Choline and Bile Acids Mixture Improved Growth Performance and Intestinal Immunity via Altering Gut Microbiota and Bacterial Metabolites in the Colon of Weaned Piglets.

Background: Choline or bile acids has many benecial roles in physiological function. However, little was known about growth performance, intestinal mucosal function and microbiota-host interactions of weaned piglets in response to choline or bile acids supplementation. This study aimed to investigate the effect of choline and bile acids mixtures (ChB) supplementation on growth performance, intestinal mucosal barrier function, gut microbiota and bacterial metabolites of weaned piglets. One hundred and twenty-eight crossbred (Duroc × Landrace × Large White) weaned piglets (initial body weight: approximately 8 kg; 21 d of age) were randomly allocated to four different dietary treatments(a control diet (Control) and the other three groups were control diet supplemented with 800 mg/kg choline chloride (choline), 500 mg/kg bile acids (bile acids) or 800 mg/kg choline chloride plus 500 mg/kg bile acids (ChB), respectively) and for 28-d feeding trail. Results: ChB signicantly increased average daily gain (ADG) and reduced feed/gain (F/G) ratio, associated with elevation of lipase activity and total bile acids level in ileal digesta compared with control diet. Additionally, ChB altered colonic microbiota by increasing the relative abundance of Lactobacillus and Faecalibacterium , and decreasing the relative abundances of unidentied-Clostridiales , Parabacteroides and Unidentied-Ruminococcaceae , when compared with control diet. Meanwhile, ChB increased the butyrate level and decreased the production of bile acid proles in the colonic digesta. Besides, feeding ChB improved gut immunity, as reected by increasing the abundance of IL-10 , FXR and mucin2 transcript, while downregulated expression of TLR4 , MyD88 , NF-κBp65 and TNF-α genes in the intestinal mucosa. Quantitative proteomics of jejunal mucosa further showed that ChB regulated the proteins that were related to inammatory response. Furthermore, the changes in the ADG and genes expression were associated with alteration of gut microbiota composition and their metabolites.


Background
It has been demonstrated that weaning stress could cause intestinal in ammation and intestinal dysfunction in piglets, leading to diarrhea, poor growth of weaned piglets [1,2]. Antibiotics that e ciently improved growth performance and reduced the diarrhea of weaned piglets, is no longer feasible due to the increasing bacterial resistance and public health concerns [3]. As a result of antibiotics prohibition recently, an urgent need to nd potential alternative for safeguarding general health of weaned piglets.
As a crucial dietary nutrient, choline has many bene cial roles in physiological function. The well-known function of choline was to emulsify fats, which extended the contact surface between lipase and fats, and accelerated lipids metabolism in the small intestine [4]. Besides, several studies recently showed that choline also exerted an anti-in ammatory effect. Dietary choline inhibited the expressions of proin ammatory biomarkers including TNF-α, IL-1β and NF-κB, while increased the abundance of anti-in ammatory cytokine genes in sh models [5,6]. To our knowledge, however, evidence to con rm the effect of dietary choline on the intestinal mucosal barrier function of mammal, especially weaned piglets was scarce.
Bile acids were produced in the liver and then released into small intestine after feed intake to promote emulsi cation of fats and accelerate lipids digestion and absorption. A majority of bile acids was reuptake in the ileum, while a small amount would reach to the colon and metabolized by resident microbiota [7].The major functions of bile acids has been shown to assist the intestinal absorption of lipids, to maintain homeostasis between gut microbiota and intestinal mucosal barrier [8]. A previous study in sh revealed that bile acid supplementation promoted lipids utilization and improve sh growth performance and feed e ciency [9]. In addition, several studies demonstrated that administration single bile acid (deoxycholic acid or chenodeoxycholic acid) could alleviate gut-associated in ammation and improve gut health, but without effect on the growth performance of weaned piglets [10,11]. Nevertheless, no established study has reported the effects of bile acids mixture on the growth performance and intestinal immune response of weaned piglets. Besides, secreted bile acids by weaned piglets were in small amounts and have limited ability to emulsify dietary fats [12]. Consequently, it may be a potential feeding strategy to supplement bile acids into diet for increasing fats utilization as well as maintaining gut health in weaned piglets.
In recently decade, microbe-host crosstalk is a particularly popular area. Gut microbiota, rapidly affected by feed dietary, play a critical role in nutritional digestion and host metabolism in human and other mammals [13,14]. As a substrate shared by the host and the gut microbiota, diet impacted host physiology through numerous bioactive bacterial metabolites. Previous studies have demonstrated the importance of bacterial metabolites such as short-chain fatty acids and bile acids for regulation of in ammatory [15,16]. Despite the potential of gut microbiota to modulate bioavailability of dietary choline and bile acids [17,18], only few studies have investigated the effect of dietary choline or bile acid supplementation on the gut microbial ecosystem [19,20].
Taken together, the data introduced above showed that little was known about growth performance, intestinal mucosal function and microbiota-host interactions of weaned piglets in response to choline or bile acids supplementation. Here, we focused on determining the effects of dietary supplementation with choline, bile acids or the two conjugates on the growth performance, microbial composition, bacterial metabolite pro les and the expression of proteins/genes related to innate immune response in the intestine.

Materials And Methods
All animal procedures used in this study were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences (authorization number GAASIAS-2016-017). All efforts were made to minimize any suffering of animals, following the Guidelines for the Care and Use of Animals for Research and Teaching.

Animals and diets.
A total of 128 weaned pigs (Duroc × Landrace × Yorkshire × Pietrain, 21 d of age) with an initial body weight of 8 kg were randomly allocated to 4 dietary treatments with 8 replicate pens per treatment, each pen containing 2 barrows and 2 gifts with a complete randomized experimental design. Control piglets (Control) were fed a basal diet, and the other groups were fed basal diet supplemented with 800 mg/kg choline chloride (choline), basal diet supplemented with 500 mg/kg bile acids (bile acids) or basal diet supplemented with 800 mg/kg choline chloride plus 500 mg/kg bile acids (ChB) for a 28-d feeding trial.
The basal diet based on pig body weight was formulated to meet nutrient recommendation of the National Research Council (NRC, 2012), and the compositions of the basal diet and nutrient pro le were presented in Table 1. All pigs had free access to feed and water throughout. At the end of study, the ADG, average daily feed intake (ADFI) and F/G ratio were calculated. One pig from each pen with medium bodyweight was selected and sacri ced after anesthesia with sodium pentobarbital (40 mg/kg BW). Digesta of intestine and colon, and craped mucosal samples of the middle duodenum, the middle jejunum, distal ileum and colon were immediately collected and frozen in liquid nitrogen, and then stored at -80°C.

Analysis of biochemical variables in intestinal digesta
The concentrations of lipase activity, total bile acids, non-free fatty acid (NEFA), total cholesterol (T-CHO) and triglyceride (TG) in digesta of duodenum, jejunum and ileum were measured using commercial kits purchased from the Nanjing Jiancheng Institute of Bioengineering, China.

Determination of SCFA concentrations
Colon content samples from weaned piglets were determined the concentrations of acetic acid, propionic acid and butyric acid by gas chromatography using a GCMS-7890B-7000D Ultra instrument (Agilent Technologies Inc.). Brie y, about 0.1 g of colon digesta of each sample was diluted with 2 mL of 25% metaphosphoric acid solution and vortexed for 2 min until the mixture was homogenized, and then 2 mL diethyl ether was added for 10 min extraction. Afterwards, this mixture was centrifuged at 4000g for 10 min at 4 °C. The collected supernatant was ltered through a 0.22-mm membrane and then analyzed using gas chromatography. Besides, an external standard calibration was used to determine the concentration of each SCFA.

Gut microbiome
Total DNA of each sample was extracted using the a QIAamp PowerFecal DNA Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. The DNA concentration and quality of each sample were detected using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scienti c, Wilmington, DE, United States). The genes of all bacterial 16S rRNA covering V3-V4 region were ampli ed with a universal forward primer 338F (50-ACTCCTRCGGGAGGCAGCAG-30) and a reverse primer 806R (50-GGACTACCVGGGTATCTAAT-30). PCR amplicons were puri ed using the Qiagen Gel Extraction Kit (Qiagen, Germany) in accordance with the manufacturer's instructions. Sequencing libraries were generated using a TruSeq® DNA PCR-Free sample preparation kit (Illumina, USA) according to the manufacturer's recommendations, and the index codes were added. The quality of library was then evaluated by a Qubit@ 2.0 Fluorometer (Thermo Fisher Scienti c, Carlsbad, CA, United States) and an Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated. Bioinformatics analysis was conducted following a recent study [21].
Quasi-targeted metabolomic analysis of bile acid pro les in colonic digesta 100 mg of colonic digesta were grinded with liquid nitrogen and then 500μL prechilled solution containing 80% methanol and with 0.1% formic acid was added. This lysate was well vortexed until the mixture was homogenized. The homogenate was incubated on ice for 5 min and then were centrifuged for 10 min (at 15000 rpm, 4°C). Then the obtained supernatant was diluted with LC-MS grade water (2:1, v/v)) and centrifuged at 15000 g for 20 min at 4°C. After centrifugation, the supernatant was collected and used for LC-MS analysis. The raw HPLC-MS/MS data were processed using the SCIEX O. After normalization to total peak intensity, the processed data were performed at metaX, where can exibly and comprehensively processed metabolomics data. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to evaluate its contribution to the classi cation. Univariate analysis (t-test) was applied to calculate the statistical signi cance (p value). The metabolites with VIP > 1 and P-value< 0.05 and fold change≥2 or FC≤0.5 were considered as signi cantly differential metabolites. R language package was used to plot clustering heat maps after the data were normalized using z-scores of the intensity areas of bile acids pro le.
Gene expression was determined by qPCR according to the method described in a previous study [22].
Gene-speci c primers were listed in Table 2. The mRNA abundance of the target genes, relative to β-actin was analyzed using the 2 −ΔΔCt method, ΔC t = C t (target gene) − C t (β-actin) and ΔΔC t = ΔC t (Treatment) -ΔC t (Control) .  Table 2 Primer sequences used in this study.

Proteomic analysis
An isobaric tag for relative and absolute quantitation (iTRAQ) analysis was carried out to detect changes in the proteomic pro le of jejunal mucosa among the four diet treatments according to the method described in a previous study [23]. A reliable protein was identi ed accordance to the screening criteria: unused>1.3, unique peptide⩾1. The identi ed protein was considered as (DEP) when the fold change was greater than 1.2 or less than 0.83, accompanied with t-test p-value less than 0.05.

Statistical Analysis
Data were expressed as means ± SEM. The data analyses of growth performance, biochemical indexes and genes expression were carried out by SPSS 20.0 software (SPSS v. 20.0, SPSS Inc., Chicago IL, USA). The other traits were performed using GraphPad Prism Version 8 (GraphPad Software, La Jolla, CA). The statistical signi cance of differences was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test, and difference was considered signi cant when p< 0.05. The correlations between gut microbiota, bacterial metabolites and genes expression were analyzed by Pearson's correlation using SPSS 20.0 software and signi cant differences were declared when p < 0.05.

Results
Diets effect on growth performance of weaned piglets As shown in Table 3, piglets fed with ChB diet gained a greater body weight and a signi cantly higher ADG than those fed control diet during the post-weaning period  Diet effects on the growth performance in weaned piglets.

Diet effect on biochemical indexes related to fats digestion in intestinal digesta of weaned piglets
As shown in Table 4, piglets fed ChB displayed a notably higher lipase activity in jejunal digesta, and signi cantly higher level of total bile acids in both jejunal and ileal digesta than those fed control diet.
However, there were no signi cant differences in lipase activity and total bile acids concentration between choline and control group or bile acids and control group. In addition, both choline and ChB supplementation signi cantly increased the NEFA content in the jejunal digesta compared to control diet.
No difference for NEFA content in the jejunal digesta was found between bile acids group and control group. However, bile acids decreased NEFA content in the ileal digesta compared with control diet.
Furthermore, choline, bile acids as well as ChB diet signi cantly decreased TG concentration in the jejunal digesta compared with control diet. Choline group or bile acids group also showed a profound reduction of TG concentration in the ileal digesta, when compared to the control group. Besides, both choline diet and bile acids diet signi cantly reduced the T-CHO level in the duodenal digesta compared to control diet.
T-CHO level in the duodenal digesta of ChB group tend to be lower than the control group, but without statistics. In the jejunal digesta, bile acids diet markedly decreased the level of T-CHO, as compared with control diet.  were identi ed in control group, choline group, bile acids group and ChB group, respectively, and 666 OTUs were shared among the four groups (Figure 1a). The diversity and richness of the colonic digesta microbiota were present in Table 5. ChB supplementation caused reduction in richness and diversity indices compared with control diet, as re ected by the reduction in Shannon, Simpson and ACE index with statistical differences. Beta-diversity analysis showed clustering of samples according to diets. In particular, the PCA results showed that ChB group and choline group were separated from control group (Figure 1b). Diet-induced changes in bacterial composition were clearly visible at the genus level. Lactobacillus and Faecalibacterium were considerably more abundant in piglets fed ChB than those in control group. In addition, the relative abundances of unidenti ed-Clostridiales, Parabacteroides and Unidenti ed-Ruminococcacace were decreased by ChB, as compared with the control diet. Piglets fed choline showed a decreased abundance of Parabacteroides compared to those fed control diet (Figure 1c, d  Table 5 Summary statistic of colonic digesta bacterial community at the 3% dissimilarity level.
Diet effect on SCFA production in weaned piglets No signi cant differences in propionate level among four groups were found (Figure 2). Choline induced an increase in the level of acetate compared with control group. Compared to piglets fed control diet, those fed ChB diet showed a profound elevation of the concentration of butyrate. However, there was not different in the concentration of butyrate among the other three groups.
Diet effect on bile acid pro les level in weaned piglets As shown in Figure 3, in the colonic digesta, levels of primary bile acids including chenodeoxycholic acid (CDCA) and 3β-ursodeoxycholic acid (3βUDCA), and secondary bile acid including hyodeoxycholic acid  Figure 4a, b, c and Table 6, there were 11,10 and19 DEPs related to innate immune system in the choline group, bile acid group and ChB group, respectively, as compared with the control group. In addition, the DEPs accumulated to NF-κB, Tolllike receptor and NOD-like receptor signaling pathway, which were highly involved in in ammation, were analyzed. Compared to the control diet, choline diet upregulated Ig-like domain-containing protein and  Table 6 Differentially expressed proteins in the pathway of immune system compared with control diet fed weaned piglets.
Diet effect on the intestinal mucosal barrier function of the weaned piglets As shown in Figure 5, there was no signi cant effect of diet treatments on the abundance of claudin-1 and occludin transcripts in the intestine. However, ChB supplementation profoundly elevated the abundance of IL-10 transcript, while signi cantly decreased MyD88, TLR4 and TNF-α genes expression in the duodenal mucosa compared to the control diet, the transcript level of NF-κBp65 tended to have the same variable trend, but without no statistics. In the jejunal mucosa, ChB diet signi cantly increased the expression of mucin2 gene, while markedly decreased abundance of MyD88, TLR4, TNF-α and NF-κBp65 transcripts. Piglets fed ChB showed a higher amount of ileal IL-10 gene expression. Choline supplementation induced a visibly higher abundance of IL-10 transcript and lower abundance of TNF-α transcript in the duodenal mucosa, lower abundance of TLR4, TNF-α and NF-κBp65 transcripts in the jejunal mucosa, when compared with those fed control diet. Additionally, bile acids supplementation signi cantly decreased the gene expressions of TNF-α and MyD88 in the duodenal mucosa, and markedly decreased the levels of TLR4, TNF-α and NF-κBp65 transcripts in the jejunal mucosa, but increased the abundance of IL-10 transcript in the ileal mucosa, as compared with the control diet.
Diet effect on the expression of FXR and TGR5 genes No difference in TGR5 gene expression in the ileal and colonic mucosa of weaned piglets was observed among four groups. However, the expression of FXR gene in the colonic mucosa was increased in choline group and ChB group compared to control group. However, no difference for this expression in ileal mucosa among the four groups was observed ( Figure 6).

Correlation analysis between gut microbiota, bacterial metabolites, ADG and intestinal genes expression
A Pearson correlation analysis was used to investigate the associations between genes expression and the abundance of the main microbial genera and their metabolites (Figure 7). The results revealed that the expression of TLR4 transcript was signi cantly negatively correlated with the abundance of Lactobacillus, while positively related to CDCA. MyD88 genes expression showed positive correlations with HDCA and UDCA, while it was negatively associated with butyrate. The abundance of TNF-a gene was positively correlated with level of UDCA. The abundance of NF-κBp65 transcript had a negative correlation with the relative abundance of Lactobacillus. FXR gene expression was signi cantly correlated with the abundance of Lactobacillus, while negatively correlated with the level of 23-NCA, HDCA and UDCA as well as the relative abundance of Parabacteroides and Unidenti ed ruminococcaceae. The abundance of IL-10 transcript showed positive association with acetate level and the relative abundance of Lactobacillus and Faecalibacterium, while showed negative correlation with HDCA, UDCA and Parabacteroides. In addition, ADG was positively correlated with the relative abundance of Lactobacillus and butyrate level.

Discussion
In the present study, bile acids and choline mixture supplementation, but not choline or bile acids alone, improved the growth performance of weaned piglets, re ecting as a marked higher ADG, a lower feed to gain ratio and a more body weight gain than control diet. The increased body weight gain and ADG by ChB was likely due to this mixture supplementation promoted dietary lipids digestion and absorption, re ecting as higher lipase activity in the jejunal digesta and higher concentration of total bile acids in the jejunum and ileum than those in control group [24,25]. Meanwhile, an augment of NEFA level and reduction in TG in the jejunal digesta further con rmed that ChB diet accelerated dietary lipids digestion.
Previous studies demonstrated that dietary choline signi cantly improved the sh speci c growth rate [26,27]. To our knowledge, there was limited study on the choline effect on the swine growth performance.
As for the effect of bile acids supplementation here, there was no impact on growth performance of weaned piglets. This nding was consistent with the previous studies that administration of CDA or CDCA had no effect on the growth performance of weaned piglets [10,11].
Evidence has shown that diets induced the changes in body weight gain by altering the composition of gut microbiota. Gut microbiota play an important role in suppression of pathogen infection, regulation of nutrient digestion and absorption, maintenance of intestinal homeostasis and immune regulation [28]. Bene cial effects of functional substances supplementation on the regulation of gut microbial composition and maintenance of host health have been reported. In the present study, choline and bile acids mixture profoundly enhanced the abundance of the genera Lactobacillus and Faecalibacterium in the colonic digesta, while decreased the abundance of the genera unidenti ed-Clostridiales, Parabacteroides and unidenti ed-Ruminococcaceae. Lactobacillus has been shown to enhance the concentrations of bile acids in the intestinal lumen and therefore promote emulsi cation and absorption of lipids [29]. This nding partly agreed with previously established study that there was a positive correlation between antimicrobial growth promoter induced body weight gain and an increase in abundance of Lactobacillus [30]. In agreement with these nding, our present study denoted that ChB increased total bile acids level in the duodenum and illume may be linked to the increased abundance of genus Lactobacillus. Moreover, a previous study found that the abundance of Parabacteroides had a signi cantly negative association with body mass index [31]. Similarly, investigators found that the abundance of Parabacteroides distasonis was relatively lower in patients with obesity and nonalcoholic fatty liver [32]. Collectively, these nding above suggested that increased ADG and body weight gain may be likely attributed to the increased abundance of Lactobacillus and the reduction in abundance of Parabacteroides induced by ChB supplementation.
Furthermore, Lactobacillus has been repeatedly reported to be crucial for prevention of pathogen infection and alleviation of intestinal in ammation [33]. A previous study demonstrated that a reduction in the abundance of Faecalibacterium in active IBD patient [34]. Prausnitzii, a specie in the genus Faecalibacterium, has been shown to not only produce butyrate, but also display an additional antiin ammatory effect in mice with colitis [35].The enrichment of Ruminococcaceae has been shown to involve in colonic mucosal in ammation, which can trigger colitis upon disruption of the barrier function of colonic epithelial cell [36]. In line with these ndings, the results in the present study indicated that the higher abundances of the bene cial bacteria including Lactobacillu and Faecalibacterium as well as the lower level of Ruminococcaceae induced by ChB supplementation might have bene cial effects on the gut health of weaned piglets.
SCFAs including acetate, propionate and butyrate, were mainly produced by bacterial fermentation of non-digestible carbohydrates in the colon [37]. These substances have been shown to play an important role in alleviation of intestinal in ammation and provide energy for the colonic epithelial cells [38].
Changes in the concentrations of SCFA, speci cally butyrate may be linked to the altered composition of gut microbiota. Herein we found that choline and bile acid mixture greatly increased butyrate level in the colonic digesta. Thus, the higher level of butyrate induced by ChB diet was supported by the increased abundance of Lactobacillus and Faecalibacterium, which were the main butyrate-producing bacteria in the colon [39].
Diet treatments here might have effects on colonic bile acid pro les characterized by a signi cant reduction in several bile acid pro les in ChB group as compared to control group. These results here were partly in line with a previous result that a lower amount of bile acids was associated with higher butyrate concentration in the porcine colon [40]. Accumulating evidence showed that abnormally higher levels of free bile acids in the colon, would disrupt colonic epithelial barrier integrity and cause epithelial cells oxidative stress and apoptosis, leading to gut dysfunction [41,42]. Previous study has reported that CDCA can induce epithelial permeability, which impaired colonic epithelial barrier integrity [43]. Furthermore, increased levels of secondary bile acids displayed harmful effects on the colonic epithelium function through activation of NF-κB [44]. Collectively, the reduction of primary bile acid (particularly CDCA) and secondary bile acid (UDCA and HDCA) induced by ChB supplementation suggested that this supplementation could reduce the cytotoxicity caused by CDCA and secondary bile acid and therefore enhanced gut barrier and alleviated in ammation.
Changes in bile acids concentrations induced by diet treatments in the colon were possible responsible for FXR activation, as it has been demonstrated that UDCA was the antagonists of FXR [43]. In the current study, ChB supplementation increased the expression of FXR gene in the colonic mucous. Increasing evidence repeatedly described FXR activation exhibited anti-in ammatory effects and protected against colitis induced by chemical, whereas FXR knock out mice showed increased susceptibility to chemical injury [45,46]. Together with these results, compelling evidence from proteomic analysis further indicated that ChB supplementation showed a e ciently anti-in ammatory role by increasing the amount of proteins related to anti-in ammatory signaling, such as FCGRT, PDP2 and NF-κB inhibitor epsilon, while signi cantly down-regulated the proteins expressions that were positively promoted in ammatory signaling, including MyD88, DDX58 and BCL10 in the intestine [47,48]. Furthermore, the decreased abundance of TNF-α transcript in duodenal and jejunal mucosa, and the elevated IL-10 gene expression in ileum in the current study may further indicated the lower in ammatory response of weaned pigs fed ChB [49]. Meanwhile, ChB suppressed TLR4-Myd88-NF-κB signal pathway, which play an important role in in ammatory response [50]. Collectively, these nding suggested that ChB e ciently inhibited the in ammatory response, which might contribute to maintaining the intestinal mucosal immune homeostasis. Furthermore, diet effect on the expression of mucin can affect intestinal function and integrity. In the present study, we found that choline and bile acids mixture signi cantly increased the abundance of mucin2, which negatively modulated in ammatory response and played an important role in maintaining homeostasis in the intestinal epithelium [51].

Conclusion
Collectively, our results in the present study indicated that dietary supplementation with choline and bile acids mixture may e ciently improve the growth performance and the intestinal mucosal immunity of weaned piglets through alteration of gut microbial composition and bacterial metabolite pro les.
Therefore, these nding suggested that choline and bile acids mixture may act as a bene cial antibiotic alternative for weaned piglets' production.

Consent for publication
Not applicable.

Availability of data and materials
The datasets analyzed in the current study are available from the corresponding author on reasonable request.

Con icts of Interest
The authors declare that they have no con ict of interest.   Diet affects short chain fatty acid level in colonic digesta of weaned piglets. Concentrations of (a) acetate, (b)propionate and (c)butyrate in the colonic contents of 28-d weaning piglets. All data are expressed as means ± SEM (n = 8). Differences were analyzed by one-way ANOVA followed by Dunnett' T test. *p < 0.05, compared with control diet.   Diets effect on the innate immunity related and tight junction related gene expressions (a) in the duodenal mucosa, (b) in the jejunal mucosa, (c) in the ileal mucosa of weaned piglets. Differences were analyzed by one-way ANOVA followed by Dunnett' T test. *p < 0.05, **p < 0.01, compared with control diet. ChB, choline and bile acids mixture.

Figure 6
Diets effect on the gene expressions of FXR and TGR5 in the ileal mucosa and colonic mucosa. Differences were analyzed by one-way ANOVA followed by Dunnett' T test. *p < 0.05, compared with