Animals across a range of species typically suffer poor welfare in an industrial setting. Poor welfare can be measured by estimating the affective state (i.e., emotion, mood, Mendl et al., 2010) of individuals, where individuals with poorer welfare often are in a more negative affective state (e.g., Harding et al., 2004; Forkman et al., 2007). For example, negative affective state measured as fearfulness (Jones, 1996; Forkman et al., 2007) increased when animals are housed in poorer conditions (e.g., Hansen et al., 1993), and is linked to higher stress hormone levels (e.g., de Haas et al., 2012). Further, being in a negative affective state can enhance negative impacts of poor welfare (Jones, 1996). Despite that animal welfare research focuses on improving animal welfare, the problems remain (Nicol, 2020). Therefore, while continuing to work on external factors that can improve animal welfare, underlying mechanisms which in turn can improve the affective state of animals, should also be explored. This route is very commonly used to improve human affective state, when medicating and reducing depression and anxiety (e.g., Hamon and Blier, 2013). Such an approach can also be successfully used to alter affective state in animals (e.g., Hymel and Sufka, 2012; reviewed by Neville et al., 2020).

Monoamines (i.e., neurotransmitters) have broadly, and across species, repeatedly been shown to affect a range of behaviour. Focusing on serotonin, this monoamine can directly or indirectly affect behaviour of relevance to animal welfare (e.g., Coppens et al., 2010; Stracke et al., 2017; Abbey-Lee et al., 2018; 2019), and experimental alteration of serotonin has successfully reduced fearfulness or related behaviours (e.g., several fish species, Winberg and Thörnqvist, 2016; mice, Mus musculus; Foltran et al., 2020; Mediterranean field crickets, Gryllus bimaculatus; Lundgren et al., 2021). Hence, due to the conserved nature of the monoaminergic systems (Gunnarsson et al., 2008), manipulation of the serotonergic system may alter fearfulness also in agricultural species.

We here focus on monoaminergic manipulation of fearfulness in chickens. The domestic chicken (Gallus gallus domesticus) is today the most numerous bird in the world and one of our most intensely farmed animals, with billions raised yearly for meat and egg production (reviewed by, e.g., Nicol, 2015; Pizzari, 2016). These chickens suffer from a variety of severe welfare problems, such as cannibalism, feather- and vent pecking (Lambton et al., 2015; Nicol, 2015). To measure their welfare, estimating level of fearfulness is commonly used to describe their negative affective state (Forkman et al., 2007; Laurence et al., 2012). Also in chickens is the serotonergic system linked to a range of behaviour (e.g., Shea et al., 1991; Dennis et al., 2013; de Haas and van der Eijk, 2018; Birkl et al., 2019), including fear-related behaviour (e.g., Hicks et al., 1975; Hennig, 1980; Phi Van et al., 2018). Therefore, with the extended aim to improve their welfare, we specifically investigate if experimental manipulation aimed to target the serotonergic system could reduce fearfulness in chickens.

Previous work on manipulations of the serotonergic system in both fowl and other species have primarily been done via drug injection (e.g., Hennig, 1980; Foltran et al., 2020; Lundgren et al., 2021) or through drinking water (Dulawa et al., 2004). Both of these approaches have their drawbacks: while injection is fast and the amount of drug given is precise, injections can cause stress to test subjects (Morton et al., 2001), may need repeated injections to keep stable levels (Hennig, 1980), and is thus impractical to large number of individuals. Administering through the drinking water has minimal stress effect on the test subject and is practical for larger number of individuals, but dosing of the drug and time of dosing will be less precise. Considering these drawbacks, we opted for an alternative approach in administering the drug in a non-invasive way, by manipulating the food given.

We chose to focus on alteration of 5-hydroxytryptophan levels (5-HTP), since this is the precursor of serotonin (where the amino acid tryptophan is metabolised into 5-HTP, which quickly is metabolised into serotonin, e.g., O'Mahony et al., 2015). We did so by experimentally manipulating the amount of 5-HTP given in feed. Increase of dietary tryptophan has resulted in higher plasma and turnover levels of serotonin in the brain of chickens (e.g., van Hierden et al., 2004b) and other animals (e.g., Winberg et al., 2001). Alteration of tryptophan in domestic fowl has previously shown to affect behaviour, such as feather pecking (Birkl et al., 2019) and aggression (Shea et al., 1991). Using a dietary manipulation allowed us to control the amount of drug each individual was given, whilst not having potentially stressful effects of injections. However, chickens have the ability to store food in their crop (Svihus, 2014), and may therefore not digest the manipulated food immediately. Thus, drug administration via food has a similar drawback as administration through water in that the timing of the dose will be less precise. As a way to get around this, we chose to study the sub-chronic effect (i.e., longer than acute, but shorter than chronic, Dulawa et al., 2004) and dose our chickens for several days in sequence.

We used this dietary manipulation of the serotonergic system on red junglefowl (Gallus gallus) hens. The red junglefowl is a growing model species for research on animal behaviour and animal welfare, and is behaviourally and cognitively similar to its descendant the domesticated chicken (reviewed by Garnham and Løvlie, 2018). Further, the red junglefowl is the main common ancestor of all domestic chickens (Fumihito et al., 1994), which should enable our findings to be general for chickens broadly and not limited to certain strains of fowl (e.g., broilers, layers).

Prior to our experimental manipulation, there was no significant difference in fearfulness between the later 5-HTP-treated hens and our control hens (5-HTP-treated: n = 24, mean ± SE = 247.71 ± 46.71; control: n = 24, mean ± SE = 277.91 ± 51.67, W = 321.5, p = 0.49; Figure 1).

However, our 5-HTP manipulation significantly lowered fearfulness in the 5-HTP-treated hens (before manipulation: n = 24, mean ± SE = 247.71 ± 46.71; after manipulation: n = 24, mean ± SE = 117.46 ± 34.28, W = 386.5, p = 0.04, Figure 1), while no such significant decrease was observed in our control hens (first test occasion: n = 24, mean ± SE = 277.91 ± 51.67; second test occasion: n = 24, mean ± SE = 190.37 ± 41.44, W = 343.5, p = 0.25, Figure 1). Further, this reduction in fearfulness was confirmed to only be significant in our 5-HTP manipulated hens, by our generalized linear model (∆fearfulness, 5-HTP-treated: n = 24, mean ± SE = 130.25 ± 43.18; ∆fearfulness, control: n = 24, mean ± SE = 87.54 ± 52.84; β = −0.07, 95% CIs = −0.09, −0.05).

We set out to test if fearfulness could be reduced via dietary manipulation of the underlying monoaminergic systems, specifically focusing on the serotonergic system. We did this by enhancing food given to individual red junglefowl hens with 5-hydroxytryptophan, for 5 consecutive days (i.e., we did a sub-chronical manipulation). We here show that this relatively short feed manipulation reduced the latency treated hens remained in tonic immobility, a common measure of fearfulness in poultry (where shorter latencies implies less fearful individuals). In our control hens were fearfulness also somewhat reduced in the second test, although not as strongly as what we observed for our experimental hens, and not significantly. The observed reduction in fearfulness in our control hens therefore confirms that habituation to the test can reduce latencies in immobility (e.g., Ratner and Thompson, 1960), and that the effect we observed in our experimental birds was mainly explained by our dietary manipulation.

We used 5-hydroxytryptophan to experimental manipulate feed given to our treatment hens. 5-hydroxytryptophan is a metabolite of tryptophan, which in turn is metabolised rapidly into serotonin (O'Mahony et al., 2015). Our finding that a manipulation set out to affect the serotonergic system affected behavioural responses, is confirmed across taxa (e.g., several fish species, Winberg and Thörnqvist, 2016; mice; Foltran et al., 2020; Mediterranean field crickets; Lundgren et al., 2021). When focusing on previous manipulation of the serotonergic system affecting fear-related behaviours, that is also confirmed across vertebrate species (e.g., humans, Hindi Attar et al., 2012; chickens; Nicol, 2015; mice; Waider et al., 2019). These findings seem to be general, independent of method of manipulation. For example, in mice, manipulated serotonin levels via injection or diet gave similar reduction in levels of brain serotonin (Foltran et al., 2020). It is promising that the use of a non-invasive method produces similar results to also more precise, invasive methods, as this should ease translation of the method to a more industrial setting.

Tryptophan is an essential amino acid obtained only through food. Poultry feed already have some percentage tryptophan (e.g., van Hierden et al., 2004b; Barua et al., 2021), and this percentage can relatively easily be enhanced further (e.g., Winberg et al., 2001), thus should be possible to increase commercially. However, there are several aspects that should be clarified before this commercial step can be taken. Firstly, it should be confirmed that dietary manipulation of 5-HTP indeed increased serotonin levels in treated hens (as shown previously in chickens, van Hierden et al., 2004b), and whether this resulted in increased peripheral serotonin levels (in plasma), and/or in the central nervous system (brain). The vast majority of serotonin (ca 95%) is synthesised in the gut and circulated in the blood (see reference in, e.g., Yan et al., 2018). The synthetic cascade is similar in the gut as in the brain (tryptophan—5-hydroxytryptophan–serotonin). However, since peripheral serotonin cannot cross the brain-blood barrier (Mann et al., 1992), serotonin synthesis in the gut and in the brain has two separate pathways and the function of serotonin can differ in the two separate networks. Across vertebrates, serotonin in both networks can affect aspects of relevance to welfare (O'Mahony et al., 2015), and to poultry welfare more specifically (e.g., feather pecking, van Hierden et al., 2004a; van Hierden et al., 2004b; de Haas and van der Eijk, 2018). Yet, the role of serotonin does not need to be parallel in plasma and brain. For example, peripheral serotonin levels were unaltered, while serotonin levels in the brain were affected by comparing broilers given probiotic diet vs. control birds (Yan et al., 2018). How and why our dietary manipulation affected behaviour, is currently unknown, and investigation of whether plasma or brain serotonin levels changed could give hint to the underlying mechanism through which our dietary manipulation acted. Further, even if the underlying mechanism to observed behavioural alteration is known, further work needs to consider that serotonin manipulation via food will not result in individual dosing. Thus, both the preferred dose, and the acceptable span of levels individuals obtained dependent on differential food intake, must be explored further. This is because dose response curves of serotonin and responses to the same fearfulness test we here used, the tonic immobility test, shows that doses can produce quite varying and non-linear responses (Hennig, 1980). In addition, alteration of feed can cause amino acid imbalances. Further, high levels of serotonin can cause serotonin syndrome in which a range of abnormal behaviours can be observed, such as head weaving, hyperactivity and twitching (reviewed by, e.g., Haberzettl et al., 2013).

On a slightly different note, yet of relevance for animal welfare, manipulation of serotonin levels in domestic fowl can reduce feather pecking (e.g., van Hierden et al., 2004a; de Haas and van der Eijk, 2018). Feather pecking is a large welfare problem in the poultry industry, and serotonin manipulations can reduce certain aspects of feather pecking (van Hierden et al., 2004b). Therefore, further work on how to enhance serotonin levels could have positive influences on chicken welfare for multiple reasons. However, the underlying mechanisms for fearfulness and feather pecking are still not fully understood, and warrant future research.

We here used a well-established behavioural test of fearfulness commonly used on poultry (not only chickens, but, e.g., quail, Jones et al., 1991). Further, the use of latency to come out of immobility after induction of tonic immobility is used as a measure of fearfulness also across other animals (reviewed by, e.g., Hennig, 1980). Yet, this measure does not always correlate with other aspects of fearfulness, or positive welfare. Longer latencies in tonic immobility has been linked with other measures of fearfulness (reviewed by, e.g., Forkman et al., 2007), but also with stress (Zulkifli et al., 1998; Kozak et al., 2019). Thus, what exactly the tonic immobility test is measuring is still debated (reviewed by, e.g., Humphreys and Ruxton, 2018). Nevertheless, the test seems to capture negative affective state. We recently showed, in the same population of junglefowl here used, that latency to remain in tonic immobility was moderately positively correlated with responses to ambiguous, intermediate cues in a cognitive judgement bias test (Garnham et al., 2022b). A judgement bias test is widely used as one of the very few confirmed methods to measure positive affective state of animals (reviewd by, e.g., Lagisz et al., 2020; Neville et al., 2020). That a low fearfulness correlated with a higher positive affective state is promising in that what we here have shown may translate to not only “less bad welfare,” but also improved welfare. However, this should be directly tested by manipulation of the serotonergic system and measure positive affective state, suggestively by the use of a judgement bias test. Suggesting such a link is also our previous findings that responses to a judgement bias test is linked to variation in the monoaminergic systems (Zidar et al., 2018; Boddington et al., 2020). In the same population as here used, we previously shown that brain gene expression of the DRD1 (dopamine receptor D1, another important neurotransmitter of the monoaminergic systems) gene, was positively correlated with positive affective state measured in a judgement bias test (Boddington et al., 2020). In layer hens, brain turnover rate of dopamine was positively correlated with positive affective state measured in a judgement bias test (Zidar et al., 2018). On the other hand, in a smaller sample of individuals from the same population of red junglefowl here used, no correlation was observed between brain gene expression of a range of serotonergic genes and latency remaining in tonic immobility (Lundgren et al., 2021). Thus, the relationship between negative and positive affective state, and its underlying mechanisms have to be explored further.

Clarification of the link between responses to tonic immobility and measured positive affective state in a judgement bias test is relevant both as it may establish how negative and positive affective states are linked, but also if tonic immobility test can be used instead of judgement bias tests. Judgement bias tests are both time consuming and take a lot of handling of the animals, as the values of negative and positive cues need to be learned (see, e.g., Zidar et al., 2018; Garnham et al., 2019). The use of a tonic immobility test is thus more time efficient, although this too needs individual handling and testing of birds. When the relationship between dietary manipulation of underlying monoaminergic systems is better understood, and if reduction in feather pecking is produced as a side-effect of serotonin enhancement, visual inspection of flocks with regard to feather pecking could perhaps act as a group-level measure of altered affective state. However, this is currently only a speculation.

Overall, the relationship between negative and positive affective states in chickens and other animals needs to be better described. We recently showed that the relationship between responses to tonic immobility and responses in the judgement bias test depends on whether chicks or adults were tested (e.g., the relationship was only found in chicks, and not in adults, Garnham et al., 2022b). This suggests that before generalizing to different ages and strains of fowl, the generality of our findings should be investigated further since our results may differ for broilers (i.e., chicks) vs. layers (i.e., adults). Further, as a first step, we here only tested females. In our population, we have previously observed no or weak effects of sex on responses to the tonic immobility test (e.g., Favati et al., 2015; Zidar et al., 2017; Lundgren et al., 2021; Garnham et al., 2022b). However, sex effects in responses to monoamine manipulation on behaviour also warrant future exploration.

We have here shown that a relatively short (5 days) experimental manipulation of diet with the aim to alter the serotonergic system of red junglefowl hens, the main ancestor of all domesticated chickens, reduced how fearful these were, measured in a tonic immobility test. Our manipulation was non-invasive through 5-hydroxytryptophan enhanced food given to the birds. Tryptophan, the precursor of serotonin (and from which 5-hydroxytryptophan in turn is metabolised), is already part of commercial chicken feed. Although still in its early stage, these aspects together produce promising results for a hopefully practical method to reduce fearfulness of also industrial birds, which in turn may improve their animal welfare. Future work should evaluate tryptophan enhanced food in industrial settings, together with the drug dosage needed to produce satisfactory levels of animal welfare.