# Do lactating ewes guide lambs to their teats by smell? Perinatal change in inguinal wax composition and lambs' reaction to it

Alary Justine, Elias Bou-Maroun, Laurence Dujourdy, Nicolas Sok, Catherine

Husson, Bruno Patris, Benoist Schaal, Alexandra Destrez

► To cite this version:

Alary Justine, Elias Bou-Maroun, Laurence Dujourdy, Nicolas Sok, Catherine Husson, et al. Do lactating ewes guide lambs to their teats by smell? Perinatal change in inguinal wax composition and lambs' reaction to it. Mammalian Biology: Zeitschrift für Säugetierkunde, 2025, 105 (4), pp.517-531. 10.1007/s42991-025-00491-1 . hal-05053678

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# Do lactating ewes guide lambs to their teats by smell? Perinatal change in inguinal wax composition and lambs' reaction to it

Justine Alary<fnref n="1" /> · Elias Bou-Maroun<fnref n="2" /> · Laurence Dujourdy<fnref n="3" />,<fnref n="4" /> · Nicolas Sok<fnref n="2" /> · Catherine Husson<fnref n="5" /> · Bruno Patris<fnref n="1" /> · Benoist Schaal<fnref n="1" /> Ⓓ · Alexandra Destrez<fnref n="1" />,<fnref n="5" /> Ⓓ

Received: 10 September 2024 / Accepted: 22 March 2025

© The Author(s) 2025

## Abstract

Amongst the array of sensory cues maternal ewes provide to their lambs, the role of odors still remains unclear in *Ovis aries*. Specifically, the communicatory potential on lambs of the scent released by the ewes' perimammary inguinal glands is little studied. It is thus worthy to increase our insight about the adaptive value of ovine inguinal glands, the chemical nature of their secretion (i.e., inguinal wax, IW) and their communicative function in the success of initial suckling episodes. A first study explores whether IW from late pregnancy ewes differs from that of postparturient ewes in macroconstituents (lipids, proteins, water) as a proxy to changes in inguinal glands' activity at the time when lambs' teat searching and sucking are initiated. Then, the composition in volatile constituents is compared between IWs sampled at both physiological stages using solid-phase micro-extraction of the headspace and gas chromatography-mass spectrometry, allowing to identify 22 odor-active compounds. A second study assessed whether newly-born lambs discriminate the odor of IW from late-pregnancy ewes from that of IW from postparturient ewes (not their own mothers). While late-gestation IW tended to differ from postpartum IW in macroconstituents and in some volatile constituents, lambs seemed to respond indiscriminately in a behavioral test of choice between paired odors although they detected them. Thus, IW of any peripartum ewe (excluding the mother) is potent to elicit olfactory attraction in lambs ahead of the first suckling experience. Ovine IW is therefore in a good position to convey odor cues or a specific chemomessage that guides lambs toward the inguinal-mammary region of their mother.

**Keywords** Sheep (*Ovis aries*) · Chemical communication · Olfaction · Behaviour · Inguinal wax · Maternal odour

## Introduction

Ovine neonates are born with all their senses functional and with sufficient ability to thermoregulate and follow their mother on their own. They were first shown to rely on vision and audition to perceive and reach their dam (e.g., Nowak 1990; Poindron et al. 1993; Vince 1993; Abdel-Moneim et al. 2009; Sèbe et al. 2010; Mora-Medina et al. 2016). But once in contact with her, they obviously depend on proximal cues, navigating the mosaic of maternal textures, temperatures and odors before locating the udder and eventually seizing a teat (Vince 1984, 1993; Vince et al. 1985; Schaal et al. 1995a, b). From the perspective of a just-born, sucking-naïve lamb, the sustained searching effort before initial colostrum feeds is far from obvious: although it is generally completed within the first two postnatal hours (Vince 1993; Dwyer and Lawrence 1999), it can last much longer, jeopardizing neonates' viability (e.g., Nowak and Poindron 2006) and, in extensive conditions, exposing them to

Handling editor: Heiko G. Rödel.

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Alexandra Destrez
alexandra destrez@agrosupdijon.fr

<footnote n="1">¹ Centre des Sciences du Goût et de l'Alimentation, CNRS, INRAE, Institut Agro, Université de Bourgogne, CSGA, 21000 Dijon, France</footnote>

<footnote n="2">² UMR PAM, Institut Agro, Université Bourgogne
Franche-Comté, Université de Bourgogne, INRAE, 1
Esplanade Erasme, 21000 Dijon, France</footnote>

<footnote n="3">³ Institut Agro Dijon, Direction Scientifique, Cellule d'Appui à
la Recherche en Sciences des Données, 21000 Dijon, France</footnote>

<footnote n="4">⁴ Laboratoire d'Informatique de Bourgogne, 21000 Dijon,
France</footnote>

<footnote n="5">⁵ Institut Agro Dijon, 21000 Dijon, France</footnote>
increased predation risk (e.g., Rowley 1969; Hewson 1984; Choquenot et al. 1997; Moberly et al. 2003).

In their quest for a teat, newborn lambs circle around the dam before reaching regions assumed to convey familiar scents (e.g., hind limbs and udder tainted with foetal fluids; Poindron et al. 1993; Schaal et al. 1995a, b). They then nuzzle under the ewe's belly, pushing their muzzle into her warmer groin region and eliciting her arched-back, hind leg-extended posture (Vince 1984, 1993; Billing and Vince 1987; Dwyer and Lawrence 1999). This typical nursing posture of the ewe favours lambs' access to the scented inguinal hotspots. Bilateral inguinal glands release there a brownish, sticky, strongly odorous secretion (Vince and Ward 1984). This so-called inguinal wax seeps downward to the right-underneath mammae, forming an odor trace from the warmest inguinal region (Vince 1984) down to the teats. When presented separately to lambs (before any suckling or exposure to suckling-related odorants after birth), inguinal wax pasted on a glass-plate elicits instant head orientation, active searching and licking, and augments respiratory rate indicative of arousal and attractiveness (Vince and Ward 1984). In addition, when lambs are transitorily anosmized (through intranasal anaesthesia), they take much more time to locate a teat (Billing and Vince 1987). Thus, olfaction is involved in the lambs' search of the teat and the dam's inguinal wax scent might convey significant cues or chemosignals to which lambs are highly reactive before any post-birth exposure.

However, the lambs' responsiveness to inguinal wax odor appears to be short-termed, at least in the sheep breed studied (i.e., clun forest) and with the variables considered so far. It is reported to be maximal before the initial colostrum feed, and rapidly declines in magnitude with accumulating sucking success (Vince and Ward 1984). The effect of inguinal wax odor in early ewe-lamb interactions appears thus temporary, lasting a matter of several hours after birth before successful suckling. Such transience in the odor-based guidance to the teats was suggested to result from the lambs' rapidly learned prioritization of auditory-visual maternal signals assumed to be more sensorially impactful and thus more readily invested with cognitive salience (Vince 1993) [thus driving most empirical research on lamb-ewe interactions on the involvement of vision and audition (e.g., Bareham 1975; Alexander 1977; Alexander and Walser 1985; Nowak 1991; Veissier et al. 1998; Nowak and Boivin 2015; Ungerfeld et al. 2021)]. To some audio-visual-oriented researchers, olfaction might even be ignored in explaining lambs' earliest social interactions and multisensory cognitions (cf. Brebner et al. 2024, for a discussion about sensory biases of researchers). However, if the compulsive nosing of lambs on the dam's body obviously mobilizes peri-oral touch (Vince 1993), it certainly also activates olfaction. Perinatal lambs' nasal chemoreception is indeed fully functional (Vince and

Ward 1984; Schaal et al. 1991, 1995a, b), and the ovine woolly or glabrous skin surfaces are rich conveyors of scented secretions (e.g., Schaffer 1940; Hayashi 1968; Lyne and Hollis 1968). Furthermore, one might assume that ovine scent-producing structures of such invariable occurrence, size and copious output as the inguinal glands (Pocock 1910; Schaffer 1940; Brown and MacDonald 1985) might not be evolutionarily conserved if functionless. However, since the pioneering work of Margaret Vince and her colleagues (e.g., Vince and Ward 1984; Vince et al. 1985; reviewed in Vince 1993), inguinal wax's communicatory potential in the parental-filial context has attracted little interest (i.e., Rietdorf 2002; Schaal 2010; Alexandre-Pires et al. 2017; Alary et al. 2023). One study suggested that inguinal wax odor facilitates artificially-reared lambs' motivation to suck and favours thriving accordingly (Alary et al. 2023). Otherwise, the first chemo-analysis of ovine inguinal wax found a complex mixture of compounds wherein alkanols and methyl esters prevail (Rietdorf 2002), while another study suggested a change in inguinal wax composition at parturition (Alexandre-Pires et al. 2017), but both studies did not assess lambs' reactivity. It is thus worthy to broaden our understanding of the mechanisms, functions and adaptive consequences of ovine inguinal glands, and of the chemical nature of inguinal secretion and its latent communicative effects between ewe and lamb.

Potential inguinal wax-based ewe-lamb chemocommunication raises indeed multiple issues of biological, chemical and ethological interest. Firstly, how does the qualitative/quantitative inguinal wax composition in macroconstituents and volatile microconstituents correlate with an ewe's physiology around lambing and lactation onset? Secondly, is the inguinal wax scent informative to suckling-inexperienced lambs? And then what might be the nature of this information: a species-wide chemosignal, an individual-specific odor cue, or multidimensional indices including various meanings at a time?

To explore the above questions, a first experiment in this study will evaluate whether inguinal wax from late-pregnancy ewes differs from the inguinal wax of postparturient ewes in macroconstituents (lipids, proteins, water) as a proxy to changes in inguinal glands' activity. Then, the composition in volatile constituents will be compared between inguinal waxes sampled at both physiological stages using headspace sampling and gas chromatography-mass spectrometry (GC-MS). In a second experiment, a paired-choice odor test will evaluate whether lambs show behavioral discrimination between these two secretions, with the prospect that, if a peripartum secretory change occurs in inguinal glands, the odor of postparturient inguinal wax might be more arousing/attractive than the odor of late-pregnant ewes. Based on prior evidence (Vince and Ward 1984; Alary et al. 2023), the lambs' responsiveness

<figure><img src="image_3.png" /></figure>
to these odor stimuli will be assessed before their initial
suckling experience, assuming that they are then maximally
reactive to inguinal wax odor.

Materials and methods

Taking place in one private and one public farms (located in Francheville and Charolles, Burgundy, France, respectively), the present study was run in the context of respective farmers' management practices for general husbandry, and specifically for breeding, lambing and artificial rearing of lambs. In both farms, husbandry practices were similar. About a month before lambing, pregnant ewes were housed in groups of 20 in a barn. At incipient parturition, they were installed in individual lambing pens (1 × 1.5 m) where they remained with their lambs for 2 days postpartum, after which they were grouped with other ewes and lambs. In both farms, the gestating ewes were fed with hay ad libitum and 800 g concentrate composed of wheat, peas and soybean. At about 2-3 weeks before lambing, the ewes were sheared at the benefit of the lambs' udder seeking behavior (e.g., Ehrhardt 2023).

In order not to overburden the farmers with the
experimenters' daily presence in the sheepfold, the two
experiments in this study were conducted on separate
farms. Thus, experiment 1 was run in 2019 in Francheville
(sampling of inguinal wax for chemical analyses), while
experiment 2 was run in 2020 in Charolles (testing the
behavioral effects of inguinal wax odor on lambs in a room
separated from the sheepfold, which was accessible only in
Charolles farm).

Regarding animal handling by the experimenters, French farmers are strictly enforced to follow animal welfare rules of French law (Code rural et de la pêche maritime, chapitre IV, articles L214-1 to L214-23), and experimenters were accordingly supervised by them.

Experiment 1: composition of inguinal wax

Sampling

As our previous study (Alary et al. 2023), inguinal wax was collected in a flock of Romane sheep (in Francheville, Burgundy, France; from November to December 2019). Pregnant ewes (n = 15; their inguinal wax being abbreviated IW-P) were sampled three weeks before the estimated lambing date, while post-parturient ewes (n = 15; IW-PP) were sampled several hours (maximum 12 h) after parturition. No obvious sign of suboptimal health was detected in the animals.

For inguinal wax sampling, the ewes were maintained in a semi-supine position during a shearing session (during 2 min at most) to expose both inguinal gland pockets that were gently scooped out with a stainless-steel spatula. Each sample (mean weight = 0.64 ± 0.43 g SD) was put in a crystal plastic tube, identified and immediately frozen to –20 °C (Alary et al. 2023), while the sampling spatula was cleaned with alcohol and then rinsed with distilled water. Part of the 15 IW-P and 15 IW-PP samples were used for headspace extraction and GC-MS analyses of volatile compounds, while 5 IW-P samples and 5 IW-PP samples were pooled to assess potential changes in inguinal wax macroconstituent between late-pregnancy and postpartum.

Macroconstituents

Moisture

Both pools of IW-P and IW-PP were dried in a programmable
air-circulating drying oven (Nüve FN500) for 24 h at 104 °C.
After this time, the pools were weighted and returned into
the oven if necessary until to obtain a constant mass. The
water content (% w/w) was defined as the ratio of the mass
of dry matter divided by the initial mass of the sample.

Fats

Soxlhet extraction was used to determine the fat content of IW-P and IW-PP (e.g., Wagner et al. 2008; Hewavitharana et al. 2020). Dry inguinal wax pools were placed in a cellulose cartridge (Gerhardt) and in a Soxlhet device composed of a room of extraction and a distillation flask (100 mL) containing a large amount of cyclohexane (70 mL, solvent extraction grade). During the reflux step, the condensed fresh solvent gradually fills the room of extraction until an overflow level when a siphon aspirates the whole content and unloads it back into the distillation flask. This operation was repeated 3 times for each inguinal wax pool. The cyclohexane was then removed to measure the mass of fat (mg) present in each dry inguinal wax pool.

Proteins

The protein content was estimated using Dumas' method (e.g., Chiacchierini et al. 2003). Dumas nitrogen analyzer (Velp NDA 701, detection limit = 3 µg N/g sample) was calibrated and used according to manufacturers' instructions in order to obtain an estimation of protein's content (in mg). The combustion and reduction oven temperatures were respectively fixed at 1030 °C and 650 °C, operated with an oxygen flow rate of 400 mL/min and an oxygen factor of 1.4 mL/mg sample. For each of both inguinal wax pools, three replicates were run.
# Volatile microconstituents

## Extraction and GC-MS analyses

The volatile constituents of inguinal wax were extracted from the headspace developing from 0.1 g of wax left in a 10-mL glass-vial incubated in a water bath set at 40 °C for 15 min. For each inguinal wax sample, the volatiles were trapped for 30 min by solid-phase microextraction (SPME) onto a triple-phase fiber (divinylbenzene/carboxen/ polydimethylsiloxane, 50/30 µm; Supelco). After extraction, the fiber was desorbed for 10 min into the GC injector. All GC runs of individual inguinal wax samples (15 IW-P and 15 IW-PP) were repeated in triplicate using an Agilent gas Chromatograph (7890A) equipped with a split/splitless injector and an Agilent MS detector (5975C-VLMSD). The GC was fitted with a DB-5MS capillary column (30 m × 0.32 mm × 0.5 µm; Agilent) with helium as the carrier gas at a rate of 1.4 mL min⁻¹. The injection temperature was set at 250 °C and carried out in splitless mode with a purge flow to split vent of 50 mL min⁻¹ at 2 min. The oven temperature was ramped from 40 to 65 °C at 2 °C min⁻¹, then from 65 to 240 °C at 5 °C min⁻¹, and finally held 5 min at 240 °C. The MS was operated in scan mode from m/z 29 to 350 with an electron impact energy of 70 eV. The ion source was set at 230 °C, and the transfer line was maintained at 240 °C. The eluting volatiles were identified by comparison of their mass spectra with those in the Willey138 and NIST libraries (NIST 2014). This involved using the retention times of a standard mixture of C7-C30 saturated alkanes (Sigma Aldrich) and comparing them with the linear retention indices (LRI) values published in the literature for GC columns of same polarity.

## Chemometric analyses

To enhance the quality and reliability of the instrumental data, standard pre-processing techniques were applied on the chromatograms (e.g., Brereton 2003). These operations included baseline correction to eliminate potential artefacts such as column bleed; alignment of chromatographic data to limit variations in flow rate due to temperature, mobile phase composition and stationary phase decomposition that can lead to shifts in retention times; normalization (Total Sum Normalization) and scaling to mitigate variations stemming from both the instrument and the sample, and eliminate potential effects of injection differences; deletion of the solvent peak at the beginning of the chromatogram; and selection of the retention time window between 7.72 and 10.46 min which corresponds to a “fingerprint” zone where the best separation between the both types of inguinal waxes was noted. A specific R package, GCalignR, was used to align and normalize the chromatograms (Ottensmann et al.

2018). Further data analyses were carried out using RStudio (v. 2023.09.1; RStudio Team 2020), R-4.2.1 (R Core Team 2022). Then, the entire chromatograms were used for the multivariate analysis (principal component analysis, PCA). Before applying the PCA, baseline corrections following a normalization (standard normal variate, SNV) were performed. Data analyses were carried out using Quasar 1.9.0 packages (Demsar et al. 2013; Toplak et al. 2021).

# Experiment 2: lamb responses to inguinal wax odor

## Animals

All testing was carried out in August 2020 in the experimental farm of the Agricultural high-school of Charolles on Romane x Charolais sheep. Lambing was watched so that all tests on lambs could be run before sucking experience, within the first hour after birth.

## Inguinal wax sampling

Inguinal wax was sampled in pregnant ewes (IW-P) while fed in head locks, and in postparturient ewes (IW-PP) in the lambing pen. For the sampling, we opted for a gentler method than in experiment 1; not positioned semi-supine to access the glands, the ewes were here standing while eating and the experimenter rubbed a swab over the inguinal glands with gloved hand (Eco nitrile PF 250 gloves, EcoShield). Brown cotton swabs (100% cotton, 10 × 10 cm, Tex), previously spread with a dab (2 g) of Vaseline® (white petroleum jelly, Merck), were used to prevent ewes' skin/mucosa irritation from friction. Vaseline was also intended to facilitate inguinal wax adhesion on the swab and to reduce desiccation of inguinal secretion due to warming the swab (see below). Sampled inguinal wax was spread on the whole swab surface by pressing the secretion inside the folded swabs, before being immediately put in air-tight plastic bags to be kept in the refrigerator (4 °C) for 12 h at most. Between each sampling, new gloves were used.

## Behavioral test

### Animals

Sixty-one just-born lambs (of 61 different ewes) were tested (between 7:00 AM and 8:00 PM). Birthweight, sex or the litter size were not decisive in their selection, but lambs were not tested after an uneasy parturition. In case of multiple pregnancies, only one lamb was chosen based on its standing ability. When the lambs were able to stand by themselves (from about 10 min after birth), they were carried to the
test room adjacent to the sheepfold. They did not engage in
suckling prior to the test.

Test setting, test device, and procedure

The testing room was close to the lambing hall, but phonically isolated from bleating ewes. Individual lambs were placed into a 120 length × 100 width × 75 cm height box lined with straw to avoid slipping. Within this test box, lambs were put in a Plexiglas device supporting the paired odor stimuli at muzzle's height of a standing lamb (Fig. 1a). The cotton swabs were heated to the body temperature of a ewe (i.e., 38–40 °C; Abdisa 2017), using a pocket heater (Rechargeable Hand Warmer, 1800 mAH, LBM) affixed bilaterally on the Plexiglas walls (Fig. 1b). In an attempt to arouse the lambs and to attenuate their separation stress, they faced a picture (40 × 58 cm) of an unfamiliar ewe while low-pitched bleats of a maternal ewe (not the mother) were played-back with a smartphone (Schaal et al. 1995a, b).

Stimuli and design

For the tests, the lambs faced a choice between a pair of
stimuli presented symmetrically. The odor stimuli consisted
in inguinal wax taken on brown cotton swabs (10×10 cm)
from pregnant and postparturient ewes (not from own
mother). Before swabbing, the cotton swabs were spread
with Vaseline (~2 g). The Control stimuli consisted in a
brown pad spread with the same amount of Vaseline, but
devoid of inguinal wax. Three pairs of stimuli pads were
assayed in three different tests with different groups of new-
born lambs to assess: (i) the relative preference between
IW-P vs. IW-PP odors (n = 20 lambs), and the absolute

preference, (ii) between IW-P odor vs. control (n = 21 lambs)
and iii) between IW-PP odor vs. control in (n = 20 lambs).

Procedure

The two-odor choice-test was adapted from similar assays perfected for human or ovine neonates (Schaal et al. 1995a, b; Klaey-Tassone et al. 2021). Before test onset, the stimuli were left on the heaters for 5 min to reach equal temperature. The lamb was positioned into the test device with the muzzle aligned on the midline between both stimuli, at about 10 cm from both of them. To control for potential side-effects, the lateral position of the stimulus pads was counterbalanced between each test. The lamb was then left free to explore both odor swabs for 5 min which were video-recorded (Hero7 Black, GoPro) for subsequent off-line analysis. Despite the lambs were taken from their mother when able to stand on four legs, we had to exclude 42% of them (n=26) that laid down during the 5-min test or showed strong signs of stress (agitation, turning opposite to the stimuli). After applying these exclusion criteria, 9 lambs remained for the IW-P odor vs. IW-PP odor assay, 15 for the IW-P odor vs. control stimulus assay, and 11 for the IW-PP odor vs. control stimulus assay. Between each two-odor test, the stimuli pads were changed, the Plexiglas device cleaned with alcohol and water, and the lining of the test-box was renewed with clean straw.

Behavioral variables: coding and definitions

The video-records of the tests were analysed in the lab by
a coder who was unaware of the nature and presentation
side of the paired stimuli. The behavior items of interest

<figure><img src="image_4.png" /><figcaption>**Fig. 1** The two-odor choice test. **a** Schema of the testing device composed of a Plexiglas wall on which two odorized pads (dark squares) are affixed. **b** Screenshot from above showing a lamb's head orientation and effective contact toward a stimulus swab</figcaption></figure>
(see below) were visualized from a computer screen and timed with the computer's internal clock through the Boris software (accuracy: 0.1 s; version 8.13, UniTo; Friard and Gamba 2016). The duration of head turning to either stimulus in a pair was coded by tracking the tip of the lamb's muzzle in the accessible space over and between both stimuli. The orientation response to a stimulus was considered positive when the muzzle was positioned toward that stimulus pad, i.e. when both nares deviated from the experimental device's midline to the right or left sides. The duration the lamb spent oriented to a given side was the sum of all orientation bouts to that side across the test.

Following the above procedure, two variables were derived from the lambs' reactivity to the paired stimuli: the durations of head orientation to either stimulus and the duration of direct muzzle contact with either stimulus. Head orientation between either stimulus in a pair was defined as any directional movement of the muzzle across the midline of the test device toward a given stimulus. Muzzle contact with either stimulus was defined as any contact of the muzzle with the pad conveying this stimulus.

## Statistical analyses

The behavioral data were not normally distributed and thus were analysed using non-parametric (two-tailed) Wilcoxon tests to compare duration of lambs' head orientation and duration of direct muzzle contact with either stimulus in the three assays. All statistical testing were carried out with the XLSTAT software (version 2015.3.01.19097; Addinsoft, Paris, France).

## Results

### Composition of inguinal wax

#### Macro-constituents

Total moisture determination indicated 21.7% and 23.3% for IW-P and IW-PP, respectively. For IW-P, the proportion in fat content totalized 9.4%, while it was 33.5% for IW-PP. Finally, the proportion in protein content amounted 27% for both IW-P and IW-PP. In sum, the pooled IW-PP samples were a bit moister and clearly richer in lipids than the pooled IW-P samples, while the protein content of inguinal wax remained unchanged between both stages.

#### Volatile olfactory compounds

The GC-MS analyses applied on the SPME extracts of inguinal wax produced chromatograms comprising about 150 visible peaks. Twenty-two compounds (illustrated in

Fig. 2) were reliably identified on the chromatograms, i.e. showing a high percentage of matching with the database values and being confirmed by comparing the calculated retention index with published ones using the same column (i.e., DB-5 nonpolar stationary phase). Table 1 gives these compounds numbered and listed in order of elution and identified by CAS number. These include mostly aldehydes (peaks # 3, 6, 12), ketones (5, 9, 21), alcohols (2, 16), phenols (19, 22), nitrogen compounds (4, 11, 20), other constituents including an aromatic (ethylbenzene), a phenone (acetophenone), a terpene (p-cymene), a furan (tetrahydrofuran), and a sulphur compound (dimethyl sulfone). These compounds occurred in both IW-P and IW-PP samples, although at different intensities (Fig. 2).

The results of the PCA are described in Fig. 3. The first two principal components account for 62% of the total variance in the data (PC1: 39.7% and PC2: 22.7%). The score plots (Fig. 3a), displaying the linear combinations of the GC data from each inguinal wax sample determined by the coefficients for each principal component, allow for the detection of potential clusters, outliers, and trends. Groupings of data on the plot indicate two or more discriminable distributions. Two areas are delineated along PC2, separating most IW-P samples from most IW-PP samples. This separation is indeed not fully exclusive, as 2–3 inguinal wax samples distribute over the IW-PP area, while 4–5 IW-PP samples are at the rim or over in the IW-P area. Thus, although inguinal wax GC profiles can be roughly grouped according to either inguinal wax categories considered, some overlap between them evades complete separation. Figure 3b represents the weights for each PC plotted against the variable number, allowing to localize the peaks which have the largest effect on either component. Peaks with RT = 7.84 min and RT = 9.30 min appear to contribute most to PC1, while peaks with RT = 9.88 and RT = 10.27 min are the most influent on PC2 (Fig. 3b). These peaks are identified as Ethylbenzene, Styrene, Heptanal and Dimethyl sulfone, respectively (Fig. 3c). In sum, although multivariate statistics indicate that some volatile compounds are related to variance between both types of inguinal wax, the individual contribution of these compounds to the whole odor blend emitted by either inguinal wax remains inconclusive.

### Lambs' responses to inguinal wax odor

Head orientation and direct muzzle contact of newly born lambs with inguinal wax odor are described in Fig. 4. As for the IW-P vs. IW-PP odor contrast, no differential response occurred in terms of duration of orientation, nor of muzzle contact with either stimulus. In the test presenting IW-P against the control, the lambs did not orient longer to either stimulus; however, when oriented to either stimulus, they spent significantly longer with their muzzle contacting the
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Representative total ion chromatograms of volatile compounds obtained after solid-phase microextraction (SPME extraction) of inguinal wax (IW) sampled from **a** a postparturient ewe (IW-PP, in red) or **b** a pregnant ewe (IW-P, in blue). Peak numbers refer to the</figcaption></figure>

IW-P swab than the control swab (V=98, p=0.030). Finally, when facing the IW-PP swab against the control swab, the lambs oriented significantly longer toward the former than to the latter stimulus (V=58, p=0.024), and tended to display longer muzzle contacts with IW-PP than with the control (V=45, p=0.074). In sum, the lambs appeared to behave indiscriminately when facing simultaneously IM-P and IW-PP odors, although they were able to detect both odors when paired with a control swab.

Discussion

The present study aimed to assess whether the odor of
inguinal wax sampled in ewes before and after parturition
was discriminable analytically by GC-MS and perceptually
by lambs' olfaction. It first evaluated whether inguinal wax
composition changed between both physiological stages,
checking the semiochemical potential of inguinal glands in
search of behaviorally-active volatiles conveyed by inguinal

compounds listed in Table 1. Note that some peak numbers are not
visible on the GC tracing due to their presence as traces with very
low areas. Also, other compounds either resulted in very small GC
peaks or could not be identified reliably

wax. It then evaluated how far lambs detect and react to
potential peripartum changes in inguinal wax composition,
testing whether postpartum inguinal wax elicits different
reactions than prepartum inguinal wax, with the hypothesis
that the former odor might be more arousing/attractive than
the latter in directing lambs' responses. It may be noted from
the outset that the inguinal waxes tested on lambs were not
the ones that were subjected to chemical analysis; the results
on the composition of inguinal waxes obtained from one
farm (experiment 1) may thus not directly map onto lambs'
reactivity to the odor of inguinal waxes sampled in the other
farm (experiment 2).

Inguinal wax composition in macroconstituents appeared
to be altered between late pregnancy and early postpartum.
While its content in lipids raised then by 24%, its protein
and water contents remained quasi-stable. This growing
amount of the fat fraction in inguinal wax may result from
an increase in skin lipid excretion during late pregnancy
and peripartum, as a consequence of hormonal alterations
of sebaceous glands' activity proper to these periods in
<table><caption>**Table 1** Volatile compounds identified in inguinal wax samples (regardless of physiological stage of donor ewes) by GC-MS (gas chromatography-mass spectrometry) after SPME (solid-phase microextraction) of their headspace</caption><thead><tr><th>RT</th><th>Peak number</th><th>Calculated RI</th><th>Confirmed RI</th><th>Identification</th><th>Compound Name</th><th>CAS number</th><th>% Database matching*</th><th>Family</th></tr></thead><tbody><tr><td>2,12</td><td>1</td><td>721</td><td></td><td>+</td><td>Furan, tetrahydro-</td><td>109-99-9</td><td>91</td><td>Furan</td></tr><tr><td>2,49</td><td>2</td><td>730</td><td></td><td>+</td><td>1-Butanol</td><td>71-36-3</td><td>86</td><td>alcohol</td></tr><tr><td>3,01</td><td>3</td><td>742</td><td>732</td><td>++</td><td>Pentanal</td><td>110-62-3</td><td>91</td><td>aldehyde</td></tr><tr><td>4,44</td><td>4</td><td>775</td><td></td><td>+</td><td>Piperidine, 1-methyl-</td><td>626-67-5</td><td>90</td><td>Piperidine</td></tr><tr><td>5,12</td><td>5</td><td>791</td><td>790</td><td>++</td><td>2-Hexanone</td><td>591-78-6</td><td>94</td><td>ketone</td></tr><tr><td>5,51</td><td>6</td><td>800</td><td>802</td><td>++</td><td>Hexanal</td><td>66-25-1</td><td>90</td><td>aldehyde</td></tr><tr><td>7,85</td><td>7</td><td>854</td><td>849</td><td>++</td><td>Ethylbenzene</td><td>100-41-4</td><td>91</td><td>Aromatic hydrocarbons</td></tr><tr><td>8,27</td><td>8</td><td>864</td><td>862</td><td>++</td><td>p-Xylene</td><td>106-42-3</td><td>93</td><td>Aromatic hydrocarbons</td></tr><tr><td>9,05</td><td>9</td><td>882</td><td>884</td><td>++</td><td>3-Heptanone</td><td>106-35-4</td><td>80</td><td>ketone</td></tr><tr><td>9,29</td><td>10</td><td>888</td><td>889</td><td>++</td><td>Styrene</td><td>100-42-5</td><td>93</td><td>Aromatic hydrocarbons</td></tr><tr><td>9,78</td><td>11</td><td>899</td><td></td><td>+</td><td>Oxime-, methoxy-phenyl-</td><td>100022-28-6</td><td>87</td><td>Oxime</td></tr><tr><td>9,87</td><td>12</td><td>901</td><td>900</td><td>++</td><td>Heptanal</td><td>111-71-7</td><td>97</td><td>aldehyde</td></tr><tr><td>10,27</td><td>13</td><td>908</td><td>909</td><td>++</td><td>Dimethyl sulfone</td><td>67-71-0</td><td>91</td><td>sulfone</td></tr><tr><td>13,1</td><td>14</td><td>960</td><td>960</td><td>++</td><td>Benzaldehyde</td><td>100-52-7</td><td>93</td><td>aldehyde</td></tr><tr><td>16,33</td><td>15</td><td>1024</td><td>1024</td><td>++</td><td>p-Cymene</td><td>874-41-9</td><td>76</td><td>Aromatic hydrocarbons</td></tr><tr><td>16,6</td><td>16</td><td>1029</td><td>1030</td><td>++</td><td>1-Hexanol, 2-ethyl-</td><td>104-76-7</td><td>78</td><td>alcohol</td></tr><tr><td>16,66</td><td>17</td><td>1031</td><td>1034</td><td>++</td><td>2-Pyrrolidinone, 1-methyl-</td><td>872-50-4</td><td>90</td><td>lactam</td></tr><tr><td>18,04</td><td>18</td><td>1064</td><td>1062</td><td>++</td><td>Acetophenone</td><td>98-86-2</td><td>91</td><td>aromatic ketone</td></tr><tr><td>18,42</td><td>19</td><td>1073</td><td>1075</td><td>++</td><td>p-Cresol</td><td>106-44-5</td><td>93</td><td>phenol</td></tr><tr><td>21,2</td><td>20</td><td>1148</td><td></td><td>+</td><td>2-Piperidinone, 1-methyl-</td><td>931-20-4</td><td>90</td><td>Piperidinone</td></tr><tr><td>22,72</td><td>21</td><td>1190</td><td>1190</td><td>++</td><td>2-Decanone</td><td>693-54-9</td><td>83</td><td>ketone</td></tr><tr><td>31,53</td><td>22</td><td>1503</td><td>1502</td><td>++</td><td>2,4-Di-tert-butylphenol</td><td>96-76-4</td><td>72</td><td>phenol</td></tr></tbody></table>

mammals (e.g., Burton et al. 1970, 1975). This rise in the lipidic fraction in inguinal wax is explained by the histological features of the ovine inguinal pockets which are abundantly endowed with sebaceous and apocrine units (Schaffer 1940; Alexandre-Pires et al. 2017). Such a peripartum change of lipids in inguinal wax may contribute to alter its physicochemical properties (fluidity) and/or local microbiological activity on it (Vanderwolf et al. 2023), potentially resulting in a different profile of volatile organic compounds (VOCs) between late gestation and early postpartum. In particular, the lipidic fraction in complex glands' secretions functions both as solvent and fixative of lipophilic molecules, reducing their evaporation rate, hence protracting their release and reducing their diffusion to a short range (Regnier and Goodwin 1977). Then, thermal variations of inguinal/mammary regions triggered by incipient lactation and at each suckling iteration (e.g., Thompson 1980; Castro-Costa et al. 2014; Bobowiec et al. 2019) may induce fluctuating variations in evaporation rates of inguinal wax VOCs. In sum, the local interplay of lipids,

temperature and microbiota in the inguinal-perimammary regions may shape the composition and fluidity of the inguinal wax matrix and regulate the odor profile that ewes present to their teat-seeking lambs.

The next issue explored in the present study relates to the chemical composition of inguinal wax effluvium and potential differentiation of inguinal wax odor sampled before and after parturition. SPME extraction of inguinal wax volatiles and GC-MS produced richly endowed chromatographic profiles, which analyses allowed identifying reliably a set of 22 volatile compounds (Table 1). An earlier GC-MS analysis of ovine inguinal wax (Rietdorf 2002) identified a complex set of 155 compounds. But Rietdorf's study did not report any compound that overlapped with those identified in the present study. This discrepancy between both analyses is explainable by contrasting extraction methods and timing of GC. While Rietdorf used pentane and methanol extractions of inguinal wax, our extraction method was selective for small molecular-weight volatile compounds. Otherwise, the Rietdorf study analyzed

<footnote>* This percentage corresponds to the similarity between the experimental mass spectrum and the one of the database.</footnote>

<footnote>** Olfactory detection threshold in air for humans (Pubchem)</footnote>

<footnote>+ Identification by comparison with mass spectrum database.</footnote>

<footnote>++ Identification by comparison with mass spectrum and retention index database.</footnote>
the chromatograms from 40 to 85 min, while in the present
study the 0-35-min window was considered. Thus, while
Rietdorf missed the volatile compounds most susceptible
to impact lambs' olfaction, the present study missed semi-
volatile or non-volatile compounds which may as well be
influential in the chemomessage conveyed by inguinal wax.
Anyway, it is clear that a more thorough chemical analysis
of ovine inguinal wax is warranted in both its volatile and
less volatile compounds.

The origin of the volatiles we detected in the inguinal wax headspace is unclear and probably complex (Charpentier et al. 2012). Some of them may not be inherent to ewes' metabolism, but derivate from plants secondary compounds, from activity of gut and skin microbiomes, or from the adsorption of pollutants (e.g., Wakayama et al. 2019). Some identified VOCs are indeed not produced by mammals, such as hydrocarbons or phenols relatable to environmental, potentially anthropogenic, sources (ethylbenzene, *p*-xylene, styrene, *p*-cresol, 2,4-di-tert-butylphenol) (e.g., Steele et al. 1994). Other compounds may be catabolized by ewes (and their ruminal microbiome) from vegetal ingesta to gather as end-products in inguinal wax (tetrahydrofuran, *p*-cymene, dimethyl sulfone, benzaldehyde, 1-methyl-pipiridine, 1-methyl-2-pyrrolidinone, 1-methyl-2-piperidinone) (e.g., Hannemann et al. 1989; He and Slupsky 2014; Benhamou et al. 2019; Li et al. 2021; Zubkov and Kouznetsov 2023). Still others may be released by commensal microbiota occurring in inguinal pockets (e.g., Archie and Theis 2011), dwelling on sebum and apocrine secretions or on products brought there from other body regions by ewes' self-directed nuzzling (saliva, mucus, preorbital secretion) or other behaviors (when laying down: contamination with feces, urine, vegetal bedding, interdigital secretions; or when laying down during/after lambing: contamination with fetal fluids, blood). However, even if some of these compounds may be extraneous to inguinal glands, it is not excluded that local mechanisms release or concentrate them in inguinal wax, engaging them into mixtures conveying potential semiochemical effects, as noted in other mammals. For example, heptanal is found in giraffes' hair (*Giraffa camelopardalis*) (Wood and Weldon 2002), frontal hair of white-tailed deer (*Odocoileus virginianus*), and interdigital scent of bontebok and blesbok (*Damaliscus pygargus*) (Burger 2005), and in human body effluvium (Curran et al. 2007). Sulfur-containing dimethyl sulfone appears ubiquitous in mammalian volatilomes (e.g., rodents: Zhang and Zhang 2011; Zhang et al. 2019. Ungulates: Bakke and Figenschou 1983; Burger 2005; de la Peña et al. 2019. Carnivores: Mattina et al. 1991; Burger et al. 2006; Apps et al. 2012. Primates: Nylander-French et al. 2020; Drabińska et al. 2021; Molinier et al. 2024). Likewise, benzaldehyde is of frequent incidence in ungulates (Wood and Weldon 2002; Burger 2005; de la Peña et al. 2019)

and carnivores (Dzięcioł et al. 2018). In brief, the different
VOCs identified in ovine inguinal wax are not uncommon in
mammals, and some have been designated as potential cues
to sexual or breeding status (1-butanol in the giant panda
(*Ailuropoda melanoleuca*) or cat (*Felis catus*): Hagey and
MacDonald 2003; Uetake et al. 2018; 2-hexanone in white-
tailed deer (*Odocoileus virginianus*): Jemiolo et al. 1995;
Dimethyl sulfone in the rat: Zhang et al. 2008; Benzaldehyde
and acetophenone in the domestic dog (*Canis lupus
familiaris*): Dzięcioł et al. 2018) or involved in mothers-
offspring interactions (Heptanal in humans or *p*-xylene in
mice: Yu et al. 2024; Lacalle-Bergeron et al. 2021). The
issue of whether these compounds and probably others, not
yet identified, inguinal wax VOCs are of any communicative
value in the ewe-lamb relationship will be answered after
applying them in long series of behavioral testing of lambs.

The PCA analysis run over the entire GC-MS data suggests that the inguinal wax from late-gestation ewes might be chemically distinguishable from inguinal wax of postparturient ewes. Although the chromatographic profiles of both inguinal wax types mostly separated along principal components, data of inguinal wax of pregnant ewes distributed within the inguinal wax of postparturient ewes cluster and vice-versa, suggesting qualitative overlap of resulting odor properties although with possible intensity differences. The fact that the 22 identified compounds occurred in both types of inguinal wax further suggests overlapping odor quality. However, the PCA allowed to identify a window on the inguinal wax chromatograms, between retention times 7.72 and 10.46 min, wherein some VOCs (i.e., ethylbenzene, styrene, heptanal and dimethyl sulfone) contributed differentially to the GC-MS data of inguinal wax from late-gestation vs. postparturient ewes. However, in absence of any data on the distinctive olfactory contribution of these compounds to the whole inguinal wax odor, no further conclusion can be drawn on whether they can alter the perceptual effect on lambs of either inguinal wax type. It may be further noted that the composition of inguinal wax from ewes within a single farm may be constrained by similarities in genetic background and environmental conditions (fodder, water, bedding, husbandry), potentially homogenizing olfactory profile between and within individuals (i.e., across physiological stages).

In line with above chemical results, newly-born lambs oriented their head alike toward the odors of inguinal wax sampled from late-pregnant and early postparturient ewes. This non-differential responsiveness cannot be explained by the lambs' random orientation or inability to detect either odor, as they positively turned toward them when presented against controls. Although Vaseline on the stimuli swabs may have hampered inguinal wax VOCs' evaporation rate (e.g., Finnerty et al. 2017) and, hence, attenuated lambs'
PC 2 (22.8%)

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Normalized abundance

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
◄ **Fig. 3** *a* Score plot of the Principal Component Analysis (PCA) showing differential grouping of the GC (gas chromatography) data for IW-P (inguinal wax of pregnant ewes, in blue) and IW-PP (inguinal wax of post parturient ewes, in red) along principal components (PC) 1 and 2. Each point represents a sample of inguinal wax. *b* Plot of the weights of the compounds (through the retention time (RT) variable) for each PC, allowing to scrutinize the GC section (from RT 7.72 to 10.46) and corresponding peaks having the largest effect on principal components. *c*. Averaged total ion chromatograms of the most differentiable GC section (from RT 7.72 to 10.46) for both types of inguinal wax samples (blue: IW-P; red: IW-PP)

responses to them, they still appeared able to discriminate
pure Vaseline from Vaseline loaded with inguinal wax. It
may thus be conservatively concluded that both inguinal wax
of pregnant ewes and inguinal wax of postparturient ewes
convey odor cues that are similarly attractive to newly-born
lambs. This outcome means that, in the present experimental
conditions, suckling-inexperienced lambs do not differentiate
inguinal wax from late-pregnancy and early postpartum ewes
despite some potential compositional variation evidenced
instrumentally. Their reactivity to inguinal wax odor does
therefore not appear to critically depend on the donor ewe's
physiological stage. Accordingly, the perinatal composition
changes of ovine inguinal gland secretion, if any, may be
too shallow to induce differential responses in the lambs;
alternatively, these perimammary glands may be active
regardless of the females' physiological state. In mammalian
species bearing precocial neonates of the follower type (Lent
1974), where the neonate is the initiator in early suckling
interactions, a continued chemomessage emission by the
mother would indeed be optimal in avoiding costly random
searching for a teat and, thus, in swiftly satisfying pressing
metabolic needs, henceforth ensuring lamb survival. In
addition, short suckling periods may reduce the postpartum
ewe-lamb dyad's immobility and related susceptibility to
predation, especially in extensive breeding conditions.
However, inguinal wax odor is not the only source of useful
information directing teat-seeking of precocious ovine
lambs. Other odor cues (i.e., from fetal fluids or delivery-
related blood; Schaal et al. 1995a, b) or non-olfactory cues
such as warmth or texture of body surface or mother's
behavior (arched-back extended leg posture) may help them
to locate the udder (Vince 1993). Clearly, the inguinal scent
must be seen in the context of a multisensory perimammary
spot evolved to attract and stimulate persistent searching for
a teat in suckling-inexperienced lambs.

Another interesting result of the present study is that
any inguinal wax odor, even if sampled from ewes which
were not the lambs' own dam (but raised on the same farm),
appeared attractive to newly-born lambs. This result is in line
with those of Vince and colleagues (e.g., Vince and Ward
1984; Vince et al. 1985; reviewed in Vince 1993), showing
that inguinal wax sampled from any (unfamiliar, unrelated)

ewe conveys odor compounds that allure lambs. Our
results extend thus to additional ovine breeds (Romane and
Romane x Charolais) the positive reaction to ovine inguinal
wax scent noted in Clun Forest lambs' regardless of the
individual identity of the donor ewe (Vince and Ward 1984).
In addition, inguinal wax odor appears to elicit attraction
unconditionally of any prior postnatal exposure to it before
behavior testing, suggesting either a prenatally-acquired
cue or the possibility of a species-specific chemosignal.
The chemical nature of this inguinal wax chemosignal, if
any, awaits further chemical analyses on, and behavioral
assays with, inguinal wax from multiple ovine breeds. In
sum, the secretion of inguinal glands from maternal ewes is
in a position to olfactorily target the mammary area in newly
born lambs despite the strong background odor of the ewe's
woolly surface.

The present study has several limitations, the first being
the small sample sizes of newly-born lambs enrolled in
the different behavioral assays. A major difficulty was to
recruit lambs before any suckling experience, leading to
exclude about 42% of them because they could not stand
stably enough for the duration of the test. Future behavioral
tests might consider breeds giving birth to lambs having
greater vitality (e.g., Dwyer and Lawrence 1999) or proxies
that do not depend on lambs’ standing ability (e.g., heart
rate, respiratory rate and amplitude, licking, sucking). In
any case, the variability of the newly-born lambs’ motor
ability could be reduced by systematically assessing their
vigor using a standardized rating scale (e.g., Matheson
et al. 2011). Secondly, inguinal wax is heterogeneous in
consistency, formed by the accretion of brownish crumbles
of more or less dehydrated wax mixed with a fresh, creamy
yellowish fraction; these two fractions should be considered
separately in future chemo-analytic and behavioral studies,
the freshest fraction being more susceptible to be affected
by recent perinatal compositional changes, if any. Thirdly,
the dosage of inguinal wax macroconstituents resulting
from the quantification of single pools of 5 prepartum
vs. 5 postpartum inguinal wax samples, related perinatal
change should be taken with caution: these dosages need
to be replicated with individual inguinal wax samples
taken at both physiological stages. Fourthly, the chemical
exploration of inguinal wax composition should be
completed in terms of sampling (in various ovine breeds
under diverse environments and feeding regimen) and
analytic methods (e.g., extending duration of GC runs to
increase compound-mass range). Fifthly, the use of Vaseline
on the cotton swabs to sample the inguinal wax applied in
the paired-choice tests was not a wise option, as it may have
interfered with the odor intensity of the original inguinal
wax, in the sense of an attenuation of VOCs’ evaporation
rate (Regnier and Goodwin 1977; Finnerty et al. 2017).
Sixthly, the tests used here in lambs presented either paired
<figure><img src="image_10.png" /><figcaption>**Fig. 4** Lambs' response durations (in seconds) to either odor stimuli presented in three different paired-choice tests opposing both inguinal waxes (IW) of pregnant (P) ewes vs. post parturient (PP) ewes (n=9 lambs for IW-P vs. IW-PP) or one of the IW with a control (n=15 lambs for IW-P vs. control and n=11 lambs for IW-PP vs. control).</figcaption></figure>

a Head orientation duration and b Direct muzzle contact duration. Boxes represent medians and first and third quartiles, whiskers represent 2.5% and 97.5% of the data distribution (#0.05 < p < 0.1; \*p < 0.05, Wilcoxon test)

inguinal waxes or inguinal waxes against a control, the first condition involving intensity-matched odors leading to non-differential response, while the second condition with intensity-mismatched stimuli led to a differential response. One cannot exclude that this odor-intensity confound might have influenced the outcome, a point that should be solved in pairing inguinal wax odor with an odor-positive control of equal intensity (at least to the human nose). Lastly, the potential uniqueness of the chemical profile of inguinal wax from pregnant/parturient ewes should be ascertained by comparison with the wax odor profile from non-pregnant ewes or even from males; optimally, inguinal wax sampling should be affected longitudinally in the same ewes before and during pregnancy, and then at lactation onset. Finally, the species-specificity of the reactogenic power of inguinal wax odor should be ascertained in presenting wax of donor ewes from various farms and breeds to lambs from various breeds and farms as well.

In conclusion, the present study indicates weak compositional alterations in the odor of inguinal wax from late pregnancy to early lactation, but these changes do not appear to differentially affect the orientation behavior of lambs in a decontextualized two-choice test. This test shows indeed that the inguinal wax of any ewe (from a

same farm environment, excluding the mother) in any perinatal stage elicits olfactory attraction in lambs prior to initial suckling experience. Ovine inguinal wax is thus in a position to convey a chemomessage that guides lambs toward the inguinal-mammary region, although this needs now to be assessed directly in the natural suckling context.

**Acknowledgements** Our sincere thanks go to Hubert et Lys Mony who welcomed us generously to their farm and provided unlimited access to their sheep. We also acknowledge Agathe Chevalier and the technicians of the Charolles agricultural high-school farm; Anne-Sophie Viaux and Salomon Beaufrère for running SPME extractions of inguinal wax and GC runs under the supervision of E. Bou-Maroun. This work was supported by Institut Agro Dijon, CNRS and Ministry of Agriculture (Paris) as doctoral funding to J.A. Finally, we thank two anonymous referees for their useful comments.

**Author contributions** A.D. and B.S. sought funding; A.D., J.A. and B.S. conceived and designed the study, with assistance from B.P., E.B-M., and N.S.; J.A. and A.D. collected the behavioral data, and J.A., C.H. and B.P. analysed them from the video recordings. N.S run the biochemical analyses; E.B-M. managed the chemical analyses, and E.B-M. and L.D. analyzed and interpreted the chemical data; B.S., J.A., and A.D. wrote the manuscript. All authors approved the final version.

**Funding** Open access funding provided by Institut Agro Dijon.

<figure><img src="image_11.png" /></figure>
## Declarations

**Conflict of interest** The authors have no conflict of interest to declare.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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