# Adult chicken hens express their affective states via bare facial skin blushing and head feather movements

Delphine Soulet, Scott A Love, Marie-Claire Blache, Céline Parias, Benoît Piégu, Gaëlle Lefort, Léa Lansade, Frédéric Lévy, Aline Bertin, Cécile Arnould

► To cite this version:

Delphine Soulet, Scott A Love, Marie-Claire Blache, Céline Parias, Benoît Piégu, et al. Adult chicken hens express their affective states via bare facial skin blushing and head feather movements. Animal Behaviour, 2025, 227, pp.123277. 10.1016/j.anbehav.2025.123277. hal-05214535

HAL Id: hal-05214535

https://hal.science/hal-05214535v1

Submitted on 19 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Adult chicken hens express their affective states via bare facial skin blushing and head feather movements

<figure><img src="image_3.png" /></figure>

Check for
updates

Delphine Soulet *,<fnref n="1" /> ID, Scott A. Love ID, Marie-Claire Blache, Céline Parias, Benoît Piégu, Gaëlle Lefort ID, Léa Lansade, Frédéric Lévy, Aline Bertin *,<fnref n="2" /> ID, Cécile Arnould *,<fnref n="2" />

INRAE, CNRS, Université de Tours, PRC, Nouzilly, France

## ARTICLE INFO

**Article history:**

Received 25 June 2024

Initial acceptance 6 September 2024

Final acceptance 6 May 2025

Available online xxx

MS. number 24-00397R

**Keywords:**

animal sentience

chicken

emotions

facial expression

feather fluffing

Gallus gallus domesticus

visual communication

In mammals, facial expressions serve as a window into a variety of affective states. Emotional facial expressions in birds have received little scientific attention. Juvenile hens showed variations in their facial display, facial redness and head feather position, depending on their affective states. When sexually mature, domestic hens develop fleshy coloured outgrowths such as the comb and the wattles, which are considered as functional visual signals of the individual's health or fertility. Here, we investigated, in adult hens, whether bare facial skin (cheeks, ear lobes, comb and wattles) redness and head feather position (sleeked/fluffed) also vary with affective states. We hypothesized little or no redness variations for the wattles and comb because of their involvement as functional signals. Twelve adult hens were filmed during naturally occurring situations and controlled tests varying in their valence and arousal level: situations associated with fear/frustration (negative valence/high arousal), situations associated with appetitive motivational states (positive valence/high arousal) and situations associated with calm/contentment (positive valence/low arousal). We showed that positive situations of low arousal were associated with the lowest redness in all areas of bare skin. A significantly higher level of redness was found in high arousal compared with low arousal positive situations, except for the comb. The highest redness was observed in all skin areas in negative situations of high arousal. Head feathers were observed more frequently fluffed in most of the positive situations and sleeked in all feed-related situations. Thus, facial skin redness, including secondary sexual characters and head feather position are potential markers of the affective states of adult hens. Studying facial displays opens new research opportunities to increase our knowledge on how avian species perceive their environment. Further research is needed to clearly differentiate between the effects of valence and arousal.

© 2025 The Authors. Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Facial expressions, mainly attributed to movement of facial muscles, are studied in various mammalian species, including primates, horses, sheep, cattle, goats, pigs, cats and dogs (Descovich et al., 2017; Lansade et al., 2018; Neethirajan et al., 2021; Waller et al., 2020). They can be used to infer affective states, as facial expressions seem to differentiate positive and negative emotions (Descovich et al., 2017; Pellon et al., 2020; Phelipon et al., 2024). For example, horses gently groomed and searching human contact have their upper lip extended, ears backward and eyes half open, while horses avoiding human

contact have contracted lips, asymmetrical ears and eyes wide open (Lansade et al., 2018). In humans, facial expressions are not the only transient change observable on the face. Variations in blood flow under the skin lead to visible changes in redness (Benitez-Quiroz et al., 2019; Thorstenson et al., 2018, 2019). These changes in facial redness seem to be associated with emotions and are referred to as blushing. Blushing refers specifically to transient facial redness arising during various emotions (Drummond et al., 2013; Keeling & Raghallaigh, 2024; Nobeyama, 2024). It differs from flushing, which refers to a more long-lasting skin redness that might expand on other body parts and can be caused by thermoregulation or pathologies (Drummond et al., 2013; Keeling & Raghallaigh, 2024; Nobeyama, 2024; Sadeghian et al., 2017). Facial colour appears to facilitate the recognition of some emotions expressed by the face. For example, an increase in redness facilitates the recognition of anger (Nakajima et al., 2017; Thorstenson et al., 2019). Also, specific redness patterns are

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: soulet.delphine@gmail.com (D. Soulet), aline.bertin@inrae.fr (A. Bertin), cecile.arnould@inrae.fr (C. Arnould).</footnote>

<footnote n="1">¹ Present address: Université de Caen Normandie (UNICAEN), Université de Rennes 1, CNRS, EthoS (Ethologie Animale et Humaine), UMR 6552, Luc-sur-Mer, France.</footnote>

<footnote n="2">² These authors contributed equally to this work.</footnote>
associated with certain emotions, such as a higher redness on the cheeks during the expression of joy (Benitez-Quiroz et al., 2018). However, perceptible changes on the face are not restricted to humans or mammals.

Many birds are able to move their facial feathers and present variations in bare facial skin colour. Body feather movements are often studied in courtship displays in the context of mate choice (Scholes & Laman, 2018; Yorzinski et al., 2013). Nonetheless, some feather movements on the head have also been observed during situations varying in emotional valence. For example, red-vented bulbuls, *Pycnonotus cafer*, and hoopoes, *Upupa epops*, erect their crest in presence of a predator (Kumar, 2010; Ruiz-Rodríguez et al., 2017), a situation associated with fear, and blue-and-yellow macaws, *Ara ararauna*, and sulphur-crested cockatoos, *Cacatua galerita*, fluff their head feathers during situations associated with calm and contentment (Bertin et al., 2018a, 2020). Rapid changes in facial colour remain barely studied in birds. However, rapid changes in facial redness, probably via haemoglobin flushing, have been observed in negative and positive affective states. For example, the head of lappet-faced vultures, *Aegypius tracheliotos*, redden during agonistic interactions (Bamford et al., 2010), while captive blue-and-yellow macaws redden on the cheeks during positive interactions with their caretaker (Bertin et al., 2018a, 2023). Depending on the authors, the change in facial skin redness in nonhuman animals is referred to as flushing or blushing. In this article, the term blushing is preferred when the described phenomenon is transient without clear association with physical activity. Overlooked so far, the combination of head feather movements and rapid changes in facial skin redness might have a function equivalent to that of facial expressions in mammals. As such, these facial displays might provide a tool to infer affective states in some avian species.

Here, we use the term affective states to include both emotions that are intense and short-lived responses following a stimulus and less intense long lasting emotional states, as defined by Mendl and Paul (2020). Affective states have a valence (positive to negative) and an arousal level (intensity variation) that represent the two axes of a bidimensional model (Mendl et al., 2010; Russell, 2003). This theoretical approach can be used to study affective states in animals by observing them in situations contrasted in emotional valence and arousal level. For example, a study on goats, *Capra hircus*, showed that the time passed with the ears backward and the tail up would inform on valence, while other ear movements (forward and horizontal), locomotion, head movements or calls would inform on arousal (Briefer et al., 2015). In previous studies, we provided a first description of changes in facial displays in juvenile domestic hens, *Gallus gallus domesticus*, of different breeds placed in situations contrasted in emotional valence and/or arousal level (Arnould et al., 2024; Soulet, Jahoui, et al., 2024). The juvenile hens presented facial skin redness variations according to the valence and arousal of the situations with lower redness consistently observed in calm states, such as preening or resting, a high redness in appetitive motivational states and the highest redness observed in fear-related states.

As in many Galliformes, the comb and wattles of hens grow and redden during sexual maturation. These fleshy outgrowths have been found to be under sexual selection for males and females and to be under natural selection mainly for a thermoregulation function (Zhao et al., 2024). They are functional visual signals communicating the quality of the individual or its social status (E. K. M. Jones & Prescott, 2000; Mukhtar & Khan, 2012; Zhao et al., 2024). For example, in female domestic fowl, comb size is related to the dominance status inside a group, the reproductive cycle (comb decreased in size in nonlaying period) or the productivity of the female (egg weight; Bradshaw, 1992; Cloutier et al.,

1996; Cornwallis & Birkhead, 2006; Mukhtar & Khan, 2012; O'Connor et al., 2011; Wilson et al., 1958). The comb colour is associated with the dominance position in a few studies but with mixed results. For example, redder comb was related to higher dominance in two studies (Bradshaw, 1992; Cloutier et al., 1996), while it was the contrary in the study of O'Connor et al. (O'Connor et al., 2011). In male red jungle fowl, *Gallus gallus*, the comb size and/or colour is associated with reproductive quality and health status (Mukhtar & Khan, 2012; Zuk et al., 1990). As such, a question arises as to whether the sexual maturation of the comb and wattles affects their capacity of rapidly changing colour as observed for the juvenile hens.

The aim of this study was to investigate variations in facial skin redness and head feather position of adult domestic hens in relation to their affective states. For this, we observed the variations in facial redness and head feathers' position of 12 sexually mature hens during seven situations associated with three kinds of affective states, based on a priori expectations regarding the literature: negative states of high arousal (situations of fear or frustration), positive states of high arousal (situations associated with appetitive motivational states) and positive states of low arousal (e.g. resting). Regarding facial skin redness, we hypothesized that the redness of nonsecondary sexual characters (cheeks and ear lobes) would vary according to affective state, similarly to what was observed previously in juvenile hens. In contrast, we hypothesized that the redness of the secondary sexual characters (comb and wattles) would not vary similarly to what was observed previously in juvenile hens because of their role in sexual selection and social status. More precisely, according to our previous work with juveniles (Arnould et al., 2024; Soulet, Jahoui, et al., 2024), we predicted a higher facial redness in negative states of high arousal than in positive states of high arousal and the lowest redness in positive states of low arousal for the cheeks and the ear lobes. For the comb and wattles, we expected little or no facial redness variations. Concerning feather position, we hypothesized that it would vary depending on the valence or arousal of some situations, as for the juveniles. We expected fluffed head feathers in resting or dustbathing and sleeked head feathers in feed-related situations.

## METHODS

### Animals and Housing

The experimental group consisted of 12 adult hens (Pekin bantam) of the same age (7 months old) individually identified by their morphological characteristics (i.e. plumage colour or comb shape). All hens were in lay, but they were never observed just after laying. They lived together in a 129 m² wooded outdoor range covered with grass, with permanent access to a hen house (width = 230 cm, length = 90 cm, height = 152 cm) in a commercial farm (Le Haut Montmartre, 37340 Cléré-les-pins, France). Water and feed were provided ad libitum. The feed (mix of seeds with minerals) was delivered early in the morning, at least 1 to 3 h before the beginning of the observations.

### Behavioural Observations

Following the methodology outlined in Soulet, Jahoui, et al. (2024) and Arnould et al. (2024), the hens were familiarized with three experimenters (A, B and C), the cameras and tripods used later, for 2 days before the experiment. Then, each hen was filmed during spontaneous situations and also controlled situations using behavioural tests. These situations were associated
with contrasted affective states according to the models proposed by Mendl et al. (2010) and Richardson et al. (2016):

(1) Calm states: affective states of positive valence with low arousal level (pos_low). This category included three spontaneous situations: Resting (lying with eyes opened or closed or crouching with eyes closed), Preening and Feeding (eating usual feed or grass; the resolution of the cameras allowed the identification of the food consumed). Resting and preening are associated with situations of calm and contentment, and they are performed optimally when there is low risk of danger (Blokhuis, 1984; Spruijt et al., 1992). Situations inducing calm are considered of low arousal by Richardson et al. (2016). Preening is also considered a comfort behaviour in poultry and is associated with a positive state (Papageorgiou et al., 2023; Zimmerman et al., 2011), dearousal and relaxation (Spruijt et al., 1992). In this experiment, preening was performed spontaneously by undisturbed hens and was often interspersed with resting. As such, preening was not attributed to stress coping. Feeding contributes to the maintenance of health and has hedonic value, with these two features being intertwined but involving different brain circuits (Saper et al., 2002). According to Saper et al. (2002), food delivered ad libitum does not require the involvement of circuits associated with novelty, attention and anticipation. Thus, we made the a priori assertion that it induced lower arousal than feeding on a very appetizing food (mealworms in our experiment).

(2) Appetitive motivational states: affective states of positive valence with high arousal level (pos_high). This category consisted of one spontaneous and one controlled situation: Dustbathing (performed on the soil of the pasture without additional substrate provided) and Rewarding Test with mealworms. As defined by Mendl et al. (2010), appetitive motivational states are associated with seeking and obtaining rewards. Dustbathing is considered as highly rewarding and induces anticipation behaviours in domestic fowl (McGrath et al., 2017; 2016). The high motivation associated with this behaviour is also seen as hens are willing to work to access a room containing a substrate for dustbathing even when not deprived (Widowski & Duncan, 2000). Even featherless chicks are motivated to dustbathe, highlighting that this behaviour is not only performed for maintenance (Vestergaard et al., 1999). Widowski and Duncan (2000) concluded that this behaviour might be associated with a state of pleasure. In addition, dustbathing contains dynamic elements such as bill-raking, wing shaking or scratching, highlighting increased activity compared with preening or resting (Grebey et al., 2020; Kruijt, 1964; van Liere, 1991). According to this literature, we classified dustbathing as a positive state of high arousal. Mealworms are classically used as a highly desired reward in domestic fowl (Zimmerman et al., 2011). In addition, hens are willing to work to feed on insects, even when not food deprived (Bruce et al., 2003). In our experiment, mealworms were only available during the test and were very appetizing. We made the a priori assertion that feeding on them induced higher arousal than feeding on the usual feed or grass (Saper et al., 2002).

(3) Frustration and fear-related states: affective states of negative valence with high arousal level (neg_high). This category consisted of two controlled situations: Frustration Test and Capture Test with manual restraint. Frustration-like situation in domestic fowl is often tested by thwarting access to a visible reward (Duncan & Wood-Gush, 1972; Zimmerman et al., 2003) and is considered a negative situation (Papini et al., 2019; Zimmerman et al., 2003). Capture by human is a frightening situation in hens (R. B. Jones, 1996), and even touching induces an increase in heart rate (Cabanac & Aizawa, 2000). As such, Frustration Test and Capture Test were associated with negative states of high arousal.

Affective states of negative valence with low arousal level were not tested in this study as it is commonly associated with sadness or depression. To induce equivalent states in birds, they should be placed in an impoverished environment or subjected to chronic stress for weeks. These kinds of experiments required a specific protocol impossible to implement with the other aims of the study.

The hens were filmed during 3 consecutive weeks (June 2022) with two Sony FDR-AX53 4K cameras, with the outdoor automatic white balance function activated. The distance between the camera and the hens varied from about 1 m to 8–9 m. Once habituated, the hens were not disturbed by being filmed. The procedure and the influence on hens' behaviour were similar to what was indicated in the study of Arnould et al. (2024; Fig. S2, 'close' situation). In this previous study, the facial skin redness of hens spontaneously resting at a distance inferior to 50 cm from the experimenters, including when in physical contact, was similar to the facial redness obtained during resting situations. For all the situations (spontaneous and controlled), the animals were observed during daylight (between 1000 hours and 1700 hours; mean temperature in the morning: 20.3 °C, in the afternoon: 25 °C, average variation: 5 °C). To ensure that enough suitable images could be extracted from the films, the spontaneous situations (Resting, Preening, Feeding, Dustbathing) were recorded as they occurred, rather than focusing on filming a single animal for a given period. We recorded at least 20 min of footage for each hen and each spontaneous situation. The three behavioural tests were conducted during the third week of observations: the Capture Test followed by the Rewarding and Frustration Tests. Each test was repeated twice to ensure the extraction of enough profile images of the head.

The Capture Test involved gently catching each hen individually by the same experimenter (A) while a second experimenter (B) filmed the profile view of the hen for 1 min. This test was repeated twice on the same day, with roles reversed between experimenters during the morning and afternoon sessions (morning: 1015 hours; afternoon: 1430 hours, temperatures are available in Table S4), resulting in 2 min of footage in total. During the capture, the hen was maintained at the waist height of the experimenter with her wings restrained by hand. The hens had not been captured by the experimenters before the test to avoid habituation to this procedure. To verify that the hens' behaviour was not affected by the repetition, the percentage of time spent trying to escape (moving the legs and trying to flap wings) during testing was extracted from the films for both tests (see Table S1).

The two other tests were carried out on the same day (Rewarding Test in the morning and Frustration Test in the afternoon; temperatures are available in Table S4) and were repeated 2 days later, with an interval of 1 day without a test. Each test lasted 2 min and 30 s, resulting in 5 min of film in total (2 x 2 min 30 s). They took place in a wire-mesh enclosure (length = 118 cm, width = 57 cm, height = 58 cm) surrounded by cardboard to prevent visual contact with conspecifics (height = 39 cm). Hens were filmed by two experimenters (A and B). The enclosure was set up the day before the tests and the hens were habituated to enter the enclosure and to eat mealworms from a transparent glass dish containing wood shavings (diameter: 70 mm, height: 40 mm). While the tests were not in progress, hens had free access to the enclosure. All hens were tested individually and freely entered the enclosure. For both tests, the glass dish was placed in the middle of the enclosure. For the Rewarding Test, the hens had free access to mealworms mixed with wood shavings in the dish. For the Frustration Test, the dish containing only mealworms was closed with a transparent glass lid so that the hens could see the worms but not eat them.
To control for the hens' motivation to feed during all tests, we measured the percentage of time spent in feeding-related behaviours: the time spent eating in, pecking on or looking at the transparent glass dish; pecking or scratching the floor or the wood shavings on the floor and walking with the head near the floor in proximity to the dish. For the Frustration Test, the time spent in pecking at the transparent glass dish and lid was considered instead of eating in (see Table S1).

## Extraction of Images and Measures of Facial Redness and Feather Position

Redness and feather position were measured from still frames of profile images of the hens, following the methodology outlined in Soulet, Jahoui, et al. (2024). The procedure used python scripts and ImageJ macros (Schindelin et al., 2012) available at https://forgemia.inra.fr/projetred/red_project.

## Extraction of Images

A deep learning model had previously been created to detect hen profiles in images by applying the python script 'DL_KeepImage_FineTuning.py'. It performs a fine tuning from network resnet50 (He et al., 2016) with weights from model ImageNet. Images used came from films recorded during a previous experiment (Arnould et al., 2024) that had been manually classified: 9013 selected images of hen profile (whole head with the cheek, ear lobe, wattle and comb visible), 10 465 images rejected (images are available on request to the authors). This deep learning model is available at https://doi.org/10.5281/zenodo.10522790.

The procedure for extracting and selecting hen profile images in this study was as follows.

1. Frames from films were evaluated with the deep learning model at interval of 2 s except for the Rewarding and Frustration Tests, where the interval was set at 1 s to obtain sufficient images (script: predict mp4.py). The model only wrote frames with correct prediction of hen profile, whatever the side of the face (right or left).

2. For each extracted profile, we conducted a manual verification step to ensure the visibility of and absence of direct lighting on all regions of interest (comb, cheek, ear lobe and wattle).

3. From the pool of profiles obtained, 20 images were randomly sampled for each hen and situation, resulting in 1680 images (pos_low = 720 images, pos_high and neg_high = 480 images each). The script used (sampling_files.py) maximized the number of films from which the images came in order to sample different days and limit the possible biases linked to weather conditions. Regarding the Capture Test, in another experiment, no significant differences in facial skin redness and feather fluffing were observed when comparing the beginning (0–20 s), middle (20–40 s) and ending (40–60 s) of the test (Soulet, Bertin, & Arnould, 2024). Thus, we sampled the 20 images without considering the moment of the tests.

## Measure of Facial Redness

An ImageJ macro (ROIwithpoints.ijm) was used to extract the mean red (R), blue (B) and green (G) values (RGB colour model) for each bare skin region of the hen face (comb, cheek, ear lobe and wattle). For this, 10 × 10 pixel squares were positioned on the four regions of interest and one on the white feathers for each image. When possible, three squares were positioned for each region: one square at each corner of the eye and one above the beak for the

cheek (areas with very few feathers which allowed to positioned the squares on bare skin); at the frontal base for the comb; in the middle of the wattle and ear lobe (Fig. 1).

Then, a correction of the white balance of the images was performed post acquisition using the white of the feathers. We ported to R a macro available on GitHub that balances RGB colour based on a selected region (Patrice Mascalchi, 2017; https://github.com/pmascalchi/ImageJ_Auto-white-balance-correction). Each channel (R, G and B) was corrected with the following formula: $V_c = V_i + mean\_white - Vweight\_i$; with $V_c$ = corrected value of the channel, $V_i$ = initial value of the channel, $mean\_white$ = mean of the R, G and B values for the white (white feathers in our experiment) and $Vwhite\_i$ = value of the channel for the white feathers.

Finally, the redness was calculated with the corrected values as $R/(R+G+B)$, following the methodology outlined in related studies (Arnould et al., 2024; Bertin et al., 2023; Passarotto et al., 2022; Pérez-Rodríguez & Viñuela, 2008; Soulet, Jahoui, et al., 2024).

## Facial Feather Position

The position of the feathers on the top of the head (Fig. 2) were defined according to the existing literature (Arnould et al., 2024; Bertin et al., 2018a, 2020), as sleeked (tip of the feathers not visible), fluffed (tip of the feathers visible) or impossible to decide. The position was visually assessed by the same experienced experimenter. A subset of images (420 images, five images per hen

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Location (black square) on the top of the head where the position of the feathers was scored sleeked (left) or fluffed (right).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Position of the squares (10 × 10 pixels) where redness was measured on the comb, cheek, ear lobe, wattle and white feathers.</figcaption></figure>
and per situation randomly selected by the same sampling script as before) were scored a second time by the same experimenter to check for the intraobserver accordance.

## Statistical Analysis

All the analyses were performed with the software R (version 4.1.2, R Core Team 2021).

The median redness of the facial skin and the feather position did not significantly differ between the two repetitions for all tests (see Tables S2 and S3). Thus, the data for the repetition of each test were pooled. Furthermore, the behaviour observed during the repetition of the Capture Test was not significantly different (the hens did not try to escape the hands of the experimenter more than 6% of time). In addition, the differences in behaviours between the repetitions for the Rewarding and Frustration Tests were small. Specifically, the birds passed at least 69% of their time expressing feeding-related behaviours until depletion of the resource during the Rewarding Test and 86% of their time expressing feeding-related behaviours directed towards the glass dish during the Frustration Test (see Table S1).

Regarding facial colour, for each region, the redness value of each hen and situation consisted of the median of the 20 randomly selected images. The statistical analyses were conducted on the three affective states resulting in two values per hen for neg_high (Capture Test, Frustration Test) and pos_high (Dustbathing, Rewarding Test), three values for pos_low (Resting, Preening, Feeding). The results by situation are available in Fig. S1–S4. As each hen was observed in multiple situations, a linear mixed model was performed (lmer function of the package lme4; Bates et al., 2015). The redness values were log2 transformed to respect the homogeneity of variance required for this model. The model included as fixed effects the regions of interest (comb, wattle, cheek, ear lobe), the three affective states (neg_high; pos_high; pos_low) and the interaction between both. The identity of the hen was included as a random effect. The requirements of the model after the log transformation (normality of the residuals and homogeneity of the variance) were confirmed using the package DHARMa (Hartig, 2022). The post hoc analysis, when necessary, was conducted using the emmeans function (package emmeans; Lenth, 2022) with *P* value adjusted by Tukey. In addition, to test the facial redness correlation between the different regions of interest for each affective state, we performed a Spearman's rank correlation test with the cor.test function.

For the feather position, the images where the position was defined as impossible to decide were removed from the statistical analysis (number of images removed: Capture Test = 22/240, new *N* = 218; Frustration Test = 15/240, new *N* = 225; Rewarding Test = 16/240, new *N* = 224; Dustbathing = 25/240, new *N* = 215; Feeding = 22/240, new *N* = 218; Resting = 27/240, new *N* = 213; Preening = 18/240, new *N* = 222). As a previous study showed that feather position differs greatly between situations (Arnould et al., 2024), they were analysed separately for each of the seven situations. A binomial generalized linear mixed model was performed including the situations as fixed effect and the identity of the hen as random effect. The glmer function of the package lme4 was used. The post hoc analysis was conducted when necessary, using the emmeans function of the package emmeans (*P* value adjusted by Tukey). In addition, to check for the intraobserver accordance, we performed a Cohen Kappa test by using the function cohen. kappa of the package psych (Revelle, 2024).

Data are expressed as median [first quartile to third quartile]. Test significance was considered at *P* ≤ 0.05.

## Ethical Note

The Ethics Committee for Animal Experimentation of Val de Loire (CEEA Vdl, CEEA - 019) provided ethical approval for this study (Protocol number: CE19 - 2022-1310 - 1), in accordance with the French and European animal experimentation regulation. The study was conducted on a commercial farm under optimal welfare conditions (protected free-range with a hen house). We chose to work with 12 adult Pekin bantam hens based on our previous work (Soulet, Jahoui, et al., 2024; Arnould et al., 2024). This breed was found to be particularly calm around humans, and the number of animals was sufficient to obtain enough images. The hens willingly entered the testing arena and were gently caught and released for the 1 min Capture Test. No fear of the experimenters was observed during the observations. Fear was only induced during the Capture Tests. At the end of the experiment, all the hens remained on the commercial farm and were sold to private individuals.

## RESULTS

### Measure of Facial Redness

The linear mixed model revealed significant effects for the three fixed effects: the regions of interest (cheek, ear lobe, wattle and comb; $F_{3, 313} = 204.37, P < 0.001$), the three affective states (neg_high, pos_high and pos_low; $F_{2, 313} = 162.61, P < 0.001$) and the interaction between the regions of interest and the affective states ($F_{6, 313} = 3.99, P < 0.001$). The cheeks, ear lobes and wattles were significantly redder in neg_high than in pos_high states and significantly redder in pos_high than in pos_low states ($P \le 0.011$ for all cases; Fig. 3). The comb was significantly redder in neg_high states than in the two other affective states ($P \le 0.002$ in both cases), for which the redness was not significantly different ($P = 0.133$). Fig. 4 illustrates the skin redness variations for one hen during the three affective states.

The facial redness between the regions of interest were positively correlated for the three affective states (Table 1). For the neg_high states, the redness of the four regions was highly correlated (>0.80), except between the wattles and ear lobes where it was 0.68 (Table 1). The correlation between the regions were lower and with a higher variability for the two positive states compared with neg_high. The variability was higher for pos_high (0.50–0.84) than for pos_low states (0.55–0.74). For all the three affective states, the lowest correlation coefficient (<0.70) involved the ear lobes (Table 1).

### Facial Feather Position

The binomial generalized linear mixed model revealed significant effects of the situations on the feather position ($\chi^2_6 = 288.10, P < 0.001$). The feather position during Resting and Dustbathing did not significantly differ ($P = 0.566$), but they were significantly more often observed fluffed than for the other situations ($P < 0.001$ for all cases, Fig. 5). The feathers were significantly more often observed fluffed during the Capture Test and Preening ($P = 0.558$ between them) than during Feeding, the Rewarding Test and the Frustration Test ($P \le 0.020$ for all cases). The feather position did not significantly differ between Feeding and the Frustration Test ($P = 0.997$) nor between the Frustration and the Rewarding Tests ($P = 0.083$). However, the feathers were significantly more often observed fluffed during Feeding than during the Rewarding Test ($P = 0.017$; Fig. 5).

The Cohen Kappa test revealed an intraobserver correlation coefficient and confidence boundaries of 0.71 (0.65–0.77).
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Redness depending on the affective states for the cheek, ear lobe, wattle and comb. Neg_high: situations of negative valence and high arousal; pos_high: situations of positive valence and high arousal; pos_low: situations of positive valence and low arousal. The box plots show the median and 25th and 75th percentiles; symbols represent individual values (two for neg_high and pos_high, three for pos_low). Different letters indicate significant differences between affective states within each facial region: *P* < 0.05.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Photographic example of skin redness variations for the same hen. Situations of negative valence and high arousal (neg_high), positive valence and high arousal (pos_high), and positive valence and low arousal (pos_low).</figcaption></figure>

<table><caption>**Table 1**
Redness correlation between the different regions of the face for the three affective states (rho values)</caption><thead><tr><th></th><th>Wattle-comb</th><th>Wattle-cheek</th><th>Wattle-ear lobe</th><th>Comb-cheek</th><th>Comb-ear lobe</th><th>Cheek-ear lobe</th></tr></thead><tbody><tr><td>Neg_high</td><td><strong>0.83</strong></td><td><strong>0.83</strong></td><td>0.68</td><td><strong>0.88</strong></td><td><strong>0.81</strong></td><td><strong>0.89</strong></td></tr><tr><td>Pos_high</td><td><strong>0.71</strong></td><td><strong>0.84</strong></td><td>0.57<sup>1</sup></td><td><strong>0.76</strong></td><td>0.50<sup>2</sup></td><td><strong>0.72</strong></td></tr><tr><td>Pos_low</td><td><strong>0.73</strong></td><td><strong>0.73</strong></td><td>0.62</td><td><strong>0.74</strong></td><td>0.55</td><td>0.65</td></tr></tbody></table>

Neg_high: situations of negative valence and high arousal; pos_high: situations of positive valence and high arousal; pos_low: situations of positive valence and low arousal. Data are presented as Spearman's rank correlation coefficient (*P* < 0.001 for all correlation except for 1: *P* = 0.005 and 2: *P* = 0.014). To differentiate the correlations highlighted in the text, correlations >0.70 are bolded.

## DISCUSSION

The facial displays of adult hens were found to change depending on the valence and/or arousal of their current situation.

All bare facial parts, including the wattles and the comb, showed significant skin redness variations in relation to the situation. The cheeks, ear lobes and wattles were redder in fear-related or frustration states, less red in appetitive motivational states and the
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Feather positions depending on the situation. Expressed in percentage of images where feathers were observed fluffed on the top of the head. The box plots show the median and 25th and 75th percentiles; symbols represent individual values. Different letters indicate significant differences: *P* < 0.05.</figcaption></figure>

least red in states related to calm and contentment. The comb was redder in fear-related or frustration states compared with the appetitive motivational states and calm and contentment states. The head feathers were more frequently fluffed in Resting and Dustbathing, two positively valanced situations.

The variations in cheeks and ear lobes redness between the different affective states were similar. Redness decreased from negative states of high arousal to positive states of high arousal and then to positive states of low arousal. This result is in accordance with previous studies on three different breeds of juvenile hens: Sussex, Pekin and Meusienne (Arnould et al., 2024; Soulet, Jahoui, et al., 2024). This is also in line with our hypothesis that facial regions with a low implication in sexual selection or social status (cheeks and ear lobes) would show redness variations depending on affective states. Even if ear lobe colour, in some cases, might intervene in mate choice in jungle fowl (Johnson et al., 1993), their implication is scarcely reported. Similarly, a low facial redness found in positive states of low arousal was described in blue-and-yellow macaws and hooded vulture, *Necrosyrtes monachus* (Bertin, Beraud, et al., 2018; Negro et al., 2006). Additionally, increased redness was found in situations of high arousal. This increase was reported for the skin around the eyes of blue-and-yellow macaws when positively interacting with their caregivers (Bertin et al., 2018a, 2023) as well as for the head of hooded vultures and the loreal areas of pied-billed grebes, *Podilymbus podiceps*, during feeding (Forbes & Ankney, 1987; Negro et al., 2006) and for the head of hooded vulture, the head and throat of Lappet-face vulture and the caruncles of male turkeys, *Meleagris gallopavo*, during agonistic contexts (Bamford et al., 2010; Negro et al., 2006). Our study also reveals variations in facial redness between positive and negative states of high arousal. Contrary to the other species described above, a fast decrease in cere redness was described in the crested caracara, *Caracara cheriway*, during frightening situations (negative state of high arousal) or in dominant individuals during agonistic interactions (Dwyer, 2014). This species shows a rapid decrease in blood flow in negative contexts, whereas it is reestablished during calm-related states (Dwyer, 2014; Negro et al., 2006). The authors suggest a signalling function of cere colour displays in intra- and interspecific interactions superimposed to the role of bare skin in thermoregulation or sexual selection. Overall, the few studies conducted so far suggest that

rapid changes in facial colour might be widespread in avian species and serve in part as a marker of affective states.

One of our main results is that wattles and comb also showed transient redness variations. In domestic hens, wattles and comb grow and redden during sexual maturation (Hardesty, 1931). Furthermore, these secondary sexual characters inform about dominance status and to intervene in mate choice in several species of Galliformes, the order including domestic and jungle fowls (Iverson & Karubian, 2017; Zhao et al., 2024). As such, we expected to find lesser or no facial skin redness variations because of affective states in these regions in adult hens. In accordance, the redness of the comb presented a lower variation compared with the other regions, differentiating only between the negative states and both positive states. But, the redness variations of the wattles differentiated between the three states similarly to the cheeks and ear lobes. These results are in accordance with those obtained with juveniles of the same breed (Arnould et al., 2024). The difference we observed between the comb and wattles might be explained by differences in the structure of the dermis or epidermis or in the number of blood vessels between these two appendices (Hardesty, 1931; Midtgård, 1984). As a limitation of our study, our sample size was small and it might not have been sufficient to obtain significant differences between the three studied affective states for the comb.

Some studies associate health and redness. For example, higher ornament's redness is associated with a better immunity in red jungle fowl (Zuk et al., 1990), red grouse (Mougeot, 2008) or red-legged partridge (Mougeot et al., 2009). However, in these cases, carotenoids pigments are widely responsible for bare part coloration and are obtained throughout the alimentation (Iverson & Karubian, 2017). Contrary to pigment coloration, the particularity of the haemoglobin's coloration is that it can vary in seconds (Arnould et al., 2024; Video S1). Furthermore, the haemoglobin's coloration can be visible through the carotenoid pigmented skin (Iverson & Karubian, 2017). The results of our study complement the knowledge on avian facial colour. The basal colour of the skin might inform on the quality and health of the individual, while the transient change could inform on the affective states. Similarly, in humans, a higher facial skin redness is associated with attractiveness and health (Stephen et al., 2012; Thorstenson, 2018); nonetheless, the haemoglobin fluctuations seem to inform about emotional states (Benitez-Quiroz et al., 2018, 2019; Nakajima et al., 2017; Thorstenson et al., 2018, 2022).

In species known to flush or blush, a high vascularization is observed in the skin of the bare parts of their face. The skin of the human face presents a high number of blood vessels close to the surface (Benitez-Quiroz et al., 2018). In crested caracaras, *Polyborus plancus*, and hooded vultures, bare facial skin shows a higher vascularization than facial skin under feathers (Negro et al., 2006). Similarly, in domestic fowl, the comb and wattles have high vascularisation (Hardesty, 1931; Midtgård, 1984). In Galliformes, one of the main functions of bare parts is supposed to be thermoregulation (Zhao et al., 2024). In line with this, comb and wattles in domestic fowl participate in heat loss (van Kampen, 1971). Nonetheless, the presence of blood vessels close to the skin surface renders visible an increase or decrease in blood flow variations, leading to a possible adaptation to a signalling function as proposed by Iverson and Karubian (2017). In accordance with this hypothesis, the eyes of nonhuman primates are particularly adapted to detect changes in colour related to haemoglobin oxygenation and/or concentration (Changizi et al., 2006), and, in human, the presence of red colour near the face increases attention towards the facial expression (Buechner et al., 2014). In domestic fowl, an experiment using pupillary reactivity shows that
domestic fowl particularly reacts to red stimulation (Barbur et al., 2002). These birds also have a large spectral sensitivity (Prescott & Wathes, 1999; Vorobeyev et al., 1998). These two characteristics arguably render the variations in skin redness salient for this species. Although additional studies are needed, the sensitivity to red coloration and high vascularization of bare facial skin lend support to a communicative function of redness variations about affective states. The transient variations in facial skin redness were found in three different breeds of hens, including the breed in this study (Arnould et al., 2024; Soulet, Jahoui, et al., 2024). Studying additional breeds varying in their basal facial colour might enable the generalization of our findings.

We found a high correlation in redness between the different regions for the three affective states. The highest correlations were observed in the negative states, while the correlations for the two positive states showed lower and more variable values. This means that, in each positive affective state, the reddening of the regions seems to be less strongly related than for the negative one, especially between ear lobes and comb or wattles. Furthermore, the region showing the lowest correlation in redness with the others was the ear lobe for all states. One can hypothesize that the ear lobe does not redden in the same manner or at the same time as the other regions. In fact, we observed that this region often seemed to be the last region to redden (personal observation). The dynamic of redness variations between facial regions could be another kind of signal of affective states but this remains to be investigated.

When the situations (Capture Test, Dustbathing, Resting etc.) were analysed separately (see Figs. S1–S4), the redness of some situations we classified into different affective states (neg_high, pos_high, pos_low), as guided by the existing literature, did not differ significantly. This was the case for Dustbathing, which did not differ from Resting, Preening and Feeding. The results for the Dustbathing situation might be explained by the alternance of more or less active phases during this behaviour (van Liere, 1991), which might induce variations in facial redness. As images with all the facial regions clearly visible were harder to obtain during active phases, the calculation of the facial redness might have included more images of less active phases. The redness for Dustbathing might be underestimated. Frustration and Rewarding Tests have been associated with different affective states but their redness did not significantly differ (see Figs. S1–S4). At the beginning of the Frustration Test, the hens were probably anticipating a rewarding situation. This could have induced a similar valence between these two situations almost at the beginning of the test. However, the hens were rapidly thwarted in their access to the reward. Nonetheless, even if the literature indicates that thwarting access to a resource induces frustration (Duncan & Wood-Gush, 1972; Mendl et al., 2010; Zimmerman et al., 2003), the real emotion felt by the animal cannot be ascertained. This could explain the absence of significant difference between Frustration and Rewarding Tests. The frustration might be transient and rapidly disappear (Mendl et al., 2010). However, this did not seem to be the case in this study as the birds were observed in feeding-related behaviours during more than two thirds of the test. Another explanation might be that the redness informs about the arousal of the situation and not the valence. In this case, the Frustration and the Rewarding Tests might have been of similar arousal. To disentangle whether redness can differentiate valence and arousal, further studies are needed. Potential avenues for investigation could include studying additional situations in each affective state, integrating some negative states of low arousal; varying the intensity of a same situation by habituating the hens to the procedure; or using physiological markers alongside facial skin redness.

As the study took place in an outdoor range, where ambient temperatures could not be controlled, we worked on a lot of images randomly selected throughout the observation period (to avoid a potential effect of the temperature). To our knowledge, there is no study showing a direct influence of the temperature on facial skin redness and we did not notice such an effect (see Fig. S5). A study found an influence of ambient temperature on facial skin temperature in domestic fowl (Kim et al., 2021). But temperatures were highly contrasted (10, 20 and 30 °C), which was not the case in our conditions. Furthermore, the phenomenon of facial skin blushing we measured is transient. The colour of the face can redden or lighten in a few seconds or minutes depending on the duration of the stimulation (Arnould et al., 2024; Video S1). As ambient temperature does not vary within this short lapse of time, it cannot explain the differences obtained in our studies. Furthermore, contrary to what might be expected, two studies showed that peripheral temperature of the comb was lower following a capture and a rewarding test (Edgar et al., 2013; Moe et al., 2012). In addition, studies on blushing in human did not find any correlation between facial skin redness and its temperature (Drummond & Lazaroo, 2012; Shearn et al., 1990), suggesting different physiological mechanisms. The physiological mechanisms underlying the reddening of the facial skin associated with affective states need to be investigated to go further in the interpretation of our results.

Regarding the head feather position, we provide a first description of feather positions in emotionally contrasted situations in adult hens. The feathers were more often fluffed during Resting and Dustbathing, than during the Capture Test and Preening, and less often fluffed during Feeding, the Frustration Test and the Rewarding Test. The birds have voluntary control over the position of their feathers with the use of one muscle for erecting and another one for depressing the feathers (Homberger & de Silva, 2003). It allows to control the degree of insulation by varying the quantity of air trapped in between. Also, the fluffing of the feathers allows the dust to enter between feathers during dustbathing or facilitate the access to the feathers during preening. Nonetheless, birds could have adapted this mechanism to other functions. Morris, for example, suggested a social function (Morris, 1956). He described, in finches, a fluffing of the head and neck feathers inducing preening by conspecifics. In addition, we suggest that feather position might inform on affective states. A study on Japanese quails highlighted a difference in feather positions during dustbathes associated with a probable difference in emotional perception of the situation (Bertin, Cornilleau, et al., 2018). Furthermore, the position of the feathers seems to be conserved between different species. The fluffing of feathers, mainly observed during positive situations (Resting, Dustbathing), is also found in other species in similar states (resting, dustbathing, reuniting with a caregiver; sulphur-crested cockatoos: Bertin et al., 2020; blue-and-yellow macaws: Bertin, Beraud, et al., 2018; Bertin et al., 2023; finches and doves: Morris, 1956; quails: Bertin, Cornilleau, et al., 2018). The low percentage of fluffed feathers observed during the Capture Test is in accordance with the results obtained in juvenile hens (Arnould et al., 2024). The Preening situation is the only situation where the fluffing of feathers varies depending on the studies. A low proportion of fluffed feathers was found for our adult hens and juvenile Meusiennes and a high proportion of fluffed feathers for the juvenile Pekins, the blue-and-yellow macaws and the sulphur-crested cockatoos (Arnould et al., 2024; Bertin et al., 2018a, 2020). Sleeked feather position was more often found in feed-related situations, whatever the affective state associated with it in our study (Feeding, the Frustration and Rewarding Tests), as it was observed in juvenile hens (Arnould et al., 2024) and parrots (Bertin et al., 2018a, 2020) during
feeding and rewarding tests. This sleeked position during foraging
might be attributed to the behaviour expressed rather than to the
affective state of the individuals. Despite the fact that feather
fluffing has thermoregulatory and plumage maintenance func-
tions, our results suggest a potential influence of the emotional
state on the position of the feathers. Further research using other
emotional situations would help to go further in the interpretation
of our results. In addition, even if we looked at the feather position
on the top of the head, hens can also move differentially the
feathers of their neck. The feather position of the top of the head,
the neck and the dynamic between them might provide comple-
mentary information about affective states.

Conclusion

This study supports the theory that facial displays, and more
specifically facial skin redness including sexual ornaments, might
serve as a window into adult hen affective states. Going beyond,
facial displays studied in relation with other dimensions such as
vocalizations or postures should expand our knowledge on avian
affective states. Despite the small sample size in our study, the
accumulating evidence from different breeds of hens strengthen
our finding of the presence of facial display variations. These re-
sults expand on our understanding of how animals perceive their
environment and open new research opportunities to explore how
birds might use facial displays to communicate, as in mammals
(Camerlink et al., 2018; de Waal, 2011).

Author Contributions

**Delphine Soulet:** Writing – original draft, Software, Formal analysis, Visualization, Resources, Data curation, Writing – review & editing, Validation, Investigation. **Scott A. Love:** Funding acquisition, Writing – review & editing, Conceptualization, Methodology. **Marie-Claire Blache:** Validation, Methodology, Data curation, Software, Investigation, Writing – review & editing, Resources, Formal analysis. **Céline Parias:** Investigation, Data curation, Writing – review & editing. **Benoît Piégu:** Writing – review & editing, Resources, Formal analysis, Validation, Methodology, Data curation, Software, Investigation. **Gaëlle Lefort:** Writing – review & editing, Resources, Validation, Methodology, Software, Formal analysis. **Léa Lansade:** Methodology, Funding acquisition, Writing – review & editing, Conceptualization. **Frédéric Lévy:** Funding acquisition, Writing – review & editing, Conceptualization, Methodology. **Aline Bertin:** Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization, Visualization, Software, Methodology, Formal analysis, Writing – review & editing, Validation, Resources, Investigation, Data curation. **Cécile Arnould:** Writing – review & editing, Validation, Resources, Investigation, Data curation, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization, Visualization, Software, Methodology, Formal analysis.

Data Availability

Data are available as Supplementary Material and python
scripts and ImageJ macros are available at https://forgemia.inra.fr/
projetred/red_project.

Declaration of Interest

All authors have no conflict of interest relevant to this article.

Acknowledgments

We thank Manuela Leduc who kindly provided access to the
birds in her living environment and took care of them (https://
lehautmontmartre.com/). We acknowledge the Région Centre-
Val de Loire and INRAE for the funding of D.S. (PhD scholarship) as
well as the metaprogram SANBA from INRAE for the funding of the
RED project (Exploration of new Emotional Biomarkers in Hens:
blushing and Immunoglobulin A). We sincerely thank the referees
and the editor for their constructive comments and suggestions,
which helped us to improve the quality and clarity of our
manuscript.

Supplementary Material

Supplementary material associated with this article is available
at https://doi.org/10.1016/j.anbehav.2025.123277.

References

Arnould, C., Love, S., Piégu, B., Lefort, G., Blache, M.-C., Parias, C., Soulet, D., Lévy, F., Nowak, R., Lansade, L., & Bertin, A. (2024). Facial blushing and feather fluffing are indicators of emotions in domestic fowl (*Gallus gallus domesticus*). *PLoS One*, *19*, Article e0306601. https://doi.org/10.1371/journal.pone.0306601

Bamford, A. J., Monadjem, A., & Hardy, I. C. W. (2010). Associations of avian facial flushing and skin colouration with agonistic interaction outcomes. *Ethology, 116*(12), 1163–1170. https://doi.org/10.1111/j.1439-0310.2010.01834.x

Barbur, J. L., Prescott, N. B., Douglas, R. H., Jarvis, J. R., & Wathes, C. M. (2002). A comparative study of stimulus-specific pupil responses in the domestic fowl (*Gallus gallus domesticus*) and the human. *Vision Research, 42*(2), 249–255. https://doi.org/10.1016/S0042-6989(01)00279-6

Bates, D., Mächler, M., Bolker, B., & Walker, S. (2015). Fitting linear mixed-effects models using lme4. *Journal of Statistical Software*, *67*(1). https://doi.org/10.18637/jss.v067.i01

Benitez-Quiroz, C. F., Srinivasan, R., & Martinez, A. M. (2018). Facial color is an efficient mechanism to visually transmit emotion. *Proceedings of the National Academy of Sciences*, *115*(14), 3581–3586. https://doi.org/10.1073/pnas.1716084115

Benitez-Quiroz, C. F., Srinivasan, R., & Martinez, A. M. (2019). Discriminant func-
tional learning of color features for the recognition of facial action units and
their intensities. *IEEE Transactions on Pattern Analysis and Machine Intelligence*,
*41*(12), 2835–2845. https://doi.org/10.1109/TPAMI.2018.2868952

Bertin, A., Beraud, A., Lansade, L., Blache, M.-C., Diot, A., Mulot, B., & Arnould, C. (2018a). Facial display and blushing: Means of visual communication in blue- and-yellow macaws (Ara ararauna)? *PLoS One*, *13*(8), Article e0201762. https://doi.org/10.1371/journal.pone.0201762

Bertin, A., Beraud, A., Lansade, L., Mulot, B., & Arnould, C. (2020). Bill covering and nape feather ruffling as indicators of calm states in the Sulphur-crested cockatoo (Cacatua galerita). *Behavioural Processes*, *178*, Article 104188. https://doi.org/10.1016/j.beproc.2020.104188

Bertin, A., Cornilleau, F., Lemarchand, J., Boissy, A., Leterrier, C., Nowak, R., Calandreau, L., Blache, M.-C., Boivin, X., Arnould, C., & Lansade, L. (2018b). Are there facial indicators of positive emotions in birds? A first exploration in Japanese quail. *Behavioural Processes*, *157*, 470–473. https://doi.org/10.1016/j.beproc.2018.06.015

Bertin, A., Mulot, B., Nowak, R., Blache, M.-C., Love, S., Arnold, M., Pinateau, A., Arnould, C., & Lansade, L. (2023). Captive blue-and-yellow macaws (Ara ararauna) show facial indicators of positive affect when reunited with their caregiver. *Behavioural Processes*, *206*, Article 104833. https://doi.org/10.1016/j.beproc.2023.104833

Blokhuis, H. J. (1984). Rest in poultry. *Applied Animal Behaviour Science*, *12*, Article 289-303.

Bradshaw, R. H. (1992). Individual attributes as predictors of social status in small
groups of laying hens. *Applied Animal Behaviour Science*, *34*(4), 359–363.
https://doi.org/10.1016/S0168-1591(05)80095-4

Briefer, E. F., Tettamanti, F., & McElligott, A. G. (2015). Emotions in goats: Mapping physiological, behavioural and vocal profiles. *Animal Behaviour*, *99*, 131–143. https://doi.org/10.1016/j.anbehav.2014.11.002

Bruce, E. H., Prescott, N. B., & Wathes, C. M. (2003). Preferred food rewards for laying hens in behavioural experiments. *British Poultry Science*, *44*(3), 345–349. https://doi.org/10.1080/0007166031000085490

Buechner, V. L., Maier, M. A., Lichtenfeld, S., & Schwarz, S. (2014). Red—take a closer look. *PLoS One*, *9*(9), Article e108111. https://doi.org/10.1371/journal.pone.0108111

Cabanac, M., & Aizawa, S. (2000). Fever and tachycardia in a bird (Gallus domesticus) after simple handling. *Physiology & Behavior*, *69*(4–5), 541–545. https://doi.org/10.1016/S0031-9384(00)00227-4
Camerlink, I., Coulange, E., Farish, M., Baxter, E. M., & Turner, S. P. (2018). Facial expression as a potential measure of both intent and emotion. *Scientific Reports*, 8(1). https://doi.org/10.1038/s41598-018-35905-3. Article 1.

Changizi, M. A., Zhang, Q., & Shimojo, S. (2006). Bare skin, blood and the evolution of primate colour vision. *Biology Letters*, 2(2), 217–221. https://doi.org/10.1098/rbl.2006.0440

Cloutier, S., Beaugrand, J. P., & Lagué, P. C. (1996). The role of individual differences and patterns of resolution in the formation of dominance orders in domestic hen triads. *Behavioural Processes*, 38(3), 227–239. https://doi.org/10.1016/S0376-6357(96)00034-4

Cornwallis, C. K., & Birkhead, T. R. (2006). Experimental evidence that female ornamentation increases the acquisition of sperm and signals fecundity. *Proceedings of the Royal Society B: Biological Sciences*, 274(1609), 583–590. https://doi.org/10.1098/rspb.2006.3757

de Waal, F. B. M. (2011). What is an animal emotion? *Annals of the New York Academy of Sciences*, 1224(1), 191–206. https://doi.org/10.1111/j.1749-6632.2010.05912.x

Descovich, K. A., Wathan, J., Leach, M. C., Buchanan-Smith, H. M., Flecknell, P., Farningham, D., & Vick, S.-J. (2017). Facial expression: An under-utilised tool for the assessment of welfare in mammals. *ALTEX*, 34(3), 409–429. https://doi.org/10.14573/altex.1607161

Drummond, P. D., & Lazaroo, D. (2012). The effect of facial blood flow on ratings of blushing and negative affect during an embarrassing task: Preliminary findings. *Journal of Anxiety Disorders*, 26(2), 305–310. https://doi.org/10.1016/j.janxdis.2011.12.012

Drummond, P. D., Minosora, K., Little, G., & Keay, W. (2013). Topical ibuprofen inhibits blushing during embarrassment and facial flushing during aerobic exercise in people with a fear of blushing. *European Neuropsychopharmacology*, 23(12), 1747–1753. https://doi.org/10.1016/j.europeuro.2013.07.013

Duncan, I. J. H., & Wood-Gush, D. G. M. (1972). Thwarting of feeding behaviour in the domestic fowl. *Animal Behaviour*, 20(3), 444–451. https://doi.org/10.1016/S0003-3472(72)80007-1

Dwyer, J. F. (2014). Correlation of cere color with intra- and interspecific agonistic interactions of crested caracaras. *Journal of Raptor Research*, 48(3), 240–247. https://doi.org/10.3356/0892-1016-48.3.240

Forbes, M. R. L., & Ankney, C. D. (1987). Hatching asynchrony and food allocation within broods of Pied-billed Grebes, *Podilymbus podiceps*. *Canadian Journal of Zoology*, 65(12), 2872–2877. https://doi.org/10.1139/z87-436

Grebecy, T. C., Ali, A. B. A., Swanson, J. C., Widowski, T. M., & Siegford, J. M. (2020). Dust bathing in laying hens: Strain, proximity to, and number of conspecifics matter. *Poultry Science*, 99(9), 4103–4112. https://doi.org/10.1016/j.psj.2020.04.032

Hardesty, M. (1931). The structural basis for the response of the comb of the Brown Leghorn fowl to the sex hormones. *American Journal of Anatomy*, 47(2), 277–323. https://doi.org/10.1002/aja.1000470204

Hartig, F. (2022). *DHARMA: Residual diagnostics for hierarchical (multi-level/mixed) regression models*. https://CRAN.R-project.org/package=DHARMA (R package version 0.4.6).

He, K., Zhang, X., Ren, S., & Sun, J. (2016). Deep residual learning for image recognition. 2016 IEEE Conference on Computer Vision and Pattern Recognition (CVPR), 770–778. https://doi.org/10.1109/CVPR.2016.90

Iverson, E. N. K., & Karubian, J. (2017). The role of bare parts in avian signaling. *Auk*, 134(3), 587–611. https://doi.org/10.1642/AUK-16-136.1

Johnson, K., Thornhill, R., Ligon, J. D., & Zuk, M. (1993). The direction of mothers' and daughters' preferences and the heritability of male ornaments in red jungle fowl (*Gallus gallus*). *Behavioral Ecology*, 4(3), 254–259. https://doi.org/10.1093/beheco/4.3.254

Jones, R. B. (1996). Fear and adaptability in poultry: Insights, implications and imperatives. *World's Poultry Science Journal*, 52(2), 131–174. https://doi.org/10.1079/WPS19960013

Jones, E. K. M., & Prescott, N. B. (2000). Visual cues used in the choice of mate by fowl and their potential importance for the breeder industry. *World's Poultry Science Journal*, 56(2), 127–138. https://doi.org/10.1079/WPS20000010

Keeling, E., & Raghallaigh, S. N. (2024). Flushing and blushing. In *Rook's Textbook of Dermatology* (pp. 1–12). John Wiley & Sons, Ltd. https://doi.org/10.1002/9781119709268.rook104

Kim, D.-H., Lee, Y.-K., Lee, S.-D., Kim, S.-H., & Lee, K.-W. (2021). Physiological and behavioral responses of laying hens exposed to long-term high temperature. *Journal of Thermal Biology*, 99, Article 103017. https://doi.org/10.1016/j.jtherbio.2021.103017

Kruijt, J. P. (1964). Ontogeny of social behaviour in Burmese red junglefowl (*Gallus Gallus Spadiceus*) Bonnaterre. *Behaviour. Supplement*, 12, 1-201.

Kumar, A. (2010). Communication value of displays and postures in red-vented bulbul *Pycnonotus cafer* (Aves: Pycnonotidae). *Journal of Threatened Taxa*, 2 (6), 919–929. https://doi.org/10.11609/jott.02272.919-29

Lansade, L., Nowak, R., Lainé, A.-L., Leterrier, C., Bonneau, C., Parias, C., & Bertin, A. (2018). Facial expression and oxytocin as possible markers of positive emotions in horses. *Scientific Reports*, 8(1), Article 14680. https://doi.org/10.1038/s41598-018-32993-z

Lenth, R. V. (2022). *emmeans: Estimated marginal means, aka least-squares means*. https://CRAN.R-project.org/package=emmeans (R package version 1.8.2).

McGrath, N., Burman, O., Dwyer, C., & Phillips, C. J. C. (2016). Does the anticipatory behaviour of chickens communicate reward quality? *Applied Animal Behaviour Science*, 184, 80–90. https://doi.org/10.1016/j.applanim.2016.08.010

McGrath, N., Dunlop, R., Dwyer, C., Burman, O., & Phillips, C. J. C. (2017). Hens vary their vocal repertoire and structure when anticipating different types of reward. *Animal Behaviour*, 130, 79–96. https://doi.org/10.1016/j.anbehav.2017.05.025

Mendl, M., Burman, O. H. P., & Paul, E. S. (2010). An integrative and functional framework for the study of animal emotion and mood. *Proceedings of the Royal Society B: Biological Sciences*, 277(1696), 2895–2904. https://doi.org/10.1098/rspb.2010.0303

Mendl, M., & Paul, E. S. (2020). Animal affect and decision-making. *Neuroscience & Biobehavioral Reviews*, 112, 144–163. https://doi.org/10.1016/j.neubiorev.2020.01.025

Midtgård, U. (1984). Density of arteriovenous anastomoses in some skin areas of the domestic fowl (gallus domesticus). *Anatomical Record*, 209(4), 455–459. https://doi.org/10.1002/ar.1092090405

Morris, D. (1956). The feather postures of birds and the problem of the origin of social signals. *Behaviour*, 9(1), 75–111. https://doi.org/10.1163/156853956X00264

Mougeot, F. (2008). Ornamental comb colour predicts T-cell-mediated immunity in male red grouse *Lagopus lagopus scoticus*. *Naturwissenschaften*, 95(2), 125–132. https://doi.org/10.1007/s00114-007-0303-6

Mougeot, F., Pérez-Rodríguez, L., Sumozas, N., & Terraube, J. (2009). Parasites, condition, immune responsiveness and carotenoid-based ornamentation in male red-Legged Partridge *Alectoris rufa*. *Journal of Avian Biology*, 40(1), 67–74.

Mukhtar, N., & Khan, S. H. (2012). Comb: An important reliable visual ornamental trait for selection in chickens. *World's Poultry Science Journal*, 68(3), 425–434. https://doi.org/10.1017/S0043933912000542

Nakajima, K., Minami, T., & Nakauchi, S. (2017). Interaction between facial expression and color. *Scientific Reports*, 7(1), Article 41019. https://doi.org/10.1038/srep41019

Neethirajan, S., Reimert, I., & Kemp, B. (2021). Measuring farm animal emotions—sensor-based approaches. *Sensors*, 21(2), 553. https://doi.org/10.3390/s21020553

Negro, J. J., Sarasola, J. H., Fariñas, F., & Zorrilla, I. (2006). Function and occurrence of facial flushing in birds. *Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology*, 143(1), 78–84. https://doi.org/10.1016/j.cbpa.2005.10.028

Nobeyama, Y. (2024). Rosacea in East Asian populations: Clinical manifestations and pathophysiological perspectives for accurate diagnosis. *Journal of Dermatology*, 51(9), 1143–1156. https://doi.org/10.1111/1346-8138.17411

O'Connor, E. A., Saunders, J. E., Grist, H., McLeman, M. A., Wathes, C. M., & Abyeyesinghe, S. M. (2011). The relationship between the comb and social behaviour in laying hens. *Applied Animal Behaviour Science*, 135(4), 293–299. https://doi.org/10.1016/j.applanim.2011.09.011

Pérez-Rodríguez, L., & Viñuela, J. (2008). Carotenoid-based bill and eye ring colouration as honest signals of condition: An experimental test in the red-legged partridge (*Alectoris rufa*). *Naturwissenschaften*, 95(9), 821–830. https://doi.org/10.1007/s00114-008-0389-5

Papageorgiou, M., Goliomytis, M., Tzamaloukas, O., Miltiadou, D., & Simitzis, P. (2023). Positive welfare indicators and their association with sustainable management systems in poultry. *Sustainability*, 15(14), Article 10890. https://doi.org/10.3390/su151410890

Papini, M. R., Penagos-Corzo, J. C., & Pérez-Acosta, A. M. (2019). Avian emotions: Comparative perspectives on fear and frustration. *Frontiers in Psychology*, 9, 2707. https://www.frontiersin.org/articles/10.3389/fpsyg.2018.02707

Passarotto, A., Rodríguez-Caballero, E., Cruz-Miralles, Á., & Avilés, J. M. (2022). Ecogeographical patterns in owl plumage colouration: Climate and vegetation cover predict global colour variation. *Global Ecology and Biogeography*, 31(3), 515–530. https://doi.org/10.1111/geb.13444

Pellon, S., Hallegot, M., Lapique, J., & Tomberg, C. (2020). A redefinition of facial communication in non-human animals. *Journal of Behavior*, 3(1). https://www.jscimedcentral.com/article/A-Redefinition-of-Facial-Communication-in-Non-Human-Animals

Phelipon, R., Ascione, A., Ruet, A., Bertin, A., Biau, S., Arnould, C., Boichot, L., & Lansade, L. (2024). Differences in behaviour, facial expressions and locomotion between positive anticipation and frustration in horses. *Applied Animal Behaviour Science*, 274, Article 106262. https://doi.org/10.1016/j.applanim.2024.106262

Prescott, N. B., & Wathes, C. M. (1999). Spectral sensitivity of the domestic fowl (*Gallus g. Domesticus*). *British Poultry Science*, 40(3), 332–339. https://doi.org/10.1080/00071669987412

Revelle, W. (2024). *psych: Procedures for Psychological, Psychometric, and Personality Research*. Northwestern University [R package version 2.4.1] version 2.4.1. https://CRAN.R-project.org/package=psych

Richardson, M., McEwan, K., Maratos, F., & Sheffield, D. (2016). Joy and calm: How an evolutionary functional model of affect regulation informs positive emotions in nature. *Evolutionary Psychological Science*, 2(4), 308–320. https://doi.org/10.1007/s40806-016-0065-5

Ruiz-Rodríguez, M., Martín-Vivaldi, M., & Avilés, J. M. (2017). Multi-functional crest display in hoopoes *Upupa epops*. *Journal of Avian Biology*, 48(11), 1425–1431. https://doi.org/10.1111jav.01402

Russell, J. A. (2003). Core affect and the psychological construction of emotion. *Psychological Review*, 110(1), 145–172. https://doi.org/10.1037/0033-295X.110.1.145

Sadeghian, A., Rouhana, H., Oswald-Stumpf, B., & Boh, E. (2017). Etiologies and management of cutaneous flushing. *Journal of the American Academy of Dermatology*, 77(3), 391–402. https://doi.org/10.1016/j.jaad.2016.12.031
Saper, C. B., Chou, T. C., & Elmquist, J. K. (2002). The need to feed: Homeostatic and hedonic control of eating. *Neuron, 36*(2), 199-211. https://doi.org/10.1016/S0896-6273(02)00969-8

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P., & Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis. *Nature Methods*, 9(7), 676-682. https://doi.org/10.1038/nmeth.2019

Scholes, E., & Laman, T. G. (2018). Distinctive courtship phenotype of the Vogelkop Superb Bird-of-Paradise Lophorina niedda Mayr, 1930 confirms new species status. *PeerJ*, 6, Article e4621. https://doi.org/10.7717/peerj.4621

Shearn, D., Bergman, E., Hill, K., Abel, A., & Hinds, L. (1990). Facial coloration and temperature responses in blushing. *Psychophysiology*, 27(6), 687–693. https://doi.org/10.1111/j.1469-8986.1990.tb03194.x

Soulet, D., Bertin, A., & Arnould, C. (2024). Première mise en évidence d'expressions faciales chez la poule domestique (Gallus gallus domesticus) (Doctoral thesis, Université de Tours). https://theses.fr/s301751.

Soulet, D., Jahoui, A., Guabiraba, R., Lansade, L., Blache, M.-C., Piégu, B., Lefort, G., Guillory, V., Quéré, P., Germain, K., Lévy, F., Love, S. A., Bertin, A., & Arnould, C. (2024). Exploration of skin redness and immunoglobulin A as markers of the affective states of hens. *Applied Animal Behaviour Science*, 274, Article 106268. https://doi.org/10.1016/j.applanim.2024.106268

Spruijt, B. M., Van Hooft, J. A., & Gispen, W. H. (1992). Ethology and neurobiology of grooming behavior. *Physiological Reviews*, 72(3), 825-852. https://doi.org/10.1152/physrev.1992.72.3.825

Stephen, I. D., Oldham, F. H., Perrett, D. I., & Barton, R. A. (2012). Redness enhances perceived aggression, dominance and attractiveness in men's faces. *Evolutionary Psychology*, 10(3), Article 147407491201000320. https://doi.org/10.1177/147407491201000312

Thorstenson, C. A. (2018). The social psychophysics of human face color: Review and recommendations. *Social Cognition*, 36(2), 247-273. https://doi.org/10.1521/soco.2018.36.2.247

Thorstenson, C. A., Elliot, A. J., Pazda, A. D., Perrett, D. I., & Xiao, D. (2018). Emotion-color associations in the context of the face. *Emotion*, 18(7), 1032-1042. https://doi.org/10.1037/emo0000358

Thorstenson, C. A., McPhetres, J., Pazda, A. D., & Young, S. G. (2022). The role of facial coloration in emotion disambiguation. *Emotion*, 22, 1604-1613. https://doi.org/10.1037/emo0000900

Thorstenson, C. A., Pazda, A. D., Young, S. G., & Elliot, A. J. (2019). Face color facilitates the disambiguation of confusing emotion expressions: Toward a social functional account of face color in emotion communication. *Emotion*, 19(5), 799-807. https://doi.org/10.1037/emo0000485

van Kampen, M. (1971). Some aspects of thermoregulation in the white leghorn fowl. *International Journal of Biometeorology*, 15(2), 244-246. https://doi.org/10.1007/BF01803906

van Liere, D. W. (1991). *Function and Organization of Dustbathing in Laying Hens*. Wageningen Agricultural University). https://core.ac.uk/download/pdf/29354689.pdf.

Vestergaard, K. S., Damm, B. I., Abbott, U. K., & BildsøE, M. (1999). Regulation of dustbathing in feathered and featherless domestic chicks : The Lorenzian model revisited. *Animal Behaviour*, 58(5), 1017-1025. https://doi.org/10.1006/anbe.1999.1233

Vorobyev, M., Osorio, D., Bennett, A. T. D., Marshall, N. J., & Cuthill, I. C. (1998). Tetrachromacy, oil droplets and bird plumage colours. *Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology*, 183(5), 621-633. https://doi.org/10.1007/s003590050286

Waller, B. M., Julle-Daniere, E., & Micheletta, J. (2020). Measuring the evolution of facial 'expression' using multi-species FACS. *Neuroscience & Biobehavioral Reviews*, 113, 1-11. https://doi.org/10.1016/j.neubiorev.2020.02.031

Widowski, T. M., & Duncan, I. J. H. (2000). Working for a dustbath: Are hens increasing pleasure rather than reducing suffering? *Applied Animal Behaviour Science*, 68(1), 39-53. https://doi.org/10.1016/S0168-1591(00)00088-5

Wilson, W. O., Woodard, A. E., Nordstrom, J. O., & Smith, A. H. (1958). Regressive changes in external indicators of laying condition following cessation of laying. *Poultry Science*, 37(1), 24-26. https://doi.org/10.3382/ps.0370024

Yorzinski, J. L., Patricelli, G. L., Babcock, J. S., Pearson, J. M., & Platt, M. L. (2013). Through their eyes: Selective attention in peahens during courtship. *Journal of Experimental Biology*, 216(16), 3035-3046. https://doi.org/10.1242/jeb.087338

Zhao, M., Kurtis, S. M., Humbel, E. A., Griffith, E. V., Liu, T., Braun, E. L., Buchholz, R., & Kimball, R. T. (2024). Bare parts in the Galliformes: The evolution of a multifunctional structure. *Royal Society Open Science*, 11(1), Article 231695. https://doi.org/10.1098/rsos.231695

Zimmerman, P. H., Buijs, S. A. F., Bolhuis, J. E., & Keeling, L. J. (2011). Behaviour of domestic fowl in anticipation of positive and negative stimuli. *Animal Behaviour*, 81(3), 569-577. https://doi.org/10.1016/j.anbehav.2010.11.028

Zimmerman, P. H., Lundberg, A., Keeling, L. J., & Koene, P. (2003). The effect of an audience on the gakel-call and other frustration behaviours in the laying hen (*Gallus gallus domesticus*). *Animal Welfare*, 12(3), 315-326. https://doi.org/10.1017/S0962728600025823

Zuk, M., Thornhill, R., Ligon, J. D., & Johnson, K. (1990). Parasites and mate choice in red jungle fowl. *American Zoologist*, 30(2), 235-244. https://doi.org/10.1093/icb/30.2.235