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

Berding, Kirsten*; Wang, Mei; Monaco, Marcia H.; Alexander, Lindsey S.; Mudd, Austin T.‡,§; Chichlowski, Maciej||; Waworuntu, Rosaline V.||; Berg, Brian M.*,||; Miller, Michael J.*,†; Dilger, Ryan N.*,‡,§; Donovan, Sharon M.*,†

Journal of Pediatric Gastroenterology and Nutrition: December 2016 - Volume 63 - Issue 6 - p 688–697
doi: 10.1097/MPG.0000000000001200
Original Articles: Nutrition
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SDC

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.

Supplemental Digital Content is available in the text

*Division of Nutritional Sciences

Department of Food Science and Human Nutrition

Piglet Nutrition and Cognition Laboratory, Department of Animal Sciences

§Neuroscience Program, University of Illinois, Urbana-Champaign

||Mead Johnson Pediatric Nutrition Institute, Evansville, IN.

Address correspondence and reprint requests to Sharon M. Donovan, Department of Food Science and Human Nutrition, University of Illinois, 339 Bevier Hall, 905 South Goodwin Ave, Urbana, IL 61801 (e-mail: sdonovan@illinois.edu).

Received 30 October, 2015

Accepted 11 March, 2016

Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal's Web site (www.jpgn.org).

This study was supported by a grant from Mead Johnson Nutrition (Evansville, IN).

S.D., M.M., and R.D. have received grant funding. S.D. has served on advisory boards, and S.D., R.D., M.W., M.M., L.A., and R.D. have consulted for Mead Johnson Nutrition. M.C., R.W., and B.B. are employees of Mead Johnson Nutrition. The remaining authors report no conflicts of interest.

What Is Known

  • Bioactive components in human milk promote enterocyte proliferation and differentiation.
  • Lactoferrin increases crypt cell proliferation and depth and intestinal lactase activity in pigs.
  • Polydextrose/galactooligosaccharides increase lactobacilli and bifidobacteria in infants.

What Is New

  • A combination of lactoferrin, milk fat globule membrane, and prebiotics increased daily body weight gain, jejunal disaccharidase activity, and ileal vasoactive intestinal peptide expression.
  • A proximal-to-distal gradient in expression of gut–brain axis markers was observed in the intestine.
  • The addition of lactoferrin, milk fat globule membrane, and prebiotics to formula modulated the microbiota of the ascending colon and feces, and reduced the proportions of opportunistic pathogens.

The early neonatal period is a critical time for intestinal maturation as the gastrointestinal (GI) tract becomes highly adaptable to environmental factors, including nutrition and the microbiota (1). Human milk is generally recognized as the optimal diet for infants, and exclusive breast-feeding is recommended for the first 6 months of life, which is a period of rapid neonatal growth and GI, immune, and cognitive development (2). Because of the beneficial effects of human milk on health and development, research has focused on identifying bioactive components present in human milk that may exert these actions. Among these components are the glycoprotein lactoferrin (Lf), milk fat globule membrane (MFGM) proteins, and human milk oligosaccharides (HMOs). Bovine Lf stimulates enterocyte proliferation (3) and affects mucosal (4) and systemic (4,5) immune development of formula-fed piglets. A recent study reported an association between human milk Lf concentration and the abundance of bifidobacteria and lactobacilli in breast-fed infants (6). MFGM provides a variety of important nutritional, digestive, and immunological benefits (7), and the addition of MFGM to infant formula resulted in serum lipid composition (8), more similar to breast-fed infants, and improved cognitive development (9). HMOs possess beneficial effects for the development of the mucosal and systemic immune system and intestinal microbiota (10). HMOs have not been commercially available in large quantities; therefore, other prebiotics that share some of the functional attributes of HMOs, such as polydextrose (PDX) and galactooligosaccharides (GOS), are added to infant formula (11,12).

Neuronal inputs throughout the intestine control motility, absorption, and secretion in the GI tract. Neurotransmitters, including tyrosine hydroxylase (TH), serotonin (5’HT), and vasoactive intestinal peptide (VIP) serve a variety of gut functions and play a regulatory role in the differentiation or function of enterocytes during early development of human intestinal mucosa (13,14). Age-related changes in neuronal innervation as well as in TH and VIP concentrations during postnatal development, suggest that the enteric nervous system (ENS) in the GI tract undergoes substantial changes in the first weeks after birth (15,16). Such a period provides a developmental window that can shape the GI function and disease later in life (17). A growing body of evidence also suggests that microbes and their metabolites can directly influence intestinal enteric neurons and play a role as key mediators of the gut–brain axis (GBA), thereby, exerting functions beyond the GI tract (18).

Emerging evidence suggests potential benefits provided by individual bioactive components in human milk; however, gaps remain regarding whether a combination of ingredients may affect the development of the GI tract, brain, GBA, and microbiome. Therefore, the goal of this study was to investigate potential mechanisms of a mixture of Lf, MFGM, and prebiotic blend on the composition of the gut microbiota, intestinal structure, and markers of the GBA in the neonatal piglet model. Outcomes related to brain and cognitive development are reported in a separate manuscript (19). Because of the positive effects of the individual ingredients on intestinal development and function, we hypothesized that piglets fed a mixture of ingredients would show improved GI development, GBA marker expression, and different composition of the developing microbiome compared with piglets fed control formula.

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METHODS

Animal Housing and Dietary Treatments

All the animal care and experimental procedures were approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee. The piglet study was conducted as described by Mudd et al (19). Briefly, vaginally delivered colostrum-deprived intact male Yorkshire piglets (n = 24) from the University of Illinois Imported Swine Research Laboratory were artificially reared over a 30-day study period.

The composition of the CONT and TEST diets are shown in Supplementary Table 1 (http://links.lww.com/MPG/A633) and described by Mudd et al (19), which were formulated and produced by Mead Johnson Nutrition (Evansville, IN) to meet the nutritional needs of piglets. Both diets contained the fatty acids docosahexaenoic acid (DHA, 91 mg/100 g diet) and arachidonic acid (ARA, 182 mg/100 g diet). The CONT diet was supplemented with MFGM-10 (2.5/100 g diet; Arla Food Ingredients, Aarhus, Denmark), GOS (3.5/100 g diet; FrieslandCampina, Zwolle, the Netherlands), PDX (1.2/100 g diet; Danisco, Terre Haute, IN), and bovine Lf (90.4% pure, 11% iron saturation; 0.3/100 g diet; Tatua Cooperative Dairy Company, Morrinsville, New Zealand) to produce the TEST diet. Both CONT and TEST milk replacer powder was reconstituted at 200 g of dry powder per 800 g of water, and piglets were fed at 285, 305, and 310 mL of reconstituted diet per kilogram of BW starting on 3, 5, and 12 days of age, respectively. At this reconstitution rate, both diets contained DHA (182 mg/L) and ARA (364 mg/L), and the reconstituted TEST treatment contained PDX/GOS (2.4 g/L and 7 g/L of PDX and GOS, respectively), Lf (0.6 g/L), and MFGM-10 (5.0 g/L).

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Sample Collection

At 31 days of age, piglets were anesthetized using a telazol:ketamine:xylazine solution (50.0 mg tiletamine plus 50.0 mg of zolazepam reconstituted with 2.50 mL ketamine [100 g/L] and 2.50 mL xylazine [100 g/L]; (Fort Dodge Animal Health, Overland Park, KS) by i.m. injection at 0.03 mL/kg of body weight. After verifying anesthetic induction, piglets were euthanized via intracardiac administration of sodium pentobarbital (86 mg/kg of body weight, Fatal-Plus, Vortech Pharmaceuticals, Dearborn, MI). Intestinal tissue and mucosa samples were collected as previously described (20) and snap-frozen in liquid nitrogen. Additional intestinal tissues were fixed in formalin and 4% paraformaldehyde (PFA). Ileal and ascending colon (AC) (the proximal one third of the colon) contents and feces were snap-frozen in liquid nitrogen and stored at −80°C for microbiota analyses. Midbrain and hippocampal samples were snap-frozen in liquid nitrogen and stored at −80°C to serve as positive controls and for standard curves for messenger RNA (mRNA) analyses.

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Mucosal Enzyme Activity

Mucosa homogenates were prepared, and lactase and sucrase activities were measured as previously described (21). Mucosal protein concentration was measured using the Bradford protein assay (Bio-Rad Laboratories, Inc, Hercules, CA). The mucosal protein concentration was expressed as milligram of protein per gram of mucosa. Lactase and sucrase activities are expressed as millimoles of glucose released per gram of protein per minute.

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Histomorphology

Intestinal histomorphology was measured using established methods (20). Digital images (20× magnification) of sections were captured using the NanoZoomer slider scanner (Hamamatsu Corporation, Bridgewater, NJ; Institute for Genomic Biology Microscopy and Imaging Facility, University of Illinois). Small intestinal villus height (μm), villus area (mm2), crypt depth (μm), crypt area (mm2), and colon cuff depth (μm) and cuff area (mm2) were measured from 10 to 15 well-oriented crypt-villus systems using AxioVision 4.8 digital image processing software (Carl Zeiss MicroImaging, Inc, Thornwood, NY).

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Neurotransmitter Abundance and Expression

Immunohistochemistry

PFA-fixed samples were sectioned into paraffin blocks at the University of Illinois, College of Veterinary Medicine. Immunohistochemical staining was performed per manufacturer's instructions. Antigen retrieval was achieved by placing slides into Antigen Unmasking solution (Vector Laboratories, Inc, Burlingame, CA) heated to 90°C for 30 minutes. Slides were stained with mouse anti-5’HT, rabbit anti-TH, rabbit anti-chromogranin A, and rabbit anti-VIP (Abcam, Cambridge, MA). After a 2-hour incubation, slides were washed in PBS and incubated with secondary antibodies containing fluoflor Alexa 488 (for serotonin), Alexa 586 (for Chromogranin A), Alexa 594 (for VIP), and Alexa 647 (for TH) (Life Technologies, Carlsbad, CA). Slides were then counter stained with 4’,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Images were captured using a Multiphoton Confocal Microscope (Carl Zeiss710, Institute for Genomic Biology, University of Illinois). Five to eight images per section per piglet were taken at 10× magnification (5’HT, EC-cells) or 20× magnification (TH, VIP) with 4 × 3 tiling at 1024 × 1024 in the x- and y-directions. Additional magnification was provided by manually enlarging specific areas within an image. Proteins were quantified with AxioVision for the quantification of immunofluorescence (Carl Zeiss, Thornwood, NY). Data for serotonin and enterochromaffin (EC)-cells are expressed as the percentage of serotonin-positive or EC-positive cells to total cells (DAPI). For VIP and TH, the data were expressed as an area (μm2), which was divided by total neuronal density (area of the nerve cluster in μm2) and multiplied by 100% to generate a percentage.

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Gene Expression by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction

Tissue mRNA expression was measured as previously described (20). Reverse transcription was performed using 0.5 μg of RNA in a 20-μL reaction volume using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Different dilutions of the RT samples were prepared and plated onto a MicroAmp optical 384-well plate (Applied Biosystems). A TaqMan Master Mix (Life Technologies), water, and a primer mix (Life Technologies) were added to the wells. The plate was analyzed using a 7900HT Fast Real-Time polymerase chain reaction (PCR) system (Applied Biosystems). Brain-derived neurotrophic factor (BDNF) (Ss03822335_s1; Life Technologies) mRNA expression was assessed in the small intestine and AC. The 60S ribosomal protein L19 (RPL 19, Ss03375624_g1; Life Technologies) was used as an endogenous control. The standard curve consisted of dilutions of pooled whole tissue hippocampal and midbrain cDNA. Normalized values were calculated by dividing the target quantity mean by the mean RPL19 quantity. The fold-difference for each measurement was determined by dividing the normalized target value (TEST) by the normalized calibrator (CONT) value.

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Microbiome Analyses

DNA Extraction

DNA was extracted from ileal (n = 21) and AC (n = 24) contents and feces (n = 24) using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA) in combination with the FastPrep-24 System (MP Biomedicals, Carlsbad, CA) as previously described (22). DNA concentration was determined with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies).

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Pyrosequencing of Bacterial 16S rRNA Genes

Amplification of the V3 to V5 regions of the bacterial 16S rRNA genes were performed with primers 341F and 907R (23). Pyrosequencing was carried out at the Research Testing Laboratory (RTL, Lubbock, TX) as previously described (24,25) using 454 Life Sciences Genome Sequencer FLX with Titanium series reagents and protocol (Roche Applied Science, Indianapolis, IN).

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Sequence Analysis

Sequences processing and analysis were performed using a bioinformatic pipeline developed at RTL. Briefly, raw sequences were denoised and chimera was checked by UCHIME (26) and trimmed with the following parameters: mean quality score >25, minimal read length >250 bp, and no primer mismatches or uncorrectable barcodes. The remaining sequences were demultiplexed and grouped into operational taxonomic units (OTUs) using the UPARSE algorithm. The centroid sequences (the longest sequences) from each OTU were selected and aligned using MUSCLE (27). The phylogenetic tree was constructed from alignment using FastTree (28), and then weighted distance matrices (29) were generated from the tree. The centroid sequences from each OTU were assigned into different taxonomic levels using SEARCH global alignment (30) against the Ribosomal Database Project (RDP) database (31). Alpha-diversity (Chao 1 and Shannon indices) was calculated using QIIME (32).

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Real-Time Polymerase Chain Reaction

Bacterial genomic DNA were analyzed for total bacteria, Bifidobacterium spp, Clostridium perfringens, Clostridium difficile, and Bacteroides fragilis using primer/probe sequences indicated in Supplementary Table 2 (http://links.lww.com/MPG/A634). Real-time qPCR was performed in 7900HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan (for B fragilis) or SYBR Green (for other bacterial groups/species) assays. All of the PCR experiments were performed in triplicate with a reaction volume of 10 μL. For SYBR Green assays, each reaction contained 5 μL of 2Χ Power SYBR Green PCR Master mix (Applied Biosystems), 1 μL bovine serum albumin (New England Biolabs, Ipswich, MA) at 1 mg/mL (final concentration 100 μg/mL), 0.5 μmol/L of each primer, and 10 ng of template DNA. The cycling conditions were 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, primer-specific annealing temperature (Supplementary Table 2, http://links.lww.com/MPG/A634) for 20 seconds, and 72°C for 45 seconds. Following amplification, a dissociation step was included to analyze the melting profile of the amplified products. The TaqMan assay for B fragilis was performed as previously described (33), except 1 μL BSA was added to the PCR reaction. The cycling conditions were 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for 20 seconds, and 72°C for 45 seconds. Standard curves (101–108 16S rRNA gene copies per reaction) were generated using purified pCR 4 TOPO-TA plasmids (Life Technologies), containing the 16S rRNA genes of Eubacterium hallii 27751 (for total bacteria), B fragilis 25285 (B fragilis), Bifidobacterium longum subsp. infantis 15697 (Bifidobacterium spp), C perfringens 13124 (C perfringens), C difficile 9689 (C difficile), and Lactobacillus rhamnosus 53103 (Lactobacillus spp). Data analysis was processed with SDS version 2.3 software supplied by Applied Biosystems. Results are presented as the number of 16S rRNA gene copies per gram of intestinal contents or feces on a wet weight basis.

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Statistical Analysis

Differences in bacterial communities between CONT and TEST piglets were evaluated with principal coordinate analysis (PCoA) and permutational multivariate analysis (PERMANOVA) of variance using distance matrices. PCoA and PERMANOVA were performed on weighted UniFrac distance using QIIME (32) and function adonis of vegan package of R (34), respectively. For adonis, distances among samples were calculated first using weighted UniFrac, and then an analysis of variance (ANOVA)-like simulation was conducted to test for group differences. Alpha-diversity and relative abundances of individual phyla and genera were examined for significant differences between diets within each of the segments using ANOVA. Relative abundances were arcsin transformed before analysis. The statistical analysis of other data was performed as ANOVA using MIXED procedure of Statistical Analysis Software (SAS) version 9.4 (SAS Institute, Cary, NC). Real-time qPCR data were log10 transformed to achieve normal distribution. Data were reported as means ± SD.

All other data were analyzed by one-way ANOVA using the MIXED procedure of SAS 9.4 (SAS Institute) to differentiate the effects of the 2 dietary treatments. Regional effects within the intestine that were independent of diet were also analyzed by a one-way ANOVA. The statistical model included replicate as a random effect, with statistical significance set at P ≤ 0.05 and trends reported when 0.05 ≤ P ≤ 0.10.

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RESULTS

Body Weight

Body weights of pigs receiving TEST diet exceeded those of pigs receiving CONT diet beginning on day 17 and continuing until trial completion (Fig. 1). The final body weight of TEST piglets (9.15 ± 0.27 kg) was higher (P = 0.005) than that of CONT piglets (8.47 ± 0.24 kg). Piglets did not exhibit any signs of sickness because of diet, although minor bouts of diarrhea did occur across all of the treatments. No feed refusals were observed for piglets on this study; thus, the daily feeding rate per kilogram of body weight corresponds to the average volumes consumed (Fig. 2).

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

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Intestinal Length and Weight

Absolute intestinal weight and length did not differ between pigs on either CONT or TEST diets. There was a tendency toward intestinal mass/per unit length (g/cm) to be greater (P = 0.09) in TEST (0.44 ± 0.043 g/cm) than in CONT (0.41 ± 0.069 g/cm) piglets. When normalized per kilogram of body weight, intestinal length was greater (P < 0.01) in CONT (133 ± 15.6 cm/kg) than in TEST (121 ± 9.2 cm/kg) piglets, whereas the ratio of the intestinal weight per kilogram of body weight did not differ between the 2 groups.

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Histomorphology

There were no statistically significant differences in small intestinal morphology between the 2 dietary treatment groups. Ileal villus length, however, tended to be greater (P = 0.08) in piglets fed the TEST diet (634.6 ± 82.1 μm) compared with piglets fed the CONT diet (578.8 ± 87.8 μm). Ascending colon cuff area was larger (P = 0.016) in piglets receiving the CONT diet (27.4 ± 3.1 mm2) compared with piglets fed the TEST diet (25.6 ± 4.6 mm2), and cuff depth tended to be deeper (P = 0.062) in the AC of piglets fed CONT diet (434.9 ± 26.4 μm) compared with TEST diet (411.9 ± 29.7 μm). The ratio of villus height-to-crypt depth tended to be higher (P = 0.06) in the ileum of piglets fed the TEST diet (3.83 ± 0.05) compared with piglets fed the CONT diet (3.73 ± 0.05) but not in the duodenum and jejunum.

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Mucosal Disaccharidase Activity

Lactase

Jejunal lactase activity (mmol glucose/g protein/min) was greater (P = 0.027) in piglets receiving the TEST diet (29.01 ± 10.02 mmol glucose/g protein/min) compared with piglets fed the CONT diet (19.3 ± 8.97 mmol glucose/g protein/min). There was no significant difference in the duodenal or ileal lactase activity between the 2 diets.

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Sucrase

Mucosal sucrase activity (mmol glucose/g protein/min) did not significantly differ in any intestinal segment between the 2 treatment diets. In the jejunum, sucrase activity tended to be higher (P = 0.099) in piglets receiving the TEST diet (44.91 ± 26.8 mmol glucose/g protein/min) compared with piglets receiving CONT diet (34.24 ± 16.04 mmol glucose/g protein/min).

The ratio of lactase-to-sucrase activity was higher in the duodenum (P = 0.001) and ileum (P = 0.03) of piglets fed the TEST diet (duodenum, 10.59 ± 12.14; ileum, 0.27 ± 0.29) compared with the CONT diet (duodenum, 7.53 ± 4.09; ileum, 0.19 ± 0.12) but did not differ between treatment groups in the jejunum.

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Intestinal Immunohistochemistry

Representative images of immunofluorescent detection of 5’HT, TH, and VIP are shown in Supplementary Figure 1 (http://links.lww.com/MPG/A632).

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Tyrosine Hydroxylase and Vasoactive Intestinal Peptide

The percentage of cells expressing TH per measured area tended to be higher (P = 0.06) in the duodenum of TEST piglets (Fig. 3A). There was no effect of diet on the TH expression in the ileum or AC nor were there regional differences in the TH expression between duodenum, ileum, and AC.

FIGURE 3

FIGURE 3

The percentage of VIP expressed per measured area was higher (P = 0.003) in the ileum for TEST piglets (Fig. 3B). There was no effect of diet on VIP expression in the duodenum or AC. With regard to regional differences, the percent of VIP-positive area to total neuronal density was greater in the small intestine (duodenum and ileum) than in AC (Fig. 3C).

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Intestinal Brain-Derived Neurotrophic Factor Expression

No differences in duodenal or ileal BDNF mRNA expressed as fold difference relative to CONT mRNA was observed between the dietary treatments (duodenum, CONT 1.00 ± 0.1 vs TEST 1.15 ± 0.1; ileum, CONT 1.00 ± 0.02 vs TEST 1.06 ± 0.02). BDNF mRNA expression did not differ between intestinal regions (duodenum 1.12 ± 0.35 vs ileum 1.00 ± 0.33).

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Microbiota Composition

After performing the quality control depletions as described above, 339,370 total sequences, with a mean of 4918 sequences per sample, were used for further bioinformatics analyses. PCoA of weighted UniFrac generated from ileum, AC, and fecal samples of CONT and TEST piglets are shown in Figure 4. PERMANOVA analysis indicated that the overall bacterial communities differed between CONT and TEST piglets in the AC (P = 0.001) and feces (P = 0.047) but not in the ileum (P = 0.679). To identify which bacteria differed between CONT and TEST piglets in the AC and feces, the sequences were classified against the RDP database (version 2.6). At the phyla level, piglets fed the TEST diet had greater (P ≤ 0.05) relative abundance of Bacteroidetes and lower (P ≤ 0.05) proportion of Proteobacteria in the AC compared with CONT piglets (Table 1). At the genus level, TEST piglets harbored higher proportions of Parabacteroides (P = 0.0001), Clostridium IV (P = 0.0015), and Lutispora (P = 0.0001), whereas CONT piglets had greater relative abundances of Mogibacterium (P = 0.0039), Collinsella (P = 0.0015), Klebsiella (P = 0.0222), Escherichia/Shigella (P = 0.0018), Eubacterium (P = 0.0011), and Roseburia (P = 0.0056) in the AC than in TEST piglets (Table 2). No differences in bacterial phyla were detected in feces (Table 2). At the genus level, however, CONT piglets had lower relative abundances of Parabacteroides (P = 0.0011), Clostridium IV (P = 0.0036), Lutispora (P = 0.0012), and Sutterella (P = 0.0293) (Table 3).

FIGURE 4

FIGURE 4

TABLE 1

TABLE 1

TABLE 2

TABLE 2

TABLE 3

TABLE 3

To compare bacterial diversity within samples, Chao 1 and Shannon indices were calculated (Supplementary Table 3, http://links.lww.com/MPG/A635). No significant differences in the Chao 1 and Shannon indices were detected between the 2 treatment groups at any sampling sites.

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Real-Time qRT-PCR for Total and Specific Groups/Species of Bacteria

The abundances of total bacteria, Bifidobacterium spp, C perfringens, C difficile, B fragilis, and Lactobacillus spp in ileum and AC contents, and feces were shown in Table 4. The density of total bacteria in AC was greater in TEST than in CONT (P = 0.034) but similar in ileum and feces. The abundances of Bifidobacterium spp, B fragilis, C perfringens, and Lactobacillus spp did not differ between the treatment groups at any intestinal segments. C difficile were not detectible in ileum and detected in AC of 1 TEST piglet (detection limit, 5.24 in Log scale). The densities of C difficile in feces were similar between CONT and TEST piglets (Table 4).

TABLE 4

TABLE 4

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DISCUSSION

To investigate how a dietary mixture more closely mimicking human milk composition affects integrated responses of the microbiome and intestinal structure and function, we studied a cow milk–based diet fortified with Lf, MFGM, and PDX/GOS in a neonatal piglet model. The key findings of the present study were that the TEST diet was well tolerated, and piglets fed the TEST diet had increased overall weight gain, greater lactase and sucrase activities in the jejunum, higher VIP concentration in the ileum, and changes in the composition of the gut microbiota.

Intestinal histomorphology has been linked to changes in digestive and absorptive capacity and daily weight gain (35). We previously reported that 14-day feeding of formula supplemented with 1.0 or 3.6 g/L bovine Lf increased enterocyte proliferation and crypt depth and area, without changes in villus height or area or disaccharidase activity (3). Herein, a tendency toward greater ileal villus length in piglets fed the TEST diet could indicate a small increase in surface area available for nutrient absorption. The observed increased ratio or villus height/crypt depth has been used in the literature as a marker for digestive capacity in the small intestine (36). The lack of other significant histomorphological observations in the present study may partially be attributable to the relatively low level of Lf provided in the diet. Likewise, the lack of changes indicates that the TEST diet was safe and supported intestinal development.

In addition to structural changes, the enzymatic capacity of the small intestine adapts to dietary changes in the early postnatal period. During development, sucrase activity increases whereas lactase activity declines in the piglet intestine (37), which is comparable to developmental changes in the human infant. A few studies also demonstrated that the inclusion of prebiotics increases enzyme activity in intestinal mucosal tissue (38,39). In the present study, greater disaccharidase activity in the intestinal mucosa of piglets fed the TEST diet was observed, suggesting improved mucosal maturation. Although blood glucose levels were not measured, the increased body weight gain could be indicative of increased nutrient absorption because digestive enzymes in the small intestine facilitate nutrient availability for the host.

The ENS is the largest and most complex subdivision of the peripheral nervous system (40). The intestinal ENS serves specific roles in the functions of the small intestine, either by directly stimulating the release of neurotransmitter from neuronal terminals (eg, VIP) or by triggering the secretion from endocrine cells (eg, serotonin) (41).

Neurotransmitters, including TH, serotonin, and VIP, serve a variety of functions in the GI tract, including regulating the differentiation and function of enterocytes during the early development of human intestinal mucosa (14,42). Animal studies have demonstrated age-related changes in neuronal innervation, as well as TH and VIP concentrations, during postnatal development, suggesting that the GI ENS undergoes substantial changes in the first weeks after birth (15,16). These changes provide a window in which optimal environmental conditions can potentially affect GI function later in life. In the present study, VIP and TH release from neuronal cell clusters and serotonin release from EC cells were detected by immunohistochemistry. The number of VIP-positive cells in the ileum and TH-positive cells in the duodenum were greater in piglets consuming the TEST diet, but no effect of diet on serotonin expression throughout the GI tract was observed. The physiological effect of increased expression of these peptides is currently unknown but could signify improved GI function through the actions of TH and VIP, improving gut motility and increasing absorptive capacity. Increased TH and VIP levels could also reflect changes in neuronal innervation of the neuromuscular layer in the GI tract, thus increasing transduction of physiological stimuli for digestive processes. Although other peripheral measures to show improved absorptive capacity were not assessed, the increased growth of piglets fed the TEST diet and the increased absorptive small intestine villi length may suggest improved nutrient use.

A novel finding of the present study is the diet-independent observation of a proximal-to-distal gradient of makers of the GBA in the GI tract. Higher numbers of EC-cells, and serotonin-, TH-, and VIP-positive cells were detected in the proximal compared with the distal intestine. To our knowledge, this is the first study to characterize regional differences of neurotransmitters in the piglet intestine. A few studies have described similar effects in other species, for example, primates or rodents (43,44). Our data aligns with these reports of higher density of neurotransmitter-secreting cells in the duodenum but contradicts with observations of high concentrations in the colon (44). The physiological effect of a regional gradient is unknown. Our observations align with observations made regarding receptor distribution in the small intestine. In the developing piglet, the 5’HT 2B receptor was 100-times higher in duodenum than other parts of intestine (45), and the duodenum has more VIP receptors in mucosa than jejunum or ileum (46). Thus, from a physiological standpoint, increased concentrations in the duodenum may support digestive and absorptive processes. In addition to the regional effect in the GI tract, the neurotransmitters assessed herein also play a key role in the gut-to-brain communication, which will be examined in future studies.

Last, we examined the effect of the TEST diet on the microbiota owing to its important role in mediating the development of GI, immune, and neural systems (47,48). Compared with CONT, TEST piglets harbored greater relative abundance of Clostridium IV and Bacteroidetes (mainly Parabacteroides) and lower proportion of Proteobacteria, such as Escherichia/Shigella and Klebsiella, in AC. A similar trend was also observed in feces. Clostridium IV belongs to the phylum Firmicutes and is the main butyrate-producing group of bacteria in the gut (49). Species that belong to Clostridium IV start to colonize the intestine of breast-fed infants during the first month of life and are strongly involved in the maintenance of overall gut function (49). Parabacteroides also benefit their host by excluding potential pathogens from colonizing the gut (50).

A higher incidence of GI and urinary tract infections was observed in formula-fed infants compared with breast-fed infants (51). Decreases in relative abundances of Escherichia/ Shigella and Klebsiella may represent health benefits of the TEST diet because these bacteria can act as opportunistic pathogens in immunocompromised subjects, such as infants. For example, Escherichia coli is known as the most common cause of urinary tract infections in infants (51), and some serotypes of E coli and Shigella are associated with infantile diarrhea (52). Klebsiella is one of the most important nosocomial pathogens, which can give rise to a wide range of disease states, including pneumonia, urinary tract infections, septicemia, meningitis, and diarrhea (53). The modulation of colonic microbiota has been reported by Hoeflinger et al (54) when younger piglets (21 days old) were fed formula supplemented with GOS and PDX alone. They did not, however, find the reduction of potential pathogenic bacteria (54). Decrease in the proportion of Escherichia/ Shigella and Klebsiella in our study may be related to the presence of MFGM in TEST diet as previous studies have reported some proteins, such as mucin and lipids (particular gangliosides), which are components of the MFGM and possess anti-pathogenic activities. For example, a bovine milk fraction containing mucin (MUC1) has shown to inhibit hemagglutination of Vibrio cholerae and E coli (55). In the context of our study, the presence of Lf in the TEST diet may further promote the reduction of pathogenic bacteria, as LF has proven antibacterial and antiviral activities (56,57). Concentrations of Salmonella in the colon and E coli throughout the intestine were lower in piglets fed milk from cows producing recombinant human LF compared with piglets fed standard cow whole milk (57,58).

In conclusion, piglets fed a mixture of LF, MFGM, and PFX/GOS achieved normal growth and intestine development. Furthermore, piglets supplemented with the nutrient blend exhibited increased jejunal enzyme activity, increased concentrations of VIP and TH in the upper intestine, and modulated composition of the gut microbiota. Although this study did not allow us to attribute specific outcomes to the individual bioactive components, these results demonstrate that this combination of ingredients was well tolerated and stimulated the development of the GI structure, function, and expression of proteins associated with the ENS.

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Acknowledgments

The authors acknowledge the efforts of Mead Johnson Nutrition employees, John Alvey and Zafir Gaygadzhiev, for assistance in formulating and manufacturing the piglet diets.

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Keywords:

disaccharidase; gut maturation; microbiota; serotonin; tyrosine hydroxylase

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