Testing detectability, attractivity, hedonic specificity, extractability, and robustness of colostrum odor-Toward an olfactory bioassay for human neonates.

Human milk odor is attractive and appetitive for human newborns. Here, we studied behavioral and heart-rate (HR) responses of 2-day-old neonates to the odor of human colostrum. To evaluate detection in two conditions of stimulus delivery, we first presented the odor of total colostrum against water. Second, the hedonic specificity of total colostrum odor was tested against vanilla odor. Third, we delivered only the fresh effluvium of colostrum separated from the colostrum matrix; the stability of this colostrum effluvium was then tested after deep congelation; finally, after sorptive extraction of fresh colostrum headspace, we assessed the activity of colostrum volatiles eluting from the gas chromatograph (GC). Regardless of the stimulus-delivery method, neonates displayed attraction reactions (HR decrease) as well as appetitive oral responses to the odor of total colostrum but not to vanilla odor. The effluvium separated from the fresh colostrum matrix remained appetitive but appeared labile under deep freezing. Finally, volatiles from fresh colostrum effluvium remained behaviorally active after GC elution, although at lower magnitude. In sum, fresh colostrum effluvium and its eluate elicited a consistent increase in newborns' oral activity (relative to water or vanilla), and they induced shallow HR decrease. Newborns' appetitive oral behavior was the most reproducible response criterion to the effluvium of colostrum. In conclusion, a set of unidentified volatile compounds from human colostrum is robust enough after extraction from the original matrix and chromatographic processing to continue eliciting appetitive responses in neonates, thus opening new directions to isolate and assay specific volatile molecules of colostrum.

Past, Present, and Future of Human Chemical Communication Research.

Although chemical signaling is an essential mode of communication in most vertebrates, it has long been viewed as having negligible effects in humans. However, a growing body of evidence shows that the sense of smell affects human behavior in social contexts ranging from affiliation and parenting to disease avoidance and social threat. This article aims to (a) introduce research on human chemical communication in the historical context of the behavioral sciences; (b) provide a balanced overview of recent advances that describe individual differences in the emission of semiochemicals and the neural mechanisms underpinning their perception, that together demonstrate communicative function; and (c) propose directions for future research toward unraveling the molecular principles involved and understanding the variability in the generation, transmission, and reception of chemical signals in increasingly ecologically valid conditions. Achieving these goals will enable us to address some important societal challenges but are within reach only with the aid of genuinely interdisciplinary approaches.

A neural marker of rapid discrimination of facial expression in 3.5- and 7-month-old infants.

Infants' ability to discriminate facial expressions has been widely explored, but little is known about the rapid and automatic ability to discriminate a given expression against many others in a single experiment. Here we investigated the development of facial expression discrimination in infancy with fast periodic visual stimulation coupled with scalp electroencephalography (EEG). EEG was recorded in eighteen 3.5- and eighteen 7-month-old infants presented with a female face expressing disgust, happiness, or a neutral emotion (in different stimulation sequences) at a base stimulation frequency of 6 Hz. Pictures of the same individual expressing other emotions (either anger, disgust, fear, happiness, sadness, or neutrality, randomly and excluding the expression presented at the base frequency) were introduced every six stimuli (at 1 Hz). Frequency-domain analysis revealed an objective (i.e., at the predefined 1-Hz frequency and harmonics) expression-change brain response in both 3.5- and 7-month-olds, indicating the visual discrimination of various expressions from disgust, happiness and neutrality from these early ages. At 3.5 months, the responses to the discrimination from disgust and happiness expressions were located mainly on medial occipital sites, whereas a more lateral topography was found for the response to the discrimination from neutrality, suggesting that expression discrimination from an emotionally neutral face relies on distinct visual cues than discrimination from a disgust or happy face. Finally, expression discrimination from happiness was associated with a reduced activity over posterior areas and an additional response over central frontal scalp regions at 7 months as compared to 3.5 months. This result suggests developmental changes in the processing of happiness expressions as compared to negative/neutral ones within this age range.

Flavor Sensing in Utero and Emerging Discriminative Behaviors in the Human Fetus.

The diet of pregnant women exposes fetuses to a variety of flavors consisting of compound sensations involving smell, taste, and chemesthesis. The effects of such prenatal flavor exposure on chemosensory development have so far been measured only postnatally in human infants. Here, we report the first direct evidence of human fetal responsiveness to flavors transferred via maternal consumption of a single-dose capsule by measuring frame-by-frame fetal facial movements. Pregnant women and their fetuses based in the northeast of England were involved in this study from 32 to 36 weeks' gestation. Fetuses exposed to carrot flavor (n = 35) showed "lip-corner puller" and "laughter-face gestalt" more frequently, whereas fetuses exposed to kale flavor (n = 34) showed more "upper-lip raiser," "lower-lip depressor," "lip stretch," "lip presser," and "cry-face gestalt" in comparison with the carrot group and a control group not exposed to any flavors (n = 30). The complexity of facial gestalts increased from 32 to 36 weeks in the kale condition, but not in the carrot condition. Findings of this study have important implications for understanding the earliest evidence for fetal abilities to sense and discriminate different flavors.

Comparative Investigation of Conventional and Innovative Headspace Extraction Methods to Explore the Volatile Content of Human Milk.

The odor of human milk induces search-like movements and oral activation in newborns, which increases their chances of taking advantage of milk intake and benefits. However, the underlying volatile fraction of human milk remains understudied. This study aimed to devise a simple method to extract a wide range of volatile compounds from small-volume human milk samples. Headspace solid phase micro-extraction (HS-SPME) with a Car/PDMS fiber and dynamic headspace extraction (D-HS) with a Tenax or a trilayer sorbent were tested because of their selective affinity for volatiles. Then, innovative variations of these methods were developed to combine their respective advantages in a one-step extraction: Static headspace with multiple SPME fibers (S-HS-MultiSPME), Dynamic headspace with multiple SPME fibers (D-HS-MultiSPME) and dynamic headspace with multiple SPME fibers and Tenax (D-HS-MultiSPME/Tenax). The extracts were analyzed by gas chromatography coupled with mass spectrometric and flame ionization detection. The relative performances of these methods were compared based on qualitative and semi-quantitative analyses of the chromatograms. The D-HS technique showed good sensitivity for most compounds, whereas HS-SPME favored the extraction of acids. The D-HS-MultiSPME/Tenax identified more than 60 compounds from human milk (some for the first time) and evidence of individual singularities. This method that can be applied to volatilome analysis of any biological fluid should further our understanding of human milk odor.

Olfaction in the Multisensory Processing of Faces: A Narrative Review of the Influence of Human Body Odors.

A recent body of research has emerged regarding the interactions between olfaction and other sensory channels to process social information. The current review examines the influence of body odors on face perception, a core component of human social cognition. First, we review studies reporting how body odors interact with the perception of invariant facial information (i.e., identity, sex, attractiveness, trustworthiness, and dominance). Although we mainly focus on the influence of body odors based on axillary odor, we also review findings about specific steroids present in axillary sweat (i.e., androstenone, androstenol, androstadienone, and estratetraenol). We next survey the literature showing body odor influences on the perception of transient face properties, notably in discussing the role of body odors in facilitating or hindering the perception of emotional facial expression, in relation to competing frameworks of emotions. Finally, we discuss the developmental origins of these olfaction-to-vision influences, as an emerging literature indicates that odor cues strongly influence face perception in infants. Body odors with a high social relevance such as the odor emanating from the mother have a widespread influence on various aspects of face perception in infancy, including categorization of faces among other objects, face scanning behavior, or facial expression perception. We conclude by suggesting that the weight of olfaction might be especially strong in infancy, shaping social perception, especially in slow-maturing senses such as vision, and that this early tutoring function of olfaction spans all developmental stages to disambiguate a complex social environment by conveying key information for social interactions until adulthood.

Odor-driven face-like categorization in the human infant brain.

Understanding how the young infant brain starts to categorize the flurry of ambiguous sensory inputs coming in from its complex environment is of primary scientific interest. Here, we test the hypothesis that senses other than vision play a key role in initiating complex visual categorizations in 20 4-mo-old infants exposed either to a baseline odor or to their mother's odor while their electroencephalogram (EEG) is recorded. Various natural images of objects are presented at a 6-Hz rate (six images/second), with face-like object configurations of the same object categories (i.e., eliciting face pareidolia in adults) interleaved every sixth stimulus (i.e., 1 Hz). In the baseline odor context, a weak neural categorization response to face-like stimuli appears at 1 Hz in the EEG frequency spectrum over bilateral occipitotemporal regions. Critically, this face-like-selective response is magnified and becomes right lateralized in the presence of maternal body odor. This reveals that nonvisual cues systematically associated with human faces in the infant's experience shape the interpretation of face-like configurations as faces in the right hemisphere, dominant for face categorization. At the individual level, this intersensory influence is particularly effective when there is no trace of face-like categorization in the baseline odor context. These observations provide evidence for the early tuning of face-(like)-selective activity from multisensory inputs in the developing brain, suggesting that perceptual development integrates information across the senses for efficient category acquisition, with early maturing systems such as olfaction driving the acquisition of categories in later-developing systems such as vision.

Male mice and cows perceive human emotional chemosignals: a preliminary study.

Olfactory cues of individuals of the same species or from different species may induce changes in behaviors and physiological reactions in mammals. However, there are few studies on the influence of human odor on animal behavior and welfare, especially those of rodents and farm animals. The present study aimed to investigate whether the odor of a stressed human (in sweat) would modify the behavior of mice and cows. We hypothesized that laboratory and farm animals can perceive human emotions though olfactory cues and that human emotional chemosignals can modify their behavioral reactions and welfare. Two odors of human axillary sweat were collected from engineering students (n = 25, 14 females and 11 males; 21.1 ± 0.7 years old, range: 19-23 years old): a "stress" odor collected after an exam and a "non-stress" odor collected after a standard class. Two experiments were then conducted to test the discrimination of these two odors by male mice (n = 20) under standard conditions and by cows (n = 10) under farm conditions. During the experiments, the behavioral responses of the animals to both odors (through a dispenser for the mice and a bucket for the cows) were observed. The mice produced significantly (p = 0.004) more fecal pellets with the stress odor dispenser than with the non-stress-odor dispenser. The cows spent significantly (p = 0.04) more time smelling the non-stress-odor bucket than control. For both species, the other behaviors observed did not differ significantly between the odors. Mice and cows seemed to perceive and react to stressful human chemosignals. Mice showed physiological reactions that indicated stress in response to the stress odor of humans, while cows showed preference reactions in response to the non-stress odor of humans. This preliminary study showed that laboratory and farm animals, such as male mice and cows, seemed to discriminate certain odors emitted by humans that were likely related to different emotions. Animals may recognize stressful human chemosignals, associate these signals with negative husbandry practices or human-animal relationships, and consequently modify their behavior.

The role of papillary skin glands in guiding mouse pups to the nipple.

The nipple odor of lactating mice (Mus musculus) plays a crucial role in attracting newborn pups and motivating them to suck milk. The characteristic odor of a lactating murine nipple is assumed to be a mixture of multiple odorous substrates, that is, milk, dam's and pups' saliva, skin glands' secretions, and amniotic fluid. The present study aimed to characterize the behavioral activity of the original odor mixture that develops over the nipples in the first 2 days postpartum. We extracted this odor mixture in water and evaluated its attractive and appetitive potencies using two behavioral assays (viz., relative attraction and oral activation assays). It resulted that the so-called nipple wash was as appetitive as fresh milk, and even more attractive than it. The behavioral potency of the nipples was shown to be specific to lactating nipples (relative to nulliparous nipples) and to be preserved for 2 weeks when stored at -80°C. Finally, we perfected a nipple deodorization procedure by inactivating the nipples' behavioral potency. We observed that such altered appetitive potency was fully restored 30 min after its washing, but without any maternal self-licking and pups' sucking, indicating that the secretions of the nipple skin glands' were sufficient to explain the success of neonatal guidance to the nipple.

Age differences in olfactory affective responses: evidence for a positivity effect and an emotional dedifferentiation.

Studies on aging and hedonic judgment of odors have never been addressed within the empirical frameworks of age-related changes in emotion which state that advancing age is associated with a reduced negativity bias and a less pronounced differentiation between hedonic valence and emotional intensity judgments. Our aim was to examine and extend these age-related effects into the field of odors. Thirty-eight younger adults and 40 older adults were asked to evaluate the hedonic valence, emotional intensity, and familiarity of 50 odors controlled for their pleasantness. Compared to younger adults, older adults rated unpleasant odorants as less unpleasant and showed an increased relationship between hedonic valence and emotional intensity ratings. This yields evidence of reduced negativity bias and emotional dedifferentiation in response to odors. Such data suggest that when faced with odors, older people exhibit a reduction of emotional dimensionality leading them to distort emotional processing in a less negative direction.

Odor-evoked hedonic contexts influence the discrimination of facial expressions in the human brain.

The influence of odor valence on expressive-face perception remains unclear. Here, three "valenced" odor contexts (pleasant, unpleasant, control) were diffused while scalp electroencephalogram (EEG) was recorded in 18 participants presented with expressive faces alternating at a 6-Hz rate. One facial expression (happiness, disgust or neutrality) repeatedly arose every 6 face pictures to isolate its discrimination from other expressions at 1 Hz and harmonics in the EEG spectrum. The amplitude of the brain response to neutrality was larger in the pleasant vs. control odor context, and fewer electrodes responded in the unpleasant odor context. The number of responding electrodes was reduced for disgust in both odor contexts. The response to happiness was unchanged between odor conditions. Overall, these observations suggest that valenced odors influence the neural discrimination of facial expressions depending on both face and odor hedonic valence, especially for the emotionally ambiguous neutral expression.

Human neonates prefer colostrum to mature milk: Evidence for an olfactory bias toward the "initial milk"?

OBJECTIVES: Colostrum is the initial milk secretion which ingestion by neonates warrants their adaptive start in life. Colostrum is accordingly expected to be attractive to newborns. The present study aims to assess whether colostrum is olfactorily attractive for 2-day-old newborns when presented against mature milk or a control. METHODS: The head-orientation of waking newborns was videotaped in three experiments pairing the odors of: (a) colostrum (sampled on postpartum day 2, not from own mother) and mature milk (sampled on average on postpartum day 32, not from own mother) (n tested newborns = 15); (b) Colostrum and control (water; n = 9); and (c) Mature milk and control (n = 13). RESULTS: When facing the odors of colostrum and mature milk, the infants turned their nose significantly longer toward former (32.8 vs 17.7% of a 120-s test). When exposed to colostrum against the control, they responded in favor of colostrum (32.9 vs 16.6%). Finally, when the odor of mature milk was presented against the control, their response appeared undifferentiated (26.7 vs 28.6%). CONCLUSIONS: These results indicate that human newborns can olfactorily differentiate conspecific lacteal fluids sampled at different lactation stages. They prefer the odor of the mammary secretion - colostrum - collected at the lactation stage that best matches the postpartum age of their own mother. These results are discussed in the context of the earliest mother-infant chemo-communication. Coinciding maternal emission and offspring reception of chemosignals conveyed in colostrum may be part of the sensory precursors of attunement between mothers and infants.

Newborn crawling and rooting in response to maternal breast odor.

A growing literature shows that perception and action are already tightly coupled in the newborn. The current study aimed to examine the nature of the coupling between olfactory stimuli from the mother and the newborn's crawling and rooting (exploratory movements of the head). To examine the coupling, the crawling and rooting behavior of 28 2-day-old newborns were studied while they were supported prone on a mobility device shaped like a mini skateboard, the Crawliskate®, their head positioned directly on top of a pad infused with either their mother's breast odor (Maternal) or the odor of water (Control). Video and 3D kinematic analyses of the number and types of limb movements and quantification of displacement across the surface revealed that newborns are significantly more efficient crawlers when they smell the maternal odor, moving greater distances although performing fewer locomotor movements. In addition, the newborns made significantly more head rooting movements in the presence of the maternal odor. These findings suggest that the circuitry underlying quadrupedal locomotion and exploratory movements of the head is already adaptable to olfactory information via higher brain processing. Moreover, the coupling between olfaction and the two action systems, locomotion and rooting, is already differentiated. As crawling enables the newborn to move toward the mother's breast immediately after birth and facilitates mother-infant interaction, the results of this study highlight the potential value of using maternal odors to stimulate mobility in infants at risk of motor delay and/or deprived of this odor when born prematurely.

Does any mother's body odor stimulate interest in mother's face in 4-month-old infants?

Little is known about the effects of olfaction on visual processing during infancy. We investigated whether and how an infant's own mother's body odor or another mother's body odor affects 4-month-old infants' looking at their mother's face when it is paired with a stranger's face. In Experiment 1, infants were exposed to their mother's body odor or to a control odor, while in Experiment 2, infants were exposed to a stranger mother's body odor while their visual preferences were recorded. Results revealed that infants looked more at the stranger's female face in presence of the control odor but that they looked more at their mother's face in the context of any mother's body odors. This effect was due to a reduction of looking at the stranger's face. These findings suggest that infants react similarly to the body odor of any mother and add to the growing body of evidence indicating that olfactory stimulation represents a pervasive aspect of infant multisensory perception.

Perinatal exposure to a dietary pesticide cocktail does not increase susceptibility to high-fat diet-induced metabolic perturbations at adulthood but modifies urinary and fecal metabolic fingerprints in C57Bl6/J mice.

BACKGROUND: We recently demonstrated that chronic dietary exposure to a mixture of pesticides at low-doses induced sexually dimorphic obesogenic and diabetogenic effects in adult mice. Perinatal pesticide exposure may also be a factor in metabolic disease etiology. However, the long-term consequences of perinatal pesticide exposure remain controversial and largely unexplored. OBJECTIVES: Here we assessed how perinatal exposure to the same low-dose pesticide cocktail impacted metabolic homeostasis in adult mice. METHODS: Six pesticides (boscalid, captan, chlopyrifos, thiachloprid, thiophanate, and ziram) were incorporated in food pellets. During the gestation and lactation periods, female (F0) mice were fed either a pesticide-free or a pesticide-enriched diet at doses exposing them to the tolerable daily intake (TDI) level for each compound, using a 1:1 body weight scaling from humans to mice. All male and female offsprings (F1) were then fed the pesticide-free diet until 18 weeks of age, followed by challenge with a pesticide-free high-fat diet (HFD) for 6 weeks. Metabolic parameters, including body weight, food and water consumption, glucose tolerance, and urinary and fecal metabolomes, were assessed over time. At the end of the experiment, we evaluated energetic metabolism and microbiota activity using biochemical assays, gene expression profiling, and 1H NMR-based metabolomics in the liver, urine, and feces. RESULTS: Perinatal pesticide exposure did not affect body weight or energy homeostasis in 6- and 14-week-old mice. As expected, HFD increased body weight and induced metabolic disorders as compared to a low-fat diet. However, HFD-induced metabolic perturbations were similar between mice with and without perinatal pesticide exposure. Interestingly, perinatal pesticide exposure induced time-specific and sex-specific alterations in the urinary and fecal metabolomes of adult mice, suggesting long-lasting changes in gut microbiota. CONCLUSIONS: Perinatal pesticide exposure induced sustained sexually dimorphic perturbations of the urinary and fecal metabolic fingerprints, but did not significantly influence the development of HFD-induced metabolic diseases.

Human olfactory communication: current challenges and future prospects.

Although anthropologists frequently report the centrality of odours in the daily lives and cultural beliefs of many small-scale communities, Western scholars have historically considered the sense of smell as minimally involved in human communication. Here, we suggest that the origin and persistence of this latter view might be a consequence of the fact that most research is conducted on participants from Western societies who, collectively, were rather old (adults), deodorized and desensitized (ODD) to various aspects of olfactory perception. The view is rapidly changing, however, and this themed issue provides a timely overview of the current state-of-the-art on human chemocommunication. Based on evolutionary models of communication, the papers cover both general mechanisms of odour production by 'senders' and odour perception by 'receivers'. Focus on specific functional contexts includes reciprocal impact of odours between infants and mothers, the role of odour in mate choice and how odours communicate emotion and disease. Finally, a position paper outlines pitfalls and opportunities for the future, against the context of the replication crisis in psychology. We believe a more nuanced view of human chemical communication is within our grasp if we can continue to develop inter-disciplinary insights and expand research activities beyond ODD people. This article is part of the Theo Murphy meeting issue 'Olfactory communication in humans'.

Olfaction scaffolds the developing human from neonate to adolescent and beyond.

The impact of the olfactory sense is regularly apparent across development. The fetus is bathed in amniotic fluid (AF) that conveys the mother's chemical ecology. Transnatal olfactory continuity between the odours of AF and milk assists in the transition to nursing. At the same time, odours emanating from the mammary areas provoke appetitive responses in newborns. Odours experienced from the mother's diet during breastfeeding, and from practices such as pre-mastication, may assist in the dietary transition at weaning. In parallel, infants are attracted to and recognize their mother's odours; later, children are able to recognize other kin and peers based on their odours. Familiar odours, such as those of the mother, regulate the child's emotions, and scaffold perception and learning through non-olfactory senses. During juvenility and adolescence, individuals become more sensitive to some bodily odours, while the timing of adolescence itself has been speculated to draw from the chemical ecology of the family unit. Odours learnt early in life and within the family niche continue to influence preferences as mate choice becomes relevant. Olfaction thus appears significant in turning on, sustaining and, in cases when mother odour is altered, disturbing adaptive reciprocity between offspring and carer during the multiple transitions of development between birth and adolescence. This article is part of the Theo Murphy meeting issue 'Olfactory communication in humans'.

Decreasing prevalence of specific anosmia to non-steroid odorants from childhood to adolescence.

Specific anosmia is defined as the inability to detect a particular odorant, despite a normal olfactory function. Previous studies reported sex-related difference in detection threshold to steroid odorants, like androstenone or androstadienone during adolescence, and boys showed an increased detection threshold with age. However, such investigations have not been performed for non-steroid odorants. Hence, the current study investigated sex- and age-related effects on the prevalence of specific anosmia in children/adolescents aged 5-14 years (n = 800) to non-steroid odorants. The detection thresholds of three non-steroid odorants (bacdanol, methylsalicylate, and 3-hydrox-2-methyl-4-pyrone) with different molecular weights were measured. Results indicate that the rate of specific anosmia for all three odorants decreases from childhood to adolescence. This decrease is independent of sex and is most pronounced for odorants with higher molecular weight compared to the odorant with lower molecular weight. Thus, the development of basic olfactory functions for these three odorants continues until adolescence. The effect of molecular weight suggests that the locus of effect is at the periphery of the olfactory system, due to changes in either olfactory receptor expression patterns or perireceptor events.

Maternal odor shapes rapid face categorization in the infant brain.

To successfully interact with a rich and ambiguous visual environment, the human brain learns to differentiate visual stimuli and to produce the same response to subsets of these stimuli despite their physical difference. Although this visual categorization function is traditionally investigated from a unisensory perspective, its early development is inherently constrained by multisensory inputs. In particular, an early-maturing sensory system such as olfaction is ideally suited to support the immature visual system in infancy by providing stability and familiarity to a rapidly changing visual environment. Here, we test the hypothesis that rapid visual categorization of salient visual signals for the young infant brain, human faces, is shaped by another highly relevant human-related input from the olfactory system, the mother's body odor. We observe that a right-hemispheric neural signature of single-glance face categorization from natural images is significantly enhanced in the maternal versus a control odor context in individual 4-month-old infant brains. A lack of difference between odor conditions for the common brain response elicited by both face and non-face images rules out a mere enhancement of arousal or visual attention in the maternal odor context. These observations show that face-selective neural activity in infancy is mediated by the presence of a (maternal) body odor, providing strong support for multisensory inputs driving category acquisition in the developing human brain and having important implications for our understanding of human perceptual development.

Attractive and appetitive odor factors in murine milk: Their fade-out time and differential cryo-preservation.

Murine milk conveys an odor factor that is both attractive and appetitive to conspecific newborns. Up to now, little is known about the temporal dynamic of this odor factor and about the stability of its behavioral activity after milk ejection. We aim to characterize the conditions in which the attractive and appetitive potency of milk to newborns is best conserved and, as a logical outcome, at standardizing conditions in which milk varies in reactogenic potency for newborns. Milk was collected and conserved in two conditions of cold (4 °C, -80 °C) for several durations (3 and 24 h, and 1, 2 and 8 months). The reactogenic potency of milk was assayed in 2 day-old mouse pups. We found that milk remains olfactorily attractive and appetitive to newborns after 3 h of storage at 4 °C, but it completely loses reactogenic potency on newborn pups after 24 h of storage at 4 °C. Storage at -80 °C preserves the behavioral activity of milk up to 1 month, but milk stored for 2 months at this temperature remains appetitive but not attractive to pups. Finally, the reactogenic potency of murine milk in pups is abolished after 8 months of storage at -80 °C. This study highlights that attractive and appetitive factors of milk appear dissociable and, in any case, highly labile. It provides, for two different storage temperatures, a temporal window in which milk remains behaviorally active on pups. These results will allow designing a contrastive chemical approach to identify the reactogenic compounds of milk.