Long-term organizing effects of prenatal exposure to testosterone or corticosterone on feather pecking

Mirjam Bakker Supervisor: Bernd Riedstra Minor Thesis Master Biology University of Groningen

Abstract Even though feather pecking is a major problem in the poultry industry the mechanisms causing this behaviour are still unclear. One hypothesis considers FP as a part of normal social behaviour, several factors influencing social behaviour could therefore also influence FP. Firstly this could be preen wax composition since several studies have shown the importance of preen wax in individual recognition. Secondly maternal hormone deposition could affect social behaviour since exposure to these hormones can have long-lasting effects on the physiology and behaviour of the offspring. Two potentially interesting hormones are testosterone (T) and corticosterone (C) because both hormones have effects on social behaviour. We predict that increased prenatal exposure to T increases FP. Increased exposure to C could either increase or decrease FP; since previous studies show contradictory results for especially the stress pathways. In this study, eggs from laying hens were exposed to either T or C. 21 weeks after hatching chickens were housed inside in groups of 3 (1 treated female, 1 control female, 1 control male) and pecking behaviour was observed. Furthermore, biometry, comb characteristics and preen wax composition were determined. The stress response was determined with a tonic immobility test. Results show that testosterone had a direct effect on feather pecking; increased prenatal exposure to T resulted in more FP. Furthermore, testosterone had an indirect effect on FP by influencing preen wax. Corticosterone had no effect on feather pecking, but this study does confirm the effect of stress and dominance on FP. In conclusion it may be profitable to select parental strains for low yolk testosterone or otherwise monitor yolk T levels of eggs. This could reduce FP or warn for a higher risk on the development of feather pecking occurring in chicks bred from high yolk T level eggs.

Introduction A major problem in the poultry industry is feather pecking (FP). These are pecks directed towards feathers of another individual. Two types of FP can be distinguished, gentle (GFP) and severe (SFP) pecking. Although GFP normally doesn’t result in substantial damage, very frequent pecking at the feathers deteriorates plumage quality. SFP is more problematic due to the pulling out of feathers. This can result in wounds, cannibalism and even death (van Horne & Achterbosch 2008; Serdaroğlu et al. 2013; Rodenburg et al. 2013). Due to feather pecking, i.e. egg weight is reduced and food intake increased. Therefore, next to decreased welfare, FP also causes economic losses (Chow & Hogan 2005; de Haas et al. 2013). Current methods to decrease the effects of injurious pecking are housing in reduced light conditions and beak trimming. Both methods bring along their own (welfare) problems: the former method impairs proper eye development (Siopes et al. 1984), the latter results in chronic pain due to damage to the nerves (Breward & Gentle 1985; Gentle et al. 1990; Quartarone et al. 2012). To increase welfare, beak trimming will be banned in the Netherlands from 2018 onwards. However, chickens with an intact beak can cause more damage to conspecifics, making it important to find a solution to this problem. Understanding the principles and mechanisms underlying the development of this behaviour are an important step in the search for a solution to prevent this behaviour in the future.

There are several hypotheses explaining why feather pecking develops. Firstly, it is considered as redirected ground pecking behaviour. In nature chickens spent on average 60% of their time on foraging behaviour (Dawkins 1989). In the intensive poultry housing systems the nutritional demands are met in just a fraction of that time, whereas the ‘need’ to perform foraging behaviour remains high. Blokhuis & Arkes (1984) showed feather pecking probably develops from the need to perform foraging behaviour. Chickens living on a floor without substrate showed a higher FP frequency compared to chickens with substrate. However, learning where to peck at an early age does not seem to prevent FP in later life: de Jong et al. (2013) showed that feather pecking could not be prevented by raising chicks in a condition with plenty of adequate foraging material. Secondly, it could be that feather pecking develops due to a lack of suitable substrate to dust bathe. Dust bathing is a primary need for birds and Vestergaard & Lisborg (1993) showed that chickens with a substrate unsuitable for dust bathing pecked more towards conspecifics, compared to chickens with a suitable substrate. This fits with Blokhuis & Arkes (1984), although they argue that it’s a lack of foraging that causes FP, not a lack of dust bathing. Thirdly, feather pecking could develop due to a lack of specific nutrients. McKeegan & Savory (2001) found that chickens showing high FP frequency also ate more feathers. When fed more nutritional food, feather pecking declined (Savory et al. 1999). On the other hand, van Krimpen et al. (2005) showed that when chickens had to work harder for their food without changing nutritional value, FP also declined. Finally, feather pecking could also be part of normal social behaviour. Riedstra & Groothuis (2002) showed that chicks pecked more towards unfamiliar individuals at such early age that it was very unlikely that the behaviour was already redirected. It could be that FP is used to identify individuals and maintain social bonds and rank order (Harrison 1965). This can explain why it is often a dominant chicken pecking towards a subordinate and why the receiver often ignores gentle pecks. Furthermore, it is known that light induced lateralization (LiL) influences social behaviour. Light reaching the embryo in the egg in the later stages of incubation lateralizes brain and behaviour. Riedstra & Groothuis (2004) showed that LiL also influenced feather pecking early in life. Light incubated animals showed increased feather pecking behaviour and where less discriminating between familiar and unfamiliar chicks than dark incubated animals. Riedstra & Groothuis (2002) also noticed that severe pecking is initially accompanied by gentle pecking. They therefore argued that both are governed by the same motivational system, but that severe pecking is an escalated version of gentle pecking. This escalation could be due to stressful environments. In poultry farming, chickens are housed in large groups. This increases the demand for social exploration, because of the large number of unfamiliar birds surrounding an individual. The amount of unfamiliar birds is thought to be stressful for chickens. It is argued that SFP arises as a mechanism to cope with this (stressful) situation (Riedstra & Groothuis 2002). It could be that different coping styles (the way an individual reacts to certain events) can explain why not every chicken shows FP (Koolhaas et al. 1999).

If feather pecking indeed is a social behaviour, it is probable that different kinds of cues that affect social behaviour are also involved in feather pecking. One such cue is individual recognition by olfaction. Recently it has become clear that olfaction is important in social behaviour and individual recognition in birds (for a review see Caro & Balthazart, 2010). An important source for olfactory signals is the preen gland (uropygial gland) located at the base of the tail feathers. This gland produces preen wax, which is used to coat feathers as part of plumage maintenance. Identification of preen wax showed that there are species and gender differences (Mardon et al. 2010; Biester et al. 2012) and also individual differences (Karlsson et al. 2010). Balthazart & Schoffeniels (1979) found that after removal of the preen gland, sexual displays are reduced, suggesting that the preen gland plays a role in mating (Johansson & Jones 2007; Hirao et al. 2009). Amo et al. (2012) showed starlings could discriminate sex of conspecifics by only using chemical cues. Chemical analyses of preen wax showed that there were sex and age differences. Going even further, Whittaker et al. (2013) found a correlation between wax composition and reproductive success, suggesting that preen wax could be used for mate choice. Sandilands et al. (2004) found that preen wax composition differed between pecked and non-pecked chickens, but only in one out of two experiments. Considering these findings, it is very probable that the preen gland is involved in social recognition in chickens and it could be possible that preen wax is a factor regulating feather pecking behaviour.

Avian eggs contain many hormones of maternal origin. Maternal hormones could influence social behaviour. It is often argued that, to increase fitness in offspring, mothers can adjust the phenotype by changing the amount of hormones she deposits (for a review see Groothuis et al., 2005). In relation to feather pecking, both testosterone (T) and corticosterone (C) are potentially interesting. Increased prenatal exposure to T affects important traits, like aggression and growth (Rubolini et al. 2005; von Engelhardt et al. 2006) and could also effect dominance by influencing comb colour (Casagrande et al. 2012; Riedstra et al. 2013). Moreover Riedstra (2003) showed that hens from a high FP (HP) line had higher T levels than the low FP (LP) line. Lateralization affects FP, as mentioned above. Both T and C have been shown to reduce LiL, at least when administered to the egg late during incubation and in pharmacological doses (Riedstra & Groothuis 2004; Rogers & Deng 2005; Freire et al. 2006). It is unknown whether C and T override the effects of LiL applied at the onset of incubation in a physiological dose. Riedstra et al. (2013) showed a near significant effect for T, for C this is unknown. An alternative pathway for testosterone or corticosterone to affect FP behaviour is via a stress related mechanism. Stress is frequently implicated in inducing the development of feather pecking behaviour. The above mentioned HP and LP line that differ in early T exposure also differ in the way they respond to stressors, both behaviourally and physiologically (Riedstra 2003). It has even been suggested that they represent two distinct coping styles (Koolhaas et al. 1999). C is also interesting in this respect since it has been shown that prenatal exposure to this hormone affects the stress response, although not in a consistent manner (Janczak et al. 2006, 2007; Hayward et al. 2006). Considering these findings, it is difficult to predict how both hormones will affect feather pecking. Increased prenatal exposure to T or C could reduce FP by removing lateralization, thereby resembling the dark incubated chicks in Riedstra & Groothuis (2004). Testosterone could also increase FP by influencing dominance rank or by resembling the HP line in Riedstra (2003). Corticosterone could influence FP by influencing stress related mechanism, but it is yet unclear in which direction.

Figure 1 Possible pathways for a treatment effect. Hormone injections could either have a direct effect on FP or an indirect effect by influencing either physical phenotype (biometry, comb colour, preen wax composition) or behavioural phenotype (stress related mechanisms). Summarizing, it is likely that aspects involved in social behaviour of poultry such as the composition of preen wax and the exposure to maternal hormones could influence feather pecking (figure 1). Considering the literature, I predict that prenatal exposure tot testosterone affects both preen wax composition and dominance rank and that via this way it affects FP behaviour. Corticosterone would mainly influence the way animals deal with stressors and affect FP via that pathway. Possible direct effects on FP will also be explored.

Methods and materials Animals and housing Eggs from laying hens (Gallus gallus domesticus) were collected from a commercial breeder (Verbeek Lunteren, the Netherlands). After drilling a small hole in the shell, eggs received an intrayolk injection of 100μl sesame oil with or without (control) dissolved hormone. For testosterone this was 75ng crystallite T, which is roughly two times the standard deviation of T measured in these eggs (Groothuis et al. 2005). For corticosterone this was 27ng. After injection the hole was sealed with candle wax. Eggs were incubated at 37.5 °C (60% humidity) and automatically turned every 3h. Light intensity was enough to induce lateralization. After hatching chicks were housed in groups of 4 (1 chick of each sex * treatment combination). After one week chickens received an additional wingclip for identification and after 35 days moved to a large outdoor roofed aviary. Twenty weeks after hatching phenotypic measurements were taken for all roosters. Twelve control males were selected for the experiment; all other males were killed using a large dose of anaesthetics. At age 21 weeks chickens were moved inside (17 ± 0.2 °C, 68 ± 2 % humidity) and housed in groups of three (1 control male, 1 control female and 1 treated female). Cages were roughly 175x95x75 cm (WxHxD) and contained sawdust as substrate. After 3 days a grid was placed over the substrate to induce feather pecking. During the whole experiment, food (a mixture of laying hen pellet and mixed grains) and water was available ad libitum. Cages were cleaned when necessary. To create a sufficient sample size, all experiments were repeated. For both hormone treatments this resulted in 2 groups (batches). An overview of the sample size for the different groups can be found in table 1.

Batch # Cages Total ♀

Corticosterone 1 12 24 2 7 14

Subtotal 2 19 38

Testosterone 1 12 24

2 9 18 Subtotal 2 21 42

Table 1 Overview of used sample size in different experiments. It was necessary to create 2 batches, because the housing was not sufficient for more than 12 groups. Due to a low sample size of treated females, it was not always possible to create all 12 groups that could be housed.

Body measurements To measure phenotypic differences (biometry), body weight (BM) and tarsus length (TL) were recorded. These measurements were taken on day -40 (age ≈ 17 weeks) for all chickens, on day -10 (age ≈ 21 weeks) for all roosters and on day 1 and day 16 for the experimental hens. On these days a preen wax sample and comb measurements were also collected. For the wax sample a cotton swab was swept over the preen gland for 10 sec. The sample was placed in a 2ml glass vial and stored at -4 °C until analysis. For the comb measurements redness of the comb was determined by using a spectrophotometer (Avantes, Eerbeek, the Netherlands). Each time three samples were taken. Brightness, hue and chroma were calculated similar to Casagrande et al. (2012). See Riedstra et al. (2013) for a complete protocol.

Figure 2 Overview of the experimental design. Selection of males was done 10 days before experiment started (only control males were kept). On day -3 animals were placed in experimental setting to habituate. On day 0 TI was preformed and on day 1 the phenotypic measurements, both only on females. Hereafter behaviour was observed. Approximately 2 weeks later, phenotypic measurements and TI were repeated. Behavioural observations During the experiment, pecking behaviour was observed for 15 minutes per cage, on average 3 times per day for 10 days total. For both corticosterone experiments this resulted in a total of 8,5h, for the first testosterone experiment 4,5h and for the second testosterone experiment 7,5h. Observations were voice recorded and processed later. Feather pecking behaviour was separated in gentle and severe pecks. For each bout, the attacker, receiver, type and number of pecks were recorded. This data was also used to determine ranking order between the two hens.

Tonic immobility The tonic immobility test (TI) was used to measure stress response (Vestergaard et al. 1993; Hughes & Duncan 1997). Chickens were tested individually. In the housing room a table was placed symmetrically in the room, directly under a lamp to avoid shadows. A chicken was taken out of its cage and placed on the back of the table. After pressing gently with one hand on the belly for 10 sec, the hand was removed and the timer was started. An induction was successful if it took the subject longer than 10s to stand up. If this was not the case, this was recorded and immobility was induced again, with a maximum of 10 trials. For all subjects time until standing up and direction of rotation was recorded. TI was only preformed during the housing experiment, for testosterone on day 0 and day 16, twice per day (once in the morning and once in the afternoon), for corticosterone only on day 0.

Gas chromatography Analysis of wax samples was performed using gas chromatography (GC). In previous research (van der Bijl 2012) it was noticed that the cotton swabs generated a lot of noise in the output. In order to maintain the same experimental conditions it was not possible to use other materials. In an attempt to reduce noise, the cotton was plucked off making sure it contained the sample taken. This sample was put in a clean 2 ml glass vial. 500μl hexane was added to each sample and shaken for 30 minutes. Using tweezers, the cotton was removed. To correct for the hexane absorbed by the cotton, it was first evaporated under a gentle flow of nitrogen gas. Subsequently, 100μl hexane was added and shaken for 1 min. This solution was transferred to a glass insert, which was placed back in the vial. All samples were placed in the auto-sampler of the gas chromatograph (GC, Agilent Technologies 7890A). For each sample 5μl was injected in the GC, which was set in splitless mode. See figure 3 for an explanation of the method. All samples were analysed together, no distinction was made for the different hormone treatments. In total there were 140 peaks found. Peaks that were positive for less than 10% of the samples were removed, resulting in 86 peaks left.

Figure 3 Method used for GC. Inlet state was 250 °C, 23,448 psi and 3 ml/min flow. The column flow was 1ml/min helium (green line). The initial oven temperature was 50 °C, which was held for 1.5 min. Hereafter the temp was first raised to 150 °C (20 °C/min) and then raised to 300 °C (4 °C/min), held for 20 min (red line). The dotted line shows the pressure during the method (adapted from Reneerkens, 2007).

Statistical analysis For both treatments there were no differences found for the repeats (ANOVA, all p-values > 0.1), therefore the data was pooled in one group per treatment. Time spend observing FP behaviour was not equal for the testosterone experiments. Therefore pecking data was corrected for this by dividing by the observation time (4,5h and 7,5h respectively). Furthermore, pecking data was not normally distributed. A square root transformation was performed to transform the Poisson distributed data. For body weight and tarsus length a repeated measures ANOVA was used to compare the different measuring dates, sex and treatment. All other data only contained females; an ANOVA was used with both treatment and dominance in the model. A principal component analysis (PCA) was performed on GC data, for testosterone and corticosterone separately, similar to van der Bijl (2012). All 86 peaks were put in the model. It resulted in 5 different components (PCs) that explained 68.1% (testosterone) and 75.6% (corticosterone) of all variation. A partial correlation analysis was performed on all data, controlling for a possible cage effect, for both experiments separately. Correlated variables where further tested using a partial correlation test, controlling for treatment and dominance. Statistical analysis was performed using SPSS 22 (IBM Corp., Armonk, NY)

Ethical note All experiments were approved by the ethical commission (DEC 5768A, D & F).

Results Body measurements For body weight and tarsus length there was a sex and time difference in both experiments (p < 0.001); males were heavier and larger than females, all subjects were heavier and larger on day 16 compared to day 0. There was no treatment or dominance effect on weight or length (table 2). Treatment or dominance had no effect on comb measurements (brightness, hue and chroma, p-values > 0.1). Testosterone had an effect on the PCA of the wax samples. For PC-2 and PC-3 there was a sex difference (p < .000 for both), for PC-4 a treatment effect for males only (p = 0.012, figure 4). For corticosterone there was a sex difference in PC-1 and PC-3 (p = 0.021 and p = 0.004 respectively) and a near treatment effect for PC 2 (p = 0.082). For all other components there was no effect (p > 0.100). In conclusion, there were no large effects of treatment on body measurements and there were also no effects of dominance on these variables

Body weight Tarsus length

1743 ± 210 99 ± 3.1 Testosterone Males p = 0.262 p = 0.173 p < 0.001 1499 ± 101 82 ± 1.7 Females p = 0.935 p = 0.340

1956 ± 148 100 ± 3.8 Corticosterone Males

p < 0.001 1450 ± 134 82 ± 2.4 Females p = 0.843 p = 0.331 Table 2 Results of body weight and tarsus length measurements. A sex difference was found (p < 0.001) in both experiments. For corticosterone no measurements were performed on treated males, therefore data contains only control males. (P-values state difference between control and treated animals) * 3

1 PC!4

!1

!3 Females Males Figure 4 Effect of testosterone on preen wax (PC-4) for control (open bars) and treated (filled bars) chickens, both females (pink) and males (blue) are shown. Only for males a treatment effect is found (p = 0.012), * p < 0.05.

Behavioural observations Dominance rank was not influenced by corticosterone (p = 0.101, 13 out of 19 dominant) or testosterone (p = 0.404, 9 out of 21 dominant). Dominant chickens directed more pecks towards the subordinate in the testosterone experiment (all p-values < 0.05), but not in the corticosterone experiment (all p-values > 0.100). Testosterone females directed more GFPs towards the subordinate compared to controls (p = 0.021). This was not reflected in the number of severe feather pecks, although SFP is correlated to GFP (R > 0.35; p < 0.05). For GFP received, there was a near significant effect (p = 0.088) for control females (see figure 5). Corticosterone had no effect on feather pecking (all p-values > 0.1). In conclusion, in both experiments treatment had no effect on dominance, but in the testosterone experiment dominance did affect pecking frequency. This was not seen in the corticosterone experiment. Gentle pecks were influenced by testosterone, but not by corticosterone. Effect of testosterone on FP Directed+to+female Received+from+female 10 * #

5 #+gentle+pecks

0

Control Control

Testosterone Testosterone Figure 5 Effect of testosterone on gentle feather pecks. Testosterone females directed more GFPs towards the other female, compared to controls. For pecks given this was significant (p = 0.021), for pecks received near significant (p = 0.088). Data was pooled for both batches. (* p < 0.05, # p < 0.1) Tonic immobility Neither hormone treatment (testosterone: p = 0.323 and 0.829 for day 0 and day 16 respectively; corticosterone: p = 0.723), nor dominance rank (testosterone: p = 0.157; corticosterone: p = 0.574) affected TI. Correlations Feather pecking was related to comb measurements in both experiments (table 3). In the T experiment, severe pecks given were lower for chickens with a higher chroma. Chickens with higher brightness received more peck from the male. In the C experiment chickens with higher hue and chroma received more gentle pecks from the other female and more severe pecks from the male. For testosterone FP was also related to preen wax (PC-2, table 4), this was for both pecks given and received. For corticosterone FP was also related to TI (table 5). Chickens that received more pecks (mostly from the rooster) spent more time immobile. Tonic immobility was related to preen wax for corticosterone (PC-2, figure 6a), but not for testosterone. In the testosterone experiment TI (day 0) is related to brightness (R = -.472, p = 0.042) and chroma (R = 0.542, p = 0.025). Chickens with higher brightness spent less time immobile, chickens with a higher chroma spent more time immobile (figure 6b). No other correlations were found (-0.4 > R < 0.4, p > 0.1). In conclusion, a relation between behaviour phenotype and physical phenotype was found. In the testosterone experiment this was between tonic immobility and comb measurements. In the corticosterone experiment this was between TI and wax analysis. Further more, a relation between FP and comb measurements (both experiments), FP and preen wax (testosterone) and FP and TI (corticosterone) were found.

Brightness Hue Chroma Total SFP R = -.532 Testosterone given p = 0.023 R = 0.522 SFP from male p = 0.031 Total pecks R = 0.516 R = -0.553

from male p = 0.034 p = 0.017 GFP from R = 0.344 R = 0.363 Corticosterone female p = 0.037 p = 0.027 R = 0.591 SFP from male p = 0.001 Table 3 Overview of all correlations between FP and comb measurements in both experiments. Only significant results are given (p < 0.05). (GFP = gentle feather pecking, SFP = severe feather pecking)

GFP towards Total pecks Total GFP Total pecks female towards female given given PC-2 R = 0.675 R = 0.534 R = 0.521 R = 0.519 p = 0.003 p = 0.027 p = 0.032 p = 0.033 SFP received Total pecks Total SFP Total pecks from female from female received received PC-2 R = -.611 R = -.543 R = -.592 R = -.505 p = 0.009 p = 0.024 p = 0.012 p = 0.039 Table 4 Correlations between preen wax (PC-2) and feather pecking in the testosterone experiment. (GFP = gentle feather pecking, SFP = severe feather pecking) Upper part is pecks directed towards another chicken, lower part is pecks received from another chicken.

GFP received SFP received Total pecks Total pecks from male from male from male received TI R = 0.347 R = 0.367 R = 0.402 R = 0.588 p = 0.035 p = 0.026 p = 0.014 p = 0.008 Table 5 Correlations between tonic immobility (TI) and feather pecking in the corticosterone experiment. All correlations were for pecks received from another chicken. (GFP = gentle feather pecking, SFP = severe feather pecking).

A B

15 2.5

1 2.0

10 1.5 Chroma 0 PC_2 1.0 Brightness 5

&1 0.5

0 0.0 0 100 200 300 400 0 100 200 300 Immobility/(sec) Brightness Immobility/(sec) Chroma Figure 6 A Correlation between TI and PC-2 (R = 0.423, p = 0.009) for the corticosterone experiment. B Correlation between TI and comb measurements (brightness and chroma) for the testosterone experiment.

Discussion The aim of this study was to see if prenatal treatment with hormones had an effect on feather pecking (figure 7). Treatment could have a direct effect, by either increasing or decreasing feather pecking (5), or an indirect effect by influencing behavioural phenotype (tonic immobility: 1-2) or by influencing physical phenotype (biometry, comb or preen wax composition: 3-4).

Figure 7 Possible pathways for a treatment effect. Hormone injections could either have a direct effect on FP or an indirect effect by influencing phenotype or stress response.

Effects of testosterone on feather pecking Testosterone had both a direct and indirect effect on feather pecking. T directly affected FP by increasing gentle pecks given to the subordinate chicken. There was a near significant effect on gentle pecks received from the dominant chicken. This is the first study to show long term organizing effects of increased prenatal exposure to testosterone. This means that maternal hormones also have effect during later life, thus marking the importance of maternal effects. Often studies only measure early effects of prenatal treatment. Our study shows the importance of also measuring effects in later life. In the poultry industry, it is severe pecking that is seen as most problematic, not the gentle pecking. Although there was no effect of T on SFP, there was a correlation between gentle and severe pecking. It could be that in this study there was not enough severe pecking to see an effect. Therefore it can still be relevant to screen eggs for yolk testosterone levels to predict the risk of feather pecking.

Testosterone indirectly affected feather pecking via physical phenotype. There was a relation between FP and preen wax composition (PC-2). There was also an effect of T on preen wax (PC-4), but only in males. In previous research by van der Bijl (2012), an effect of T on wax was also found but only in females. In this case it was for PC-1, which explains a lot more variance than PC-4 in this experiment (46,8% and 7,8% respectively). In both cases testosterone resulted in a higher value, suggesting that the wax composition of testosterone animals contained more lightweight components. Although in this experiment there is no effect of T on wax for females, the effect of treatment is still important. Furthermore, these results show that testosterone influences wax composition and that wax influences feather pecking, even though this is for different components. As argued before, it is possible that preen wax plays an important role in individual recognition. A big problem in poultry farming is the amount of chickens housed together. It is impossible for a chicken to know each individual in a barn, therefore increasing the demand for social exploration. This could be very stressful and it is possible that this is one of the reasons feather pecking occurs. If social recognition could be influenced, resulting in a decreased demand for exploration, it could result in decreased FP. Therefore it is important to further investigate not only the role of preen wax, but also the composition of individual chickens. A possible approach could be removing either olfaction or the preen gland, but a less invasive way is preferable.

Testosterone had no effect on behavioural phenotype and there was no relation between FP and behavioural phenotype. However behavioural phenotype affects FP in an indirect way. Our results show that there is a relation between tonic immobility, comb measurements and feather pecking. Chickens that showed a longer TI, had higher chroma and lower brightness. Chickens with higher chroma and lower brightness received less severe pecks, but also directed less severe pecks towards another chicken. These results suggest that chickens with higher fear show reduced feather pecking and receive fewer pecks. Earlier results by Riedstra (2003) showed that chickens from a high FP line had a longer TI than the low FP line. Also, Riedstra & Groothuis (2002) argued that SFP probably arises from GFP in more stressful situations. Both suggest that an increase in feather pecking also increases tonic immobility. Our results are contradictory. Further research is necessary to investigate the role of stress in FP.

Although testosterone affected feather pecking, there was no treatment effect on dominance. As mentioned before, dominant chickens peck more compared to subordinate chickens (as is also found in this experiment). Therefore, if testosterone chickens peck more, an increase in dominance is also expected. These results could probably be explained by the fact that not all cages showed the same activity in FP behaviour. Housing chickens in same-treatment set-ups (two control or two testosterone chickens together) could control for these activity differences.

In conclusion, testosterone affects feather pecking in different ways. Firstly there is a direct effect. Testosterone treated chickens showed more gentle feather pecking. Secondly, there are indirect effects of testosterone. There is a relation between T, physical phenotype (wax) and FP. There is also a relation between behavioural phenotype (TI), physical phenotype (Comb) and FP. See figure 8 for an overview.

Figure 8 Effect diagram for testosterone. Several effects are found, shown with red lines. The grey lines show effects that were not present in our results. Future research should investigate the effects of testosterone on feather pecking in the industry. For this it is necessary to measure hormone levels in fertilized eggs, hereafter FP behaviour can be observed. If chickens with high levels of T in yolk also peck more, it can be possible to decrease FP in farm by selecting eggs with low yolk testosterone.

Effect of corticosterone on feather pecking In the corticosterone experiment, only non-treatment related effects are found. Corticosterone had no effect on tonic immobility, feather pecking behaviour, biometric- or comb measurements. For preen-wax a near significant effect was found for PC-2. It could be that maternal corticosterone plays no role in these factors, but the role of corticosterone in feather pecking cannot be excluded. It is possible that C only has an embryonic effect. Kalliecharan & Hall (1974) showed that during embryonic development, there is an increase in C from day 12 of incubation, with C at highest concentration around day 19. Studies using prenatal injection on day 18 of incubation (i.e. Freire et al. 2006) did found effects on feather pecking. Corticosterone production is regulated by the HPA-axis. After a stressor, the hypothalamus releases corticotrophin-releasing hormone (CRH). CRH activated the anterior pituitary gland, which releases adrenocorticotrophic hormone (ACTH). ACTH activates the adrenal gland, which produces several glucocorticoids, including corticosterone. If maternal hormones could manipulate sensitivity of the HPA-axis, it is possible this would also affect feather pecking. It looks like maternal corticosterone does not affect the HPA-axis, but other hormones (i.e. cortisol, another glucocorticoid) could be potentially interesting. For this more research is necessary.

Still, an effect of prenatal exposure to corticosterone on feather pecking cannot be excluded due to the near significant effect of C on preen wax. The used method to identify wax components results in lots of noise. This noise could mask effects of C on preen wax. A cleaner method could control for this. Furthermore it is possible that different behaviours tests are necessary to show a prenatal effect of C on stress. It is recommended to use more stress related behaviour tests in future studies.

This experiment shows that feather pecking is affected by several factors. Both behavioural- and physical phenotype affected FP, and there also was a relation between both (see figure 9). No effect of dominance is found in this experiment. As argued before, comb colour is a signal of dominance. In this experiment, increased chroma and hue resulted in more FP. Therefore the role of dominance in regulating FP cannot be excluded.

Figure 9 Effect diagram for corticosterone. Several effects were found, shown by orange arrows. The small arrow stands for a near significant effect. Grey arrows stand for no effect. Overall, we can conclude that feather pecking is influenced by several factors. Therefore this is a multifactorial behaviour, which makes it harder to find the underling cause of FP. Our research shows that both behavioural and physical phenotype plays a role in regulating FP. Testosterone had both a direct and indirect effect on FP, this is not seen in the corticosterone experiment. Selection on eggs with low yolk T could be a solution to decrease feather pecking, but probably more alterations are necessary to remove the problem of FP. References

AMO, L., AVILÉS, J. M., PAREJO, D., PEÑA, A., RODRÍGUEZ, J. & TOMÁS, G. 2012. Sex recognition by odour and variation in the uropygial gland secretion in starlings. The Journal of animal ecology, 81, 605–13.

BALTHAZART, J. & SCHOFFENIELS, E. 1979. Pheromones are involved in the control of sexual behaviour in birds. Die Naturwissenschaften, 66, 55–6.

BIESTER, E.-M., HELLENBRAND, J., GRUBER, J., HAMBERG, M. & FRENTZEN, M. 2012. Identification of avian wax synthases. BMC biochemistry, 13, 4.

BLOKHUIS, H. J. & ARKES, J. G. 1984. Some observations on the development of feather-pecking in poultry. Applied Animal Behaviour Science, 12, 145–157.

BREWARD, J. & GENTLE, M. J. 1985. Neuroma formation and abnormal afferent nerve discharges after partial beak amputation (beak trimming) in poultry. Experientia, 41, 1132–1134.

CARO, S. P. & BALTHAZART, J. 2010. Pheromones in birds: myth or reality? Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology, 196, 751–66.

CASAGRANDE, S., COSTANTINI, D., DELL’OMO, G., TAGLIAVINI, J. & GROOTHUIS, T. G. G. 2012. Differential effects of testosterone metabolites oestradiol and dihydrotestosterone on oxidative stress and carotenoid-dependent colour expression in a bird. Behavioral Ecology and Sociobiology, 66, 1319–1331.

CHOW, A. & HOGAN, J. A. 2005. The development of feather pecking in Burmese red junglefowl: the influence of early experience with exploratory-rich environments. Applied Animal Behaviour Science, 93, 283–294.

DAWKINS, M. 1989. Time Budgets in Red Junglefowl as a Baseline for the Assessment of Welfare in Domestic Fowl. Applied Animal Behaviour Science, 24, 77–80.

DE HAAS, E. N., KEMP, B., BOLHUIS, J. E., GROOTHUIS, T. & RODENBURG, T. B. 2013. Fear, stress, and feather pecking in commercial white and brown laying hen parent-stock flocks and their relationships with production parameters. Poultry science, 92, 2259–69.

DE JONG, I., REUVEKAMP, B. & GUNNINK, H. 2013. Can substrate in early rearing prevent feather pecking in adult laying hens? Animal Welfare, 22, 305–314.

FREIRE, R., VAN DORT, S. & ROGERS, L. J. 2006. Pre- and post-hatching effects of corticosterone treatment on behavior of the domestic chick. Hormones and behavior, 49, 157–65.

GENTLE, M. J., WADDINGTON, D., HUNTER, L. N. & JONES, R. B. 1990. Behavioural evidence for persistent pain following partial beak amputation in chickens. Applied Animal Behaviour Science, 27, 149–157.

GROOTHUIS, T. G. G., MÜLLER, W., VON ENGELHARDT, N., CARERE, C. & EISING, C. 2005. Maternal hormones as a tool to adjust offspring phenotype in avian species. Neuroscience and biobehavioral reviews, 29, 329–52.

HARRISON, C. 1965. Allopreening as Agonistic Behaviour. Behaviour, 24, 161–209.

HAYWARD, L. S., RICHARDSON, J. B., GROGAN, M. N. & WINGFIELD, J. C. 2006. Sex differences in the organizational effects of corticosterone in the egg yolk of quail. General and comparative endocrinology, 146, 144–8. HIRAO, A., AOYAMA, M. & SUGITA, S. 2009. The role of uropygial gland on sexual behavior in domestic chicken Gallus gallus domesticus. Behavioural Processes, 80, 115–120.

HUGHES, B. O. & DUNCAN, I. J. H. 1997. The influence of strain and environmental factors upon feather pecking and cannibalism in fowls. British poultry science, 13, 525–547.

JANCZAK, A. M., BRAASTAD, B. O. & BAKKEN, M. 2006. Behavioural effects of embryonic exposure to corticosterone in chickens. Applied Animal Behaviour Science, 96, 69–82.

JANCZAK, A. M., HEIKKILÄ, M., VALROS, A., TORJESEN, P., ANDERSEN, I. L. & BAKKEN, M. 2007. Effects of embryonic corticosterone exposure and post-hatch handling on tonic immobility and willingness to compete in chicks. Applied Animal Behaviour Science, 107, 275–286.

JOHANSSON, B. G. & JONES, T. M. 2007. The role of chemical communication in mate choice. Biological reviews of the Cambridge Philosophical Society, 82, 265–89.

KALLIECHARAN, R. & HALL, B. 1974. A developmental study of the levels of progesterone, corticosterone, cortisol, and cortisone circulating in plasma of chick embryos. General and comparative endocrinology, 372, 364–372.

KARLSSON, A.-C., JENSEN, P., ELGLAND, M., LAUR, K., FYRNER, T., KONRADSSON, P. & LASKA, M. 2010. Red junglefowl have individual body odors. The Journal of experimental biology, 213, 1619–24.

KOOLHAAS, J. M., KORTE, S. M., DE BOER, S. F., VAN DER VEGT, B. J., VAN REENEN, C. G., HOPSTER, H., DE JONG, I. C., RUIS, M. A & BLOKHUIS, H. J. 1999. Coping styles in animals: current status in behavior and stress-physiology. Neuroscience and biobehavioral reviews, 23, 925–35.

MARDON, J., SAUNDERS, S. M., ANDERSON, M. J., COUCHOUX, C. & BONADONNA, F. 2010. Species, gender, and identity: cracking petrels’ sociochemical code. Chemical senses, 35, 309–21.

MCKEEGAN, D. E. . & SAVORY, C. . 2001. Feather eating in individually caged hens which differ in their propensity to feather peck. Applied Animal Behaviour Science, 73, 131–140.

QUARTARONE, V., ROCCA, G. DELLA & PASSANTINO, A. 2012. Beak trimming and other pain’s sources in laying hens and broilers: welfare problems and medico-legal aspects. Large Animal Review, 18, 245–252.

RENEERKENS, J. W. H. 2007. Functional aspects of seasonal variation in preen wax composition of sandpipers (Scolopacidae). PhD Thesis, University of Groningen.

RIEDSTRA, B. 2003. Development and social nature of feather pecking. PhD Thesis, University of Groningen.

RIEDSTRA, B. & GROOTHUIS, T. G. . 2002. Early feather pecking as a form of social exploration: the effect of group stability on feather pecking and tonic immobility in domestic chicks. Applied Animal Behaviour Science, 77, 127–138.

RIEDSTRA, B. & GROOTHUIS, T. G. . 2004. Prenatal light exposure affects early feather-pecking behaviour in the domestic chick. Animal Behaviour, 67, 1037–1042.

RIEDSTRA, B., PFANNKUCHE, K. A. & GROOTHUIS, T. G. G. 2013. Increased exposure to yolk testosterone has feminizing effects in chickens, Gallus gallus domesticus. Animal Behaviour, 85, 701–708.

RODENBURG, T. B., VAN KRIMPEN, M. M., DE JONG, I. C., DE HAAS, E. N., KOPS, M. S., RIEDSTRA, B. J., NORDQUIST, R. E., WAGENAAR, J. P., BESTMAN, M. & NICOL, C. J. 2013. The prevention and control of feather pecking in laying hens: identifying the underlying principles. World’s Poultry Science Journal, 69, 361–374.

ROGERS, L. J. & DENG, C. 2005. Corticosterone treatment of the chick embryo affects light- stimulated development of the thalamofugal visual pathway. Behavioural brain research, 159, 63–71.

RUBOLINI, D., ROMANO, M., MARTINELLI, R. & SAINO, N. 2005. Effects of elevated yolk testosterone levels on survival, growth and immunity of male and female yellow-legged gull chicks. Behavioral Ecology and Sociobiology, 59, 344–352.

SANDILANDS, V., POWELL, K., KEELING, L. & SAVORY, C. J. 2004. Preen gland function in layer fowls: factors affecting preen oil fatty acid composition. British Poultry Science, 45, 109–115.

SAVORY, C. J., MANN, J. S. & MACLEOD, M. G. 1999. Incidence of pecking damage in growing bantams in relation to food form, group size, stocking density, dietary tryptophan concentration and dietary protein source. British poultry science, 40, 579–84.

SERDAROĞLU, M., KOKNAROGLU, H. & AKUNAL, T. 2013. Animal welfare: An animal science approach. Meat Science, 95, 821–827.

SIOPES, T. D., TIMMONS, M. B., BAUGHMAN, G. R. & PARKHURST, C. R. 1984. The effects of light intensity on turkey poult performance, eye morphology, and adrenal weight. Poultry science, 63, 904–9.

VAN DER BIJL, W. 2012. The relationships between feather pecking, prenatal hormones & preen wax composition. Minor Thesis, University of Groningen.

VAN HORNE, P. L. M. & ACHTERBOSCH, T. J. 2008. Animal welfare in poultry production systems: impact of EU standards on world trade. World’s Poultry Science Journal, 64, 40–52.

VAN KRIMPEN, M. M., KWAKKEL, R. P., REUVEKAMP, B. F. J., VAN DER PEET-SCHWERING, C. M. C., DEN HARTOG, L. A. & VERSTEGEN, M. W. A. 2005. Impact of feeding management on feather pecking in laying hens. World’s Poultry Science Journal, 61, 663–686.

VESTERGAARD, K. & LISBORG, L. 1993. A model of feather pecking development which relates to dustbathing in the fowl. Behaviour, 126, 291–308.

VESTERGAARD, K., KRUIJT, J. & HOGAN, J. 1993. Feather pecking and chronic fear in groups of red junglefowl: their relations to dustbathing, rearing environment and social status. Animal behaviour, 45, 1127–1140.

VON ENGELHARDT, N., CARERE, C., DIJKSTRA, C. & GROOTHUIS, T. G. G. 2006. Sex-specific effects of yolk testosterone on survival, begging and growth of zebra finches. Proceedings. Biological sciences / The Royal Society, 273, 65–70.

WHITTAKER, D. J., GERLACH, N. M., SOINI, H. A., NOVOTNY, M. V. & KETTERSON, E. D. 2013. Bird odour predicts reproductive success. Animal Behaviour, 86, 697–703.