# I. Le mouton, un modèle d'intérêt pour l'étude de l'environnement précoce :

## 1. Généralités

Le mouton (Ovis aries) est un ongulé marchant sur deux doigts. Il appartient à la famille des Bovidés, plus précisément à la sous-famille des Caprinés et au genre Ovis. Le mouton est un mammifère herbivore, et plus particulièrement un ruminant. C'est l'un des premiers animaux domestiqués par l'Homme. L'élevage ovin en France représente un cheptel d'environ 7 millions d'animaux (Agreste, 2021). Les types d'élevages peuvent être variés : en plein air, en bergerie ou encore en transhumance. Cet élevage est utilisé pour la production de viande, de lait (notamment pour la production de fromages), de laine ou encore de peau (pour obtenir du cuir appelé "basane"). Il existe actuellement plus de 200 races différentes dans le monde (Sheep Breeds, 2021). Ces races peuvent être classées en différents groupes : les races à faibles effectifs (Ex : Berrichon de l'Indre), précoces (Ex : Ile de France), d'herbage (Ex : Bleu du Maine), Mérinos (Ex : Mérinos de Rambouillet), prolifiques (Ex : Romane), laitières (Ex : Lacaune) ou encore rustiques (Ex : Limousine) (Perret, 1986).

## 2. Écologie comportementale

Du point du vue de l'écologie comportementale, le mouton est un animal grégaire, il vit en groupe avec une hiérarchie sociale. Cependant, en dehors des périodes de reproduction, les mâles vivent dans un troupeau distinct de celui des femelles et des jeunes (Main et al., 1996; Ruckstuhl & Neuhaus, 2002). Le mouton est une espèce nidifuge ; le nouveau-né naît dans le groupe de femelles à un stade de maturité avancée. En effet, le poids du cerveau de l'agneau à la naissance représente déjà 50% du poids du cerveau d'un adulte. De plus, leurs capacités sensorielles (visuelle, olfactive et auditive) et motrices sont fonctionnelles dès la naissance (McIntosh et al., 1979). L'agneau va donc pouvoir rapidement se lever et suivre sa mère (Fahmy & Robert, 1997; Lent, 1974). Jusqu'au sevrage qui a lieu environ 4 mois après la naissance, l'agneau se nourrit principalement en tétant sa mère. A partir de la puberté (vers l'âge de 6 mois), les agneaux vont avoir tendance à s'éloigner de leur mère (Gaudin et al., 2018) pour passer plus de temps avec leurs congénères (de même sexe et de même âge) (Shillito Walser & Williams, 1986). Vers l'âge de 24 mois, les mâles vont quitter le groupe
tandis que les femelles vont rester dans ce groupe tout en se détachant de leur mère (Franzmann & Geist, 1972).

### 3. La race Ile de France

La race Ile de France est une race issue du croisement entre des moutons Dishley et des Mérinos de Rambouillet. Le poids moyen est de 110 à 150 kg pour les béliers et 70 à 90 kg pour les brebis. Cette race est particulièrement appréciée pour sa vitesse de croissance rapide, son adaptation facile au désaisonnement ainsi que pour sa laine (Perret, 1986). De façon générale, en élevage, l'agnelage a lieu une fois par an.

### 4. Conditions d'élevage et expérience précoce :

Lors d'une visite en élevage ovin, il est courant de trouver deux techniques d'élevage des agneaux. Ils peuvent être élevés avec leur mère et nourris avec le lait maternel ou sans leur mère nourris avec un lait artificiel.

#### a. Agneaux en allaitement maternel :

Lorsque les agneaux sont élevés avec leur mère (Figure 10), ils vont pouvoir ingérer le colostrum. L'ingestion de colostrum est importante et va apporter des anticorps maternels à l'agneau (Nowak & Poindron, 2006). Ces anticorps vont lui permettre de mieux se défendre face à des potentielles infections. Cette ingestion de colostrum va également permettre de créer un lien

entre la mère et son petit et favoriser la reconnaissance de la mère par son petit (Val-Laillet et al., 2004, 2006). Cette reconnaissance est importante puisque les soins maternels sont prodigués exclusivement à leur progéniture, c'est la sélectivité (Nowak, 1999). Les facteurs facilitant la reconnaissance de l'agneau diffèrent s'il est à distance ou à proximité de sa mère.

<figure><img src="image_27.png" /><figcaption>Figure 10 : Photos d'agneaux élevés avec leur mère</figcaption></figure>
Si l'agneau est à distance de sa mère, ce sont les signaux acoustiques et visuels qui vont faciliter la reconnaissance (se met en place 12 heures après la naissance) tandis que lorsque l'agneau est proche de sa mère, ce sont les critères olfactifs qui vont être privilégiés pour la reconnaissance (se met en place 4 heures après la naissance) (Nowak, 1999). Cette reconnaissance et cette préférence ont également lieu dans le sens de l'agneau envers sa mère (Nowak et al., 1997). De plus, des agneaux qui mangent des aliments aromatisés en présence de leur mère avant le sevrage, mangent plus de cet aliment après le sevrage que les agneaux qui ont mangé cet aliment aromatisé avec d'autres brebis ou qui ont mangé des aliments non aromatisés avec leur mère (Saint-Dizier et al., 2007). Cette expérience montre que le développement des préférences alimentaires des agneaux dépend notamment de l'observation du comportement de la mère. Il est cependant important que l'agneau ait accès, en complément, aux indices olfactifs caractérisant la nourriture (Saint-Dizier et al., 2007). L'expérience précoce et le lien avec la mère semblent donc être des paramètres importants pour le bon développement (comportemental, immunologique, ...) de l'agneau.

b. Agneaux en allaitement artificiel :

<figure><img src="image_28.png" /><figcaption>Figure 11 : Photo d'agneaux âgés de 3 jours en allaitement artificiel</figcaption></figure>

Dans les élevages laitiers, l'agneau est séparé de sa mère pour être élevé en allaitement artificiel. Cependant, même en dehors des élevages laitiers, il arrive parfois que les agneaux ne puissent pas être élevés avec leur mère. Ils sont alors élevés en allaitement artificiel (Figure 11). Cet élevage en allaitement artificiel est notamment indispensable pour sauver des agneaux dont la mère n'est pas maternelle, pour sauver des agneaux dont la mère est malade (par exemple, en cas de mammite) ou encore lorsqu'ils sont plus de 2 ou 3 par portée et que la mère ne peut pas tous les nourrir. Cependant, cet élevage en allaitement artificiel a un impact sur le développement des agneaux.
Dans ce type d'élevage, les agneaux sont élevés en groupe avec leurs congénères de même âge mais sans figure maternelle. Contrairement au lien qui se crée entre la mère et son petit, aucun lien affectif spécifique ne se développe entre les agneaux du groupe (Gaudin, 2015). Un lien affectif sélectif se développe seulement lorsque les agneaux sont élevés en binôme avec un congénère particulier, ce qui favorise une proximité entre les deux agneaux. Ce lien créé semble être différent de celui créé entre une mère et son agneau, il ne correspond pas à un lien d'attachement (Gaudin, 2015). L'allaitement artificiel a également des effets délétères sur la santé des agneaux (Latham & Mason, 2008) notamment en induisant une mortalité plus élevée qu'en élevage sous la mère. Il a également été montré que l'allaitement artificiel entraîne une différenciation plus tardive de l'hypophyse (Love et al., 2021).

Les agneaux en allaitement artificiels semblent donc avoir un développement comportemental et cérébral différent des agneaux élevés en allaitement maternel.

## 5. Comment contrecarrer les effets de l'allaitement artificiel ?

Dans le but de contrecarrer les effets de l'allaitement artificiel, des études ont été menées afin d'enrichir le milieu d'élevage des agneaux. Une possibilité pour enrichir le milieu et pallier l'absence de figure maternelle est d'ajouter un objet dans le milieu. Des macaques rhésus élevés avec un objet explorent de façon plus importante un environnement nouveau s'ils sont en présence de cet objet (Harlow, 1959). Lorsqu'ils sont placés dans un environnement nouveau sans l'objet familier, leur réaction de détresse est moins forte qu'une séparation maternelle (Reite et al., 1978). Ces études semblent donc montrer que chez les primates non humains, le lien qui s'établit avec un objet est moins fort que le lien établit avec la mère. De plus, Cairns (1966) a montré qu'il était possible de créer un lien entre un agneau et un objet large (tel qu'un ballon) ou une figure non maternelle (brebis autre que la mère ou chien), le lien crée entre un objet et un agneau étant moins fort que le lien créé avec une figure sociale. Gaudin et al. (2018) a montré que des agneaux en allaitement artificiel peuvent créer un lien affectif avec les congénères avec lesquels ils sont élevés alors qu'ils ne créent pas de lien fort avec l'objet avec lequel ils sont élevés. Pour les veaux laitiers, des essais ont été menés pour utiliser des vaches nourrice (Brunet et al., 2016). Ces vaches nourrices vont allaiter plusieurs veaux à la fois et donc permettre de ne pas recourir à l'allaitement artificiel. Cependant, cette technique peut être limitée par le refus de la vache nourrice d'accepter un veau qui n'est pas le sien, il faut donc tenter l'adoption le plus tôt
possible après la naissance. Cet allaitement avec une nourrice et non en allaitement artificiel
favoriserait la croissance des veaux (Brunet et al., 2016).

6. Objectif de l'étude :

Pour contrecarrer les effets délétères de l'allaitement artificiel, différentes méthodes ont été
testées pour enrichir le milieu de vie des animaux. Dans cette étude, nous voulons enrichir
le milieu grâce à la diffusion de musique classique et étudier l'impact de cette diffusion chez
des agneaux Ile de France en allaitement artificiel. L'hypothèse est que la diffusion de
musique peut aider à contrecarrer les effets délétères de l'allaitement artificiel. Plus
généralement, l'objectif est d'étudier l'impact de l'expérience précoce sur les
comportements adaptatifs.

Pour réaliser cette étude, nous avons choisi de considérer la musique comme une stimulation
auditive sans *a priori* sur le sens particulier que la musique pourrait avoir pour l'animal. Ce
parti a été choisi après une première recherche bibliographique relativement peu fructueuse
chez les mammifères d'élevage alors que les témoignages sont nombreux dans les médias.
Dans ce contexte, nous avons entrepris la rédaction d'un article de revue qui, après avoir
rappelé quelques généralités sur les sons, met en lumière, chez les vertébrés, les similarités
anatomiques permettant de percevoir les sons. Cette revue illustre ensuite des exemples
d'usage de la musique auprès de mammifères d'élevage ou vivant en captivité.
II. Stimulation auditive chez les animaux

1. The use and impact of auditory stimulations in animals

Article soumis pour publication le 26 mars 2021
dans Journal of Interdisciplinary Methodologies and Issues in Sciences
En révision
<figure><img src="image_29.png" /></figure>

# The use and impact of auditory stimulation in animals

Guillaume UBIEMA<fnref n="1" />,#, Marine SIWIASZCZYK<fnref n="1" />,#, Céline PARIAS<fnref n="1" />, Roman BRESSO<fnref n="2" />, Christophe HAY<fnref n="3" />, Baptiste MULOT<fnref n="4" />, Scott A. LOVE<fnref n="1" />,*, Elodie CHAILLOU<fnref n="1" />,*

*Correspondance : elodie.chaillou@inrae.fr; scott.love@inrae.fr

# authors with equal contribution

DOI : 10.18713/JIMIS-ddmmyy-v-a

Soumis le 26 mars 2021 – Jour Mois-en-lettres Année

Volume : 9 – Année : AAAA

Titre du numéro: **Methods to assess the effects of sensory stimulations on wellness**

Éditeurs : Sandra Perez, Gabriel Gandolfo

## Abstract

Music can cause pleasant sensations in humans whereas some noises can cause discomfort. The effects of music and noise have also been somewhat studied in animals, showing different impacts. In this review we aim to illustrate the differences and similarities between animals, in terms of their sensitivity to auditory stimuli (noise or music), by first recalling some generalities about the physical characteristics of sound and the biological bases of hearing. Second, based on the studies reported in this review, we conclude that ambient noise is harmful and/or stressful, and that musical sounds can take many forms with a large range of impacts in animals. Finally, we present two practical examples of the use of music with animals (one in the context of a zoo and the other in cattle breeding) and an example of an experiment designed to understand the impact of music on neonate lambs. These three examples highlight how music can help to improve animal welfare.

## Keywords

acoustic ; welfare ; music

<footnote n="1">¹ PRC, INRAE, CNRS, IFCE, Université de Tours, France</footnote>

<footnote n="2">² Musicoprod, 72500 Dissay-sous-Courcillon, France</footnote>

<footnote n="3">³ La maison d'à côté, 41350 Montlivault, France</footnote>

<footnote n="4">⁴ ZooParc de Beauval & Beauval Nature, 41110 Saint-Aignan, France</footnote>
# I INTRODUCTION - GENERALITIES ABOUT THE ACOUSTIC PROPERTIES OF AUDITORY STIMULI AND THE BIOLOGICAL BASES OF HEARING

## 1.1 Acoustic properties of sound

Sound is a physical phenomenon, defined as a mechanical vibration propagating as a longitudinal and periodic wave within a material medium. It is characterized by:

- the amplitude of pressure change expressed in Pascal (Pa),

- the wavelength (λ) which is the distance over which the wave's shape repeats,

- the frequency expressed in Hertz (Hz) which is the number of pressure variations per second,

- the period which is the time interval between two identical and successive vibratory states in which the wave propagates,

- and the propagation speed which depends on the medium (Hansen et al., 2017).

The decibel (dB) is the unit used to measure loudness. In humans, the audibility threshold is set at 0 dB, discomfort being observed with a sound of 110 dB and pain with a sound of 130 dB (https://www.ncbi.nlm.nih.gov/books/NBK390300/).

A large variety of sounds are found in the environment. Based on their frequency profile, it is possible to discriminate between pure and complex sounds (Fig. 1A). Pure sounds are composed of a single sinusoid, whereas complex sounds are composed of several different frequencies. The discriminant between music and noise is in the organization of these different frequencies. For musical sounds, the frequencies are multiples of the fundamental frequency and are often referred to as “harmonics” (Fig. 1B). For noise, the frequencies can also be numerous but without specific organization with each other (Fig. 1C). Thus, noise is a non-periodic sound, which is often perceived as unpleasant or harmful (Walker et al., 2011). By analogy with white light and its visible spectrum, there is also the concept of white and colored noises. Based on physical properties, white noise is a theoretical construct of a signal covering an infinite bandwidth and flat power spectral density over the frequency range relevant to the environment (Zhivomirov
<figure><img src="image_30.png" /><figcaption>Figure 1: Waveform of different sounds. A. Pure sound. B. Musical sound, *f* is the fundamental frequency (also named as the 1st harmonic), 2*f* is the 2nd harmonic (1/2 of the fundamental frequency), 4*f* is the 4th harmonic (1/4 of the fundamental frequency). C. Noise.</figcaption></figure>

2018). The colored noises, such as violet, blue, pink or red, are distinguished according to their power spectral density and the spectral slope rate (for more details, see table 1 in Zhivomirov 2018). Like musical sounds, ambient noise, white noise and ecological noise could have an impact on health, behavior or the physiology of animals and humans. When investigating this impact it is important to consider the hearing, or audible, range of the species.

## 1.2 Hearing range

Sound frequencies are classified according to the audible range, that of humans being between 31 Hz and 17,600 Hz, conventionally described as being between 20 Hz to 20,000 Hz, at a sound level of 60 dB in air (Jackson et al., 1999). According to the human hearing range, sounds with frequencies below 20 Hz are classified as infrasound (Leventhall, 2007) and those with frequencies above 20,000 Hz are classified as ultrasound (Carlsen, 1975). However, the idea that a sound with a frequency below 20 Hz is inaudible is disputed (Leventhall, 2007). Indeed, by increasing the sound level (more than 80 dB), it is possible to hear sounds with frequencies below the 20 Hz threshold, this capacity also depending on the age of the person (Leventhall, 2007). Then, to evaluate the impact of the different types of auditory stimulations in non-human animals, it is necessary to take into consideration the hearing range of each species (Fig. 2). For example, the bottlenose dolphin (*Tursiops truncatus*) is able to distinguish sounds from 100 Hz to ultrasounds up to a frequency of 150,000 Hz (Houser and Finneran, 2006) while the humpback whale (*Megaptera novaeangliae*) and the elephant (*Elephas maximus*) are able to detect infrasound as low as 15 (Tubelli et al., 2018) and
17 Hz (Heffner and Heffner, 1980), respectively. This capacity to detect infrasound is not restricted to large mammals as it has been shown that chickens (*Gallus gallus domesticus*) are also able to detect infrasound as low as 9 Hz (Hill et al., 2014).

<figure><img src="image_31.png" /><figcaption>Figure 2: Distribution of hearing ranges defined in Human (red) compared to other mammals (blue). The hearing ranges on terrestrial animals were established at 60 dB: Elephant: (Heffner and Heffner, 1980); Cat, Cattle, Dog, Goat, Human, Sheep, Pig (Heffner 1998); Non-human Primate: (Heffner 2004).; Chicken (Hill et al., 2014). Note that Chicken' hearing range was extended from infrasound (9.1 Hz) like Elephant and Humpback whale. Carp (Vetter et al., 2018); Bottlenose dolphin (Houser and Finneran, 2006); Humpback whale (Tubelli et al., 2018).</figcaption></figure>
**1.3 Biological basis of hearing**

**1.3.1 The auditory system**

Hearing is a complex biological system that analyses auditory stimuli through acoustical (frequencies spectra) and spatial (localization of the stimuli) information. Anatomically, the auditory system includes a peripheral part with the outer, middle and inner ear, and a central part with a specific neuronal network (Evans 1992). The reception and amplification of auditory signals are performed by the outer and middle ear; the transmission of signal frequencies is performed from the middle ear to the inner ear, including the cochlea. The cochlear nerve encodes the auditory signals, according to their frequencies, through the hair cells (mechanosensitive cells) from the cochlea. This first step of the hearing process, involving the peripheral auditory system, allows to translate acoustic oscillations into spatio-temporal patterns of neuronal discharges (Evans, 1992). The second step concerns the central auditory system with the neuronal network starting from the cochlear nucleus to successive brainstem and thalamic structures, before integration by auditory cortex (Evans, 1992; Pickles, 2015; Fig. 3). Briefly, neuronal discharge from the cochlear nucleus is transmitted to the lateral and median olivary nuclei through bilateral projections. After this first relay, the signal is transmitted through ipsilateral projections (lateral lemniscus tract) from the olivary nuclei to the inferior colliculi. The last relay from the inferior colliculi to the auditory cortex involves the medial geniculate body of the thalamus (Evans, 1992; Šuta et al., 2008). Throughout these ascending neural paths, integration is

<figure><img src="image_32.png" /></figure>

ensured during each relay to refine the neuronal encoding of the auditory information presented to
the cortex (Pickles, 2015).
Figure 3: Schematic representation of the neuronal network of the auditory system from cochlea (inner ear) to the auditory cortex. Based on mammal brain anatomy: LSO, lateral part of the superior olivary nucleus; MSO, median part of the superior olivary nucleus; IC, inferior colliculi; MGB, medial geniculate body.

The examination of the auditory system in different vertebrates shows that it presents similarities in its organization with a peripheral auditory system with external part, middle ear and inner ear, and a central peripheral auditory system, including brainstem, thalamic and cortical projections (Itatani and Klump, 2017; Köppl, 2011; Ladich and Winkler, 2017; Reis et al., 2017; Roberto et al., 1989; Rouiller and Welker, 2000; Šuta et al., 2008). However, across vertebrates anatomical differences are also observed for the external auditory system, the upstream part of the tympanic membrane (Ladich and Winkler, 2017). Amphibians have an external tympanic membrane and no real external organ. Fishes have no outer or middle ear, their role being partially performed by the swimbladder. Birds and land mammals possess external auditory canals to conduct acoustic oscillations to the tympanic membrane. In cetaceans, external auditory canals and tympanic membrane are present but do not conduct the acoustic oscillations. Whatever the species, most of the similarities are observed in the inner ear organization (Ladich and Winkler, 2017). It is interesting to note that the anatomo-functional organization of the peripheral auditory system enables a species to hear the sound frequencies produced by that species (Ladich and Winkler, 2017). In other words, the frequency hearing range of a species corresponds to the frequency range of sounds produced by the species. Concerning the central auditory system, most of the brain structures identified in humans have also been anatomically identified in other mammals or their homologous structures in other vertebrates (birds, fishes, etc.; Oertel, 1999; Rouiller and Welker, 2000). The existence of a central auditory system throughout the animal kingdom is not sufficient to confirm the ability of animals to perceive, analyze, recognize and/or interpret all types of sounds such as music. However, behavioral studies have highlighted the ability of different vertebrates to use vocal communication.

### 1.3.2 Acoustic communication and emotion in animals

Animals can communicate with each other using vocalizations (Sebe et al., 2008). Vertebrates have different repertoires of vocalizations that are used for communicating in social contexts and/or their emotional state. For example, 11 types of vocalizations have been identified in guinea pigs, some of which can lead to a behavioral response in conspecifics (Berryman, 1981), 21 in cats, which differ according to context (Tavernier et al., 2020) and a dozen in elephants, which vary with age (Soltis, 2009). Ewes can discriminate between their own offspring and other lambs based on vocalization alone (Sebe et al., 2008). For cattle (Green et al., 2018) and sheep (Guesdon et al., 2015, 2012), two gregarious species, vocalizations can also reflect the emotional state of individuals, particularly in situations of social separation. These observations are not specific to terrestrial mammals, since
vocalizations are essential in most bird species, particularly songbirds (Filippi et al., 2019) and are present in marine mammals (Erbe et al., 2016), amphibians (Velásquez, 2014), crocodilians (Vergne et al., 2009) and fishes (Ladich and Winkler, 2017). Several authors have hypothesized that non-human animal vocalizations can be compared to human language and music, on the basis that these different sound productions have rhythm (Ravignani et al., 2019) and a temporal profile (Filippi et al., 2019). Such a hypothesis is all the more conceivable since it can be based on biological foundations such as animal hearing ability (see part 1.2) and strong similarities in the auditory system throughout the non-human animal kingdom (see part 1.3).

Considering the universality of both the auditory system and vocal communication, it is legitimate to postulate the existence of a universal effect of music within the animal kingdom, in particular as a means of improving welfare, a use that has been reported several times in different media<fnref n="2" /> and more rarely in research journals (Alworth and Buerkle, 2013; Dhungana et al., 2018). To address this question, we propose to consider auditory stimuli along two axes: auditory stimuli qualified as noise and auditory musical stimuli. For each of these, we will review the scientific literature that examines its influence on animal emotional responses<fnref n="3" />.

## II IMPACT OF NOISE EXPOSURE ON DIFFERENT WELFARE-RELATED PARAMETERS

The French Ministry of Ecology defines noise as,

"an acoustic phenomenon producing an auditory sensation considered unpleasant or disturbing. Excessive noise has effects on the organs of hearing (physiological dimension), but can also disturb the body in general, especially sleep and behavior (psychological dimension). (...) Noise pollution can result from three main sources: transport, neighbors and activities."<fnref n="4" />

The World Health Organization has developed preventive strategies to reduce noise exposure (WHO, 2018). From a physical point of view, noise is defined as a complex, non-periodic sound. In non-human animals, various studies have demonstrated the harmful effects of noise on the health and welfare of farm animals or animals kept in zoos.

<footnote n="2">² https://www.livescience.com/33780-animal-music-pets.html ; https://www.youtube.com/watch?v=_2raNqztPX0 ; https://www.youtube.com/watch?v=1ZW4Ska8gvA ; https://www.petmd.com/blogs/thedailyvet/aobrien/2013/march/music-influences-milk-production-in-cows-29895 ; https://www.sciencesetavenir.fr/nature-environnement/la-musique-adoucit-les-vaches_4441</footnote>

<footnote n="3">³ As best as possible, we will specify the information relating to animals and/or to experimental plan which could influence the emotional responses in addition to the auditory stimuli (gender, age, breed...).</footnote>

<footnote n="4">⁴ https://www.ecologique-solidaire.gouv.fr/bruit-et-nuisances-sonores</footnote>
## 2.1 Ambient noise

The impact of ambient noise is examined according to the animals' habitat. For example, farm animals are exposed to many different types of sounds throughout their life, especially within their housing from machinery, human intervention and motor vehicles, during transport, and in slaughterhouse. This noise is generally of high intensity and can cause deleterious behavioral and neurophysiological effects to the animal during chronic or sudden exposure. In sheep (1-3 years of age), it was found that the addition of ambient noise (90-100 dB) during transport did not change their heart-rate, salivary cortisol levels or behavior (Hall et al., 1998). This indicates that the extra noise did not further stress, or appease, the sheep in this already stressful situation. In contrast, the noise associated with the handling (human voices, metal-on-metal clanging, etc.; 85-95 dB) of crossbred beef heifers increases their heart-rate and movement (Waynert et al., 1999). However, these increases in heart-rate and movement were lower after five days of exposure, suggesting that the animals had gotten used to the noise presented and its significance had changed. Evaluation of chronic exposure to ambient noise was studied in growing castrated male Landrace pigs (Otten et al., 2004). At 12 weeks of age, individuals were submitted to a 120 min-period of broadband noise (mixture of noises between 0 and 23 kHz, 90 dB) either daily or every 2-3 days for four weeks. In this experimental context, the pigs exposed to noise every day had delayed daily weight gain, and alteration of their corticotrope axis (higher plasma cortisol and adrenal corticotropic hormone, ACTH), which is involved in the response to stress (Otten et al., 2004). In pigs exposed to noise every 2-3 days, higher levels of glucocorticoid receptor binding in the hippocampus and alteration at the adrenal levels of the corticotrope axis such as modification of the cell densities in the adrenal medullary were observed (Kanitz et al., 2005). Together, these results suggest a negative emotional impact of acute or chronic noise-exposure in farm animals. Similar conclusions have been made in dogs who were exposed to an alarm for 3-mins (75 dB, 0.25-8 kHz) and subsequently displayed muscle contraction, piloerection and salivation, associated with increased heart rate and blood pressure, and an increased plasma ACTH, noradrenaline and cortisol (Engeland et al., 1990). Consequences of noise exposure have also been studied in chickens, this animal model presenting interest for evaluating the impact of *in ovo* exposure (Donofre et al., 2020; Kathpalia et al., 2019; Sanyal et al., 2013). As for farm mammals and dogs, noise exposure produced stress as indicated by increased corticosterone in 42-day-old broilers (acute exposure of 10 min at 80 dB and 100 dB, Chloupek 2009), and heterophilic/lymphocyte ratio in 36-week-old hens (exposure of 60 min/day at 90 dB (Campo et al., 2005). *In ovo* exposure to noise (110 dB, 30-3000 Hz, for 15 min/hour from day 10 to hatching; Kathpalia et al., 2019; Sanyal et al., 2013) lead to increased noradrenaline plasma at 1-day-old, impaired spatial orientation with decreased expression markers of maturation in
hippocampus (synaptophysine, postsynaptic density-95: Sanyal et al., 2013; Brain-derived neurotrophic factor: Kathpalia et al., 2019). Together these studies affirm that farm animals (mammals and birds) are sensitive to noise exposure (90-110 dB) and that noise has a stressful character that can alter the animal's health from a physiological, behavioral, neurobiological and developmental point of view. Moreover, these alterations are also observed at intensities below the human discomfort threshold (110 dB).

## 2.2 White noise

Although ambient noise has been shown to have adverse health effects, some noise may have positive effects. This is the case with white noise. This sound occurs when a variety of sounds of different frequencies are played at a constant level. In clinical uses, white noise is thought to facilitate sleeping after artery surgery (Williamson, 1992). It also improves learning in healthy adults (Angwin et al., 2017) and could be a possible therapeutic option for children with attention deficit hyperactivity disorder (Pickens et al., 2018). It has also been found to improve performance in cognitive tasks in adult monkeys (*Macaca arctoides*) exposed to white noise (80 dB) for 15 min before the task (Carlson et al., 1997) and also to have a calming effect on infant Japanese macaques (5-27 days old) in stressful situation (Kawakami et al., 2002). However, the impact of white noise is not always beneficial. Whereas 2-day-old hen chicks, spent more time in a food-conditioned place, they spent less time in white noise-conditioned place (Jones et al., 2012), suggesting that white noise can also be an aversive stimulus during a conditioned place preferences test.

## 2.3 Ecological sounds

Other noises such as so-called "ecological" sounds, which consist of auditory stimuli in relation with the animal's natural environment, can also be beneficial to animals. In particular, rainforest sounds, containing sounds of animals (e.g. birds, primates, elephants) and forests (e.g. rain, river, wind), have been used in several studies. The sound of a rainforest has a tendency to increase social interactions and resting time on captive gorillas by masking ambient noise (8-41 years old; Wells et al., 2006). But this effect is questioned in adult gorillas since some studies have reported increased agitation in 12-23 years old gorillas (Ogden et al., 1994) while others have reported no effect on anxiety behavior (Brooker, 2016). These differences between studies could be due to the different experimental designs (duration of exposure, animal housing, etc.) or to interindividual variability.

On the basis of the studies reported in section 2, it is difficult to conclude whether or not exposure to ambient noise, white noise or environmental noise has adverse or positive effects, since these noises were broadcast at different intensities. Nevertheless, at equivalent sound level (60-80 dB), it seems
that exposure to white noise (ocean waves) or conspecifics vocalizations would be more beneficial
than exposure to ambient noise (noises truck, train...) at least in chickens (Campo et al., 2005;
Donofre et al., 2020). But controlling the sound level parameter alone is not sufficient to compare the
effects of exposure to these different noises. In addition to the duration, or even suddenness, or
frequency of exposure, it is also important to consider all the characteristics of auditory stimulation.
In particular, as we will see in the following section, exposure to musical sounds will not have the
same effects depending on the level, of course, but also on the instruments, the harmonies and the
tempo.

III IMPACT OF MUSIC EXPOSURE ON DIFFERENT WELFARE-RELATED PARAMETERS

**3.1 How to define music?**

Music is one of the oldest forms of art and is an integral part of our daily lives. Music could be defined
as an aggregation of notes or as a succession of notes. But, for André Manoukian, French pianist,
jazzman and composing author, this succession of notes could be considered like music when this
sound creates a “sound sensation”<fnref n="5" />. This transformation is done when the notes are submitted to a
rhythmic arrangement. However, our ear is able to discriminate dissonances and consonances, a
phenomenon based on the notion of beat, when several frequencies are heard simultaneously.
Consonances lead to a calming effect and dissonances to a feeling of being aggressed, harmony being
the art of combining both sensations, in occidental music. A. Manoukian defined “the harmony”
(accordance) as a low proportion of frequencies between the “G” (440 Hz) and the octave (880 Hz),
in other words when the proportion is equal to a whole number. For songwriter Frank Zappa<fnref n="6" />, written
music on a score becomes a musical experience when converted into “twirling air molecules”; then
each air disturbance caused voluntarily is seen as a composition. Moreover, for this composing author,
because each (physical) component of a sound could be perceptible (audible), any sound can be
consumed as music. For both A. Manoukian and F. Zappa, music has the capacity to create a personal
sensation.

According to the diverse components of music such as intensity, tempo, pitch, timbre, nuances, and
instruments, and because not all individuals are sensitive to the same music (or component), the large
panel of impacts of music exposure is not surprising.

Music exposure can provoke different emotional states in human beings, which can have both
beneficial and harmful effects on health. In particular, it has been shown that music can be used as a

<footnote n="5">⁵ Comments from his column on the radio show “Par Jupiter”, France Inter, 2020-08-26 “Le Cluster”.</footnote>

<footnote n="6">⁶ Zappa par Zappa, juin 2005, Eds De L’Archipel, 396p, ISBN 2840877159 (original edition 1989).</footnote>
therapeutic element in the emergence of a new discipline: music therapy. It can regulate
cardiovascular parameters (Koelsch and Jäncke, 2015), relieve pain (Cheung et al., 2018; Lee, 2016),
neurodegenerative diseases (García-Casares et al., 2018) and psychiatric disorders such as depression,
anxiety, schizophrenia, sleep disorders and dementia (Wang and Agius, 2018). In this context, many
authors also advocate that music has beneficial effects on animals. By analogy with the idea of a
“sound sensation” proposed by A. Manoukian and F. Zappa, examining the impact of musical
exposure on animals requires considering their emotional reaction and the parameters that
characterize musical sound such as intensity, tempo or instrument.

## 3.2 Sound level

To our knowledge, very few studies have compared the impact of different sound levels of music on animals, i.e. a study comparing two different exposure intensities of the same piece of music. In subadult meat chicks, the impact of music exposure was studied with two styles of music (dinner vs. rock'n roll) at two different sound levels (70 vs. 85 dB). Whatever the level applied, chicks exposed to music expressed stress-related behavior from the first exposure and expressed jerking head motion after two weeks of exposure (Christensen and Knight, 1975). The sound level chosen for an experiment is often chosen as a function of the ambient noise level in the environment. For example, rainbow trout or common carp reared in tanks (with recirculating water system) are exposed to ambient noise levels close to 122 dB and the impact of music exposure was evaluated at a level of 130 dB (Papoutsoglou et al., 2013, 2010, 2007). In these different studies, the animals from the control conditions were exposed to ambient noise such as animal voices and fans (Campo et al., 2005), the noise of chickens (Christensen and Knight, 1975) or machines (Papoutsoglou et al., 2013, 2010, 2007) at a level smaller than those used for music exposure. In this context, two questions could be asked concerning the reported stressful impacts of music described. In chickens, for example: is it the consequence of sound level (75 dB in Campo et al., 2005; 70 and 85 dB in Christensen and Knight, 1975) or the consequence of novelty or unknown noise. In chicks, prenatal exposure to music has been evaluated at two different levels. At 110 dB, prenatal music exposed chicks (from E10) performed better in spatial orientation, learning and memory at 1 day old than prenatal noise exposed chicks (Sanyal et al., 2013). Similar effects were reported with a level of 65 dB (Chaudhury et al., 2010). At 110 dB as well as 65 dB, the cognitive impacts of prenatal music exposure are associated with neuronal modifications in auditory nuclei (Alladi et al., 2005, 2002; Kathpalia et al., 2019) and hippocampus (Chaudhury et al., 2010, 2009, 2008, 2006; Chaudhury and Wadhwa, 2009; Sanyal et al., 2013), through different actors of neogenesis. However, depending on the study and the variable measured, these effects were also found when chicks were prenatally exposed to species-specific sounds at the same level.
In conclusion, the impact of music sound level is not clear since it was evaluated in interaction with other environmental factors such as light intensity (i.e. Papoutsoglou et al., 2010), including the frequency factor that differs between music and control noise (i.e. Kathpalia et al., 2019). Perhaps the main precaution to take when testing the impact of music exposure is to present it at a level equivalent to that of the control noise (ambient noise). Finally, even within a piece of music, the sound level can vary. These variations in music are called nuances. They are expressed by indications ranging from pianissimo to fortissimo, and crescendo and decrescendo, nuances to which we must also add the notion of tempo.

### 3.3 Tempo

Tempo is the speed at which a piece of music is played and is expressed as the number of beats per minute (bpm). Tempo lower than 40 bpm is named largissimo and tempo higher than 188 bpm is named prestissimo. Based on the work from Karageorghis and Terry (Karageorghis and Priest, 2012), in humans, tempo, musicality, cultural impact<fnref n="7" /> and association<fnref n="8" /> are the four characteristics of the piece of music that contribute to its impact on motivational and physiological responses (heart rate at rest, during physical exercise). Among these factors, tempo is suggested to be the most relevant. Several studies aimed to demonstrate a significant relationship between tempo and heart rate at rest or during exercise (Karageorghis et al., 2006; Navarro et al., 2018; van Dyck et al., 2017).

In animals, a famous YouTube video<fnref n="9" /> of Snowball, a 12-year-old male cockatoo "dancing in rhythm" to music, led Patel and colleagues to study this phenomenon in 2009 (Patel et al., 2009a). Then, Snowball was subjected to a tempo of 108 bpm (moderato – allegretto) that was increased and decreased. Snowball was found to be able to synchronize to the tempo and even with variations (Patel et al., 2009b). This ability to synchronize the motor activity with music tempo was also observed in a 1-year old female lion (Cook et al., 2013).

The question of tempo was also examined with studies of preference. For example, adult marmosets and tamarins expressed a preference for slow tempo (65 bpm, adagio) compared to faster tempo (370 bpm, prestissimo; Mc Dermott and Hauser, 2007). At the same prestissimo (200 bpm), 4 week old piglets were more often seen walking, standing or tail wagging compared to piglets exposed to adagio (65 bpm; Li et al., 2019). This suggests that fast tempi would stimulate piglets and in a positive way since tail wagging is an indicator of positive emotion in pigs (Reimert et al., 2013). Finally,

<footnote n="7">⁷ "Cultural impact concerns the pervasiveness of the music within society or a sub-cultural group" (Karageorghis and Priest, 2012).</footnote>

<footnote n="8">⁸ "association refers to the extra-musical associations that may be evoked" (Karageorghis and Priest, 2012) such as a film.</footnote>

<footnote n="9">⁹ https://www.youtube.com/watch?v=N7IZmRnAo6s</footnote>
young and adult gorillas were more active when they were exposed to music, with fast tempi (106-111 bpm) inducing higher activity than slow tempi (33-57 bpm) (Brooker, 2016).

It is therefore certain that tempo has a considerable influence on behavior and that a fast tempo increases animal activity. However, increased activity does not necessarily indicate well-being and positive emotion. In some cases, it may express emotional stress and a desire to escape. Further research is therefore necessary to conclude more precisely whether a tempo has a positive or negative effect on the animals.

## 3.4 Musicality

Musicality refers to harmony and to melody. As seen above (part 3.2.), musicality is suggested to play an important role in the motivational impact of music (Karageorghis and Priest, 2012). To take into account this factor, we need to investigate the capacity of animals to be sensitive to harmony and to discriminate instruments and/or voice in a piece of music. Harmony is the combination of successive musical sounds. In theory, “harmony is the science which regulates the formation and sequence of chords. [...] We noticed that superimposed melodic lines frequently provided chords of the type: C-E-G. It has been recognized that a string which vibrates, a ball which is struck, generate sounds in the same chords.”<fnref n="10" /> Consonant sounds (chords) are recognized by individuals as a unity, this cohesion and stability produces a pleasant listening sensation. Conversely, dissonant sounds give an impression of instability and an unpleasant listening sensation. Depending on culture, lifestyle and habits, it is therefore easy to understand that what seems consonant to one individual will seem dissonant to another. In this context, how could we define a consonant and dissonant sound for animals? Some authors addressed this question in European starlings (age unknown) (Hulse et al., 1995), adult Java sparrow (Watanabe et al., 2005) and 6 to 7 years old Japanese macaques (Izumi, 2000) by using operant conditioning tests. All found that the animals were able to distinguish between consonant and dissonant sounds. Moreover, there is an “innate” preference for consonant sounds in infant chimpanzee (17 weeks old) (Sugimoto et al., 2010) and in subadult chicks (2 months old) (Chiandetti and Vallortigara, 2011).

Furthermore, some authors examined whether instrument and voice in a piece of music are differently perceived by animals. In this aim, effects of different types of music on social behavior were examined in chimpanzees (Videan et al., 2007). The animals (male and female between 4 and 48 years old) were subjected to known classical (Pavarotti), easy-listening (Enya) vocal or instrumental music.

<footnote n="10">¹⁰ Definition from the collective book « Initiation à la musique. A l’usage des amateurs de musique et de radio », Ed. du Tambourinaire, 1935, 418pp, (personal translation).</footnote>
These pieces of music also differed by their tempo (95-170 bpm for classical vocal and 50 to 90 bpm for easy-listening vocal). The authors concluded that instrumental music was more effective at increasing affiliative behaviors whereas vocal music was more effective at decreasing agonistic behaviors (Videan et al., 2007). Dogs presented with classical music (100-143 bpm) spent more time sleeping and less time vocalizing than dogs presented with heavy metal music (102-151 bpm), who spent more time body shaking (Kogan et al., 2012). This suggests a calming effect of classical music and an excitatory or stressful effect of heavy metal (Kogan et al., 2012). Because the characteristics such as sound level, harmony are unknown, it is difficult to fully understand these differences between types of music in the case of animals. However, they do support the idea that animals are able to express a preference between different types of music.

## 3.5 Music preferences

As mentioned before (3.1.), chimpanzees (Sugimoto et al., 2010) and chicks (Chiandetti and Vallortigara, 2011) are able to express preferences for consonant sounds compared to dissonant sounds. But, when animals are subjected to different pieces of music and to silence, individuals do not necessarily express the same preference - preferences are personal. For example, in four adult Java sparrows, two preferred Bach to Schoenberg or silence, and Vivaldi to Carter or silence. The two others preferred silence to Bach or Schoenberg. Interestingly, when preference was tested against noise, one of these two latter birds ran away from noise (Watanabe and Nemoto, 1998). Musical preferences have also been reported in growing piglets (Li et al., 2019) and adult tamarins and marmosets (Mc Dermott and Hauser, 2007). In milking cows, a study reported that the use of country music is efficient for conditioning cows on voluntary approach to an automatic milking system (Uetake et al., 1997).

The evidence of positive or negative impacts of auditory stimulations on animal physiology, immune system and behavior needs to be further investigated. Whereas harmful impacts of high levels of noise are reported in a large panel of species, positive impacts of music are more questionable but appear more evident in birds and primates than in other species. However, music is being more and more used in animal experimental facilities, for indoor reared farm animals or in zooparks. In the final section of this review, two examples of the practice of using music with animals (one in the context of a zoo and the other in cattle breeding) will be highlighted as well as an example of an experiment designed to understand the impact of music on motherless lambs reared with formula-milk.
# IV EXAMPLES OF PRACTICE

## 4.1 Preventing stress with music at the ZooParc de Beauval<fnref n="11" />

At the ZooParc de Beauval, the use of auditory stimulation began in 2001-2002 with the installation of speakers in the African savannah building that is dedicated to herbivores. These species are very reactive. Because of their gregarious nature, all the animals in the different groups could react sharply in a crowd movement after a sudden, loud or unknown noise. In addition to caregivers and veterinarians, the animals are exposed throughout the day to visitors, maintenance and construction workers. Caregivers first used radio with music and talk shows before changing to streaming music playlists. In this way, the animals were subjected to a large panel of music styles with different variations of rhythm and pitch. At night, background music was stopped to allow the animals to have a resting period with lesser ambient noise than during the day. The sound level of the auditory stimulations was neither measured nor fixed. The aim being that the level was sufficient to mask a large panel of ambient noise (metallic doors, engines...) but to be not too loud, at the risk of compromising the communication and the safety of the people working with the animals.

After the introduction of music the caregivers observed that the animals were more likely to keep calm and quiet when they were working in the building. For example, the Nyala (*Tragelaphus angasii*) housed in the Hippopotamus building with ambient music are less stressed than the Sitatunga (*Tragelaphus spekii*) housed on their own in an old building without music. Because of the positive impact of the auditory stimulation, it was also applied in the Asian savannah building since 2011-2012.

However the use of music is not applied to all species at the ZooParc de Beauval because not all species are as reactive to specific events as herbivores. In these other cases, specific protocols have been developed to handle individuals safely when necessary. For example, non-human primates are trained for simple medical procedures; carnivores such as lions, and tigers are not handled within the group; and species living in tropical buildings are presented with environmental sounds such as waterfalls and bird vocalizations.

Such a music exposure is therefore beneficial for the animal welfare whether it is a direct or indirect effect.

<footnote n="11">¹¹ Information collected during the phone interview with Baptiste Mulot, head of veterinarian (ZooParc de Beauval, Saint-Aignan, Loir-et-Cher), done the 21st of September 2020 by E. Chaillou and M. Siwiaszczyk.</footnote>
## 4.2 Music used in Wagyu breeding in the aim to produce a very high quality of meat<fnref n="12" />

Christophe Hay discovered the Kobe meat in Japan and testifies never having felt such as a sensation on the palate. Determined to produce meat of this quality in France, he first worked with a Wagyu beef breeder but noticed that the quality of the meat was irregular. Therefore, he decided to buy his own herd of Wagyu in 2019 (now composed of 52 animals) in order to take care of animal well-being, control each stage of breeding and therefore the quality of the meat. His herd is raised by one of his childhood friends, in natural meadows. The breeding method of Wagyu is very specific. First, animals spend 36 months in meadows and then 9 months in a park of 15 m². The animals still have visual contacts with their conspecifics and access to cattle brushes. During these 9 months, all feeding is controlled and made up of 65% of flax and cereals. The aim of Christophe Hay is to respect the animal and to produce the best meat. Thus specific attention is paid to the well-being of these animals to keep them as calm and relaxed as possible. In this aim, and to provide a "presence" in their park when the breeder is not there, they are presented with calm music from a yoga playlist. To provide the animals with rest time the music is not continuous throughout the entire day. Instead it is played for 2 hours in the morning and 2 hours in the afternoon. The animals are also massaged with brushes by the breeder for 30-45 minutes every day. These enrichments have been observed to indeed relax the animals and to therefore produce high quality meat, which is well marbled. The efficiency of this practice is demonstrated by the reaction of the Wagyu. This breed is known to be reactive and afraid by many situations, such as human contact. Interestingly, the rearing conditions developed by Christophe Hay and his friend, modify the valence of human which is spontaneously and voluntary approached by the individuals.

In this practice, music is included in a large panel of breeding practices, all of them playing a role in the well-being of the Wagyu. To maintain the best conditions possible throughout the life of the animal, Christophe Hay and his colleagues hope to be authorized to stream music during transport from the farm to the slaughterhouse. In this context, the animals could be maintained in a novel and stressful environment (track, slaughterhouse) with a known element, the music.

## 4.3 The use of music to counteract the negative impacts of motherless rearing conditions<fnref n="13" />

The aim of this study was to investigate the possibility that music exposure could counteract the negative impacts of maternal deprivation in sheep, which also results in formula-milk feeding rather than breastfeeding. This rearing condition is known to alter development in several mammal species.

<footnote n="12">¹² Information collected during the phone interview with Christophe Hay, Michelin-starred chef (La maison d'à côté, Montlivault, Loir-et-Cher, France) done the 22nd of September 2020 by E. Chaillou and M. Siwiaszczyk.</footnote>

<footnote n="13">¹³ This study was designed to test our hypothesis on male and female. Male lambs were born between the 4th and 6th of March, were subjected to the experimental design the 11th. The study was stopped the 16th of March due to the Covid-19 pandemic, which means no females were included.</footnote>
In lambs, this practice is commonly used in milk production and to save lambs when they cannot be reared by their mother. Our hypothesis was that music stimulates lambs which could be more active when they are exposed to music.

### 4.3.1 Method

To test our hypothesis, we designed a protocol in which male lambs were split into two groups at 7±1 days of age. In the first week of life the lambs were separated from their mother at 24 h after birth (allowing colostrum ingestion) and subsequently trained to drink formula-milk with rubber teats. Both groups, control (CONT, n=10) and music (MUS, n=10), were reared in two distinct parks (length 5 m, width 3 m, height 1.7 m) equipped with video monitoring. To avoid noise pollution between the groups the parks were located in two distant rooms. The walls of the both parks were covered with honeycomb boxes to improve sound diffusion and the ground was covered with straw. Each park was equipped with a water-drinker, 3 rubber teats for *ad libitum* access to formula-milk, a trough containing hay, a trough containing granules, heating light to prevent hypothermia, and traffic cones for allow playing enrichment (Fig. 4). Light was switched on from 8:30 am to 6:00 pm. The MUS park was equipped with two speakers set at 1.7 m high, for music streaming and the CONT park was equipped with two dummy speakers set at the same height. In CONT group, the sound level of ambient noise varied between 30 and 71 dB. In the MUS group, the level of noise (ambient and music) varied between 31 and 66 dB.

<figure><img src="image_33.png" /></figure>
Figure 4: Organization of the two distinct parks of control lambs (CONT) and of music-exposed lambs (MUSIC). Note that the CONT park is equipped with two dummy speakers (orange arrows) to simulate the presence of speakers as in the MUSIC park (yellow arrows). For the rest, the both parks present exactly the same equipment: trough with hay, through with granules, 3 rubber treats, heating light, honeycomb boxes and traffic cone (*).

Playlists were created with extracts from radio broadcasts "Radio Classique". These extracts mainly contained instrumental or vocal pieces, and some spoken extracts (announcing of pieces of music). The music streaming was performed for 45 min every 90 min from 10:30 am to 5:15 pm (total of 3:45 hours a day).

Audiovisual recording was performed continuously from 7:30 am to 18:30 pm. Behavioral observations (Table 1) were realized for three days every 10 sec on four distinct 5-min phases: before and after the 1st music streaming of the day (10:30 am), and before and after the last music streaming of the day (17:15 pm). For each lamb, 12 phases were observed (30 scans per phase) and data have been expressed in mean ± SD of number of times a behavior was observed (the maximum value being 30). The first observation (obs1) was done about two hours after the arrival of lambs in their respective park and human interventions (blood samples, weight...).

The impact of music exposure was tested by ANOVA repeated measures (# observation) with independent factor (CONT vs MUS) followed by post-hoc analyses corrected by Holm.

<table><thead><tr><th>Behavior</th><th>Definition</th></tr></thead><tbody><tr><td><strong>Posture</strong></td><td></td></tr><tr><td>Stand up</td><td>Lamb is standing on all fours</td></tr><tr><td>Lying down</td><td>Lamb's sternum is in contact with floor</td></tr><tr><td><strong>Locomotion</strong></td><td></td></tr><tr><td>Walk</td><td>Progress slowly</td></tr><tr><td>Run</td><td>Progress fast</td></tr><tr><td><strong>Exploration</strong></td><td>Lamb is standing, nose in contact with sniffing movements of a part of park (walls, floor...)</td></tr><tr><td><strong>Feeding</strong></td><td></td></tr><tr><td>Formula-milk</td><td>Lamb is feeding to the automatic milk supplier</td></tr><tr><td>Water</td><td>Lamb is drinking to the water-drinker</td></tr></tbody></table>
<table><caption>Table 1: Ethogram used for behavioral observations</caption><tr><td>Hay</td><td>Lambs is eating hay</td></tr><tr><td>Granules</td><td>Lamb is eating granules to the trough</td></tr></table>

### 4.3.1 Results

Looking at the mean number of times a behavior was expressed (Table 2), we noted that the highest occurrence was found for lying down posture, that the activity was related to the time of day (AM vs. PM observation sessions), and that the activity profiles differed throughout the three days and between groups. Statistical analyses confirmed these results (Table 2), suggesting that music impacts maintenance behaviors soon after three days of exposure. Group effects were found for locomotion and exploration, and interactions were found for all behaviors studied.

<table><caption>Table 2: Results of the statistical analysis</caption><thead><tr><th>Behaviors</th><th>Group effect (CONT vs MUSIC)</th><th>Observation effect (repeated measures)</th><th>Factors interactions (Group x Observation)</th></tr></thead><tbody><tr><td>Lying down posture</td><td>p = 0.54<br>F<sub>1,20</sub> = 0.40<br>η<sup>2</sup> = 7.7 x 10<sup>-4</sup></td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 36.57<br>η<sup>2</sup> = 0.41</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 28.31<br>η<sup>2</sup> = 0.32</td></tr><tr><td>Locomotion</td><td>p = 0.002<br>F<sub>1,20</sub> = 12.41<br>η<sup>2</sup> = 0.052</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 8.03<br>η<sup>2</sup> = 0.20</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 6.13<br>η<sup>2</sup> = 0.15</td></tr><tr><td>Exploration</td><td>p = 0.002<br>F<sub>1,20</sub> = 12.70<br>η<sup>2</sup> = 0.021</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 44.91<br>η<sup>2</sup> = 0.44</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 27.66<br>η<sup>2</sup> = 0.28</td></tr><tr><td>Feeding</td><td>p = 0.114<br>F<sub>1,20</sub> = 2.72<br>η<sup>2</sup> = 0.009</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 11.00<br>η<sup>2</sup> = 0.22</td><td>p &lt; 0.001<br>F<sub>11,220</sub> = 15.84<br>η<sup>2</sup> = 0.31</td></tr></tbody></table>

The two first days, control lambs were seen lied down more often during morning than afternoon (significant statistical differences were found between obs. 1 and 3, 2 and 4, 5 and 7, 6 and 8). The profile observed in music lambs is different since they were always seen lying down during the four observations of the day 1 (Fig. 5). The 2nd day, control and music lambs presented similar profiles and we noticed that during afternoon the control lambs were more often seen lying down than the music lambs (significant statistical differences were found between both groups for obs. 7 and 8). The day 3, music lambs seemed to be more active since they were less often seen lied down than the control lambs.

According to their age (7 days the day 1 of the observations), lambs were very rarely seen in locomotion (less than 5 times out of 30 scans per observation), this activity being more frequent
afternoon (obs. 3, 4, 7, 8, 11, 12) than morning (obs. 1, 2, 5, 6, 9, 10). Consistent with number of time
lambs seen lying down, control lambs were more often seen in locomotion the afternoon than music
lambs (significant statistical differences between control and music lambs for the obs. 3 and 4). From
the 2nd day, the both groups presented the same profiles for locomotion.

While locomotion was rarely found throughout the scans, exploration was found more than 20 and
feeding behavior more than 10 times out of 30 scans (Fig. 5). The first day, only some control lambs
were seen in exploration and it was only during afternoon (significant statistical differences between
obs. 1, 2 and obs. 4). Music lambs were more often seen in exploration than controls (significant
statistical differences between control and music lambs for obs. 7 and 8). The profile for feeding
activity was similar to thus found for exploration: its frequency was higher in the afternoon than in
the morning and increased throughout the study. Moreover the profiles between control and music
lambs were inverted from the 1st to the last day of the study (Fig. 5): day 1 control lambs were more
often seen eating than music lambs (significant statistical differences for the obs. 4) and day 3 the
contrary was found (significant statistical differences for the obs. 11 and 12).
<figure><img src="image_34.png" /><figcaption>Locomotion</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>Exploration</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>Feeding</figcaption></figure>

<figure><img src="image_37.png" /></figure>

Figure 5: Mean number of times control (n = 11 in blue) and music (n = 11 in orange) were observed in lying down posture, locomotion, exploration or feeding for 30 scans (every 10 seconds for 5-min period) done morning (AM) or afternoon (PM) during three successive days when the lambs were 1 week old.
### 4.3.2 Preliminary conclusions

Regardless of the studied behavior, it can be concluded that the lambs are more active in the afternoon than in the morning, and that exposure to music has not modified this organization.

When the lambs were not seen lying down, they were in exploration or feeding activities, and the number of times animals were active was higher on day 3 compared to days 1 and 2. This finding could be explained by the age of the animals (10 days at the end of our study).

The interpretation of day 1 is difficult and could be contradictory for each of the 2 groups. The high level of activity observed in the control lambs could be interpreted as stress or on the contrary as well-being since the animals expressed exploration and feeding behaviors. For lambs exposed to music, their inactivity can be interpreted as a certain well-being since they slept without disturbance when exposed to music. But, again, the interpretation of this result can be totally different and the inactivity can be seen as stress-related behavior such as immobility.

The differences found on days 2 and 3 are consistent with our hypothesis: lambs exposed to music are more active than controls (evidenced by exploration and feeding behaviors).

Altogether, these preliminary observations suggest that the conditions of music exposure used in our experimental design were able to modify the maintenance and exploratory behaviors after only three days of exposure.

## IV CONCLUSION AND REFERENCES

This review of the literature highlights that different kinds of sounds can impact differently on a large panel of functions, such as individual health, behavioral and physiological indicators of stress, or welfare, in humans as well as throughout the animal kingdom. While there are clear differences in the hearing ranges between vertebrates, there are also universalities in some parts of the auditory system, from its external part to the auditory neuronal network, and in an ability to communicate vocally and express emotions. This evidence provides support for the hypothesis that an ability to perceive and be impacted by music could be widespread in the animal kingdom.

The research reviewed shows that noise can have either negative or positive impacts on animals depending on the type of noise they are exposed to, the level of the noise and on other as yet unknown factors. More specifically to music, level, tempo and musicality appear to be important factors in determining its impact on an animal. However, more research systematically varying these factors in a variety of species is required to better understand their individual and interacting impact.

The examples provided here also highlight that, in practice, various stakeholders believe that music provides positive animal welfare benefits; while the limited scientific research reviewed indicates that several factors (duration, sound level, tempo, musicality etc.) should be considered carefully to ensure
that the impact is indeed positive. In this aim, two complementary fields of investigation could be
conducted, both of which require an interdisciplinarity approach. The first field of investigation would
aim to describe the acoustic environment of animals, especially in captive/indoor conditions (zoo,
livestock or pets), according to auditory stimulations and auditory abilities of a species. This first
field of investigation exists and could progress by associating bioacoustics, neuroscience, ethology
and comparative / evolutionary disciplines. The second field of investigation concerns the impact of
music exposure to animal welfare. Studies performed in this aim are rare, not always published,
whereas practices already exist in breeding and zoos. Developing participatory sciences with people
who use music with animals, ethologists, animal caregivers, breeders, veterinarians is a relevant
approach to evaluate the impact of music on animal welfare.

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**Acknowledgements**

This project is supported by Region Centre Val de Loire and INRAE Centre Val de Loire.
Experimentation was supported by the APR Neuro2Co grant (2018-2022), the PhD (Marine
Siwiaszczyk) salary is covered by the APR Neuro2Co grant, Beauval Nature and INRAE (Phase
department), internship gratification (Guillaume Ubiema) was covered by the APR Neuro2Co grant
from the Region Centre Val de Loire. The authors thank the UEPAO unit (animal facilities) for taking
care of the animals during the experimentation.

Nous remercions l'équipe d'Episciences pour son support et les collègues de JDMDH pour la première version de cette charte graphique.
2. Talking sheep – “Bah, Bah”
Talking sheep - “Bah, Bah”

Marine Siwiaszczyk¹, Scott A. Love¹, Elodie Chaillou¹

¹Unité de Physiologie de la Reproduction et des Comportements (PRC), INRAE, CNRS, IFCE, Université de Tours, 37380 Nouzilly, France

Article soumis pour publication le 30 avril 2021
dans Frontiers for Young Minds

Le journal Frontiers for Young Minds est un journal scientifique dédié aux enfants. La relecture se fait par un groupe d'enfants encadré par un professeur.
**Abstract**

If you've ever been out to the countryside in the spring, you might have heard sheep bleating to their lambs. Sheep also bleat when they are separated from the flock or stressed in some other way. To us, all these bleats sound very similar. But do you think they also sound similar to the lambs? Or do you think they know whose mother is calling and what they are saying? Scientists try to interpret the bleats of sheep by observing their behavior when they hear these sounds. They study the sound waves of recorded bleats to identify each sheep's unique voice and even how they are feeling. They also investigate the brain to find out what's going on inside their head when they hear these sounds. Studies show that sheep really can recognize each other's voices and communicate vocally.

**Keywords:** Behavior - Sound Analysis - Brain - Vocalization – Communication

**Communicating with sound**

People communicate with each other by talking but do you think animals can also talk to each other? You probably answered, “no”. But do you think animals can *communicate* with each other? If you have ever lived with a pet then you probably know that they are even capable of communicating with *us*. A dog can ask you to throw a stick just by dropping it at your feet. A cat can ask for food by rubbing against your leg. These animals are using behavior to communicate information - “throw the stick” and “give me food”. If you don't do what they are asking, the dog might bark and the cat might meow to insist that they really want you to do it, now. Barking and meowing are examples of animals using sounds produced by the mouth (like talking) to communicate. By observing animals in farms, zoos or in nature, ethologists have discovered that many animals are able to communicate using sounds, called vocalizations or bleats for sheep.

**Interpreting sheep sounds from their behavior**

To interpret the meaning of sheep vocalizations, ethologists often study their bleats and their behavior at the same time using video cameras and microphones. Observing sheep in this way, ethologists showed that an ewe and her lamb can communicate using what they called low- and high-bleats. Low-bleats are emitted with a closed mouth, when a ewe and her lamb are close to each other, during caregiving moments such as suckling or licking. High-bleats are emitted with the mouth wide open, when a ewe and her lamb are separated. It's a bit like when parents call their children who are too far away! These high-bleats are also emitted by adult sheep when they are separated from their herd, when they are stressed and even when their food arrives late! These observations allow us to interpret
low-bleats as comforting vocalizations and high-bleats as distress calls. But scientists don't only *observe* animals, they also create experiments using behavioral tests to investigate how sheep use bleats to communicate.

The two-choice behavioral test helps us to understand whether animals can tell the difference between two sounds. If you heard a dog bark and a cat meow you could easily say which was the dog. If you heard a ewe bleat and a lamb bleat, could you correctly say which was the ewe? These are both examples of two-choice tests in which you are asked to give your answer by speaking. But sheep cannot *tell* us the answer, so how do they respond in a two-choice test?

One method that scientists use is to present the bleats in different locations, one on the left and one on the right (Figure 1). When the sheep hear the bleats they can behave in different ways. Scientists count how many times the sheep chooses (walks towards and stays near) each location (sound). In one study lambs had to choose between the bleat of their mother in one location and the bleat of an unfamiliar ewe in a different location (1). The lambs could not see or smell the ewes, they could only hear them. The results showed that lambs chose the sound of their mother much more than the sound of the unfamiliar ewe. So we know that lambs prefer the bleats of their own mother and also that they can tell the difference between the bleats of different sheep. From a similar test we also know that sheep prefer the bleats of their friends who live in the same barn more than the bleats of unknown sheep.

<figure><img src="image_38.png" /><figcaption>**Figure 1.** Schematic drawing of a two-choice behavioral test. In this type of test, lambs hear two bleats coming from different locations and can show that it prefers one of the bleats by walking towards and staying near it.</figcaption></figure>

1) A lamb can hear the bleats of two ewes that they cannot see. One ewe is its mother (blue) and the other is an unfamiliar ewe (orange). 2) The bleats of the mother (blue) and unfamiliar ewe (orange) are presented from speakers in different locations. Scientists count the number of times that the lamb walks towards each bleat location.
How do you think the sounds are presented in these tests? Well there are two different ways: 1) the bleats can be made by sheep present but not visible during the test or 2) the bleats can be recorded with a microphone long before and presented using speakers during the test (Figure 1). One of the problems with the bleats being made by actual sheep is that the sheep can “say” whatever they want. Scientists can’t control their use of high-bleats or low-bleats. So to test whether lambs preferred high- or low-bleats it was necessary to use speakers. These speakers are used to playback only high-bleats from one location and only low-bleats from a different location (1). This test actually showed that lambs did not have a preference, maybe they liked both. Another good reason to record and playback the bleats is that we can also get interesting information from analyzing their sound waves.

## Analyzing the bleat sound wave

Sound waves have physical properties that can be measured and analyzed. One important physical property of a sound is its amplitude. If you hear a sound with a high amplitude, it will be loud. If you hear a low amplitude sound, it will be quieter. Similarly, high-bleats are louder than low-bleats because they have a higher amplitude. These bleats also have different frequencies, which is another important physical property. High-bleats have a higher frequency than low-bleats. To understand what different frequencies actually sound like you could imagine the sound of a whistle (high frequency) and the sound of thunder (low frequency). Bioacousticians study amplitude, frequency and other physical properties of sounds to identify their acoustical signature.

When you talk on the telephone, you can recognize the person from their voice. You can do this because each person's voice has a vocal signature. Like a fingerprint a person's vocal signature is unique to them. It is a unique combination of physical sound properties. Earlier we learned that sheep can tell the difference between the bleats of different sheep. So how do you think this is possible? By studying the physical properties of sheep vocalizations, bioacousticians have shown that sheep also have vocal signatures that can be used to tell them apart (2).

It is not only an individual's identity that is stored in a combination of these properties, it could also be what the animal is feeling. Scientists are currently trying to link how an animal is feeling with a unique combination of these physical properties. If successful, they could develop a computer program that can detect different types of bleats by analyzing the combination of sound properties. This is interesting because we could automatically detect, for example, when an animal on a farm is in pain (3). The farmer could then be alerted and go to help the animal.
## What is the role of the sheep brain in vocal recognition?

Lots of research is still needed for neuroscientists to answer this question. But by studying sheep brain anatomy and what we know about the human brain, it is possible to hypothesize the role of the brain in sheep communication.

The first role of the brain is sound perception through the auditory system (Figure 2). This system includes the ear with its external and internal parts and specific brain structures. The reception of the sound is performed by the ear. The physical properties of sound (e.g., frequency) are transmitted to the eardrum and the cochlea. The cochlea encodes the sound into an electrical signal. This transformation is based on the frequencies of the sound and is done by the cochlear hair cells. These hair cells are very thin and fragile and can be broken if you listen to music that is too loud. This electrical signal flows through the cochlear nerve, the cochlear nucleus, the inferior colliculi, the medial geniculate nucleus to finally arrive in the auditory cortex. We know where these regions are in the sheep brain (Figure 2) but there is no evidence of the role they play in sheep communication. The second role of the brain is the interpretation and recognition of sounds. Here, several other parts of the brain are involved, such as the amygdala, known to combine different sensory information (olfactory, visual and auditory), and the hippocampus, known to have a major role in memory. These two roles of the brain enable sheep to learn different sensory information from their lambs and to recognize them. To help neuroscientists better understand the role of the brain in sheep communication, they can use imagery techniques that are beginning to be used with farm animals. These techniques allow us to see how the brain functions. The most famous being electrophysiology and functional magnetic resonance imaging (4).

<figure><img src="image_39.png" /><figcaption>**Figure 2.** Sound perception through the auditory system (red arrows). The first organ of the auditory system is the external ear, which localizes the sound (“bah”). Ear canal (brown) protects the eardrum. Sound is amplified by the eardrum</figcaption></figure>
(purple) then encoded by the cochlea (orange) into an electrical signal. This signal is transmitted to the brain through the cochlear nerve, cochlear nucleus, inferior colliculi and geniculate nucleus before arriving at the auditory cortex (yellow). In sheep, the cochlear nucleus, the inferior colliculi and the geniculate nucleus have been identified by neuroanatomy studies whereas the auditory cortex has not been clearly identified.

## Conclusion

Sheep, like other animals, can communicate using sound. By studying their behavior ethologists have shown that sheep can tell apart one sheep from another by only listening to their bleats. They can do this because each sheep has its own vocal signature contained within its bleat. The sound wave of bleats can also contain information about how a sheep is feeling. Bioacousticians are trying to find this information to help, for example, farmers detect when their animals are in pain. Neuroscientists investigate the structure and function of the brain to understand its role in vocal communication. To fully understand sheep communication it is important for ethologists, bioacousticians and neuroscientists to work together. This will help us to understand sheep more globally. The more we know about the lives of sheep the more chance we have at improving the lives of this farm animal.

## Glossary

**Communication:** the sharing of information.

**Ewe:** adult female sheep.

**Suckling:** the lamb feeds on its mother's milk.

**Ethologist:** a scientist who studies animal behavior.

**Bioacoustician:** a scientist who studies the sounds produced by animals and the anatomy used to produce (e.g., the mouth), hear (e.g., the ear) and interpret (e.g., the brain) them.

**Neuroscientist:** a scientist who studies the brain.

## References

1. Sèbe F, Duboscq J, Aubin T, Ligout S, Poindron P. Early vocal recognition of mother by lambs: contribution of low- and high-frequency vocalizations. Anim Behav. 2010 May;79(5):1055-66.

2. Searby A, Jouventin P. Mother-lamb acoustic recognition in sheep: a frequency coding. Proc R Soc Lond B Biol Sci. 2003 Sep 7;270(1526):1765-71.

3. McCloughlin MP, Stewart R, McElligott AG. Automated bioacoustics: methods in ecology and conservation and their potential for animal welfare monitoring. J R Soc Interface. 2019 Jun
28;16(155):20190225.

4. Amanamba U, Sojka A, Harris S, Bucknam M, Hegdé J. A Window Into Your Brain: How fMRI Helps Us Understand What Is Going on Inside Our Heads. Front Young Minds. 2020;8(484603).
### III. Impact de la musique sur les agneaux élevés en allaitement artificiel

#### 1. Résumé de l'article

L'élevage ovin a recours à l'allaitement artificiel des agneaux séparés précocement de leur mère et nourris avec un lait artificiel. Cette pratique nuit au développement du jeune qui présente de nombreuses altérations comportementales, endocriniennes et de sa santé. Pour contrecarrer les effets délétères de l'allaitement artificiel, plusieurs techniques ont été testées telles que l'utilisation de nourrices (Brunet et al., 2016) ou l'ajout d'objet dans l'enclos d'élevage en tant qu'enrichissement du milieu (Gaudin et al., 2018). Un élément potentiel d'enrichissement du milieu n'a pas encore été étudié : la diffusion de musique. En effet, la diffusion de musique a montré des effets positifs chez différentes espèces animales telles que le chimpanzé (Howell et al., 2003) ou le cochon (J. Li et al., 2021; X. Li et al., 2019). Grâce à ces études, nous nous posons la question d'un effet bénéfique de la diffusion de musique pour contrecarrer l'absence de la mère pour les agneaux en allaitement artificiel. Notre hypothèse est que la diffusion de musique classique va aider à contrecarrer les effets délétères de l'allaitement artificiel et les modifications neurobiologiques induites. La diffusion de musique pourrait améliorer le bien-être des agneaux, modifier leur réactivité émotionnelle ainsi que leur morphologie cérébrale.

Pour tester cette hypothèse, nous avons comparé deux groupes de 14 agneaux chacun (7 mâles et 7 femelles). Le premier groupe d'agneau est élevé en allaitement artificiel conventionnel tandis que le deuxième groupe est élevé en allaitement artificiel avec un environnement enrichi par la diffusion de musique classique. Pour évaluer la croissance des agneaux, nous avons réalisé des pesées et un relevé d'état sanitaire hebdomadaires. Pour évaluer le bien-être des agneaux, nous avons étudié leur comportement, leur réactivité émotionnelle ainsi que leur motivation sociale. Enfin, nous avons étudié l'impact de la diffusion de musique sur la morphologie à l'aide de l'IRM.

La diffusion de musique classique n'a pas d'impact sur l'état sanitaire des agneaux. Cependant, le poids corporel des agneaux exposés à la musique est inférieur à celui des agneaux non exposés. Les analyses comportementales n'ont pas permis de comprendre cette différence. Des différences comportementales ont été relevées entre les deux groupes, indiquant un effet de la musique avec des agneaux exposés à la musique qui mangent plus et qui vont être vus moins souvent debout en train d'explorer. La diffusion de musique classique a impacté deux régions cérébrales : le noyau caudé qui est plus petit chez les agneaux exposés à la musique et le noyau géniculé médial qui est plus gros pour les agneaux exposés à la musique. Lors des tests de réactivité émotionnelle, la musique semble
avoir un effet apaisant pour les agneaux exposés, effet qui n'apparait pas pour les agneaux non exposés. Toutes les différences observées au cours de l'expérience montrent que la musique a des effets sur les agneaux, exposés ou non à la musique pendant l'élevage, mais ne permettent pas de conclure quant à ses effets positifs.
2. Impact of classical music on emotional reactivity and brain morphology of lambs bred by mother-deprivation with milk replacement

*En préparation*
# Impact of classical music on emotional reactivity and brain morphology of lambs bred by mother-deprivation with milk replacement

Marine Siwiaszczyk<fnref n="1" />, Céline Parias<fnref n="1" />, Raymond Nowak<fnref n="1" />, Frédéric Lévy<fnref n="1" />, Mélody Morisse<fnref n="1" />, Quentin Derian<fnref n="1" />, Olivier Lasserre<fnref n="2" />, Didier Dubreuil<fnref n="2" />, Scott A Love<fnref n="1" />, Elodie Chaillou<fnref n="1" />

## Abstract

It is common practice in sheep breeding to raise lambs without their mother and for them to be fed with milk replacement. This practice is deleterious for the development of the lambs who present behavioral, endocrine and health alterations. To counteract the deleterious effects of this mother-deprivation with milk replacement, several techniques have been tested such as the use of nurses or the addition of objects in the breeding enclosure as environmental enrichment. A potential element of environmental enrichment has not yet been studied: the diffusion of music. Indeed, the diffusion of music has shown positive effects in several animal species such as the chimpanzee or the pig. We hypothesize that the diffusion of classical music may help to counteract the deleterious effects of mother-deprivation with milk replacement in lambs. Playing music could improve the well-being of lambs, modify their emotional reactivity as well as their brain morphology. To test this hypothesis 28 lambs were bred by conventional mother-deprivation with milk replacement but only half were exposed to a diffusion of classical music as an environmental enrichment. During breeding, music leads to a lower body weight and to modified behavior. Music-exposed lambs were more often seen in eating behavior and less often seen in exploration. During emotional reactivity tests, music appeared to have a calming effect on music-exposed lambs, which was not evident for unexposed lambs. Overall the experiment showed that music had effects on the lambs but it did not clearly show positive effects on well-being.

**Keywords:** behavior – lambs – MRI – music – reactivity

## Acknowledgements

We are grateful to all the members of the Unité Expérimentale de Physiologie Animale de l'Orfrasière for taking care of the lambs. We also wish to thank PIXANIM for the MRI acquisitions and the Laboratoire de phénotypage-endocrinologie for the hormone assays.

<footnote n="1">¹CNRS, IFCE, INRAE, Université de Tours, PRC, Nouzilly, France</footnote>

<footnote n="2">²CNRS, IFCE, INRAE, Université de Tours, UEPAO, Nouzilly, France</footnote>
Introduction

Sheep (Ovis aries) are common farm animals used for meat and milk production. In conventional dairy farms, lambs are bred by mother-deprivation with milk replacement, whereas for meat production lambs are bred with their mother. However, even for meat production, lambs can be bred by mother-deprivation with milk replacement. This is the case when the lamb's mother is not maternal, is sick or has too many lambs to raise. When the lambs are raised with their mother, they ingest her colostrum, essential for passive immunity transfer to the lambs (Hernandez et al., 2009; Khan & Ahmad, 1997; Nowak & Poindron, 2006). This colostrum ingestion also helps to create a link between the mother and her lamb and promote the recognition of the mother by the lamb (Val-Laillet et al., 2004, 2006). This recognition is important since maternal care is provided exclusively to their own lamb, it is selectivity (Nowak, 1999a). The factors that facilitate recognition of a lamb by its mother differ depending on whether it is near or far from the mother. If the lamb is at a distance from its mother, it is the acoustic and visual signals that facilitate recognition (takes place 12 hours after birth) while when the lamb is close to its mother, it is olfaction that is favored for recognition (takes place 4 hours after birth) (Nowak, 1999b). This recognition and preference also take place in the reciprocal direction, from the lamb towards its mother (Nowak et al., 1997). Previous studies have shown that mother-deprivation with milk replacement has a negative effect on the development of the lambs. Lambs bred by mother-deprivation with milk replacement rather than being fed by their mother have an altered behavioral repertoire (Napolitano et al., 2008), health (Latham & Mason, 2008) and endocrine axis (Gaudin et al., 2018). Mother-deprivation with milk replacement influences the maturation of brain regions such as the caudate nucleus. The volume of the caudate nucleus is smaller for the mother-deprived lambs than for the lambs bred with their mother (Love et al., 2021). The parameters of microstructural data of the brain are also impacted by the mother-deprivation with milk replacement (Fractional Anisotropy (FA) of grey and white matter) (Love et al., 2021). Moreover, the method of breeding has an impact on the bond that the lamb creates with its congeners (Gaudin et al., 2018). This impact is different depending on the attachment partner (mother, sibling, other lambs in the flock) (Gaudin et al., 2018). To counteract the deleterious effect of artificial breastfeeding, various techniques have been tested, such as the use of nurses (Brunet et al., 2016) or the addition of objects in the pen to enrich the environment (Gaudin et al., 2018). Moreover, the addition of objects is little efficient because the attachment of the lamb for this object is weak (Gaudin et al., 2018). One potential environmental enrichment that has not been investigated is music, despite its positive impact on animals reported in scientific literature or by its use in breeding or zooparc (Ubiema et al., 2021). In captive chimpanzees, music reduced agitated and aggressive behaviors and
increased social behavior (Howell et al., 2003). In a group of Western Lowland Gorillas, music with
a low tempo reduced their anxiety behavior (Brooker, 2016). Chicks exposed to music in the
embryonic stage have improved their spatial orientation, learning and memory (Sanyal et al., 2013).
However, the potential benefits of music for farm animals have been little studied. In piglets, exposure
to music after weaning facilitated their playing behavior (de Jonge et al., 2008). The behavioral
response of piglets to musical stimulation differs depending on the tempo or the musical instruments
(X. Li et al., 2019). Diffusing Mozart songs also induced a low basal cortisol level (Li et al., 2021).
Moreover, in practice, radio has been used in zoos to reduce stress and reactivity of animals (Ubiema
et al., 2021). In this context, we question the positive impact of music to counteract the absence of
the mother in lambs raised by mother-deprivation with milk replacement.

We hypothesized that the diffusion of classical music could help to counteract the deleterious effects of mother-deprivation with milk replacement and the underlying neurobiological modification. The diffusion of classical music could improve the well-being of the lambs, modify their emotional reactivity and brain morphology.

To test our hypothesis, we compared two groups of lambs. The first was raised by mother-deprivation
with milk replacement and the second by mother-deprivation with milk replacement with the
diffusion of classical music as an enrichment. To evaluate the influence of classical music on lamb
growing, body weight and sanitary state were recorded weekly from birth to 2.5 months of age.
Behavioural activity, emotional reactivity and social motivation were examined in the aim to evaluate
the influence of music diffusion on lambs' well-being. In addition to behavioral observations, we
measured plasma levels of cortisol and oxytocin throughout the breeding and during emotional
reactivity assessment. Finally, we examined the impact of music diffusion on brain morphology using
magnetic resonance imaging (MRI).

**Material & Methods**

Ethics

All procedures were performed in accordance with the European directive 2010/63/EU for animal
protection and welfare used for scientific purposes and approved by the local ethical committee for
animal experimentation (CEEA VdL, Tours, France, authorization N°23520-2020010714121242v3).
At the end of the experiment all remaining animals returned to the UEPAO.
## Animals and housing

Experiments were conducted on 28 Ile de France lambs (14 males and 14 females) born between 08/30/2021 and 09/02/2021 at the "Unité Expérimentale de Physiologie Animale de l'Orfrasière" (UEPAO, INRAE Val de Loire, France; https://doi.org/10.15454/1.5573896321728955E12). Lambs were separated from their mother 48 hours after birth allowing colostrum ingestion and divided into two groups based on gender (7 females and 7 males / group), body weight at birth (3.65 kg ± 0.42 for the No music group and 3.67 kg ± 0.44 for the Music group) and sibling. Each group was housed in identical enclosures (10 m x 2 m) made of 1.80 m-high plywood to avoid visual or direct contact with caregivers outside the care periods. Both were equipped with 2 water-drinkers, 2 hay racks, 2 troughs containing granules and 6 rubber teats connected to an automatic feeder (EuroLouve or Forster) providing *ad libitum* access to milk replacement (Lactiprestige Ovicabr' securit: 23% proteins, 25% fat, 6.5% ash, 3.5% water, 0.15% cellulose, 0.8% calcium, 0.6% phosphore, 0.7% sodium) at constant temperature (43°C) and constant concentration (200 g/L). For the first five days of the experiment, lambs were trained to drink formula-milk with rubber teats 4 times per day by different caregivers until they could feed on their own. To prevent hypothermia two red heating lamps were placed in each enclosure for the first 14 days of the experiment. To avoid noise pollution between the groups the enclosures were located in two distant rooms. The walls of both enclosures were covered with honeycomb boxes (29.5 cm x 29.5 cm) to improve sound diffusion and the ground was covered with straw. Traffic cones were placed in enclosures to allow playing enrichment. The light was on between 8:30 a.m and 05:30 p.m.

## Music streaming

Four speakers (YAMAHA NS-AW194) were suspended above the Music enclosure where music was played using a dual audio amplifier Micro AMP 2 mkII and a scheduler MY MUSIC PLAYER LE de iDAL (Interactive Digital Audio Line). In the control enclosure, 4 dummy cardboard speakers were suspended to simulate speakers and ensure identical environments for the two groups. The music played corresponds to classical music podcasts from the Radio Classique station (https://www.radioclassique.fr). Classical music was therefore varied (with or without singers, with different rhythms). Music was played everyday throughout the whole experiment, 5 times a day in periods of 45 minutes (between 10:30 a.m and 11:15 a.m, 12:00 p.m and 12:45 p.m, 01:30 p.m and 02:15 p.m, 03:00 p.m and 03:45 p.m, 04:30 p.m and 05:15 p.m) at a maximum volume of 75 dB under the speakers. At the most distant corners, the volume was around 60 dB. The mean level of ambient
noise was around 30-35 dB in both enclosures and the mean level of automatic feeder was around 75 dB.

## Blood sample, weight and sanitary status

During the breeding period, once a week, weight, sanitary state and blood samples were carried out for each lamb. Sanitary conditions were noted according to the cleanliness of the posterior, the presence or absence of diarrhea, runny nose and eye, cough and injuries. Blood samples were immediately put in ice until being centrifuged for 30 min at 4°C and 3800 RPM. Plasmas were collected and stored at -20°C until assayed of cortisol and oxytocin. Moreover, blood samples were performed to assay plasma cortisol and oxytocin and, then, measure the hormonal basal levels during breeding and evaluate emotional reactivity.

## Behavioral measurements

Cameras (Vivotek FE8174V) were installed on the ceiling to record lambs behavior every day between 8:30 a.m and 05:30 p.m. Human interventions were carried out in the morning before 10 a.m, the behavior was analyzed on periods without human intervention. Behaviors were analyzed using Boris software (Friard & Gamba, 2016). 5 periods of 5 minutes were sampled for analysis: two periods in the morning (1 period with music (10:50-10:55) and the other without music (11:35-11:40), one at noon (13:50-13:55) and two in the afternoon (1 period without music (16:05-16:10) and the other with music(16:50-16:55)). For each 5 min period, every 10 seconds (i.e. 31 observations), the posture, the location in the enclosure and the behavior were analyzed for each lamb according to the ethogram described in table 1. Observations were made by two observers who were trained in the same way. They each made half of the observations for each group.

<table><thead><tr><td>Activity</td><td>Description</td></tr></thead><tbody><tr><td>Food - milk</td><td>The lamb is drinking milk, teat in mouth</td></tr><tr><td>Food - solid</td><td>The lamb is eating granules or hay</td></tr><tr><td>Exploration</td><td>The lamb is walking with its head down or smelling the straw or scratching or touching and standing on an object</td></tr></tbody></table>
<table><caption>Table 1: Description of each activity measured with Boris during breeding.</caption><tr><td>Locomotion</td><td>The lamb is walking with its head up or running or jumping or riding or stepping back</td></tr><tr><td>Grooming</td><td>The lamb is nibbling or scratching or shaking itself or rubbing against an object</td></tr><tr><td>Immobile</td><td>The lamb is not eating, exploring, moving or grooming</td></tr><tr><td>Location</td><td>The lamb has both front legs in a defined zone</td></tr></table>

## Hormones assay

Hormones assay were realized by enzyme immunoassay (EIA) at the "Laboratoire de phénotypage-endocrinologie" (UMR PRC, INRAE Val de Loire, France).

### Cortisol

Cortisol was measured on 25µL aliquots of undiluted plasma and all samples were analysed in the same assay. The limit of detection of the assay was 4ng/ml. The intra-asssay coefficient of variation for quality control plasma containing 30ng/ml was 5.2%.

### Ocytocin

The concentration of oxytocin in plasma was determined by EIA after molecular extraction using Sep-Pack C18 cartridges (Waters). For the quality control containing 40pg/ml oxytocin, the intra-assay coefficient of variation was 18.4%.

## MRI

MRI of the brain was performed with a 3 Teslas Siemens Magnetom Verio® scanner (Erlangen, Germany) located at the PIXANIM platform, INRAE Nouzilly, France.

At 6 weeks of age, MRI was acquired for each lamb of the two groups. The experiment took place over 7 days. Each day, 4 lambs (2 Music and 2 No Music with 1 male and 1 female / group) were selected to be tested individually. Before and after the anesthesia, the 4 lambs were grouped together in a room so that they were never alone and thus avoided the stress of social isolation.
## Anesthesia

Before each acquisition, lambs were placed under general anesthesia to prevent stress and avoid motion artifacts. Anesthesia was induced by an intra-venous co-injection of rompun® and ketamine®. After this injection and the loss of muscular tonus, a tracheal tube was inserted to maintain the anesthesia with Isoflurane®. The drug doses and tracheal tube sizes were adapted to each animal based on age and body weight. The duration of anesthesia was limited to 2.5 hours, during which cardiac rhythm and SpO2 levels were continuously monitored. At the end of the acquisition, Isoflurane® was stopped and the tracheal tube removed after the first signs of autonomous breathing.

3 lambs died during the anesthesia period (two in the No Music group and one in the Music group).

## MRI acquisition

During acquisition lambs laid on their stomachs (prone position) with a 4-channel Siemens FLEX coil wrapped around their head and, for their comfort and safety, were covered with a cloth and noise-reducing headphones (BOLDfonic, Cambridge Research Systems). T₁-weighted (T₁w) images were acquired with the 3D-MPRAGE (Magnetisation Prepared Rapid Acquisition with Gradient Echo) sequence with the following parameters: Repetition Time, TR=2500ms; Echo Time, TE=3.18ms; Bandwidth, BW=150Hz/pixel; Inversion Time, TI=900ms; Flip Angle=12°; Slice Thickness=0.5mm; Field of View, FOV=192x192mm; matrix=384x384; in plane resolution 0.5x0.5mm; Number of Excitations, NEX=2; Integrated Parallel Acquisition, iPAT=2; acquisition time, TA=18m37s.

## Study-specific Template Creation

The T₁w images acquired for each lamb were used to create a study-specific template using the antsMultivariateTemplateConstruction2.sh function of the ANTS toolkit (Avants et al. 2008; Avants et al. 2011; Avants and Gee 2004). Briefly, template construction involved:

1) Creating an initial target template by averaging the T₁w images;

2) Pairwise normalization of each T₁w image to the target template;

3) Create new target template by averaging the normalized images output during step 2;

4) Transform the new target template by the average inverse affine transform and the average inverse warp field multiplied by a scaling factor (0.2);
5) Iterate steps 2 to 4 four times.

## Regions of Interest

Regions of interest (ROI) were segmented manually on the study-specific template: whole-brain; brain subdivisions: telencephalon, diencephalon, mesencephalon, pons, medulla oblongata, spinal cord, cerebellum and brain regions: caudate nucleus, periaqueductal gray, amygdala, olfactory bulb, hippocampus, medial geniculate nucleus, inferior colliculi and cochlear nucleus. The transforms calculated to create the template from each individual were then used to transform each of these ROIs to the individual's brain space. For each lamb, the total brain volume (mm³) and the volume of each segmented region were calculated (number of voxels in the region multiplied by the volume of a voxel, (0.5 mm)³). To remove the potential confound of intersubject variability of total brain volume, the region analysis was conducted on relative region volumes. The relative volume of each region, expressed as a proportion, was calculated by dividing its volume by the individual's total brain volume and multiplying by 100.

## Reactivity and motivation tests

We maintained exactly 7 days between MRI acquisition and the reactivity and motivation tests to allow lambs to recover from anaesthesia and to avoid bias due to possible interaction between anaesthesia and behavioural tests.

### Reactivity test

Each day, 4 lambs (2 males and 2 females) were selected (2 of each group) to be tested. Each lamb was tested individually. A blood sample was taken before leaving their breeding enclosure to measure the basal levels of cortisol and oxytocin. The testing arena consisted of a 2 x 4 m rectangle delimited by wooden panels 2 m high and cut into 8 equal zones (1 m²). The test was divided into three 10-minute phases and was carried out in the morning:

- A phase of social isolation during which the lamb was alone in the testing area in silence. This environment was unknown to the lamb (SI).

- A phase of social isolation with music during which the lamb was alone in the testing area with classical music played by two speakers behind the wall of zones 7 and 8 (SI+M).
- A phase of social isolation with a known passive person known to the lamb sitting against the wall between zones 7 and 8 in silence (SI+H).

Between the SI+M and SI+M phases, a blood sample was taken to measure the levels of cortisol and oxytocin.

For each phase, the behavior and the location in the testing area were analyzed continuously for each lamb according to the ethogram described in table 2. During the experiment, the number of bleats and excrement were counted; all other parameters were analyzed in the video recordings.

<table><caption>Table 2: Description of each activity measured with Boris during reactivity test.</caption><thead><tr><td>Activity</td><td>Description</td></tr></thead><tbody><tr><td>Location</td><td>The lamb has both front legs in the zone 1 - 8</td></tr><tr><td>Escape</td><td>The lamb jumps against the wall or puts its front legs on the wall</td></tr><tr><td>Sniffing</td><td>The lamb scents with physical contact the wall or the ground for a minimum of two secondes</td></tr><tr><td>Locomotion</td><td>The lamb walks and puts both front legs in another zone</td></tr><tr><td>Bleating</td><td>Number of bleats</td></tr><tr><td>Lying</td><td>The lamb is lying on the floor for a minimum of 2 secondes</td></tr><tr><td>Immobile</td><td>The lamb is not moving for a minimum of 2 secondes</td></tr><tr><td>Excrements</td><td>Number of urines and feces</td></tr></tbody></table>

## Motivation test

This test took place in the afternoon of the reactivity test and the lambs were tested in the same order as for the reactivity test. The testing arena was the same as for the reactivity test. However, in zones 1 and 2, the wall was open and there were 4 unknown lambs of the same age on the other side. The wall was open but barred, which meant that the lambs could touch but not join the lambs on the other side of the open barrier. The test was divided into four 2-minute phases:

- A phase during which the lamb was alone in the testing area (apart from the congeners) in silence (Known Environment, KE).
- A phase during which the lamb was alone in the testing area (apart from the congeners) with classical music played by two speakers behind the wall of zones 7 and 8 (Music, M).

- A phase during which the music was off and a construction cone was placed against the wall along the wall between zones 7 and 8. This cone was similar to the one present in the lamb pen during breeding (Known Object, KO).

- A phase in which a known passive person sat against the wall between zones 7 and 8 with no music (Human, H).

For each phase, the behavior and the location in the testing area were analyzed continuously for each lamb according to the ethogram described in table 3. During the experiment, the number of bleats and excrements were counted; all other parameters were analyzed in the video recordings.

<table><caption>Table 3: Description of each activity measured with Boris during motivation test.</caption><thead><tr><td>Activity</td><td>Description</td></tr></thead><tbody><tr><td>Location</td><td>The lamb has both front legs in the zone 1 - 8</td></tr><tr><td>Escape</td><td>The lamb jumps against the wall or puts its front legs on the wall</td></tr><tr><td>Sniffing</td><td>The lamb scents with physical contact the wall or the ground for a minimum of two secondes</td></tr><tr><td>Locomotion</td><td>The lamb walks and puts both front legs in another zone</td></tr><tr><td>Immobile</td><td>The lamb is not moving for a minimum of 2 secondes</td></tr><tr><td>Lying</td><td>The lamb is lying on the floor for a minimum of 2 secondes</td></tr><tr><td>Contact with the congeners</td><td>The lamb is in physical contact with the wall where the congeners are located (any part of the body)</td></tr><tr><td>Bleating</td><td>Number of bleats</td></tr><tr><td>Excrements</td><td>Number of urines and feces</td></tr></tbody></table>

## Statistics

All statistical tests were conducted using R software (R Core Team, 2020). Data are presented as mean +/- std error and individual values.
Weight

For the weight, mixed-effect ANOVA with 3 factors were conducted: Day of observation as a within factor and Group (Music vs No Music) and Gender (Male vs Female) as between factors. If the Gender was not significant, mixed-effect ANOVA with 2 factors were conducted: Day of observation as a within factor and Group as a between factor.

Health status

Statistics were conducted only for cleanliness of the posterior and the presence or not of diarrhea. Cleanliness of the posterior was tested using Wilcoxon test and the diarrhea was tested using Fisher test.

Cortisol and Oxytocin assay

For the cortisol and oxytocin level, mixed-effect ANOVA with 3 factors were conducted: Day of
collect as a within factor and Group (Music vs No Music) and Gender (Male vs Female) as between
factors. ANOVA assumptions were checked using Shapiro-Wilk normality test (function shapiro_test
from the rstatix package) and Levene's test for homogeneity of variance (function levene_test from
the rstatix package).

MRI analysis

For the brain volume, mixed-effect ANOVA with 2 factors were conducted: Group (Music vs No Music) and Gender (Male vs Female) as between factors. If the Gender was not significant, t-test were conducted between the two groups.

For the relative volume of brain subdivisions and brain regions, mixed-effect ANOVA with 3 factors were conducted: Hemisphere (Left vs Right) as a within factor and Group (Music vs No Music) and Gender (Male vs Female) as between factors. If the Gender was not significant, mixed-effect ANOVA with 2 factors were conducted: Hemisphere as a within factor and Group as a between factor.
## Behavioral measurements

The behaviors "Stand and Explore", "Lay down and Rest", "Food", "Food - Milk" and "Food - Solid" were tested using mixed-effect ANOVA with 3 factors: Day of observation as a within factor and Group (Music vs No Music) and Gender (Male vs Female) as between factors. If the Gender was not significant, mixed-effect ANOVA with 2 factors were conducted: Day of observation as a within factor and Group as a between factor.

## Reactivity test

The locomotion, number of bleats, number of escape attempts, time spent immobile, sniffing time and lying time were tested using mixed-effect ANOVA with 3 factors: Phase (SI vs SI-M vs SI-H) as a within factor and Group (Music vs No Music) and Gender (Male vs Female) as between factors. If the Gender was not significant, mixed-effect ANOVA with 2 factors were conducted: Phase as a within factor and Group as a between factor. The number of excrement was tested with a Wilcoxon test.

## Motivation test

The locomotion, number of bleats, number of escape attempts, time spent motionless, sniffing time, contact time with congeners and lying time were tested using mixed-effect ANOVA with 3 factors: Phase (SI vs SI-M vs SI-H) as a within factor and Group (Music vs No Music) and Gender (Male vs Female) as between factors. If the Gender was not significant, mixed-effect ANOVA with 2 factors were conducted: Phase as a within factor and Group as a between factor. The number of excrement was tested with a Wilcoxon test.

# Results

## Breeding

### Weight (Figure 1)

The weight was unaffected by the gender of the lambs (F = 1.9669, p > 0.05). There was a significant interaction between the Group and the Day of weight (F = 15.541, p < 0.001). The weight of Music
lambs was significantly less than the weight of No Music lambs in the last weeks of the experiment (day of observation: 30/09, 07/10 and 14/10).

<figure><img src="image_40.png" /><figcaption>Figure 1: Individual and mean (± standard error) body weights (kg) measured throughout the breeding (top) and separated for gender (bottom).</figcaption></figure>

## Sanitary status

There was no significant difference between the two groups for the cleanliness of the posterior or diarrhea. Cases of cough were very rarely observed whatever the group.

## Cortisol assay (Figure 2)

The cortisol level was unaffected by the gender of the lambs (F = 0.043, p > 0.05). There was a significant interaction between the Group and the Day of collect (F = 2.205, p < 0.05). The cortisol
levels were significantly higher for the Music lambs than the No Music lambs only on 23/09 and 14/10.

<figure><img src="image_41.png" /><figcaption>Figure 2: Mean (± standard error) and individual plasma cortisol level measured throughout the breeding (left) and the overall mean per group (right).</figcaption></figure>

*Oxytocin assay (Figure 3)*

The oxytocin level was unaffected by the gender of the lambs (F = 2.353, p > 0.05). There was a significant interaction between the Group and the Day of blood sample (F = 3.231, p < 0.05). The levels of oxytocin were significantly lower for the Music lambs than the No Music lambs only at days 09/09, 16/09, 23/09 and 14/10.
<figure><img src="image_42.png" /><figcaption>Figure 3: Mean (± standard error) and individual plasma oxytocin level measured throughout the breeding (left) and the overall mean per group (right).</figcaption></figure>

## Behavioral measurements

*Impact of music diffusion evaluated by comparing No Music and Music group according to the global activity throughout the breeding*

The behavior “Stand and Explore” was unaffected by the gender of the lambs (F = 2.044, p > 0.05) (Figure 4). There was a significant interaction between the Group and the Day of observation: over the entire breeding period, the No Music lambs were more often seen standing up exploring than the Music lambs (F = 9.547, p = 0.005). However, the differences were not always in the same order according to the Day of observation (F = 7.240, p < 0.001).
<figure><img src="image_43.png" /></figure>

Figure 4: Mean (± standard error) cumulative occurrence of “Stand and explore” measured throughout the breeding (top) and the overall mean per group (bottom left) and gender (bottom right).

The behavior “Lay down and Rest” was unaffected by the gender of the lambs (F = 3.858, p > 0.05) (Figure 5). The number of times the lambs were seen lying down and resting decreased over time until they reached a plateau (F = 16.438, p < 0.001). Over the entire breeding period, the No Music lambs were less often seen lying down resting than Music lambs. However, the differences were not always in the same order according to the Day of observation (F = 4.150, p < 0.001).
<figure><img src="image_44.png" /></figure>

Figure 5: Mean (± standard error) cumulative occurrence of “Lay down and Rest” measured throughout the breeding (top) and the overall mean per group (bottom left) and per gender.

The total food intake was unaffected by the gender of the lambs (F = 0.026, p > 0.05). Over the entire experiment, the Music lambs were seen eating more often than the No Music lambs (9.301, p = 0.005). This group effect varied throughout the experiment, the differences were not always in the same order according to the Day of observation (F = 5.426, p < 0.001) (Figure 6).
<figure><img src="image_45.png" /></figure>

Figure 6: Mean (± standard error) cumulative occurrence of “Total Food” measured throughout the breeding (left) and the overall mean for each group (right). The “Total Food” represents the mean total number of times that the lambs were observed to be either drinking milk or eating solid food. The dashed line represents the day the barriers were opened giving access to the entire enclosure.

The milk consumption was unaffected by the gender of the lambs (F = 1.780, p > 0.05). Over the entire experiment, the lambs of the Music group were seen drinking milk more often than once of the No Music group (F = 13.171, p = 0.001). This milk consumption decreased over time (F = 6.999, p < 0.001) (Figure 7).

<figure><img src="image_46.png" /></figure>
Figure 7: Mean (± standard error) cumulative occurrence of “Milk” measured throughout the breeding (left) and the overall mean for each group (right). The dashed line represents the day the barriers were opened giving access to the entire enclosure.

There was an interaction between the gender and the group for the consumption of solid food (F = 4.664, p = 0.041): the females of the Music group were seen more often eating solid food than the females of the No Music group, whereas no difference was found between No Music and Music males. The consumption of solid food tended to increase over time (F = 4.624, p < 0.001) (Figure 8).

<figure><img src="image_47.png" /></figure>

Figure 8: Mean (± standard error) cumulative occurrence of “Solid Food” measured throughout the breeding (left) and the overall mean for each group and gender (right).

Impact of music diffusion evaluated by comparing periods with and without music in the Music group

The behavior “Stand and Explore” was unaffected by the gender of the lambs (F = 0.213, p = 0.652). The Music lambs were more often seen standing up exploring during periods without music than periods with music (F = 22.737, p < 0.001) (Figure 9).
<figure><img src="image_48.png" /></figure>

Figure 9: Mean (± standard error) cumulative occurrence of “Stand and explore” measured throughout the breeding period with or without music (left) and the overall mean for each period (right).

The behavior “Lay down and Rest” was unaffected by the gender of the lambs (F = 0.713, p = 0.415). The Music lambs were more often seen lying down and resting during periods with music than periods without music (F = 11.352, p = 0.005). Moreover, the time spent lying down and resting decreased over time until they reached a plateau (F = 11.810, p < 0.001) (Figure 10).

<figure><img src="image_49.png" /></figure>

Figure 10: Mean (± standard error) cumulative occurrence of “Lay Down and Rest” measured throughout the breeding period with or without music (left) and the overall mean for each period (right).
The total food intake was unaffected by the gender of the lambs (F = 0.668, p = 0.430). The Music lambs were more often seen eating during periods without music than periods with music (F = 15.415, p = 0.002).

The milk consumption was unaffected by the gender of the lambs (F = 0.410, p = 0.534). The Music lambs were more often seen drinking milk during periods without music than periods with music (F = 34.248, p < 0.001) (Figure 11).

<figure><img src="image_50.png" /><figcaption>Figure 11: Mean (± standard error) cumulative occurrence of “Drink milk” measured throughout the breeding period with or without music (left) and the overall mean for each period (right).</figcaption></figure>

The consumption of solid food was unaffected by the gender of the lambs (F = 2.095, p = 0.173) and no difference between periods with or without music were found (F = 0.01, p = 0.972).

### MRI analysis:

#### Brain volume

The brain volume was unaffected by the gender of the lamb (F = 0.764, p > 0.391). The t-test did not show a significant difference between the two groups (t = 1.975, p = 0.065). However, subjective observation of the data indicates that the brain volume of the Music group (84,51 ± 8,024 cm³) was smaller than the brain volume of the No Music group (89,661 ± 5,089 cm³).
<figure><img src="image_51.png" /><figcaption>Relative volumes of brain subdivisions (Figure 12)</figcaption></figure>

Figure 12: Mean (± standard error) and individual relative volumes (% of total brain volume) in the left and right hemispheres for both the No Music and Music lambs. Each region is displayed as a colored overlay on the T<sub>1</sub>w study-specific template.

The relative volume of the telencephalon was the only subdivision affected by the gender of the lambs: its relative volume was larger for the females than the males (F = 8.217, p = 0.009). The relative volumes of the telencephalon, the pons and the medulla oblongata were significantly bigger in the left than in the right hemisphere. There was no significant difference between groups or hemispheres for the diencephalon, the mesencephalon and the cerebellum.
<table><caption>Relative volumes of brain regions (Table 4)</caption><thead><tr><th rowspan="2">Brain Region</th><th colspan="2">No Music lambs</th><th colspan="2">Music lambs</th><th colspan="3">Statistical values</th></tr><tr><th>Left-H</th><th>Right-H</th><th>Left-H</th><th>Right-H</th><th>Group</th><th>Hemispher<br>re</th><th>Interactio<br>n</th></tr></thead><tbody><tr><td>Caudate nucleus</td><td><u>0.836 ±</u><br><u>0.05</u></td><td><u>0.843 ±</u><br><u>0.038</u></td><td>0.784 ±<br>0.044</td><td>0.793 ±<br>0.037</td><td><b>F=10.87</b><br><b>1</b><br><b>p=0.003</b></td><td>F=2.006<br>p=0.169</td><td>F=0.015<br>p=0.903</td></tr><tr><td>Periaqueductal<br>gray</td><td>0.135 ±<br>0.01</td><td>0.136 ±<br>0.006</td><td>0.138 ±<br>0.111</td><td>0.135 ±<br>0.009</td><td>F=0.071<br>p=0.762</td><td>F=0.232<br>p=0.634</td><td>F=0.855<br>p=0.364</td></tr><tr><td>Amygdala</td><td>0.499 ±<br>0.03</td><td>0.498 ±<br>0.032</td><td>0.499 ±<br>0.017</td><td>0.508 ±<br>0.026</td><td>F=0.271<br>p=0.607</td><td>F=2.415<br>p=0.133</td><td>F=2.748<br>p=0.11</td></tr><tr><td>Olfactory bulb</td><td><b>0.397 ±</b><br><b>0.035</b></td><td>0.382 ±<br>0.031</td><td><b>0.402 ±</b><br><b>0.055</b></td><td>0.376 ±<br>0.05</td><td>F=0.000<br>3<br>p=0.987</td><td><b>F=16.517</b><br><b>p=0.0004</b></td><td>F=1.234<br>p=0.277</td></tr><tr><td>Hippocampus</td><td>0.519 ±<br>0.045</td><td>0.518 ±<br>0.044</td><td>0.516 ±<br>0.04</td><td>0.516 ±<br>0.031</td><td>F=0.028<br>p=0.868</td><td>F=0.037<br>p=0.850</td><td>F=0.007<br>p=0.934</td></tr><tr><td>Medial<br>geniculate<br>nucleus</td><td><b>0.055 ±</b><br><b>0.005</b></td><td>0.049 ±<br>0.005</td><td><u>0.057 ±</u><br><u>0.004</u></td><td><u>0.053 ±</u><br><u>0.005</u></td><td><b>F=4.597</b><br><b>p=0.042</b></td><td><b>F=72.632</b><br><b>p&lt;0.0001</b></td><td><b>F=4.713</b><br><b>p=0.04</b></td></tr><tr><td>Inferior<br>colliculus</td><td><b>0.03 ±</b><br><b>0.003</b></td><td>0.029 ±<br>0.003</td><td><b>0.031 ±</b><br><b>0.003</b></td><td>0.031 ±<br>0.004</td><td>F=1.06<br>p=0.313</td><td><b>F=9.122</b><br><b>p=0.006</b></td><td>F=2.609<br>p=0.119</td></tr><tr><td>Cochlear<br>nucleus</td><td>0.028 ±<br>0.002</td><td>0.278 ±<br>0.002</td><td>0.028 ±<br>0.002</td><td>0.029 ±<br>0.002</td><td>F=0.544<br>p=0.468</td><td>F=0.388<br>p=0.539</td><td>F=0.838<br>p=0.369</td></tr><tr><td>Telencephalon</td><td><b>28.264 ±</b><br><b>0.519</b></td><td>27.56 ±<br>0.519</td><td><b>28.318 ±</b><br><b>0.44</b></td><td>27.775 ±<br>0.365</td><td>F=0.743<br>p=0.397</td><td><b>F=47.39</b><br><b>p&lt;0.0001</b></td><td>F=0.8<br>p=0.379</td></tr><tr><td>Diencephalon</td><td>2.394 ±<br>0.099</td><td>2.371 ±<br>0.099</td><td>2.389 ±<br>0.102</td><td>2.387 ±<br>0.088</td><td>F=0.025<br>p=0.876</td><td>F=1.910<br>p=0.179</td><td>F=1.459<br>p=0.238</td></tr><tr><td>Mesencephalon</td><td>1.417 ±<br>0.072</td><td>1.416 ±<br>0.071</td><td>1.44 ±<br>0.091</td><td>1.437 ±<br>0.085</td><td>F=0.566<br>p=0.459</td><td>F=0.035<br>p=0.853</td><td>F=0.016<br>p=0.9</td></tr><tr><td>Pons</td><td><b>0.351 ±</b><br><b>0.029</b></td><td>0.334 ±<br>0.023</td><td><b>0.361 ±</b><br><b>0.027</b></td><td>0.336 ±<br>0.017</td><td>F=0.768<br>p=0.389</td><td><b>F=14.877</b><br><b>p=0.0007</b></td><td>F=0.608<br>p=0.443</td></tr><tr><td>Medulla<br>Oblongata</td><td><b>1.077 ±</b><br><b>0.074</b></td><td>1.045 ±<br>0.069</td><td><b>1.113 ±</b><br><b>0.074</b></td><td>1.052 ±<br>0.052</td><td>F=1.241<br>p=0.276</td><td><b>F=7.302</b><br><b>p=0.012</b></td><td>F=0.66<br>p=0.424</td></tr><tr><td>Cerebellum</td><td>4.129 ±<br>0.343</td><td>4.133 ±<br>0.237</td><td>4.245 ±<br>0.323</td><td>3.997 ±<br>0.189</td><td>F=0.012<br>p=0.914</td><td>F=3.251<br>p=0.083</td><td>F=3.458<br>p=0.075</td></tr></tbody></table>

Table 4: Relative region volumes (% of total brain volume) and statistical values. Mean relative volumes ± standard errors are displayed for each hemisphere and group. Underlined values highlight the group with the larger volume; bold font highlights the hemisphere with the larger volume.
The relative volume of the studied regions were not affected by the gender of the lambs. The relative volume of the caudate nucleus was significantly smaller in the Music lambs than in the No Music lambs (F = 10.871, p = 0.003). There was an interaction between the groups and hemispheres for the medial geniculate nucleus (F = 4.713, p = 0.04): its relative volume was larger for the Music group than the No Music group only in the right hemisphere. The relative volume of the olfactory bulb and the inferior colliculi was larger in the left than in the right hemisphere. There was no significant difference (Group or Hemisphere) for the periaqueductal gray, amygdala, hippocampus and cochlear nucleus.

## Reactivity test

### *Hormonal responses* (Figure 13)

#### Cortisol assay

The cortisol level was unaffected by the gender of the lambs for the reactivity test (F = 0.069, p > 0.05). There was no significant difference between the groups for the cortisol level (F = 0.518, p > 0.05). However, the cortisol level was significantly higher after the social isolation phase with music than the cortisol level in the breeding enclosure (F = 93.185, p < 0.001).

#### Oxytocin assay

The oxytocin level was unaffected by the gender of the lambs for the reactivity test (F = 1.565, p > 0.05). The oxytocin levels were significantly lower for the lambs exposed to classical music than the control lambs before the test and after the phase where classical music was played (F = 11.162, p < 0.01). This difference was already observed during breeding. There was no significant difference between the two phases (F = 1.713, p > 0.05).
<figure><img src="image_52.png" /><figcaption>Figure 13: Mean (± standard error) plasma cortisol level measured during the reactivity test (top, left) with gender influence (top, right) and plasma oxytocin level measured during the reactivity test (bottom, left) with gender influence (bottom, right).</figcaption></figure>

## Behavioral responses

The locomotion, evaluated by the number of crossed zones, was unaffected by the gender of the lambs ($F = 0.373, p = 0.548$). There was a significant interaction between the group and the phase ($F = 6.364, p = 0.003$): the locomotion was bigger for the Music group than for the No Music group only during the SI phase (Figure 14). The number of escape attempts was unaffected by the gender of the lambs ($F = 1.093, p = 0.308$). There was a significant interaction between the group and the phase for the number of escape attempts ($F = 4.271, p = 0.02$): the number of escape attempts was significantly bigger for the Music group than for the No Music group only for the SI phase (Figure 14).
<figure><img src="image_53.png" /></figure>

Figure 14: Mean (± standard error) and individual cumulative occurrence of locomotion (number of times the lamb changed zones) and (left) and escape attempts (right) during each phase of the reactivity test. SI = Social Isolation phase; SI-M = Social Isolation phase with music diffusion; SI-H = Social Isolation with the presence of a passive man.

The number of bleats was significantly higher for females than males (F = 12.030, p = 0.002) and was significantly different between the three phases: the number of bleats was higher during the SI phase than during the SI+M and SI+H phase and was higher in the SI+M phase than during the SI+H phase. The number of bleats decreased across the phases (Figure 15).

<figure><img src="image_54.png" /></figure>

Figure 15: Mean (± standard error) and individual cumulative occurrence of bleats for female (left) and male (right) lambs during each phase of the reactivity test. SI = Social Isolation phase; SI-M = Social Isolation phase with music diffusion; SI-H = Social Isolation with the presence of a passive man.
The time spent immobile was unaffected by the gender of the lambs (F = 0.820, p = 0.376). The time spent immobile was not different between groups (F = 0.015, p = 0.904) but was significantly different between phases (F = 9.197, p = 0.00046): it was significantly shorter during the SI phase than the SI+M and SI+H phases (Figure 16).

The sniffing time was unaffected by the gender of the lambs (F = 0.352, p = 0.560). The sniffing time was not different between groups (F = 1.127, p = 0.300) but was significantly different between phases (F = 31.696, p < 0.001): it was significantly shorter during the SI+H phase than during the SI and SI+M phases (Figure 16).

<figure><img src="image_55.png" /><figcaption>Figure 16: Mean (± standard error) and individual cumulative occurrence of immobility and (left) and sniffing (right) during each phase of the reactivity test. SI = Social Isolation phase; SI-M = Social Isolation phase with music diffusion; SI-H = Social Isolation with the presence of a passive man.</figcaption></figure>

The time lying on the floor was affected by the gender of the lambs (F = 4.500, p = 0.047): males spent significantly more time lying on the floor than females. However, there was no group or phase difference for the time lying on the floor.

There were no interaction, group or phase differences for the number of excrements.

Subjective observation of the time spent in each zone showed that the lambs were more often located in zones 7 and 8 when the familiar human was present, the Music lambs being more often in these zones than the No music lambs (Figure 17).
<figure><img src="image_56.png" /></figure>

Figure 17: Mean time (in seconds) spent in each zone during each phase of the reactivity test. SI = Social Isolation phase; SI-M = Social Isolation phase with music diffusion; SI-H = Social Isolation with the presence of a passive man.

## Motivation test

The locomotion was unaffected by the gender of the lambs (F = 0.086, p = 0.772). There were no Group or Phase differences for the locomotion (Figure 18).

The number of bleats was unaffected by the gender of the lambs (F = 0.305, p = 0.586). There was an interaction between Group and Phase factors for the number of bleats (F = 3.275, p = 0.026) (Figure 18).

The time spent immobile was unaffected by the gender of the lambs (F = 0.216, p = 0.647). This time spent immobile increased during the experiment: it was significantly longer in KO phase than in KE and M phases. It was also significantly longer in the H phase than in the KO phase. However, it was not different between groups (F = 0.180, p = 0.676) (Figure 18).

The sniffing time was unaffected by the gender of the lambs (F = 0.067, p = 0.798). The sniffing time was significantly different between phases (F = 9.288, p < 0.001): it was significantly longer for the M phase than the SI phase and was also longer for the H phase than for the KO phase. However, there was no difference between groups (F = 1.136, p = 0.298) (Figure 18).
<figure><img src="image_57.png" /><figcaption>Figure 18: Mean (± standard error) and individual cumulative occurrence of locomotion and (top left) and bleats (top right) or duration (in seconds) of time spent immobile (bottom left) and sniffing (bottom right) during each phase of the motivation test. KE = Known Environment; M = Music; KO = Known Object; H = Human.</figcaption></figure>

The contact time with congeners was unaffected by the gender of the lambs ($F = 0.950, p = 0.341$).

The contact time was significantly different between phases ($F = 21.529, p < 0.001$): this contact time decreased with time.

There were no interaction, group or phase differences for the number of excrements.

The examination of the time spent in each zone showed that the lambs were more often located in zones 7 and 8 during music diffusion and when the familiar human was present (Figure 19).
<figure><img src="image_58.png" /><figcaption>Figure 19: Mean time (in seconds) spent in each zone during each phase of the motivation test. KE = Known Environment; M = Music; KO = Known Object; H = Human.</figcaption></figure>

Discussion

Global impact of music during breeding

No impact of music exposure was noticed for the lambs' sanitary status. However, the presence of diarrhea and dirty posterior was observed regardless of group. The sanitary status of our lambs bred by mother-deprivation with milk replacement is in accordance with the literature and indicates that their sanitary status was degraded (Belanche et al., 2019; Napolitano et al., 2008).

The body weight of the No Music group was superior to the weight of the Music group from day 28. Two hypotheses may explain this difference: the No Music lambs eat more than the Music lambs or the Music lambs have a higher activity than the No Music lambs. However, neither of these hypotheses were validated during the experiment. That said, the examination of the feeding behavior of Music lambs showed that they ate and explored less during music diffusion than during the periods of no music, whereas it was the contrary for “lay and rest” behavior. These observations suggest that music diffusion could interrupt the activities of lambs, which become more immobile with music. To explore this idea further, our perspective is to analyse behaviors in longer periods with and without music diffusion. Moreover, in order to understand where this difference comes from, it would be
interesting to explore the metabolism of lambs and examine their body composition, in particular the
rate of fat and muscle mass growth.

Working with young lambs bred by mother-deprivation with milk replacement, two main behavioral
changes were expected: the modification of feeding behavior with the access to solid food 10 days
after birth, and the decreased time spent lying down, the time in exploration increasing with the age
of lambs. We did observe these two phenomena in our study but with some differences according to
the music diffusion. First, Music lambs were the first to eat the solid food a couple of days before the
No music lambs. Next, because the solid food access was associated with an access to the entire
enclosure, we also observed an impact on exploration and resting behaviors. As expected, the time
spent “Lay down and Rest” decreased over time, certainly due to the fact that the lambs develop and
acquire autonomy.

The examination of the behavioral activities throughout the breeding period highlighted an impact of
music diffusion on behavioral activities but without clear increased well-being. Perhaps even on the
contrary, the music could have disrupted the lambs behavior. Indeed, by comparing the phases with
music and without music, we observed that lambs spent less time in exploration and ate less when
music was diffused, whereas they spent more time in laying and resting posture. Further examination
of this latter posture should allow us to discriminate whether lambs are in tonic immobility posture
(in stress) or in a relaxation posture.

The examination of basal hormonal profiles of oxytocin and cortisol could help us to interpret the
impact of music diffusion. The cortisol level was not different between the two groups except for two
days (where the cortisol level was higher for the Music group than the No Music group). Because we
do not observe an evident and constant impact of music on basal cortisol level, we conclude, with this
parameter, that the music exposure had neither a positive nor a negative effect to counteract the
absence of mother in lambs. However, we noticed high basal levels of plasma cortisol for both groups.
In rhesus macaque (Clarke, 1993) and sheep (Moberg et al., 1980), it has been shown to have a high
level of cortisol after birth that decreases during the following weeks. In our study, the level of cortisol
did not decrease. Thus, our results are in accordance with the literature indicating that mother-
deprivation with milk replacement could lead to an early stressful situation resulting in deregulation
of the HPA axis (Gaudin, 2015). Concerning oxytocin, our study highlighted higher basal levels of
plasma oxytocin in No Music lambs than in Music lambs. Because specific oxytocin plasma levels
(high or low) cannot be used as reliable biomarkers of a specific emotional state (Sippel et al., 2017),
it is difficult to conclude about the impact of music on lambs' well-being. In Gaudin's study (Gaudin
et al., 2018), elevated plasma oxytocin levels were observed in maternally deprived lambs. This high
level of oxytocin was interpreted as being due to the absence of a maternal figure. In our study, Music
lambs had lower basal levels of plasma oxytocin that could be interpreted in two opposite ways: a diminution of chronic stress due to absence of mother or an increase of chronic stress.

In this context, based on our hypothesis, our results did not suggest that music was able to counteract the deleterious impact of the absence of the mother or to increase lamb well-being (table 5).

<table><caption>Table 5: Summary of the differences between No Music and Music group during breeding</caption><thead><tr><th colspan="3">Breeding period</th></tr><tr><th>Variables</th><th>Expected data</th><th>Observed data</th></tr></thead><tbody><tr><td>Body weight</td><td>Music ≥ No Music</td><td>Music &lt; No Music</td></tr><tr><td>Sanitary status</td><td>Music ≥ No Music</td><td>Music = No Music</td></tr><tr><td>Stand and Explore</td><td>Music &gt; No Music</td><td>Music &lt; No Music</td></tr><tr><td>Lay down and Rest</td><td>Music &lt; No Music</td><td>Music &lt; No Music</td></tr><tr><td>Food intake</td><td>Music &gt; No Music</td><td>Music &gt; No Music</td></tr><tr><td>Milk drinking</td><td>Music &gt; No Music</td><td>Music &gt; No Music</td></tr><tr><td>Solid Food intake</td><td>Music &gt; No Music</td><td>Music &gt; No Music</td></tr><tr><td>Oxytocin</td><td>Music ≥ No Music</td><td>Music &lt; No Music</td></tr><tr><td>Cortisol</td><td>Music ≤ No Music</td><td>Music &gt; No Music</td></tr></tbody></table>

## Impact of music on brain morphology

The diffusion of music early after birth may have modified the development of brain regions or brain structures specifically involved in auditory perception, in locomotor activity or cognition. Based on our results, the impact of music diffusion, after about 2 months of exposure, is limited to the caudate nucleus and the medial geniculate nucleus.

The relative volume of the caudate nucleus was smaller for the Music group than the No Music group. It has previously been reported that the caudate nucleus is larger for lambs bred with their mother
(Love et al., 2021), probably due to high locomotor and cognitive activity. In our experiment, the No Music lambs were seen more often standing up and exploring than the Music group: this locomotor difference may be at the origin of the difference in caudate nucleus relative volume. The relative volume of the medial geniculate nucleus was larger for the Music group than for the No Music group in the right hemisphere. As the medial geniculate nucleus is involved in the auditory system (Henkel, 2018), this difference in relative volume of the medial geniculate nucleus may be due to the diffusion of music during breeding. Music may promote the development of this brain region. This part of our work would benefit from further investigation by studying other MRI parameters (signal intensity, FA...) and by studying the dynamics of specific brain structure development. Indeed, it has been observed that some impact of early experience could be transitory (Love et al., 2021).

*Emotional reactivity*

Our hypothesis was that if music is able to improve well-being, lambs exposed to music would be less sensitive to stress. We predicted that Music lambs would present lower stress responses than the No music lambs (table 6). Our observations did not confirm our hypothesis since music-exposed lambs presented similar high plasma cortisol levels, high numbers of bleats, higher escape attempts and locomotor activity than the No Music lambs, suggesting that music-exposed lambs were more stressed by social isolation. As we expected, the presence of the familiar human appeased the lambs of both groups. Interestingly, and as predicted, music diffusion during social isolation appeased exclusively the Music lambs. During this phase, Music lambs presented lower occurrences of locomotion and escape attempts than during the social isolation, this calming effect of music was not observed for No Music lambs. However, we noticed that, for both groups, females bleated more than males, which is consistent with the literature indicating that females are more stressed than males (Viérin & Bouissou, 2003). This result can be confirmed by the fact that the males also spent more time lying down during the test than the females, indicating that the males were less stressed than females. We also noticed that the animals from both groups spent more time close to music and human during the second and third phases showing that No Music lambs were also attracted by music (table 6).
<table><caption>Table 6: Summary of the differences between No Music and Music group during reactivity test</caption><thead><tr><th colspan="7">Reactivity test</th></tr><tr><th rowspan="2">Variables</th><th colspan="3">Expected data</th><th colspan="3">Observed data</th></tr><tr><th>SI</th><th>SI+M</th><th>SI+H</th><th>SI</th><th>SI+M</th><th>SI+H</th></tr></thead><tbody><tr><td>Cortisol</td><td>Music ≤ No music</td><td>Music &lt;&lt; No Music</td><td></td><td>Music = No Music</td><td>Music = No Music</td><td></td></tr><tr><td>Oxytocin</td><td>Music ≥ No music</td><td>Music &gt;&gt; No music</td><td></td><td>Music &lt; No Music</td><td>Music &lt; No Music</td><td></td></tr><tr><td>Locomotion</td><td>Music ≤ No music</td><td>Music &lt;&lt; No Music</td><td>Music = No Music</td><td>Music &gt; No music</td><td>Music = No Music</td><td>Music = No Music</td></tr><tr><td>Bleats</td><td>Music ≤ No music</td><td>Music &lt;&lt; No Music</td><td>Music = No Music</td><td>Music = No Music</td><td>Music = No Music</td><td>Music = No Music</td></tr><tr><td>Escape</td><td>Music ≤ No music</td><td>Music &lt;&lt; No Music</td><td>Music = No Music</td><td>Music &gt; No music</td><td>Music = No Music</td><td>Music = No Music</td></tr><tr><td>Lay down</td><td>Music &gt; No Music</td><td>Music &gt; No Music</td><td>Music = No Music</td><td>Music = No Music</td><td>Music = No Music</td><td>Music = No Music</td></tr><tr><td>Location (close to the stimulus Music or Human)</td><td></td><td>Music &gt;&gt; no music</td><td>Music = no music</td><td></td><td>Music = no music</td><td>Music &lt; no music</td></tr></tbody></table>

## Motivation

No difference was found between the Music and No Music lambs during this test except in the presence of the passive human. The number of bleats decreased during the H phase only for the No Music group. Music lambs may be less sensitive to human presence in the arena test and prefer unknown congeners. In this test, we also noticed that the animals were observed in the zones close to the stimuli when they were present (music and human), confirming the interest for music even for lambs that were not previously exposed to it.

## Conclusion

To our knowledge this is the first investigation of the possibility that music could help to counteract the negative consequences of mother-deprivation and milk-replacement early after birth.

Preliminary results obtained in male lambs after one week of exposure at the age of 7-10 days suggested that music was able to modify behavior (Ubiema et al., 2021). Our study confirms this
preliminary conclusion but does not show a clear positive impact on lambs' well-being. While music seems to have a calming effect on socially isolated lambs exposed to music during early life, it does not seem to have a positive impact during breeding. Positive impact was reported in several species (Ubiema et al., 2021) and also recently described in pigs (J. Li et al., 2021). In this latter study, music has an impact on behavioral response and cortisol levels in growing pigs, for which exposure to music started at the age of 40 days. In our study, music started only a few days after birth and lasted for about 2.5 months, which may be too early to detect its effect. Contrary to our preliminary study, lambs were exposed to a novel environment with music, an unknown sound, immediately after being separated from their mother. These environmental changes were maybe too abrupt and disturbed the lambs, too young to be exposed to music.

In most studies, only one unique song is played throughout the experiment (Aoun et al., 2005; J. Li et al., 2021; Sutoo & Akiyama, 2004). However, we made the choice to diffuse podcasts of varied classical music (rhythm, presence of singing or not etc.) to avoid boring the animals. It could be interesting to study the impact of only one song and to test other types of music. It has been shown that, in mice, “New born” music has an impact on stress but not classical or pop music (Van Loo et al., 2004). Moreover, some human voices were kept in the podcasts, because we wanted to test a situation that could be easily translated in farms - simply turning on the radio. If the animals have associated human voices heard during the music phases with the caregiver's voices, this could explain some differences between Music and No music lambs found in reactivity and motivations tests when a passive human was present.

To go further, it would be relevant to compare the effect of music to other ambient and natural noises or to the noises of congeners as it has been tested in some other studies (Cloutier et al., 2000; Wells et al., 2006). However, our study demonstrates that lambs are not insensitive to classical music. First, we noticed that in stressful situations, lambs were attracted to music, which could be an indicator that music could have a calming effect. Second, for Music lambs, there is no doubt that behavioral activity was modified. At last, we noticed that it was less difficult to anesthetize the music-exposed lambs than the no music lambs. In this context, it may be interesting to repeat this experience to increase the groups' size.
## Author contributions

MS, CP, RN, FL, SL, EC contributed to protocol design; MS, CP, MM, OL, DD, SL, EC performed data acquisition; CP, OL, DD gave animal care; QD segmented the MRI images; MS, CP conducted behavioural observation of video recordings; MS, SL, EC performed data analysis and interpretation; MS, SL, EC wrote the first draft.

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Hypothèse phylogénie : les animaux sauvages
# I. Les animaux de la faune sauvage, un modèle d'intérêt pour l'étude de la phylogénie

Comme nous l'avons vu en introduction générale, les comportements adaptatifs des animaux font l'objet d'une sélection constante au cours de l'évolution. Du point de vue de l'écologie comportementale, les comportements qui ont fait l'objet d'une sélection tout au long de l'évolution concernent l'habitat, la socialité ou l'alimentation des animaux. En neurosciences, ce cadre théorique a été repris dans des travaux de neuroanatomie comparée pour comprendre l'évolution du cerveau humain (Herculano-Houzel, 2019; Mota et al., 2019). Ces travaux ont examiné différents paramètres rendant compte de l'anatomie cérébrale, comme les volumes de matière grise corticale et de matière blanche sous corticale, et n'ont pas mis en évidence de relation universelle commune à toutes les espèces de mammifères (Mota et al., 2019). Au contraire, il semblerait même que de tels paramètres sont communs à différentes classes de mammifères (Herculano-Houzel, 2019). A l'échelle neuranatomique, notre équipe a constaté que l'organisation des connexions de la PAG décrites chez la brebis adulte présentait des différences notables avec celle décrite chez le rat ou le chat mais qu'elle était proche de celle décrite chez le lapin ou le singe écureuil (Menant et al., 2018). Cette différence a été interprétée sur la base de l'écologie comportementale des différentes espèces, à savoir que la brebis, le lapin et le singe écureuil sont des proies alors que le rat et le chat sont des prédateurs.

C'est dans ce contexte que nous avons initié une approche de biologie comparée pour tester notre hypothèse sur la phylogénie. Nous avons pu envisager une telle démarche grâce à notre partenaire Beauval Nature et le ZooParc de Beauval qui a collecté les encéphales lors des autopsies des animaux décédés de cause naturelle dans le zooparc. A l'image du don du corps pour les recherches en biologie humaine, la disponibilité des pièces anatomiques n'est pas prévisible et les approches proposées sont essentiellement qualitatives et descriptives. Pour initier notre travail, sur l'ensemble des pièces anatomiques prélevées, nous avons sélectionné dans un premier temps des espèces herbivores (sitatunga, bongo, cerf-souris, maki-catta) et des espèces carnivores prédatrices (jaguar, tigre, otarie). Bien qu'étant nés en captivité, les animaux expriment la plupart des comportements de l'espèce en particulier lorsque les enclos sont adaptés à leurs besoins en termes d'habitat et d'enrichissement (caches alimentaires par exemple). Ainsi, il existe toujours une hiérarchie sociale dans les groupes de félins ou de primates, qui s'exprime notamment lors de la distribution alimentaire. Quant aux comportements de chasse propres aux prédateurs, même s'ils ne peuvent s'exprimer comme en milieu naturel, il peut arriver que les animaux reproduisent leurs comportements de chasse avec de petites proies telles que des pigeons ou des rongeurs égarés dans les enclos ! S'agissant des herbivores, même
s'ils ne sont plus soumis à la prédation dans leurs enclos, ils présentent toujours un comportement craintif, notamment face à l'être humain, ainsi que leur caractère grégaire.

Dans ce travail, je présenterai brièvement les caractéristiques générales relatives à chaque spécimen étudié avant de présenter la méthodologie utilisée et les premiers résultats comparatifs.

## 1. Caractéristiques générales des spécimens

Les spécimens présentés ici sont :

- 3 ruminants : le sitatunga, le bongo, le cerf-souris

- 1 primate omnivore à tendance frugivore : le maki-catta

- 3 carnivores : le tigre, le jaguar, l'otarie.

<figure><img src="image_59.png" /><figcaption>Figure 12 : Photo d'un groupe de sitatungas prise au ZooParc de Beauval (Saint Aignan, France)</figcaption></figure>

Le sitatunga (Tragelaphus spekii) ou Guib d'eau (Figure 12), de la famille Bovidae, du genre Tragelaphus est une antilope semi-aquatique (Sitatunga (guib d'eau) : description + photos, 2019; Sitatunga - Wikipédia, 2021). C'est un herbivore qui a la capacité de se nourrir en étant semi ou totalement immergé dans l'eau. Il se nourrit à la fois d'herbes, de feuilles, de plantes aquatiques et de fruits. Cette antilope se trouve en Afrique centrale mais aussi

Afrique de l'Est et Afrique de l'Ouest. Son habitat se trouve dans les grandes forêts africaines ainsi que dans les zones humides marécageuses. Du point de vue de la socialité, le sitatunga est un animal social et grégaire qui vit en troupeau de quelques individus, essentiellement constitué de femelles et de jeunes. Comme pour les ovins, les mâles vivent entre eux, et rejoignent les femelles en période de reproduction. Il existe un dimorphisme sexuel au niveau du pelage avec des mâles bruns foncés tandis les femelles présentent un pelage de couleur fauve. Les deux genres portent des rayures et des taches blanches sur leur pelage. La taille des individus diffère aussi avec le genre. Les mâles pèsent entre 70 et 110 kg, mesurent entre 150 et 160 cm pour une hauteur au garrot de 100 à 120 cm et possèdent des cornes en spirale pouvant mesurer jusqu'à 90 cm. Les femelles sont plus petites puisqu'elles mesurent environ 130-140 cm pour une hauteur de garrot d'environ 80-90 cm pour un poids d'environ 45 à 65 kg, elles ne possèdent pas de cornes. La durée de gestation est de 7 à 8 mois pour des portées de 1 à 2 petits maximum. Les petits arrivent à maturité sexuel à 1 an pour les femelles et plus tardivement pour les mâles, vers 1 an et demi. Leur longévité est d'environ 19 ans.
Le bongo (*Tragelaphus eurycerus*) de la famille *Bovidae*, du genre *Tragelaphus* est une grande antilope (Figure 13) (Bongo, 2021; Bongo (animal) — Wikipédia, 2021; Bongo : Fiche descriptive complète + photos, 2019). C'est un herbivore qui se nourrit de jeunes pousses d'arbustes, de bambous, de racines et de graminées. Son habitat se trouve principalement en Afrique équatoriale (pour la sous espèce

*Tragelaphus eurycerus eurycerus*) et plus rarement dans une zone montagneuse du Kenya (pour la sous espèce *Tragelaphus eurycerus isaaci*). C'est un animal social et grégaire qui vit en groupe familial de 5 à 7 individus et parfois seulement en couple. Les animaux présentent un pelage roux avec des rayures verticales et des taches blanches, ainsi qu'une crête de poils au niveau de la colonne vertébrale. Les mâles pèsent entre 240 et 405 kg tandis que les femelles pèsent entre 210 et 235 kg. Ils mesurent entre 170 et 250 cm avec une hauteur au garrot de 125 cm. Les mâles mais aussi les femelles possèdent des cornes en spirales pouvant mesurer jusqu'à 1 m. La durée de gestation est de 9 à 10 mois pour des portées de 1 à 2 petits maximum. Les petits arrivent à maturité sexuelle à 20 mois. Leur longévité est d'environ 17 ans dans la nature et 19 ans en captivité.

<figure><img src="image_60.png" /><figcaption>Figure 13 : Photo d'un bongo (Tragelaphus eurycerus)</figcaption></figure>
<figure><img src="image_61.png" /><figcaption>Figure 14 : Photo d'un cerf souris (Tragulus javanicus) prise au ZooParc de Beauval (Saint-Aignan, France)</figcaption></figure>

Le cerf souris (Tragulus javanicus) ou Petit kanchil de Java de la famille Tragulidae, du genre Tragulus est un herbivore (Figure 14) (Cerf-souris – Tragulus kanchil, 2021; Tragulus javanicus — Wikipédia, 2021). Il se nourrit d'herbes, de fruits, de jeunes pousses, de fruits mais aussi parfois d'insectes. Ce mammifère se trouve seulement sur l'île de Java en Indonésie, dans les forêts tropicales. C'est un animal qui vit en couple et parfois en solitaire lorsqu'il devient plus âgé. Il mesure 45 cm avec une hauteur au garrot de 20 à 25 cm et pèse environ 2 kg. Cela fait du cerf souris l'un des plus petits ongulés qui existe. Il ne possède pas de bois mais le mâle possède deux grandes canines qui

sortent de sa bouche et qui vont servir lors de combats. La durée de gestation est d'environ 150 jours pour des portées de 1 à 2 petits maximum. Les petits arrivent à maturité sexuelle à 4 ou 5 mois. Lors d'observations menées au ZooParc de Beauval, nous avons noté la capacité du cerf-souris à du toilettage grâce à sa longue langue (Figure 15).

<figure><img src="image_62.png" /><figcaption>Figure 15 : Photo d'un cerf souris se toilettant (ZooParc de Beauval)</figcaption></figure>
Le maki catta (Lemur catta) de la famille Lemuridae, du genre Lemur est un primate lemuriforme (Figure 16) (Lémur catta — Wikipédia, 2021; Maki Catta, 2021). Il est omnivore à tendance frugivore. Ce lémurien est présent exclusivement à Madagascar, plus précisément au sud et sud-ouest de l'île. Il vit dans des fourrées épineuses ou des forêts sèches. C'est une espèce sociale qui vit en groupe d'une vingtaine d'individus ; souvent composé d'une femelle dominante et d'un seul mâle. Ils sont de couleur brun-gris sur le dos, gris clair sur la croupe et les membres, et blanche sur le poitrail. Sa queue mesure environ 60 cm et est annelée de noir et blanc, elle sert de balancier pour sauter dans les arbres. Ses membres ont 5 doigts terminés par un ongle plat et les mains ont des pouces opposables. Il mesure environ 40 cm et pèse entre 2,3 et 3,5 kg. La durée de gestation est d'environ 4

mois pour des portées de 1 à 2 petits maximum. Les petits arrivent à maturité sexuelle vers l'âge de
2-3 ans. La longévité du maki catta est de 20 à 25 ans.

<figure><img src="image_63.png" /><figcaption>Figure 16 : Photo d'un maki catta (Lemur catta)</figcaption></figure>

<figure><img src="image_64.png" /><figcaption>Figure 17 : Photo d'un jaguar (Panthera onca)</figcaption></figure>

Le jaguar (Panthera onca) de la famille des Felidae, du genre Panthera est un grand félin (Figure 17) (Jaguar: Description du félin d'Amérique du Sud + photos, 2021; Jaguar — Wikipédia, 2021). C'est un carnivore, chasseur opportuniste qui va manger à la fois des grandes proies (cervidés, serpents capibaras etc...) et des proies plus petites (grenouilles, poissons, tortues etc...). Le jaguar se trouve en

Amérique centrale et Amérique du Sud (du Mexique au Paraguay). Il vit essentiellement dans la forêt tropicale mais en présence d'étendues d'eau, c'est donc un très bon nageur. C'est une espèce solitaire et territoriale, les mâles et les femelles se rencontrent seulement pour l'accouplement. Leur pelage est jaune tacheté. Les femelles mesurent entre 1,10 et 1,70 m pour une hauteur au garrot de 70 cm et pèsent entre 40 et 70 kg. Les mâles sont plus imposants, ils mesurent entre 1,40 et 1,85 m et pèsent
entre 55 et 100 kg. La durée de gestation est de 91 à 111 jours pour une portée de 2 à 4 petits. Les petits arrivent à maturité sexuelle à l'âge de 2-3 ans pour les femelles et 3-4 ans pour les mâles. Leur durée de vie est d'environ 15 ans en milieu naturel et 23 ans captivité.

Le tigre (*Panthera tigris*) de la famille *Felidae*, du genre *Panthera* est un grand félin (Figure 18) (*Tigre : description complète du félin* + photos, 2020; *Tigre — Wikipédia*, 2021). C'est un carnivore qui chasse de grosses proies telles que le cerf, le sanglier ou le buffle. Il se retrouve dans toute l'Asie (Inde, Népal, Bangladesh, Indonésie etc...). Il vit dans les forêts tropicales, les mangroves mais aussi dans les marécages.

C'est un très bon nageur capable de traverser des rivières. C'est une espèce solitaire et territoriale. Son pelage est orange avec des rayures noires verticales. Sa taille et son poids varient en fonction de la sous-espèce : ils peuvent mesurer de 1,40 à 3,30 m pour une hauteur au garrot de 80 cm à 1 m et peser entre 65 et 185 kg pour les femelles et 180 à 320 kg pour les mâles. La durée de gestation est de 3 à 4 mois pour une portée de 2 à 3 petits. Les petits arrivent à maturité sexuelle à l'âge de 3 ans pour les femelles et entre 3 et 6 ans pour les mâles. Leur durée de vie est d'environ 15 ans en milieu naturel et jusqu'à 25 ans en captivité.

<figure><img src="image_65.png" /><figcaption>Figure 18 : Photo d'un tigre (Panthera tigris)</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>Figure 19 : Photo d'un groupe d'otaries de Californie (Zalophus californianus)</figcaption></figure>

L'otarie de Californie (*Zalophus californianus*) ou lion de mer de Californie, de la famille *Otariidae* et du genre *Zalophus*) est un mammifère marin carnivore, plus particulièrement piscivore (Figure 19) (*Lion de mer de Californie : description* + photos, 2019; *Otarie de Californie — Wikipédia*, 2021).

L'otarie se nourrit de poissons tels que le saumon, les anchois ou

encore les pieuvres. Elle se trouve dans l'océan Pacifique, au niveau des côtes Ouest de la Californie,
de l'Alaska jusqu'au Costa Rica. L'otarie de Californie vit près des côtes et également près de zones fortement peuplées telles que les ports. C'est une espèce sociale et grégaire qui vit en colonies composées de dizaines d'individus. Leur pelage est brun foncé, les mâles ont en plus une coloration plus claire au niveau du ventre et des flancs. Il existe un important dimorphisme sexuel au niveau de la taille avec les mâles qui mesurent entre 2 m et 2,50m et pèsent de 200 à 300 kg tandis que les femelles mesurent de 1,50 à 2 m et pèsent entre 50 et 100 kg. La durée de gestation est de 11 à 12 mois pour une portée de 1 à 2 petits maximum. Les petits arrivent à maturité sexuelle entre 3 et 5 ans pour les femelles et 5 à 6 ans pour les mâles. Leur durée de vie est de 15 à 25 ans.

## II. Méthodologie

### 1. Pièces anatomiques

Les cerveaux des sept spécimens ont été prélevés lors de l'autopsie systématique pratiquée après le décès constaté des animaux du ZooParc de Beauval (Saint-Aignan, France). Les décès naturels sont constatés lors des visites matinales des équipes, les dépouilles sont alors transportées et maintenues en chambre froide jusqu'à l'autopsie qui a lieu en fin de matinée ou en fin de journée selon la disponibilité des équipes vétérinaires. Sur la base de l'expérience du laboratoire d'anatomie et de l'association du don du corps (CHRU Bretonneau, Université de Tours), nous savons qu'au-delà de 12 heures après le décès, si la dépouille n'est pas conservée à +4°C, les organes commencent à se dégrader. Il a donc été convenu, en amont, du projet que seules les pièces pouvant être prélevées dans ce délai de 12 heures seraient collectées.

Lors de l'autopsie, les dépouilles ont été pesées, les encéphales ont été prélevés et immergés dans des flacons prêts à l'emploi contenant du formol 4% et conservés à 4°C pour être fixés. Après plusieurs semaines de fixation, les encéphales ont été pesés.

### 2. Acquisitions IRM

Au minimum 2 jours avant l'acquisition IRM, les encéphales ont été réhydratés dans une solution de tampon phosphate afin de récupérer du signal puis immergés dans du Fluorinert.

En fonction de la taille du cerveau, les acquisitions ont été réalisées avec des scanners IRM et des antennes adaptés. Les cerveaux du cerf souris, du maki catta, du sitatunga, du bébé jaguar et du bébé tigre étant de petite taille, ils ont été imagés avec un scanner IRM préclinique Bruker BioSpec de 11,7
Tesla (Gmax=780mT/m, slew-rate=9500T/m/s) associé à une antenne émettrice-réceptrice Bruker.
Les cerveaux du bongo et de l'otarie de Californie étant de taille plus grosse, ont été imagés avec un
scanner IRM clinique Siemens de 7 Tesla (Gmax=50mT/m, slew-rate=333T/m/s) associé à une
antenne Nova Médical 1Tx/32Rx.

Pour les cerveaux imagés avec le scanner IRM préclinique 11,7 Tesla, une acquisition anatomique a été réalisée en utilisant une séquence 2D Écho de Spin (SE) pondérée en T₂. La résolution a été adaptée en fonction de chaque individu : 175 µm pour le cerf souris, 150 µm pour le maki catta, 200 µm pour les bébés tigre et jaguar ainsi que pour le sitatunga. Les autres paramètres de la séquence étaient : TE/TR = 16/9000ms, flip angle = 90°, 9-10 excitations, 1 répétition. La durée d'acquisition était d'environ 5 heures pour chaque pièce anatomique. Pour les cerveaux de bongo et d'otarie de Californie imagés avec le scanner IRM clinique 7 Tesla, une acquisition anatomique a été réalisée en utilisant une séquence 3D SPACE pondérée en T₂. La résolution d'acquisition était de 500 µm. Les autres paramètres de la séquence étaient : TE/TR = 193/3200ms, 1 excitation, 1 écho, flip angle = 120°. La durée d'acquisition était d'environ 55 minutes.

Les images RM ont été réorientées selon l'axe CA-CP avant d'être segmentées.

### 3. Segmentation des images IRM

#### a. Encéphale

A partir des images RM, chaque encéphale a été segmenté semi automatiquement. Un seuil a été appliqué pour sélectionner autant de tissu cérébral que possible tout en excluant l'arrière-plan de l'image ce qui a permis de créer des masques de chaque encéphale. Ces masques ont ensuite été corrigés manuellement sur le logiciel de segmentation ITK-Snap pour s'assurer que tout le tissu cérébral était inclus dans le masque.

#### b. Régions d'intérêt

Pour chaque cerveau, des régions d'intérêts ont été délimitées manuellement à l'aide du logiciel ITK-Snap. Les régions segmentées sont la PAG et le noyau caudé.
### III. Résultats

#### 1. Caractéristiques morphologiques des spécimens

Le tableau 2 présente les différentes caractéristiques morphologiques des spécimens étudiés :

<table><caption>Tableau 2 : Tableau indiquant les différentes caractéristiques des spécimens étudiés : âge, poids corporel, poids et volume de l'encéphale</caption><thead><tr><th>Espèce animale</th><th>Âge</th><th>Poids corporel (kg)</th><th>Poids encéphale (g)</th><th>Volume encéphale (mm³)</th></tr></thead><tbody><tr><td>Bongo</td><td>2 ans</td><td>150</td><td>386</td><td>353600</td></tr><tr><td>Cerf souris</td><td>1 an</td><td>1,438</td><td>15</td><td>12080</td></tr><tr><td>Cerf souris</td><td></td><td></td><td></td><td></td></tr><tr><td>Jaguar</td><td>1 mois</td><td>3,1</td><td>66,2</td><td>56280</td></tr><tr><td>Maki catta</td><td>-</td><td>2,75</td><td>29</td><td>25590</td></tr><tr><td>Otarie</td><td>-</td><td>212</td><td>399,99</td><td>377800</td></tr><tr><td>Sitatunga</td><td>2 ans et 9 mois</td><td>28,12</td><td>138,8</td><td>-</td></tr><tr><td>Tigre</td><td>- (bébé)</td><td>1,145</td><td>59,9</td><td>52680</td></tr></tbody></table>

<figure><img src="image_67.png" /><figcaption>Figure 20 : Répartition des différentes espèces animales en fonction du poids de leur encéphale et de leur poids corporel</figcaption></figure>
La représentation du poids des encéphales en fonction du poids corporel montre que plus l'animal est grand, plus son encéphale est lourd (Figure 20). Cette relation semble linéaire pour nos individus. Dans la littérature, il a été montré que chez les mammifères, le poids de l'encéphale est déterminé à 90 % par le poids corporel (Dicke & Roth, 2016). Cependant, à l'échelle de chaque espèce, certaines respectent plus ou moins cette relation. Le cerveau du dauphin, du chimpanzé ou de l'Homme est plus gros qu'attendu tandis que le cerveau de l'hippopotame ou de la baleine bleue est plus petit que ce que laisse penser cette relation.

<figure><img src="image_68.png" /><figcaption>Figure 21 : Répartition des différentes espèces animales en fonction du volume (mm3) et du poids (kg) de leur encéphale</figcaption></figure>

La représentation du volume en fonction du poids de l'encéphale semble également linéaire bien que deux cerveaux (tigre et jaguar) soit des cerveaux de juvéniles qui ne sont donc probablement pas matures. L'immaturité de ces deux encéphales est perceptible à l'examen des images RM peu contrastées (Figure 21) pour lesquelles les faisceaux de fibres blanches sont moins visibles que pour les spécimens adultes.

## 2. Morphologie des régions d'intérêt

Pour chaque spécimen, l'encéphale a été segmenté ainsi que la PAG et le noyau caudé (Figure 22 à Figure 27).
<figure><img src="image_69.png" /></figure>

Figure 22 : Coupes axiales (gauche), coronales (milieu) et sagittales (droite) d'un encéphale de bongo superposées avec le masque cérébrale (en haut) et les régions d'intérêt (au milieu) focalisées sur la PAG (en vert). La rangée du bas présente la reconstruction 3D du masque cérébral. A = Avant, S = Supérieur, P = Postérieur, R = Hémisphère droit. Images acquises avec un scanner IRM 7 Tesla à NeuroSpin.
<figure><img src="image_70.png" /></figure>

Figure 23 : Coupes axiales (gauche), coronales (milieu) et sagittales (droite) d'un encéphale de cerf souris superposées avec le masque cérébrale (en haut) et les régions d'intérêt (au milieu) focalisées sur la PAG (en vert). La rangée du bas présente la reconstruction 3D du masque cérébral. A = Avant, S = Supérieur, P = Postérieur, R = Hémisphère droit. Images acquises avec un scanner IRM 11.7 Tesla à NeuroSpin.
<figure><img src="image_71.png" /><figcaption>Figure 24 : Coupes axiales (gauche), coronales (milieu) et sagittales (droite) d'un encéphale de jaguar juvénile superposées avec le masque cérébrale (en haut) et les régions d'intérêt (au milieu) focalisées sur la PAG (en vert). La rangée du bas présente la reconstruction 3D du masque cérébral. A = Avant, S = Supérieur, P = Postérieur, R = Hémisphère droit. Images acquises avec un scanner IRM 11.7 Tesla à NeuroSpin</figcaption></figure>
<figure><img src="image_72.png" /></figure>

Figure 25 : Coupes axiales (gauche), coronales (milieu) et sagittales (droite) d'un encéphale de maki catta superposées avec le masque cérébrale (en haut) et les régions d'intérêt (au milieu) focalisées sur la PAG (en vert). La rangée du bas présente la reconstruction 3D du masque cérébral. A = Avant, S = Supérieur, P = Postérieur, R = Hémisphère droit. Images acquises avec un scanner IRM 11.7 Tesla à NeuroSpin
<figure><img src="image_73.png" /><figcaption>Figure 26 : Coupes axiales (gauche), coronales (milieu) et sagittales (droite) d'un encéphale d'otarie superposées avec le masque cérébrale (en haut) et les régions d'intérêt (au milieu) focalisées sur la PAG (en vert). La rangée du bas présente la reconstruction 3D du masque cérébral. A = Avant, S = Supérieur, P = Postérieur, R = Hémisphère droit. Images acquises avec un scanner IRM 7 Tesla à NeuroSpin.</figcaption></figure>
<figure><img src="image_74.png" /><figcaption>Figure 27 : Coupes axiales (gauche), coronales (milieu) et sagittales (droite) d'un encéphale de tigre superposées avec le masque cérébrale (en haut) et les régions d'intérêt (au milieu) focalisées sur la PAG (en vert). La rangée du bas présente la reconstruction 3D du masque cérébral. A = Avant, S = Supérieur, P = Postérieur, R = Hémisphère droit. Images acquises avec un scanner IRM 11.7 Tesla à NeuroSpin</figcaption></figure>
Quelle que soit l'espèce considérée, la forme du noyau caudé comme celle de la PAG sont très similaires. L'examen des volumes relatifs de ces structures (Tableau 3) suggère que la PAG occupe un volume dans l'encéphale plus petit que le noyau caudé chez toutes les espèces. Chez le tigre et le jaguar, la PAG occupe un volume environ deux fois plus petit que le noyau caudé. Pour le bongo et le cerf souris, les volumes occupés par la PAG et le noyau caudé sont relativement égaux. Chez le maki catta et l'otarie, le volume occupé par la PAG semble être cinq fois plus petit. Suite à ces observations, il semble donc que le volume occupé par la PAG et par le noyau caudé diffère selon les espèces étudiées.

<table><caption>Tableau 3 : Récapitulatif du volume de l'encéphale, de la PAG, du noyau caudé ainsi que du pourcentage du volume de la PAG et du noyau caudé en fonction du volume de l'encéphale pour chaque espèce</caption><thead><tr><td>Animal</td><td>Volume de l'encéphale (mm³)</td><td>Volume de la PAG (mm³)</td><td>Volume du noyau caudé (mm³)</td><td>% du volume de la PAG en fonction du volume de l'encéphale</td><td>% du volume du noyau caudé en fonction du volume de l'encéphale</td></tr></thead><tbody><tr><td>Bongo</td><td>353600</td><td>704,5</td><td>993,65</td><td>0,199</td><td>0,281</td></tr><tr><td>Maki catta</td><td>25590</td><td>22,55</td><td>198,9</td><td>0,088</td><td>0,777</td></tr><tr><td>Cerf souris</td><td>12080</td><td>57,68</td><td>63,48</td><td>0,477</td><td>0,525</td></tr><tr><td>Otarie</td><td>377800</td><td>214,1</td><td>2157,5</td><td>0,057</td><td>0,571</td></tr><tr><td>Tigre</td><td>52680</td><td>106,6</td><td>303,15</td><td>0,202</td><td>0,575</td></tr><tr><td>Jaguar</td><td>56280</td><td>153,18</td><td>271,1</td><td>0,272</td><td>0,482</td></tr></tbody></table>

## IV. Améliorations de la préparation des pièces anatomiques

### 1. Gestion des prélèvements

Dans les spécimens présentés ici, tous les encéphales ont été extraits de la boîte crânienne pour être immergés directement dans le formol. Cette manière de procéder rend la manipulation des tissus compliquée de par leur fragilité. En effet, lors de l'extraction, la boîte crânienne est découpée avec un craniotomie (petite scie circulaire) qui selon l'épaisseur du crâne de l'individu peut engendrer des lésions sur l'encéphale (« coup de scie »). Par ailleurs, l'encéphale est prélevé sans être fixé par perfusion (cf. modalité de prélèvement des encéphales de caille), ce qui le fragilise d'autant plus notamment lorsqu'il est placé dans le contenant pour la fixation. A cette étape, l'encéphale peut être déformé (cas de l'otarie) en prenant par exemple la forme de son contenant. Enfin, la fragilité du tissu « frais » rend sa manipulation délicate, les différentes parties de l'encéphale pouvant être séparés lors
de l'extraction de la boîte crânienne ou de l'immersion dans le formol (cas du sitatunga non présenté ici).

Afin d'améliorer cette étape cruciale, il a été décidé de ne plus extraire les encéphales des têtes et de réaliser une fixation sur les têtes dépecées. Cela permet de maintenir le tissu en place dans la boite crânienne. Cette nouvelle méthode de fixation est toujours en cours de validation, puisqu'elle pose également des problèmes de fixation, le liquide ne parvenant pas forcément à rentrer parfaitement dans les tissus. La solution idéale serait de réaliser des perfusions pour réaliser une fixation optimale. Cependant, cette technique demanderait des aménagements ainsi qu'une certification des bâtiments pour la sécurité des personnels et de l'environnement.

## 2. Préparation des pièces pour acquisition :

Lors des segmentations des régions anatomiques des IRMs acquises *ex vivo* (cailles et animaux issus de la faune sauvage), nous avons été confrontés à la difficulté de segmenter le système ventriculaire. Ce dernier contient le liquide cérébrospinal qui présente un contraste noir sur des images pondérées en T₁ et acquises *in vivo*. Sur les images acquises *ex vivo*, en pondération T₂, le système ventriculaire présente un contraste noir ou blanc, cette différence étant liée à la présence ou non d'air dans les ventricules. L'air peut pénétrer dans les ventricules lors du prélèvement, lors du rinçage des pièces anatomiques ou lors du transfert des pièces dans le milieu d'acquisition (fluorinert®). La présence de la boîte crânienne n'empêche pas la pénétration de l'air dans les ventricules qui se vident probablement de leur contenu lors des manipulation successives. Nous l'avons constaté pour la segmentation du template de l'encéphale de caille, avec des prises de contraste totalement différentes pour le ventricule latéral entre deux coupes successives.

Confrontés au même problème avec les pièces anatomiques humaines, nos partenaires du laboratoire d'anatomie (iBrain, Inserm) ont mis en place un système d'agitation sous vide pour les bains de fixation, de rinçage ou d'immersion dans le tampon d'acquisition. De cette manière, la pénétration des liquides dans le système ventriculaire est améliorée, ce qui garantit une prise de contraste homogène.

## V. Perspectives

Sur les images actuelles, nous allons poursuivre la segmentation de structures clefs comme l'hippocampe, l'amygdale et extraire pour chacune de ces structures les tractogrammes. Le recalage