Feeding Mode, but Not Prebiotics, Affects Colonic Microbiota Composition and Volatile Fatty Acid Concentrations in Sow-Reared, Formula-Fed, and Combination-Fed Piglets.

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 contents were 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.

Safety evaluation of 3'-siallylactose sodium salt supplementation on growth and clinical parameters in neonatal piglets.

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.

Dietary Human Milk Oligosaccharides but Not Prebiotic Oligosaccharides Increase Circulating Natural Killer Cell and Mesenteric Lymph Node Memory T Cell Populations in Noninfected and Rotavirus-Infected Neonatal Piglets.

Background: Human milk oligosaccharides (HMOs) have antimicrobial and immunomodulatory actions. It has previously been reported that these oligosaccharides contribute to the reduced duration of rotavirus-induced diarrhea in pigs.Objective: We measured the effects of HMOs and prebiotic oligosaccharides on immune cell populations from noninfected and rotavirus-infected pigs. We hypothesized that dietary HMOs would modulate systemic and gastrointestinal immunity.Methods: Colostrum-deprived newborn pigs were fed formula, formula with 4 g HMOs/L (2'-fucosyllactose, lacto-N-neotetraose, 6'-sialyllactose, 3'-sialyllactose, and free sialic acid), or formula with 3.6 g short-chain galactooligosaccharides/L and 0.4 g long-chain fructooligosaccharides/L. On day 10, half of the pigs were infected with the porcine rotavirus strain OSU. Peripheral blood mononuclear cell (PBMC), mesenteric lymph node (MLN), and ileal Peyer's patch immune cell populations were assessed with the use of flow cytometry 5 d postinfection. Interferon-γ (IFN-γ)-producing cells were assessed with the use of Enzyme-Linked ImmunoSpot assay.Results: Infection changed immune cell populations with more systemic natural killer (NK) cells, memory effector T cells, and major histocompatibility complex II+ cells in infected than noninfected pigs (P < 0.06). Regardless of infection status, HMO-fed pigs had nearly twice as many PBMC NK cells, 36% more MLN effector memory T cells, and 5 times more PBMC basophils than formula-fed pigs (P < 0.04). These populations were intermediate in pigs fed prebiotics. PBMCs from HMO-fed noninfected pigs had twice as many IFN-γ-producing cells as did those from formula-fed noninfected pigs (P = 0.017). The PBMCs and MLNs of formula-fed noninfected pigs had 3 times more plasmacytoid dendritic cells (pDCs) than those of HMO-fed noninfected and formula-fed infected pigs (P < 0.04). In the MLNs, the formula-fed noninfected pigs had more macrophages, pDCs, and mature DCs (P < 0.04) but fewer immature DCs than HMO-fed noninfected pigs (P = 0.022).Conclusions: Dietary HMOs were more effective than prebiotics in altering systemic and gastrointestinal immune cells in pigs. These altered immune cell populations may mediate the effects of dietary HMOs on rotavirus infection susceptibility.

Prebiotics and Bioactive Milk Fractions Affect Gut Development, Microbiota, and Neurotransmitter Expression in Piglets.

OBJECTIVE: This study tested the hypothesis that the addition of prebiotics and 2 functional milk ingredients to infant formula would maintain normal growth and gut development, and modify microbiota composition and neurotransmitter gene expression in neonatal piglets. METHODS: Two-day-old male piglets (n = 24) were fed formula (CONT) or formula with polydextrose (1.2 g/100 g diet), galactooligosaccharides (3.5 g/100 g diet), bovine lactoferrin (0.3 g/100 g diet), and milk fat globule membrane-10 (2.5 g/100 g diet) (TEST) for 30 days. On study day 31, intestinal samples, ileal and colonic contents, and feces were collected. Intestinal histomorphology, disaccharidase activity, serotonin (5'HT), vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH) were measured. Gut microbiota composition was assessed by pyrosequencing of the V3-V5 regions of 16S rRNA and quantitative polymerase chain reaction. RESULTS: Body weight of piglets on TEST was greater (P ≤ 0.05) than CONT on days 17 to 30. Both groups displayed growth patterns within the range observed for sow-reared pigs. TEST piglets had greater jejunal lactase (P = 0.03) and higher (P = 0.003) ileal VIP expression. TEST piglets tended to have greater (P = 0.09) sucrase activity, longer (P = 0.08) ileal villi, and greater (P = 0.06) duodenal TH expression. Microbial communities of TEST piglets differed from CONT in ascending colon (AC, P = 0.001) and feces (P ≤ 0.05). CONT piglets had greater relative abundances of Mogibacterium, Collinsella, Klebsiella, Escherichia/Shigella, Eubacterium, and Roseburia compared with TEST piglets in AC. In feces, CONT piglets harbored lower (P ≤ 0.05) proportions of Parabacteroides, Clostridium IV, Lutispora, and Sutterella than TEST piglets. CONCLUSIONS: A mixture of bioactive ingredients improved weight gain and gut maturation, modulated colonic and fecal microbial composition, and reduced the proportions of opportunistic pathogens.

Bovine osteopontin modifies the intestinal transcriptome of formula-fed infant rhesus monkeys to be more similar to those that were breastfed.

BACKGROUND: Osteopontin (OPN) is a multifunctional protein found in human milk at high concentration. OBJECTIVE: The impact of supplemental bovine OPN on growth, body composition, and the jejunal transcriptome was assessed. METHODS: Newborn rhesus monkeys were randomly assigned to be breastfed (n = 4) or to receive formula [formula fed (FF), n = 6] or formula supplemented with 125 mg/L of bovine OPN (bOPN, n = 6) for 3 mo. Jejunal mRNA was extracted and subjected to microarray analysis. RESULTS: Growth was similar among all the treatment groups, but breastfed monkeys were ∼25% leaner at 3 mo. Pairwise comparisons demonstrated that 1017 genes were differentially expressed between breastfed and FF groups, 217 between breastfed and bOPN groups, and 119 between FF and bOPN groups. The data were also analyzed with the use of weighted gene coexpression network analysis, which revealed 6 modules of coexpressed genes that differed among the 3 treatments. Nearly 50% of genes were assigned to one module in which breastfed differed from FF and bOPN expression was intermediate. This module was enriched for genes related to cell adhesion and motility, cytoskeletal remodeling, wingless and integration site signaling, and neuronal development. Most of these canonical pathways centered on integrins, which are receptors for OPN. CONCLUSIONS: The intestinal transcriptome of breastfed and FF monkeys differs, but bovine OPN at levels similar to human milk shifts gene expression profiles to be more similar to breastfed monkeys.

Mode of delivery and early nutrition modulate microbial colonization and fermentation products in neonatal piglets.

Colonization of the intestinal microbiota after birth plays an important role in development of the neonatal gastrointestinal and immune systems. Two key environmental factors that influence the colonization pattern are delivery mode and nutrition. In this study, the impact of delivery mode and nutrition on microbial colonization and metabolic activity was investigated in the pig model. Vaginally (VD) or caesarean- (CD) delivered piglets were sow-reared (SR) or fed formula alone (FF) or with 4 g/L prebiotics [1:1 ratio of short-chain fructo-oligosaccharides (scFOS) and polydextrose (PDX); FP]. Intestinal contents were collected on d 7 and 14. SR piglets harbored different microbial populations from FF and FP piglets in ileum and ascending colon (AC). On d 7, FF piglets had a greater abundance of Clostridium XIVa in AC, but lower total bacteria, Clostridium XIVa, and Lactobacillus spp. in ileum and Fecalibacterium prausnitzii in AC compared with FP piglets. On d 14, total bacteria were more abundant in FP than FF piglets. Butyrate, isobutyrate, valerate, and isovalerate concentrations in AC were greater in SR piglets compared with FF or FP piglets. At both sampling days, acetate concentrations in AC were similar between the SR and FF groups, whereas propionate was higher in the SR compared with FF group. Delivery mode also significantly affected microbial populations. Bacterial densities differed in AC for Bacteroides-Prevotella at d 7 and Clostridium XIVa at d 14, being higher in VD piglets. Correspondingly, VD piglets had higher propionate in ileum and propionate and butyrate in AC compared with CD piglets. Our results indicate that both delivery mode and nutrition affect microbial composition and metabolic activity. Supplementation of scFOS/PDX to formula modulates microbial colonization and produces a SCFA pattern closer to that of SR piglets.

Natural killer cell populations and cytotoxic activity in pigs fed mother's milk, formula, or formula supplemented with bovine lactoferrin.

BACKGROUND: Natural killer (NK) cells are components of the innate immune defense system, and their levels differ between breast and formula-fed (FF) infants. Lactoferrin (Lf) modulates NK cell cytotoxicity ex vivo. We hypothesized that dietary bovine Lf (bLf) would increase NK cell populations and cytotoxicity. METHODS: Piglets were sow-reared (SR), FF, or 1 g/l bLf-fed (LF) for 21 d. NK cells (CD3(-)CD4(-)CD8(+)) in blood (peripheral blood mononuclear cells (PBMCs)), spleen, and mesenteric lymph node (MLN) were determined by flow cytometry. PBMC NK cells were tested for cytotoxic activity against target K562 cells ex vivo in the presence of media (unstimulated), interleukin-2, or bLf. NK cell mRNA expression was determined by reverse transcription-quantitative PCR. RESULTS: SR and LF piglets had more NK cells in MLN (P = 0.0097) and spleen (P = 0.0980) than FF piglets. In PBMCs, SR piglets had more NK cells than FF piglets (P = 0.0072); LF piglets were intermediate and not different from FF or SR piglets. NK cell intelectin-2 mRNA expression was 2.5-fold higher (P = 0.0095) in LF than SR or FF piglets. NK cells in SR piglets exhibited greater (P < 0.0001) cytotoxic activity than those in LF or FF piglets, which was supported by greater perforin mRNA expression. CONCLUSION: Dietary bLf increased blood NK cell populations and NK Lf receptor expression but not NK cell cytotoxicity.

Human milk oligosaccharides inhibit rotavirus infectivity in vitro and in acutely infected piglets.

Human milk (HM) is rich in oligosaccharides (HMO) that exert prebiotic and anti-infective activities. HM feeding reduces the incidence of rotavirus (RV) infection in infants. Herein, the anti-RV activity of oligosaccharides was tested in an established in vitro system for assessing cellular binding and viral infectivity/replication, and also tested in a newly developed, acute RV infection, in situ piglet model. For the in vitro work, crude HMO isolated from pooled HM, neutral HMO (lacto-N-neotetraose, LNnT; 2'-fucosyllactose) and acidic HMO (aHMO, '-sialyllactose, 3'-SL; -sialyllactose, -SL) were tested against the porcine OSU strain and human RV Wa strain. The RV Wa strain was not inhibited by any oligosaccharides. However, the RV OSU strain infectivity was dose-dependently inhibited by sialic acid (SA)-containing HMO. 3'-SL and 6'-SL concordantly inhibited (125)I-radiolabelled RV cellular binding and infectivity/replication. For the in situ study, a midline laparotomy was performed on 21-d-old formula-fed piglets and six 10 cm loops of ileum were isolated in situ. Briefly, 2 mg/ml of LNnT, aHMO mixture (40% 6'-SL/10 % 3'-SL/50 % SA) or media with or without the RV OSU strain (1 x 10(7) focus-forming units)were injected into the loops and maintained for 6 h. The loops treated with HMO treatments þ RV had lower RV replication, as assessed by non-structural protein-4 (NSP4) mRNA expression, than RV-treated loops alone. In conclusion, SA-containing HMO inhibited RV infectivity in vitro; however, both neutral HMO and SA with aHMO decreased NSP4 replication during acute RV infection in situ.

Formula supplementation with human and bovine milk oligosaccharides modulates blood IgG and T-helper cell populations, and ex vivo LPS-stimulated cytokine production in a neonatal preclinical model.

Introduction: Human 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. Methods: Pigs (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. Results: Serum 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-1ß 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. Discussion: The 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.

Addition of polydextrose and galactooligosaccharide to formula does not affect bacterial translocation in the neonatal piglet.

OBJECTIVES: The aim of the study was to determine the effect of polydextrose (PDX) and galactooligosaccharide (GOS) on bacterial translocation (BT) in neonatal piglets. MATERIALS AND METHODS: Piglets (n = 36) were randomized 12 hours after birth to receive total enteral nutrition (TEN) as formula; TEN + GOS (4 g/L), TEN + PDX (4 g/L), or TEN + GOS + PDX (2 g/L each) for 7 days or were supported by total parenteral nutrition (TPN) as a positive control for BT (n = 8). Blood, spleen, liver, and mesenteric lymph node (MLN) samples were cultured for aerobic and anaerobic bacteria. Colon microbiota 16S rDNA was measured by polymerase chain reaction. Myeloperoxidase activity and tumor necrosis factor-α expression were measured in ileum and ascending colon. RESULTS: Among the enterally fed groups, no difference was seen in the Lactobacillus and Bacteroides 16S rDNA copies per gram of colonic contents, yet total bacterial levels were lower (P < 0.05) in the TEN + GOS group compared with TEN alone. Bacteria were detected in the blood, liver spleen, and MLN of TPN piglets. In contrast, bacterial counts were predominantly detected in the MLN of TEN piglets, at much lower levels than in TPN, and levels were not affected by GOS and PDX addition. TPN piglets had elevated (P < 0.05) ileal myeloperoxidase activity and a trend in elevated ascending colon tumor necrosis factor-α expression (P = 0.1). CONCLUSIONS: PDX and GOS added to formula do not induce BT in healthy piglets. Low levels of bacteria in MLN of healthy neonatal piglets may reflect mucosal sampling rather than pathological BT.

Whey Protein Lipid Concentrate High in Milk Fat Globule Membrane Components Inhibit Porcine and Human Rotavirus in vitro.

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.

Combination-Feeding Causes Differences in Aspects of Systemic and Mucosal Immune Cell Phenotypes and Functions Compared to Exclusive Sow-Rearing or Formula-Feeding in Piglets.

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.

Bovine Milk Oligosaccharides and Human Milk Oligosaccharides Modulate the Gut Microbiota Composition and Volatile Fatty Acid Concentrations in a Preclinical Neonatal Model.

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.

Evaluation of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis on Growth, Organ Weights, and Intestinal Development of Piglets.

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.

Early postnatal exposure to di(2-ethylhexyl) phthalate causes sex-specific disruption of gonadal development in pigs.

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.

Noninvasive stool-based detection of infant gastrointestinal development using gene expression profiles from exfoliated epithelial cells.

We have developed a novel molecular methodology that utilizes stool samples containing intact sloughed epithelial cells to quantify intestinal gene expression profiles in the developing human neonate. Since nutrition exerts a major role in regulating neonatal intestinal development and function, our goal was to identify gene sets (combinations) that are differentially regulated in response to infant feeding. For this purpose, fecal mRNA was isolated from exclusively breast-fed (n = 12) and formula-fed (n = 10) infants at 3 mo of age. Linear discriminant analysis was successfully used to identify the single genes and the two- to three-gene combinations that best distinguish the feeding groups. In addition, putative "master" regulatory genes were identified using coefficient of determination analysis. These results support our premise that mRNA isolated from stool has value in terms of characterizing the epigenetic mechanisms underlying the developmentally regulated transcriptional activation/repression of genes known to modulate gastrointestinal function. As larger data sets become available, this methodology can be extended to validation and, ultimately, identification of the main nutritional components that modulate intestinal maturation and function.

Encapsulation of tributyrin by gamma-cyclodextrin: Complexation, spray drying, and in vitro fermentation.

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.

Evaluation of 6'-Sialyllactose Sodium Salt Supplementation to Formula on Growth and Clinical Parameters in Neonatal Piglets.

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.

Analysis of gut microbiome, nutrition and immune status in autism spectrum disorder: a case-control study in Ecuador.

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-ß1 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.

Hormonal correlates of ontogeny in baboons (Papio hamadryas anubis) and mangabeys (Cercocebus atys).

This study investigates the relationship between serum hormone levels and morphometrics during ontogeny in olive baboons (Papio hamadryas anubis) and sooty mangabeys (Cercocebus atys), to test hypotheses about the endocrine regulation of species size differences. First, we expect that levels of hormones and binding proteins predict size change during ontogeny in both species. Second, a high level of integration among the hormones and binding proteins analyzed is expected, with the implication that they act in combination to influence the development of body size and shape. Utilizing a mixed longitudinal sample, we compare change in 18 different measurements, which reflect overall size growth as well as growth in length and circumference, with levels of six growth-related hormones and binding proteins. We examine the relationship between hormone and binding protein levels and morphometrics, using multivariate analyses and "arithmetically-estimated" velocity curves of hormones, binding proteins, to characterize how the endocrine factors analyzed relate to growth. Results suggest that levels of these endocrine factors can be used to predict local and overall growth during ontogeny and that integration between multiple hormone axes is indicated. While important for growth in both species, ontogenetic changes in hormone and binding protein levels are more tightly correlated with changes in morphometric measurements in baboons than mangabeys. These results have important implications for understanding why some smaller-bodied species have higher absolute growth-related hormone levels than larger-bodied species.