# Olfactory awareness in lambs assessed through habituation-dishabituation and approach-withdrawal tests

Izïa Larrigaldie, Fabrice Damon, Bruno Patris, Benoist Schaal, Alexandra Destrez

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Izïa Larrigaldie, Fabrice Damon, Bruno Patris, Benoist Schaal, Alexandra Destrez. Olfactory awareness in lambs assessed through habituation-dishabituation and approach-withdrawal tests. Applied Animal Behaviour Science, 2025, 291, pp.106734. 10.1016/j.applanim.2025.106734. hal-05157357

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https://institut-agro-dijon.hal.science/hal-05157357v1

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# Olfactory awareness in lambs assessed through habituation-dishabituation and approach-withdrawal tests

Izïa Larrigaldie<sup>a, *, 1</sup> ●, Fabrice Damon<sup>a</sup>, Bruno Patris<sup>a</sup>, Benoist Schaal<sup>a</sup>, Alexandra Destrez<sup>a, b</sup>

## ARTICLE INFO

### Keywords:

- Sheep (Ovis aries)

- Dog (Canis familiaris)

- Olfaction

- Behavioral tests

- Wool odor

- Fecal odor

## ABSTRACT

Assessing olfaction in lambs presents methodological challenges due to their precocial development and extreme sensitivity to isolation and novelty. Previous research studied lambs' olfactory reactivity, particularly to odors from conspecifics, their responses to other odor sources remaining underexplored. To begin addressing this gap, we implemented two tests to evaluate olfactory discrimination in lambs using two distinct odors: dog feces odor (DO) and ovine wool odor (OO). First, after validating the procedure on 10 Suffolk ewes with the same odorous stimuli, a habituation-dishabituation (H-D) test run on 6-day-old lambs (Suffolk, n = 26, 14 females) consisted in repeating 4 times one odor for habituation, followed by a single presentation the other, novel odor for dis-habituation (presentation order of OO/DO being counterbalanced across animals). The ewes and lambs habituated to both odors, exhibiting a significant decline in sniffing duration; they also dishabituated to novel odors, attesting their clear discrimination between them. Second, an approach-withdrawal test using boxes scented with these same stimuli was run a group of lambs p (Suffolk, age: 24-day, n = 21). In the approach test, lambs spent significantly more time near objects scented with unfamiliar conspecific wool than those scented with canine feces. Both tests confirmed that lambs clearly discriminate both odors. While the habituation-dishabituation test proved effective in assessing odor discrimination, interpretation of the approach test results was more complex under the given experimental conditions.

## 1. Introduction

Behavioral responses to odors are notably difficult to quantify in lambs. The sensory system of these precocial newborns appears to be dominated by vision and audition, which explains why research on lamb behavior and cognition has predominantly focused on visual and auditory reactivity (Abdel-Moneim et al., 2009; Porter et al., 1995; Porter and Ligout, 2004; Searby and Jouventin, 2003). Additionally, measuring lambs' responses individually is complicated because of their extreme sensitivity to isolation from dam or peers (Mora-Medina et al., 2016; Schichowski et al., 2008). Moreover, lambs respond negatively to novel environments or novel objects in their environment (Beck et al., 2021; Burritt and Provenza, 1997), although the presence of familiar conspecifics can serve as an immediate buffer against such stressors (Carbajal and Orihuela, 2001; Da Costa et al., 2004; Guesdon et al., 2015; R. H. Porter et al., 1995). Despite the fact that these previous studies have

documented neonate lambs' responsiveness to olfactory cues, primarily to odors from, or associated with, familiar conspecifics (e.g., Alary et al., 2023; Lévy et al., 2020; Ligout et al., 2004), little is known about how lambs detect and respond to unfamiliar odors. The present study seeks to determine whether lambs can discriminate and exhibit affective behavioral responses to unfamiliar odors – such as interest, detection, attraction or avoidance. Specifically, we assess their reactions a scent from an unknown conspecific versus a scent from a unfamiliar species.

To maximize their olfactory responsiveness, we exposed lambs to odorant substrates known to elicit strong and contrasted emotional reactions in adult sheep, namely ovine wool and canine feces. Sheep wool does consistently elicit investigative and approach behaviors, and has previously been used to assess olfactory preferences and social learning in sheep (e.g., Baldwin and Meese, 1977; Vince and Ward, 1984; Zambra et al., 2021). In contrast, the odor of canine feces has been used to assess avoidance responses in sheep (Arnould et al., 1998; Pfister et al., 1990;

<footnote><sup>a</sup> Development of Olfactory Cognition and Communication Lab, Centre des Sciences du Goût et de l'Alimentation, CNRS, Institut Agro, Inrae, Université Bourgogne Europe, Dijon 21000, France</footnote>

<footnote><sup>b</sup> Institut Agro, Dijon 21000, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: izia.larrigaldie@u-bourgogne.fr (I. Larrigaldie).</footnote>

<footnote n="1">1 03.80.68.16.52</footnote>
Zambra et al., 2021) or olfaction functionality after experimental anosmization (Poindron et al., 1974). In general, adult sheep avoid food in presence of canid feces, even when starving (Pfister et al., 1990), and they display increased locomotion and agonistic behaviors when exposed to such odors (Zambra et al., 2021).

Most research has focused on adult sheep (Arnould et al., 1998; Wang et al., 2023; Zambra et al., 2021), typically in food choice tests (Arnould et al., 1998; Arnould and Signoret, 1993; Pfister et al., 1990; Wang et al., 2023). Studies assessing neonate or young ruminants' responses to non-food-related odors in non-food contexts remain limited. For example, calves exposed to coyote scent – compared to those exposed to ambient air – displayed heightened startle responses and sustained elevated heart rate (Adcock and Tucker, 2020), indicating that they react to predator odors before any exposure. Thus, across developmental stages, domestic species can detect and respond to odor cues from conspecifics, cues from conspecifics and both human and nonhuman heterospecifics. However, investigating further such odor-based responses requires to implement methodologies that avoid interference between familiar food-related odors and the target non-familiar odor cues (e.g., Larrigaldie et al., 2024).

Another constraint to consider when studying gregarious ungulates is the social context of the test setting. Shy ovines, in particular, benefit from social companions when isolated, as their stress being reduced when individuals can maintain visual, olfactory, and/or auditory contact with conspecifics (Da Costa et al., 2004; Guesdon et al., 2015; Rault, 2012). Similarly, social partners can alleviate stress induced by novel environments (R. H. Porter et al., 1995). The social context and individual social status (age, sex, reproductive stage) also influence sheep behavioral responses to aversive stimuli. For example, Hutson (1982) demonstrated that flight behaviors vary when adult sheep face a threat (sudden stimulus); their flight response depends on whether they are alone or in a group. Phenomenon confirmed by González et al. (2013), who demonstrated that sheep in groups display slower movements and fewer escape attempts relative to those alone or in smaller groups. Given such social influences on stress and threat perception, it is critical to assess how lambs respond to olfactory stimuli both individually and within their social group.

This study consists in two experiments applying odor stimuli known to be discriminable and to convey distinct emotional valence for adult sheep: ovine wool and canine feces. Experiment 1 relied on habituation-dishabituation processes to confirm adult ewes' olfactory discrimination of both substrates, and secondarily their ability to elicit differential emotional responses in the present conditions. The same paradigm was then applied to lambs, hypothesizing that adult responses might serve as a reference to interpret lambs' reactivity, keeping open the possibility of age-specific behavioral patterns. Based on earlier studies with adult sheep, we predicted that lambs would discriminate canine feces odor from ovine wool odor, and display more aversive behaviors toward the former than the latter.

Experiment 2 deployed an approach/withdrawal test to examine lambs' behavioral responses to both odor sources disposed within a group setting. Unlike Experiment 1, in which individuals were tested in isolation, this experiment presented odor stimuli within the lambs' familiar environment, minimizing physical/social disturbances and eliminating direct human handling. In these conditions, two predictions can be proposed: i) either lambs' repulsive behaviors to dog odor might be less pronounced in social group than in isolation, due to potential social modulation of fear responses; or ii) dog odor might elicit stronger repulsion in group condition.

# 2. Animals, materials, and methods

## 2.1. Ethics statement

The present study was approved by the Animal Experimentation Ethics Committee of the University of Burgundy (under authorization

#105). It took place from 03/03-24/04/2023 in a commercial farm located in northern Burgundy (Francheville, France). The study was run according to the farmer's management practices in breeding, lambing and artificial rearing of lambs. French farmers are strictly enforced to follow the Rural Code that regulates animal welfare (Code rural et de la pêche maritime, chapter IV. Articles L214-1 to L214-23). Thus, the experimenters were under the constant supervision of the farmer and his associates when they handled the animals during behavioral tests.

## 2.2. Animals and housing

Ewes (Suffolk breed; n = 150) were typically raised on pasture. With approaching parturition, pregnant ewes (not synchronized by hormonal treatment) were sheltered in a closed, naturally lit barn on straw-lined concrete floor. After delivery, ewes and their lambs were kept for 24 h in 2 × 1.5-m lambing pens to warrant colostrum intake. Following initial maternal interactions, all lambs underwent standard postnatal procedures (umbilical care, weighing, and ear-tagging). Some lambs were allocated to artificial rearing due to multiple births (i.e., more than two lambs per ewe). To mitigate the risk of insufficient milk yield in ewes bearing triplets or larger litters, surplus lambs were removed from their dams after receiving colostrum, housed in small groups in separate pens, and reared artificially. These lambs were closely monitored for health and growth throughout the rearing period. In accordance with the farm's breeding management protocol, a maximum of two lambs remained with each ewe, while the selection of surplus lambs for artificial rearing was based on ensuring body weight homogeneity among those left under maternal care. As births progressed, some lambs were selected based on the previously mentioned criteria, bringing the number of nursing lambs to 40 by the end of the lambing period. These artificially fed lambs were housed together in a 22-m² open-space nursery within the barn (Fig. 1a) until weaning. The nursery pen was equipped with a milk dispenser (EOO 32 heated mixer, Förster-Technik GmbH, Engen, Germany) providing the milk replacer ad libitum (Agnodor Tradition+, Bonilait, Chasseneuil-du-Poitou, France; 22 % proteins, 23 % lipids, 0.05 % cellulose, and 8.0 % cinders). The milk feeder automatically diluted the milk powder at 42 °C, to deliver milk to the lambs at 38 °C through three rubber nipples. Lambs also had unrestricted access to clay, as well as water and hay.

## 2.3. Behavioral tests

Two behavioral tests were designed. The habituation-dishabituation test aimed to assess the animals' discrimination of both odor substrates, and secondarily to seek some hint about their emotional reactions to them. The approach-withdrawal test aimed to measure whether the animals spontaneously reacted in terms of proximity to these substrates placed in their everyday environment. For both tests, the experimenters refrained from smoking or wearing any scented products prior to the tests. They wore a same standardized green overall over their clothes from the beginning to the end of the tests. For all manipulations, both experimenters also wore nitrile gloves (Kimtech, Purple Nitrile).

### 2.3.1. Test of habituation-dishabituation on adult ewes

Prior to the main experiment with lambs, the experimental setup aimed to assess the behavioral effectiveness of the selected odor stimuli, as well as the validity of the experimental design to induce habituation-dishabituation. The test was run on 10 postparturient Suffolk ewes (mean age ± standard deviation (SD) = 2 ± 1.4 years) isolated in their lambing pens, familiarized to experimenters' presence and odor-presentation stick for one standardized 3-min session. Two female experimenters conducted the study: Experimenter 1 preparing the odor sticks and operating the camera, while Experimenter 2 presented the odor sticks to the ewes. To maintain blinding, Experimenter 2 (who was consistent throughout the study) was unaware of the odor condition. During the testing itself, Experimenter 1 remained silent at about 3 m
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** The odor exposure setting for the Habituation-Dishabituation and approach tests: for the spatial design of the test pens set up for the two tests (a), with a focus on the Habituation-Dishabituation test pen and the presentation of the olfactive stimulus (b), with perforated barriers allowing audiovisual contact with conspecifics represented by dashed lines fort the two test pens. And a focus on the odor-dispensing stick (c) and the boxe (d).</figcaption></figure>

from the lambing pen. Between each ewe, the odor sticks were cleaned with water, but the sticks used for dog odor- vs. sheep odor-presentation were kept consistent throughout the study.

The ability of individual ewes to discriminate between dog odor (DO) and ovine odor (OO) was assessed through a sequential habituation-dishabituation (H-D) test (Aviles-Rosa et al., 2020; Coronas-Samano et al., 2016; Larrigaldie et al., 2024). During the habituation phase, ewes were exposed to an odorant (O1, habituation stimulus) across 3 30-sec trials (O1.1, O1.2, and O1.3) separated by 10-sec inter-trial intervals. Then, the presentation of a different odorant (O2, dishabituation or test stimulus) for 30 sec constituted the dishabituation phase. A decline in behavioral responsiveness was expected over the habituation trials, whereas an increase was anticipated upon exposure to the dishabituation stimulus (Aviles-Rosa et al., 2020; Coronas-Samano et al., 2016). The H-D test for each individual ewe lasted approximately 2.5 min. Based on prior research on sheep reactivity to predator feral odors (Poindron et al., 1974; Wang et al., 2023; Zambra et al., 2021), the order of odor presentation was not counterbalanced across subjects; specifically, ovine odor was always used for habituation, while dog odor was consistently presented for dishabituation.

### 2.3.2. Habituation-dishabituation test procedure for lambs

In this test, the same lambs participated in both the habituation and dishabituation phases and all individuals involved in this procedure were reared under the same conditions (artificial-rearing). Prior testing, all lambs were familiarized to the experimental conditions in being

individually introduced into the test pen and positioned onto the lap of Experimenter 1 (Fig. 1b), who gently stroked them for 7 min (Coulon et al., 2015; Serrapica et al., 2017); this procedure was repeated once daily for a total of five repetitions per animal. By the time of testing, each lamb had undergone familiarization to the experimenter's handling for a cumulative total of 35 min (following Coulon et al.'s (2015) procedure). Experimenters 1 and 2 performed the same tasks than in those in above pre-test with adult ewes (see 2.3.1).

The H-D test involved 28 Suffolk lambs (aged 6 ± 0 days; 14 females), which were tested individually, isolated from the group by slated barriers allowing visual, auditory and olfactory contact with peers. The test pen was set up in the animals' familiar enclosure in the breeding barn (Fig. 1b). Lambs exhibiting signs of excessive stress (frequent high-pitched bleating, jumping, or escape attempts) were removed from the study (n = 2). After this screening, 26 lambs (13 males, 13 females) were involved.

During the habituation phase, lambs were exposed to an odorant (O1) presented across 4 consecutive 30-sec trials (O1.1, O1.2, O1.3, and O1.4), with 10-sec inter-trial intervals. Then, following a 10-sec pause, a novel odorant (O2) was delivered for 30 sec for dishabituation. The odor presentation order was counterbalanced across lambs, while being constrained by existing agricultural practices regarding the assignment of lambs to the artificial rearing regime. Thus, the odor presentation order was counterbalanced with DO in habituation vs. OO in dishabituation for 13 lambs (9 females, 4 males) and OO in habituation vs. DO in dishabituation for 13 other lambs (5 females, 8 males). Since
lambs were not in the same condition as the ewes, which may have been
affected by lambing 24 h before the test (Sharafeldin et al., 1971), the
habituation phase could be extended. Thus, the procedure run for the
lambs was different from the one run for the adult ewes in both number
of repeated presentations for habituation and odor counterbalancing
between habituation/dishabituation. The 26 lambs were tested between
March 3 and April 16, 2023.

2.3.3. *Approach/withdrawal test*

Just after the H-D test, a group of 21 artificially-reared lambs (Suffolk
breed, mean age = 24.1 ± 2.9 days; among which 8 had participated in
the H-D test) were tested in their habitual pen (Fig. 1a) by the same
experimenters who run previous H-D tests. Therefore, no additional
familiarization to the setting was necessary here, except for two boxes
placed into the pen during the 5 days preceding the approach test. The
test comprised four unblinded sessions in which all individuals in the
group were followed by scan-sampling (Lehner, 1998). Observations
were conducted over two days in the morning (between 8:30 and 11:30
AM) and afternoon (between 1:30 and 4:30 PM), the first day aiming to
assess the effects of control condition, while the second day was to assess
the effects of odor condition. The majority of their locomotor activity
occurred during the daytime (Piccione, Giannetto, Casella, et al., 2010),
with intensity fluctuations throughout this period (Piccione, Giannetto,
Fazio, et al., 2010), this design allowed thus for the assessment of po-
tential time-of-day effects on responses to a familiar object, whether
scented or unscented.

The lambs were either exposed to two control boxes or to two scented
boxes (DO vs. OO). Both experimental boxes were placed at 5-m distance
within the pen, in separate areas (A1 and A2; Fig. 1a). Each observation
session involved 3 h of scan-sampling, during which behavioral items
were recorded every 5 min (resulting in 37 observational points per test,
from T0 to T180). The odorized boxes were designated as “DO box” for
the dog odorized box and as “OO box” for the ovine odorized box. The
positions of the boxes were alternated during the various observation
sessions (morning, afternoon) to test the potential spatial effect (A1 or
A2). The surface of the boxes was cleaned with water after each testing
session. The approach-withdrawal tests were run between April 16 and
24, 2023.

2.4. Odor stimuli

The odor stimuli consisted in dog feces, assumed representative of a
predator odor (negative valence; Arnould et al., 1998; Arnould and
Signoret, 1993; Pfister et al., 1990; Poindron et al., 1974; Zambra et al.,
2021), and sheep wool, representative of a non-predator odor (neutral or
positive valence; e.g., Baldwin and Meese, 1977; Vince and Ward, 1984;
Zambra et al., 2021). The two types of odorant substrates were stored at
-20°C for one week until testing. Canine feces were collected from a 9
year-old female dog (poodle/terrier crossbreed; spayed with uterus and
ovaries removed) fed an omnivorous diet (dry dog food; Royal Canin
Mini Adult 8 + containing 28% animal-based ingredients and 7.8%
plant-based ingredients). Ovine wool odor was sampled differently ac-
cording to the test (see below). Both the dog and the ewes providing the
odor stimuli were unfamiliar to the tested lambs. The odor stimuli were
presented using distinct methods in the habituation-dishabituation test
(Experiment 1) and the approach-withdrawal test (Experiment 2).

For the habituation-dishabituation test, for WO the odorant sub-
strates were presented as cotton pads (8 × 9.5 cm, Tetra Médical,
Annonay, France) rubbed either on the back of ewes (along the spine,
excluding rump) from another flock from the same farm (Romane, 6
year-old, not pregnant), allowing the collection of the odorous and
greasy substance known as suint, also referred to lanolin (Bonsma and
Starke, 1934; Cholewinska et al., 2020). For DO, the odorant substrates
were also presented as cotton pads rubbed on the dog feces. The odor-
ized pads were then folded inward for visual uniformity and affixed with
scentless tape (Micropore, 3 M, St Paul, USA) to the end of a 42,5 cm

plastic stick (Fig. 1c).

For the approach-withdrawal test, plain odorous substrates were
used. To increase the intensity of odors, multiple dog fecal samples were
pooled and a batch of wool from multiple ewes (Romane breed) was
collected during a shearing session. These samples were placed into
white, opaque plastic boxes (60 × 25 x 35.5 cm, Fig. 1d) locally used as
enrichment objects for artificially-reared lambs. The boxes were perfor-
rated by multiple holes (diameter: 0.7 mm) to release the odors of either
85 g of dog feces or 120 g of sheep wool (minimally required quantities
based on the experimenters’ nose). Considering their relatively stable
odor intensity over 8 h, the odor substrates in the boxes remained un-
changed throughout a day of test.

2.5. *Dependent variables*

2.5.1. *Habituation-dishabituation test*

The behavior of lambs was recorded on video (GoPro HERO8 Black) for subsequent frame-by-frame analysis on a computer using the BORIS software (version 8.13, UniTo; 2016). Video analysis was run by a single coder who was blind to the odor conditions. Several behavioral categories were coded as defined in Table 1. These included the lamb's attraction to the odor stick, which was coded based on investigative behaviors such as visuo-olfactory exploration, muzzle contact, and oral grasping. Additionally, behavioral indicators of emotional states were quantified in response to the odor stimulus, including negative reactions such as bleating, jolts, escapes, micturition and defecation. Based on previous studies (Boissy et al., 2011; Guesgen et al., 2016; Reefmann et al., 2009; Tamioso et al., 2017) ear positions were further quantified as a proxy to the lambs' emotional dispositions (for detailed definitions, see Table 2). All above behavioral items were decoded as both occurrence and duration across the five 30-second odor presentation trials (four habituation trials followed by one dishabituation trial).

2.5.2. *Approach-withdrawal test*

Six items of lamb's behavior (defined in Table 3) were recorded using
real-time scan-sampling with the aim to evaluate the degree of their (dis)
interest/attraction/aversion towards the stimuli (DO or OO) emitted
from the boxes. Two experimenters, each assigned to a specific box and
positioned within their designated area (Fig.c), recorded the number of
lambs performing one or more ethogram items. Thus, attraction/with-
drawal was assessed in terms of spatial proximity to a target box
(proximal: 0-50 cm; distal: > 50 cm), and in terms of visuo-olfacto-
tactile included five items: "Lying around box", "Standing around
box", "Standing on box", "Sniffing box" and "Taping head and/or foot
against box". In a second step these behavioral items were grouped into
two categories: interest/attraction (including "Lying around box",
"Standing around box", "Standing on box", "Sniffing box" and "Taping
head and/or foot against box") and disinterest/aversion (indicating
behavior outside the designated area) relative to the target odorized
area. This allowed us to obtain the total number of lambs showing in-
terest or not in the box, at each recorded time point during the scan
sampling.

2.6. *Data analyses*

2.6.1. *Habituation-dishabituation test*

The random assignment of lambs to odor presentation order (DO-OO
or OO-DO) resulted in an unequal distribution of males and females
across conditions, preventing us to consider sex as a factor. As previously
described, the H-D test consisted of two phases: 1/ *habituation*, which
involved presenting the same stimulus four times, ranging from trial
O1.1 (first habituation trial) to trial O1.4 (fourth habituation trial), and
2/ *dishabituation*, represented by trial O2. The same procedure was
applied to the ewes, except that there were only three presentations
during the habituation phase (O1.1 to O1.3). Following Arbuckle et al.
(2015) and Yang and Crawley (2009), these two phases were analyzed
<table><caption>**Table 1**

Definitions of the behavioral items coded during the Habituation-Dishabituation test, with their assumed underlying emotional categories and related references.</caption><thead><tr><th>Pattern of actions</th><th>Definition</th><th>Variables (unit)</th><th>Emotional categories</th><th>References</th></tr></thead><tbody><tr><td>Vocalization</td><td>High-pitched bleating with open mouth.</td><td>Occurrence</td><td>Negative stress, opposition</td><td>(Da Costa et al., 2004; Greiveldinger et al., 2007; Guesdon et al., 2015)</td></tr><tr><td>Micturition</td><td>Visible micturition and associated typical posture</td><td>Occurrence</td><td>High frequency: anxious state</td><td>(Monk et al., 2018)</td></tr><tr><td>Defecation</td><td>Visible defecation</td><td>Occurrence</td><td>High frequency: anxious state</td><td>Monk et al., (2018)</td></tr><tr><td>Jolting</td><td>Visible transient contractions of shoulder and/or of posterior occurring with bending of legs or moving legs away from each other.</td><td>Occurrence</td><td>Fear/negative stress in response to suddenness</td><td>Greiveldinger et al., (2007)</td></tr><tr><td>Attempted escape</td><td>The animal tries to free itself from the experimenter's embrace, wiggling and struggling.</td><td>Occurrence</td><td>Fear/negative stress</td><td>(Bouissou et al., 1996; Romeyer and Bouissou, 1992)</td></tr><tr><td>Turning to experimenter</td><td>Whole head turned 90° towards the experimenter, nostrils facing experimenter's chest.</td><td>Occurrence, duration (s)</td><td>Stimulus disinterest</td><td>Viérin and Bouissou, (2002)</td></tr><tr><td>Sniffing experimenter</td><td>Animal turns nostrils towards workwear of experimenter and sniffs it.</td><td>Occurrence, duration (s)</td><td></td><td></td></tr><tr><td>Chewing experimenter's workwear</td><td>Animal turns head to workwear of experimenter and chews it.</td><td>Occurrence, duration (s)</td><td></td><td></td></tr><tr><td>Sniffing the stick</td><td>Head and ears facing stick, nose &lt; 5 cm from stick</td><td>Occurrence, duration (s)</td><td>High proportion: interest, preference</td><td>Alary et al., (2023)</td></tr><tr><td>Contacting stick</td><td>Animal touches the stick with muzzle or head.</td><td>Occurrence, duration (s)</td><td>Low proportion: disinterest, avoidance</td><td></td></tr><tr><td>Oral grasp of stick</td><td>stick in mouth, with obvious chewing motions.</td><td>Occurrence, duration (s)</td><td></td><td></td></tr><tr><td>Sniffing pad</td><td>Head and ears facing the pad, nose less than 5 cm from pad</td><td>Occurrence, duration (s)</td><td></td><td></td></tr><tr><td>Contact with pad</td><td>Animal touches pad with muzzle or head.</td><td>Occurrence, duration (s)</td><td></td><td></td></tr><tr><td>Oral grasp of pad</td><td>pad in mouth, with obvious chewing motions.</td><td>Occurrence, duration (s)</td><td></td><td></td></tr></tbody></table>

separately. Specifically, i) data from trial O1.1 were compared to those of trial O1.4 (or O1.3, for ewes) to assess whether repeated exposure to the stimulus resulted in a decrease in responsiveness; indicating a loss of interest, and ii) data from trials O1.4 (or O1.3, for ewes) were compared to those from trial O2 to evaluate a potential rebound of interest in response to the novel stimulus, which indicates the ability to discriminate between the habituated and novel stimuli.

Two statistical outliers across all variables were identified using the Median Absolute Deviation (Leys et al., 2013), i.e., mean score > 3 MAD) and were dropped from subsequent analyses. Therefore, the final statistical analysis of the H-D test included 14 females and 12 males. Both the occurrences and durations of behavioral observations (i.e., behavior items and ear positions) were analyzed using a mixed model with restricted maximum likelihood estimation (REML), incorporating animals as random factor, and stimulus order in the H-D test (DO-OO or OO-PO), and odors (DO and OO) as fixed factor for the analyses of the habituation and dishabituation responses. Bonferroni corrections were used for multiple comparisons. Some variables showed deviations from normality in model residuals. We nevertheless used linear mixed model to analyze the data as recommended by Knief and Forstmeier (2021), as 1) linear mixed model are fairly robust to non-normality, 2) deviations from normality usually do not bias regression coefficients, 3) and non-normality of residuals does not impair hypothesis testing (type I error rate is kept at the desired low rate). For the test on adult ewes, as none of the variables followed a normal distribution and the test included only one order of presentation (OO followed by DO), the presentations (O1.1 vs. O1.3 and O1.3 vs. O2.1) were compared using Friedman tests. All analyses were made using Jamovi (α = 0.05, The Jamovi project, 2023, version 2.4; R core team 2022; Gallucci, 2019).

### 2.6.2. Approach-withdrawal test

Since, in this testing situation, the effects of the control and odor conditions were measured on different days, the data were analyzed separately. For the control condition, we investigated the effects of box positioning, session timing (am vs. pm), and time progression on lamb exploration behavior applying a Quasi-Poisson model (to address overdispersion in count data).

Model diagnostics confirmed the appropriateness of the quasi-Poisson model, with no violation of key assumptions, including the absence of excessive overdispersion (φ = 1.078). A Quasi-Poisson model was applied likewise to investigate the effects of odor condition, session timing, and time progression on lamb exploration behavior for the odor condition. The model diagnostics confirmed that the Quasi-Poisson model was appropriate, with no violations of key assumptions (dispersion = 0.795). All these analyses were run with R Core Team (2022).

## 3. Results

All results are presented as follows: mean ± standard deviation (SD).

### 3.1. Olfactory habituation-dishabituation test on ewes

**Habituation** – Ewes sniffed the pad conveying the ovine wool odor significantly longer during the first than during the third presentation (O1.1: 7.48 ± 8.29 sec vs. O1.3: 2.02 ± 2.95 sec, $\chi^2(1) = 10$, p = 0.0016). Similarly, they touched the odor pad with their muzzle longer during the first presentation compared to the third one (O1.1: 3.84 ± 6.46 sec vs. O1.3: 0.76 ± 1.55 sec, $\chi^2(1) = 4.5$, p = 0.023). Stimulus repetition did not affect the duration of contact with the stick it-self (O1.1: 0.32 ± 1.02 sec vs. O1.3: 0.00 ± 0.00 sec, $\chi^2(1) = 1$, p = 0.317).

Additionally, ewes held their ears horizontal longer at the end than at the beginning of the habituation phase (O1.1: 0.91 ± 1.37 sec vs. O1.3: 5.36 ± 5.35 sec, $\chi^2(1) = 5.44$, p = 0.019). None of the other measured behaviors, including "contact sniffing stick", "oral grasping stick" and "oral grasping pad", or the other ears' positions showed significant habituation.

**Dishabituation** – Following repeated exposure to ovine wool odor, ewes showed significant dishabituation in response to the novel dog feces odor, as indicated by increased duration of sniffing (O2: 5.33
<table><caption>**Table 2**
Positions of ear pinnae in sheep: operational definitions in relation with assumed emotional states (and associated references).</caption><thead><tr><th>Ear position</th><th>Definitions</th><th>Variables (units)</th><th>Emotional categories</th><th>References</th></tr></thead><tbody><tr><td>Forward/up<br>front of head<br>back of head</td><td>Ear pointing forward at an angle > 30° from the perpendicular.</td><td>Occurrence, duration (s)</td><td>Negative response such as irritation</td><td>Boissy et al., (2011)<br>Reefmann et al., (2009)</td></tr><tr><td>Horizontal/axial ears<br>front of head<br>back of head</td><td>Ears perpendicular to croup axis.</td><td>Occurrence, duration (s)</td><td>Positive emotion or neutral state</td><td>Tamioso et al. (2017)<br>Boissy et al. (2011)</td></tr><tr><td>Backwards<br>front of head<br>back of head</td><td>Ear pointing backwards at an angle > 30° from perpendicular.</td><td>Occurrence, duration (s)</td><td>Negative state such as fear or pain</td><td>Boissy et al., (2011)<br>Guesgen et al., (2016)</td></tr><tr><td>Asymmetric<br>front of head<br>back of head</td><td>Right and left ear in different position.</td><td>Occurrence, duration (s)</td><td>Negative state</td><td>Reefmann, 2009</td></tr><tr><td>Changing position</td><td>Number of times individuals change ear position</td><td>Occurrence</td><td>High frequency: negative state<br>Low frequency: positive state</td><td>Reefmann, 2009<br>Tamioso et al., (2017)<br>Guesgen et al., (2016)</td></tr></tbody></table>

± 5.35 sec vs. O1.3: 2.02 ± 2.95 sec, $\chi^2$ (1) = 5.44, p = 0.019) and touching pad (O2: 2.28 ± 2.36 sec vs. O1.3: 0.76 ± 1.55 sec, $\chi^2$ (1) = 8, p = 0.004). Additionally, they held their ears in horizontal position more frequently at the end of habituation phase compared to the new odor presentation (O1.3: 1.20 ± 0.91 times vs. O2.1: 0.60 ± 0.69 times, $\chi^2$ (1) = 5, p = 0.025). None of the other variables, i.e. duration/frequency of "sniffing stick," "oral grasping stick," "touching pad" or "oral grasping pad", or ear positioning variables was significantly altered at dishabituation.

In sum, adult ewes displayed signs of dishabituation with the canine odor after being successfully habituated to the sheep wool odor, displaying an increasing sign of neutral/positive emotional state toward this latter odor.

## 3.2. Habituation-dishabituation test on lambs

**Habituation** – First, as expected, the lambs sniffed at the odor pads significantly more during trial 1 than during trial 4, in terms of both duration (O1.1: 6.63 ± 4.71 sec vs. O1.4: 2.27 ± 2.14 sec, $F_{(1,24.4)}$ = 21.45, p < 0.001; Table 4) and frequency (O1.1: 2.35 ± 1.41 times vs. O1.4: 1.19 ± 0.80 times, $F_{(1,48)}$ = 12.97, p < 0.001; Table 4). Secondly, the duration of contact with pads decreased between trials 1 and 4 (O1.1: 2.77 ± 3.49 sec vs. O1.4: 0.55 ± 1.49 sec, $F_{(1,48)}$ = 8.53, p = 0.005; Table 4), as did the frequency of contact occurrences with them (O1.1: 1.54 ± 1.39 times vs. O1.4: 0.57 ± 1.17 times, $F_{(1,25.1)}$ = 7.36, p = 0.011; Table 4).

The presentation stick was also sniffed less during trial 4 than trial 1 (in duration: O1.1: 2.83 ± 2.36 sec vs. O1.4: 1.34 ± 2.99 sec, $F_{(1,25)}$ = 4.9, p = 0.035; in frequency: O1.1: 2 ± 1.33 times vs. O1.4: 0.80 ± 1.50 times, $F_{(1,25.2)}$ = 14.52, p < .001; Table 4). Beyond mere sniffing, additional behavioral items were subject to habituation. Thus, the lambs' ear pinnae positions varied along the four trials, with significantly longer 'ears forward' postures (indicative of a negative state; Table 2) during trial 1 than during trial 4 (23 ± 5.32 sec vs. 20.2 ± 8.50 sec, $F_{(1,25)}$ = 4.74, p = 0.035). Without any effect of the odor being observed (DO: 21.19 ± 7.20 sec vs. OO: 22.04 ± 7.23 sec, $F_{(1,37.5)}$ = 0.49, p = 0.489).

More ever, a general odor effect to the testing materials and experimenters was noted (see Table 4). Indeed, over the whole habituation phase, the sniffing response differed by habituating odor: DO pads were sniffed at longer than OO pads regardless of exposure trial (DO: 5.56 ± 4.54 sec vs. OO: 3.34 ± 3.68 sec, $F_{(1,28.6)}$ = 5.30, p = 0.028), and this contrast in olfactory interest was specially noted from trial 1 (O1.1) as DO induced longer sniffing relative to OO (DO: 8.61 ± 3.50 sec vs. OO: 4.64 ± 3.51 sec, $F_{(1,24.4)}$ = 5.30, p = 0.031; see Fig. 2a). Further, the rate of olfactory habituation between trials 1 and 4 differed between DO and OO: while DO pads induced a significant decrease in both sniffing duration (O1.1: 8.61 ± 1.16 sec vs. O1.4: 2.50 ± 1.45 sec, $F_{(1,24.4)}$ = 21.45, p < 0.001) and frequency (O1.1: 2.61 ± 1.12 times vs O1.4: 1.07 ± 0.42 times, $F_{(1,48)}$ = 12.97, p = 0.030), a non-significant change in the same direction occurred for OO pads in terms of either sniffing
<table><caption>**Table 3**
Definitions of the behavioral items coded during the Approach-withdrawal test, with their assumed underlying emotional categories and related references.</caption><thead><tr><th>Pattern of actions</th><th>Definition</th><th>Variables</th><th>Emotional categories</th><th>References</th></tr></thead><tbody><tr><td>Lying around box</td><td>The animal is lying on the ground within a 50 cm radius of the box.</td><td>Number of individuals</td><td>Low fear level, animal relaxed.</td><td>(Bœ et al., 2006; Viérin and Bouissou, 2002)</td></tr><tr><td>Standing around box</td><td>The animal stands within a 50 cm radius of the box.</td><td>Number of individuals</td><td>Low fear level, positive emotional state.</td><td>Viérin and Bouissou, (2002)</td></tr><tr><td>Standing on box</td><td>The animal places at least two or more feet on the box, standing on it.</td><td>Number of individuals</td><td>Low fear level, exploration, positive emotional state.</td><td>Viérin and Bouissou, (2002)</td></tr><tr><td>Sniffing box</td><td>The animal directs its head toward the box, with its nose 5-10 cm from the box and/or one of its holes.</td><td>Number of individuals</td><td>Curiosity, positive or negative emotional state</td><td>(Vandenheede et al., 1998)</td></tr><tr><td>Taping head and/or foot against box</td><td>The animal intentionally makes contact with the box by performing back-and-forth horizontal movements with its head and/or vertical movements with its paws.</td><td>Number of individuals</td><td>Behavior associated with play.</td><td>(Chapagain et al., 2014)</td></tr><tr><td>Outside the box area</td><td>The animal is outside the box area, at a distance greater than 50 cm.</td><td>Number of individuals</td><td>Avoidance, disinterest.</td><td></td></tr></tbody></table>

duration (O1.1: 4.64 ± 2.28 sec vs. O1.4: 2.03 ± 2.06 sec, F(1,24.4) = 3.45, p = 0.365) or frequency (O1.1: 2.21 ± 1.53 times vs. O1.4: 1.31 ± 0.94 times, F(1,48) = 0.63, p = 0.325). Also, lambs displayed an unexpected behavior differentiated by odor, which was not seen in adult ewes: those protruded their tongue more frequently (OO: 1.22 ± 1.15 times vs. DO: 0.360 ± 0.76 times, F(1,33.3) = 8.43, p = 0.006, Fig. 2b) and longer toward OO pads than toward DO pads (OO: 1.94 ± 1.91 sec vs DO: 0.64 ± 1.86 sec, F(1,30.1) = 5.57, p = 0.024). This behavior was characterized by an active extension of the tongue out of the oral cavity, with an anteroposterior and sometimes bilateral movement (Fig.A in supplementary materiel). Tongue protrusion could occur either before

or after any olfacto-tactile interaction with the pads, but never during. During this behavior, the tongue made contact with the upper lip without elevation of the latter or the nostril region (Fig.A in supplementary materiel). The extension of the tongue outside the oral cavity was not specifically associated with particular head movements.

Lastly, muzzle orientation toward the experimenter during habituation was affected by the odor: when facing OO, lambs increasingly turned towards the experimenter between trials 1 and 4 (O1.1: 0.38 ± 0.53 vs. O1.4: 0.69 ± 0.75 times; F(1,27.1) = 4.61, p = 0.040), though no such effect was noted for DO (O1.1: 0.50 ± 0.67 times vs. O1.4: 0.65 ± 1.50 times).

Finally, it may be noted that no significant effect of the odor stimulus (DO or OO) were noted for any of the other behavioral categories analyzed along the habituation procedure, especially those indexing aversion.

**Dishabituation** – Regardless of the dishabituating odor or the odor to which they had been habituated, the lambs exhibited a significant increase in olfactory interest when exposed to the novel odor: thus, sniffing duration increased between the last habituation trial and the dishabituation trial (O1.4: 2.24 ± 2.14 sec vs. O2: 5.57 ± 4.93 sec; F(1,48) = 9.62, p = 0.003; Table 5; Fig. 3a). Similarly, the frequency of sniffing bouts increased toward the novel stimulus in comparison to the habituated stimulus (O1.4: 1.19 ± 0.80 times vs. O2: 1.96 ± 1.68 times; F(1,48) = 5.01, p = 0.034; Table 5), and the lambs longer contacted with their muzzles the pad bearing the novel odor (O1.4: 0.55 ± 1.49 sec vs. O2: 2.09 ± 3.35 sec; F(1,48) = 4.35, p = 0.042; Table 5; Fig. 3b).

More ever, an odor effect to the pads was also noted. Indeed, regarding tongue extension outside the oral cavity, a significant global interaction effect between the presentation and the nature of the odor was observed (F(1,48) = 4.75; p = 0.034). The lambs habituated to DO stuck out their tongue with licking-like motions more frequently when dishabituated with OO (O1.4: 0.23 ± 0.59 times vs. O2: 0.76 ± 1.24 times). Contrastedly, lambs habituated to OO protruded their tongue less frequently when dishabituated with DO (O1.4: 1 ± 1.08 times vs. O2: 0.38 ± 0.76 times).

To sum up, 6-day-old lambs appeared to somehow habituate in four trials to an unfamiliar setting composed of unknown experimenters and novel devices, among which pads impregnated with OO or DO. They showed more olfactory interest (sniffing) to the odor pads at trial one than at trial four of the habituation sequence, investigating longer DO pads than OO pads. Lastly, the lambs' habituation rate was clearer to DO than to OO, with an attenuation of negative affective state when facing DO, as reflected in ear posture. The dishabituation trials showed clearly that the lambs discriminated OO from DO, with a significant rebound in sniffing between the habituated and the novel stimulus regardless of the latter's nature. For the tongue extension behavior, the dishabituation response depended, however, on the habituation odor.

<table><caption>**Table 4**
Descriptive (means ± SD) and statistical main results of lambs in the habituation phase of the habituation-dishabituation test.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Odor presentation</th><th rowspan="2">Odor presentation p-values</th><th rowspan="2">Odor p-values</th><th rowspan="2">Presentation*Odor p-values</th></tr><tr><th>O1.1</th><th>O1.4</th></tr></thead><tbody><tr><td>D_Sniffing pad</td><td>6.63 ± 4.71</td><td>2.27 ± 2.14</td><td><strong>.0001</strong><sup>***</sup></td><td><strong>.028*</strong></td><td>.075</td></tr><tr><td>D_Contact with pad</td><td>2.77 ± 3.49</td><td>0.55 ± 1.49</td><td><strong>.005</strong><sup>**</sup></td><td>.818</td><td>.607</td></tr><tr><td>D_Oral grasp of pad</td><td>0.54 ± 1.32</td><td>1.07 ± 3.95</td><td>.451</td><td>.067</td><td>.450</td></tr><tr><td>D_Sniffing the stick</td><td>2.83 ± 2.36</td><td>1.34 ± 2.99</td><td><strong>.035*</strong></td><td>.637</td><td>.187</td></tr><tr><td>D_Contacting the stick</td><td>0.93 ± 1.23</td><td>0.52 ± 1.49</td><td>.240</td><td>.783</td><td>.290</td></tr><tr><td>D_Tongue Extension</td><td>1.56 ± 2.26</td><td>1.02 ± 1.66</td><td>.274</td><td><strong>.025*</strong></td><td>.978</td></tr><tr><td>O_Sniffing pad</td><td>2.35 ± 1.41</td><td>1.19 ± 0.80</td><td><strong>.0007</strong><sup>***</sup></td><td>.932</td><td>.428</td></tr><tr><td>O_Contact with pad</td><td>1.54 ± 1.39</td><td>0.57 ± 1.17</td><td><strong>.012*</strong></td><td>.706</td><td>.847</td></tr><tr><td>O_Oral grasp of pad</td><td>0.23 ± 0.51</td><td>0.11 ± 0.32</td><td>.379</td><td><strong>.004</strong><sup>**</sup></td><td>.379</td></tr><tr><td>O_Sniffing the stick</td><td>2.00 ± 1.33</td><td>0.80 ± 1.50</td><td><strong>.0008</strong><sup>***</sup></td><td>.647</td><td>.329</td></tr><tr><td>O_Contacting the stick</td><td>0.92 ± 1.26</td><td>0.38 ± 0.89</td><td><strong>.048*</strong></td><td>.765</td><td>.280</td></tr><tr><td>O_Tongue Extension</td><td>1.00 ± 1.16</td><td>0.61 ± 0.94</td><td>.174</td><td><strong>.006</strong><sup>**</sup></td><td>.704</td></tr></tbody></table>

D: durations; O: number of occurrences; * p < .05, ** p < .01, *** p < .001; O1.1 and O1.4 refer to the 1st and 4th trials in the habituation procedure
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Mean duration of pad sniffing during the first presentation phase (O1.1) in lambs as a function of the presented stimulus (a), and average occurrence of tongue protrusion during the habituation phase in lambs as a function of the presented stimulus (b). O1.1 and O1.4: first and fourth presentation of habituation odor, respectively. Boxplots show the median, first, and third quartiles with error bars which represent standard errors. The average is represented by the red bridges, with error bars which represent standard errors, the other colored dots represent individuals; REML*: p < 0.05 and **: p < 0.01.</figcaption></figure>

<caption>**Table 5**</caption>

Descriptive (means ± SD) and statistical main results of lambs in the dishabituation phase of the habituation-dishabituation test.

<table><thead><tr><th rowspan="2"></th><th colspan="2">Odor presentation</th><th rowspan="2">Odor presentation p-values</th><th rowspan="2">Odor p-values</th><th rowspan="2">Presentation*Odor p-values</th></tr><tr><th>O1.4</th><th>O2</th></tr></thead><tbody><tr><td>D_Sniffing pad</td><td>2.27 ± 2.14</td><td>5.57 ± 4.93</td><td>.003**</td><td>.350</td><td>.622</td></tr><tr><td>D_Contact with pad</td><td>0.55 ± 1.49</td><td>2.09 ± 3.35</td><td>.042*</td><td>.691</td><td>.919</td></tr><tr><td>D_Oral grasp of pad</td><td>1.07 ± 3.95</td><td>0.93 ± 2.64</td><td>.886</td><td>.286</td><td>.226</td></tr><tr><td>D_Sniffing the stick</td><td>1.34 ± 2.99</td><td>1.34 ± 4.43</td><td>.523</td><td>.423</td><td>.678</td></tr><tr><td>D_Contacting the stick</td><td>0.52 ± 1.49</td><td>0.78 ± 2.55</td><td>.638</td><td>.364</td><td>.697</td></tr><tr><td>D_Tongue Extension</td><td>1.02 ± 1.66</td><td>0.64 ± 1.17</td><td>.307</td><td>.055</td><td>.172</td></tr><tr><td>O_Sniffing pad</td><td>1.19 ± 0.80</td><td>1.96 ± 1.68</td><td>.034*</td><td>.659</td><td>.799</td></tr><tr><td>O_Contact with pad</td><td>0.57 ± 1.17</td><td>1.34 ± 1.99</td><td>.078</td><td>.367</td><td>.210</td></tr><tr><td>O_Oral grasp of pad</td><td>0.11 ± 0.32</td><td>0.27 ± 0.60</td><td>.259</td><td>.259</td><td>.569</td></tr><tr><td>O_Sniffing the stick</td><td>0.81 ± 1.50</td><td>1.00 ± 0.89</td><td>.576</td><td>.911</td><td>.742</td></tr><tr><td>O_Contacting the stick</td><td>0.38 ± 0.90</td><td>0.58 ± 0.94</td><td>.464</td><td>.660</td><td>.883</td></tr><tr><td>O_Tongue Extension</td><td>0.61 ± 0.94</td><td>0.94 ± 1.27</td><td>.885</td><td>.470</td><td>.034</td></tr></tbody></table>

D: durations; O: number of occurrences; * p < .05, ** p < .01; O1.4 and O2 refer to the 4st and 5th trials in the habituation-dishabituation procedure

### 3.3. *Approach-withdrawal test*

#### 3.3.1. *Control condition*

Models results indicate that lambs exhibited a slight preference for the boxes placed in area A1 over those placed in area A2, with an

incidence rate ratio (IRR) of 0.5 meaning that they were approximately 0.5 times less likely to explore the boxes when presented in area A2 than when presented in area A1 (t = -2.20; p = 0.029).

The session timing (morning vs. afternoon) also influenced significantly the lambs' exploration rates of the boxes (t = -3.63, p < 0.001).
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Mean duration of pad sniffing during the dishabituation phase (4 30-sec trials) in lambs whatever the stimulus (a), and average duration of pad-contact during the habituation phase in lambs regardless of the stimulus (b). O1.4 and O2: fourth presentation of habituation odor and first presentation of dishabituation odor, respectively. Boxplots show the median, first, and third quartiles with error bars which represent standard errors. The average is represented by the red bridges, and the other colored dots represent individuals; REML *: p < 0.05 and **: p < 0.01.</figcaption></figure>

The number of lambs interacting with them decreased indeed in the afternoon sessions compared to the morning sessions (Fig. 4a). Further, the boxes' positions (A1 vs. A2) and session timing (morning vs. afternoon.) did not interact (p = 0.482), indicating that the effect of spatial position of the boxes is similar during both session timings. However, the lambs' exploration of the boxes decreased significantly in the afternoon session (IRR = 0.48, p < 0.001, Fig. 4a). Moreover, a quadratic time effect was noted in the afternoon (IRR = 1.53, p = 0.010), suggesting that exploration was initially high before declining at a faster rate later in the session (Fig. 4a). In contrast, the morning session did not show such a significant quadratic trend (p = 0.41), implying a more stable exploration pattern in mornings (Fig. 4a).

### 3.3.2. Odor condition

Models results indicate that lambs exhibited a higher proportion of interactions for the OO box over the DO box, with an IRR of 10, meaning that they were approximately 10 times more likely to explore OO box than DO box (t = 6.51; p < 0.001). The session timing (morning vs. afternoon) alone did not significantly influence the lambs' exploration rates of either boxes (p = 0.512), suggesting that they did not explore

one or the other box more during one session over the other. Much like in the control conditions, the lambs' exploration decreased significantly in the afternoon sessions (IRR = 0.78, p = 0.018, Fig. 4b). Finally, a quadratic time effect emerged also in the afternoon sessions (IRR = 1.53, p < 0.001), suggesting that lambs' exploration first increased before declining at a faster rate later during the session (Fig. 4b). In contrast, the morning sessions did not show a significant quadratic trend of box exploration (p = 0.179), implying a stable exploration pattern (Fig. 4b).

These findings highlight a notable influence of odor cues on lambs' spatial behavior within their familiar pen, while also suggesting that their exploration drive is dynamic and changes nonlinearly over time, particularly in the afternoon sessions.

## 4. Discussion

This study examined whether neonate lambs can discriminate between the odor of an unfamiliar dog (DO) and that of an unfamiliar wool conspecific (WO). First, the discrimination of DO and WO by lambs was assessed using two processes measurable through the habituation-dishabituation (HD) paradigm. Second, the influence of these odors on
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Predicted number of lambs exploring the boxes every 5 min over the 3-hour survey, based on condition and session, using a Quasi-Poisson model in the control (a) and odor (b) conditions. The solid and dashed lines represent the model's predicted values for each session: solid lines correspond to the morning session (A.M.), and dashed lines to the afternoon session (P.M.). The turquoise line represents the measurements for area 1 (A1), while the light pink line corresponds to area 2 (A2) for the control condition. Red indicates the measurements for the dog-odorized box (DO) and blue for the ovine-odorized box (OO) in the odor condition. The parameter φ corresponds to the dispersion parameter, indicating a good model fit when close to 1. The shaded areas around each line depict the 95 % confidence intervals.</figcaption></figure>

the lambs' flock reaction was examined in the approach-withdrawal test. By 6 days of age, artificially reared lambs were able to differentiate the odor of the feces from an unfamiliar dog from the odor of wool from unfamiliar sheep. While we expected lambs to exhibit aversive responses (e.g., escape attempts, freezing, or characteristic ear positions) to dog versus sheep odors, this was not the case under the present conditions: dog feces did not clearly elicit negatively valanced responses. We have only observed that lambs tested in groups exhibited some avoidance behavior toward the predator odor.

Following and our predictions and the results of earlier studies (Arnould et al., 1998; Arnould and Signoret, 1993; Pfister et al., 1990;

Poindron et al., 1974; Wang et al., 2023; Zambra et al., 2021), adult ewes displayed habituation in terms of a decreasing olfactory interest (actualized in sniffing) toward repeatedly presented odor pads, and then dishabituation upon delivery of a novel odor (which, in the present experiment, was always dog fecal odor), as revealed by an increased sniffing. These findings confirm that adult ewes do detect and discriminate the odors of ovine wool and of canine feces, both stemming from unfamiliar donors, supporting the validity of the present experimental protocol to assess *olfactory discrimination* in adult ewes. On the side of responses reflecting the animals' affective responses, the outcome of the habituating-dishabituating test was more discrete. Thus, the growing
olfactory disinterest toward the repeated odor stimulus during habituation went with an increasingly frequent horizontal posture of ears, a proxy of neutral-to-positive emotional state (Tamioso et al., 2017; Boissy et al., 2011). However, after successful habituation to OO, the presentation of the novel DO did not induce any significant change in the other emotional items assumed to provide a metric of alertness or aversion. Therefore, at least in the present conditions of the H-D procedure, this phase of the test appears less sensitive to assess whether odors convey differential emotional content to adult ewes.

Six-day-old lambs also displayed habituation to conspecific wool odor by altering their responses toward them over four exposure repetitions. They also habituated to a radically different kind of smell emitted from canine fecal odor. Both the duration/frequency of sniffing/ touching the pads/sticks decreased, suggesting reduction of attention/ interest to the odor stimuli and their support. Thus, lambs do habituate to the repetition of very different unfamiliar odor qualities. During the habituation procedure, lambs also displayed emotion-related responses beyond mere exploration. Specifically, they spent less and less time with their ears oriented forward when exposed to the odor stimuli, an indication of progressive reduction of negative affective state (Boissy et al., 2011; Reefmann et al., 2009) to the novelty of the setting and target stimuli. However, the nature of the odor stimulus is not without effect on the dynamics and form of their responses during habituation. Indeed, lambs exposed to DO sniffed the compress for a longer duration than those exposed to OO, and the overall habituation effect observed was primarily driven by the habituation of lambs exposed to DO. This is evidenced by the fact that the decrease in both the duration and frequency of olfactory exploration in lambs exposed to OO was not significant. Lambs may also have perceived the conspecific nature of WO, which could have induced a buffering effect, thereby reducing their behavioral responses (Carbajal and Orihuela, 2001; Da Costa et al., 2004; Guesdon et al., 2015; R. H. Porter et al., 1995). Another response item of the lambs was unexpected when referring the ewes' repertoire: they differentiated OO from DO during the habituation phase in that they protruded their tongue longer and more frequently with licking-like motions to the ovine wool odor relative to the canine fecal odor. The fact that these licking-like actions were more intensive toward the pads conveying OO than toward those conveying DO may be interpreted as reflecting greater chemosensory interest or appetence to conspecific wool odor than to canine feces odor. Indeed, given that artificially reared lambs can exhibit consummatory behaviors toward inappropriate objects (Napolitano et al., 2006), they may have suppressed their consummatory response toward the DO pad due to perceiving it as aversive. The fact that lambs may have perceived the conspecific nature of WO, as well as its origin from adult individuals, could have also triggered behaviors from the mother-young bonding repertoire. For instance, they may have exhibited nipple-seeking behavior, such as tongue extension beyond the oral cavity. That being said, lambs appear to discriminate both stimuli already from their first presentations during the habituation phase, with signs of more positive appetence to OO than to DO. Results consistent with previous research in adult sheep (Arnould et al., 1998; Arnould and Signoret, 1993; Pfister et al., 1990; Poindron et al., 1974; Wang et al., 2023; Zambra et al., 2021).

The significant rebound in sniffing elicited by either dishabituation odor further proves that lambs, like adult ewes, do detect and discriminate both odor stimuli. As both odor substrates were alternatively used as habituation and dishabituation stimuli, OO-DO and DO-OO contrasts can be compared at dishabituation. Both orders of odor presentation along the whole habituation procedure were effective in releasing differential responses to the dishabituation odor. Specifically, the lambs' licking-like response was enhanced when dishabituated with OO and lowered when dishabituated with DO. If this licking-like response is taken as an index of appetence, then one may logically deduce that OO is more appetent to lambs than DO. While this greater relative appetence for OO over DO was somehow predicted, we also expected lambs to exhibit aversive responses – such as escape attempts, freezing or

characteristic ear positions – to DO relative to OO. However, if under the present conditions DO elicited less positive/neutral responses than OO, it did not elicit clearly negatively-valenced responses. Indeed, the greater tongue extension observed with OO could be explained by the fact that the stronger odor intensity of the DO pad might have inhibited consummatory behaviors, leading lambs to sniff it for longer durations while losing interest in the OO pad more quickly, as indicated by their increased orientation toward the experimenter.

However, the approach-withdrawal test resulted in a different outcome. In these conditions, lambs also behaved differently toward DO vs. OO: fewer lambs stayed close to the box emitting DO compared to the box emitting OO. Thus, lambs moving freely in their familiar pen reveal that they detect and differentiate two contrasting odors by staying more or less close to their respective sources, and by interacting with them. Even directing their interactions toward a rarely explored area when it contained the enrichment scented with conspecific odor. However, the approach-withdrawal responses of lambs to either odor box was modulated by its position the space of the pen, by diurnal timing (Piccione et al., 2010) or even by the agricultural practices implemented on the farm (Piccione et al., 2011). Thus, for future applications of this test, it might be better to place the target odor stimuli in selected areas where the lambs display either quietness and rumination, or exploration and play, to verify whether they disrupt their habitual behavior.

The observation of lambs' aversive responsiveness to DO in the approach-withdrawal test, but not in the habituation test may be a result of the social context. Group dynamics play a crucial role in threat perception in gregarious species (Gray and Webster, 2023; Smith, 1986) and lambs may have reacted more robustly when surrounded by conspecifics. Both tests differ indeed by the presence of a familiar experimenter, who may have provided a sense of security to artificially-raised lambs (Coulon et al., 2015; Guesdon et al., 2015; Serrapica et al., 2017), reducing aversive reactions to any stress agent, specifically here to DO, in the habituation-dishabituation test. At the same time, differences in odor presentation (small pads vs. larger odor sources), individual variation in reactivity, or methodological constraints could have also contributed to the observed differences between the tests. In particular, chemosensory variations underlying this differential response by lambs may reside non-exclusively in the odors' quality (wool vs. feces), intensity (DO > OO), or origin (conspecific vs. heterospecific), a point to be elucidated by future experiments. Accordingly, the odorization of the pads in the habituation test might have conveyed less olfactory impact than the amount applied in the approach test. In addition, the expected aversive effect of the donor dog's fecal odor may be minimized by its diet poor in animal-sourced components (Scherer and Smee, 2016).

Another potentially influential factor differentiating both test is lambs' age (6 vs. 24 day in the H-D and A-W test, respectively). Vigi-lance and flight distance change indeed with age in sheep (Dwyer, 2004): younger animals maintaining closer proximity to conspecifics and exhibiting stronger following behavior, while older animals are generally less reactive to aversive stimuli. Finally, the sex ratio of the groups tested in either test were different, potentially creating an imbalance in reactivity due to well-known sex difference in ovine stress responses (Dwyer, 2004; Vandenheede and Bouissou, 1993; Wang et al., 2023; Zambra et al., 2021). Finally, as young animals deprived of maternal care are generally less capable of coping with stress (Fish et al., 2004), it would be of interest to test lambs reared by their mothers, to determine whether the behavioral responses of interest measured here differ from those observed under our experimental conditions. All above points must be seen as limitations of the present study, in need of consideration in forthcoming studies.

## 5. Conclusion

The habituation-dishabituation test conducted on adult ewes and on artificially-reared lambs demonstrated their ability to discriminate between dog feces and ovine wool odors, chosen for their radically
different odor impact and valence. This test appears thus to be a valid method for assessing young sheep's differential reactivity to odor substrates bearing contrasting valences. The second approach-withdrawal test faced methodological challenges under our conditions, particularly in accounting for confounding factors such as diurnal timing and spatial preferences. Despite these challenges, this test offers valuable insights into lambs' responses to non-food odors in a social context, and highlights the potential for scent-based enrichments to encourage exploration in underutilized areas. Minimizing spatial biases and expanding the range of odor sources could further enhance its reliability. Future research should aim to explore sheep's olfactory abilities in a non-food context, investigating which odors they can detect in their environment and how their responses vary based on interactive factors such as familiarity, novelty, sex, and social context.

### CRediT authorship contribution statement

**Izía Larrigaldie:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Bruno Patris:** Writing – review & editing, Validation, Data curation, Conceptualization. **Fabrice Damon:** Writing – review & editing, Validation, Methodology, Formal analysis, Conceptualization. **Benoist Schaal:** Writing – review & editing, Validation, Supervision, Conceptualization. **Alexandra Destrez:** Writing – review & editing, Validation, Supervision, Methodology, Funding acquisition, Data curation, Conceptualization.

### Ethics

All procedures concerning the animals were approved by the Dijon Animal Experimentation Ethics Committee (CEEA; under #105).

### Funding

Acknowledgements are expressed to the MESR, the Université Bourgogne Europe and the Ecole doctorale Environnement-Santé who funded the doctoral grant to I. Larrigaldie; Institut Agro Dijon and CNRS funded the study; EARL L'Abrepin funded the study animals and their husbandry.

### Declaration of Competing Interest

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

### Acknowledgements

We sincerely thank Lys, Eloi, and Hubert Mony for welcoming us and providing the opportunity to conduct our experiments on their farm.

### Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.applanim.2025.106734.

### Data availability

The data that support the findings of this study are not openly available and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at Centre des Sciences du Goût et de l'Alimentation in Dijon, France.

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