The diesters of 1,2-benzenedicarboxylic acid (phthalic acid), or phthalates, are multifunctional chemicals used in personal care products, medications, and plastics. Phthalate metabolites are also found in human milk and infant formula. However, their impact on intestinal development and microbiota composition in early life is understudied. Herein, the effect of di-(2-ethylhexyl) phthalate (DEHP) on growth, intestinal morphology, enzyme activity, and microbiota composition was assessed. Piglets (two-day-old, n = 24) were randomized to receive either 20 mg (DEHP20) or 200 mg (DEHP200)/kg body weight (BW)/day in corn oil. A control group (CON, 0 mg phthalate) received the same volume of corn oil as DEHP200. After 21 days, tissue and urine samples were collected. DEHP did not affect weight gain. Urinary DEHP metabolite concentrations increased in a dose-dependent fashion. Jejunal villus length was significantly shorter in the DEHP200 than CON and DEHP20, while villus area was smaller in DEHP200 than DEHP20 but not CON. Crypt depth and area were higher in DEHP200 than DEHP20, but neither differed from CON. Additionally, jejunal sucrase activity was higher in the DEHP200 than CON. Bacterial alpha diversity differed significantly between DEHP groups and CON in the ascending colon, while beta diversity revealed significant differences between DEHP200 and CON and DEHP20. The abundance of Christensenellaceae R-7 group, Romboutsia , Lachnospiraceae UCG-004, Odoribacter , and Sphaerochaeta , among others, differed in DEHP-exposed vs. CON animals. Thus, daily exposure to phthalates during infancy changes the villus structure and disaccharidase activity in the small intestine and these changes may be modulated by the colonic bacterial community.

Human milk is a source of oligosaccharides that promote the growth of beneficial bacteria. Bifidobacterium longum subsp. infantis, a dominant species in breastfed infants, has the capacity to utilize milk oligosaccharides. Herein, the effects of 2'fucosyllactose (2'FL), B. infantis Bi-26 (Bi-26), and a combination thereof on piglet gut microbiota composition and volatile fatty acid (VFA) concentrations were assessed. Fifty-two intact male pigs were provided ad libitum access to a nutritionally-adequate milk replacer without (CON) or with 1.0 g/L 2’FL (FL) from postnatal day 2 to 34/35. Pigs were further stratified to receive either 12% glycerol or Bi-26 in glycerol orally, 109 colony-forming unit/day (BI and FLBI). Ascending colon (AC) and rectal contents were collected. Gut microbiota profiles were assessed by 16S rRNA gene sequencing and real-time PCR and VFA were determined by gas chromatography. Neither 2'FL nor Bi-26 affected the overall microbiota composition (P > 0. 05); however, alpha diversity and the relative abundances of bacterial genera were influenced by the treatments. Shannon indices were lower in AC of piglets fed Bi-26 (P = 0.048). Proportions of Clostridia UCG-014, Lachnoclostridium, Christensenellaceae R-7 group and Anaerovoracaceae family XIII AD3011 group were lower, while Faecalibacterium was higher in AC of piglets receiving 2'FL (P < 0.05). Bi-26 decreased (P < 0.05) colonic abundances of Parabacteroides, Fusobacterium, Butyricimonas and uncultured Prevotellaceae. In rectal contents,7 bacterial genera were impacted by 2'FL and 3 by Bi-26 (P < 0.05). Interactive effects were observed for several bacterial genera and acetate concentrations (P < 0.05). In AC, Lachnospiraceae CAG-56 was higher in CON than all other groups and Allisonella was lower in BI piglets vs. CON. Rectal contents Bacteroides was higher in BI piglets than CON. Compared to CON, acetate concentrations were higher in AC of FL piglets (P < 0.05). 2'FL and Bi-26 supplemented to milk replacer exerted individual and synbiotic influences on gut bacterial composition, and 2'FL alone increased specific VFA concentration, demonstrating its prebiotic potential. DuPont Nutrition and Biosciences.

IntroductionThe oligosaccharide 2′-fucosyllactose (2′-FL) is a predominant component of human milk, serving as a prebiotic for gut microbiota and influencing immune development in infants. Bifidobacterium longum subspecies infantis (B. infantis) is a commensal bacterium found in breastfed infants. Both 2′-FL and a specific strain of B. infantis, Bi-26™, are commercially available. This study investigates the potential synbiotic relationship between 2′-FL and Bi-26™ on immune development.MethodsTwo-day-old piglets (n = 53) were randomized in a 2 × 2 design, receiving either a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2′-FL (FL). Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26™ (109 CFU) (BI and FLBI) orally once daily. On postnatal day (PND) 34/35, animals were euthanized, and blood was collected for serum cytokine analysis. Additionally, peripheral blood mononuclear cells (PBMCs) were isolated for ex vivo stimulation and flow cytometry analysis. Serum and ex vivo cytokines were analyzed using a multivariate model. All other outcomes were analyzed using a two-way ANOVA, considering prebiotic and probiotic fixed effects. The significance level was set at a p value <0.05, with trends reported for 0.05 < p < 0.1.ResultsImmune cell populations in PBMCs were unaffected by the experimental treatment. However, serum interleukin (IL)-1RA, IL-1β, IL-12, and IL-18 were all higher (p < 0.05) in the FL group than in the CON group. In isolated PBMCs, lipopolysaccharide (LPS) stimulation resulted in higher IL-1RA and a trend for higher IFN-γ secretion in the FL group vs. the CON group.Conclusion2′-FL stimulates a balanced cytokine profile in healthy piglets without changing immune cell populations. When immune cells are stimulated ex vivo with LPS, 2′-FL primes T-cells for a proinflammatory response, which is moderated by co-administration of Bi-26™.

IntroductionHuman milk contains structurally diverse oligosaccharides (HMO), which are multifunctional modulators of neonatal immune development. Our objective was to investigate formula supplemented with fucosylated (2’FL) + neutral (lacto-N-neotetraose, LNnt) oligosaccharides and/or sialylated bovine milk oligosaccharides (BMOS) on immunological outcomes.MethodsPigs (n=46) were randomized at 48h of age to four diets: sow milk replacer formula (CON), BMOS (CON + 6.5 g/L BMOS), HMO (CON + 1.0 g/L 2’FL + 0.5 g/L LNnT), or BMOS+HMO (CON + 6.5 g/L BMOS + 1.0 g/L 2’FL + 0.5 g/L LNnT). Blood and tissues were collected on postnatal day 33 for measurement of cytokines and IgG, phenotypic identification of immune cells, and ex vivo lipopolysaccharide (LPS)-stimulation of immune cells.ResultsSerum IgG was significantly lower in the HMO group than BMOS+HMO but did not differ from CON or BMOS. The percentage of PBMC T-helper cells was lower in BMOS+HMO than the other groups. Splenocytes from the BMOS group secreted more IL-1β when stimulated ex vivo with LPS compared to CON or HMO groups. For PBMCs, a statistical interaction of BMOS*HMO was observed for IL-10 secretion (p=0.037), with BMOS+HMO and HMO groups differing at p=0.1.DiscussionThe addition of a mix of fucosylated and sialylated oligosaccharides to infant formula provides specific activities in the immune system that differ from formulations supplemented with one oligosaccharide structure.

Butyrate, a volatile fatty acid (VFA) produced by gut bacteria, regulates gut barrier function and immune response. However, the incorporation of butyrate into foods is challenging. Tributyrin (TB), a triglyceride with 3 butyrate molecules, represents an alternative to butyrate. Herein, the effects of encapsulated TB with gamma-cyclodextrin (ɣ-CD/TB) on gut microbiota composition and VFA concentrations were assessed in dextran sulfate sodium (DSS)-induced colitis piglets. Two-day-old piglets (n = 59) were randomized to three diets: control (CON) [Advance Baby Pig Liqui-Wean], CD [CON + 9.0 mM ɣ-CD], or CDTB [CON + 9.0 mM ɣ-CD/TB]. Half of the piglets in each group received DSS (1.25 g/kg body weight) from postnatal day (PND) 14 to 18. Ileal (IL), ascending colon (AC) and rectal (RC) contents were collected at PND 27. Microbiota were assessed by 16S rRNA gene sequencing and VFA were measured by gas chromatography. Permutational multivariate analysis of variance showed diet affected the beta diversity in IL (P = 0.045) with CON differing from CD or CDTB. Relative abundances of ileal Weissellaand Actinobacillus were higher and Turicibacter were lower in CON than CD or CDTB piglets (P < 0.05). While diet did not affect overall bacterial composition in AC and RC, it impacted proportions of some bacterial genera. Bacteroides was lower, Holdemanella and Ruminococcus were higher in AC of CON than CDTB. Rectal Bacteroides were lower and UCG-010 were higher in CON compared to CDTB (P < 0.05). Colitis influenced beta diversity in AC and RC (P = 0.002). Compared to piglets without colitis, proportions of 10 bacterial genera were lower and 6 were higher in AC, while 9 were lower and 11 were higher in RC of colitis piglets (P < 0.05). Diet impacted alpha diversity in IL and AC with observed features in IL, Shannon and evenness indices and phylogenetic diversity (Faith PD) in AC being higher in CON than CDTB (P < 0.05). Colitis increased Faith PD in RC (P = 0.004). For VFA, colitis decreased isobutyrate and isovalerate in IL and increased acetate, propionate, butyrate and valerate in RC, regardless of diet. Encapsulated ɣ-CD/TB modulates the bacterial community along the piglet intestinal tract, while colitis influences both the microbiota composition and VFA concentrations. NIFA.

Human milk is rich in oligosaccharides that influence intestinal development and serve as prebiotics for the infant gut microbiota. Probiotics and 2’-fucosyllactose (2’-FL) added individually to infant formula have been shown to influence infant development, but less is known about the effects of their synbiotic administration. Herein, the impact of formula supplementation with 2’-fucosyllactose (2’-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26), or 2’-FL + Bi-26 on weight gain, organ weights, and intestinal development in piglets was investigated. Two-day-old piglets (n = 53) were randomized in a 2 × 2 design to be fed a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2’-FL. Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26 (109 CFU) orally once daily. Body weights and food intake were monitored from postnatal day (PND) 2 to 33/34. On PND 34/35, animals were euthanized and intestine, liver and brain weights were assessed. Intestinal samples were collected for morphological analyses and measurement of disaccharidase activity. Dry matter of cecum and colon contents and Bifidobacterium longum subsp. infantis abundance by RT-PCR were also measured. All diets were well tolerated, and formula intake did not differ among the treatment groups. Daily body weights were affected by 2’-FL, Bi-26, and day, but no interaction was observed. There was a trend (p = 0.075) for greater total body weight gain in CON versus all other groups. Jejunal and ascending colon histomorphology were unaffected by treatment; however, there were main effects of 2’-FL to increase (p = 0.040) and Bi-26 to decrease (p = 0.001) ileal crypt depth. The addition of 2’-FL and/or Bi-26 to milk replacer supported piglet growth with no detrimental effects on body and organ weights, or intestinal structure and function.

Inflammatory bowel diseases are common in adults, and their incidence is increasing in children. Butyrate, a short chain fatty acid, can improve gut health, but its bitterness limits incorporation into foods. Herein, the efficacy of tributyrin (TB), a butyrate analog, encapsulated with ɣ-cyclodextrin (ɣ-CD) on growth and intestinal inflammation in a piglet model of dextran-sodium sulfate (DSS)-induced colitis was investigated. Two-day-old piglets were randomized to three diets: control (formula alone, CON, N = 41), CD (CON + 9.0 mM ɣ-CD, N = 41), or CDTB (CON + 9.0 mM ɣ-CD/TB, N = 43). Half of the piglets in each group received DSS (1.25 g/kg body weight) from postnatal day (PND) 14 to 18. Blood and intestinal samples were collected on PND 19 and 27. IL-6 and TNF-α concentration in serum and colon mucosa were assessed by ELISA and IL-6, TNF-α, IL-10 and IFN-γ gene expression in colonic tissue were measured by qPCR. A quantitative histological grading system was used to assess 4 independent parameters: epithelial lining, cell infiltration, crypt damage and extent of the intestinal section affected. Average daily weight gain (WG) between PND2-10 and 10–19 were affected by diet (P < 0.05), while colitis led to lower WG from PND19-27. A diet and colitis interaction indicated that after PND22 CDTB-COL piglets weighed less than CON and CD (P = 0.051). Diet had no impact on the descending colon (DC) morphology in the colitis animals, but the severity of the DC inflammation increased from PND19 and 27 (P < 0.0001). TNF-α and IL-6 concentrations were elevated in the ascending colon (AC) at PND19, but by PND27 concentrations were similar to non-colitis levels. A similar pattern was found in the DC, except that IL-6 remained elevated on PND27. IL-6, TNF-α and IFN-γ gene expression were significantly higher in the DC of colitis vs. non-colitis animals on PND27. Diet and colitis had no effect on serum TNF-α and IL-6. The piglet as a model of DSS colitis is a feasible alternative to rodents. Dietary ɣ-CD/TB reduced growth rate and did not ameliorate the inflammatory process of DSS-induced colitis in neonatal pigs. Herein, oral administration of encapsulated TB did not reduce DSS-induced tissue damage or inflammation, however, additional studies are needed to confirm these observations. NIFA, ISDA 2017–67,017-26,519.

Di(2-ethylhexyl) phthalate (DEHP) is a chemical commonly used as a plasticizer to render polyvinyl chloride products more durable and flexible. Although exposure to DEHP has raised many health concerns due to the identification of DEHP as an endocrine disruptor, it is still used in consumer products, including polyvinyl chloride plastics, medical tubing, car interiors, and children's toys. To investigate the impact of early life exposure to DEHP on the ovary and testes, newborn piglets were orally dosed with DEHP (20 or 200 mg/kg/day) or vehicle control (tocopherol-stripped corn oil) for 21 days. Following treatment, ovaries, testes, and sera were harvested for histological assessment and measurement of steroid hormone levels. In male piglets, progesterone and pregnenolone levels were significantly lower in both treatment groups compared to control, whereas in female piglets, progesterone was significantly higher in the 20 mg group compared to control, indicating sex-specific effects in a non-monotonic manner. Follicle numbers and gene expression of steroidogenic enzymes and apoptotic factors were not altered in treated ovaries compared to controls. In DEHP-treated testes, germ cell migration was impaired and germ cell death was significantly increased compared to controls. Overall, the results of this study suggest that neonatal exposure to DEHP in pigs leads to sex-specific disruption of the reproductive system.

Background: The milk fat globule membrane (MFMG) is a complex milk component that has been shown to inhibit rotavirus (RV) binding to cell membranes in vitro. Herein, a whey protein lipid concentrate high in MFGM components (WPLC) and whey protein concentrate (WPC; control) were screened for anti-infective activity against porcine OSU and human Wa strains of RV in both the African Green Monkey kidney (MA104) and the human colorectal adenocarcinoma (Caco-2) cell lines. Materials and Methods: Confluent cells were exposed to OSU or Wa RV in the presence of WPLC or WPC (control) at 0, 0.1, 0.5, 1.0, 2.5, or 5 mg/ml. Infectivity was detected by immunohistochemistry and expressed as % inhibition relative to 0 mg/ml. WPLC efficacy over WPC was expressed as fold-change. One-way ANOVA analyzed data for the independent and interactive effects of concentration, test material, and RV strain. Results: Both WPLC and WPC exhibited concentration-dependent inhibition of human Wa and porcine OSU RV infectivity in MA104 and Caco-2 cells (p < 0.0001). WPLC was 1.5–4.8-fold more effective in reducing infectivity than WPC. WPLC efficacy was independent of RV strains, but varied between cell lines. WPLC and WPC at concentrations ≥0.5 mg/mL were most effective in reducing human Wa RV infectivity in MA104 cells (p < 0.0001). Conclusions: WPLC decreased infectivity of two strains for RV which differ in their dependency on sialic acid for binding to cells. Inhibition was observed in the most commonly used cell type for RV infectivity assays (MA104) and an intestinal cell line (Caco-2). An effect on virus infectivity might be a potential mechanisms of action contributing to beneficial effects of supplementation of infant formula with MGFM reducing the risk of infections and consequently diarrhea incidence in infants.

Human milk contains both prebiotic oligosaccharides and live bacteria, which are thought to bring health benefits to breastfed infants. Herein, we investigated the impact of formula supplementation with 2'-fucosyllactose (2'FL) and Bifidobacterium longum subsp. infantis (Bi-26) alone or in combination on growth, organ weights, and intestinal development of neonatal piglets. Two-day-old intact male piglets (N = 53) were randomized to be fed a nutritionally-adequate milk replacer ad libitum without (CON) or with 1.0 g/L 2'FL (FL). Pigs were further stratified to receive either 12% glycerol solution alone or Bi-26 (109 CFU) in glycerol orally once daily (BI and FLBI). Body weights and food intake were monitored from postnatal day (PND) 2 to 33/34. On PND 34/35, animals were euthanized, intestine, liver and brain weights were assessed, and intestinal samples were collected for morphological analyses and disaccharidase activity. Dry matter of intestinal contents was also measured. Growth and food intake were analyzed as a 3-way, repeated-measures ANOVA with fixed effects of prebiotic, probiotic, and day, whereas all other variables were analyzed by a 2-way ANOVA with fixed effects of prebiotic and probiotic. Level of significance was set at P ≤ 0.05 and trends are reported at 0.05 > P < 0.1. All diets were well tolerated and food intake did not differ among the treatment groups. Daily body weights were affected by 2’FL, Bi26, and day, but no interaction was observed. However, there was a trend (p = 0.075) for greater body weight gain in CON vs. all other groups. No differences were observed for intestine, liver, or brain weight per kg body weight, jejunal or ileal lactase or sucrase activities, or fecal dry matter among the groups. Histomorphological outcomes in jejunum, ileum, and ascending colon were similar in all groups, except for a trend (p = 0.069) for larger ileal crypt volume in FL vs. CON piglets. The addition of 2'FL and/or Bi-26 to milk replacer supported piglet growth with no detrimental effects on body and organ weights, or intestinal structure and function. DuPont Nutrition & Biosciences.

Combination feeding (human milk and formula) is common and influences immune development compared to exclusive breastfeeding. Infant formulas contain prebiotics, which influence immune development. Herein, immune development of combination-fed (CF), sow-reared (SR) and formula-fed (FF) piglets, and the effect of prebiotics was tested. Piglets (n = 47) were randomized to: SR, FF, CF, FF+prebiotic (FP), and CF+prebiotic (CP). FP and CP received formula with galactooligosaccharides and inulin (4 g/L in a 4:1 ratio). CF and CP piglets were sow-reared for until d5 and then rotated between a sow and formula every 12 h. On day 21, piglets received an intraperitoneal injection of lipopolysaccharide 2 h prior to necropsy. Immune cells from blood, mesenteric lymph nodes (MLN), and spleen were phenotyped. Classical (nitric oxide synthase) and alternative (arginase activity) activation pathways were measured in isolated macrophages. Serum IL-6 and TNF-α were measured by ELISA. SR piglets had lower (p < 0.0001) CD4+ T-helper cells and higher (p < 0.0001) B-cells in PBMC than all other groups. CP piglets had higher (p < 0.0001) arginase activity compared to all other groups. FF piglets had higher (p < 0.05) IL-6 compared to both CF and SR, but were similar to FP and CP. Thus, CF, with or without prebiotics, differentially affected immunity compared to exclusively fed groups.

Probiotics and 2’fucosyllactose (2’FL) have been shown to affect immune development in infants, but less is known about their synbiotic administration. Herein, the effects of 2’FL and Bifidobacterium longum subsp. infantis (Bi-26) on immune development was investigated in the young pig. Male piglets (N = 53 were provided ad libitum access to milk replacer without (CON) or with 1g/L 2’FL (FL) from postnatal day 2 to 34/35. Pigs were then stratified to receive Bi-26 prepared in glycerol stock (109 CFU) or glycerol stock alone (BI and FLBI). Blood, mesenteric lymph nodes (MLN), and ascending colon (AC) and rectal (RC) contents were collected. MLN and peripheral blood mononuclear cells (PBMC) immune cells were quantified by flow cytometry. Immunoglobulins (Igs) were measured by ELISA. PBMC and MLN cells were stimulated ex vivo with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) for 72 h. Cytokines in serum and ex vivo cell supernatants were measured by multiplex assay. Cytokine data were analyzed by multivariate LCA-model. All other data were analyzed by a 2-way ANOVA with fixed effects of prebiotic and probiotic. sIgA tended (P = 0.07) to be higher in RC than AC, with no treatment effect. Serum IgG and IgM and MLN and PBMC immune cells were unaffected by treatment. Serum, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-10, IL-12 and IL-18 were all higher in FL than CON (p < 0.05). In unstimulated PBMC, IL-18 was lower in FL, BI and FLBI than CON (p < 0.05). In LPS-stimulated PBMCs, IFNγ was higher in FL and IL1-RA was higher in FLBI than CON (p < 0.05). LPS had no effect on MLN cytokines; but, PHA increased IL-17 in FL and FLBI versus CON (p < 0.05). Compared to CON, serum cytokines involved in Th1 T-cell differentiation, IL-2, IL-12 and IL-18 were higher in FL piglets. This was countered by the increased production of IL-4 and IL-10. IL-1RA also increased, potentially balancing higher IL-1α and IL-1β in FL piglets. These differences were not observed in FLBI. LPS stimulated IFNγ secretion by PBMC from FL, whereas cells from FLBI secreted more IL-1RA. These findings suggest that dietary 2’FL primes T-cells for proinflammatory cytokine secretion, which is modulated by co-administration of Bi-26. DuPont Nutrition and Biosciences.

Milk oligosaccharides (OS) shape microbiome structure and function, but their relative abundances differ between species. Herein, the impact of the human milk oligosaccharides (HMO) (2′-fucosyllactose [2′FL] and lacto-N-neotetraose [LNnT]) and OS isolated from bovine milk (BMOS) on microbiota composition and volatile fatty acid (VFA) concentrations in ascending colon (AC) contents and feces was assessed. Intact male piglets received diets either containing 6.5 g/L BMOS (n = 12), 1.0 g/L 2′FL + 0.5 g/L LNnT (HMO; n = 12), both (HMO + BMOS; n = 10), or neither (CON; n = 10) from postnatal day (PND) 2 to 34. Microbiota were assessed by 16S rRNA gene sequencing and real-time PCR, and VFA were measured by gas chromatography. The microbiota was affected by OS in an intestine region-specific manner. BMOS reduced (p < 0.05) microbial richness in the AC, microbiota composition in the AC and feces, and acetate concentrations in AC, regardless of HMO presence. HMO alone did not affect overall microbial composition, but increased (p < 0.05) the relative proportion of specific taxa, including Blautia, compared to other groups. Bacteroides abundance was increased (p < 0.05) in the AC by BMOS and synergistically by BMOS + HMO in the feces. Distinct effects of HMO and BMOS suggest complementary and sometimes synergistic benefits of supplementing a complex mixture of OS to formula.

Butyrate is a short-chain fatty acid (SCFA) known for support in gastrointestinal (GI) health. Tributyrin (TB) could be used as an alternate source of butyrate. The objectives of this study were to encapsulate TB using gamma-cyclodextrin (CD) by spray-drying and to investigate the physicochemical and the fermentation properties of TB/CD complex. The TB/CD complex precipitated in water with an average stoichiometry of 1:1.3 of TB:CD. At a 1:2 molar ratio of TB:CD, TB was fully retained in the spray-dried TB/CD complex. The spray-dried TB/CD complex showed crystalline structure, supported by both X-ray diffraction spectra and scanning electron microscopy images. The TB/CD complex at 1:2 molar ratio was fermented and several SCFAs, including butyrate, were produced in an in vitro test using piglets' ileal and colonic contents. A dose-dependent increase in the butyrate concentration in both ileum and ascending colon was observed. Approximately, 426 and 1189 µmole butyrate was produced per gram of TB/CD powder at 9 mM treatment in ileum and ascending colon, respectively. Thus, the production of the TB/CD complex using spray drying is feasible and the complex has the potential for food applications to improve intestinal health. PRACTICAL APPLICATION: The findings in this study can be applied to produce encapsulated tributyrin with gamma-cyclodextrin efficiently using spray-drying. The TB/CD complex was highly fermentable and caused an increase in the butyrate concentration in both ileum and ascending colon, which can be incorporated in foods to enhance butyrate delivery to the GI tract to assist gut health.

Most studies on autism spectrum disorder (ASD) risk factors have been conducted in developed countries where ethnicity and environment are different than in developing countries. We compared nutritional status, immune response and microbiota composition in mestizo children with ASD with matched controls in Ecuador. Twenty-five cases and 35 controls were matched by age, sex and school location. The prevalence of under- and overweight was higher in children with ASD. Nutritional differences were accompanied by abnormal food habits and more frequent gastrointestinal symptoms in children with ASD. Also, greater serum concentrations of TGF-β1 were observed in children with ASD. Finally, there was greater alpha diversity and abundance of Bacteroides (2 OTUs), Akkermansia, Coprococcus and different species of Ruminococcus in ASD children.

Oligosaccharides are complex, non-digestible glycans found in large abundance in human milk. The abundance and the profile of bovine milk oligosaccharides and bovine milk based in infant formula differ from those in human milk. Recently, some human milk oligosaccharides (HMOs) have been supplemented to infant formula, however, not all forms have been available in large scale. The objective of the study was to investigate the dose-dependent effects of an enzymatically-synthesized 6′-sialyllactose (6′-SL) sodium salt supplemented to swine milk replacer on growth, hematological parameters, and organ microscopic assessment in our pre-clinical neonatal pig model. Two-day-old male and female pigs (n = 47) were provided one of four experimental diets for 21 days. Diets were formulated to contain 0 (CON), 300 (LOW), 600 (MOD), or 1200 (HIGH) mg/L of 6′-SL sodium salt. On days 8 and 22, samples were collected for hematological and histological analyses. Supplemental 6′-SL sodium salt at all doses supported growth and development comparable to those observed in control animals. In addition, serum chemistries, hematology, and organ microscopic structure were unaffected by 6′-SL (p > 0.05). Thus, addition of enzymatically-synthesized 6′-SL to a milk replacer formula supported growth and clinical outcomes similar to the control formula in the neonatal piglet.

ObjectivesRapid weight gain over the first 12 months of life is a risk factor for childhood obesity. While macronutrient concentrations in human milk (HM) or infant formula (IF) have been associated with rapid weight gain or weight-for-length Z-scores later in infancy, few studies evaluate milk intake. Therefore, the objective was to assess how nutrient intake at 6 weeks of age, across multiple feeding modes, influences growth over the first year of life.MethodsData were collected from 222 healthy mother-infant pairs enrolled in the STRONG Kids 2 Cohort. Twenty-four hour test weighing was conducted to measure milk intake volume and a human milk sample was collected at 6 weeks postpartum. Milk samples were analyzed for total protein (Bradford assay), fat (total lipid extraction), and carbohydrate (Orcinol assay). Mode of feeding at 6 weeks, servings per day of HM and/or IF, and formula type were reported by mothers. For exclusively breastfeeding infants, HM macronutrient concentrations and total intake were used to calculate nutrient intake. For those receiving IF, the numbers of feedings from HM and/or from IF and formula nutrition information were also used to calculate nutrient intake. Infant length and weight collected at study visits were used to calculate weight-for-length Z-scores (WFL-Z). Mixed linear models were used to measure associations between macronutrient intake, total calories, total milk intake and WFL-Z at 3 months and 12 months of age. Models were controlled for 6-week feeding mode, breastfeeding duration, and timing of solid food introduction.ResultsAt 3 months, WFL-Z was associated with total milk (P = 0.03) and caloric intake (P = 0.03) as well as intake of fat (P = 0.02), carbohydrate (P = 0.05), and protein (P = 0.03). WFL-Z at 12 months was associated with protein (P = 0.02) and fat (P = 0.04) intakes at 6 weeks but no longer associated with other measures of nutrient intake.ConclusionsOur results support previous findings suggesting that high protein intake in infancy contributes to obesity risk. While milk intake was only quantified once, data show that nutrient intake at 6 weeks-of-age influences future growth. More research is needed to understand other lifestyle and nutritional factors between 3 and 12 months that contribute to growth trajectories and weight during infancy.Funding SourcesNational Dairy Council, NIH RO1DK107561, The Gerber Foundation, The Doris Kelley Christopher Foundation.

Background: Many infants consume both human milk and infant formula (combination-fed); however, little is known about how combination-feeding affects the gut microbiota or prebiotic fermentation compared to formula feeding. Objectives: We investigated the impact of feeding mode and prebiotics on bacterial colonization and volatile fatty acid (VFA) concentrations. Methods: Newborn piglets (Large White and Landrace) were randomly assigned to 5 groups (n = 6/group): formula-fed (FF), formula-fed with prebiotics (FP), sow-reared (SR), combination-fed (CF), and combination-fed with prebiotics (CP). SR piglets remained with the sows 24 h/d. FF and FP were fed formula or formula with galactooligosaccharide and inulin (4 g/L in a 4:1 ratio). CF and CP were sow-reared for 5 d and then rotated between the sow and formula-feeding every 12 h. Ascending colon contentswere collected at day 21. The microbiota was analyzed by pyrosequencing and denaturing gradient gel electrophoresis (DGGE). VFAs were determined by gas chromatography. Results: Distance-based redundancy analysis of DGGE and pyrosequencing data separated microbiota of FF from CF and SR. CF differed from SR by DGGE, but only a trend (P = 0.09) by pyrosequencing. Bacterial composition of CF was more similar to SR than FF. No bacterial genera in CF significantly differed from SR; however, 9 genera differed between CF and FF, including Lactobacillus, Clostridium XIVa, and Fusobacterium. VFA concentrations were similar between CF and SR, while isovalerate and isobutyrate were 2-fold greater (P < 0.05) in CF than FF. Neither microbiota nor VFA profile was affected by prebiotic supplementation. Conclusions: Microbial colonization patterns and VFA profiles of CF piglets were more similar to SR piglets than FF piglets. Prebiotics did not affect piglet bacterial composition and/or VFA concentrations relative to the main feeding modes (FF and CF). Thus, partial exposure to breast milk can be beneficial for microbiota development of FF neonates.

Sialyllactose (SL) is an abundant oligosaccharide in human milk with health benefits that include intestinal maturation, gut microbiota modulation, and cognitive development. Recent technological advances support large scale production of different forms of sialyllactose, which will enable their use as a food ingredient. The objective of the study was to investigate the dose-dependent effects of novel enzymatically-synthesized 3'-sialyllactose (3'SL) sodium salt supplemented to swine milk replacer on growth, hematological parameters and tissue histology in a pre-clinical neonatal pig model. Forty-five two-day-old male and female pigs were provided one of four experimental diets for 21 days. Diets were formulated to contain 0 (CON), 140 (LOW), 200 (MOD) or 500 (HIGH) mg/L of 3'SL sodium salt. Samples were collected on days 8 and 22 of the study for hematological and histological analyses. The addition of 3'SL sodium salt to formula at all doses was well-tolerated by neonatal piglets and supported growth and development comparable to those observed in the CON group. In addition, serum chemistries as well as hematology and organ microscopic structure were unaffected by 3'SL (p > 0.05). These data provide supportive evidence for the safety of supplementation of this enzymatically-synthesized 3'SL sodium salt to human infant formula.

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.