A deficient serotonergic system is associated with psychopathological behaviors in various species, among which, feather pecking (FP) in chickens. Deficiency in the serotonergic system can predispose birds to develop FP, while the serotonergic system is affected in birds that feather peck. Serotonin (5-HT) can further influence dopamine (DA) activity. Lines with high FP tendency generally have low central 5-HT and DA turnovers at a young age, but high turnovers at an adult age in brain areas involved in somato-motor regulation and goal-directed behavior. Agonizing 5-HT1A and 5-HT1B receptors increases FP, while antagonizing D2 receptor reduces FP. Genetic associations exist between FP, 5-HT1A and 5-HT1B receptor functioning and metabolism of 5-HT and DA. Birds with deficient functioning of the somatodendritic 5-HT1A autoreceptor and 5-HT metabolism appear predisposed to develop FP. Birds which feather peck often eat feathers, have low whole-blood 5-HT, different gut-microbiota composition and immune competence compared to non-peckers. FP and feather eating likely affect the interaction between gut microbiota, immune system and serotonergic system, but this needs further investigation.

Feather pecking (FP), a damaging behavior where laying hens peck and pull at feathers of conspecifics, is multifactorial and has been linked to numerous behavioral and physiological characteristics. The gut microbiota has been shown to influence host behavior and physiology in many species, and could therefore affect the development of damaging behaviors, such as FP. Yet, it is unknown whether FP genotypes (high FP [HFP] and low FP [LFP] lines) or FP phenotypes (i.e., individuals differing in FP, feather peckers and neutrals) differ in their gut microbiota composition. Therefore, we identified mucosa-associated microbiota composition of the ileum and cecum at 10 and 30 wk of age. At 30 wk of age, we further identified luminal microbiota composition from combined content of the ileum, ceca, and colon. FP phenotypes could not be distinguished from each other in mucosa-associated or luminal microbiota composition. However, HFP neutrals were characterized by a higher relative abundance of genera of Clostridiales, but lower relative abundance of Lactobacillus for the luminal microbiota composition compared to LFP phenotypes. Furthermore, HFP neutrals had a higher diversity and evenness for the luminal microbiota compared to LFP phenotypes. FP genotypes could not be distinguished from each other in mucosa-associated microbiota composition. Yet, FP genotypes could be distinguished from each other in luminal microbiota composition. HFP birds were characterized by a higher relative abundance of genera of Clostridiales, but lower relative abundance of Staphylococcus and Lactobacillus compared to LFP birds. Furthermore, HFP birds had a higher diversity and evenness for both cecal mucosa-associated and luminal microbiota compared to LFP birds at adult age. In conclusion, we here show that divergent selection on FP can (in)directly affect luminal microbiota composition. Whether differences in microbiota composition are causal to FP or a consequence of FP remains to be elucidated.

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in life 17. These findings suggest that the gut microbiota influences behavioural and physiological characteristics in poultry and could therefore influence a bird's ability to cope with environmental and social challenges, such as those encountered in animal production systems. Excessive damaging behaviours are indicative of an animal's inability to cope with a restrictive environment and are frequently seen in production animals. Feather pecking (FP) in chickens is one such damaging behaviour, which involves hens pecking and pulling at feathers of conspecifics, thereby reducing animal welfare and productivity 18. Feather pecking is multifactorial and has been linked to numerous behavioural characteristics, such as fearfulness, stress and activity, as well as physiological characteristics, such as serotonergic, dopaminergic and immune systems 19-21. Since behavioural and physiological systems that are related to FP are also affected by the gut microbiota, microbiota might play a role in the development of FP. Indeed, lines selected for high FP (HFP) and low FP (LFP) differ in behavioural responses, stress response, activity, central serotonergic and dopaminergic activity, peripheral serotonin, innate and adaptive immune characteristics 22-28. Moreover, the HFP and LFP lines differ in intestinal microbial metabolites and microbiota composition determined from caecal droppings and intestinal luminal content 29-31. These findings point to a relationship between the gut microbiota and FP, however, it is unknown whether the gut microbiota influences the development of FP. Therefore, this study aims to identify the effects of early-life microbiota transplantation on FP and behavioural and physiological characteristics related to FP in lines divergently selected for FP (HFP and LFP lines). We further identify the effects of microbiota transplantation on microbiota composition. We hypothesize that microbiota transplantation results in recipients adopting a similar behavioural profile as that seen in the donor line. For example, LFP birds receiving HFP microbiota show more FP and more active behavioural responses compared to LFP birds receiving LFP microbiota or control treatment. Results High and low feather pecking transplantation pools had distinct microbiota composition. Gut microbiota was collected from adult chickens of the HFP and LFP lines that were shown to differ in microbiota composition 31. Transplantation pools were made per line and could be distinguished from each other in terms of microbiota composition using a principal component analysis (PCA) (Fig. 1A). The orders of Clostridiales and Lactobacillales had the highest relative abundance in both pools. The HFP pool had a higher relative abundance of

14Feather pecking (FP) is a major welfare and economic issue in the egg production industry. 15Behavioural characteristics, such as fearfulness, have been related to FP. However, it is unknown how 16 divergent selection on FP affects fearfulness in comparison to no selection on FP. Therefore, we 17 compared responses of birds selected on low (LFP) and high feather pecking (HFP) with birds from 18 an unselected control line (CON) to several behavioural tests (i.e. novel object (NO), novel 19 environment (NE), open field (OF) and tonic immobility (TI)) at young and adult ages. Furthermore, 20 the relation between actual FP behaviour (i.e. FP phenotypes) and fearfulness is not well understood. 21 Therefore, we compared responses of birds with differing FP phenotypes. Feather pecking phenotypes 22 of individual birds were identified via FP observations at several ages. The number of severe feather 23 pecks given and received was used to categorize birds as feather peckers, feather pecker-victims, 24 victims or neutrals. Here we show that HFP birds repeatedly had more active responses (i.e. they 25 approached a NO sooner, vocalized sooner and more, showed more flight attempts and had shorter TI 26 durations), which could indicate lower fearfulness, compared to CON and LFP birds at both young 27 and adult ages. Within the HFP line, feather peckers had more active responses (i.e. they tended to 28show more flight attempts compared to victims and tended to walk more compared to neutrals), 29 suggesting lower fearfulness, compared to victims and neutrals. Thus, in this study high FP seems to 30 be related to low fearfulness, which is opposite to what previously has been found in other 31 experimental and commercial lines. This stresses the need for further research into the genetic and 32 phenotypic correlations between FP and fearfulness in various populations of chickens, especially in 33 commercial lines. Findings from experimental lines should be used with caution when developing 34 control and/or prevention methods that are to be applied in commercial settings. Furthermore, activity 35 and/or coping style might overrule fearfulness within the HFP line, as HFP birds and feather peckers 36 within the HFP line had more active responses. This might indicate a complex interplay between 37 fearfulness, activity and coping style that could play a role in the development of FP. 38 (LP) FP line (Korte et al., 1997). HP chicks showed a longer duration of freezing, and vocalized and 51 walked later in an open field (OF) test than LP chicks, but no difference was found in tonic 52 immobility (TI) duration (Jones et al., 1995). In a commercial line comparison, fewer Rhode Island 53Red (RIR) birds moved away from a novel object (NO) than White Leghorn (WL) birds at adult age 54 and WL birds had more feather damage, indicating that WL birds were more fearful and showed more 55 FP than RIR birds (Uitdehaag et al., 2008). On an individual level Rodenburg et al. (2004) found a 56 strong negative correlation between OF activity at a y...