# Impacts of digestibility stalls on the welfare of growing
bulls

I. Veissier, R. Bellagi, P. Nozière, R. Baumont

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

## To cite this version:

I. Veissier, R. Bellagi, P. Nozière, R. Baumont. Impacts of digestibility stalls on the welfare of growing bulls. Animal, 2025, 19 (5), pp.101513. 10.1016/j.animal.2025.101513. hal-05070758

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# Impacts of digestibility stalls on the welfare of growing bulls

I. Veissier *, R. Bellagi, P. Nozière, R. Baumont

Université Clermont Auvergne, INRAE, VetAgro Sup, UMR Herbivores, 63122 Saint-Genès-Champanelle, France

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

## ARTICLE INFO

**Article history:**

Received 23 September 2024

Revised 1 April 2025

Accepted 3 April 2025

Available online 11 April 2025

**Keywords:**

Behaviour

Cattle

Hair cortisol

Restraint

Stress

## ABSTRACT

Diet digestibility and nitrogen balance are essential indicators for studies on ruminant nutrition. Both these indicators are conventionally assessed over 1– to 2-week periods when animals are restrained in so-called digestibility stalls that largely restrict their movements. Here, we aimed to assess the welfare of bulls in digestibility stalls and to check whether stall-related welfare impacts could be limited by shortening the duration of the stay in the stall. Sixteen Charolais bulls accommodated in a large group pen were confined in digestibility stalls for 2 weeks, in two repetitions spaced 2.5 months apart. Urine and faeces were collected daily for 10 days from Day 7 in stalls. From 7 days before the bulls entered the stalls to 7 days after they exited, we measured their feed intake daily and monitored their posture (standing vs lying) and main activity patterns (eating, ruminating, other activity, inactivity) using activity sensors. External lesions (hair losses, wounds) were assessed once a week during the same period. Cortisol was determined in hair from the tail tuft at 3-week intervals from before entering the stalls to after 10 or 15 days in the stalls. Bulls responded to the digestibility stalls by eating lower amounts of feed, spending more time standing or inactive and less time eating or ruminating than when they were in the group pen. The increase in time spent inactive and the decrease in time spent eating were stronger in Repetition 2 than in Repetition 1. The time spent inactive decreased over the first few days in the stalls then increased again over the course of days when urine and faeces were collected. Bulls were more likely to be affected by lesions once they were in the stalls. Hair cortisol increased when the bulls were in the stalls, with a more marked increase after a short stay in Repetition 2. In conclusion, we find definitive evidence that digestibility and nitrogen measurements in digestibility stalls are constraining for animals, and bulls do not appear to habituate to time in the stalls. Confinement in the stalls as well as handling while in the stalls can both have negative welfare impacts.

© 2025 The Author(s). Published by Elsevier B.V. on behalf of The animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Implications

We confirm that confinement in digestibility stalls has welfare impacts on bulls. Both moving into the stalls and staying in the stalls appear to be stressful, especially when faeces and urine are collected. There were no signs that the animals habituate to time in stalls, so previous exposure to stalls – with the view that animals would adapt – appears to be counterproductive. We recommend increasing the amount of room given to the animals, finding ways to cause the least possible disturbance during the collection of urine and faeces, and ensuring that in-stall animals are handled as gently as possible.

## Introduction

Diet digestibility and nitrogen balance are essential indicators for studies on nutrition, especially in ruminants which can utilise

feeds that humans cannot eat but are poor nitrogen utilizers. Assessing these two indicators requires precise measurement of feed intakes and nutrient excretions in urine and faeces separately. This is conventionally performed by confining animals in 'digestibility stalls' that enable separated collection of urine and faeces (Hristov et al., 2019, Danesh Mesgaran et al., 2020). The confinement period can last from 1 week (for diet digestibility measurements) to 2 weeks (for nitrogen balance measurements). Such procedures carry ethical concerns, because digestibility stalls largely restrict animals' room for movement, allowing them only to lie down and stand up.

Digestibility stalls were designed to cause as little discomfort as possible to adult cattle while allowing collection of urine and faeces (Veissier et al., 2021). These stalls enable animals to share visual contact with conspecifics, the floor is covered by a foam mat to ensure lying comfort, and the side walls can be folded down for urine collection while reducing the risk of injury to the animal or the experimenter. Nevertheless, the digestibility stalls may not totally eliminate the welfare impacts of confinement, handling to get the animals into the stalls, or exposure to a novel environment,

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: Isabelle.veissier@inrae.fr (I. Veissier).</footnote>
and social isolation, all of which that have been found stressful in other circumstances (Veissier et al., 1989, Boissy and Bouissou 1995, Herskin et al., 2007, Chen et al., 2016, Woiwode et al., 2016) but have not been documented specifically in digestibility stalls.

In a companion paper, Bellagi et al. (in revision) investigated the minimum number of days needed to house bulls inside the stalls for reliable nitrogen balance measurements. The first – scientific – objective of the present study was to assess potential welfare impairments in bulls in digestibility stalls. Although welfare results from both the absence of suffering and the presence of positive experiences (Dawkins, 1980, Duncan, 2005, Mellor, 2016), we focused our analysis on negative aspects which could be evidenced by a reduction of feed intake, behavioural changes (posture and main activity), cortisol release in hair and lesions. The second – practical – objective was to check whether stall-related welfare impacts could be limited by a shorter stay than is conventionally practised to measure digestibility or nitrogen balance (10 vs 15 days). Indeed, if the main welfare impacts of digestibility stalls are due to confinement, then a shorter in-stall stay would benefit the animals. Alternatively, if the main welfare impacts of digestibility stalls are due to handling when being moved to the stalls, then there would be no benefit of shortening the stay in the stalls, and if the poor welfare is mainly caused by having to face a new situation, then, the animals should stabilise their behaviours after a few days of habituation, with no subsequent welfare impact.

## Material and methods

### Study design

This study used the experimental set-up from Bellagi et al. (in revision). Briefly, 16 Charolais bulls aged 314 ± 5.7 days at the start of the experiment were divided into two batches of eight bulls each. The bulls had been hot disbudded when they were 3-4-week-old. They were grouped in a deep-litter pen (5 × 7.7 m) in a barn except when they were moved to the digestibility room that contained eight digestibility stalls (Fig. 1). The barn and the room received natural light and were artificially lighted when needed. There was no light between 1800 and 0700 h. The barn and the room were naturally ventilated thanks to large openings on one side equipped with wind-breakers (nets in the barn and plastic bands in the digestibility room), and air vents in the ceiling; no drafts were noticed. The temperature in the barn and the digestibility room was about 10-12 °C. Each batch was subjected to a 2-week stay in the digestibility stalls (one stall per bull) in two repetitions set 2.5 months apart. For each repetition, we considered a 30-day period: 7 days during which the bulls were accommodated in the group pen (Phase A, Days 1-7), 1 day when they were moved to the stalls (Day 8), 5 days of adaptation to the stalls (Phase B, Days 9-13), 9 days when digestibility measurements were performed (Phase C, Days 14-22), 1 day when the bulls were moved back to the group pen (Day 23), and the 7 subsequent days (Phase D, Days 24-30) (Table 1). Cortisol was determined after 10 days in-stall (short duration, Repetition 1 - Batch 2 and Repetition 2 - Batch 1) or 15 days in-stall (long duration, Repetition 1 - Batch 1 and Repetition 2 - Batch 2). Two diets differing in CP content were distributed, each to half of the bulls in a batch, but these diets did not affect feed intake (8.08 vs 8.02 kg/d of DM intake in the high-protein vs low-protein diets; SE, 0.13; *P* = 0.84) and so the diet factor was not considered in this paper. The observational unit was the bull.

### Procedure for digestibility and nitrogen balance assessment

The bulls were fed ad libitum a total mixed ration based on grass silage and concentrate (55:45 on a DM basis). The amount of feed offered to each bull was adjusted weekly to ensure a minimum of 10% of refusals. On the day the bulls were moved to the digestibility stalls, each bull was weighed, then held in a restraint cage where it was fitted with a neck collar, and finally led to the digestibility stall with one person behind and one ahead guiding the bull with a rope.

We used eight digestibility stalls manufactured by Chambron Gautier® (Pont-du-Château, France; Fig. 1). To reduce constraints on the animals, the stall can be length- and width-adjusted to size of the animal (up to 2.10 × 2.80 m), the floor is covered with a foam mat, and the animal can see its neighbours in adjacent and facing stalls (description in Veissier et al., 2021). The eight stalls were placed in two rows of four stalls set face-to-face, in a walled space separate from the rest of the barn. When in stalls, the bulls were tethered with a 1 m-long chain attached to the neck collar. Stall width was 2.10 m when bulls first entered the stalls, then reduced progressively by 0.20 m per day from Days 9 to 12 to ease the collection of faeces. Final stall width was 1.30 m.

Diet digestibility and nitrogen balance were assessed from Day 13 until Day 22 (see the companion paper by Bellagi et al., in revision). The assessment was based on feed intake and materials excreted in urine and faeces. For an assessment on a given Day n, the urine and faeces were collected on Day n + 1. From a behavioural point of view, we thus considered Day 13 as still part of Phase A (adaptation). Faeces were collected in a container located under the stall. Urine was collected using a 50 cm-wide harness positioned on the animal's belly (from the evening of Day 11) connected to a 30 L bottle outside the stall. The presence of caretakers was increased during measurements (Phase C) compared to the adaptation phase (Phase B): in the morning, the caretakers made the bulls stand up if they were lying down to check that there were no faeces that had dropped onto the mat instead into the faeces container (any faeces found on the mat were to be collected); at various intervals during the day, they checked that the belly hardness was in place and functioning.

### Measurements

#### Bull weight gain, feed intake, and water consumption

The average daily weight gain of the bulls was calculated from their weight on the days they were moved in and out of the digestibility stalls. Feed intake was measured for each individual animal throughout the experiment by weighing feed offered and feed refused. Samples of the feed offered and refusals were oven-dried at 103 °C for 48 h to measure the DM content. Feed intake was expressed in DM intake. The water consumption of bulls was monitored daily thanks to consumption meters fitted on individual drinkers (Fig. 1) and checked at 0900 h every day on days when the measures of digestibility were performed (Phase C).

#### Behaviour

The behaviour of the bulls was monitored from Day 1 to Day 30. Behavioural data for Days 8 and 23 were discarded because the bulls were partly in the group pen and partly in stalls. The bulls were fitted with activity sensors (Axel Medria, ITK/Medria, France) based on triaxial accelerometers mounted on their neck collar (description in Bouchon et al., 2025). The system can distinguish the animal's posture (standing vs lying) and its activity pattern (inactivity, eating, ruminating, other activity) with an accuracy of 83% for standing/lying, 86% for inactivity, 85% for ruminating, and
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Experimental set-up for bull digestibility measurements. Top, digestibility room; Bottom, digestibility stall (©HTournadre in Veissier et al., 2021).</figcaption></figure>

77% for eating (Bouchon et al., 2025). Inactivity, ruminating and other activities can occur when the animals are standing or lying. The sensor system provides the posture and activity pattern in which the animal spent the most time during 5-min slots (.csv file). From the 288 slots obtained for each day, we then calculated the percentage of time spent in each posture and each activity pattern for each day thanks to the R-Shiny AUTOMNE application (Bouchon et al., 2025).

## Lesions

The skin of the bulls was examined once a week throughout the experiment. Skin lesions were classified according to the Welfare

Quality (2009) protocol, as either hair loss, when hair was lost but the skin was not damaged, or other lesions, when the skin was damaged (inflammation, scratches). Each time a lesion was observed, we noted its location (head, neck, shoulder, foreleg, back, belly, rump, hindleg).

## Hair cortisol

To obtain an estimate of the cumulative stress imposed on the bulls, we determined cortisol in hair from the tail tuft, which is an area where hair grows fast. Around each repetition, the hair was shaved three times at 3-week intervals, so as to determine cortisol only on recently-grown hair. The hair from the first shaving
<caption>**Table 1**</caption>

Measurements performed on bulls. The bulls were weighed on the days they were moved in and out of digestibility stalls. The feed intake and the behaviour (posture, activity) were monitored over 30-day periods, i.e. 1 week in a group pen before the bulls were moved to digestibility stalls (Phase A), 2 weeks in the stalls (Phase B, first 5 days; Phase C, following 9 days when urine and faeces were collected every morning), and the following week when back in the group pen (Phase D). Water consumption was measured during Phase C. Hair cortisol content was measured twice, at a 3-week interval, with the second measurement performed after 10 or 15 days in the stalls (Short vs Long duration in stalls). Caretakers checked for observable external lesions once a week.

<table><thead><tr><th rowspan="2">Phase</th><th rowspan="2">Day</th><th rowspan="2">Weight</th><th rowspan="2">Feed intake</th><th rowspan="2">Behaviour</th><th rowspan="2">Water consumption</th><th>Short</th><th>Long</th><th rowspan="2">Lesions</th></tr><tr><th colspan="2">Cortisol</th></tr></thead><tbody><tr><td rowspan="4">A</td><td>-10</td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td></tr><tr><td>-3</td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td></tr><tr><td>-4</td><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td></tr><tr><td>1</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">In</td><td>2</td><td></td><td>x</td><td>x</td><td></td><td></td><td>x</td><td></td></tr><tr><td>3</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>4</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>5</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>6</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td>x</td></tr><tr><td>7</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="8">B</td><td>8</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>9</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>10</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>11</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>12</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td>x</td></tr><tr><td>13</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td></tr><tr><td>15</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td></tr><tr><td rowspan="10">C</td><td>16</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>17</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>18</td><td></td><td>x</td><td>x</td><td>x</td><td>x</td><td></td><td></td></tr><tr><td>19</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td>x</td></tr><tr><td>20</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td></tr><tr><td>21</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td></tr><tr><td>22</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td></tr><tr><td>23</td><td>x</td><td></td><td></td><td>x</td><td></td><td>x</td><td></td></tr><tr><td rowspan="8">D</td><td>24</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>26</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td>x</td></tr><tr><td>27</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>29</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>30</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr></tbody></table>

was discarded. The hair from the second shaving was used to assess the basal level of cortisol. The hair from the third shaving was used to assess the cumulative stress experienced in the digestibility stalls. The third shaving was performed on Day 18 vs Day 23, corresponding to 10 vs 15 days in-stall (short vs long duration). The first and the second shavings were performed six and 3 weeks before the third one. The hair samples were placed in aluminum foil-wrapped envelopes, stored at room temperature, and sent to the Department of Clinical Science of the Swedish University of Agricultural Sciences (SLU, Sweden) for cortisol determination.

Cortisol was determined using an ELISA technique according to the method described in Tamminen et al. (2021). Briefly, hair samples were washed with isopropanol, because the hair may be soiled by faeces and urine which contain cortisol or its metabolites (Möstl et al., 2002, Otten et al., 2022). The isopropanol was then allowed to evaporate by leaving the samples at room temperature under a fume hood for 3 days. Residual dirtiness of the hair was assessed visually by counting particles and assessing the hair aspect (shiny vs dry or dull). For this study, we distinguished clean vs dirty samples, with clean samples containing less than five particles of dirt in 50 mg hair and with shiny hair. Then, 50 g samples of hair were ground and then added with 1.2 mL methanol to extract the cortisol. After a first centrifugation at 7 000 g for 2 min, 0.8 mL of the supernatant was extracted and re-centrifuged at 10 000 g for 5 min. After evaporation of methanol at 38 °C under a fume hood, 150 µLof phosphate-buffered saline (0.01 M PBS, pH 7.4) was

added to the dried samples. Cortisol was finally determined using ELISA kits designed for salivary cortisol (Salimetrics assay #1-3002, Salimetrics Europe Ltd) following the manufacturer's instructions. Each sample was assayed in duplicate. The CV for low (0.10 pg/ mg) and high (0.99 pg/mg) controls was 4.37 and 5.57%, respectively. To facilitate readability, we refer to the results of the assays as 'cortisol concentration', even though they may include some metabolites of cortisol from faeces despite having washed the hair.

## Statistical analysis

All statistical analysis was performed using SAS statistical software (SAS Institute Inc., 2013, Cary, NC). To meet the conditions for ANOVA, the proportions of time spent in each activity were arcsine-transformed, and cortisol concentrations were log-transformed. For these transformations, we added 1 to the initial values so as not to have null values. We used the mixed procedure to analyse fixed effects (see below) with the bull as random factor (applied to the intercept). We used the option 'variance components' for the covariance matrix as it was associated with the lowest AIC and BIC. Batch and Batch × Repetition interaction were not considered in the final models as they had no significant effects. When included in a model, the Day was used as a covariate to highlight trends with time. For daily weight gain in stalls, we included only Repetition (1 or 2) as fixed factor. For water intake, we considered Repetition and Day as fixed factor, and their interaction. For behaviour and feed intake, we considered Repetition, Phases (A,
B, C, D), Day and interactions between Phase and Day and between Repetition and Phase as fixed effects. For cortisol, we considered Cleanliness of the hair sample (clean vs dirty), Time (first vs second cortisol determination), Duration in-stall (10 vs 15 days), Repetition, and interactions between Time and Duration and between Time, Duration and Repetition. Normality and homogeneity of residuals were checked graphically. Means were compared with Tuckey adjustment.

Frequency of lesions was analysed using a Chi-2 test. Due to the low prevalence of hair losses and other lesions – especially in Repetition 1 – we compared the number of animals affected by a lesion (hair loss or other) in Phase A or the 2 weeks before vs in Phases B, C, or D in Repetition 2.

We calculated Pearson correlations between cortisol response (difference between the second and the first cortisol determination within each repetition) and behaviour in Phases B and C.

The null hypothesis was rejected when $P \le 0.05$. To facilitate interpretation of the results of mixed models, F values and probabilities of effects refer to the models on arcsine-transformed or log-transformed data (when these transformations were used) but estimates of means and SEs calculated on non-transformed data are shown in figures or text.

## Results

Only significant results are reported, unless otherwise indicated. Values not shown in tables or figures are mentioned in the text. One bull was sick during Phase C of Repetition 1; The data on weight, feed and water intake and behaviour were excluded from the analysis but we kept the cortisol and lesion data because the second hair sample was taken before the animal went sick and we considered that sickness did not affect lesions.

### Bull weight gain, feed intake, and water consumption

On the day they were moved to the stalls, the bulls weighed an average of 414.6 ± 4.1 kg at Repetition 1 and 515.3 ± 6.4 kg) at Repetition 2. On the day they were moved out of the stalls, they weighed an average of 423.3 ± 5.5 kg) at Repetition 1 and 532.4 ± 7.7 kg at Repetition 2. The daily weight gain was higher on Repetition 2 than on Repetition 1 (1.14 ± 0.140 vs 0.585 ± 0.144 kg/d, $P = 0.004$).

The bulls ate less feed when in-stall (Phases B and C, 7.94 ± 0.172 and 8.09 ± 0.150 kg/d) than when in the group pen (Phases A and D, 8.45 ± 0.152 and 8.61 ± 0.152 kg/d), with no difference between Phases B and C or between Phases A and D (Table 2 and Fig. 2). In addition, feed intake increased over the course of Phases B and D (+0.138 ± 0.070 and + 0.298 ± 0.036 kg/d) but

remained stable over the course of Phases A and C (0.013 ± 0.051 and 0.008 ± 0.044 kg/d). Bulls ate less feed in Repetition 1 than in Repetition 2 (7.86 ± 0.145 vs 8.68 ± 0.144 kg/d). There was an interaction between Phase and Repetition: there were no differences between Phases A and D or between Phases B and C, whatever the repetition; In Repetition 1, Phases B and C differed from Phase D but not from Phase A whereas in Repetition 2, Phases B and C differed from Phase A, but not from Phase D.

During the digestibility measurements in stalls (Phase C), the bulls drank an average of 19.5 L of water per day (SE, 0.72 L) at Repetition 1 and 23.7 L per day (SE, 0.76 L) at Repetition 2. There was a tendency for water consumption to decrease over the days (– 0.19 L/day, $P = 0.06$).

### Behaviour

Bulls responded to the digestibility stalls by modifying their time budget (Table 2 and Fig. 2).

The bulls spent more time standing when in-stall (Phases B and C, 67.4 ± 1.06 and 65.6 ± 0.65% time) than when in the group pen (Phases A and D, 61.8 ± 0.70 and 62.3 ± 0.69% time), with no difference between Phases B and C or between Phases A and D. No differences were noticed between repetitions, except that on Repetition 1, Phases D and B did not differ from each other and on Repetition 2, Phase C did not differ from any other phase (Supplementary Fig. S1).

The bulls spent more time inactive when in-stall (Phases B and C, 20.4 ± 1.03 and 19.7 ± 0.83% time) than when in the group pen (Phases A and D, 12.3 ± 0.85 and 13.2 ± 0.85% time), with no difference between Phases B and C or between Phases A and D. Percentage of time spent inactive decreased over the course of Phase B (–0.92 ± 0.52%/day) but increased over the course of Phases C and D (± 0.62 ± 0.32%/day and + 0.44 ± 0.26%/day, respectively) and showed little variation over the course of Phase A (–0.03 ± 0.38%/day). Bulls spent less time inactive in Repetition 1 than in Repetition 2 (15.8 ± 0.78 vs 17.0 ± 0.79% time). More specifically, bulls tended to be less inactive during Phase C in Repetition 1 than in Repetition 2 (38.2 ± 1.26 vs 41.0 ± 1.25% time, $P = 0.06$).

The bulls spent less time eating when in-stall (Phases B and C, 12.3 ± 0.95 and 14.5 ± 0.75% time) than when in the group pen (Phases A and D, 15.7 ± 0.78 and 17.2 ± 0.77% time), especially in Phase B that differed from all other phases. Percentage of time spent eating increased over the course of Phase A (+0.45 ± 0.34/day) but decreased over the course of Phase C (–0.62 ± 0.29/day) and showed little variation over the course of Phases B and D (–0.01 ± 0.47 and –0.28 ± 0.24%/day). The decrease in time spent eating in Phases B and C compared to Phases A and D was stronger in Repetition 2 than in Repetition 1: Time spent eating did not vary

<caption>**Table 2**</caption>

Results of the mixed models on behaviour and feed intake. Bulls (n = 16) were moved into digestibility stalls in two repetitions. Within each repetition: Phase A, before being moved into the stalls (1 week), Phase B, first 5 days in the stalls; Phase C, the following 9 days in-stall, when urine and faeces were collected each morning; Phase D, after being moved out of the stalls and back into the group pen (1 week).

<table><thead><tr><th rowspan="2">Item</th><th colspan="2">Phase</th><th colspan="2">Day</th><th colspan="2">Phase*Day</th><th colspan="2">Repetition</th><th colspan="2">Repetition × Phase</th></tr><tr><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th></tr></thead><tbody><tr><td>Postures and activities (%time)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Standing</td><td>5.0</td><td>**</td><td>0</td><td>0.92</td><td>1.1</td><td>0.33</td><td>2.84</td><td>0.09</td><td>2.64</td><td>*</td></tr><tr><td>Inactive</td><td>26.1</td><td>***</td><td>0.3</td><td>0.60</td><td>9.6</td><td>***</td><td>0.24</td><td>0.63</td><td>3.24</td><td>*</td></tr><tr><td>Ruminating</td><td>6.26</td><td>***</td><td>1.2</td><td>0.26</td><td>2.1</td><td>0.10</td><td>1.95</td><td>0.16</td><td>0.85</td><td>0.47</td></tr><tr><td>Eating</td><td>5.68</td><td>***</td><td>0.8</td><td>0.37</td><td>4.9</td><td>**</td><td>0</td><td>1.00</td><td>6.41</td><td>***</td></tr><tr><td>Other activity</td><td>5.1</td><td>**</td><td>0</td><td>0.99</td><td>4.5</td><td>**</td><td>3.43</td><td>0.06</td><td>1.81</td><td>0.14</td></tr><tr><td>Intake (kg DMI/d)</td><td>7.08</td><td>***</td><td>23.8</td><td>***</td><td>10.8</td><td>***</td><td>124</td><td>***</td><td>5.15</td><td>**</td></tr></tbody></table>

Abbreviations: F = Fisher values; DMI = DM intake.

<footnote>* $P < 0.05$.</footnote>

<footnote>** $P < 0.01$.</footnote>

<footnote>*** $P < 0.001$.</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Variations in daily behaviour and feed intake when bulls are moved to digestibility stalls. Phase A, 1 week in a group pen before being moved to the stalls; Phase B, 5 days in the stalls without collection of urine and faeces; Phase C, the following 9 days in stalls when urine and faeces were collected each morning; Phase D, 1 week after being moved back into the group pen (means and SE, n = 16 bulls observed during two repetitions). Abbreviation: DMI = DM intake.</figcaption></figure>

between phases in Repetition 1 (14.6 ± 0.71, 13.2 ± 1.10, 15.6 ± 0.87, and 16.2 ± 0.91% time in Phases A, B, C, and D, respectively) but in Repetition 2, it was significantly different in Phases B and C (11.5 ± 1.13 and 13.3 ± 0.86% time) compared to Phases A and D (16.9 ± 0.92 and 18.2 ± 0.91% time) (*P* < 0.01).

The bulls spent less time ruminating when in-stall (Phases B and C, 25.3 ± 1.17 and 23.5 ± 1.00% time) than when in the group pen (Phases A and D, 30.3 ± 1.02 and 30.2 ± 1.02% time), with no difference between Phases B and C or between Phases A and D. Time spent ruminating showed no change within phases nor between repetitions.

There was an overall Phase effect on the time spent on other activities, but after Tuckey adjustment, none of the pairwise differences between phases were significant. Percentage of time spent on other activities increased over the course of Phase B (+1.25 ± 0.52%/d) but decreased over the course of all other phases, especially Phase D (-0.25 ± 0.37, -0.24 ± 0.32 and -0.68 ± 0.27% time in Phases A, C, and D, respectively).

## Lesions

In Repetition 1, none of the bulls were affected by hair losses, but three bulls were affected by skin lesions other than hair losses (inflammation, scratches) during Phase A or the 2 weeks before and one of them had an additional lesion in Phase B.

In Repetition 2, hair losses affected two bulls in Phase A or the 2 weeks before and six other bulls during the subsequent phases. Most cases of hair loss were located on the shoulders and rump. In Repetition 2, skin lesions other than hair losses affected two bulls in Phase A or the 2 weeks before and six other bulls during subsequent phases, i.e. two bulls in Phases B and D, two bulls in Phase C, and two bulls in Phase D. In Repetition 2, the number of animals affected by lesions (hair loss or other) increased from before being put in-stall (Phase A or the 2 weeks before) to after being moved to the stall (Phases B, C, and D) (Chi-2 = 4.57, *P* = 0.03).
<caption>**Table 3**</caption>

Results of the mixed models on hair cortisol. Bulls (n = 16) were moved into digestibility stalls in two repetitions. Within each repetition: Phase A, before being moved into the stalls (1 week), Phase B, first 5 days in the stalls; Phase C, the following 9 days in-stall, when urine and faeces were collected each morning; Phase D, after being moved out of the stalls and back into the group pen (1 week).

<table><thead><tr><th rowspan="2">Item</th><th colspan="2">Cleanliness</th><th colspan="2">Time</th><th colspan="2">Duration</th><th colspan="2">Repetition</th><th colspan="2">Time × Duration</th><th colspan="2">Time × Duration × Repetition</th></tr><tr><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th><th>F</th><th>P</th></tr></thead><tbody><tr><td>Hair cortisol</td><td>11.4</td><td>**</td><td>33.4</td><td>***</td><td>0.42</td><td>0.53</td><td>4.57</td><td>**</td><td>2.97</td><td>0.09</td><td>4.57</td><td>**</td></tr></tbody></table>

Abbreviation: F = Fisher values.

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Hair cortisol content of bulls submitted to digestibility stalls in two repetitions. T0, before being moved into the stalls; T1, after a short (10 days) or long (15 days) stay in the stalls. When T1 corresponded to a short stay in Repetition 1, it corresponded to a long stay in Repetition 2, and vice versa (adjusted means and SE, n = 8 bulls per Duration (Short vs Long stay) across each repetition. Bars with no common letters differ significantly (P < 0.05), except for T1 short duration between the repetitions and for long duration between T1 Repetition 1 and T0 Repetition 2 (P < 0.10).</figcaption></figure>

## Hair cortisol

Dirty hair contained more cortisol than clean hair (12.0 ± 0.88 vs 10.4 ± 1.01 pg/mg, Table 3). Hair cortisol content increased from the first to the second determination (6.03 ± 0.94 vs 16.4 ± 0.86 pg/mg, Fig. 3). The increase was more marked when the bulls had stayed in the stall for a shorter duration, especially during Repetition 2.

## Correlations between variables

We did not find significant correlations between cortisol response and time spent in each activity or feed intake in Phases B and C (P > 0.05; Supplementary Figs. S2 and S3).

## Discussion

Bulls responded to being put in digestibility stalls by releasing cortisol and showing changes in behaviour and feed intake. These responses did not cross-correlate. Cortisol accumulates in hair, and so hair cortisol level is used as a measure of stress experienced during the preceding days or weeks (Gonzalez-de-la-Vara et al., 2011, Tallo-Parra et al., 2015, Heimbürge et al., 2020). Here, hair cortisol content increased when the bulls were in the digestibility stalls. However, the increase was not more marked after a long vs a short stay (here 10 vs 15 days). Hair cortisol therefore may reflect the stress of being moved to the stall rather than staying in the stall. It is unlikely that this stress was due to the novelty of the stalls, because the stress responses did not decrease but rather increased in Repetition 2. Therefore, the increase in hair cortisol when the bulls were in the digestibility stalls is likely due, at least

in part, to handling or to the change from having free movement in the group pen to being individually constrained in a stall. Alternatively, the release of cortisol could be due to staying in stalls, with the cortisol response decreasing over time. Indeed reduced cortisol secretion during prolonged exposure to a stressful situation is thought to prevent the body from being exposed to high levels of cortisol for long periods of time (Knights and Smith, 2007). In our study, the hair cortisol response was not associated with behavioural responses, suggesting separate causal factors. Behavioural responses appear to be due to being in the stalls rather than being moved to the stalls (see below). We therefore suggest that the hair cortisol response was mainly due to being moved to the stalls.

The time bulls spent in digestibility stalls increased the likelihood of lesions, including hair loss patches, inflammation, or scratches. Most of the hair loss cases were located on the shoulders or rump, and so were presumably due to friction against the stall partitions. Although none of the lesions were serious, they nevertheless confirm that the stalls strongly hindered the animals' movements.

Bulls modified their behaviour when they were moved into the digestibility stalls. When in the stalls, the bulls spent more time standing, presumably because lying was restricted by the narrowness of the stall – even when it was 2.1 m wide –, as already reported in calves accommodated in crates (Le Neindre, 1993). In addition, the bulls spent more time inactive and less time eating or ruminating when in-stall than when they were in the group pen. They also ate less when in-stall than when in the group pen. Most of these responses – except time spent standing – decreased over the course of the first few days in-stall: time spent inactive decreased over the days whereas feed intake increased until the collection of urine and faeces started. In parallel, time spent on

<footnote>** P < 0.01.</footnote>

<footnote>*** P < 0.001.</footnote>
activities other than eating or ruminating increased. These time-
course changes suggest a degree of habituation to the stalls as long
as the bulls were not disturbed by further handling, which concurs
with earlier findings that cattle and pigs need about a week to
adapt to new environments (Dantzer, 1973, Veissier et al., 1989).
Time spent standing remained stable over the first few days spent
in-stalls, suggesting no habituation, presumably because the width
of the stalls was reduced by 20 cm per day for 5 days (from 2.10 to
1.30 m).

The habituation pattern was reversed when the collection of
urine and faeces started. When collection started, time spent inac-
tive (that had decreased during the first few days in-stall)
increased while time spent performing other activities (that previ-
ously increased) decreased. In addition, time spent eating (that had
previously been stable) decreased whereas feed intake (that had
previously increased) remained stable. These results suggest that
the bulls were disturbed by the collection of urine or faeces. Sam-
ple collection was done in the morning, before feeding. At that
time, the bulls were usually lying down, so the experimenters
had to make them stand up so that they could collect any faeces
that remained on the floor of the stalls. In addition, the caretakers
visited the bulls several times during the day to check that the har-
ness used for collecting urine was in place and to collect faeces that
had not dropped in the faeces container. We suspect that making
the animal stand up was stressful, especially due to the narrowness
of the stall that makes movements difficult. Reducing the number
of days of measurements for the determination of digestibility and
nitrogen balance would thus bring some improvement in welfare.
Disturbances may also be reduced if the experimenters wait until
the animals are standing up before collecting urine and faeces.
The waiting time may be short if the experimenter is present when
the feed is distributed, as feed distribution usually prompts the
animals to stand up.

Finally, bulls did not habituate between repetitions. The cortisol
response to time in the stalls was more marked in Repetition 2.
Furthermore, the increase in time spent lying inactive and the
decrease in time spent eating and in feed intake when bulls were
in-stalls (compared to in the group pen) were also stronger in Rep-
etition 2. Rather than habituating to being in digestibility stalls, the
bulls appeared to become more responsive to it, as if they antici-
pated what was going to happen.

In conclusion, we find clear evidence that digestibility and
nitrogen measurements in digestibility stalls affect the welfare
of animals. Confinement in the stalls as well as handling while
in the stalls can both have negative welfare impacts. There were
no signs that the bulls habituate to time in stalls, so previous
exposure to stalls – with the view that the animals would adapt
– appears to be counterproductive. We recommend increasing
the amount of room given to the animals, finding ways to cause
the least possible disturbance during the collection of urine and
faeces (e.g. by intervening right after feed distribution), and
ensuring that in-stall animals are handled as gently as possible.
We believe that reducing stresses would not only improve the
welfare of the animals but also improve the accuracy of the
digestibility and nitrogen balance measurements by inducing a
more stable pattern of behaviour – including eating behaviour
– and limiting the effects of physiological stress responses on
metabolism.

Supplementary material

Supplementary Material for this article (https://doi.org/10.
1016/j.animal.2025.101513) can be found at the foot of the online
page, in the Appendix section.

Ethics approval

The protocol for this study was evaluated by the Ethics Commit-
tee for Animal Experimentation of the Auvergne-Rhône-Alpes
Region and approved by the French Ministry of Higher Education,
Research and Innovation (Authorisation number: APAFIS #
16194-2016101016361277 v6 issued on 14th January 2019).

Data and model availability statement

The data/models were not deposited in an official repository.
Data are available on request.

Declaration of Generative AI and Al-assisted technologies in the
writing process

During the preparation of this work the author(s) did not use
any AI and Al-assisted technologies.

Author ORCIDs

Isabelle Veissier: https://orcid.org/0000-0002-8497-5395.

Rahma Bellagi: https://orcid.org/0000-0002-1134-9342.

Pierre Nozière: https://orcid.org/0000-0003-1727-8984.

René Baumont: https://orcid.org/0000-0002-3802-0651.

CRediT authorship contribution statement

I. Veissier: Writing – review & editing, Writing – original draft, Visualisation, Supervision, Methodology, Investigation, Formal analysis, Conceptualisation. R. Bellagi: Writing – review & editing, Writing – original draft, Validation, Investigation, Formal analysis, Data curation. P. Nozière: Writing – review & editing, Validation, Supervision, Resources, Investigation, Data curation, Conceptualisation. R. Baumont: Writing – review & editing, Validation, Supervision, Resources, Project administration, Investigation, Funding acquisition, Data curation, Conceptualisation.

Declaration of interest

The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgements

We thank the personnel of the INRAE's Herbipôle experimental
unit (https://doi.org/10.15454/1.5572318050509348E12), specifi-
cally Sébastien Alcouffe, Dominique Chassaignes and Pascal Faure
for their technical support, and Nathalie Thollon for extracting
and processing the data from activity sensors. We also thank Karin
Alvåsen and the KV-lab joint laboratory at the SLU Department of
Clinical Sciences for performing the hair cortisol determinations.
We thank Metaform for checking and amending the English writ-
ing and Rodolphe Loubaton from VetAgro-Sup for his advice on
statistics.

Financial support statement

The study was supported by the SmartCow project funded by
the European Commission (Horizon H2020 research and innova-
tion programme, grant agreement No. 730924). The work falls
within the thematic area of the French government IDEX-ISITE ini-
tiative 16-INDEX-0001 (CAP 20-25), and specifically the IRC-SAE (international research centre on sustainable agroecosystems).

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