors, including low dietary TRP (Savory, 1998), also point towards serotonergic involvement in the development of FP behaviour.

Since 5-HT cannot cross the blood-brain barrier, central 5-HT synthesis depends on the availability and transport of its precursor, TRP, in the brain (Leathwood and Fernstrom, 1990). TRP competes with other large neutral amino acids (LNAAs) for active transport by the large amino acid transporter system (Markus et al., 2000). Consequently, decreased peripheral TRP relative to other LNAAs results in a lower TRP influx into the central nervous system (see chapter 1.4). This phenomenon is demonstrated by the dietary acute tryptophan depletion (ATD) method, in which an orally-administered amino acid mixture, lacking TRP that reduces 5-HT levels in the brain (Young et al., 1989; Hood et al., 2005), is used as a non-invasive research tool to assess the involvement of the 5-HT system in human psychiatric disorders. To this end, Birkl et al. (2017) showed for the first time that an ATD mixture in laying hens effectively reduces the ratio of TRP to all LNAA by 70% of baseline levels, three hours after administration.

It was previously shown that the 5-HT system might modulate the social behaviour problem of FP in laying hens (Kops et al., 2017). Chapter 2) of this thesis has demonstrated that ATD can reduce peripheral TRP-levels in laying hens. Yet, it is unclear whether ATD would result in an increase of SFP in a group setting. Hence, this was studied in the first experiment of this chapter. The second experiment used a modified, delayed reward task in an operant chamber to assess pecking behaviour in laying hens; the number of pecks at an illuminatable pecking key were compared before and after ATD treatment in individuals categorized based on their phenotypic FP behaviour. Human studies have shown that participants perform more poorly with a dysphoric distractor in a proofreading task after ATD treatment, possibly due to ATD causing a shift in attention to the distractor (Harmer et al., 2009). As such, we hypothesized that ATD-treated birds in an operant conditioning chamber would shift their attention to a dysphoric or frustrating stimulus (a 5-second delay in reward dispense), as shown by increasing the number of pecks at the pecking key during the delay period. Finally, we hypothesized that this effect of ATD would be most pronounced in hens phenotypically identified as active feather peckers.

3.2 Materials and methods

3.2.1 Animals and housing

Sexually mature birds with a tendency to engage in FP were required for this experiment. One hundred and sixty non-beak-trimmed White Leghorn laying hens were raised in 10 identical aviary pens (16 birds per pen) at Arkell Research Station at the University of Guelph. The high (HFP) and low (LFP) FP, and unselected control birds (UC) in the present study originated from a selection experiment (Kjaer et al., 2001) in which birds of one line were divergently selected based on high and low levels of FP behaviour. Each pen contained 4 HFP, 3 LFP, and 9 UC line birds. Birds were kept in identical pens littered with wood shavings (9 birds/m2; 15 cm perch length/bird at 90 cm above the ground; 125 x 31 cm platforms, 65 cm above the ground; 120 cm2 nest/bird; 10 cm food trough length/bird; 10 nipple drinkers). Cameras (Samsung SNO-5080R, IR, Samsung Techwin CO., Gyeongi-do Korea) were mounted on the ceiling above the

entrance of each pen. Food, commercial laying hen mash, (analytical feed specifications are provided in 6. Appendix (Table 6)) and water were provided ad libitum. The hens were kept in a ventilated, windowless room, on a 14:10 h light:dark cycle, with a light intensity of 25 lx at animal level, and an average daily room temperature of 20°C. The pens were separated by opaque boards to prevent physical and visual contact with birds from other pens (see Kozak et al., 2016, for more details on housing).

3.2.2 Operant chamber test equipment

The custom-made operant chamber and computer (Med Associates, St. Albans, VT, USA) used in this experiment were kept in a testing room a short distance from the home pens. The polycarbonate operant chamber measured 52 L x 50.5 W x 56 H cm. One lighted pecking key (red light) was presented 36 cm above the ground (2.5 cm diameter). The feeder was accessible through a rectangular hole (13.5 L x 4.5 H cm) in the center of the test panel wall, 25 cm above the floor. The feeder was covered by a lid, which was manually opened and closed by the experimenter; food was only accessible when the lid was open. A LED house light was placed on top of the box to indicate the start of a trial and would automatically turn off at the end of each session. The operant chamber was controlled using the Trans IV program (Med Associates, St. Albans, VT, USA). The number of rewards received and the number of pecks on the pecking key were automatically recorded and saved. A camera (JVC GC-PX100BU HD Everio) was set up in front of the operant chamber to record each session.

3.2.3 Acute tryptophan depletion and balanced control capsule

The amino acid mixtures used for ATD and Balanced Control (BC) treatments were composed of the following 12 amino acids (Evonik, Essen, Germany): Arginine (0.145g), Cysteine (0.081g), Glycine (0.208g), Histidine (0.035g), Isoleucine (0.135g), Leucine (0.17g), Lysine (0.145g), Methionine (0.062g), Phenylalanine (0.098g), Threonine (0.098g), Tyrosine (0.08g), Tryptophan (0.033g for Balanced Control, 0g for ATD), and Valine (0.145g). The resulting mixture of 1.435g was homogenized and

aliquoted into two 1g gelatin capsules (1.5 x 0.5cm). For more details, see Birkl et al. (2017).

3.2.4 Tryptophan–deficient amino acid mixtures to groups of laying hens – feather pecking

For identification, the birds were fitted with numbered silicone “backpacks”, which consisted of two silicone squares (14.5cm x 6cm x 0.2cm) resting on the back of the bird, secured with two plastic-coated clothesline wires, which looped under the wings and attached to the silicone via eyelets (Harlander-Matauschek and Häusler, 2009).

We used gelatin capsules (CapsulCN® Int. CO., LTD size 00; cubage 0,95 cc), filled with a mixture of hard-boiled, chopped eggs and commercial feed to habituate the birds to swallowing the capsules. Each bird was provided with five capsules while being held in a holding pen (dog crate), for a max. of 10 minutes. This was done three times on three consecutive days. Birds were brought back into their home pens after they had consumed all five capsules, as on the second day all birds consumed the five capsules within less than a minute. As outlined in Figure 3, laying hens were treated (ATD/BC) and observed (baseline/treatment) over a period of one week. Baseline refers to birds who have not received any treatment prior to observations. Treatment consisted of ATD and BC treatment. Pens were assigned to a counterbalanced order of capsule consumption: pens 1 to 5 received two ATD capsules first, and pens 6 to 10 received two BC capsules first. The ATD/BC capsules were administered to all hens, beginning at 7:00 a.m. The time of capsule administration for each pen was recorded to ensure that video analysis would commence exactly three hours after capsule administration. All occurrences (Altmann, 1974) of FP interactions from the videos were observed and classified as gentle (repeated pecks at the tips and edges of feathers without removal of the entire feather) or severe (forceful pecking and/or removal of a feather) (Savory, 1995). Repeated pecks directed at the same individual were recorded as one bout. A bout ended when there were no pecks for 4 seconds (Zeltner et al., 2000). The instigator and receiver of each interaction were noted. Baseline (10 minutes) and ATD/BC (10 minutes) video observations took place three hours post-capsule

administration, which is when peripheral TRP depletion peaks (Birkl et al., 2017). The video observer was blinded to the treatments.

3.2.5 Tryptophan–deficient amino acid mixtures to laying hens in an operant chamber – operant pecking

Out of the 160 birds used for the prior experiment, a sub-group was selected for operant chamber testing based on their FP phenotypes. For phenotyping, birds were observed in their home pens twice a day (10 minutes between 9:00 a.m. and 11:00 a.m., 10 minutes between 2:00 p.m. and 4:00 p.m.), two times per week, over a period of 4 weeks. For phenotypic categorization, we adapted the phenotyping scheme of Daigle et al., (2015); in the present experiment a feather pecker (P) was defined as a hen that delivered gentle and/or severe feather pecks more than 10 times in at least two 10- minute video sessions, and received fewer pecks than the number of pecks they delivered. A recipient was defined as a hen that received more than 10 pecks in at least two 10-minute video sessions and pecked less than three times in any 10-minute video session. A bystander was defined as a hen that neither pecked nor received pecks in two subsequent 10-minute video sessions. Consequently, we created three distinct groups based solely on phenotype and independent of genetic lines: 10 feather pecker birds (5 UC, 3 L, 2H), 10 bystander birds (8 UC, 2 H) and 10 recipients (7UC, 2 L, 1H). After habituating the 30 chosen birds to the operant chamber for three days, birds were then trained on a fixed ratio (FR) 1 schedule of reinforcement for another three days, whereby they pecked at an illuminated red key for immediate delivery of a high-quality food reward (chopped, hard-boiled egg). Birds were not food-deprived for habituation, training or testing. One training session consisted of 20 rewards. To pass the FR 1 training phase, birds needed to meet the criterion of being able to peck the key and subsequently receive the food reward 15 times (75%) in a row on two consecutive sessions. All birds surpassed the training criteria within 25 trials. After the FR 1 training phase was completed, all hens completed training for the 5-second delay test sessions (Schaal et al., 1998). Each session lasted 4 minutes in which the maximum number of theoretically obtainable rewards per session was 32. During sessions, a key peck resulted in a 2-second-long period of reward delivery, only if 5 seconds had elapsed

from the key peck without reward (Figure 3, 4). All birds learned this task before the ATD/BC testing phase to control for learning effects. The criterion to pass the delay task was for the birds to obtain at least 10 rewards in two consecutive sessions before moving on to the testing phase. During the testing phase, the first bird received the ATD/BC treatment at 7:00 a.m., with a 15-minute delay between birds to allow sufficient time for testing. Half of the birds (15) were given two ATD capsules, and the other half were given two BC capsules. Testing on the 5-second delay schedule was performed at the point of maximal tryptophan depletion (3 hours after capsule administration). The test was identical to the 5-second delay schedule in the training phase. All 30 birds were tested on the first day with 15 birds subjected to ATD and 15 birds to BC (counterbalanced). 24 hours later, all 30 birds were tested, ATD and BC treatment reversed (Figure 3). For administration as for testing, a 15-minute break between birds allowed for testing all birds at exactly 3 hours post treatment. The number of times the pecking key became illuminated and the number of pecks delivered at the key by each hen for both the FR 1 immediate-reward and 5-second delay schedules were recorded automatically by the MED-PC IV Software. Additionally, all hens were videotaped during all sessions to count the number of non-key pecks (i.e. pecks directed at the wall surrounding the key).

3.2.6 Statistical analysis

To analyze feather pecking behaviour after dietary ATD versus BC treatment in the home pens, we used a Proc Glimmix procedure in SAS (SAS Institute Inc., Cary, NC 2016) based on the mixed modeling approach for randomized experiments. We analyzed the effect of treatment (ATD/BC) and line (HFP, LFP, UC), and their interactions on pecking behaviour (gentle FP and severe FP bouts performed by each individual in 10 minutes) as the response variable, with baseline levels of FP behaviour prior to treatment as a covariate. Data were fitted with a Poisson distribution. Due to repeated measurements being taken on the same group of animals and their pens (random effect), an autoregressive covariance structure of order 1 was fitted with individuals and pens used as random effects. The degrees of freedom were adjusted using the Kenward-Roger method. To analyze the number of key pecks during the FR 1

and 5-second delay schedules, as well as the number of non-key pecks, we used a Proc Glimmix procedure with treatment (ATD, BC) and phenotype (pecker, recipient, bystander), and their interactions as fixed effects, while line, day of testing, and individual were used as random effects. Later, as no effect of day of testing was found, we removed this from the model. We used baseline pecking during the 5-second delay task, performed by each individual, as a co-variate to better account for individual differences to baseline performance. The data were fitted using a Poisson distribution. Estimated means ± standard errors of pecks per minute are reported.

3.3 Results

3.3.1 Effects of ATD/BC on feather-pecking behaviour in group housing

There was a significant effect of dietary treatment, in which ATD-treated birds performed 28.77% more gentle FP bouts than BC birds (F (1,313) = 7.85, P < 0.005). There was no effect of line and no interaction between dietary treatment and line on the number of gentle FP bouts. Severe FP hardly occurred during home pen observations. Therefore, no meaningful statistical analysis could be performed with respect to this behaviour.

3.3.2 Effects of ATD/BC on performance in the operant chamber

The maximum number of theoretically obtainable rewards (n = 32) within the 4-minute testing period was not reached by any of the subjects (mean = 19.8 ± 2.1 SD, maximum

= 26, minimum = 10). Neither significant dietary treatment (F(1, 1) = 1.18, P < 0.5; ATD:

3.07 ± 0.0 vs. BC: 2.98 ± 0.06 pecks/min) nor phenotype (F(2, 1) = 0.23, P < 0.8; P: 3.06

± 0.07; R: 2.99 ±0.07; B: 3.04 ± 0.07pecks/min) effects, nor interactions (F(21, 1) = 0.138, P < 0.9) were observed for the number of rewarded key pecks (= rewards obtained). There was a significant dietary treatment effect in which ATD-treated birds performed

4.89% more 5-second delay key pecks than BC birds (F(1,213) = 12.87, P < 0.0017). The number of 5-second delay key pecks were significantly impacted by the treatment and phenotype interaction (F(21, 21) = 24.51, P < 0.0001), where P hens delivered the highest number of 5-second delay key pecks during ATD, and the lowest number of pecks

during BC treatment (4.37 ± 0.19 vs. 3.61 ± 0.21; t = -7.89, P < 0.0017), and vice versa in recipient birds (3.82 ± 0.21 vs. 4.13 ± 0.23; t = 2.40, P = 0.03). Bystanders were unaffected by the treatment (4.07 ± 0.21 vs. 3.92 ± 0.2; t = -1.41, P = 0.17).

3.4 Discussion

To the best of our knowledge, the present study is the first to examine the effect of ATD treatment on 1) FP behaviour of laying hens selected for high and low propensities of FP in a group setting, and 2) the key pecking performance of feather peckers (P), recipients (R) and bystanders (B) in an operant chamber with a delayed reward schedule.

In the home pens, ATD treatment increased the overall number of gentle FP bouts for all genotypes of the hens. We observed between 2 to 8 bouts per hour (0.33 to 1.3 bouts per 10 minutes/bird), which compares well to other studies; e.g. The Laywel project (www.laywel.eu) and Blokhuis et al., (2007), who summarised studies that reported highly variable rates of FP between birds (from 0.5 pecks/bird/hour to over 30 pecks/bird/hour), with great variation between hybrids. Severe FP was not triggered at all after ATD compared to the BC treatment, which further supports the notion of FP as a heterogeneous behaviour, comprised of GFP and SFP. This result supports part of our hypothesis and confirms studies by van Hierden et al. (2004) and Savory et al. (1998), which found that the 5-HT system has a role to play in GFP behaviour. Riedstra and Groothuis (2004) stated that severe FP is embedded in bouts of GFP, and that there may be a common underlying motivation and/or neurobiological basis between these two forms of pecking behaviour. Yet, there is also evidence that severe FP and GFP may result from different underlying motivations (Dixon et al., 2008). However, even though imbalances in the 5-HT system may constitute a common risk factor in the development of gentle and severe FP, ATD might not be sufficient to cause severe FP episodes. Our birds were not assigned to a specific stress treatment in the current experiment. Therefore, whether ATD alone was an insufficient stressor, and whether a more severe stressor in combination with ATD would increase the risk of severe FP in a group housing situation, needs further investigation. No differences in propensity to

develop GFP due to ATD were associated with genetic line. Whether a longer observation period would have allowed to observe more SFP behaviour remains unclear but seems unlikely. Firstly, the observed numbers of bouts in this study compare well to other studies (see above e.g. Laywel project), and secondly, other studies with similar regimes of observations have been able to identify SFP (e.g. Kjaer, 2009), using the same genetic lines as used in this present thesis. Nonetheless, the observation regime is given some more critical thought in Chapter 5) of this thesis.

We hypothesized that the HFP genotype, selected for FP activity, would be more vulnerable, due to potential heritable neurochemical deficits (Kjaer, 2009), leading to higher numbers of FP bouts after ATD. While studies in humans point towards differential effects of ATD on behaviour between healthy and unhealthy populations (Booij et al., 2003), we did not observe that birds of the HFP genotype constituted a vulnerable population that responded more sensitively to ATD in their performance of FP in a social setting. However, a more severe ATD treatment and/or an additional stressor might have induced higher rates of gentle and/or severe FP in these genetically susceptible individuals. The possibility remains that the HFP birds developed FP under the ATD treatment first, which was observed and then mimicked by the rest of the group out of frustration (Cloutier et al., 2002). The rest of the group could also have been stressed by the active peckers running around to find recipients, with this inherent stressor causing them to begin pecking one another, in turn (Jones et al., 2004). However, whether the HFP birds started FP under the ATD treatment and triggered a chain reaction, causing a shift in the behaviour of their conspecifics, was not analyzed in the present study, but might be an interesting avenue of future investigations.

Overall, in the operant chamber, rewarded key pecks were unaffected by ATD/BC treatment or phenotype (pecker, recipient, bystander). This indicates that all birds (who were not food-deprived) were similarly motivated to obtain a high-quality food reward, irrespective of treatment. This may give rise to the concern that some of the birds were not completely motivated to obtain rewards. Interestingly, video observations showed that rather than spending 2 seconds eating from the feeder, some hens learned to spend that time using their beaks to push the food out from the feeder onto the ground,

in order to freely consume the food even after access to the feeder was removed. This might explain why the hens did not achieve the maximum number of rewards. ATD increased the number of 5-second delay key pecks, with its most pronounced effect on P birds, also increased the number of 5-second delay non-key pecks. Our birds were subjected to a test situation in which they were only able to access a high-quality food reward for a short period of time by constantly moving and shifting their attention between the key and feeder. Considering this challenging procedure, the increased number of 5-second delay key and non-key pecks could be seen as stress-related pecking behaviour. Yet, experiments involving punishments for delayed pecks will have to elucidate whether these pecks result from an inability to withhold a response. Additionally, from research conducted on humans, it is well understood that ATD is more effective in vulnerable individuals, namely individuals with past or present behavioural disorders linked to an underlying dysfunction of the 5-HT system, such as depression (Roiser et al., 2007). Although, we did not measure 5-HT biochemical parameters in our birds, P birds may have been more vulnerable to the ATD treatment and/or the testing stress, explaining the higher number of 5-second delay key and non- key pecks performed by these birds. Additionally, it may be assumed that after ATD, P birds performed more poorly with the 5-second delay period compared to the BC treatment because they perceived the delay as a form of distractor stimulus from the reward. This suggests that ATD may have shifted their attention to the distracting delay period, causing them to peck at the perceived distractor – manifesting in this case as unrewarded pecks on and around the key and feeder. However, whether the higher number of pecks seen in P birds was due to this attentional shift towards a perceived distractor, or due to frustration, cannot be determined based on the present results, and requires further investigation.

While the present study is a good starting point into the investigation of the relationship between the 5-HT system and FP, it is limited by the fact that we did not collect and analyze baseline and ATD- or BC-related plasma TRP and metabolite levels. Hence, the present results need to be viewed with caution, keeping in mind that neither blood- levels of AA nor central levels of AA were measured. Therefore, we cannot conclude that behavioural changes were caused by changes in central TRP availability. However,

the method of ATD treatment that we used has been previously shown to robustly decrease blood TRP in laying hens 3 hours after application (Birkl et al., 2017). With regards to the magnitude and duration of effects of the dietary ATD treatment, it needs to be noted that a more prolonged depletion might have led to more pronounced effects in the group housing situation and/or during the operant learning task. However, the present study provides important starting-point data for future studies involving prolonged TRP depletion. Additionally, brief dietary ATD treatment procedures may not capture the potentially complex relationships between the 5-HT system and behaviour. Likewise, we need to consider that “direct evidence that ATD decreases extracellular 5- HT concentrations is still lacking in mammals, and that several studies provide support for alternative underlying mechanisms of ATD in mammals”, as recently outlined by van Donkelaar et al. (2011) and Young (2013). Similarly, the same consideration should be made for avian species (Birkl et al., 2017), calling for caution when attributing the ATD results solely to 5-HT mechanisms.

In summary, although the ATD technique has been used extensively in psychiatric research over recent decades, and it continues to be a popular method in behavioural disorder studies of the 5-HT system in humans and mammals, the present study detected, for the first time, an effect of dietary ATD on pecking behaviour in laying hens living in a group setting, as well as their operant learning behaviour. In the future, ATD could be of potential value to further elucidate the complex interplay between the 5-HT system and alternative neurobiological mechanisms, its dietary manipulation by ATD treatments, and FP in laying hens. However, as outlined in the previous chapter, effects of ATD on central levels of TRP/5-HT in birds must be investigated first. Until a significant reduction of central TRP/5-HT levels by the here presented ATD-protocol can be confirmed, the results of this experiment remain speculative in terms of the underlying mechanisms.

Acknowledgement

This study was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC DG), and by the Ontario Ministry of Agriculture, Food and Rural Affairs

(OMAFRA). Jacqueline Chow and Peter McBride received Undergraduate Student Research Awards (NSERC USRA) to test birds in the operant chamber and to observe video recordings. We thank the Evonik AG, especially Dr. Ariane Helmbrecht, for providing the amino acid mixtures free of charge. We would also like to thank Michael Ross for his assistance in setting up the operant chamber, and the Arkell Research Station animal care attendants for taking care of the birds.

3.5 References

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3.6 Figures

Figure 2 Schematic representation of experimental timeline for the acute tryptophan depletion (ATD)/balanced control (BC) group housing and operant chamber testing schedule

4 Figure

Timeline of Timelineof 5

delay second reward. schedule of

Delayed key-pecks across phenotypes after ATD and UC treatment 5 4,5 4 3,5 3 2,5 ATD 2 UC

Pecks/minute 1,5 1 0,5 0 P R B

Figure 5 Key peck/minute (mean ± SE) during the delay across all three phenotypes after ATD and UC treatment; Feather peckers (P), recipients (R) and bystanders (B).

4 The role of the kynurenine pathway in the development of

feather pecking in domestic chicken lines3

Abstract

Research into the role of tryptophan (TRP) degradation away from the serotonergic to the kynurenine (KYN) pathway by stimulating the brain-endocrine-immune axis system interaction has brought new insight into potential etiologies of certain human behavioural and mental disorders. TRP is involved in damaging interactions such as feather pecking behaviour (FP) in birds kept for egg laying. Therefore, our goal was to determine the effect of social disruption stress on FP and the metabolism of the amino acids TRP, phenylalanine (PHE), tyrosine (TYR), their relevant ratios, and related biomarkers of immune activation in adolescent birds selected for and against FP behaviour.

We used 160 laying hens selected for high (HFP) or low (LFP) FP activity and an unselected control line (UC). Ten pens with 16 individuals each (4 HFP birds; 3 LFP birds; 9 UC birds) were used. At 16 weeks of age, we disrupted the groups twice in 5 pens by mixing individuals with unfamiliar birds to simulate social stress. Blood plasma was collected before and after social disruption treatments, to measure amino acid concentrations and related immune biomarkers. Birds’ FP behaviour was recorded before and after social disruption treatments. HFP birds performed significantly more FP and had lower KYN/TRP ratios than LFP or UC birds. We detected significantly higher FP activity and significantly lower plasma PHE/TYR ratios and a trend to lower KYN/TRP in socially disrupted compared to control pens. Thus, for the first time, our study highlights a role of the kynurenine pathway in the developmentof FP, therefore

3 An edited version of this chapter has been published in Frontiers in Veterinary Sciences, with the following authors: Birkl, P., Forsythe, P., Gostner, J.M., Kjaer, J.B., Kunze, W., McBride, P., Fuchs, D. and Harlander-Matauschek, A.

this pathway might be a novel therapeutic target to prevent or treat FP in millions of birds kept for egg laying.

4.1 Introduction

Birds kept for egg-laying face many serious animal welfare problems. One of the most challenging issues is feather pecking (FP). Feather pecking (FP) is an oral behaviour, mostly categorized into gentle (GFP) and severe (SFP) FP, in which individuals peck repetitively at another bird's feather cover (Blokhuis et al., 2007). The act often results in feathers or parts of feathers being broken off, plucked/pulled (Neal, 1956) and subsequently ingested (Harlander-Matauschek et al., 2007; 2008; 2009a), causing significant skin injuries to the victim (Savory, 1995). Other than feather-picking in pet birds, such as in psittacines (Jenkins, 2001), which is mainly self-directed, FP in birds kept for egg laying takes place in a social context between two birds.

On commercial farms, these birds live in a densely populated environment with large groups of thousands of female individuals (Van Staaveren et al., 2018). Under natural conditions, social groups typically consist of one male with up to 20 females (McBride et al., 1969; Wood-Gush, 1971) demonstrating the dramatic contrast between natural and commercial group sizes. In these groups, it is unlikely for birds to adequately recognize/remember individuals as these social environments constantly change. More specifically, a social environment such as this may have strong adverse effects on behavioural functioning, producing abnormally high levels of FP (Bilčıḱ and Keeling, 2000). FP behaviour may naturally shape the social environment of the involved birds, where feather peckers may cause social stress, and social stress causes FP within a group due to social instability (Cheng et al., 2002, Birkl et al., 2018a). Moreover, feather peckers appear less social, displaying less motivation to join a group (Jones and Hocking, 1999) and developing pronounced stress reactions to increased social contact (Kjaer and Jorgensen, 2011).

The neurobiological mechanisms that underlie FP are not well understood. It has been suggested that the avian serotonergic (5-HT) system (de Haas and van der Eijk, 2018) and the precursor tryptophan (TRP) are intimately linked to FP. Furthermore, genetic

factors (Kjaer et al., 2001) such as candidate genes linked to the 5-HT system (Biscarini et al., 2010), lower forebrain 5-HT turnover levels (van Hierden et al., 2002; 2004), and nutritional factors, including low dietary TRP (Savory et al., 1999) also point towards serotonergic system involvement in the development of FP behaviour.

Stressful situations can lower peripheral and brain TRP levels by stimulating the immune system and activating the HPA-axis (Miura et al., 2008). Therefore, the concept of TRP metabolism alterations elicited by the immune system is of growing interest for understanding TRP/5-HT-related behaviours or dirorders such as depression (Widner et al., 2002). TRP is degraded in two major pathways: the first is the kynurenine (KYN) pathway, which is initiated by the enzyme indolamine 2,3-dioxygenase-1 (IDO), and the second is the 5-HT/serotonergic pathway, which is initiated by TRP hydroxylase (TPH) (Stone et al., 2013). Stressful events and immunological challenges induce IDO activity, which metabolizes TRP to KYN, which may be depleting the system of TRP to produce 5-HT in the mammalian brain (Konsman et al., 2002). Additionally, 5-HT cannot cross the blood-brain barrier; central 5-HT synthesis depends on the availability and transport of its precursor, TRP, into the brain (Leathwood and Fernstrom, 1990). TRP competes with other large neutral amino acids (LNAAs) for active transport by the large amino acid transporter system (Markus et al., 2000). Consequently, decreased peripheral TRP relative to other LNAAs results in a lower TRP influx into the central nervous system. The KYN/TRP ratio reflects IDO activity in mammals (Schroecksnadel et al., 2006), where lower KYN/TRP ratios were found in people with major depressive disorders (Umehara et al., 2017). Additional to TRP metabolism, in mammals, pro-inflammatory cascades were found to be associated with disturbed phenylalanine hydroxylase (PAH) activity (Neurauter et al., 2008), which metabolizes phenylalanine (PHE) to tyrosine (TYR) and is reflected by the PHE/TYR ratio, linked to the neurotransmitters adrenalin, noradrenalin, and dopamine. In our previous work, we have established that PAH activity (measured by the ratio of PHE/TYR) may play a role in the development of injurious pecking behaviour, mostly aggressive pecking (Birkl et al., 2017)., but it is unlcear whether PAH activity does play a role in GFP or SFP.

The present study aimed to investigate the effect of social stress resulting from social disruption on (i) FP activity, (ii) metabolism of the amino acids TRP, PHE, TYR, their relevant ratios, and (iii) related biomarkers of immune activation in adolescent birds selected for and against FP behaviour. We hypothesized that social disruption treatment is associated with increased FP, an accelerated TRP metabolism, shifts to PHE/TYR metabolism, and increased neopterin production, which is a sensitive marker of immune activation in humans and primates (Fuchs et al., 1993).

4.2 Materials and methods

4.2.1 Animals and housing

The birds in the present study originated from a selection experiment in which birds were divergently selected for based on FP activity, high (H) and low (L), and an unselected control line (UC) (Kjaer et al., 2001). Laying hens in their 16th week of life, were housed in ten pens with 16 individuals each with housing conditions being identical to the ones described in chapter 3). Pens offered space for 9 adult birds/m2, 15 cm perch length/bird at 90 cm above the ground, 125 x 31 cm plastic slatted platforms 65 cm above the ground, 10 cm food trough length/bird, and 10 nipple drinkers. Wood shavings were used as floor bedding material. Cameras (Samsung SNO-5080R, IR, Samsung Techwin CO., Gyeongi-do Korea) were ceiling-mounted within each pen above the entrance. The birds were kept in a ventilated, windowless room with a light intensity of 25 lx at animal level. Management of the temperature and lighting schedule during rearing was according to commercial management guidelines and are more closely described in chapter 3).

4.2.2 Social disruption stress treatment

Ten days before the social disruption treatment, the birds (14 weeks of age) were individually tagged with numbered soft silicone plates fastened to the backs of the birds using elastic straps around their wings, which served to identify birds on video observations (Harlander-Matauschek et al., 2009). At 16 weeks of age, we disrupted five pens by mixing individuals with unfamiliar birds from other pens to simulate social

stress. The other five pens remained undisrupted (control pens). For each social disruption treatment, each pen was split into four groups of four individuals. The groups of four individuals were taken out of their five “home” pens and distributed into other pens where they encountered 12 unfamiliar individuals. Three days after social disruption treatment, the procedure was repeated to increase the effects of social stress by disrupting the newly established social environment once again. With each disruption, control groups were caught and released into a different pen (group remained undisrupted) as a sham treatment. In summary, five treatment groups received a social disruption treatment and five controlled groups received no social disruption treatment. All groups were moved out of their home pens to avoid “home pen” advantage.

4.2.3 Behavioural observations

Behavioural observations, based on the sampling procedure of Kjaer (2009), of each pen were undertaken 2 days before social disruption treatment to record baseline (pre- treatment) behaviour (10 minutes in the morning (9:00 h) and 10 minutes in the afternoon (14:00 h)). Pens were again observed for 10-minute periods at the following intervals after social disruption treatment (post-treatment behaviour): two minutes, one hour, and on the following day for 10 minutes in the morning (9:00 h) and 10 minutes in the afternoon (14:00 h). During the observation periods, all occurrences (Altmann, 1974) of FP were recorded. FP was subdivided into gentle and severe FP pecks as described in Bilcik and Keeling (2000). FP analysis of video-footage was conducted by one individual observer. FP behaviour is reported in bouts/hour calculated for each point in time, averaged for each treatment-group/genotype prior to the statistical analysis.

4.2.4 Body weight

Each birds’ bodyweight was measured one day before (at 16 weeks of age) and three weeks after (at 19 weeks of age) the social disruption stress.

4.2.5 Blood measurements

Blood samples were taken from each bird one day before the first treatment, prior to feeding (baseline data) and four days after the second social disruption treatment. The wing vein of each bird was punctured with a 21-gauge butterfly needle to draw 3 mL of blood per animal into an EDTA tube. All blood collections were performed between 9:00-12:00h. Individual birds were sampled at the same time point on both blood collection days.

Blood samples were stored on ice until centrifuged for 10 minutes at 4°C and 2500 rpm (1500g) for plasma separation. Plasma was then aliquoted into 1.5mL vials and stored at -20°C until shipping on dry ice for processing for aromatic amino acids (AAA), Tryptophan (TRP), Tyrosine (TYR), Phenylalanine (PHE) and large neutral amino acids (LNAA); Threonine, Methionine, Valine, Leucine, Isoleucine and Histidine analysis performed on a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters, Manchester, UK) at the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre (SickKids Hospital, Toronto, Canada).

The remaining plasma samples were used to analyze the concentrations of free TRP and KYN as well as concentrations of PHE and TYR by high-performance liquid chromatography (HPLC) on a ProStar Varian system (USA) using rp-18 columns (LiChroCART 55-4, 3µm grain size; Merck, Germany) and acetic-sodium acetate buffer (pH=4) as eluent (flow-rate: 0.9 mL/min) according to the protocol described earlier (Neurauter et al., 2013) at the Biocentre of the Medical University in Innsbruck, Austria. Three-nitro-L-TYR (Sigma Aldrich, Austria) was used as an internal standard. TRP and KYN standards were purchased from Sigma-Aldrich (Austria). KYN and 3-nitro-L-TYR were detected by UV-absorbance at 360 nm wavelength (Shimadzu SPD-6A UV detector, Austria). TRP was detected by its fluorescence with an excitation wavelength of 286 nm and an emission wavelength of 366 nm (ProStar 360 detector, Varian, USA). The sensitivity of the measurements was 0.1 µmol/L TRP and 0.5 µmol/L KYN. HPLC and UPLC methods of amino acid measurements showed strong positive correlations

(Birkl et al., 2018b). Neopterin was measured by ELISA (BRAHMS Diagnostics, Hennigdorf, Germany).

4.2.6 Statistical analysis

To analyze the effect of social disruption stress on behaviour and physiology, we used a Proc Glimmix procedure in SAS (SAS Institute Inc., Cary, NC, 2016). The effect of social disruption stress treatment (disruption/no disruption) and line (H, L, UC), and their interactions on FP behaviour, body weight and blood measurements as the response variable, with baseline measurements before the treatment as covariates, was measured. In the random statement, we accounted for repeated measurements on pens and mixing the lines within the pens. We used Poisson distribution for behavioural data, a Gaussian distribution for body weight and blood measurements, and a compound symmetry structure was fitted to the model. The degrees of freedom were adjusted using the Kenward-Roger method. Due to zero-inflated pecking data (counts), GFP and severe FP were merged into one category. Normal distribution of residuals was tested by employing a Proc Univariate procedure. Data are presented as LSM ± SE unless otherwise stated.

4.3 Results

Baseline (pre-social disruption treatment) and post-social disruption treatment absolute plasma level concentrations and relevant ratios for domestic chicken lines genetically selected for FP behaviour are listed in Table 2, 3 and 4.

4.3.1 Effects of social disruption and genetics on FP behaviour and body weight

The occurrence of FP behaviour (mean ± SD bouts per hour) was significantly affected by the social disruption treatment (F1,152 = 20.58, P = 0.001), with socially disrupted groups showing 33 % more FP than undisrupted groups (4.6 ± 3.8 vs. 3.1 ± 3.3) across all lines (see Figure 6). There was a significant effect of genetic line (F2,152 = 53.30, P = 0.001), where H birds performed 68% more FP (8.6 ± 3.7) compared to L (2.7 ± 1.8; t = - 7.38; P < 0.0001) and 44% more than UC birds (4.8 ± 1.8; t = - 10.12; P < 0.0001, see

Figure 5). There was a significant effect of social disruption treatment (F 2,153 = 6.36, P = 0.01) on body weight gain (384.36 ± 17.91g) which was greater in disrupted compared to non-disrupted groups (320.73 ± 17.75g; t = - 2.52, P < 0.0127) across all lines over a period of 3 weeks (see Figure 7), with no differences in weight gain between lines.

Social disruption treatment tended to increase blood TRP concentrations (F1,152 = 3.4, P = 0.07), where TRP concentrations tended to be higher in socially disrupted (103.25 ± 1.48 μmol/L) than non-disrupted groups (99.4 ± 1.5 μmol/L; t = - 84; P = 0.07) across all lines. L birds (103.72 ± 2.17 μmol/L) had significantly higher blood TRP concentrations than UC birds (98.48 ± 1.25 μmol /L; t = - 2.08; P = 0.04); however, L birds did not differ from H birds (101.77 ± 1.89 μmol/L).

There was a significant effect of genetic line (F 2,152 = 8.03, P = 0.0005), which could be attributed to lower TRP/Σ AAA ratios in UC birds (0.288 ± 0.002) compared to L birds (0.3 ± 0.004; t = - 1.97, P < 0.05) and H birds (0.311 ± 0.004 t = - 3.92, P < 0.0001).

TRP/Σ LNAA concentrations were not significantly different between disrupted and non- disrupted groups (disrupted: 0.088 ± 0.001 vs. non-disrupted: 0.086 ± 0.001; F1,152 = 2.82, P = 0.09), or genetic line (H birds: 0.085 ± 0.001; L: 0.089 ± 0.0002; UC: 0.09 ±

0.001 5; F2,152 = 1.3, P = 0.27), and no significant interactions were observed for TRP/Σ LNAA concentrations.

There was a significant effect of social disruption treatment on PHE/TYR (F1,152 = 15.51, P = 0.0001), where socially disrupted birds had 8.5% lower PHE/TYR ratios (0.65 ± 0.01 μmol/ μmol) compared to birds in non-social disrupted groups (0.7 ± 0.009 μmol/μmol;t

= 3.94; P < 0.0001) across all lines. There was a significant effect of line (F 2,152 = 17.27, P = 0.001), where H birds had 15 % higher PHE/TYR ratios (0.74 ± 0.01 μmol/ μmol) compared to L (0.63 ± 0.01 μmol/ μmol ; t = - 5.34; P < 0.0001) and 11% higher PHE/TYR ratios compared to UC birds (0.65 ± 0.001 μmol/ μmol ± 0.005; t = - 5.13; P < 0.0001).

4.3.2 Effects of social disruption and genetics on biomarkers of immune activation

KYN concentrations were not affected by social disruption (disrupted: 0.35 ± 0.03

μmol/L vs. non-disrupted: 0.35 ± 0.03 μmol/L; F1,152 = 0.00, P = 0.94; Table 3); or genetic line effects (H birds: 0.33 ± 0.03 μmol/L; L: 0.34 ± 0.04; UC: 0.37 ± 0.02 5; F2,152 = 0.524, P = 0.59), and no interactions were observed for KYN concentrations (Table 3).

There was a tendency for social disruption treatment to decrease KYN/TRP (F1,152 = 3.41, P = 0.07), where social disrupted birds had 7.43 % lower KYN/TRP ratios (3.24 ± 0.1 μmol/ mmol) compared to birds in non-social disrupted groups (3.5 ± 0.099 μmol/ mmol; t = 1.85; P < 0.07) across all lines (Table 3). H birds, (3.2 ± 0.13 μmol/ mmol) had significantly lower KYN/TRP ratios (9.35%) compared to UC birds (3.53 ± 0.08 μmol/ mmol ; t = 2.15; P < 0.03; Table 3).

No significant differences were observed between neopterin levels in social disrupted (2.9 ± 0.06 nmol/L) and non-social disrupted groups (2.7 ± 0.06 nmol/L; Table 3), or between lines (H birds: 2.88 ± 0.08 nmol/L, L birds: 2.78 ± 0.096 nmol/L, UC birds: 2.82 ± 0.05 nmol/L; Table 3).

4.4 Discussion

In the present study, we investigated social disruption stress in three chicken lines genetically selected for FP behaviour, and examined effects on the FP response and the TRP/KYN metabolic pathway. As far as we are aware, no previous study reported avian TRP catabolism rate and FP behaviour after a socially stressful situation in domestic birds, nor has the TRP/KYN pathway been compared between genetic lines selected for or against FP behaviour.

The disruption of an established social environment produced an increase in FP behaviour when compared to control groups. The social disruption treatment meant that a group of familiar birds were taken out of their home pen and at the same time new birds were being introduced to them; thus, this treatment includes both a resident group

faced with unfamiliar birds and a new group incorporating into an existing group. FP in this context could reflect frustration (Grigor et al., 1995; Lindberg and Nicol, 1996) after disruption of an established social environment (social bonds/relationships were built throughout the 16 weeks and then disrupted). Indeed, the exposure to stress is a critical risk factor for developing FP-behaviour in chickens (El-Lethey et al., 2000). However, Riedstra and Groothuis (2002) stated that FP could develop from a normal bird-to-bird social exploration, where chicks direct their pecks preferentially towards unfamiliar birds (Zajonc et al., 1975), which also helps to maintain a social relationship by keeping intruders out and the established group intact (Wood-Gush and Rowland, 1973). It remains possible that the birds in our study developed pecking at the feather cover of other birds to socially explore unfamiliar birds, meaning a “normal basal level of allopreening” (Blokhuis, 1986, Vestergaard et al., 1993). So, while it is the working hypothesis of this thesis that SFP is a redirected foraging behaviour, one should not discount the possibility that social exploration, in this scenario of mixing unfamiliar birds, may have contributed to the increase in GFP in the present study. However, the relative importance and whether there exist possible interactions between social exploration and redirected foraging behaviour remain unknown.

Higher FP levels in HFP birds demonstrated the influence of genetic background on FP behaviour. No interaction between genetic line and social disruption stress was found. This result was in contrast to our prediction. Social disruption stress did not make HFP birds more susceptible to developing FP behaviour than LFP and UC birds, even though these genetic lines (Kjaer et al., 2001) show behavioural and physiological differences in response to stressful laboratory test situations; specifically, responses of the HPA- axis (Kjaer and Guemene, 2009). Also, the relative sympathetic and parasympathetic nervous system activation responses have been shown to be more reactive in these birds (Kjaer and Joergensen, 2011).

Social disruption treatment tended to increase blood TRP levels across all lines, and the effect was significant in LFP birds. Blood levels of TRP are dependent on nutrition, metabolic rates and interactions between metabolites of amino acids, carbohydrates, and lipids (Gannon and Nuttall, 2010). We cannot rule out that different amounts of feed

consumed by certain individuals may have affected blood TRP levels, or whether the release of stress hormones such as corticosterone increased protein-breakdown, increasing TRP-levels in plasma. Interestingly, birds in disrupted groups gained more weight, likely due to increased feed intake, which might have impacted blood TRP levels in these birds. Stress can elevate brain TRP levels (Miura et al., 2008). The uptake of TRP into the brain is not determined by the absolute TRP concentrations, but rather by the ratio of TRP to other relevant amino acids. One mechanism regulating TRP uptake into the brain is by an elevation of the TRP/AAA or TRP/ LNAA ratios, which favor TRP uptake in the brain and thereby increases brain TRP levels (Fernstrom and Wurtman, 1972) and potential TRP related behaviour. However, we did not observe elevated TRP/AAA or TRP/LNAA ratios after social disruption treatment in the present study meaning that the present study does not allow to conclude that there exists a link between social stress and TRP availability.

In addition to higher FP activity in social disrupted groups and HFP birds, birds in disrupted social environments tended to have lower KYN/TRP ratios, and HFP birds had significantly lower KYN/TRP ratios compared to UC birds. Stress is likely to impact TRP metabolism, suggesting KYN pathway involvement since IDO is activated by glucocorticoids and proinflammatory cytokines (Miura et al., 2008). The enzyme IDO is a biological mediator of inflammation, and KYN/TRP ratios are reliable indicators of IDO (Schroecksnadel et al., 2006). However, whether the differences in the KYN/TRP ratios were enough to alter brain 5-HT and impact FP in the present study is unknown. Moreover, humans with major depressive disorders (MDD) have lower KYN/TRP ratios than control groups (Umehara et al., 2017). Following antidepressant treatment, KYN concentrations in humans were elevated, and a trend towards elevated KYN/TRP ratios was found (Umehara et al., 2017; Myint et al., 2013). On the other hand, patients with medical conditions such as colorectal cancer often present with increased KYN/TRP ratios, and the lowering of tryptophan due to its increased catabolism was found to be associated with depression (Huang et al., 2002). However, whether HFP birds and birds in socially disrupted groups with lower KYN/TRP ratios and a higher number of FP pecks were in a depressive-like state cannot be determined by the present results, and requires further investigation. In the present study, for the first time in the domestic

adolescent laying hen, the KYN pathway of TRP metabolism was considered. Hence, it is noteworthy that blood plasma concentrations of KYN measured in adolescent birds appear much lower (Table 3 and 4) than what has been found in human studies (1.78 ± 0.04, n = 100; see Geisler et al. (2015)) and TRP levels higher (Table 3 and 4) compared to humans (67.4 ± 1.02, n = 100; EM ± SE; values are taken from Geisler et al. (2015)). It can therefore be assumed that significant differences exist in the functioning and regulation of the avian TRP metabolism that may need further investigation.

For groups with social disruption treatment, PHE/TYR ratios were significantly lower than in the non-disrupted groups. PHE is an essential amino acid that can be converted to TYR, the precursors of catecholamines and thyroid hormones (Neurauter et al., 2008; Haroon et al., 2012). TYR has been shown to be an important amino acid for producing proteins which participate in host defense activities, where their role of synthesis increases several fold under stress (Jahoor et al., 2006). Stressful events can increase PAH activity, decrease PHE/TYR ratios, and increase dopaminergic activity in mammals (Pani et al., 2000). Although we did not measure corticosterone as a marker for physiological stress response in the present study, previous studies have found higher corticosterone levels in FP birds (van Hierden et al., 2004), suggesting the low PHE/TYR ratio in blood plasma of birds in social-disrupted groups (and higher FP levels) could indicate a stress-mediated increase in PAH. Indeed, Birkl et al. (2017) measured low PHE/TYR ratios in birds kept in groups with aggressive pecking/negative social interaction problems (psychological stressor) and in birds with an early onset of egg-laying (physiological stressor), suggesting a stress-mediated increase in PAH. Interestingly, lower PHE/TYR ratios in athletes performing exhaustive exercises were interpreted as more probable to produce higher catecholamine levels, which may compensate, at least partly, for mood lowering intensive exercise (stressor) programs (Strasser et al., 2016). Whether birds in socially disrupted groups with lower PHE/TYR ratios produce more catecholamines and increase brain dopaminergic activity needs further investigation.

The development of body weight was significantly affected by social disruption treatment; surprisingly, birds in socially disrupted groups gained more weight. In humans, facing stressors might increase or decrease food intake (Demori and Grasselli, 2016). In the case of chronic stress or if there are multiple sequential stressors, glucocorticoid levels can be maintained chronically high, leading to increased feeding and consequent obesity (Spencer and Tilbrook, 2011). We did not measure feed intake in the present study. Nevertheless, the weight gain in socially disrupted birds suggests higher feed consumption. Considering the social disruption treatment has strong adverse chronic effects on psychological (increased FP) and physiological (amino acid metabolism) function, birds could have, as shown in rat models for obesity research (Pecoraro et al., 2004), consumed more food. In mammals, palatable food can activate the dopaminergic pathway where high levels of dopamine help to extinguish the activity of the HPA axis (Volkow et al., 2011). Similar considerations could be hypothesized in the present study: social disruption treatment triggered pecking at the feather cover of other birds, where birds might pluck and eat feathers (Harlander-Matauchek et al., 2006; 2007), leading to positive post-ingestive feedback and making feathers highly palatable (Harlander-Matauschek et al., 2009). Additionally, feather ingestion increases the conversion of feed into body weight (Harlander-Matauschek and Bessei, 2005). Together, the highly palatable feathers and glucocorticoids (high pen specific stress levels in socially disrupted groups) might have activated the dopaminergic circuit reward system (they cannot or will not stop), indicated by the low PHE/TYR ratio, to peck at, pluck and eat palatable feathers and feed. However, this hypothesis needs testing.

Avian plasma neopterin concentrations could be detected in adolescent birds, albeit at much lower concentrations than in humans. Even so, neither social disruption treatments nor genetic differences between lines impacted neopterin levels. A more chronic and severe social stressor, such as experienced by broiler chickens during crating (Bedanova et al., 2014), may have led to different neopterin results. However, there are doubts on whether the analysis by Bedanova et al., 2014 was conducted in a correct and reproducible fashion (Dr. Dietmar Fuchs, pers. Communication). Neopterin as an immune marker in avian species would be useful, as avian immune activation is often difficult to assess due to the limited availability of cytokine detection assays.

A potential limitation of the present study is that the measured peripheral plasma blood levels of amino acids and their specific metabolites might not be representative of their levels in the CNS. However, it has been shown in human and other mammals that there are strong associations between blood plasma and cerebrospinal fluid levels of KYN/TRP and PHE/TYR (Curzon, 1979; Gal and Sherman, 1980). A further limitation of our study is that we cannot rule out that feed/water consumption may have affected the metabolite levels.

In the present study, birds were kept in enriched environments (elevated platforms, perches, nest boxes), in comparison to commercial housing systems, and birds could avoid negative social interactions. Also, the stress level induced in the experiment is likely much less than would be expected under similar conditions in commercial situations (large groups and high stocking densities), in terms of severity and duration, which might have impacted behavioural and physiological measurements. However, birds in the present study were given the opportunity to have a fixed social environment for the first 16 weeks of life, in a relatively small group. In comparison, commercial situations do not permit any fixed social environment throughout their entire life. So, social stress may be a more chronic and severe impact for birds in the present study compared to commercial contexts. Further, the number of pens per treatment was relatively small, and larger studies are needed to confirm the results.

In conclusion, our research provides insight into the influence of social stress on the avian aromatic amino acid pathway. For the first time, a link between the KYN pathway and FP was considered. Neopterin and TRP/KYN was introduced as a potentially useful tool to measure stress responses in laying hens. By investigating the role that inflammatory processes might play in FP, this study could be a starting point to look at various immunological aspects of treatment to reduce, stop or prevent FP in birds kept for egg laying.

Acknowledgment

This study was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC DG), by the Ontario Ministry of Agriculture, Food and Rural Affairs

(OMAFRA) and the Egg Farmers of Canada (EFC). Jacqueline Chow and Peter McBride received Undergraduate Student Research Awards (NSERC USRA) to analyse video recordings.

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4.6 Tables

Table 3 Descriptive statistics: Mean ± Standard Error of the Mean (SEM) for biological markers before (PRE) and after (POST) social disruption treatment in disrupted and undisrupted groups.

Disrupted Undisrupted Undisrupted Disrupted Baseline

Baseline PRE POST PRE POST

Tryptophan (μmol/L) 105.69 ± 9.20 98.50 ± 13.13 101.13 ± 12.64 98.32 ± 10.65

Kynurenine (μmol/L) 0.34 ± 0.09 0.35 ± 0.08 0.30 ± 0.09 0.36 ± 0.29

Kyn/Trp (μmol/mmol) 32.48 ± 0.784 35.50 ± 0.76 28.65 ± 0.793 31.88 ± 0.89

Neopterin (nmol/L) 2.7 ± 0.42 2.74 ± 0.44 2.88 ± 0.48 2.81 ± 0.60

Tyrosine (μmol/L) 199.87 ± 44.40 197.75 ± 41.72 208.38 ± 36.45 207.12 ± 32.54

Phenylalanine (μmol/L) 133.08 ± 22.26 135.37 ± 21.05 133.73 ± 13.79 132.15 ± 11.85

Phe/Tyr (mol/mol) 0.67± 0.13 0.71 ± 0.11 0.63 ± 0.09 0.62 ± 0.09

TRP/AAA 0.36 ± 0.05 0.30 ± 0.04 0.33 ± 0.04 0.28 ± 0.03

TRP/LNAA 0.09 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 0.09 ± 0.01

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Disrupted Undisrupted Undisrupted POST Disrupted POST Baseline PRE UC line Baseline PRE (n=45) (n = 45) (n = 45) (n= 45)

Tryptophan 104.03 ± 9.2a 96.76 ± 13.4b 103.64 ± 10.3a 99.78 ± 10.1ab (μmol/L)

Kynurenine 0.34 ± 0.1ab 0.35 ± 0.1ab 0.30 ± 0.1a 0.40 ± 0.4b (μmol/L)

Kyn/Trp 33.0 ±.7.3ab 36.0 ± 8.22a 29.3 ± 7.6b 34.6 ± 10.3ab (μmol/mmol)

Neopterin 27.0 ± 4.2 31.8 ± 5.8 29.6 ± 6.8a 32.2 ± 2.4a (nmol/L)

Tyrosine (μmol/L) 204.79 ± 29.4 204.73 ± 35.4 221.07 ± 32.6 220.36 ± 32.5

Phenylalanine 134.10 ± 19.9 136.63 ± 18.8 140.36 ± 13.5 138.72 ± 12.1 (μmol/L)

Phe/Tyr 0.66 ± 0.1 0.67 ± 0.1 0.64 ± 0.1 0.64 ± 0.1 (mol/mol)

TRP/AAA 0.34 ± 0 0.28 ± 0 0.33 ± 0 0.28 ± 0

TRP/LNAA 0.09 ± 0 0.09 ± 0 0.10 ± 0 0.09 ± 0

Undisrupted Disrupted Undisrupted POST Disrupted POST LFP line Baseline PRE Baseline PRE (n=15) (n = 15) (n = 15) (n= 15)

Tryptophan 110.79 ± 8.6 102.87 ± 11.1 105.60 ± 17.9 105.33 ± 12.1 (μmol/L)

Kynurenine 0.34 ± 0.1 0.36 ± 0.1 0.28 ± 0.1 0.32 ± 0.1 (μmol/L)

Kyn/Trp 30.6 ± 6.6ab 35.3 ± 7.4a 26.3 ± 5.4b 30.5 ± 7.1ab (μmol/mmol)

Neopterin 26.9 ± 3.4 31.3 ± 4.2 28.8 ± 0.50 31.1 ± 2.7 (nmol/L)

Tyrosine (μmol/L) 220.87 ± 47.6 219.22 ± 39.9 231.19 ± 40.5 233.59 ± 29.9

Phenylalanine 132.78 ± 19.3 138.38 ± 8.5 139.81 ± 13.8 137.70 ± 12.9 (μmol/L)

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Phe/Tyr 0.61 ± 0.1 0.65 ± 0.1 0.62 ± 0.1 0.59 ± 0.1 (mol/mol)

TRP/AAA 0.36 ± 0a 0.30 ± 0b 0.33 ± 0ab 0.30 ± 0b

TRP/LNAA 0.09 ± 0 0.09 ± 0 0.09 ± 0 0.09 ± 0

Undisrupted Disrupted Undisrupted POST Disrupted POST HFP line Baseline PRE Baseline PRE (n=20) (n = 20) (n = 20) (n= 20)

Tryptophan 105.58 ± 8.8ab 99.14 ± 12.7b 108.34 ± 12.6a 104.82 ± 9.7ab (μmol/L)

Kynurenine 0.35 ± 0.1ab 0.34 ± 0.1ab 0.37 ± 0.1a 0.32 ± 0.1b (μmol/L)

Kyn/Trp 32.7 ± 10.0 34.5 ± 6.8 34.2 ± 8.9 30.9 ± 6.0 (μmol/mmol)

Neopterin 2.87 ± 0.5 3.21 ± 0.4 2.88 ± 0.5 3.17 ± 0.3 (nmol/L)

Tyrosine (μmol/L) 173.03 ± 17.6ab 165.95 ± 8.3b 194.32 ± 32.7a 189.17 ± 18.5ab

Phenylalanine 130.98 ± 23.2 130.28 ± 25.8 135.10 ± 14.1 133.33 ± 9.4 (μmol/L)

Phe/Tyr 0.73 ± 0.2ab 0.80 ± 0.1a 0.71 ± 0.1b 0.71 ± 0.1b (mol/mol)

TRP/AAA 0.39 ± 0.1a 0.34 ± 0ab 0.37 ± 0ab 0.32 ± 0b

TRP/LNAA 0.09 ± 0 0.08 ± 0 0.09 ± 0 0.08 ± 0

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Table 5 Mean concentrations [nmol/ml] of amino acids and Standard Deviation (SD) before and after social disruption: Tyrosine (TYR), Phenylalanine (PHE), Tryptophan (TRP), Glycine (GLN), Gultamic acid (GLU), Valine (VAL), Leucine (LEU) and Isoleucine (ILEU). Line; UC= unselected control, LFP low feather pecking, HFP = high feather pecking. Baseline values (week 16) and values after treatment (week 17) are presented.

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Treatment: TYR PHE TRP GLC Undisrupted

week week week week week week week week Line SD SD SD SD SD SD SD SD 16 17 16 17 16 17 16 17

UC (n=45) 205 29 205 35 134 35 137 19 114 13 95 14 971 148 949 181

LFP (n=15) 221 48 219 40 132 19 138 8 124 9 107 10 926 122 979 130

HFP (n=20) 173 23 166 26 131 18 130 8 118 12 100 12 967 125 983 114

Treatment: GLU VAL LEU ILEU Disrupted

week week week week week week week week Line SD SD SD SD SD SD SD SD 16 17 16 17 16 17 16 17

UC (n=45) 204 33 224 39 371 67 417 84 368 61 174 35 155 29 393 79

LFP (n=15) 216 31 236 27 410 78 465 65 403 63 198 29 173 31 444 52

HFP (n=19) 198 25 213 29 437 69 453 64 424 58 190 27 183 30 423 49

Treatment: TYR PHE TRP GLC Disrupted

week week week week week week week week Line SD SD SD SD SD SD SD SD 16 17 16 17 16 17 16 17

UC (n=45) 221 33 220 32 140 14 139 12 118 13 101 10 1022 143 914 126

LFP (n=15) 231 40 234 30 140 14 138 13 123 22 110 14 1020 98 920 123

HFP (n=19) 194 33 189 19 135 14 133 9 121 17 102 10 1007 140 938 128

Treatment: GLU VAL LEU ILEU Disrupted

week week week week week week week week Line SD SD SD SD SD SD SD SD 16 17 16 17 16 17 16 17

UC (n=45) 210 27 217 33 361 59 423 64 361 51 182 27 152 25 400 52

LFP (n=15) 213 25 223 31 387 86 449 76 394 75 195 32 165 37 429 64

HFP (n=19) 198 32 201 24 405 59 452 43 404 53 197 18 173 26 427 36

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4.7 Figures

FP- bouts across genotypes 14 12 10 8 H birds

6 L birds

bouts/hour -

FP 4 UC birds 2

0 Genetic line

Figure 6 FP-bouts/hour (mean ± SD) after treatment between High (H) Low (L) feather- pecking birds and unselected control birds (UC)

FP- bouts disrupted vs undisrupted

9 8 7 6 5 Disrupted

4 bouts/hour

- Undisrupted

3 FP 2 1

0 Treatment

Figure 7 FP-bouts/hour (mean ± SD) after treatment between Disrupted and Undisrupted birds

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Weight-gain: Disrupted vs Undisrupted 450

400 350 300

250 gain [g]

- Disrupted 200 Undisrupted

150 weight 100

50 0 Treatment Figure 8 Body-weight gain in [g] (mean ± SD) of disrupted and undisrupted groups, three weeks post-mixing

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5 General Discussion

5.1 Introduction

The aim of this thesis was to investigate which roles are played by acutely lowered peripheral TRP and social disruption, in the development of SFP in laying hens. Previous work showed that birds selected for high or low levels of SFP differ in behavioural and physiological characteristics (reviewed in Sections 1.3 and 1.4). This represented the foundation of this thesis and genetic lines selected for their propensity to perform SFP were used as a model.

TRP (the precursor of 5-HT) has been shown to play a role in the development of FP (e.g. van Hierden et al., 2004). However, direct, acute manipulation of peripheral TRP- availability for central 5-HT synthesis in association with SFP has not been investigated so far. Hence, Chapter 2 developed a protocol for nutritional depletion of TRP based on existing protocols for rodents and humans (Sections 1.5 and 1.6).

In Chapter 3) this protocol was tested on birds in a group housing or operant conditioning situation. This was to investigate whether acute depletion of peripheral TRP would be associated with an increased occurrence of SFP or operant pecking.

However, TRP is not only the precursor of 5-HT but also key in the KYN pathway. In this pathway, the rate-limiting enzymes TDO or IDO-1/2 convert TRP to KYN and TDO can be activated by glucocorticoids or by hepatic TDO (Oxenkrug, 2010). Pro-inflammatory cytokines induce IDO1 and increase TRP breakdown indicated by KYN/TRP ratio (Ball et al., 2007). TRP conversion into KYN limits the availability for 5-HT synthesis.

The PHE-TYR pathway is the starting point of the biosynthesis of noradrenergic, adrenergic and dopaminergic neurotransmitter. This pathway is controlled by the enzyme PAH which activity is reflected by the PHE/TYR ratio (Neurauter et al., 2008). PAH activity has been reported to be sensitive to stress (Strasser et al., 2016). Stress has been identified as one of the risk factors to SFP-development (Section 1.3.2). In commercial laying hen husbandry, an inadequate social environment and disruption of

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social structures can be assumed to constitute a severe stressor to the birds (Bestman et al., 2009).

Hence, Chapter 4 looked at the role of social disruption on SFP and whether disruption would be accompanied by increased PHE/TYR turnover. Also, increased activity of the kynurenine pathway, was investigated as an expression of physiological stress. We further predicted that this increase of TRP conversion towards the kynurenine pathway would be paralleled by a shift of TRP/LNAA in plasma. This would be in the direction of lower TRP/LNAA ratios, which would indicate increased competition of LNAA towards TRP at the blood-brain-barrier. As a result reduced TRP availability would exist for central 5-HT synthesis (Fernstrom and Wurtmann, 1971).

The purpose of the following section is to identify conclusions and discuss steps required to gain an even better understanding of the interplay of stress (social and nutritional), amino acid metabolism and FP behaviour.

5.2 Chapter 2) and 3): ATD increases FP and operant pecking

Increase of dietary TRP has previously been shown to reduce FP (van Hierden et al., 2004; Savory et al., 1998), but contradictory reports also exist (Dewart, 2014). As indicated in Chapter 1), concentrations of TRP levels between these previous studies are not comparable. Particularly, in the case of Savory (1998) and van Hierden et al, (2004), TRP levels were excessively higher than in commercial diets (~10 fold) and birds between studies were not of the same age. Recently, more attention has been paid to TRP levels in layer feed, such as the studies of Helmbrecht et al., (2015) and, Katthak and Helmbrecht (2018) which have suggested that TRP levels for commercial layers may need to be reconsidered and possibly increased, to meet the birds’ requirements at peak-reproduction. However, the only recent work attempting to link dietary TRP levels to FP behaviour in commercial birds, found no link (Dewart, 2014). The notion that TRP supplementation counteracts FP can therefore not be viewed as firmly established at this point. Hence, studies replicating TRP-supplementation with commercial layers during egg-laying and with practically-relevant concentrations of TRP are needed.

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In this thesis, for the first time, an acute depletion of TRP was tested as a direct and short-term manipulation of peripheral TRP levels (ATD). In rodents and mammals, ATD is a well-established non-invasive method to reduce peripheral TRP in order to study behaviour linked to the 5-HT system. Therefore, ATD was adapted for laying hens (Chapter 2) to study the effects of an acute reduction of peripheral TRP on SFP and on pecking in an operant chamber. After showing that this recipe could deplete plasma levels of TRP, birds were treated with ATD, and the effects of ATD were observed in a social group housing situation as well as in an operant conditioning chamber (Chapter 3).

In Chapter 3) an increase of GFP after ATD has been reported as well as increased pecking during the unrewarded phase of the operant trial. This change in behaviour is hypothesized to result from a decreased availability of TRP for central 5-HT synthesis (Young, 2013). However, further research is needed to demonstrate that a reduction of TRP in avian plasma, to the extent presented in Chapter 2), does, in fact, result in central TRP and 5-HT reduction.

Observations of birds in their home pens showed an effect of ATD on GFP activity, but genotypes were not affected. This could be due to birds selected for FP behaviour could be most affected by ATD. Hence an increase in FP activity would be observed primarily in birds selected for FP. However, this observation could not be confirmed during the trial. SFP was also hardly observed, possibly since the ATD treatment did not constitute a trigger severe enough to spark SFP. As discussed in Chapter 3), the 5-HT system may constitute a common risk factor in the development of GFP and SFP. However, ATD might not be sufficient to cause severe FP episodes. Experimental birds in Chapter 3) were not assigned to a specific stress treatment. Therefore, it is unclear whether ATD alone was an insufficient stressor, and whether a more severe stressor in combination with ATD would increase the risk of severe FP in group housed birds. This may be an interesting topic for future research (see Section 5.4). Whether a small number of birds (genetically predisposed to FP) initiated SFP before our video observations remains speculative. Yet, an increase in GFP behaviour was observed post ATD in home pens, which requires further investigation.

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For the operant conditioning experiment, phenotype was used as a fixed effect (ntotal= 30, with 10 active feather peckers, 10 bystanders and 10 recipients), rather than genetic line. This ensured that birds who performed FP were tested in contrast to non-FP birds, regardless of their genotype. As a result, the phenotype groups were not homogeneous regarding genotypes and therefore, the latter was considered a random effect. An experimental set-up that tests genotypes (with phenotype as a random effect) against one another could be useful to further elucidate the role of genotype in sensitivity to ATD. Matching pheno- and genotype by only choosing genetic feather peckers who have been confirmed to perform FP may also appear as an option. This approach would avoid contradictory directions of phenotype and genotype. However, it would not allow for discerning the two factors from one another, and it has been shown recently that such pheno- genotype categorization can be difficult (von der Eijk et al., 2018). For this experiment alternative explanations for the increased pecking during the delay phase of FP birds post ATD (e.g. motivational frustration), cannot be excluded.

In the experiment, pecks during the delay phase were not punished by a negative stimulus (unpleasant noise/extended delay period). It would certainly be worthwhile to test birds differing in their propensity to perform FP in an operant conditioning paradigm that involves punishment for premature pecks. This would allow elucidating whether FP or non-FP birds express different levels of motor-control. From the results, in the absence of punishment of early pecks, it cannot be concluded whether birds were trying but failing to withhold a response. Without punishment of premature-responses, numerous pecks at the key prior to illumination may have resulted from superstitious behaviour- where birds are reinforced for several pecks by delayed rewards instead of being (only) rewarded to withhold a response or punished for not withholding a response (Blough, 1959). Yet, a recent study testing birds in a go/no-go task indicated that both FP- and non-FP birds have the ability to withhold from making a response (Heinsius et al., 2019). In that study the absence of a reward served as a punishment.

Implementing an aversive form of punishment (e.g. extended delay before reward or audio-stimulus), might pose some challenges. For example, it will be crucial to ensure that a punishment does not lead birds to express superstitious behaviour or to learning

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extinction. Hence, such a trial would need careful validation and well-thought-out pilot studies to identify a punishment that is suitable for the trial and the species.

Another interesting research avenue could be to conduct consumer demand studies to investigate whether SFP birds consume more neurotransmitter precursors PHE or TRP. It has been shown that birds performing high levels of SFP pay a higher maximum price for feathers as a reinforcement (Harlander-Matauschek et al., 2006). Using PHE/TRP supplemented feed rations as a reinforcement might yield similar results. This could either support or speak against the notion that SFP is linked to an underlying PHE/TRP deficiency.

A shortcoming of Chapter 3) is that plasma levels of TRP were not obtained pre- and post- ATD administration. This was assumed to be justifiable in order to reduce stress in the birds (blood sampling before behaviour testing) and due to financial considerations. Nonetheless, it would be crucial for a future study to obtain pre- and post-testing blood samples to determine TRP levels in a trial that involves both sham blood-collection treatments and handling without an actual blood-collection. Hence, the results presented in this thesis and associations between ATD-treatment and changes in behaviour need to be viewed with caution. In mammals, it is established that strong associations exist between cerebrospinal fluid levels of KYN/TRP and PHE/TYR (Curzon, 1979; Gal and Sherman, 1980). Future experiments in birds are needed to confirm that nutritional depletion of TRP results in changes in central TRP/5-HT levels. Hence, a follow-up study is recommended to measure central levels of TRP and 5-HT post-ATD. This could either be done via microdyalisis (e.g. see Kops et al., 2014) or by sacrificing birds post-ATD treatment as previously done in rodent-studies (e.g. Biskup et al., 2012).

The neurobiological basis of SFP could be better understood by investigating which brain-regions are most affected by such a treatment, or for SFP birds in general. For example, if SFP represents a malfunctional stereotypic behaviour, it could be expected to find evidence of a dysfunction in the dorsal striatum of the basal ganglia in birds (Medina et al., 1999) analogous to mammals (Garner and Mason, 2002). It could also

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be expected that such a CNS-dysfunction be accompanied by increased levels of perseverative behaviour. Yet, no association between SFP birds and increased levels of perseverative behaviour were found (Kjaer et al., 2015). Nor were these birds (SFP and non-SFP) found to differ in terms of their ability to withhold from making a response in a go/no-go task (Heinsius et al., 2019). How an actual punishment for false responses (e.g. aversive audio stimulus) may influence these results remains unclear.

In addition to nutritional manipulation of TRP-availability, there are pharmacological procedures that directly impact the serotonergic system. These would present themselves as ideal ways to study SFP and a potential involvement of 5-HT. One procedure would be a selective serotonin reuptake inhibitor (SSRI) such as fluoxetine, which is used in anti-depressants such as Cipralex ©, Felicium ©, Fluctine © or Prozac ©. In the past, pharmacological studies on FP birds have shown that S-15525, a somatodendritic 5-HT agonist increased FP behaviour (van Hierden et al., 2004). However, there is no study that tested whether FP can be treated by administration of SSRI, hence eliciting the opposite effect of a 5-HT agonist. This information would be of great relevance to better understand the role of 5-HT in FP. If FP could be treated by SSRI administration, as reported for stereotypic behaviour in other animals (e.g. Poulsen et al., 1996), this would provide further evidence that FP might be malfunctional. Ideally, this would be tested in experimental lines and commercial hybrids- to elucidate whether either or both genotypes respond to SSRI treatment with the expected reduction in SFP behaviour. Such future experiments could help in better understanding the nature and relationship between SFP and the serotonergic system.

However, the developed protocols introduced in this thesis provide an excellent basic approach on which future work can be built upon to further study the relationship between FP and the 5-HT system, utilizing operant conditioning paradigms.

5.3 Chapter 4) Social disruption increases FP

Stress has been shown to represent a risk factor in the development of SFP (see Section 1.3.2). In practice, birds are moved from rearing to layer barns when they reach adolescence (Bestman et al., 2009). The social mixing that occurrs during that time

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constitutes a severe stressor to the birds and social stress is known to have long-lasting negative effects on animals including compromising their immune-system (Proudfoot and Habing, 2015). Yet, it is unclear to what extent social stress may contribute to the development of SFP and whether such a behavioural changes may be accompanied by a physiolgical response, such as immune-responsive amino acid metabolism (TRP/KYN and PHE/TYR).

In Chapter 4) it was therefore tested whether social disruption would increase SFP. Behaviour was observed, and blood samples were analysed to investigate whether this disruption and a potential increase in FP would be paralleled by a shift in essential amino acid metabolism (TRP and PHE).

It was hypothesized that social disruption would result in stronger expression of TRP metabolism towards the kynurenine pathway (increased IDO1 activity) and expected to observe a decrease of TRP/LNAA ratio, as an indicator of available TRP in the periphery. It was also hypothesized that social disruption would result in increased PHE/TYR conversion (increase PAH1 activity). Both enzymes (IDO1/TDO and PAH1) have been reported as indicators of an immune response to stress (Gibney et al., 2014; Strasser et al., 2016) and along these lines we also expected to measure increased neopterin levels as an indicator of an immune response (Murr et al., 2002).

The results demonstrated that birds who were subjected to social disruption indeed showed increased GFP activity but no SFP was observed. An increase in KYN production was observed in disrupted groups. It is speculated that the stress treatment of mixing unfamiliar birds during adolescence for two times might have been too mild to spark SFP based on exposure to a sustained and severe stressor. In Section 1.3.2 it has been postulated that intensive and long-lasting stress may spark the development of SFP, which was the intention of the experiment in Chapter 4). However, as discussed in chapter 4) this treatment might have been mitigated by the overall enriched environment and, up to the experiment, stable social environment (compared to commercial systems). It may therefore be worthwhile to experiment with a more severe social stressor (e.g. more consecutive social disruptions and less enriched

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environment). Until such an experiment is conducted, we cannot conclude whether SFP did rarely occur due to the stressor being too mild or the specific stressor being irrelevant to the development of SFP. Also, such a follow-up experiment, could further investigate the role that GFP plays as a potential precursor to SFP or GFP may develop independent thereof, as discussed in Chapter 1).

The measurements of KYN and TRP were conducted peripherally not on central blood plasma. Hence, the results do not allow for any interpretation of TRP availability in the central nervous system. While an increase of KYN production may have occurred in the periphery, the consequences of such a shift on the central availability of TRP are uncertain. The results concerning TRP/LNAA ratios, which are indicative of central TRP availability (Fernstrom and Wurtmann, 1971), call for caution, as no effect of social disruption on this ratio was observed.

Therefore, it is suggested that future studies investigate 1) the effects of peripheral TRP metabolism changes on central availability of TRP and 2) whether the lack of a response of TRP/LNAA was due to the stressor being not severe enough, or, whether the 0-hypothesis holds and there is ultimately no effect of social disruption on TRP/LNAA ratio.

Another important aspect to take into account when interpreting data related to TRP metabolism is the complexity of the TRP pathways and factors at play when it comes to the availability of TRP for central uptake. The greatest factor in consuming dietary TRP is protein synthesis (Badawy, 2017). Future studies investigating the effects of stressors on SFP would benefit from also measureing to which extent a stressor alters protein synthesis and the effects of such possible alterations on TRP metabolic pathways.

Also, increased KYN production itself cannot explain how central nervous processes are altered. For example, alternatively to biasing TRP availability, increased KYN production could mean increased quinolinic acid production, causing neurotoxic effects in the central nervous system (Stone and Connick, 1985). Therefore, to come closer to a functional explanation of TRP metabolism and its connection to FP, some metabolites and enzymes along the kynurenine pathway would have to be studied in the central

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nervous system. Future studies should also expand on investigating the major TRP pathway (KYN) and its branches towards the neurotoxic metabolite quinolinic acid and the neuroprotective metabolite kynurenine acid (Stone and Connick, 1985). While not neglecting the three minor pathways being (1) hydroxylation (serotonergic pathway), (2) decarboxylation (tryptamine) and (3) transamination (indolepyruvic acid) (Badawy, 2017). While much attention has already been given to endogenous TRP metabolites, such as the hormone and neurotransmitter 5-HT (e.g., van Hierden et al., 2014), other endogenous metabolits, such as melatonin (in the pineal gland and gut), has received little attention to date.

While it was not within the scope of this thesis to investigate the role of commensal microorganisms (the microbiome) in the gastro-intestinal tract, in the development of SFP, this may prove a very important research avenue in the future. In humans the gut- microbiome is influenced by early life events such as natural vs cesarean birth (Dominguez-Bello et al., 2016), breast-feeding vs formula-feeding (Guaraldi and Salvatori, 2012), the environment and diet of infants (Azad et al., 2013). It is well understood that the microbiome plays an important role in the development of an animal’s immune system and shapes the development of the 5-HT-ergic system (O´Mahoney et al., 2015). TRP cannot only be consumed by commensal bacteria (producing TRP metabolites such as indole, skatole and tryptamine), but also be synthesized, which further complicates the mass balance of TRP (O’Mahoney et al., 2015). Recent work has suggested differences in the composition of the microbiome between FP and non-FP birds (Meyer et al., 2013; Birkl et al., 2018; van der Eijk et al., 2019). Work from mammalian research (Forsythe et al., 2016) could be useful in better understanding the bidirectional communication between the gut and the CNS via hormonal, nervous, cytokine or microbial metabolite-dependent processes. A better understanding of these complex pathways may lead to practical implications to counteract FP, such as via the development of microbiome-targeting neutraceuticals e.g. probiotics. Considering that bacteria in the GI-tract might consume and/or synthesize TRP within the host, the gut-microbiome may play an important role at the most basic levels of TRP availability to the host (O´Mahoney et al., 2015). Also, more fundamental research will be needed to better understand differences between TRP/5-

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HT metabolism in birds and mammals. For example, while orthologs for TRP-degrading enzymes IDO-2 and TDO exist in the chicken, it lacks an ortholog for IDO-1 (van Staaveren and Harlander, 2019). In humans, IDO-2 only accounts for 3-5% of IDO-1 enzymatic activity (Ball et al., 2007). In summary, the results of this thesis indicate that social stress and accompanying changes in the TRP/KYN pathway may represent promising research avenues for both applied and fundamental research. Both approaches will be needed in gaining a better understanding of neurobioligcal mechanisms associated to SFP.

5.4 General considerations and limitations

This section shall discuss limitations and address general considerations to support future investigations into the issue of SFP.

Firstly, do underlying neurobiological differences between SFP birds and non-SFP constitute a dysfunctional biological system (van Staaveren and Harlander, 2019)? To this date, it is unclear at which point adverse effects of SFP might disrupt normal function. Research investigating if SFP represents malfunctional behaviour, possibly comparable to stereotypic behaviour (e.g. repetitive, perseverative) in other species (Mason and Garner, 2006), is still scarce (Kjaer et al., 2015; Heinsius et al., 2019). Overall, the molecular mechanisms that induce SFP behaviour, making it persistent in some birds and inconsistent in others (Daigle et al., 2015) is still a puzzle. Therefore, given the limited understanding of underlying mechanisms involved in causing SFP, no such mechanism can be attributed to represent the cause of SFP (van Saaveren and Harlander, 2020). More fundamental research into relevant neurobiological mechanisms is needed to gain understanding beyond the level of risk factors or processes associated to SFP.

1) Limits of behavioural observations

Since SFP was rarely observed in the conducted video-observations the question arises whether the behaviour was hardly occurring or simply missed. Also, in Chapter 4) SFP and GFP data was grouped together for analysis. Considering the assumed

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heterogeneity of GFP and SFP, the exclusion of the very few instances of SFP would have been preferable in retrospect. However, since the ommission of the very few SFP- datapoints from the analysis would have been justified based on their numerical insignificance, the same insignificance can be assumed for these scant occurrences of SFP as a source of error in the performed analysis. The recordings and analysis of the video-observations are described for each chapter but in general one home-pen observation consisted of 10 minute intervals (one in the morning and one in the afternoon). As described in the experimental chapter, this sampling method was chosen based on the work of Kjaer (2009). However, video observation is a critical element of this study and in this case may warrant some additional and critical consideration as a potential limitation: Before the collection of experimental data was started, home-pens were video-recorded for entire length of the light-schedule. These recordings were skimmed to find out more about the daily routine, not only of the birds but also in relation to management. Observations were timed to not interfere with daily disruptions of the birds by feeding and egg collection (08:00-10:00 am) or afternoon-checks (03:00- 04:00 pm). Also, early morning recordings (06:00-08:00 am) were avoided since birds were mostly observed feeding or in nest-boxes for egg-laying. Additionally, all home- pen observations were analysed by one trained observer per experiment.

2) Experimental lines versus commercial lines

Since the birds used for the experiments of this thesis stem from an experimental selection program (Kjaer and Sørensen, 2001), the theoretical possibility arises that such birds may express a different sub-form of SFP than commercial hybrids (e.g. with or without consumption of feathers). However, the same issue would arise when working with a particular commercial line. Also, the genetic background of commercial hybrid lines is subjected to constant changes due to company-specific genetic programs (Dr. Harlander, pers. Communication). At best, experiments investigating SFP would therefore work with both experimentally selected birds and commercial hybrids who also show FP behaviour. Experiments comparing commercial to experimental lines in their behaviour and physiology could bring clarity to the notion that birds experimentally selected for SFP express the same kind of behaviour (for the same or similar reasons)

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as commercial hybrids. Studies comparing FP birds of commercial lines with those created by selecting for FP are, however, still missing.

3) Development of SFP: How does it start?

The question of whether SFP precedes feather-eating or vice versa remains unanswered. If SFP constitutes a malfunctional behaviour it would be expected that birds engage in SFP prior or independently eating feathers. If SFP develops as an adaptive or maladaptive behaviour, it would be likely to see feather eating preceding SFP- once easily obtainable feathers from the ground have all been consumed, birds may then resort to pluck feathers from conspecifics. Both hypotheses need testing by detailed behavioural observations of commercial and experimental birds in their early life and throughout their reproductive cycle (laying period).

5.5 Conclusion

In conclusion, this thesis has developed and tested an ATD protocol for laying hens (Chapter 2), which still requires validation with respect to effects on central TRP/5-HT levels. Laying hens divergently selected for FP were subjected to ATD (nutritional stress). The result was an increase in GFP in home pens as well as increased unrewarded pecking activity in the operant chamber (Chapter 3), yet no effect was observed for SFP. Finally, the effects of social disruption treatment were tested on the birds in non-cage housing (Chapter 4). This latter experiment revealed a positive link between GFP and social disruption, however, no increase in SFP was observed. For this, novel methods were employed for measuring a physiological stress response in birds (immune activation), linked to the metabolism of the monoamine-precursors TRP and PHE. The sensitivity of these measurements to the stress treatment was partially demonstrated.

Chapter 5) provides a synthesis of the experimental chapters, emphasizing on limitation and directions for future research. This future work should involve operant testing where birds are punished for premature responses and investigate the relationship between GFP and SFP, e.g. via a more severe stress-treatment. Furthermore, detailed

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behavioural observations, pharmacological studies and more fundamental research regarding the kynurenine pathway in birds is suggested. Also, the need for comparative studies involving commercial flocks is emphasized. This thesis hopefully also inspires further work on investigating the complex relationship between TRP metabolism, immune activation and the development of SFP in laying hens.

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5.6 References

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Appendix

Table 6 Analyzed nutrient composition of diet fed to FP and non-FP birds

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Analytical diet specifications

WEIGHT 100.00 [kg] M.E. POULTRY 2,886.30 [kcal/kg] CRUDE PROTEIN 18.07 [%] ARGININE 1.12 [%] LYSINE 0.89 [%] METHIONINE 0.38 [%] TSAA 0.64 [%] TRYPTOPHAN 0.19 [%] THREONINE 0.63 [%] GLYCINE 1.01 [%] HISTIDINE 0.46 [%] LEUCINE 1.60 [%] ISOLEUCINE 0.70 [%] PHENYLALANINE 0.86 [%] PHENYL + TYRO 1.47 [%] VALINE 0.86 [%] GLYC. + SERINE 1.72 [%] PROLINE 1.14 [%] CRUDE FAT 5.77 [%] LINOLEIC ACID 1.97 [%] CRUDE FIBRE 1.83 [%] ADF 2.91 [%] NDF 7.63 [%] DRY MATTER 89.38 [%] CALCIUM TOTAL 4.22 [%] PHOS.TOTAL 0.65 [%] PHOS AV. POULTRY 0.44 [%] SODIUM 0.18 [%] CHLORIDE 0.27 [%] POTASSIUM 0.61 [%] MAGNESIUM 0.17 [%] SULPHUR 0.22 [%] IRON 209.50 [MG/kg] MANGANESE 119.25 [MG/kg] ZINC 118.36 [MG/kg] COPPER 12.71 [MG/kg] IODINE 1.50 [MG/kg] SELENIUM 0.45 [MG/kg] COBALT 0.03 [MG/kg] FLUORINE 9.90 [MG/kg] VIT. A 15.00 [KIU/kg]

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VIT. D3 4.50 [KIU/kg] VIT. E 60.00 [IU/kg] MENADIONE (VIT. K) 3.75 [MG/kg] VIT. B12 37.50 [MCG/kg] RIBOFLAVIN 14.25 [MG/kg] NIACIN 75.00 [MG/kg] CHOLINE 1.11 [G/kg] D-PANTOTHENIC ACID 24.00 [MG/kg] PYRIDOXINE 6.75 [MG/kg] THIAMINE 3.75 [MG/kg] FOLIC ACID 3.75 [MG/kg] BIOTIN 225.00 [MCG/kg] XANTHOPHYLLS 12.83 [MG/kg]

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