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International audience; Limited studies in humans and in animal models have investigated the neurotoxic risks related to a gestational exposure to diesel exhaust particles (DEP) on the embryonic brain, especially those regarding monoaminergic systems linked to neurocognitive disorders. We previously showed that exposure to DEP alters monoaminergic neurotransmission in fetal olfactory bulbs and modifies tissue morphology along with behavioral consequences at birth in a rabbit model. Given the anatomical and functional connections between olfactory and central brain structures, we further characterized their impacts in brain regions associated with monoaminergic neurotransmission. At gestational day 28 (GD28), fetal rabbit brains were collected from dams exposed by nose-only to either a clean air or filtered DEP for 2 h/day, 5 days/week, from GD3 to GD27. HPLC dosage and histochemical analyses of the main monoaminergic systems, i.e., dopamine (DA), noradrenaline (NA), and serotonin (5-HT) and their metabolites were conducted in microdissected fetal brain regions. DEP exposure increased the level of DA and decreased the dopaminergic metabolites ratios in the prefrontal cortex (PFC), together with sex-specific alterations in the hippocampus (Hp). In addition, HVA level was increased in the temporal cortex (TCx). Serotonin and 5-HIAA levels were decreased in the fetal Hp. However, DEP exposure did not significantly modify NA levels, tyrosine hydroxylase, tryptophan hydroxylase or AChE enzymatic activity in fetal brain. Exposure to DEP during fetal life results in dopaminergic and serotonergic changes in critical brain regions that might lead to detrimental potential short-term neural disturbances as precursors of long-term neurocognitive consequences.

Resume Objectifs Selon le concept des « origines developpementales de la sante et des maladies », l’obesite maternelle predispose la descendance aux maladies chroniques. Pour de multiples raisons, une perte de poids preconceptionnelle est recommandee aux femmes obeses. Cependant, les avantages et les potentiels inconvenients pour l’enfant a naitre sont encore mal etudies. Methodes Nous avons evalue le developpement fœtal et analyse l’expression de modificateurs epigenetiques et de genes metaboliques dans le foie et le placenta. Les descendants nes de mere « controle », « obese » ou « perte de poids » ont ete soumis apres le sevrage a un regime controle ou hyperlipidique. Nous avons suivi les parametres metaboliques et les comportements olfactifs jusqu’a l’âge adulte. Resultats Les fœtus de mere obese presentent une restriction de croissance et une alteration de l’expression de modificateurs epigenetiques de la voie d’acetylation des histones. Les phenotypes metaboliques et olfactifs (obesite, dyslipidemie, intolerance au glucose et faible motivation et capacite a trouver une recompense alimentaire) s’expliquent principalement par le regime post-sevrage, mais l’obesite maternelle a un effet notable de conditionnement dependant du sexe : les mâles nes de mere obese developpent une obesite accrue par rapport a leurs congeneres. La perte de poids preconceptionnelle normalise les phenotypes fœtaux et metaboliques decrits ci-dessus, mais certains genes restent differentiels. De plus, elle a des effets inattendus sur les performances olfactives : la sensibilite olfactive peripherique et la motivation induite par le jeune sont reduites. Conclusions Nos resultats confirment les avantages d’une perte de poids preconceptionnelle pour la sante metabolique des descendants, mais revelent de potentiels effets indesirables tels qu’observes pour le comportement olfactif.

International audience; Adaptations to the ever changing social and physical environment are necessary, sometimes involving the stress response. Olfaction is a chemical sense allowing the detection of molecules in the air, and many species rely on it for social recognition and fundamental functions such as feeding or reproduction. This non-exhaustive literature review addresses three topics at the intersection of sensory biology and stress biology, in order to raise awareness and enlighten our understanding of animal behaviour. First, we show that stress and associated emotions, suchas fear, can alter odour perception, from the sensor (peripheral nervous system) to the interpretation of the sensory input (brain). This can be of relevance for understanding the behavioural consequences of negative experiences and depression-like states. Second, odours involved in animal chemical communication can signal danger in the form of predators or stressed conspecifics, and in turn induce stress in recipient animals, as a formof chemical empathy. Finally, we review some of the evidence for the use of odours as stress-relief agents. There is no clear evidence of treatment efficacy and it might be that under the labels of inhalation aromatherapy, aromachology or pheromone therapy, a lack of perspective has led to the conflation of various biological phe-nomena: an alleged spontaneous olfactory effect on stress relief, environmental enrichment or the recall of positive emotional states. Careful experimental design and critical analysis are paramount to clearly evaluate this promising intervention. The world around us shapes our emotions, which in turn affect our perception. There might be a huge animal welfare potential to harvest from improved knowledge in the studies of stress andolfaction interactions, this will eventually allow us to show more empathy to animals in our care.

Rats detect and use odorant molecules as a source of information about their environment. Some of these molecules come from conspecifics, and many arise as by-products from microbial activity. Thus, compared to conventionally housed rats, germ-free rats are raised in an environment with fewer odorants, but this reduction is rarely quantified. Using gas chromatography-mass spectrometry, we found that germ-free rat faeces samples contained half as many volatile molecules than conventional rat faeces (52 vs 109 (±2.4) molecules; P < 0.001) and overall these were only 12% as abundant. We then investigated if odours from female germ-free rats in oestrus would have pro-erectile effects in conventional male rats. For this aim, conventionally housed Brown Norway (BN) rats (n = 16) with sexual experience with either Fischer or BN females, were exposed to four different odour types: faeces from germ-free Fischer rat in oestrus, faeces from conventional rats in oestrus and di-oestrus (either from Fischer or BN), and a control (either 1-hexanol or male rat faeces). The number of penile erections per test as well as the duration of freezing behaviour was significantly higher with the oestrous odours (germ-free and conventional) compared to the control, with intermediate responses to the di-oestrous faeces. The findings indicate that, despite a significantly reduced composition in terms of volatiles compared to conventionally housed rats, the faeces of germ-free rats contain sufficient odorants to evoke sexual responses in conventional male rats. Oestrous odours of rats thus appear not to be of microbial origin.

International audience; The present study examined in lambs whether exposure to flavors derived from pregnant mother‘s diet and transferred to amniotic fluid (AF) could induce a preference for artificial milk containing one of these flavors. To test this hypothesis, cumin was added to the maternal diet in the last month of gestation. Preference for artificial milk containing p-cymene, one of the chemosensory compounds of cumin, was tested within the first two days after birth in maternally deprived lambs born from mothers fed a cumin-flavored diet (Cumin group), or an unflavored diet (Control group). Aromatic profile of AF from cumin-fed mothers was analyzed by GC-MS/MS to determine whether p-cymene could be detected. While the control group avoided the flavored artificial milk on day 1, the Cumin group did not and showed a preference for the cumin-scented formula on day 2. GC-MS/MS profile of AF revealed that four of the main volatile cumin compounds, p-cymene, p-cymenene, β-pinene and γ-terpinene were present in variable amounts in all samples, p-cymene being the most frequently detected. These findings indicate that newborn lambs can memorize flavors from the mother's diet present in AF and that prenatal experience influences their preference for an artificial milk containing one specific flavor.

The olfactory mucosa holds olfactory sensory neurons directly in contact with an aggressive environment. In order to maintain its integrity, it is one of the few neural zones which are continuously renewed during the whole animal life. Among several factors regulating this renewal, endothelin acts as an anti-apoptotic factor in the rat olfactory epithelium. In the present study, we explored whether endothelin could also act as a proliferative factor. Using primary culture of the olfactory mucosa, we found that an early treatment with endothelin increased its growth. Consistently, a treatment with a mixture of BQ123 and BQ788 (endothelin receptor antagonists) decreased the primary culture growth without affecting the cellular death level. We then used combined approaches of calcium imaging, reverse transcriptase-quantitative polymerase chain reaction and protein level measurements to show that endothelin was locally synthetized by the primary culture until it reached confluency. Furthermore, in vivo intranasal instillation of endothelin receptor antagonists led to a decrease of olfactory mucosa cell expressing proliferating cell nuclear antigen (PCNA), a marker of proliferation. Only short-term treatment reduced the PCNA level in the olfactory mucosa cells. When the treatment was prolonged, the PCNA level was not statistically affected but the expression level of endothelin was increased. Overall, our results show that endothelin plays a proliferative role in the olfactory mucosa and that its level is dynamically regulated. This study was approved by the Comité d’éthique en expérimentation animale COMETHEA (COMETHEA C2EA -45; protocol approval #12-058) on November 28, 2012.

International audience; Early sensory experience, such as exposure to maternal or other environmental factors, is considered to influence neurocognitive development and behaviors. In many species, exposure to odorants during pregnancy or lactation impacts the morpho-functional development of the olfactory circuitry with changes in olfactory sensitivity, feeding behavior and food preferences at birth or later. However, few studies have investigated the impact of a perinatal exposure to odorants on the anxiety-like behavior of animals to stressfull stimuli. Here, we exposed mice to heptaldehyde (HEP) during pregnancy and lactation and measured the anxiety-like behavior of their offspring to stress-inducing novel stimuli at weaning in presence or absence of odorants. We applied a combined social and maternal separation as a stressor and measured the anxiety-like behavior in an open field (OF) in presence of two odorants, HEP or alpha-pinene (AP) as a control odorant. Although the presence of the odorant during the social separation did not influence anxiety-like behavior, we found that, if mice born to non-odorized mothers exhibited a decreased exploratory behavior in the presence of both odorants, the effect was restricted to AP for the mice perinatally exposed to HEP. These results show that anxiety-like behaviors during a stress-inducing event could be reduced by the presence of a familiar odorant. We propose that the recall of an early olfactory experience could contribute to the improvement of animal welfare in various situations associated with husbandry practices.

International audience; In this selective review, examples are given of how different animal species use different sensory modalities in different ways. These examples serve to illustrate that we should not forget to take sensory abilities of the animals that we manage into account to ensure or improve their welfare. As humans we use predominantly vision and hearing, and do not always consider the housing and handling of animals as it is perceived by them. Through published studies on the use of sight, hearing, touch, and smell by different species, this review shows that these sensory modalities, alone or in combination, are pivotal for appropriate management of animals in our care. This includes exposure to and use of appropriate stimuli, as well as removal of potentially stressful sensory inputs. Such measures are important not only for the validity of research results, but also for improved animal welfare and production.

Sofiane Safi-Stibler a obtenu une bourse de voyage de la Société Française de Nutrition pour présenter ses travaux lors de ce congrès. Sofiane Safi-Stibler a obtenu un prix de présentation orale lors du congrès de la SF-DOHaD à Grenoble en 2018 afin de présenter ses travaux au cours du Congrès International de la DOHaD 2019 à Melbourne.Sofiane Safi-Stibler a obtenu une bourse de voyage de la Société Française de Nutrition pour présenter ses travaux lors de ce congrès. Sofiane Safi-Stibler a obtenu un prix de présentation orale lors du congrès de la SF-DOHaD à Grenoble en 2018 afin de présenter ses travaux au cours du Congrès International de la DOHaD 2019 à Melbourne.; International audience; Background/AimsMaternal obesity is associated with fertility disorders, obstetric complications, and development of metabolic syndrome in offspring. The recommendation to overweight or obese women is to lose weight before pregnancy, but the impact of these prescriptions on the offspring health is insufficiently studied. Previous results on late-term mice fetuses have shown that maternal obesity leads to fetal growth restriction and to the modification of epigenetic machinery-related gene expression in fetal liver and placenta. Moreover, fetuses from the preconceptional weight loss maternal group normalize their growth, but some fetal genes stay differentially expressed.MethodWe analysed the metabolic phenotype of offspring born to control (CTRL), obese (OB) or weight loss after diet-induced obesity (WL) mothers. To highlight a possible conditioning effect, offspring were either fed a control (CD) or a high fat diet (HFD). Their metabolism and olfactory behavior were monitored for up to 6 months. Then, we analyzed the metabolome of three tissues involved in food intake and nutrient management (liver, olfactory bulb and hypothalamus) in male adult offspring.ResultsMultiple Factorial Analysis integration of offspring phenotypic data showed a major influence of the post-weaning diet and pointed a difference in HFD-fed males according to their maternal group (OB vs WL). This confirmed a sex-dependant metabolic conditioning by the maternal environment. The olfactory sensitivity, measured by electro-olfactogram, was reduced in WL male offspring, whatever the postnatal diet.The metabolomics study annotated 278, 258 and 200 metabolites in the liver, olfactory bulb and hypothalamus respectively. Again, the post-weaning diet had a major effect in the three tissues but interestingly, the maternal group also influenced the hepatic metabolome of adult offspring.ConclusionsThese data, integrating metabolism, olfactory behavior and metabolome, provide new and original information on the effects of preconceptional maternal metabolic status in the offspring health conditioning upon diet challenges.

Anne Gabory a reçu le prix du meilleur poster. Cette distinction a été offerte par l'Institute for Neuroscience, D-HEST, ETH Zurich.; Effect of maternal obesity and preconceptional weight loss on foeto-placental growth and offspring health in mice: expression of epigenetic modifiers at the interface with metabolism. Epigenetic Inheritance Symposium 2019

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International audience; Most associative learning tests in rodents use negative stimuli, such as electric shocks. We investigated if young rats can learn to associate the presence of an odour with the experience of being tickled (i.e. using an experimenter’s hand to mimic rough-and-tumble play), shown to elicit 50 kHz ultrasonic vocalisations (USVs), which are indicative of positive affect. Male, pair-housed Wistar rats (N = 24) were all exposed to two neutral odours (A and B) presented in a perforated container on alternate days in a test arena. Following 60s of exposure, the rats were either tickled on days when odour A (n = 8) or odour B (n = 8) was present, or never tickled (n = 8). When tickled, rats produced significantly more 50 kHz USVs compared to the days when not being tickled, and compared to control rats. The level of anticipatory 50 kHz USVs in the 60s prior to tickling did not differ significantly between the tickled and control rats. As a retrieval test following the odour conditioning, rats were exposed successively in the same arena to three odours: an unknown neutral odour, extract of fox faeces, and either odours A or B. Compared to controls, 50 kHz USVs of tickled rats increased when exposed to the odour they had previously experienced when tickled, indicating that these rats had learned to associate the odour with the positive experience of being tickled. In a test with free access for 5 min to both arms of a T-maze, each containing one of the odours, rats tickled with odour A spent more time in the arm with this odour. This work is the first to test in a fully balanced design whether rats can learn to associate an odour with tickling, and indicates that positive odour conditioning has potential to be used as an alternative to negative conditioning tests.

International audience; In the olfactory epithelium, olfactory sensitive neurons and their axons are surrounded by glia-like cells called sustentacular cells, which maintain both the structural and ionic integrity of the olfactory mucosa. We have previously found that endothelin-1 (ET-1) can uncouple sustentacular cell gap junctions in vitro similarly as carbenoxolone, a known gap junction uncoupling agent. The role of gap junctions in odorant transduction remains controversial and we explored here if ET-1 naturally produced by the olfactory mucosa could impact odorant detection. Using calcium imaging on olfactory mucosa explant, we first confirmed that ET-1 uncouples gap junctions in an olfactory mucosa preparation preserving the tissue integrity. We next measured the olfactory epithelium responses to odorant stimulation using electro-olfactogram recordings. While the amplitude of the response was not modified by application of ET-1 and carbenoxolone, its repolarizing phase was slower after both treatments. We finally examined the behavioral performances of rat pups in an orientation test based on maternal odor recognition after intranasal instillations of ET-1 or carbenoxolone. While rat pups performances were decreased after ET-1 treatment, it was unchanged after carbenoxolone treatment. Overall, our results indicate that ET-1 modulates olfactory responses at least partly through gap junction uncoupling.

ARTICLE IN PRESS; International audience; At the interface of the environment and the nervous system, the olfactory mucosa (OM) is a privileged pathway for environmental toxicants and pathogens towards the central nervous system. The OM is known to produce antimicrobial and immunological components but the mechanisms of action of the immune system on the OM remain poorly explored. IL-17c is a potent mediator of respiratory epithelial innate immune responses, whose receptors are highly expressed in the OM of mice. We first characterized the presence of the IL-17c and its receptors in the OM. While IL-17c was weakly expressed in the control condition, it was strongly expressed in vivo after intranasal administration of polyinosinic–polycytidylic (Poly I:C), a Toll Like Receptor 3 agonist, mimicking a viral infection. Using calcium imaging and electrophysiological recordings, we found that IL-17c can effectively activate OM cells through the release of ATP. In the longer term, intranasal chronic instillations of IL-17c increased the cellular dynamics of the epithelium and promoted immune cells infiltrations. Finally, IL-17c decreased cell death induced by Poly(I:C) in an OM primary culture. The OM is thus a tissue highly responsive to immune mediators, proving its central role as a barrier against airway pathogens.

Background/Aims: Particles in the nm size (nanoparticles, NP, or ultrafine particles) are ubiquitous in our environment through air pollution. Feto-placental NP distribution and physiopathological consequences of maternal inhalation of pure or complex NP mixtures have been poorly explored. Method: Pregnant rabbit females were exposed to diluted diesel exhaust (DE), a major component of airborne pollution in large European towns, at levels close to human exposure, with a mean NP size of 69nm. Exposure was performed over two hours, 5 days a week, throughout gestation. Feto-placental and postnatal effects over two generations were explored. In a second experiment, pregnant rabbits inhaled 11.5nm uncoated gold NP (Au-NP) at 132+/-38μg/m3 in the same exposure conditions. Effects on fetal phenotype and placental function were analysed. Results: DE affected placental function and were transferred to the fetal circulation but only minor signs of fetal growth retardation were observed. Nevertheless, exposure affected placental perfusion, offspring brain function and metabolism with differences related to sex, as well as 2nd generation placental function. Exposure to Au-NP did not affect fetal growth but affected maternal erythrocyte volume and induced sex-specific alterations in placental gene expression as well as DNA lesions in maternal bone marrow and fetal kidneys of males.Conclusions: Airborne exposure to NP can affect maternal health but also placental function and post-natal metabolic and neuronal development without directly disturbing fetal growth in rabbits.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice., Author summary DMXL2 gene dysfunction underlies various human diseases, including breast cancer, non-syndromic hearing loss, and polyendocrine-polyneuropathy syndrome, demonstrating the large range of potential actions of DMXL2. We show here that Dmxl2 expression is crucial for survival in mice, as neonates die within hours of birth when this gene is inactivated. The transmission of olfactory information is affected, leading to an absence of suckling and impaired feeding. Severe hypoglycemia is also observed in male neonates. We observed Dmxl2 expression in several organs, including the brain, heart and adrenal glands, potentially corresponding to some of the phenotypes observed in Dmxl2-deficient pups. We also described Dmxl2 expression in the reproductive tracts and gonads and showed that Dmxl2 inactivation specifically in the testes has a significant effect on the initial waves of spermatogenesis, resulting in very low levels of sperm production at puberty. Our results suggest that DMXL2 deficits should be considered in men with impaired fertility, as pathogenic variants of this gene may be associated with male infertility in humans.

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Play is a putatively positive experience and of key interest to the study of affective state in animals. Rats produce 50kHz ultrasonic vocalisation (USVs) during positive experiences, including social play and tickling. The tickling paradigm is intended to mimic social play resulting in positively valanced ultrasonic vocalisation (USV) production. We tested two hypotheses on the relationship between tickling and play: that tickling would increase play behaviour or that play behaviour would increase in anticipation of tickling, and that tickling would share some specific properties of play (rebound and emotional contagion of unexposed cage mates). Male Wistar rats (N=64, with 32 rats/cohort) of 28 days of age were housed in pairs with one rat assigned to be tickled and one as the non-tickled control. Production of 50kHz USVs and hand-following behaviour was measured. Prior to handling, solitary and social play was recorded for 5 minutes in the home cage. A two-day break in tickling was used to assess a potential rebound increase in responses to tickling. Only one rat within each cage was handled to assess emotional contagion through changes in the behaviour of the cage-mate. Solitary but not social play increased prior to tickling relative to controls (p = 0.01). There were marked differences between cohorts; tickled rats in C2 produced less 50kHz USVs than those in C1 (p = 0.04) and overall, C2 rats played less than rats in C1 (social p = 0.04 and solitary p < 0.001) and had a lighter start weight on arrival (p = 0.009) compared with cohort 1 (C1). In C1, there was evidence of rebound in USV production (p < 0.001) and a contagious effect of tickling reflected by increased hand-following in cage mates (p = 0.02). We found a positive relationship between start weight and USV responses to tickling (Rs = 0.43, p < 0.001), suggesting that the divergence in USV production may be due to developmental differences between cohorts. The results suggest that the relationship between tickling and play is complex in that tickling only affected solitary and not social play, and that tickling responses showed rebound and contagion effects on cage-mates which were specific to cohort responses to tickling.

According to the &ldquo, developmental origins of health and disease&rdquo, (DOHaD) concept, maternal obesity predisposes the offspring to non-communicable diseases in adulthood. While a preconceptional weight loss (WL) is recommended for obese women, its benefits on the offspring have been poorly addressed. We evaluated whether preconceptional WL was able to reverse the adverse effects of maternal obesity in a mouse model, exhibiting a modification of foetal growth and of the expression of genes encoding epigenetic modifiers in liver and placenta. We tracked metabolic and olfactory behavioural trajectories of offspring born to control, obese or WL mothers. After weaning, the offspring were either put on a control diet (CD) or a high-fat (HFD). After only few weeks of HFD, the offspring developed obesity, metabolic alterations and olfactory impairments, independently of maternal context. However, male offspring born to obese mother gained even more weight under HFD than their counterparts born to lean mothers. Preconceptional WL normalized the offspring metabolic phenotypes but had unexpected effects on olfactory performance: a reduction in olfactory sensitivity, along with a lack of fasting-induced, olfactory-based motivation. Our results confirm the benefits of maternal preconceptional WL for male offspring metabolic health but highlight some possible adverse outcomes on olfactory-based behaviours.

The many functions of astrocytes, such as glutamate recycling and morphological plasticity, enable them to stabilize synapses environment and protect neurons. Little is known about how they adapt to glucocorticoid-induced stress, and even less about the influence of dietary factors. We previously showed that omega-3 polyunsaturated fatty acids (ω3PUFA), dietary fats which alleviate stress responses, influence the way astroglia regulate glutamatergic synapses. We have explored the role of docosahexaenoic acid (DHA), the main ω3PUFA, in the astroglial responses to corticosterone, the main stress hormone in rodents to determine whether ω3PUFA help astrocytes resist stress. Cultured rat astrocytes were enriched in DHA or arachidonic acid (AA, the main ω6PUFA) and given 100 nM corticosterone for several days. Corticosterone stimulated astrocyte glutamate recycling by increasing glutamate uptake and glutamine synthetase (GS), and altered the astrocyte cytoskeleton. DHA-enriched astrocytes no longer responded to the action of corticosterone on glutamate uptake, had decreased GS, and the cytoskeletal effect of corticosterone was delayed, while AA-enriched cells were unaffected. The DHA-dependent anti-corticosterone effect was related to fewer glucocorticoid receptors, while corticosterone increased DHA incorporation into astrocyte membranes. Thus, DHA helps astrocytes resist the influence of corticosterone, so perhaps promoting a sustainable response by the stressed brain. We show that corticosterone increases the glutamate recycling capacity of rat cortical astrocytes in culture, and alters their morphology, which may be detrimental in the long term. Increasing the membrane incorporation of docosahexaenoic acid (DHA), the main omega-3 in brain, reduces the amount of glucocorticoid receptors (GR) and prevents the effects of corticosterone. This may help the astrocytes maintain a functional phenotype in chronic stress situations.

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International audience; The tickling paradigm was developed to mimic social play in rats. This study investigated whether tickling shares properties of conspecific play, namely rebound and emotional contagion. 64 adolescent (28 days) male Wistar rats, split into two batches, were housed in pairs and assigned to treatment (tickling) or control (neutral handling). One animal within each cage (n = 32, 16 per batch) was handled. During handling, frequency modulated ultrasonic vocalisation (FM USV) production and hand-following (HF) was measured. To study rebound, animals were given a two-day break from handling. To study contagion, we measured the responses of handled animals and their paired cage mate in the elevated plus maze and during home-cage anticipatory play. Cage mates were also subjected to a handling test on the final day. If contagion was present, a relationship between handled animal and cage mates would be expected. There were significant batch effects in FM USV response but not in HF response. In batch 1 (B1), tickled animals produced more FM USVs (F1,89.1 = 36.18, p < 0.001) and showed increased HF compared with controls (F1,88.7 = 16.46, p < 0.001), both measures indicating that tickled animals were in a more positive emotional state. In batch 2 (B2), treatment had no effect on FM USVs (F1,94= 0.44, p = 0.508). However, treatment had the same effect on HF (F1,188 = 0, p = 0.948). Irrespective of batch effects, there was no significant rebound effect in USVs or HF, as neither measure significantly increased after the two-day non-handling period (USVS; F1,188 = 1.08, p = 0.303; HF; F1,188 = 0.98, p = 0.325). There was evidence of contagion on HF as overall, cage-mates of tickled animals hand-followed for longer (F1,9 = 11.59, p = 0.002). This study suggests that tickling induced a behavioural change in the cage-mate although this was independent of changes in anxiety and of other changes in positive affect.

International audience; The detection of food odors by the olfactory system, which plays a key role in regulating food intake and elaborating the hedonic value of food, is reciprocally influenced by the metabolic state. Fasting increases olfactory performance, notably by increasing the activity of olfactory bulb (OB) neurons. The glutamatergic synapses between olfactory sensory neurons and mitral cells in the OB glomeruli are regulated by astrocytes, periglomerular neurons, and centrifugal afferents. We compared the expansion of astroglial processes by quantifying GFAP-labeled areas in fed and fasted rats to see whether OB glomerular astrocytes are involved in the metabolic sensing and adaptation of the olfactory system. Glomerular astroglial spreading was much greater in all OB regions of rats fasted for 17 hr than in controls. Intra-peritoneal administration of the anorexigenic peptide PYY3-36 or glucose in 17 hr-fasted rats respectively decreased their food intake or restored their glycemia, and reversed the fasting-induced astroglial spreading. Direct application of the orexigenic peptides ghrelin or NPY to OB slices increased astroglial spreading, whereas PYY3-36 resulted in astroglial retraction, in agreement with the in vivo effects of fasting and satiety on glomerular astrocytes. Thus the morphological plasticity of OB glomerular astrocytes depends on the metabolic state of the rats and is influenced by peptides that regulate food intake. This plasticity may be part of the mechanism by which the olfactory system adapts to food intake.

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International audience; The tickling paradigm is a well-established model used to elicit positive emotional states in rats, but there is little evidence on how pre-existing characteristics (e.g. anxiety and physical condition) influence the response of individuals to tickling. 64 adolescent (age 28 days) male Wistar rats, (split into two batches) were housed in pairs and assigned to treatment (tickling) or control (neutral handling). One animal within each cage (n = 32, 16 per batch) was handled. During handling, frequency modulated ultrasonic vocalisation (FM USV) production and approach behaviour in the form of hand-following (HF) was measured. All animals were run through the elevated plus maze (EPM) as a measure of anxiety during the pre (EPM1) and post-(EPM2) handling phases. There were significant batch effects in body weight, anxiety and tickling responses. In batch 1 (B1), tickled animals produced more FM USVs (F1,89.1 36.18, p < 0.001) and showed increased HF compared with controls (F1,88.7 = 16.46, p < 0.001), both measures indicating that tickled animals were in a more positive emotional state. In batch 2 (B2), treatment had no effect on FM USVs (F1,94= 0.44, p = 0.508). However, there was no difference in HF between the batches (F1,188 = 0, p = 0.948). Compared with B1, B2 tickled animals produced fewer FM USVs (F1,94 = 6.98, p < 0.001), with all animals showing increased anxiety (EPM1; F1,188 = 51.22, p < 0.001 and EPM2; F1,188 = 51.22, p < 0.001), a lower starting body weight (F1,188 = 50.25, p < 0.001) and reduced average daily gain (F1,188 = 35.95, p = 0.008). FM USV production showed an inverse relationship with anxiety (EPM1; F1,188 = 10.82, p = 0.001 and EPM2; F1,188 = 21.58, p < 0.001) and a relationship with physical condition (start weight; F1,188 = 5.73, p = 0.018 and average daily gain; F1,188 = 11.40, p < 0.001). This is the first study to investigate the effects of pre-existing characteristics on responses to tickling. Despite being sourced from the same supplier, rats showed pre-existing differences in anxiety and physical condition which may have affected responses to the tickling protocol.

International audience; TMT (2,5-dihydro-2,4,5-trimethylthiazoline) is known as a component of fox feces inducing fear in rodents. However, no recent chemical analyses of fox feces are available, and few studies make direct comparisons between TMT and fox feces. Fox feces from 3 individuals were used to prepare 24 samples to be analyzed for the presence of TMT using gas chromatography-mass spectrometry (GC-MS). When TMT was added in low amounts (50–2000 nmol/g), TMT was detected in 10 out of 11 samples. When no TMT was added, TMT was detected in only 1 out of 13 samples. In a second experiment, we tested the behavioral response of male Brown Norway (BN) and Wistar rats to either fox feces, a low amount of TMT (0.6 nmol) or 1-hexanol. TMT induced freezing in the rats, but fox feces induced significantly more freezing episodes and longer total duration of freezing in both rat strains. In experiment 3, male BN rats were exposed over several days to fox feces, rat feces, 1-hexanol, cadaverine, 2-phenylethylamine, and TMT, one odor at a time. Fox feces induced significantly more freezing episodes of a longer total duration than any of the other odors, with rat feces and 1-hexanol giving rise to the lowest amount of freezing. This finding, together with our inability to verify the presence of TMT in fox feces, indicates that the concentration of TMT in our fox feces samples was below 50 nmol/g. It may also be that other compounds in fox feces play a role in its fear-inducing properties.

The event of giving birth is an essential part of animal production. In dairy cattle production, there are substantial economical and welfare related challenges arising around the time of parturition, and hence increased focus on efficient management of the calving cow. Drawing on the research literature on pre-partum maternal behavior, this review compares cattle to other members of the ungulate clade with the aim of understanding the biological basis of bovine pre-partum behavior with main emphasis on dairy cows. Ultimately, this knowledge may be used in future development of housing systems and recommendations for management of calving cows. Maternal pre-partum behavior varies among species, but the final goal of ungulate mothers is the same: ensuring a calm parturition and optimal environment for the onset of post-partum maternal behavior by locating an appropriate birth site, with low risk of predators, disturbances and mistaken identity of offspring. Features of chosen birth sites vary among species, and depend largely on the environment, as ungulate females display a considerable ability to adapt to their surroundings. However, within commercial housing conditions in dairy production, the animals’ ability to adapt behaviorally appears to be challenged. Confinement alongside high stocking densities leave little room to express birth-site selection behavior, posing a high risk of agonistic social behavior, disturbances, and mis-mothering, as well as exposure to olfactory cues influencing both pre- and post-partum maternal behavior. Dairy cows are thus exposed to several factors in a commercial calving environment, which may thwart their maternal motivations and influence their behavior. In addition, pre-partum cattle may be more affected by olfactory cues than other ungulate species (e.g. sheep) because they are attracted to birth fluids already before calving. Hence, providing dairy cows with an environment where they can perform the maternal behavior they are motivated for, may aid a calm and secure calving and provide optimal surroundings for post-partum maternal behavior. Future research should focus on designing motivation-based housing systems allowing freedom to express pre-partum maternal behavior, and investigate in more detail the effects of the environment on the welfare of calving cows and their offspring.

International audience; Stressed animals have an increased risk of health and welfare problems, thus methods for easy and early stress detection is important for appropriate animal management. Using the ability of rats to distinguish between faeces odours from stressed and non-stressed conspecifics, we investigated whether rats could detect stress status in another species (the chicken), which would suggest a commonality in odorous stress signatures across species. We carried out four experiments to investigate the existence of stress-specific odours. In the first experiment using a T-maze, male Brown Norway (BN) rats (n = 12) were found to sniff the faeces samples from stressed rats and chickens less relative to the samples from non-stressed individuals (P < 0.05). In the second experiment, where odours were presented in an arena one at a time, male BN rats (n = 16) sniffed faeces samples from stressed rats and chickens for longer than those from non-stressed controls (P< 0.05). Within each test, the same responses to stress odours were seen independent of species of origin. This suggests that both in rats and chickens stress gives rise to specific volatile organic compounds (VOCs). In a third experiment, faeces from chickens, which had been stressed or non-stressed at hatching and subsequently exposed or not to acute stress at two weeks of age were tested on male BN rats (n = 18). These rats were also tested with faeces from non-stressed and acutely stressed rats as well as herb odour (1-hexanol) used as control. Number of freezing episodes was higher when rats were exposed to any of the samples originating from stressed individuals compared to that observed with herb odour (P < 0.05). Also, defensive burying was more likely to occur when rats were exposed to faeces from chickens stressed at hatching (P < 0.05). Finally, a fourth trial analysed faecal samples from non-stressed and acutely stressed rats using gas chromatography coupled with mass spectrometry (GC-MS), and identified ten VOCs potentially involved in the distinctive smell detectable in faeces from acutely stressed rats. These findings confirm the existence of stress-specific odours in rats and indicate that, although not necessarily identical, a similar type of odour may be present in stressed poultry. In addition, this odour could be detected by rats in chicken faeces collected almost two weeks after the birds had been exposed to a stressful event. Our results suggest that patterns of VOCs may have the potential to be used as a tool for early, non-invasive screening of stress status in animals.

Appendix A. Supplementary data: Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.03.066.; International audience; We report a dose-dependent detection of androstenone in solution, as one of the boar taint compounds, based on related OR7D4 olfactory receptors immobilized on a gold electrode through their 6-His tag and NTA-copper complex, as visualized through fluorescence microscopy. Square wave voltammetry (SWV) is for the first time, the method used to monitor the olfactory receptor/odorant recognition process. The relative variation of the Cu(I)-Cu(II) current peak increases linearly versus log (concentration of androstenone) from 10(-14) M to 10(-4) M, in buffer solution. Negative tests were performed, using an unrelated odorant, helional, itself a ligand of OR 1740. Cross-selectivity was also tested after immobilization of OR 1740.

International audience; Multi-tier aviary systems are becoming more common in organic egg production. The area on the tiers can be included in the net area available to the hens (also referred to as usable area) when calculating maximum indoor stocking densities in organic systems within the EU. In this article, results on egg production, laying behaviour and use of veranda and outdoor area are reported for organic laying hens housed in a multi-tier system with permanent access to a veranda and kept at stocking densities (D) of 6, 9 and 12 hens/m2 available floor area, with concomitant increases in the number of hens per trough, drinker, perch and nest space. In a fourth treatment, access to the top tier was blocked reducing vertical, trough and perch access at the lowest stocking density (treatment D6x). In all other aspects than stocking density, the experiment followed the EU regulations on the keeping of organic laying hens. Laying percentage was significantly lower (P

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Master

Olfaction plays a key role in the elaboration of the hedonic value of foods and the regulation of food intake. Reciprocally, the detection of food odors is influenced by the metabolic state. Fasting increases olfactory performances, notably by increasing neuronal activity in the olfactory bulb (OB). Within the OB glomeruli, the glutamatergic synapses between olfactory sensory neurons and mitral cells are regulated by astrocytes, periglomerular neurons and centrifugal afferents. Astrocytes, which support mecanisms of metaplasticity in the brain, may drive the metabolic regulation of the olfactory response in the OB. To test whether OB glomerular astrocytes are involved in the metabolic sensing of the olfactory system, we compared the astroglial processes expansion by quantification of the GFAPlabelled area in fed and fasted rats.Astroglial spreading was markedly increased in fasting rats, as compared to fed rats, in all regions of the OB after 17h-fasting. Intra-peritoneal administration of the anorexigenic peptide PYY3-36 or glucose in 17h-fasted rats respectively decreased their food intake or restored their glycemia and both reversed the increased astroglial deployment induced by fasting. Direct application of orexigenic peptides ghrelin and NPY on OB acute slices, resulted in an increase of the astroglial deployment, whereas application of PYY3-36 oppositely resulted in astroglial retraction within the glomeruli. These results are in close agreement with the effects of fasting or satiation on the morphology of astrocytes.The deployment of the glomerular astroglial processes clearly varies according to the metabolic state of the rats, and is influenced by food intake regulatory peptides. This plasticity may participate in the adaptation of the olfactory sensitivity to food intake.; L’olfaction participe à l’élaboration de la valeur hédonique des aliments et à la régulation de la prise alimentaire. Réciproquement, la détection des odeurs alimentaires est influencée par le statut métabolique. Le jeûne augmente les performances olfactives, notamment en augmentant l'activité neuronale dans le bulbe olfactif (BO). Au sein des glomérules du BO, les synapses glutamatergiques entre les neurones sensoriels olfactifs et les cellules mitrales sont régulées par des astrocytes, des neurones périglomérulaires et des afférences centrifuges. En tant que partenaires synaptiques, les astrocytes sont à l’origine de mécanismes de métaplasticité dans le système nerveux central, qui pourrait participer à la régulation métabolique de la réponse olfactive au niveau du BO. Afin de tester si les astrocytes glomérulaires du BO sont impliqués dans la détection du statut métabolique par le système olfactif, nous avons comparé le déploiement des prolongements astrocytaires glomérulaires, par quantification de l’aire occupée par la GFAP chez des rats nourris et mis à jeun.Le déploiement astrocytaire est nettement augmenté chez les rats à jeun par rapport aux rats nourris, dans toutes les régions du BO dès 17h de jeûne. L'administration intra-peritoneale du peptide anorexigène PYY3-36 ou de glucose chez des rats à jeun diminue leur prise alimentaire ou restaure leur glycémie respectivement, et abolit dans les deux cas l'augmentation du déploiement astrocytaire induite par le jeûne. L'application directe des peptides orexigènes ghréline et NPY sur des tranches de BO entraîne une augmentation du déploiement astrocytaire alors que l'application de PYY3-36 entraîne une rétraction astrocytaire au sein des glomérules. Ces résultats concordent avec les variations de la morphologie des astrocytes, observées respectivement en situation de jeûne ou de satiété.Le déploiement des prolongements astrocytaires glomérulaires varie donc en fonction de l'état métabolique des rats, et il est influencé par les peptides régulant la prise alimentaire. Cetteplasticité pourrait participer à l'adaptation de la sensibilité olfactive à l’état métabolique des individus.

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National audience

In mammals, the main olfactory system displays all the structural and functional characteristics at the last third of the gestational stage. Thus, the fetus is able to detect, to discriminate, but also to memorize the odorants present in the amniotic fluid in which it bathes. In the rodent, these odors memorized in utero are fundamental for the survival of the newborn at birth by allowing it to trend itself towards food sources. Afterwards, odorants are guiding the newborn to olfactory and food choices and are promoting subsequent learning. It is known that the mother's diet varies the olfactory keys of the biological fluids (amniotic fluid, milk) and thus modifies the nature of the olfactory sources encountered by the fetus and then the newborn in the perinatal period. The neuroanatomic and functional consequences of this impregnation are the subject of recent studies. The objective of this thesis was to characterize the effects of perinatal odor exposure on the maturation and functioning of the olfactory system in relation to the development of olfactory preferences and to a stressing challenge at weaning. For that, we set up a model of perinatal odor exposure through maternal feeding in the mI7-GFP murine transgenic strain expressing the olfactory receptor I7 coupled with the Green Fluorescent Protein (GFP). We evaluated the neuroanatomic, molecular and behavioral consequences on the pups before weaning, and their evolution over time by focusing our efforts on the postnatal stages 3 (PND3), PND12-14 and PND21. Due to the fragility of the transgenic strain, we worked on mI7-GFP mice pups crossfostered by CD1 mice mothers raised under the same conditions after having validated the presence of heptanal in the amniotic fluid of mI7-GFP mice and the milk of CD1 mice by GC-MSMS. We characterized the effects of perinatal odor exposure on the maturation and functioning of the olfactory system in relation to the olfactory preference until weaning (mI7-GFP mice pups under CD1 adoptive mothers) and on the stress reaction to maternal separation at weaning (CD1 mice pups under biological mother). Our results show that perinatal exposure to heptanal leads to a significant increase in the number of I7 glomeruli in the olfactory bulb (OB) associated to a slight modification of the tissue homeostasis in the olfactory mucosa (OM) and to subtle differencies in heptanal sensitivity and preferences, that are amplified at PND21. From a molecular point of view, these effects are associated to a down-regulation of the expression of the I7 receptor and genes of neuronal signaling and an odorantspecific decrease in EOG response which may highlight a modification of the cellular dynamics. Finally, the effect of perinatal exposure to heptanal on the response to stressing conditions after maternal separation was assessed by recording CD1 mice pups grown under the same conditions than fostered mi7 mice on odorized and non odorized open-field. We showed that the reaction of odorized mice in the open field at PND21 is attenuated when the odorant is present in the environment, compared to non odorized mice that display anxiety-like behavior. Overall, this study demonstrates the consequences of a perinatal odorant exposure in the young, in terms of behavior (olfactory preference and anxiety), structural and molecular plasticity of the olfactory system, on a transgenic strain for which we had little available data.; Chez les mammifères, le système olfactif principal est fonctionnel dans le dernier tiers du stade gestationnel. Le fœtus est donc capable de détecter avec sensibilité, discriminer, et mémoriser les odorants présents dans le liquide amniotique dans lequel il baigne. Chez le rongeur, ces odorants mémorisés in utero sont fondamentaux pour la survie du nouveau-né à la naissance en lui permettant de s’orienter vers la mamelle et de déclencher la tétée. Par la suite, ces odorants vont guider le nouveau-né dans ses choix olfactifs et alimentaires et favoriser ses apprentissages ultérieurs. Or, on sait que l’alimentation de la mère fait varier les clés olfactives des fluides biologiques (liquide amniotique, lait) et donc modifie la nature des sources olfactives rencontrées par le fœtus, puis le nouveau-né dans la période périnatale. Dans le but d’évaluer l’impact d’une exposition odorante périnatale sur le fonctionnement et la maturation du système olfactif en relation avec le développement de préférences médiées par l’olfaction et la modulation du stress au moment du sevrage, nous avons mis en place un modèle d’exposition odorante périnatale via l’alimentation maternelle dans une lignée transgénique murine exprimant le récepteur olfactif I7 couplé à une protéine fluorescente, dont l’odorant préférentiel est l’heptanal. Après validation de la présence d’heptanal dans le liquide amniotique et le lait de ces souris par GC-MS-MS, nos résultats montrent que l’exposition périnatale à l’heptanal entraine une augmentation significative du nombre de glomérules I7 présents au niveau du bulbe olfactif à PN12 par rapport aux animaux contrôles sans modification notable de l’homéostasie tissulaire ou de la transcription du gène I7 au niveau de la muqueuse olfactive. Ce changement structural important au niveau des projections axonales du bulbe olfactif s’accompagne d’un faible effet sur le comportement olfactif des souriceaux dont les capacités de discrimination ne sont que très faiblement améliorées par rapport aux souriceaux non exposés. A PN21, l’effet de l’exposition odorante sur le nombre de glomérules I7 est atténué, mais reste significatif. A ce stade, les souriceaux odorisés montrent une attraction olfactive pour l’heptanal par rapport au groupe contrôle non exposé, bien que l’effet sur un choix entre aliment odorisé ou pas soit plus contrasté. D’un point de vue moléculaire, ces effets sont associés à une diminution de l’expression du récepteur I7, et de gènes de la signalisation neuronale pouvant traduire une modification de la dynamique cellulaire. Et comme le montrent les résultats obtenus en électro-olfactogramme à cet âge, la sensibilité de la muqueuse olfactive vis-à-vis de l’heptanal est diminuée alors qu’elle ne l’est pas pour d’autres composés odorants. Ces travaux montrent que l’exposition périnatale à un odorant s’accompagne d’effets précoces sur le système olfactif qui impactent son organisation et d’effets sur les choix olfactifs qui se renforcent au cours du développement. Enfin, l’effet d’une exposition périnatale à l’heptanal sur la réponse à des conditions stressantes après séparation maternelle au moment du sevrage a été évaluée en enregistrant des souriceaux CD1 élevés dans les mêmes conditions d’odorisation que les souriceaux mI7-GFP adoptés, en open-field odorisé ou non. Nous avons montré que la réaction de stress des souriceaux à PN21, exposés en périnatal à l’heptanal, dans l’open field, est atténuée lorsque l’heptanal est présent dans l’environnement, comparé à la réaction des souriceaux non exposés qui affichent un comportement d’anxiété. Dans l’ensemble, cette étude a donc permis de mettre en évidence les conséquences en terme d’effets comportementaux (préférences médiées par l’olfaction et diminution du stress), structuraux et moléculaires, d’une exposition odorante périnatale chez le jeune, à l’aide d’une lignée transgénique pour laquelle nous disposions de peu de données.

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International audience; We assessed whether the ratio of dietary n-6/n-3 polyunsaturated fatty acids (PUFA) during egg formation engenders transgenerational maternal effects in domestic chicks. We analyzed yolk lipid and hormone concentrations, and HPA-axis activity in hens fed a control diet (high n-6/n-3 ratio) or a diet enriched in n-3 PUFAs (low n-6/n-3 ratio) for 6 consecutive weeks. Their chicks were tested for neophobia during the first week of life. We found higher corticosterone metabolites in droppings of hens fed the diet enriched in n-3 and significantly higher concentrations of yolk progesterone, androstenedione, and estradiol in their eggs compared to controls. Chicks of hens fed the n-3 enriched diet showed a lower body mass at hatch than controls and expressed higher neophobia when exposed to a novel object. These results add support to the hypothesis that the nutritional state of female birds produces variation in yolk hormone levels and engender maternal effects.

National audience; Le diagnostic et le suivi de pathologies, dont les cancers et les pathologies infectieuses, représentent des enjeux majeurs de santé publique. Les odeurs corporelles sont caractéristiques de chaque individu, et dépendent de son profil génétique et de son état physiopathologique. La présence d'odeurs corporelles spécifiques a été identifiée dans plusieurs pathologies (divers cancers, tuberculose, et autres pathologies y compris non infectieuses ...). Celles-ci peuvent être détectées par des animaux, dont les capacités olfactives sont très développées, tant du point de vue de la sensibilité de la détection des odeurs que de sa spécificité. Les composés odorants volatils constituent des biomarqueurs qui peuvent également être détectés et identifiés par des nez électroniques, constitués de réseaux de senseurs à large spectre dont les propriétés physicochimiques sont modifiées en présence d'odeur. Toutefois, il peut être difficile pour les nez électroniques de concurrencer le système olfactif animal optimisé au cours de l'évolution. Contrairement aux nez électroniques, les biosenseurs olfactifs sont des dispositifs hybrides et bioinspirés de détection d'odeurs, utilisant comme éléments sensibles des protéines de liaison aux odeurs, des cellules exprimant des récepteurs olfactifs à leur membrane, ou des récepteurs olfactifs eux-mêmes, dont c'est la fonction intrinsèque de lier et discriminer les odorants à très faible concentration. L'élaboration de tels supernez bioélectroniques miniaturisés, multiplexés, vise la mesure quantitative directe de ces biomarqueurs odorants, en particulier pour des applications biomédicales.

The sense of smell is likely to influence the behaviour of domestic and captive animals in a wide range of management and housing situations. In domestic cattle, there may be unexploited potential for using odours and olfaction in the management; however, published studies on bovine olfactory capacity are scarce. By applying an olfactory Habituation/Dishabituation test developed for rodents, this study aimed to assess olfactory ability in cattle. Twenty-three cows (n = 10) and heifers (n = 13) were tested with three different odours (orange juice, liquid coffee and tap water as an odourless control) presented in a test bucket. The test was conducted on individual animals in their home pen and consisted of each odour being presented twice in a row for 2 min each with an inter-trial pause of 2 min. Following another 2-min pause without odour the animal was presented with a new odour, with order of odour presentation balanced among animals. Duration of sniffing (muzzle in proximity or contact with) the test bucket as well as the occurrence of licking or biting the test bucket were recorded by direct observation. All animals sniffed an odour (i.e. the test bucket) less when presented for the second time (habituation; all 1st vs. 2nd presentations: P < 0.001). Sniffing duration increased at the subsequent presentation of a new odour (dishabituation; all 2nd vs. 1 st presentations: P < 0.001). All animals sniffed coffee and orange longer than water (water vs. coffee: P < 0.001 and water vs. orange: P < 0.001), but they also sniffed coffee longer than orange (P = 0.021). Licking or biting behaviour occurred only when presented with coffee or orange (13 out of 23 animals for both samples). The test showed that cows and heifers were able to distinguish between different complex odours (coffee and orange juice), and that the animals showed increased interest for one of the odours (coffee). This is the first example of a Habituation/Dishabituation test being adapted for and applied to cattle. The test may require further development, but represents the first steps towards exploring the possibility of using odours when adapting or enriching the environment in which we keep cattle.