Breast milk is the optimal form of nutrition for the human infant. In the U.S., 77% of women initiate breastfeeding immediately after birth, but by 6 months post-partum, only 43% are still breastfeeding with only 13% being exclusively breastfed (CDC, 2009). The remaining 30% of mothers are using formula supplemented with breast milk or “combined feeding” (CF) (CDC, 2009). Many infants who are receiving formula as their primary of source of nutrition are deprived of beneficial compounds naturally existing in human milk. One of the groups of bioactive components is the human milk oligosaccharides (HMO). This complex group of structures has many beneficial effects for the infant including promoting the growth of Bifidobacteria, acting as anti-adhesive and anti-microbial factors against pathogens, modulating immune development, and perhaps even providing nutrients for brain development (Bode, 2012). Prebiotics are routinely added to infant formula to mimic the actions of HMOs. In addition to increasing rates of formula feeding, the proportion of infants being born by cesarean section is also on the rise with rates being approximately 30% in the U.S. (Hamilton BE, 2009; Betrán AP, 2007; Mayor S, 2005).Route of delivery can influence colonization of intestinal microbiota. Vaginally-delivered (VD) infants acquire microbes from their mother’s vaginal canal and perineum, while cesarean-derived infants obtain more microbes from the environment, including the mother’s skin (Biasucci et al, 2008, Dominguez-Bello et al, 2010).The microbes that colonize the newborn intestine influence other functions of the gut, including mucosal immune development (Mshviladadze and Neu, 2010). Herein, we have three aims to investigate the effects of these early life events on neonatal intestinal, microbial, and immune development.Our first aim was to investigate the effect of route of delivery on microbial development and intestinal structure and function. Neonatal piglets were delivered either vaginally (VD) or by cesarean (CD) sow-reared for 21 days and then moved to cages and fed a weaning diet until d28. Samples were collected at d3, d14, d21 and d28 to examine effects of time. Mode of delivery did not affect digestive enzyme activity. Lactase and sucrase enzyme activities were measured. Lactase activity was variable over time (P<0.05), and sucrase activity increased over time (p<0.05). Both of these results were not unusual and reflect normal development in the piglet. Intestinal permeability was measured using the Ussing chamber. Route of delivery affect some of these parameters. CD piglets had greater glucose and glutamine transport at d14 compared to VD piglets. VD piglets also had increased jejunal basal Isc compared to CD piglets at all time points. In both the ileum and duodenum, 28-d-old piglets showed significant electrophysiological changes when compared to other time points, which s may be related to weaning. Resistance was increased at d28 in the duodenum compared to all other days, signaling that tight junction permeability was decreased. Intestinal histomorphology, villus height and crypt depth were evaluated and both day and mode of delivery had an effect, with VD piglets having longer villi than CD piglets and villi being longer at d14 than any other time point. Although, there were no significant differences in weight gain over time between the two groups, a slight separation in growth started to occur at d14 with the CD piglets slowing their rate of weight gain compared to VD piglets. These two results, in addition to the increased glucose and glutamine transport at d14, may be connected. Bacterial diversity was evaluated using Terminal Restriction Fragment Length Polymorphism (T-RFLP). There was no effect of route on this parameter. However, day did have an effect with the colonic communities separating out by day. Bacterial densities in the ileum and AC were quantified using qPCR and both universal and group specific primers. In the ileum, densities of total bacteria, Clostridium cluster XIVa and Lactobacillus were stable between d3 and 21, then decreased at d28. The numbers of ileal Bifidobacterium and Bacteroides-Prevotella did not change over time. In the AC, populations of total bacteria and Clostridium cluster XIVa increased from d3 till 14, were stable until d21, then decreased at d28. The numbers of Bifidobacterium and Lactobacillus were stable prior to weaning, and then increased and decreased, respectively, at d28. The densities of Bacteroides-Prevotella increased from d3 to d14 and were stable until d28.The products of bacteria fermentation, short chain fatty acids (SCFA), were affected by day, but not by route of delivery. Ileal butyrate concentrations decreased over time, reaching its lowest point at d28. Ileal acetate and propionate concentrations varied over time. In the ascending colon (AC), concentrations of all three short chain fatty acids (SCFA) remained stable through d21 and then increased at d28. Concentrations of branch chain fatty acids (BCFA) were only different in the AC, with isobutyrate and isovalerate being greatest at d14 and lowest at d28. Valerate concentrations in the AC were lower at d3 compared to all other days. In summary, changes in both intestinal parameters as well as microbiota were observed over time, many related to changes in diet at weaning. Route of delivery did not have the marked affects as previously hypothesized. This could be related to all the piglets being sow reared which may have lessened the impact of route of delivery on microbial colonization and in turn the other parameters of intestinal structure, function and permeability. The second aim of this dissertation focused on the effect of route of delivery and diet on intestinal function and immunity. Piglets were born either vaginally or through cesarean section and were fed one of three diets: formula, formula+prebiotic, or sow milk. The prebiotics chosen were polydextrose and scFOS (2g/L of each). The study duration was 14 days with a collection time point at d7 and d14. Dissacharidase and peptidase enzymes were examined in jejunal and ileal tissues. In the jejunum, neither day nor diet had an effect on lactase activity, but day had an effect on sucrase activity with d14 piglets having higher sucrase activity than d7. In the ileum, both day (p<0.0001) and diet (p<0.0001) had an effect on lactase activity with formula fed (FF)piglets having highest lactase activity followed by formula+prebiotic (FP) piglets, and thensow reared (SR). Lactase activity was also higher at d14 than d7. Ileal sucrase activity was also impacted by both day (p<0.0001) and diet (p<0.0001) with FF piglets having higher sucrase activity than both FP and SR piglets and activity was higher at d14 compared to d7.Aminopeptidase N (APN) was measured in both jejunum and ileum, and Dipeptidylpeptidase IV (DPPIV) was measured in jejunum and ileum, as well as serum. In the jejunum, both day (p=0.0023) and diet (p=0.0003) were significant, with d7 piglets having higher APN activity than d14. Both FF and FP piglets had higher APN activity compared to SR. In ileum, APN also had both day (p=0.047) and diet (p<0.0001) effects with FF piglets having significantly higher activity than FP and SR. Jejunal DPPIV showed effects of diet (p<0.0001) with FF piglets having higher activity than FP and SR. In ileum, both diet (p<0.0001) and day (p=0.0182) had effects with FF piglets having higher activity than FP and SR. There were no significant effects in blood. Ileal sections were stained for CD3+ T-lymphocyte populations. There was an effect of diet (p=0.0085) and day (p<0.0001) on population of CD3+ cells in the ileum. All groups had significantly more CD3+ cells at d14 compared to d7 and SR had significantly more cells compared to FF with FP being intermediate. Ileal cytokine expression was measured to evaluate Th1, Th2 and Treg cytokine expression. There were no differences between diet or day for this parameter. In summary, route of deliver did not affect intestinal structure or function, cytokine secretion or CD3+ cell populations in the intestine. The prebiotics, however, did show some effects with peptidase activity in both the jejunum and ileum as well as with CD3+ cell staining in the ileum.Formula+prebiotic piglets had more similar overall intestinal peptidase activity to SR piglets compared to FF. The addition of the prebiotic may have helped to modulate this result. However, FP piglets had significantly lower CD3+ cell expression in the ileum compared to SR.While the prebiotic creates similar effects to breast milk in some arenas, it still is not a complete replacement. The final aim of this dissertation focused on the effects of combined feeding and prebiotics on immune response and the colonic transcriptome. We developed a novel piglet model to investigate these effects. Newborn piglets were randomized into 5 groups: sow-reared (SR), formula fed (FF), formula+prebiotic (FP), combined fed (CF), and combined fed +prebiotic (CP) (n=6 per group). SR remained with the sow 24h/day and FF/FP were fed a sow milk replacer with or without prebiotics (GOS and Inulin; 2g/L each). CF/CP piglets were sow-reared for 5d and were then rotated between the sow and respective formula feeding group every 12h. On d21, piglets were intraperitoneally injected with 10ug/kg body weight of LPS. Four hours after infection, blood, mesenteric lymph node (MLN), spleen (SPL), and AC were collected and analysis performed. Immunity was tested by investigating both immune cell profiles and macrophage activation. T-cells, B-cells, dendritic cells, and macrophages were isolated, stained, and measured using a flow cytometer. In blood, SR showed significantly (p<0.0001) lower percentage of CD4+CD8- T-helper cells, while in the MLN they were intermediate between CP (p=0.0264) and all other diet groups. However, SR had a significantly (p<0.0001) higher percentage of B-cells compared to all other diet groups in blood. There were no significant differences between diet groups with dendritic cell or macrophage populations.Both acute phase response pro-inflammatory cytokines, IL-6 and TNF-α, were measured using ELISA. FF piglets had a much higher IL-6 response than both CF and SR diet groups. There was no significant difference in immune reaction to TNF-α. In addition, macrophage activation pathways, classical versus alternative, were also tested in both MLN and SPL tissue. Both nitric oxide synthase (classical) and arginase (alternative) enzyme activities were evaluated. There were no significant differences between diet groups when measuring nitric oxide synthase activity, but CP group did have significantly (p<0.0001) higher expression of arginase in MLN compared to all other diet groups. Combined feeding with prebiotic did have some effect in response to immune stimulus and seemed to encourage an anti-inflammatory immune profile as evidenced by the significantly higher arginase production which is caused by secretion of anti-inflammatory cytokines: IL-4, IL-10, IL-13 (Dewals et al, 2010). Gene expression of FF, CF, and SR piglets was assessed using a porcine gene microarray (Agilent). Among the 25,273 probes on the microarray, 449 were differentially expressed (FDR p<0.2). Bioinformatic analyses using MetaCore, revealed that the most significant GeneGo pathway maps were related to apoptosis, carbohydrate metabolism, lipid metabolism and neuronal pathway synthesis. Based on the pathways generated and the corresponding heatmap, CF and SR may have more similar gene expression patterns with genes related to lipid and carbohydrate metabolism, and apoptosis. FF and CF may have more similar gene expression patterns with genes related to glucose transport.More research is needed in this area to see how these differences affect future health outcomes.
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Early nutrition affects intestinal development and immune response in the neonatal piglet
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