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.

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.

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.

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.

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.

Newborns prefer the odor of milk and nipples from females matched in lactation age: Comparison of two mouse strains.

Newborn mice are attracted to mammary odor cues carried in murine milk and nipple secretions. However, murine milk odor is not equally attractive along lactation. The present study focuses on the differential response of 2day-old mouse pups of C57Bl/6 (C) and Balb/C (B) strains to the odor of milk (Experiment 1) and nipples (Experiment 2) that are matched/unmatched in terms of pup's age or strain. In Experiment 1, C and B pups were tested in a series of tests simultaneously opposing either murine milk and a blank (water), or two milks collected in early and late lactation (lactation days 2 and 15, respectively) from females belonging to their own or the other strain. Results showed that C and B pups were attracted to the odor of the different milks regardless of the lactation age and the strain of the donor female. Nevertheless, C and B pups preferred the odor conveyed by early- than late-lactation milk of either strain. Moreover, early-lactation milk from C females was more attractive than early-lactation milk from B females for pups of either strain. In Experiment 2, differential nipple grasping response of C and B pups was measured when they were exposed to nipples of females in early or late lactation. The proportion of C pups that grasped a nipple was greater when they were exposed to a nipple in early lactation regardless of the strain of the donor females, whereas the proportion of B pups that grasped a nipple was greater when they were exposed to a nipple in early lactation, but only from own strain. Thus, newborn mice prefer the odor of milk and nipples from females that are matched in lactation age. This result is discussed in terms of reciprocally adaptive mechanisms between lactating females and their newborn offspring.

The response of newly born mice to odors of murine colostrum and milk: unconditionally attractive, conditionally discriminated.

It is a general rule that milk conveys chemosensory cues that are attractive to mammalian neonates. This study investigated whether compositional fluctuations in milk along lactation induce variations in newborn mouse pups' (Mus musculus, strain BALB/c) attraction to milk odor. Pups differing in suckling experience were exposed to the odor of milk sampled from females varying in lactational stage. Immediately after birth, suckling-inexperienced (P0) and suckling-experienced (P0suck ) pups were assayed in a series of paired-choice tests contrasting murine milk [of lactation days 0, 3, 15 (abridged L0, L3, L15, respectively)] and a blank (water) to evaluate olfactory detection and attraction of milk odor. Preference tests further paired these milk two-by-two to assess their relative attraction. Results showed first that P0 and P0suck pups detect and positively orient to any milk odor. When L0 is presented against L15 milk, P0 pups orient for a similar duration towards these odor stimuli, whereas P0suck pups spend more time toward the odor of L0 than of L15 milk. Finally, P0suck pups orient similarly to odors of L0 milk collected before/after the first suckling episode (L0 and L0suck , respectively), but the odor of L0 milk was more attractive than that of L3 milk. Thus, mouse pups' positive orientation toward the odors of murine colostrum (assumed to correspond to L0/L0suck milk) and later-lactation milk appears unconditional of previous suckling experience, whereas their ability to discriminate or display preference between milk differing in lactation stage appears conditional on postnatal exposure effects.

Testing smell when it is really vital: behavioral assays of social odors in the neonatal mouse.

The initial interactions of mouse newborns with their mother are crucial for their survival. These interactions rapidly end in the pups reaching nipples and getting milk. While we realize that olfaction is clearly prevailing in the success of these first suckling episodes, we still understand little about the nature and range of the natural odorants involved. Here we non-exhaustively describe some experimental principles and methods to assay the behavior of newly born and infant mice exposed to different odor stimuli from conspecifics. Testing neonatal and young mice with chemostimuli which they are evolutionarily or developmentally canalized to detect may be a productive way to trace unanticipated odor signals. Moreover, testing neonates also may also lead to characterize unsuspected strategies of murine females to produce and release odor messages.

Orientation of newborn mice to lactating females: identifying biological substrates of semiochemical interest.

Among mammals, odor-based communication between females and infants is decisive for neonatal survival. So far, the nature of odor substrates involved in the localization of the mother and their nipples is unknown in mice. The present study aims: (1) to evaluate the specific attractive value of lactating females to newborn mice, (2) to localize the abdominal region that is most attractive to pups, and (3) to identify odor substrates that support such attraction. Results showed that 5-6-day-old mice roam preferentially over the abdomen of lactating females than the abdomen of non-lactating females. In lactating females, pups are more attracted to abdominal areas comprising nipples. The blend of odor substrates from nipples, as well as separate sources presumed to compose it, viz. milk, maternal saliva and pup saliva, were detectable and equivalently attractive to pups. The equivalent attraction of these different odor substrates may derive either from overlap in chemical constituents, or from associative learning during nursing.

How does a newly born mouse get to the nipple? Odor substrates eliciting first nipple grasping and sucking responses.

It is a mammalian female strategy to emit odor cues and signals that direct their inexperienced newborns to the nipple, and optimize their initial sucking success and, hence, viability. Here, natural odorous substrates that contribute to nipple grasping were investigated in mice, a species that has not been much scrutinized on this topic. The response of pups toward the nipples of lactating females (LF) versus nonlactating females (NLF) were first assessed right after watched birth, before and after the first suckling experience, and at 1 day old, after more extended suckling experience. It appeared that only nipples of LF induced grasping at these early ages, leading to take NLF as the baseline setting to present various odor substrates sampled from LF, viz. amniotic fluid, murine milk, LF saliva, pup saliva, LF urine, and an odorless control stimulus (water). Results indicate that: (1) only amniotic fluid and fresh milk induced nipple grasping before the first suckling experience; (2) LF saliva started inducing grasping after the first suckling experience; (3) pup saliva released grasping after 24-36 hr of suckling experience; finally (4) neither LF urine, nor water induced any nipple grasping. In conclusion, the activity of amniotic fluid and murine milk on neonatal pup behavior before any postnatal suckling experience suggests that either prenatal learning and/or predisposed olfactory mechanisms do operate, while the behavioral activation due to maternal and infantile salivas clearly depends on postnatal exposure.

An odor timer in milk? Synchrony in the odor of milk effluvium and neonatal chemosensation in the mouse.

Mammalian newborns exhibit avid responsiveness to odor compounds emanating from conspecific milk. Milk is however developmentally heterogeneous in composition as a function of both evolved constraints and offspring demand. The present study aimed to verify whether milk odor attractivity for neonates is equally distributed along lactation in Mus musculus (Balb-c strain). Therefore, we exposed pups varying in age to milk samples collected from females in different lactational stages. The pups were assayed at postnatal days 2 (P2), 6 (P6) and 15 (P15) in a series of paired-choice tests opposing either murine milk and a blank (water), or two samples of milk collected in different stages of lactation [lactation days 2 (L2), 6 (L6), and 15 L15)]. Pups of any age were able to detect, and were attracted to, the odor of the different milk. When milk from different lactational stages were simultaneously presented, P2 pups oriented for a similar duration to the odors of L2 and of L6 milk, but significantly less to the odor of L15 milk. Next, P6 pups roamed equivalently over L2 and L6 milk odors, but still less over the odor of L15 milk. Finally, P15 pups explored as much L15 milk odor as the odors of both L2 and L6 milk. This developmental shift in milk attractivity is discussed in terms of changing chemosensory properties of milk and of shifting chemosensory abilities/experience of pups.

Long-lasting memory for an odor acquired at the mother's breast.

Whether neonatal odor memory can persist into toddlerhood and influence behaviors that tap processes related to cognition (attention and exploration), motivation (choice and consumption), and emotion (hedonic processing) remains under-researched. Using a quasi-experimental longitudinal design, we examined whether an odor experienced at the mother's breast can be retained at 7 and 21 months. The prescribed prophylactic use of a camomile-scented balm defined two groups: infants exposed (CaE) or never exposed (CaNE) to camomile odor. At 7 months, exploratory responses to three similar objects differing in odor (including camomile) were analyzed. At 21 months, three tasks were used to assess toddlers' (i) facial responses; (ii) exploratory responses to three similar, but differently odorized objects; and (iii) choices between two bottles carrying different odors. CaE infants displayed preferential responses for camomile odor at both ages in every task. In contrast, CaNE infants behaved either randomly or more negatively to camomile odor. This study indicates that early odor memories acquired during breastfeeding can be reactivated and influence behavioral processes until at least toddlerhood.

Mammary olfactory signalisation in females and odor processing in neonates: ways evolved by rabbits and humans.

Mammalian females have long been known to release olfactory attraction in their offspring. Mammary odor cues control infant state, attention and directional responses, delay distress responses, stimulate breathing and positive oral actions, and finally can boost learning. Here, we survey female-offspring odor communication in two mammalian species - European rabbits and humans - taken as representatives of evolutionary extremes in terms of structure and dynamics of mother-infant relations, and level of neonatal autonomy. Despite these early psychobiological differences, females in both species have evolved mammary structures combining multiple sources of endogenous and exogenous odorants, and of greasy fixatives, conferring on them a chemocommunicative function. To process these mammary chemosignals, neonates have co-evolved multiple perceptual mechanisms. Their behaviour appears to be driven by plastic mechanism(s) calibrated by circumstantial odor experience in preceding and current environments (fetal and postnatal induction of sensory processes and learning), and by predisposed mechanisms supported by pathways that may be hard-wired to detect species-specific signals. In rabbit neonates, predisposed and plastic mechanisms are working inclusively. In human neonates, only plastic mechanisms could be demonstrated so far. These mammary signals and cues confer success in offspring's approach and exploration of maternal body surface, and ensuing effective initial feeds and rapid learning of maternal identity. Although the duration of the impact of these mammary signals is variable in newborns of species exposed to contrasting life-history patterns, their functional role in setting on infant-mother interaction in the context of milk transfer can be crucial.

CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions.

Rats and mice exhibit a spontaneous attraction for lipids. Such a behavior raises the possibility that an orosensory system is responsible for the detection of dietary lipids. The fatty acid transporter CD36 appears to be a plausible candidate for this function since it has a high affinity for long-chain fatty acids (LCFAs) and is found in lingual papillae in the rat. To explore this hypothesis further, experiments were conducted in rats and in wild-type and CD36-null mice. In mice, RT-PCR experiments with primers specific for candidate lipid-binding proteins revealed that only CD36 expression was restricted to lingual papillae although absent from the palatal papillae. Immunostaining studies showed a distribution of CD36 along the apical side of circumvallate taste bud cells. CD36 gene inactivation fully abolished the preference for LCFA-enriched solutions and solid diet observed in wild-type mice. Furthermore, in rats and wild-type mice with an esophageal ligation, deposition of unsaturated LCFAs onto the tongue led to a rapid and sustained rise in flux and protein content of pancreatobiliary secretions. These findings demonstrate that CD36 is involved in oral LCFA detection and raise the possibility that an alteration in the lingual fat perception may be linked to feeding dysregulation.