Dietary prebiotics produce favorable changes in the commensal gut microbiome and reduce host vulnerability to stress-induced disruptions in complex behaviors such as sleep. The mechanisms for how prebiotics modulate stress physiology remain unclear; however, emerging evidence suggests that gut microbes and their metabolites may play a role. This study tested if stress and/or dietary prebiotics (Test diet) alter the fecal metabolome; and explored if these changes were related to sleep and/or gut microbial alpha diversity. Male F344 rats on either Test or Control diet were instrumented for electroencephalography biotelemetry measures of sleep/wake. After 5 weeks on diet, rats were either stressed or remained in home cages. Based on untargeted mass spectrometry and 16S rRNA gene sequencing, both stress and Test diet altered the fecal metabolome/microbiome. In addition, Test diet prevented the stress-induced reduction in microbial alpha diversity based on PD_Whole_Tree, which has been previously published. Network propagation analysis revealed that stress increased members of the neuroactive steroidal pregnane molecular family; and that Test diet reduced this effect. We also discovered links between sleep, alpha diversity, and pyrimidine, secondary bile acid, and neuroactive glucocorticoid/pregnanolone-type steroidal metabolites. These results reveal novel microbial-dependent metabolites that may modulate stress physiology and sleep.

Background: Exposure to stressful stimuli dysregulates inflammatory processes and alters the gut microbiota. Prebiotics, including long-chain fermentable fibers and milk oligosaccharides, have the potential to limit inflammation through modulation of the gut microbiota. To determine whether prebiotics attenuate stress-induced inflammation and microbiota perturbations, mice were fed either a control diet or a diet supplemented with galactooligosaccharides, polydextrose and sialyllactose (GOS+PDX+SL) or sialyllactose (SL) for 2 weeks prior to and during a 6-day exposure to a social disruption stressor. Spleens were collected for immunoreactivity assays. Colon contents were examined for stressor- and diet- induced changes in the gut microbiome and metabolome through 16S rRNA gene sequencing, shotgun metagenomic sequencing and UPLC-MS/MS.Results: Stress increased circulating IL-6 and enhanced splenocyte immunoreactivity to an ex vivo LPS challenge. Diets containing GOS+PDX+SL or SL alone attenuated these responses. Stress exposure resulted in large changes to the gut metabolome, including robust shifts in amino acids, peptides, nucleotides/nucleosides, tryptophan metabolites, and B vitamins. Multiple B vitamins were inversely associated with IL-6 and were augmented in mice fed either GOS+PDX+SL or SL diets. Stressed mice exhibited distinct microbial communities with lower abundances of Lactobacillus spp. and higher abundances of Bacteroides spp. Diet supplementation with GOS+PDX+SL, but not SL alone, orthogonally altered the microbiome and enhanced the growth of Bifidobacterium spp. Metagenome-assembled genomes (MAGs) from mice fed the GOS+PDX+SL diet unveiled genes in a Bifidobacterium MAG for de novo B vitamin synthesis. B vitamers directly attenuated the stressor-induced exacerbation of cytokine production in LPS-stimulated splenocytes.Conclusions: Overall, these data indicate that colonic metabolites, including B vitamins, are responsive to psychosocial stress. Dietary prebiotics reestablish colonic B vitamins and limit stress-induced inflammation.

Nutritional interventions targeting the microbiota-gut-brain axis are proposed to modulate stress-induced dysfunction of physiological processes and brain development. Maternal separation (MS) in rats induces long-term alterations to behaviour, pain responses, gut microbiome and brain neurochemistry. In this study, the effects of dietary interventions (milk fat globule membrane [MFGM] and a polydextrose/galacto-oligosaccharide prebiotic blend) were evaluated. Diets were provided from postnatal day 21 to both non-separated and MS offspring. Spatial memory, visceral sensitivity and stress reactivity were assessed in adulthood. Gene transcripts associated with cognition and stress and the caecal microbiota composition were analysed. MS-induced visceral hypersensitivity was ameliorated by MFGM and to greater extent with the combination of MFGM and prebiotic blend. Furthermore, spatial learning and memory were improved by prebiotics and MFGM alone and with the combination. The prebiotic blend and the combination of the prebiotics and MFGM appeared to facilitate return to baseline with regard to HPA axis response to the restraint stress, which can be beneficial in times where coping mechanisms to stressful events are required. Interestingly, the combination of MFGM and prebiotic reduced the long-term impact of MS on a marker of myelination in the prefrontal cortex. MS affected the microbiota at family level only, while MFGM, the prebiotic blend and the combination influenced abundance at family and genus level as well as influencing beta-diversity levels. In conclusion, intervention with MFGM and prebiotic blend significantly impacted the composition of the microbiota as well as ameliorating some of the long-term effects of early-life stress.

Introduction: Milk fat globule membrane (MFGM) is a protein- and phospholipid-rich membrane that surrounds the lipid droplet in milk. We have previously reported that a diet composed of a combination of prebiotics, bovine MFGM (bMFGM), and lactoferrin (bLf) supported brain development in young pigs. Due to the growing interest of its potential benefits in neurodevelopment, the present study focused on the effects of dietary bMFGM alone using the pig as a translational model.Methods: Male pigs were provided ad libitum access to milk replacer with added whey protein-lipid concentrate (source of bMFGM) at 0 (CONT), 2.5 (MFGM-2.5), or 5 (MFGM-5.0) g/L from postnatal day (PND) 2 to 31. Blood was collected from pigs at PND 15 and 31, and pigs underwent behavioral testing using the novel object recognition task starting at PND 25. At PND 31, magnetic resonance imaging was conducted and animals were subsequently euthanized for tissue collection.Results: No group differences in body weight gain or milk intake were observed. At PND 31, few group differences were detected in absolute and relative brain volumes, brain water diffusivity outcomes, or behavioral parameters using the novel object recognition task. Serum lipoprotein was higher in pigs receiving diets with added dietary bMFGM compared with the CONT group. Serum cholesterol and high-density lipoprotein significantly higher (all P < 0.05) in the MFGM-2.5 compared with the CONT group. However, cholesterol concentrations within the brain prefrontal cortex and hippocampus did not differ among dietary groups.Conclusion: In this pig model, dietary supplementation with bMFGM was well-tolerated and supported growth and dietary intake similar to the control formula. Added dietary bMFGM was associated with increased serum lipoprotein, but no group differences in early brain cholesterol concentrations, macrostructure, microstructure, or recognition memory pigs at 31 days of age. Further examination of longitudinal brain development and myelination in the pig, particularly at later ages/maturation, is warranted.

Early life nutrition is critical for brain development. Dietary prebiotics and bioactive milk fractions support brain development by increasing plasticity and altering activity in brain regions important for cognition and emotion regulation, perhaps through the gut-microbiome-brain axis. Here we examined the impact of a diet containing prebiotics, lactoferrin, and milk fat globule membrane (test diet) on beneficial gut bacteria, basal gene expression for activity and plasticity markers within brain circuits important for cognition and anxiety, and anxiety-related behavior in the open field. Juvenile male F344 rats were fed the test diet or a calorically matched control diet beginning postnatal day 24. After 4 weeks on diets, rats were sacrificed and brains were removed. Test diet significantly increased mRNA expression for cfos, brain derived neurotropic factor, and the GluN1 subunit of the NMDA receptor in the prefrontal cortex and reduced cfos mRNA within the amygdala. Diet-induced increases in fecal Lactobacillus spp., measured using selective bacterial culture, positively correlated with altered gene expression for cfos and serotonin receptors within multiple brain regions. In a separate cohort of juvenile rats, 4 weeks of the test diet increased time spent in the center of the open field, a behavior indicative of reduced anxiety. These data demonstrate that early life diets containing prebiotics and bioactive milk fractions can adaptively alter genes in neural circuits underlying emotion regulation and impact anxiety-related behavior.

Background: Major risk factors for necrotizing enterocolitis (NEC) include premature birth and formula feeding in the context of microbial colonization of the gastrointestinal tract We previously showed that feeding formula composed of lactose vs. corn syrup solids protects against NEC in preterm pigs; however, the microbial and metabolic effects of these different carbohydrates used in infant formula has not been explored. Objective: Our objective was to characterize the effects of lactose- and corn syrup solid-based formulas on the metabolic and microbial profiles of preterm piglets and to determine whether unique metabolomic or microbiome signatures correlate with severity or incidence of NEC. Design/methods: Preterm piglets (103 days gestation) were given total parenteral nutrition (2 days) followed by gradual (5 days) advancement of enteral feeding of formulas matched in nutrient content but containing either lactose (LAC), corn syrup solids (CSS), or 1:1 mix (MIX). Gut contents and mucosal samples were collected and analyzed for microbial profiles by sequencing the V4 region of the 16S rRNA gene. Metabolomic profiles of cecal contents and plasma were analyzed by LC/GC mass spectrometry. Results: NEC incidence was 14, 50, and 44% in the LAC, MIX, and CSS groups, respectively. The dominant classes of bacteria were Bacilli, Clostridia, and Gammaproteobacteria. The number of observed OTUs was lowest in colon contents of CSS-fed pigs. CSS-based formula was associated with higher Bacilli and lower Clostridium from clusters XIVa and XI in the colon. NEC was associated with decreased Gammaproteobacteria in the stomach and increased Clostridium sensu stricto in the ileum. Plasma from NEC piglets was enriched with metabolites of purine metabolism, aromatic amino acid metabolism, and bile acids. Markers of glycolysis, e.g., lactate, were increased in the cecal contents of CSS-fed pigs and in plasma of pigs which developed NEC. Conclusions: Feeding formula containing lactose is not completely protective against NEC, yet selects for greater microbial richness associated with changes in Bacilli and Clostridium and lower NEC incidence. We conclude that feeding preterm piglets a corn syrup solid vs. lactose-based formula increases the incidence of NEC and produces distinct metabolomic signatures despite modest changes in microbiome profiles.

Background: Sialyllactose (SL) is a highly abundant oligosaccharide in human milk that has been shown to influence intestinal maturation and cognitive development and exert bifidogenic effects on the gut microbiota. The SL content of infant formula is significantly less than that of human milk, therefore there is interest in determining the effect of supplementing SL to infant formula at the levels in human milk on neonatal outcomes. Objective: The aim of this study was to investigate the effect of varying doses of dietary SL compared with a milk replacer formula on weight gain, gastrointestinal development, and microbiota composition in piglets. Methods: Thirty-eight intact male piglets were randomly assigned to 1 of 4 experimental diets from 2 to 32-33 d of age. Diets were formulated to contain SL at 0 mg/L (CON), 130 mg/L (LOW), 380 mg/L (MOD), or 760 mg/L (HIGH). At 32-33 d of age, blood was collected for serum chemistry and blood cellular analyses, and coagulation time. Immediately after humane killing, the small intestine was excised and intestinal segments fixed for quantification of mucin-producing goblet cells and morphologic analysis. In addition, mucosal disaccharide activity was assessed. Colonic luminal contents and feces were collected for measurement of pH, dry matter, volatile fatty acids, and the microbiota. Results: SL at <= 760 mg/L supported normal growth, intestinal development, and enzyme activity as well as serum chemistries and hematology (P > 0.05). In addition, SL supplementation did not affect overall microbiota structure and diversity in ascending colon contents and feces, but had minor effects on the relative abundances of specific microbes. Conclusions: The findings in this study demonstrate that SL addition to a prebiotic-containing formula was well-tolerated by neonatal piglets, supported normal growth, and did not result in any adverse effects on serum chemistries or intestinal development.

Sialic acid (SA) is an integral component of gangliosides and signaling molecules in the brain and its dietary intake may support cognitive development. We previously reported that feeding sialyllactose, a milk oligosaccharide that contains SA, alters SA content and diffusivity in the pig brain. The present research sought to expand upon such results and describe the effects of feeding sialyllactose on recognition memory and sleep/wake activity using a translational pig model. Pigs were provided ad libitum access to a customized milk replacer containing 0 g/L or 380 g/L of sialyllactose from postnatal day (PND) 2–22. Beginning on PND 15, pigs were fitted with accelerometers to track home-cage activity and testing on the novel object recognition task began at PND 17. There were no significant effects of diet on average daily body weight gain, average daily milk intake, or the gain-to-feed ratio during the study (all p ≥ 0.11). Pigs on both diets were able to display recognition memory on the novel object recognition task (p < 0.01), but performance and exploratory behavior did not differ between groups (all p ≥ 0.11). Total activity and percent time spent sleeping were equivalent between groups during both day and night cycles (all p ≥ 0.56). Dietary sialyllactose did not alter growth performance of young pigs, and there was no evidence that providing SA via sialyllactose benefits the development of recognition memory or gross sleep-related behaviors.

Objectives: Previous studies have shown that dietary prebiotics have the potential to improve memory, alter social behavior, and reduce anxiety-like behaviors in rodents. The present research sought to expand upon such results and describe the effects of feeding prebiotics early in life on cognition and neurochemistry using a translational piglet model. Methods: Pigs were provided customized milk replacer containing 2 g/L each of polydextrose (PDX) and galactooligosaccharide (PDX/GOS) or 0 g/L (Control) from postnatal day (PND) 2-33. Beginning on PND 25, pigs were tested on the novel object recognition (NOR), novel location recognition (NLR), and backtest tasks to measure recognition memory and response to restraint stress. At study conclusion pigs were euthanized and intestine, blood, and brain tissues were collected and analyzed. Results: PDX/GOS-fed pigs demonstrated recognition memory on the NOR task (P < 0.001) whereas Control pigs did not (P = 0.184). Additionally, PDX/GOS-fed pigs visited the novel and sample objects more frequently (all P < 0.05) while spending less time per visit exploring the sample object (P = 0.028) than Control pigs. Volatile fatty acids (VFAs) were decreased in the ascending colon (P < 0.012), whereas butyrate tended to be higher in blood (P = 0.080) and lower in the hippocampus (P = 0.061) of PDX/GOS-fed pigs. PDX/GOS-fed pigs exhibited lower serotonin (P = 0.016) in the hippocampus. Conclusion: These findings suggest that early life consumption of PDX/GOS supports recognition memory as measured by NOR while modulating the concentrations of VFAs in the colon, blood, and brain, as well as hippocampal serotonin. [GRAPHICS] .

Oligosaccharides are the third most abundant component in human milk and modulate neonate microbiota, gut and immune development. This study investigated potential dose‐dependent effects of sialyllactose (Lacprodan SAL‐10 ® , SL) on growth and gut development of neonatal piglets. Beginning at 2 d of age, 38 vaginally‐derived male piglets (mean body weight (BW) 1.64 ± 0.04 kg) were randomized to diets formulated to contain: control (CONT) [0 mg SL/L milk replacer], low (LOW) [130 mg SL/L], moderate (MOD) [380 mg SL/L], and high (HIGH) [760 mg SL/L]. Diets contained 4 g/L of a 1:1 mixture of polydextrose and galactooligosaccharides. Diets were prepared at 20% (wt/vol) and piglets received 285 mL and 325 mL per kg BW on d2–5 and 5–33, respectively. On d32 or 33, piglets were euthanized and samples were collected. Outcomes were analyzed using a one‐way ANOVA; significance was set at p ≤ 0.05 and trends at 0.05 < p < 0.10. Dietary SL was well tolerated regardless of dose and growth patterns were similar among the groups. Weight gain between d2 and 33 did not differ among the groups and the average gain was 4.23 ± 0.29 kg (CONT: 4.19 kg ± 0.42; LOW: 4.08 kg ± 0.64; MOD: 4.43 kg ± 0.69; HIGH: 4.22 kg ± 0.68). Similarly, intestinal length and weight, villus and crypt morphology in the small and large intestine and the number of acidic sulfated mucin‐secreting goblet cells did not differ among the groups. Short‐chain and branched‐chain fatty acid profiles in colonic contents were similar between all four groups. No differences in complete blood counts were observed and serum clinical chemistry values were similar (minerals, electrolytes, protein, enzymes, kidney function, liver function and acid:base balance and prothrombin time), except for activated partial thromboplastin time (aPTT) and glutathione dehydrogenase (GLDH). APTT (seconds) in HIGH (12.6 ± 0.5) was lower (p<0.02) than CONT (14.1 ± 0.2), LOW (13.8 ± 0.4) and MOD (13.6 ± 0.2). GLDH (U/L) in CONT (0.9 ± 0.1) was lower (p<0.02) than LOW (1.3 ± 0.1) and MOD (1.5 ± 0.2) and tended (p<0.09) to be lower than HIGH (1.2 ± 0.08). Due to the low magnitude of differences and lack of a clear dose response, the differences in aPTT were not clinically significant. In addition to a lack of dose‐response in our study, normal values for other liver enzymes suggest that slightly higher GLDH values are also unlikely of clinical significance. In conclusion, dietary SL at up to 760 mg/L was well tolerated and supported similar growth and intestinal development as the CONT formula in neonatal piglets. Support or Funding Information Mead Johnson Nutrition

Nutritional interventions targeting the microbiota‐gut‐brain axis may be able to modulate stress‐induced dysfunction of physiological processes and brain development. Maternal separation (MS) stress of rat pups is a robust model of early life stress that induces long‐term alterations to behavior and brain neurochemistry. In this study the effects of milk fat globule membrane (Lacprodan MFGM‐10®, 15 g/kg) and a polydextrose / galactooligosaccharide (7 g/kg each) prebiotic blend were evaluated on early life stress induced alterations. The maternal separation protocol was conducted as described in O'Mahony et al., 2009. Rats were separated from their mothers for 3 h /day from postnatal day (PND) 2 to 12. Starting at weaning (PND 21), both non‐separated (NS) and maternally separated (MS) offspring were randomized into separate experimental groups and were provided drinking water with or without supplementation of MFGM, prebiotic blend or a combination of both. Visceral sensitivity was assessed using the colorectal distension test conducted at PND 79. MS rats demonstrated visceral hypersensitivity to colorectal distension reflected by a lower pain threshold and a higher number of pain behaviours when compared to NS rats. This increase in sensitivity in MS rats was ameliorated by MFGM (p=0.01) and also the combination of MFGM and the prebiotic blend (p=0.045). Furthermore, MS rats showed significant impairments in spatial and reference memory in the Morris water maze, which was carried out between PND 61–65. Cognitive performance in this test was improved by the prebiotic blend (p=0.007) and MFGM alone (p=0.019) as well as the combination of both (p<0.0001). Interestingly, the combination of MFGM and prebiotic reversed the impact of early life stress on the mineralocorticoid receptor (MR) expression in the hippocampus, which is associated with negative feedback of the hypothalamic pituitary adrenal (HPA) axis. In conclusion, feeding MFGM and prebiotics to rats ameliorated the visceral pain sensitivity and cognitive impairment caused by MS. The changes to MR expression indicate that regulation of the negative feedback of the HPA axis may be partially involved. Further studies are needed to confirm this as well as elaborate on the specific mechanism of actions related to gut and brain neurochemistry.Support or Funding InformationSupported by Science Foundation Ireland and Mead Johnson Nutrition.

Stress exposure can produce disruptions in the sleep cycle that may contribute to altered cognitive performance. Prebiotics can selectively enhance select gut microbial species that may promote favorable response to stressrobustness. Our previous studies showed that a milk oligosaccharide, sialyllactose, supported normal microbial communities (similar to non‐stressed animals) and behavioral responses during stressor exposure, potentially through effects on the gut microbiota–brain axis. The aim of the current study was to determine whether early life dietary oligosaccharides would modulate the sleep cycle and protect sleep architecture following stress. We hypothesized that diet containing a blend of prebiotics (galactooligosaccharides, GOS+polydextrose, PDX) and/or sialyllactose (Lacprodan SAL‐10 ® ; SL) would mitigate stress induced alterations of electrophysiological markers of sleep. Male Sprague Dawley rats (postnatal day (P)21) were placed on one of the experimental diets for 30d: a) SL [2.2g/kg], b)GOS+PDX [7g/kg each], c) GOS+PDX [7g/kg each] + SL [2.2g/kg] or d) Control[cellulose as fiber source]. Telemetry devices were implanted on P64 to examine the differences in electroencephalograph (EEG) and electromyograph (EMG)measurements. Baseline 24h EEG recordings were performed on P84. Rats were exposed to an acute stressor (inescapable stress) on P85 in order to examine the potential protective effects of our experimental diets on stress‐induced sleep/wake cycle. An additional group of Control animals remained undisturbed(Control Unstressed). There were no differences in food intake and body weight gain among the groups. There was a normal EEG distribution of power across the 24 h period under baseline conditions for all diets. During baseline, GOS+PDX+SL group had decreased waketheta frequency compared to Control Unstressed group (P<0.05). All stressed groups slept more (both NREM and REM) compared to the Control Unstressed group. Test diet groups had less overall waking following stress compared to the control animals. Rats on GOS+PDX diet had more overall NREM (NREM consolidation)compared to Control Unstressed animals (P<0.05). REM alpha and beta power were increased in rats on GOS+PDX and SL diets compared to control animals during baseline (prior to stress exposure) and following stress (P<0.05). Overall, these data demonstrate that an early life diet containing GOS+PDX and/or SL can increase NREM sleep consolidation and mitigate stress‐induced sleep disruptions in adulthood. Ongoing microbiome analysis might provide further insights on the mechanism of actions of this novel dietary intervention. Support or Funding Information Supported by Mead Johnson Nutrition.

A prebiotic blend of polydextrose and galactooligoasaccaride (PDX‐GOS) represents a mixture of long‐ and short‐chain oligosaccharides. Emerging evidence in rodent models suggests dietary prebiotics are capable of modulating the gut microbiota and animal behavior. The present research set out to describe the effects of feeding PDX‐GOS early in life on cognition, exploratory behaviors and brain neurochemistry using a piglet model. Piglets were provided customized milk replacer containing 2 g/L each of PDX and GOS (TEST) or 0 g/L (CONT) from postnatal day (PND) 2–33. Beginning at PND 25, piglets were tested on the novel object recognition (NOR), novel location recognition, and tonic immobility tasks to measure recognition memory and response to restraint stress. At study conclusion, piglets were euthanized, and intestine and brain tissues were collected. No effects of diet were observed for lengths or weights of intestinal tissues (P > 0.119). Concentrations of volatile fatty acids (acetate, propionate, butyrate) were decreased in the ascending colon of pigs in the TEST group (P < 0.012), but unchanged in the cecum (P > 0.383). Piglets consuming the TEST diet were able to demonstrate NOR after a 48 h delay (P < 0.001), but piglets consuming the CONT diet could not (P = 0.184). Additionally, piglets consuming the TEST diet visited both the novel and sample objects more frequently (all P < 0.05), spent less time per visit exploring the sample object (P = 0.028), and exhibited greater total movement during the sample phase (P < 0.001) than those on the CONT diet. Neither group was able to complete the novel location recognition task after a delay of 24 h (all P > 0.320), and there were no effects of diet observed for outcomes in the tonic immobility task (all P > 0.537). Concentrations of serotonin were lower (P = 0.016) in the hippocampus of pigs fed the TEST diet and tended to be lower (P = 0.055) in the striatum of those same pigs. These findings suggest that early‐life consumption of PDX‐GOS results in prebiotic effects in the ascending colon, subtly alters concentrations of catecholamines in the brain and supports recognition memory. We believe this is the first account of non‐digestible prebiotic fibers displaying simultaneous effects in the gut and brain of neonatal piglets. Such findings are likely the result of interactions within the gut‐microbiome‐brain axis, signifying dietary intervention as a possible mechanism to impact both gut and brain development and function in early life.Support or Funding InformationThis project was supported by Mead Johnson Nutrition.

Sialic acid (SA) is a key component of gangliosides and neural cell adhesion molecules important during neurodevelopment. Human milk contains SA in the form of sialyllactose (SL) an abundant oligosaccharide. To better understand the potential role of dietary SL on neurodevelopment, the effects of varying doses of dietary SL on brain SA content and neuroimaging markers of development were assessed in a newborn piglet model. Thirty-eight male pigs were provided one of four experimental diets from 2 to 32 days of age. Diets were formulated to contain: 0 mg SL/L (CON), 130 mg SL/L (LOW), 380 mg SL/L (MOD) or 760 mg SL/L (HIGH). At 32 or 33 days of age, all pigs were subjected to magnetic resonance imaging (MRI) to assess brain development. After MRI, pig serum and brains were collected and total, free and bound SA was analyzed. Results from this study indicate dietary SL influenced (p = 0.05) bound SA in the prefrontal cortex and the ratio of free SA to bound SA in the hippocampus (p = 0.04). Diffusion tensor imaging indicated treatment effects in mean (p < 0.01), axial (p < 0.01) and radial (p = 0.01) diffusivity in the corpus callosum. Tract-based spatial statistics (TBSS) indicated differences (p < 0.05) in white matter tracts and voxel-based morphometry (VBM) indicated differences (p < 0.05) in grey matter between LOW and MOD pigs. CONT and HIGH pigs were not included in the TBSS and VBM assessments. These findings suggest the corpus callosum, prefrontal cortex and hippocampus may be differentially sensitive to dietary SL supplementation.

Sialyllactose is a milk oligosaccharide composed of a sialic acid conjugated to a lactose molecule that is present in human, bovine and porcine milks. Animal studies have demonstrated a variety of biological effects associated with provision of dietary sialyllactose, including gut maturation, immune function and increasing brain sialic acid levels. This study aimed to examine the dose response effects of sialyllactose (Lacprodan SAL‐10®; SL) on piglet brain development. Beginning at 2 d of age, 38 (n = 9–10 per treatment) vaginally‐derived male piglets received one of four diets formulated to contain: control (CONT) [0 mg SL/L milk replacer], low (LOW) [130 mg SL/L], moderate (MOD) [380 mg SL/L], and high (HIGH) [760 mg SL/L]. All diets contained 4 g/L of a 1:1 mixture of polydextrose and galactooligosaccharides. At 32 or 33 d of age, piglets were subjected to magnetic resonance imaging (MRI) procedures to assess macrostructural and microstructural brain development. All outcomes were analyzed using a one‐way ANOVA to assess differences between dietary treatments, significant outcomes were accepted at P ≤ 0.05 and trends at 0.05 < P < 0.10. Absolute brain volumes were not different (P > 0.05) between dietary treatments. Moreover, assessment of ten individual brain regions did not yield differences (P > 0.05) due to diet in absolute or relative volume measures. Axial, mean and radial diffusivity are used to assess white matter integrity and have been shown to change with age. We measured changes in these variables in different brain regions associated with feeding different levels of dietary SL. Diffusion tensor imaging revealed differences due to dietary treatment in measures of axial diffusivity (P = 0.002), mean diffusivity (P = 0.004), and radial diffusivity (P = 0.007) of the corpus callosum. In each of these outcomes, piglets provided the MOD diet exhibited the highest diffusivity measures compared with all other dietary treatments. Mean diffusivity (P = 0.075) and radial diffusivity (P = 0.051) in the left hippocampus tended to differ by dietary treatment, with piglets provided the MOD diet exhibiting the highest diffusivity measures. Recent evidence in piglets suggests provision of SL resulted in increased corpus callosum sialic acid. While the results presented herein do not assess sialic acid concentrations of the corpus callosum, our results corroborate recent evidence suggesting sensitivity of the corpus callosum to provision of dietary SL. The differences in corpus callosum MRI measures in response to dietary SL merit future research to assess the impact of SL on specific aspects of corpus callosum development and associated functions.Support or Funding InformationThis project was supported by Mead Johnson Nutrition.

Fatty acids are pivotal for growth and development of the neonate, and the brain is especially susceptible to their presence or absence. There is interest in understanding the mechanisms by which specific fatty acids in the circulation influence development, but relatively little research has explored medium chain fatty acids (MCFA) in this context. Therefore, the objective of this exploratory study was to identify novel relationships whereby circulating MCFA levels modulate hippocampal metabolites. Beginning at 2 d and continuing until 30 d of age, 24 vaginally‐derived male piglets were provided custom milk replacer formulated to meet piglet nutrient requirements. At 30 d of age, piglets underwent magnetic resonance spectroscopy (MRS) procedures to quantify metabolite concentrations in the hippocampus. Following MRS, samples from the right hippocampus and serum were harvested from pigs at 31 d of age for lipidomic profiling. Linear regressions were used to assess the relationship between serum MCFA and hippocampal MRS metabolites, as well as between serum MCFA and hippocampal FA. Subsequently, a mediation model was used to investigate whether the relationships between serum MCFA and hippocampal MRS metabolites were mediated by hippocampal FA. The mediation analysis revealed that hippocampal free dihomo‐γ‐linolenic acid (FA‐20:3n6) partially mediated the relationship between serum free lauric acid (FA‐12:0) and brain glycerophosphocholine plus phosphocholine (GPC‐PCh). First, increased levels of serum FA‐12:0 related to decreased levels of hippocampal FA‐20:3n6 (P = 0.011). Second, increased levels of serum FA‐12:0 related to decreased brain GPC‐PCh (P = 0.002). We observed a significant (P < 0.05; 95% CI: −0.245 to −0.019) indirect path of the mediation (i.e., effect of serum FA‐12:0 though hippocampal FA‐20:3n6 on brain GPC‐PCh). Additionally, the direct path (i.e., effect of serum FA‐12:0 on brain GPC‐PCh, accounting for hippocampal FA‐20:3n6) was also significant (P < 0.05; 95% CI: −0.435 to −0.012). Therefore, hippocampal FA‐20:3n6 partially mediated the relationship between serum FA‐12:0 and brain GPC‐PCh. Overall, these results suggest the levels of circulating FA‐12:0 may be related to hippocampal metabolism of previously unrelated complex lipid moieties. Future research should test for direct effects of altering circulating FA‐12:0 on these metabolites and associated roles in brain development and functions. Support or Funding Information This project was funded by Mead Johnson Nutrition.

Early life is a period of significant brain development when the brain is at its most plastic and vulnerable. Stressful episodes during this window of development have long-lasting effects on the central nervous system. Rodent maternal separation (MS) is a reliable model of early-life stress and induces alterations in both physiology and behaviour. Intriguingly, the gut microbiota of MS offspring differ from that of non-separated offspring, suggesting a mechanistic role for the microbiota-gut-brain axis. Hence, we tested whether dietary factors known to affect the gut microbiota alter the neurobehavioural effects of MS. The impact of consuming diet containing prebiotics polydextrose (PDX) and galactooligosaccharide (GOS) alone or in combination with live bacteria Lactobacillus rhamnosus GG (LGG) from weaning onwards in rats subjected to early-life MS was assessed. Adult offspring were assessed for anxiety-like behaviour in the open field test, spatial memory using the Morris water maze, and reactivity to restraint stress. Brains were examined via PCR for changes in mRNA gene expression. Here, we demonstrate that diets containing a combination of PDX/GOS and LGG attenuates the effects of early-life MS on anxiety-like behaviour and hippocampal-dependent learning with changes to hippocampal mRNA expression of genes related to stress circuitry, anxiety and learning.

There are extensive bidirectional interactions between the gut microbiota and the central nervous system (CNS). Dietary interventions that impact the microbiota, such as prebiotics and milk oligosaccharides, have been shown to reduce risk of many deleterious effects of stressor exposure. However, the mechanisms through which the microbiota influence the host are not well understood. We tested whether exposure to a social stressor, called social disruption (SDR), results in metabolomic changes in the colon, and whether prebiotics (blend of galactooligosaccharides (GOS) and polydextrose (PDX)) and/or milk oligosaccharide sialyllactose (Lacprodan SAL‐10®, SL) support a normal metabolome in the presence of stress. Male mice (postnatal day (P) 48–64) were placed on one of the experimental diets for 14d: a) SL [2.2g/kg], b) GOS+PDX [15g/kg each] + SL [2.2g/kg] or c) Control [cellulose as fiber source]. Mice were then randomly assigned to either the non‐stressed control group or to the Social Disruption Stressor (SDR Stressor) condition. SDR stressor entails repeated social defeat for 2 hrs per day on 6 consecutive days. Metabolites in the colonic contents were then assessed using LC/MS, while serum cytokine levels were assessed by ELISA. Mice fed a control diet and exposed to the stressor showed significant differences in 116 out of 529 detected metabolites, compared to control diet non‐stressed mice. This was partly due to significant reductions in dipeptides and amino acids, as well as significant increases in nucleotides and sphingolipids. Interestingly, stress‐exposed mice fed diets enriched with prebiotics (either a combination of GOS, PDX, and SL or SL alone) showed similar changes in dipeptides, nucleotides, and sphingolipids, but also showed increases in polyunsaturated fatty acids and endocannabinoids, as compared with the non‐stress groups. Polyunsaturated fatty acids and endocannabinoids can have anti‐inflammatory effects, thus we determined whether the experimental diets would attenuate stressor‐induced increases in inflammatory cytokines. Serum IL‐6 and IL‐1β were significantly increased in stressor‐exposed mice, but this effect tended to be attenuated in mice fed both treatment diets (diet × stress condition interactions, p‐values = 0.081 and 0.056, respectively). This study demonstrates that dietary prebiotics and milk oligosaccharides can impact the colonic metabolome to potentially attenuate stressor‐induced immunomodulation.Support or Funding InformationSupported by Mead Johnson Nutrition

Severe, repeated or chronic stress produces negative health outcomes including disruptions of the sleep/wake cycle and gut microbial dysbiosis. Diets rich in prebiotics and glycoproteins impact the gut microbiota and may increase gut microbial species that reduce the impact of stress. This experiment tested the hypothesis that consumption of dietary prebiotics, lactoferrin (Lf) and milk fat globule membrane (MFGM) will reduce the negative physiological impacts of stress. Male F344 rats, postnatal day (PND) 24, received a diet with prebiotics, Lf and MFGM (test) or a calorically matched control diet. Fecal samples were collected on PND 35/70/91 for 16S rRNA sequencing to examine microbial composition and, in a subset of rats; Lactobacillus rhamnosus was measured using selective culture. On PND 59, biotelemetry devices were implanted to record sleep/wake electroencephalographic (EEG). Rats were exposed to an acute stressor (100, 1.5 mA, tail shocks) on PND 87 and recordings continued until PND 94. Test diet, compared to control diet, increased fecal Lactobacillus rhamnosus colony forming units (CFU), facilitated non-rapid eye movement (NREM) sleep consolidation (PND 71/72) and enhanced rapid eye movement (REM) sleep rebound after stressor exposure (PND 87). Rats fed control diet had stress-induced reductions in alpha diversity and diurnal amplitude of temperature, which were attenuated by the test diet (PND 91). Stepwise multiple regression analysis revealed a significant linear relationship between early-life Deferribacteres (PND 35) and longer NREM sleep episodes (PND 71/72). A diet containing prebiotics, Lf and MFGM enhanced sleep quality, which was related to changes in gut bacteria and modulated the impact of stress on sleep, diurnal rhythms and the gut microbiota.

In pediatric nutrition, there is increasing interest in identifying lipid fractions and specific fatty acids (FA) that are important for neonatal development. Throughout infancy, brain growth is highly dynamic and requires particular FA to ensure a proper developmental trajectory. Previous research has focused on the relationship between serum and brain FA. However, the relationship between neurodevelopmental markers and FA found in serum and brain has yet to be explored. A better understanding of these relationships may allow researchers to identify novel mechanisms of brain development. Thus, the objective of this exploratory study was to determine the relationship between serum FA, brain FA, and magnetic resonance imaging (MRI) measures of neurodevelopment in neonatal piglets. Beginning at 2 d and continuing until 30 d of age, 24 vaginally‐derived male piglets were provided custom milk replacer formulated to meet piglet nutrient requirements. At 30 d of age, piglets underwent magnetic resonance spectroscopy (MRS) procedures to quantify metabolite concentrations in the hippocampus. Following MRS, the right hippocampus and serum were harvested from pigs at 31 d of age for lipidomic profiling. Linear regressions were used to assess the relationship between serum FA and hippocampal MRS metabolites, as well as between serum FA and hippocampal FA. Subsequently, a mediation model was used for serum FA that significantly related to both hippocampal FA and hippocampal MRS metabolites. A bootstrapping method drawing 5,000 samples with replacement from the dataset was implemented to estimate a sampling distribution (95% confidence interval) for the indirect and direct mediation effects. The mediation analysis revealed that hippocampal phosphatidylcholine stearic acid (PC‐18:0) fully mediated the relationship between serum triacylglycerol eicosanoic acid (TG‐20:0) and hippocampal myo‐inositol (MI) concentrations. First, increased serum TG‐20:0 levels were related to increased levels of hippocampal PC‐18:0 (P = 0.002). Second, increased serum TG‐20:0 levels were related to decreased concentrations of hippocampal MI (P = 0.016). The indirect mediation path (i.e., effect of serum TG‐20:0 levels on hippocampal MI concentration through hippocampal PC‐18:0 levels) was also significant (P < 0.05; 95% CI: −2.15 – −0.18). The direct mediation pathway (i.e., effect of serum TG‐20:0 on hippocampal MI concentration, accounting for hippocampal PC‐18:0) was not significant (P > 0.05; 95% CI: −1.84 – 0.69). Thus, hippocampal PC‐18:0 levels fully mediated the relationship between serum TG‐20:0 and hippocampal MI. Previous research suggests brain phospholipid levels rise and MI concentrations decline early in life. This study is the first to link these two observations, while also offering a possible relationship whereby a specific serum fatty acid may be involved. Future work should focus on testing direct relationships between serum TG‐20:0 and hippocampal PC‐18:0.Support or Funding InformationThis project was funded by Mead Johnson Nutrition.