Short Administration of Combined Prebiotics Improved Microbial Colonization, Gut Barrier, and Growth Performance of Neonatal Piglets.

This study was conducted to investigate the effects of short administration with the combination (GMF) of galactooligosaccharides (GOS), milk fat globule membrane (MFGM), and fructooligosaccharides (FOS) on microbiota, intestinal barriers, and growth performance of neonatal piglets. Sixteen newborn piglets were divided into two groups: GMF group and CON group; GMF solution (5 mL) and saline (5 mL) were, respectively, administered to piglets in the GMF group and CON group once a day during the first week after birth. The results showed that GMF administration improved the growth performance of neonatal piglets on day 8 and day 21, coupled with the enriched genus Lactobacillus on day 8 and the increased genera norank_f__Muribaculaceae, Christensenellaceae_R-7_group, Enterococcus, and Romboutsia on day 21. Additionally, GMF administration increased luminal acetate and propionate levels, upregulated the gene expressions of intestinal tight junctions (Occludin, Claudins, and ZO-1), mucins (Mucin-1, Mucin-2, Mucin-4, and Mucin-20), and cytokines (TNF-α, IL-1β, and IL-22) while decreased the plasma diamine oxidase (DAO) level on day 21. The correlation analysis showed a positive relationship between the colonized beneficial microbiota and the modified intestinal barrier genes. In conclusion, the first week administration of GMF facilitated the colonization of beneficial bacteria, promoted intestinal development by enhancing microbiota-associated intestinal barrier functions, and improved the growth performance of the piglets during the whole neonatal period. Our findings provide guidelines for combined prebiotics application in modulating the microbial colonization and intestinal development of the neonates.

Introduction

After delivery, the structure and functionality of the gastrointestinal tract of piglets rapidly adapt to the transition from parenteral nutrition (via placenta) to enteral nutrition (colostrum/milk), accompanied with the dynamic microbial colonization.[1] The initial microbiota establishment in the neonatal period laid the critical foundations for long-lasting health outcomes,[2] which engaged in multiple interactions with intestinal development, metabolic homeostasis, and immunological defense.[3] Therefore, early neonatal gut development and microbial colonization must be the window opportunity for whole lifelong health.[4]

Because the postnatal gut microecology is highly plastic and susceptibly affected by nutrients intake and surrounding environment during the neonatal period,[5] superior intestinal integrity and functions are imperative for host health and growth.[6] Tight junctions (i.e., Claudins, Occludin) and mucins build up the primary barrier, and beneficial gut microbes (i.e., Lactobacillus) participate in the intestinal barrier establishment by selectively competing for microbial colonization and closely interacting with immunological defense.[7] What is more, colonized microbiota participates in the nutritional metabolism and the microbial metabolites also contribute to the intestinal barrier integrity.[8] Thus, optimal bacteria colonization in the early life impresses the barrier functions, leading to crucial importance for intestinal development and neonatal growth.

Galactooligosaccharides (GOS) and fructooligosaccharides (FOS) are prebiotics selectively utilized by host microorganisms conferring the prebiotic role in health benefits.[9] Research studies on GOS and FOS showed the preference on beneficial bacteria colonization, such as Lactobacillus,[10,11] as well as the intestinal development and immunological response.[12] What is more, milk fat globule membranes (MFGMs), originated from milk, exhibit a nutritional bioactivity in metabolic regulation and intestinal homeostasis.[13] MFGM was also reported to change the microbiota composition and enhance the intestinal barrier function.[14,15] Based on the benefits of the single prebiotic, the combination of different prebiotics was proposed to be synergistic.[16] The blend of GOS and FOS showed a similar effect on the metabolic activity of the microbial flora as mother milk oligosaccharides.[17] The mixture of GOS and MFGM intervention was reported to improve the neurodevelopment and microbial population of young piglets.[18] However, the combined effects of GOS, MFGM, and FOS on microbial composition and intestinal development remained unclear.

Considering the significance of the early neonatal period as well as the predominance of these three prebiotics, the aim of this research is to investigate the effects and underlying mechanisms of combined GOS, MFGM, and FOS administration during the first week on the microbial colonization, intestinal barrier functions, and growth performance of the neonatal piglets of the whole neonatal period.

Results

Effects of GMF Administration during the First Week on the Growth Performance of Neonatal Piglets on Day 8 and Day 21

As shown in Table , GMF administration in early life significantly increased the body weight of piglets on day 8 and day 21 (P < 0.05) and the average daily gain of piglets during the neonatal period (P < 0.05).

Table 1

Effects of GMF Administration during the First Week on the Growth Performance of the Neonatal Piglets on Day 8 and Day 21a

items	CON	GMF	P value
body weight (kg)
day 8	2.33 ± 0.08	2.62 ± 0.08	0.022
day 21	5.68 ± 0.15	6.18 ± 0.13	0.024
average daily gain (g)
day 1–8	116.43 ± 9.08	149.39 ± 10.62	0.041
day 1–21	197.86 ± 6.66	221.56 ± 5.54	0.019

CON, piglets in the CON group administered with saline; GMF, piglets in the GMF group administered with the combination of GOS, MFGM, and FOS (n = 8).

Effects of GMF Administration during the First Week on the Microbiota Composition of Neonatal Piglets on Day 8 and Day 21

To investigate the early differences of the bacteria community between piglets from CON and GMF groups, the microbial diversity, composition, and differences were assessed by 16S rRNA high-throughput sequencing.

On day 8 (Figure ), the α-diversity showed that GMF administration decreased the Sobs index (P < 0.05) (Figure A) without altering the Shannon index (Figure B). For the β-diversity, principal coordinates analysis (PCoA) showed significant differences between the CON group and the GMF group (Figure C). The bar plots of the community showed that Firmicutes and Bacteroidetes were dominated phyla in piglets of both the CON group and the GMF group (Figure D), as well as the genus Bacteroides, followed by Lactobacillus (Figure E). Differential microbiota of piglets demonstrated that GMF administration significantly enriched the genus of Lactobacillus, whereas it reduced unclassified_f__Lachnospiraceae (P < 0.05) (Figure F). Furthermore, linear discriminant analysis effect size (LEfSe) analysis also confirmed the significantly elevated Lactobacillus in piglets of the GMF group (Figure G).

Figure 1

Effects of GMF administration during the first week on fecal microbiota composition of the neonatal piglets on day 8. α-diversity (Sobs index and Shannon index) (A,B); β-diversity of PCoA based on unweighted unifrac distances (C); microbial composition at the phylum and genus levels (D,E); differential microbial composition based on Wilcoxon rank sum test (F), values are means with their standard errors represented by horizontal bars; LEfSe analysis at the genus level (G), LDA score >4; *P < 0.05; **P < 0.01; CON, piglets in the CON group administered with saline; GMF, piglets in the GMF group administered with the combination of GOS, MFGM, and FOS (n = 5).

On day 21 (Figure ), GMF administration increased the Sobs index (Figure A) and Shannon index (Figure B). PCoA revealed significant differences between the CON group and the GMF group (Figure C). As for the microbial composition, the phyla of Firmicutes and Bacteroidetes (Figure D) and the genus of Bacteroides were most dominant in both the CON group and the GMF group (Figure E). Microbial differences at the genus level indicated the increased norank_f__Muribaculaceae, Enterococcus, Christensenellaceae_R-7_group, and Romboutsia and decreased [Eubacterium]_coprostanoligenes_group in the GMF group (P < 0.05) (Figure F). LEfSe analysis declared the upregulation of the genera norank_f__Muribaculaceae, Enterococcus, Romboutsia, Ruminococcaceae_UCG-002, Christensenellaceae_R-7_group, Marvinbryantia, CHKCI001, and unclassified_k_norank_d_Bacteria in the GMF group (Figure G).

Figure 2

Effects of GMF administration during the first week on fecal microbiota composition of the neonatal piglets on day 21. α-diversity (Sobs index and Shannon index) (A,B); β-diversity of PCoA based on unweighted unifrac distances (C); microbial composition at the phylum and genus levels (D,E); differential microbial composition based on the Wilcoxon rank sum test (F), values are means with their standard errors represented by horizontal bars; LEfSe analysis at the genus level (G), LDA score >4; *P < 0.05, **P < 0.01, ***P < 0.001; CON, piglets in the CON group administered with saline; GMF, piglets in the GMF group administered with the combination of GOS, MFGM, and FOS (n = 5).

Effects of GMF Administration during the First Week on Microbial Functional Profiles of Neonatal Piglets on Day 8 and Day 21

To further explore the functional profiles of the bacterial community, we applied Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) by using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Figure ).

Figure 3

Effects of GMF administration during the first week on microbial functional profiles of the neonatal piglets on day 8 and day 21. Microbial functional profiles of the neonatal piglets on day 8 (A) and day 21 (B). Values are means with their standard errors represented by horizontal bars; *P < 0.05; **, P < 0.01; CON, piglets in the CON group administered with saline; GMF, piglets in the GMF group administered with the combination of GOS, MFGM, and FOS (n = 5).

On day 8, the results showed that GMF administration increased the genes involved in glycolysis/gluconeogenesis, glycerolipid metabolism, mitogen-activated protein kinase (MAPK) signaling pathway, endocytosis, isoflavonoid biosynthesis, and caffeine metabolism while decreased the genes associated with porphyrin and chlorophyll metabolism and nitrogen metabolism (P < 0.05) (Figure A).

On day 21, GMF intervention markedly enriched the genes related to the methane metabolism, arginine, and proline metabolism, oxidative phosphorylation, phenylalanine, tyrosine, and tryptophan biosynthesis, butyrate metabolism, lipid biosynthesis proteins, propionate metabolism, valine, leucine and isoleucine degradation, beta-alanine metabolism, phenylalanine metabolism, tryptophan metabolism, and RNA polymerase, limonene, and pinene degradation but dropped genes connected with other ion-coupled transporters and other transporters (P < 0.05) (Figure B).

Effects of GMF Administration during the First Week on Intestinal Short-Chain Fatty Acids and Their Receptors of Neonatal Piglets on Day 21

In order to evaluate the microbial metabolites of neonatal piglets after GMF administration, we measured the intestinal short-chain fatty acids (SCFAs) levels and gene expressions of G protein-coupled receptors (GPRs) in piglets on day 21. The propionate concentration in ileum and acetate concentration in colon were significantly higher in GMF piglets than those in CON piglets (P < 0.05) (Figure A). What is more, the gene expressions of GPR41 in ileum and GPR41 and GPR43 in the colon of piglets were remarkably upregulated after GMF administration (P < 0.05) (Figure B).

Figure 4

Effects of GMF administration during the first week on intestinal SCFAs and their receptors of the neonatal piglets on day 21. Intestinal luminal SCFAs concentrations (A) and GPRs (B). *P < 0.05. CON, piglets in the CON group administered with saline; GMF, piglets in the GMF group administered with the combination of GOS, MFGM, and FOS (n = 5).

Effects of GMF Administration during the First Week on Intestinal Barriers of Neonatal Piglets on Day 21

To identify the intestinal villi structural development, the barrier functions, and gut permeability after GMF administration, the gut morphological structure, barrier-associated gene expressions in mucosa, and plasma DAO level were measured. In the ileum, the gene expressions of tight junction proteins (E-cadherin and ZO-1) (Figure A), mucins (Mucin-1, Mucin-2, and Mucin-4) (Figure B), and IL-22 (Figure C) were elevated in piglets from the GMF group (P < 0.05). Similarly, the gene expressions of tight junction proteins (Occludin, Claudin-1, and ZO-1) (Figure D), Mucin-20 (Figure E), and cytokines (TNF-α and IL-1β) (Figure F) were also upregulated in the colon of piglets from the GMF group (P < 0.05). Furthermore, the plasma DAO level was significantly declined in the GMF group (P < 0.05) (Figure G).

Figure 5

Effects of GMF administration during the first week on intestinal barrier-associated gene expressions and gut permeability of the neonatal piglets on day 21. Intestinal barrier-associated gene expressions in ileal mucosa (A–C) and colonic mucosa (D–F); plasma DAO level (G); values are means with their standard errors represented by vertical bars; *P < 0.05; CON, piglets in the CON group administered with saline; GMF, piglets in the GMF group administered with the combination of GOS, MFGM, and FOS; DAO, diamine oxidase; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-10, interleukin 10; IL-22, interleukin 22; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α; ZO-1, zonula occluden 1 (n = 5).

Spearman Correlation Analysis between Differential Bacteria and Intestinal Barrier-Associated Genes of the Neonatal Piglets on Day 8 and Day 21

To confirm the relationship of microbiota alteration and associated barrier functions, the spearman correlation analysis was performed. The results showed that the enriched Lactobacillus on day 8 was positively related with gene expressions of TNF-α, Mucin-4, Claudin-1, and IL-1β, whereas the genus unclassified_f__Lachnospiraceae was negatively associated with gene expressions of ZO-1, TNF-α, Mucin-1, and Mucin-4 (P < 0.05) (Figure A). On day 21, the elevated genera of norank_f__Muribaculaceae, Christensenellaceae_R-7_group, Enterococcus, and Romboutsia showed positive connection with almost all barrier function genes (P < 0.05) (Figure B).

Figure 6

Correlation analysis between differential bacteria and intestinal barrier-associated genes of the neonatal piglets. Spearman correlation coefficients of the impacted bacteria on day 8 (A) and day 21 (B) with intestinal barrier-associated genes: orange color represents the positive correlations, and green color represents the negative correlations; *P < 0.05; **P < 0.01; ***P < 0.001.

Discussion

After birth, the newborn neonates experience rapid changes in intestinal functions and the gut morphological maturation, coupled with diverse microbial colonization during the neonatal period, which served as vital foundations for promoting health and growth in piglets.[19] Accumulating research studies have reported that early nutritional intervention performs advantageous effects on intestinal integrity and functions via shaping gut microbiota.[20] In the present study, we found that orally administration with the combination of GOS, MFGM, and FOS to neonatal piglets during the first week motivated the beneficial bacteria colonization, which associated with intestinal barrier functions enhancement and contributed to improve the growth performance during the whole neonatal period.

Emerging research studies highlighted the growth-enhancing benefits of functional additives in pigs.[21] GOS and FOS are prebiotics which can be fermented by gut microbiota to produce SCFAs, playing advantageous roles for intestinal development and growth.[22,23] In addition, MFGM is a protein–lipid complex surrounding the milk fat globules, reported to accelerate the growth of infants and piglets.[24] In the present study, GMF administration during the first week, with the homogeneous average daily feed intake (ADFI) of the creep feed (73.47 vs 69.27 g; SEM = 11.15, P = 0.732), improved the growth performance of piglets during the whole neonatal period, indicating the positive and durable effects of combined GOS, MFGM, and FOS on growth promotion.

Much insight has focused on the diet-microbiota crosstalk, and diet emerges as a pivotal determinant of the gut microbiota community structure and function.[25] Previous studies showed that dietary GOS and FOS interventions modulated the microbial community in the neonatal period.[26] Furthermore, MFGM fractions can be utilized by the biofilms to exert antimicrobial and anti-inflammatory effects on animals.[27] In the current study, GMF administration for neonatal piglets enriched the genus Lactobacillus on day 8, which is consistent with the previous research studies.[28] Additionally, Lactobacillus was known as one of the beneficial microbes in the neonatal intestines contributing to program the microbial composition and balance the metabolism in the early life of piglets.[29] In addition, the increased genera norank_f__Muribaculaceae, Enterococcus, Christensenellaceae_R-7_group, and Romboutsia were detected on day 21 after GMF administration. Recent studies demonstrated that the family of Muribaculaceae, also known as family S24-7, was tightly associated with carbohydrate degradation.[30] We supposed that GOS and FOS might be degraded into SCFAs and other metabolites, which helped to improve the intestinal development and regulate the microbial population.[31,32]Enterococcus was a ubiquitous group of commensal bacteria with great relevance to health.[33] Besides, Enterococcus faecalis was regarded as probiotics to modulate the intestinal flora and gut health in piglets.[34] The genus of the Christensenellaceae_R-7_group, dominated in piglets after GMF administration, was also reported to deliver benefits for health.[35] In addition, Romboutsia was identified as the key components to utilize FOS for microbiota modulation.[36]

Increasing evidence pointed out that luminal microbiota served as a mediator of dietary impact on the host metabolic status.[37] Gut microbes participated in numerous nutrient metabolisms, such as carbohydrates, lipids, and proteins,[38] and several metabolic diseases were associated with the microbial dysbiosis.[39] Previous research studies declared that GOS and FOS changed the microbial community and their associated metabolism.[40,41] In the present study, results based on the PICRUSt analysis of microbial functional profiles on day 8 displayed the promotion for the glycolipid metabolism, gluconeogenesis metabolism, and MAPK signaling pathway after GMF administration, from which it can be inferred that the activated MAPK pathway might generate more energy-related metabolites for piglets to facilitate the health status and growth performance.[42] Moreover, microbial functional profiles on day 21 that are involved in the metabolism of functional amino acids, such as tryptophan, valine, leucine, and arginine, conveyed varieties of beneficial effects in the immunological defense and intestinal development.[43] Meanwhile, from the motivation of butyrate metabolism and propionate metabolism on day 21, it could be inferred that GMF administration during the first week might shape the microbial colonization in the early life and durably modulate the microbial populations and their metabolic status on day 21. The microbial metabolites, such as SCFAs, could not only serve as energy resources for epithelial cells[44] but also stimulate the expressions of their receptors to activate the downstream pathway to improve the intestinal development,[45] which consequently contributed to the better growth performance.

Beneficial bacteria colonization can assist in improving the intestinal barrier integrity and functions.[46] The spearman correlation analysis affirmed the indispensable role of the enriched beneficial microbes in improving the intestinal barrier functions of piglets. On the one hand, the colonized bacteria could directly influence physiological and homeostatic status in the host by crosstalking with epithelial cells through redox signaling.[47] On the other hand, the combination of GOS, MFGM, and FOS could be utilized by the luminal microbes, and their metabolites might enhance the intestinal integrity and barrier functions.[48] Actually, tight junctions are most important components responsible for maintaining the paracellular permeability.[49] In the present study, the upregulation of tight junction protein genes (E-cadherin, Occludin, Claudin-1, and ZO-1) indicated the enhancement of the physical barrier.[50] The chemical barrier is a primary dynamic mucus layer with secreted mucins covering the epithelium, and the elevated mucin genes (Mucin-1, Mucin-2, Mucin-4, and Mucin-20) in this study illuminated the well-established mucus layer for preventing pathogen invasion.[51] The immunological barrier protects the host from infection by secreting cytokines and antimicrobial peptides.[52] In the present study, the first week GMF administration upregulated the gene expressions of IL-1β, TNF-α, and IL-22 in piglets on day 21. In addition to the functions in inflammation, IL-1β and TNF-α, at low levels, were reported to have beneficial homeostatic functions, such as host defense against pathogens.[53,54] IL-22 was a cytokine of the IL-10 family and well known for its antimicrobial and tissue-protective properties, which contributed to the pathogen clearance and microbial community modulation.[55] What is more, DAO was originally located in epithelial cells, and the lower content of DAO in plasma after GMF administration represented the adverse intestinal permeability and improved barrier functions,[56] which ultimately contributed to the growth enhancement of piglets.

Conclusions

Oral administration of combined GOS, MFGM, and FOS to the neonatal piglets during their first week after birth facilitated the beneficial bacteria colonization (Lactobacillus, Entercoccus, and Romboutsia), increased luminal SCFA levels, and enhanced the intestinal barrier functions by elevating the gene expressions of tight junctions (Occludin and ZO-1), mucins (Mucin-2 and Mucin-4), and cytokines (IL-1β and IL-22), which contributed to the growth performance of piglets during the whole neonatal period. Our findings will provide important insights into the application of GOS, MFGM, and FOS in modulating gut microecology of neonates during their early life.

Materials and Methods

Animals and Experimental Design

The animal experimental protocols and sampling procedure were approved by the China Agricultural University Animal Care and Use Committee (AW07040202-1, Beijing, China). Totally, 16 newborn piglets (Duroc × Landrace × Yokshire) with an average birth weight of 1.52 ± 0.04 kg were obtained from 16 litters (one piglet in each litter) and then divided into the CON group and the GMF group. Piglets in the GMF group were orally administered 5 mL of GMF solution (GOS/MFGM/FOS = 62.2%:35.2%:2.6%, provided by the Beijing Sanyuan Foods Co. Ltd, Beijing, China) with a dose of 1.2 g/kg body weight during the first week after birth. The purities of GOS, MFGM, and FOS were, respectively, 90, 95, and 93% (w/w) on dry matter. Similarly, piglets in the CON group were orally administered the same dose of physiological saline. The piglets started to receive a commercial creep feed from day 8 postpartum and had free access to sow milk and water throughout the whole neonatal period. The health statuses of piglets were monitored daily, and their body weights were recorded on day 21. The characteristics of lactating sows in the CON group and the GMF group were similar, including body weight (253.38 vs 250.75 kg; SEM = 10.50, P = 0.806), backfat thickness (2.46 vs 2.47 mm; SEM = 0.40, P = 0.937), and litter size (12.88 vs 12.63; SEM = 0.61, P = 0.688). All of the sows during lactation were fed the same lactating diet (Table S1) with approximately 6 kg per day.

Sample Collection

On day 8 and day 21 of the neonatal period, feces of piglets were collected and snap-frozen in liquid nitrogen for microbiota analysis. On day 21, five neonatal piglets per group (approximate to the average body weight of each group) were selected for sampling. The blood samples were collected from the jugular vein, and plasma was cautiously collected after centrifuging at 3000g at 4 °C for 10 min. After being humanely euthanized, mid-ileal and mid-colonic segments were fixed in the formalin for morphology analysis, and the mucosa and luminal digesta from ileum and colon were quickly obtained and frozen in liquid nitrogen for gene expression and SCFA measurement. All samples were stored at −80 °C until further analysis.

Plasma Diamine Oxidase Measurement

The plasma diamine oxidase (DAO) level was measured by ELISA according to the manufacturer’s instructions (Beijing Sino-UK Institute of Biological Technology, Beijing, China).

Intestinal Morphological Analysis

Intestinal samples were removed from 10% phosphate-buffered formalin, dehydrated through a graded ethanol series (70 to 100%), cleared with xylene, and then embedded in paraffin wax. Serial sections (5 μm thickness) were cut by a LEICA RM2135 rotary microtome (Leica Microsystems GmbH, CA, U.S.) and stained with hematoxylin and eosin. A minimum of 15 intact and well-oriented villi and their associated crypts from each segment were measured at 100× magnifications under bright field on a Zeiss Axio Imager microscope (Carl Zeiss Microscopy LLC, NY, U.S.). The villus height was measured from the tip of the villi to the villus crypt junction, and the crypt depth was defined as the depth of the invagination between adjacent villi.

Bacterial DNA Extraction, 16S rRNA Gene Amplification, and Sequencing

Five fecal samples of neonatal piglets in each group on day 8 and day 21 were randomly selected for total genomic DNA extraction, which was conducted by using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Tübingen, Germany) according to the manufacturer’s protocol. The V3–V4 region of the 16S rRNA gene was amplified using universal primers of 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplified products were detected by 2% agarose gel electrophoresis and then purified by an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, California, United States). A Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was subsequently used to quantify amplified products. Purified PCR products were pooled into equimolar amounts and sequenced on the Illumina HiSeq 2500 platform to generate paired end reads of 300 bp.

Analysis of Sequencing Data

Raw paired-end reads were strictly analyzed using QIIME (version 1.9). In brief, the low-quality sequences with a length of <220 or >500 nt, an average quality score of <20, and sequences containing >3 nitrogenous bases were removed. UPARSE (version 7.0) was used to cluster remaining high-quality sequences into OTUs with 97% similarity, and chimeric sequences were removed using UCHIME. The taxonomy assignment of OTUs was conducted with the RDP classifier against the SILVA 16S rRNA gene database (Release132) with a confidence threshold value as 0.70. The data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com).

Luminal SCFAs Concentrations Measurement

SCFAs including acetate, propionate, and butyrate in luminal contents were quantified using an ion chromatograph. In brief, 0.5 g of digesta samples was weighed and dissolved in 8 mL of ultrapure water to homogenize and then centrifuged at 5000g for 10 min. After this, the supernatants were diluted as 1: 50 and filtered through a 0.22 μm membrane and then subjected to an ion chromatography system (DIONEX ICS-3000, Thermo Fisher Scientific, Waltham, Massachusetts, United States) for SCFA measurement.

RNA Isolation, cDNA Synthesis, and Real-Time Quantitative PCR

Total RNA of mid-ileal and mid-colonic mucosa was extracted by the Trizol reagent (Invitrogen, United States) by following the protocol, whereas cDNA was obtained by using a Prime Script RT Kit (Takara, Kusatsu, Shiga, Japan). RT-qPCR was performed according to the SYBR Premix Ex Taq II instructions (Takara, Kusatsu, Shiga, Japan), and the reaction was conducted on a Light Cycler System (Roche, South San Francisco, California, United States). Primers for RT-qPCR are listed in Table S2 and were synthesized by Generay Company (Shanghai, China). Amplifications were performed in triplicate for each sample. The relative expression of target genes to that of the reference gene (GAPDH) was calculated according to the 2–ΔΔ method.

Statistical Analysis

The data were analyzed by SPSS 20.0 (IBM, United States), and the results were shown as the mean ± SEM (standard error of mean). The Wilcoxon rank-sum test was applied for the analysis of microbial differences. The Spearman rank correlation coefficient was used for the evaluation for the correlation analysis. In other analyses, the Student’s t-test was used for determining the statistical differences and GraphPad Prism (version 7, GraphPad Software, United States) was used for the graphical representations. All statistical analyses were considered significant at P < 0.05.