Proteobacteria abundance during nursing predicts physical growth and brain volume at one year of age in young rhesus monkeys.

Over the last decade, multiple studies have highlighted the essential role of gut microbiota in normal infant development. However, the sensitive periods during which gut bacteria are established and become associated with physical growth and maturation of the brain are still poorly defined. This study tracked the assembly of the intestinal microbiota during the initial nursing period, and changes in community structure after transitioning to solid food in infant rhesus monkeys (Macaca mulatta). Anthropometric measures and rectal swabs were obtained at 2-month intervals across the first year of life and bacterial taxa identified by 16S rRNA gene sequencing. At 12 months of age, total brain and cortical regions volumes were quantified through structural magnetic resonance imaging. The bacterial community structure was dynamic and characterized by discrete maturational phases, reflecting an early influence of breast milk and the later transition to solid foods. Commensal microbial taxa varied with diet similar to findings in other animals and human infants; however, monkeys differ in the relative abundances of Lactobacilli and Bifidobacteria, two taxa predominant in breastfed human infants. Higher abundances of taxa in the phylum Proteobacteria during nursing were predictive of slower growth trajectories and smaller brain volumes at one year of age. Our findings define discrete phases of microbial succession in infant monkeys and suggest there may be a critical period during nursing when endogenous differences in certain taxa can shift the community structure and influence the pace of physical growth and the maturational trajectory of the brain.

Variation in spatial organization of the gut microbiota along the longitudinal and transverse axes of the intestines.

Elucidation of the mechanisms by which the microbiota-gut-brain axis influences behavior requires understanding the anatomical relationship of bacteria with mucosal elements. We herein report that microbes were mainly associated with food or fecal matter in the intestinal lumen. In the small intestine, bacterial density increased from proximal-to-distal levels and was much higher in the large intestine. A mucus layer was present between the mucosal epithelium and fecal boluses in the large intestine, but not between food and the mucosal epithelium in the small intestine. In contrast, in all intestinal regions lacking food or fecal boluses, the lumen was small, or absent, and contained little or no bacteria or mucus. The association of bacteria with food was tested in the small intestine by examining the effect of fasting on it. Bacterial density was equivalent in the ileum of fasted and fed mice, but fasting greatly reduced the amount of food containing bacteria, suggesting the amount of bacteria was reduced. Critically, this study provides evidence that the vast majority of the microbiota in the intestines are associated with the food matrix thereby raising questions regarding how the gut microbiota can potentially signal the brain and influence behavior. Given their spatial location within the lumen, which keeps them at a great distance from neuronal elements in the mucosa, combined with immune and mucus barriers, microbiota more likely to influence behavior through secretion of bacterial products that can traverse the spatial difference to interact with gut neurons and not through direct physical association.

Voluntary binge-patterned alcohol drinking and sex-specific influences on monoamine-related neurochemical signatures in the mouse gut and brain.

BACKGROUND: Altered monoamine (i.e., serotonin, dopamine, and norepinephrine) activity following episodes of alcohol abuse plays key roles not only in the motivation to ingest ethanol, but also physiological dysfunction related to its misuse. Although monoamine activity is essential for physiological processes that require coordinated communication across the gut-brain axis (GBA), relatively little is known about how alcohol misuse may affect monoamine levels across the GBA. Therefore, we evaluated monoamine activity across the mouse gut and brain following episodes of binge-patterned ethanol drinking. METHODS: Monoamine and select metabolite neurochemical concentrations were analyzed by ultra-high-performance liquid chromatography in gut and brain regions of female and male C57BL/6J mice following "Drinking in the Dark" (DID), a binge-patterned ethanol ingestion paradigm. RESULTS: First, we found that alcohol access had an overall small effect on gut monoamine-related neurochemical concentrations, primarily influencing dopamine activity. Second, neurochemical patterns between the small intestine and the striatum were correlated, adding to recent evidence of modulatory activity between these areas. Third, although alcohol access robustly influenced activity in brain areas in the mesolimbic dopamine system, binge exposure also influenced monoaminergic activity in the hypothalamic region. Finally, sex differences were observed in the concentrations of neurochemicals within the gut, which was particularly pronounced in the small intestine. CONCLUSION: Together, these data provide insights into the influence of alcohol abuse and biological sex on monoamine-related neurochemical changes across the GBA, which could have important implications for GBA function and dysfunction.

Lyticase Facilitates Mycobiome Resolution Without Disrupting Microbiome Fidelity in Primates.

BACKGROUND: Microbiome research has expanded to consider contributions of microbial kingdoms beyond bacteria, including fungi (i.e., the mycobiome). However, optimal specimen handling protocols are varied, including uncertainty of how enzymes utilized to facilitate fungal DNA recovery may interfere with bacterial microbiome sequencing from the same samples. METHODS: With Institutional Animal Care and Use Committee approval, fecal samples were obtained from 20 rhesus macaques (10 males, 10 females; Macaca mulatta). DNA was extracted using commercially available kits, with or without lyticase enzyme treatment. 16S ribosomal RNA (bacterial) and Internal Transcribed Spacer (ITS; fungal) sequencing was performed on the Illumina MiSeq platform. Bioinformatics analysis was performed using Qiime and Calypso. RESULTS: Inclusion of lyticase in the sample preparation pipeline significantly increased usable fungal ITS reads, community alpha diversity, and enhanced detection of numerous fungal genera that were otherwise poorly or not detected in primate fecal samples. Bacterial 16S ribosomal RNA amplicons obtained from library preparation were statistically unchanged by the presence of lyticase. CONCLUSIONS: We demonstrate inclusion of the enzyme lyticase for fungal cell wall digestion markedly enhances mycobiota detection while maintaining fidelity of microbiome identification and community features in non-human primates. In restricted sample volumes, as are common in limited human samples, use of single sample DNA isolation will facilitate increased rigor and controlled approaches in future work.

Maternal and Breast Milk Influences on the Infant Gut Microbiome, Enteric Health and Growth Outcomes of Rhesus Monkeys.

OBJECTIVES: Gut bacteria play an essential role during infancy and are strongly influenced by the mode of birth and feeding. A primate model was used to investigate the benefits of exposure to the mother or conversely the negative impact of early nursery rearing on microbial colonization. METHOD: Rectal swabs were obtained from rhesus macaques born vaginally and mother-reared (MR, N = 35) or delivered primarily via cesarean-section and human-reared (HR, N = 19). Microbiome composition was determined by rRNA gene amplicon sequencing at 2, 4, and 8 weeks of age and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs used to assess influences on functional metabolic pathways in the gut. Growth trajectories and incidence of diarrheic symptoms were evaluated. RESULTS: The microbial community structure was different between MR and HR infants with respect to phylogeny and abundance at all 3 ages. When examining dominant phyla, HR infants had a higher Firmicutes-to-Bacteroidetes ratio. At the genus level, breast milk-dependent commensal taxa and adult-typical genera were more abundant in MR infants. This difference resulted in a corresponding shift in the predicted metabolic effects, specifically for microbial genes associated with metabolism and immune function. HR infants had faster growth trajectories (P < 0.001), but more diarrheic symptoms by 6 months postnatal (P = 0.008). CONCLUSIONS: MR infants acquired adult-typical microbiota more quickly, and had higher levels of several beneficial commensal taxa. Cesarean-delivered and formula-fed infants had different developmental trajectories of bacterial colonization. Establishment of the gut microbiome was associated with an infant's growth trajectory, and implicated in the subsequent vulnerability to Campylobacter infections associated with diarrhea in infant monkeys.

Interactions Between Stress and Sex in Microbial Responses Within the Microbiota-Gut-Brain Axis in a Mouse Model.

OBJECTIVE: Animal models are frequently used to examine stress response, but experiments seldom include females. The connection between the microbiota-gut-brain axis and behavioral stress response is investigated here using a mixed-sex mouse cohort. METHODS: CF-1 mice underwent alternating days of restraint and forced swim for 19 days (male n = 8, female n = 8) with matching numbers of control animals at which point the 16S rRNA genes of gut microbiota were sequenced. Mixed linear models accounting for stress status and sex with individuals nested in cage to control for cage effects evaluated these data. Murine behaviors in elevated plus-maze, open-field, and light/dark box were investigated. RESULTS: Community-level associations with sex, stress, and their interaction were significant. Males had higher microbial diversity than females (p = .025). Of the 638 operational taxonomic units detected in at least 25% of samples, 94 operational taxonomic units were significant: 31 (stress), 61 (sex), and 34 (sex-stress interaction). Twenty of the 39 behavioral measures were significant for stress, 3 for sex, and 6 for sex-stress. However, no significant associations between behavioral measures and specific microbes were detected. CONCLUSIONS: These data suggest sex influences stress response and the microbiota-gut-brain axis and that studies of behavior and the microbiome therefore benefit from consideration of how sex differences drive behavior and microbial community structure. Host stress resilience and absence of associations between stress-induced behaviors with specific microbes suggests that hypothalamic-pituitary-adrenal axis activation represents a threshold for microbial influence on host behavior. Future studies are needed in examining the intersection of sex, stress response, and the microbiota-gut-brain axis.

Symposium review: Microbial endocrinology-Why the integration of microbes, epithelial cells, and neurochemical signals in the digestive tract matters to ruminant health.

The union of microbiology and neurobiology, which has been termed microbial endocrinology, is defined as the study of the ability of microorganisms to produce and respond to neurochemicals that originate either within the microorganisms themselves or within the host they inhabit. It serves as the basis for an evolutionarily derived method of communication between a host and its microbiota. Mechanisms elucidated by microbial endocrinology give new insight into the ways the microbiota can affect host stress, metabolic efficiency, resistance to disease, and other factors that may prove relevant to the dairy industry.

Social Influences on Prevotella and the Gut Microbiome of Young Monkeys.

OBJECTIVE: Our aim was to evaluate the bacterial profiles of young monkeys as they were weaned into peer groups with a particular focus on Prevotella, an important taxon in both human and nonhuman primates. The weaning of infants and increased social contact with peers is a developmental stage that is likely to affect the gut microbiome. METHODS: Gut bacteria were assessed in 63 rhesus monkeys living in social groups comprised of 4 to 7 individuals. Two groups were assessed prospectively on day 1 and 2 weeks after rehousing away from the mother and group formation. Ten additional groups were assessed at 2 weeks after group establishment. Fecal genomic DNA was extracted and 16S ribosomal RNA sequenced by Illumina MiSeq (5 social groups) and 454-amplicon pyrosequencing (7 social groups). RESULTS: Combining weaned infants into small social groups led to a microbial convergence by 2 weeks (p < .001). Diversity analyses indicated more similar community structure within peer groups than across groups (p < .01). Prevotella was the predominant taxon, and its abundance differed markedly across individuals. Indices of richness, microbial profiles, and less abundant taxa were all associated with the Prevotella levels. Functional Kyoto Encyclopedia of Genes and Genomes analyses suggested corresponding shifts in metabolic pathways. CONCLUSIONS: The formation of small groups of young rhesus monkeys was associated with significant shifts in the gut microbiota. The profiles were closely associated with the abundance of Prevotella, a predominant taxon in the rhesus monkey gut. Changes in the structure of the gut microbiome are likely to induce differences in metabolic and physiologic functioning.

Low Lactobacilli abundance and polymicrobial diversity in the lower reproductive tract of female rhesus monkeys do not compromise their reproductive success.

The lower reproductive tract of nonhuman primates is colonized with a diverse microbiota, resembling bacterial vaginosis (BV), a gynecological condition associated with negative reproductive outcomes in women. Our 4 aims were to: (i) assess the prevalence of low Lactobacilli and a BV-like profile in female rhesus monkeys; (ii) quantify cytokines in their cervicovaginal fluid (CVF); (iii) examine the composition and structure of their mucosal microbiota with culture-independent sequencing methods; and (iv) evaluate the potential influence on reproductive success. CVF specimens were obtained from 27 female rhesus monkeys for Gram's staining, and to determine acidity (pH), and quantify proinflammatory cytokines. Based on Nugent's classification, 40% had a score of 7 or higher, which would be indicative of BV in women. Nugent scores were significantly correlated with the pH of the CVF. Interleukin-1ß was present at high concentrations, but not further elevated by high Nugent scores. Vaginal swabs were obtained from eight additional females to determine microbial diversity by rRNA gene amplicon sequencing. At the phylum level, the Firmicutes/Bacteroidetes ratio was low. The relative abundance of Lactobacilli was also low (between 3% and 17%), and 11 other genera were present at >1%. However, neither the microbial diversity in the community structure, nor high Nugent scores, was associated with reduced fecundity. Female monkeys provide an opportunity to understand how reproductive success can be sustained in the presence of a diverse polymicrobial community in the reproductive tract.

Gut Microbiota and a Selectively Bred Taste Phenotype: A Novel Model of Microbiome-Behavior Relationships.

OBJECTIVES: The microbiota-gut-brain axis is increasingly implicated in obesity, anxiety, stress, and other health-related processes. Researchers have proposed that gut microbiota may influence dietary habits, and pathways through the microbiota-gut-brain axis make such a relationship feasible; however, few data bear on the hypothesis. As a first step in the development of a model system, the gut microbiome was examined in rat lines selectively outbred on a taste phenotype with biobehavioral profiles that have diverged with respect to energy regulation, anxiety, and stress. METHODS: Occidental low and high-saccharin-consuming rats were assessed for body mass and chow, water, and saccharin intake; littermate controls had shared cages with rats in the experimental group but were not assessed. Cecum and colon microbial communities were profiled using Illumina 16S rRNA sequencing and multivariate analysis of microbial diversity and composition. RESULTS: The saccharin phenotype was confirmed (low-saccharin-consuming rats, 0.7Δ% [0.9Δ%]; high-saccharin-consuming rats, 28.1Δ% [3.6Δ%]). Regardless of saccharin exposure, gut microbiota differed between lines in terms of overall community similarity and taxa at lower phylogenetic levels. Specifically, 16 genera in three phyla distinguished the lines at a 10% false discovery rate. DISCUSSION: The study demonstrates for the first time that rodent lines created through selective pressure on taste and differing on functionally related correlates host different microbial communities. Whether the microbiota are causally related to the taste phenotype or its correlates remains to be determined. These findings encourage further inquiry on the relationship of the microbiome to taste, dietary habits, emotion, and health.

Gut Microbiota and Brain Function: An Evolving Field in Neuroscience.

There is a growing appreciation of the importance of gut microbiota to health and disease. This has been driven by advances in sequencing technology and recent findings demonstrating the important role of microbiota in common health disorders such as obesity. Moreover, the potential role of gut microbiota in influencing brain function, behavior, and mental health has attracted the attention of neuroscientists and psychiatrists. At the 29(th) International College of Neuropsychopharmacology (CINP) World Congress held in Vancouver, Canada, in June 2014, a group of experts presented the symposium, "Gut microbiota and brain function: Relevance to psychiatric disorders" to review the latest findings in how gut microbiota may play a role in brain function, behavior, and disease. The symposium covered a broad range of topics, including gut microbiota and neuroendocrine function, the influence of gut microbiota on behavior, probiotics as regulators of brain and behavior, and imaging the gut-brain axis in humans. This report provides an overview of these presentations.

Microbial Endocrinology: An Ongoing Personal Journey.

The development of microbial endocrinology is covered from a decidedly personal perspective. Specific focus is given to the role of microbial endocrinology in the evolutionary symbiosis between man and microbe as it relates to both health and disease. Since the first edition of this book series 5 years ago, the role of microbial endocrinology in the microbiota-gut-brain axis is additionally discussed. Future avenues of research are suggested.

Staphylococci, Catecholamine Inotropes and Hospital-Acquired Infections.

Patients in hospital intensive care units have long been recognized as being at high risk for developing infections from bacteria, fungi, and viruses from within the hospital locality. Risk factors for development of nosocomial infections have usually focussed on the patient's physical condition and the number and type of invasive medical procedures administered. Using the staphylococci as its focus, this chapter presents recent evidence that some of the medications routinely used in the treatment of acutely ill patients may also be a risk factor for the development of nosocomial infections.

Exposure to a social stressor disrupts the community structure of the colonic mucosa-associated microbiota.

BACKGROUND: The microbiota of the mammalian gastrointestinal (GI) tract consists of diverse populations of commensal bacteria that interact with host physiological function. Dysregulating these populations, through exogenous means such as antibiotics or dietary changes, can have adverse consequences on the health of the host. Studies from laboratories such as ours have demonstrated that exposure to psychological stressors disrupts the population profile of intestinal microbiota. To date, such studies have primarily focused on prolonged stressors (repeated across several days) and have assessed fecal bacterial populations. It is not known whether shorter stressors can also impact the microbiota, and whether colonic mucosa-associated populations can also be affected. The mucosa-associated microbiota exist in close proximity to elements of the host immune system and the two are tightly interrelated. Therefore, alterations in these populations should be emphasized. Additionally, stressors can induce differential responses in anxiety-like behavior and corticosterone outputs in variant strains of mice. Thus, whether stressor exposure can have contrasting effects on the colonic microbiota in inbred C57BL/6 mice and outbred CD-1 mice was also examined. RESULTS: In the present study, we used high throughput pyrosequencing to assess the effects of a single 2-hour exposure to a social stressor, called social disruption (SDR), on colonic mucosa-associated microbial profiles of C57BL/6 mice. The data indicate that exposure to the stressor significantly changed the community profile and significantly reduced the relative proportions of two genera and one family of highly abundant intestinal bacteria, including the genus Lactobacillus. This finding was confirmed using a quantitative real-time polymerase chain reaction (qPCR) technique. The use of qPCR also identified mouse strain-specific differences in bacterial abundances. L. reuteri, an immunomodulatory species, was decreased in stressor-exposed CD-1 mice, but not C57BL/6 mice. CONCLUSIONS: These data illustrate that stressor exposure can affect microbial populations, including the lactobacilli, that are closely associated with the colonic mucosa. Because the lactobacilli can have beneficial effects on human health, stressor-induced reductions of their population could have important health implications.

Microbial endocrinology and the microbiota-gut-brain axis.

Microbial endocrinology is defined as the study of the ability of microorganisms to both produce and recognize neurochemicals that originate either within the microorganisms themselves or within the host they inhabit. As such, microbial endocrinology represents the intersection of the fields of microbiology and neurobiology. The acquisition of neurochemical-based cell-to-cell signaling mechanisms in eukaryotic organisms is believed to have been acquired due to late horizontal gene transfer from prokaryotic microorganisms. When considered in the context of the microbiota's ability to influence host behavior, microbial endocrinology with its theoretical basis rooted in shared neuroendocrine signaling mechanisms provides for testable experiments with which to understand the role of the microbiota in host behavior and as importantly the ability of the host to influence the microbiota through neuroendocrine-based mechanisms.

Linking norepinephrine production and performance to diet-induced low-grade, chronic inflammation in the intestine of broilers.

Maintenance of intestinal health is critical to successful poultry production and one of the goals of the poultry production industry. For decades the poultry industry has relied upon the inclusion of antibiotic growth promoters (AGP) to achieve this goal and improve growth performance. With the removal of AGPs, the emergence of chronic, low-level gut inflammation has come to the forefront of concern in the poultry industry with the diet being the primary source of inflammatory triggers. We have developed a dietary model of low-grade, chronic intestinal inflammation in broilers that employs feeding a high nonstarch polysaccharides (NSP) diet composed of 30% rice bran to study the effects of this inflammation on bird performance and physiology. For the present studies, we hypothesize that the low-grade chronic inflammation causes neurons in the intestinal enteric nervous system to secrete neurochemicals that activate immune cells that drive the inflammation and negatively affect bird performance. To test our hypothesis, 1-day-old broiler chickens were weighed and divided into 2 dietary regimes: a control corn-soybean diet and a group fed a high NSP diet (30% rice bran). At 7-, 14-, 21-, and 28-d posthatch (PH), birds were weighed, fecal material collected, and 5 birds were sacrificed and sections of duodenal and cecal tissues excised, and duodenal and cecal contents collected for ultra-high performance liquid chromatography analyses (UHPLC). UHPLC revealed 1000s-fold increase in the concentration of norepinephrine (NOR) in birds fed the high NSP diet compared to the control fed birds. Further, the fecal concentrations of NOR were also found to be significantly elevated in the birds on the NSP diet throughout all time points. There were no differences in weight gain nor feed conversion from 1 to 14 d PH, but birds fed the high NSP diet had significantly reduced weight gain and feed conversion from 14 to 28 d PH. The results revealed that a dietary-induced low-grade chronic inflammatory response increased NOR production in the gut which negatively affected bird performance. This study suggests that neuroimmune pathways may serve as a mechanistic target for the development of new interventions to decrease the incidence of chronic inflammation and thereby benefit performance.

Cold stress initiates catecholaminergic and serotonergic responses in the chicken gut that are associated with functional shifts in the microbiome.

Climate change is one of the most significant challenges facing the sustainability of global poultry production. Stress resulting from extreme temperature swings, including cold snaps, is a major concern for food production birds. Despite being well-documented in mammals, the effect of environmental stress on enteric neurophysiology and concomitant impact on host-microbiome interactions remains poorly understood in birds. As early life stressors may imprint long-term adaptive changes in the host, the present study sought to determine whether cold temperature stress, a prominent form of early life stress in chickens, elicits changes in enteric stress-related neurochemical concentrations that coincide with compositional and functional changes in the microbiome that persist into the later life of the bird. Chicks were, or were not, subjected to cold ambient temperature stress during the first week post-hatch and then remained at normal temperature for the remainder of the study. 16S rRNA gene and shallow shotgun metagenomic analyses demonstrated taxonomic and functional divergence between the cecal microbiomes of control and cold stressed chickens that persisted for weeks following cessation of the stressor. Enteric concentrations of serotonin, norepinephrine, and other monoamine neurochemicals were elevated (P < 0.05) in both cecal tissue and luminal content of cold stressed chickens. Significant (P < 0.05) associations were identified between cecal neurochemical concentrations and microbial taxa, suggesting host enteric neurochemical responses to environmental stress may shape the cecal microbiome. These findings demonstrate for the first time that early life exposure to environmental temperature stress can change the developmental trajectory of both the chicken cecal microbiome and host neuroendocrine enteric physiology. As many neurochemicals serve as interkingdom signaling molecules, the relationships identified here could be exploited to control the impact of climate change-driven stress on avian enteric host-microbe interactions.

Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this hypothesis, we developed an in vivo chronic wound, diabetic mouse model to determine the ability of the opportunistic pathogen, Pseudomonas aeruginosa, to cause biofilm-associated infections. Utilizing this model, we observed that diabetic mice with P. aeruginosa-infected chronic wounds displayed impaired bacterial clearing and wound closure in comparison with their non-diabetic littermates. While treating diabetic mice with insulin improved their overall health, it did not restore their ability to resolve P. aeruginosa wound infections or speed healing. In fact, the prevalence of biofilms and the tolerance of P. aeruginosa to gentamicin treatment increased when diabetic mice were treated with insulin. Insulin treatment was observed to directly affect the ability of P. aeruginosa to form biofilms in vitro. These data demonstrate that the chronically wounded diabetic mouse appears to be a useful model to study wound healing and biofilm infection dynamics, and suggest that the diabetic wound environment may promote the formation of biofilms. Further, this model provides for the elucidation of mechanistic factors, such as the ability of insulin to influence antimicrobial effectiveness, which may be relevant to the formation of biofilms in diabetic wounds.

Probiotics function mechanistically as delivery vehicles for neuroactive compounds: Microbial endocrinology in the design and use of probiotics.

I hypothesize here that the ability of probiotics to synthesize neuroactive compounds provides a unifying microbial endocrinology-based mechanism to explain the hitherto incompletely understood action of commensal microbiota that affect the host's gastrointestinal and psychological health. Once ingested, probiotics enter an interactive environment encompassing microbiological, immunological, and neurophysiological components. By utilizing a trans-disciplinary framework known as microbial endocrinology, mechanisms that would otherwise not be considered become apparent since any candidate would need to be shared among all three components. The range of neurochemicals produced by probiotics includes neurochemicals for which receptor-based targets on immune and neuronal elements (intestinal and extra-intestinal) have been well characterized. Production of neurochemicals by probiotics therefore allows for their consideration as delivery vehicles for neuroactive compounds. This unifying microbial endocrinology-based hypothesis, which may facilitate the selection and design of probiotics for clinical use, also highlights the largely unrecognized role of neuroscience in understanding how microbes may influence health.

Catecholamine concentrations in duck eggs are impacted by hen exposure to heat stress.

Rapid "fight-or-flight" responses to stress are largely orchestrated by the catecholamines. Moreover, catecholamines and catecholamine precursors are widely recognized to act as interkingdom signaling molecules among host and microbiota, as well as to serve as chemotactic signals for bacterial foodborne pathogens. While albumen and yolk concentrations of glucocorticoids have received extensive attention as non-invasive indicators of hen response to stress, little is known regarding the impact of the hen's stress response on in ovo catecholamine and catecholamine precursor concentrations. The aim of the present study was to determine norepinephrine and L-dopa concentrations in albumen and yolk of eggs laid by hens maintained under normal or heat stress conditions. Norepinephrine and L-dopa concentrations were also measured in oviductal tissue. Breeder ducks (∼35 weeks/age) were kept under normal (22°C) conditions or subjected to cyclical heat stress (35°C day/29.5°C night) for 3 weeks. Eggs (n = 12 per timepoint/group) were collected on a weekly basis. Hens were sacrificed at baseline or after 3 weeks of heat stress for oviductal tissue collection. Albumen, yolk, and oviduct concentrations of norepinephrine and L-dopa were determined using ultra high-performance liquid chromatography with electrochemical detection. Norepinephrine and L-dopa were detected in oviductal tissue as well as egg albumen and yolk. Norepinephrine concentrations were elevated (p < 0.05) in the yolk of eggs laid by the heat stress group compared to those of the control group. Norepinephrine concentrations in albumen were elevated (p < 0.05) in the heat stress group compared to control group at week 2. L-dopa concentrations were not significantly affected (p > 0.05) by heat stress in albumen, yolk, or oviductal tissue. Together, the present study provides the first evidence of the stress neurohormone, norepinephrine, in duck eggs and identifies that hen exposure to heat stress can affect in ovo norepinephrine concentrations. These data highlight the potential utility of in ovo catecholamine concentrations as non-invasive measures of the hen's response to stress, as well as warrants future research into whether hen deposition of stress-related neurochemicals into the egg could serve as a chemotactic signal in the vertical transmission of foodborne pathogens.