Effects of fermented feeds and ginseng polysaccharides on the intestinal morphology and microbiota composition of Xuefeng black-bone chicken.

Fermented feeds contain abundant organic acids, amino acids, and small peptides, which improve the nutritional status as well as the morphology and microbiota composition of the intestine. Ginseng polysaccharides exhibit several biological activities and contribute to improving intestinal development. Here, Xuefeng black-bone chickens were fed a basal diet fermented by Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus plantarum, and Enterococcus faecium, with or without ginseng polysaccharides. The 100% microbially fermented feed (Fe) and 100% microbially fermented feed and ginseng polysaccharide (FP) groups showed significantly increased villus height and villus height to crypt depth ratio, and decreased crypt depth in the jejunum. In the 100% complete feed and ginseng polysaccharide (Po) group, the villus height to crypt depth ratio was significantly increased, crypt depth was reduced, and villus height remained unaffected. Next, we studied the intestinal microbial composition of 32 Xuefeng black-bone chickens. A total of 10 phyla and 442 genera were identified, among which Firmicutes, Proteobacteria, and Bacteroidetes were the most dominant phyla. At the genus level, Sutterella and Asteroleplasma abundance increased and decreased, respectively, in the FP and Po groups. Sutterella abundance was positively correlated to villus height and villus height to crypt depth ratio, and negatively correlated to crypt depth, and Asteroleplasma abundance was positively correlated to crypt depth and negatively correlated to villus height to crypt depth ratio. At the species level, the FP group showed significantly increased Bacteroides_vulgatus and Eubacterium_tortuosum and decreased Mycoplasma_gallinarum and Asteroleplasma_anaerobium abundance, and the Po group showed significantly increased Mycoplasma_gallinarum and Asteroleplasma_anaerobium abundance. Moreover, bacterial abundance was closely related to the jejunum histomorphology. Asteroleplasma_anaerobium abundance was positively correlated with crypt depth and negatively correlated with villus height to crypt depth ratio. Mycoplasma_gallinarum abundance was positively correlated to villus height, and Bacteroides_vulgatus and Eubacterium_tortuosum abundance was positively correlated with villus height to crypt depth ratio and negatively correlated with crypt depth. Therefore, fermented feeds with ginseng polysaccharides may be used as effective alternatives to antibiotics for improving intestinal morphology and microbial composition.

Introduction

It is well known that antibiotic growth promoters are added to animal feed for livestock growth promotion. However, the overuse of antibiotics has led to antibiotic resistance in animal microbial populations and raises the risk of transfer of antibiotic resistance genes to the human microbiota [1], thereby posing a threat to global public health. Hence, the use of antibiotics in animal feeds has been prohibited in many countries [2], which has led to a decline in animal production and an increase in the risk of food-borne infections in consumers due to higher rates of infections in livestock [3]. In order to solve the problems associated with the ban of antibiotics in livestock production, researchers are working towards finding new alternatives to antibiotics, such as the use of feed fermented by microbes. In general, the basal diet that fermented with probiotics has been extensively studied due to its benefits of increasing nutrient bioavailability and nutritional value [4]. Many researchers have reported that the use of probiotics for feed fermentation improves growth performance [4], meat quality [5], ileal amino acid digestibility [6], and immune function [7] in broilers. Other alternatives include addition of biological and functional additives to the diet, such as moringa, mulberry, Chinese herbal medicine, tea polyphenol, dietary fiber, and ginseng polysaccharides. Among them, ginseng polysaccharides, due to their high content of polysaccharides, peptides, saponins, and other active substances, can be used as functional feed additives to promote food intake [8], improve immunity [9], and decrease the amount of abdominal fat and serum cholesterol [10] in broilers. Therefore, if ginseng polysaccharide is mixed with microbially fermented feed, it may have a positive impact on the development of new alternative antibiotics. To date, however, no studies have focused on evaluating the effect of the combined use of these two substances in livestock and poultry diet. Xuefeng black-bone chicken is the most famous native breed in the Hunan province of China. It is greatly favored by people because its meat contains high contents of protein, vitamins, and amino acids [11], which has led to its increased demand. However, its slow growth, long feeding period, and risk of disease during the breeding process largely limits the development of the Xuefeng black-bone chicken industry. In the present study, we aimed to investigate the effect of feed fermented by microbes and ginseng polysaccharide on Xuefeng black-bone chicken intestinal morphology and microflora population. Our findings not only help solve the problems associated with the ban of antibiotics in livestock production, but also contribute to the development of new, effective alternatives to antibiotics, and provide a new scheme for improving intestinal health in Xuefeng black-bone chicken.

Materials and methods

Chickens, diet, and experimental design

For microbial fermentation of feed, the basal diet was mixed with probiotics, including Bacillus subtilis (5 × 108 colony forming units/g), Saccharomyces cerevisiae (5 × 107 colony forming units/g), Lactobacillus plantarum (3 × 108 colony forming units/g), and Enterococcus faecium (5 × 108 colony forming units/mL) (inoculum proportion = 1:1:3:3 v/v, inoculum size = 3% v/v, moisture = 45% w/v, and temperature = 35 ± 1°C). Additionally, molasses (2%) was added and mixed thoroughly using a feed-stuff mixer. The fermented feed was kept in a polyethylene bag with a one-way air valve during the fermentation process, and fermented by anaerobic fermentation for 20 days. The probiotic strains were obtained from the probiotics collection of Guangdong Institute Microbiology (Guangzhou, China).

Xuefeng black-bone chicken, a native breed of China, has excellent quality of meat with high contents of protein, vitamins, and amino acids, and hence was selected for this experiment. A total of 400 Xuefeng black-bone chickens (one-day-old and balanced for body weight) were used in a completely randomized design for a 7-d adaptation period and a 150-d experimental period. The chickens were randomly divided into 4 groups (4 replicates per group and 25 chickens in each replicate): 100% complete feed group (Cn group), 100% microbially fermented feed group (Fe group), 100% complete feed and ginseng polysaccharide (200 g/t) group (Po group), and 100% microbially fermented feed and ginseng polysaccharide group (FP group). In our paper, the Ginseng polysaccharides were extracted from the roots of ginseng with hot water, precipitated by 80% ethanol and deproteinated based on our previous method [12]. Their nutritional requirements were met according to the National Research Council feeding standards (NRC, 1994) and basic diet formulations and nutrient composition are shown in S1 Table. Chickens were raised in floor pens, and placed into separate floor pens with 20 individuals per pen. All birds received feed and fresh water ad libitum throughout the experiment. Feed was removed from the pen 24 hours before sampling. All animal procedures were approved by the Animal Care Committee at South China Agricultural University, and the experimental individuals were anaesthetized prior to exsanguination according to the University’s guidelines for animal research.

Data collection and sampling

On day 150, 2 broiler chickens were randomly selected from each pen (8 broiler chickens/treatment) and euthanized with an overdose of CO2 [13]. The intestinal tract was immediately removed. Tissue samples of the jejunum were obtained and gently flushed with 0.9% saline to remove the intestinal contents and fixed in 10% formalin for histomorphological analysis. Subsequently, the jejunum contents (8 broiler chickens/treatment) were collected in sterile 1.5 mL tubes and stored at –80°C until DNA isolation.

Intestinal histomorphology

The intestinal samples were processed according to the method of Thompson et al. [14]. In brief, the intestinal samples were dehydrated with increasing concentrations of ethanol, cleared with xylene (Surgipath Medical Industries, Richmond, IL), and embedded in paraffin wax (Thermo Fisher Scientific, Kalamazoo, MC). Cross sections (5 μm) were stained with hematoxylin and eosin (GeneCopoeia, Rockville, MD). The stained sections were dehydrated with ethanol, cleared with xylene, and mounted with DPX mountant based on the method of Jiang et al. [15]. ImageJ software (National Institutes of Health, USA) was used to determine the morphometric measurements of villus height and crypt depth of the jejunum using an Olympus BX40 F-3 microscope (Olympus Cooperation, Tokyo, Japan) attached to a digital video camera (Q-imaging, 01-MBF-200R-CLR-12, SN: Q32316, Canada) [16].

DNA extraction, 16S rRNA gene sequencing, and annotation analysis

Total genomic DNA from the jejunum contents was extracted using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the specifcations of the manufacturers. The DNA samples were tested for integrity using 1% agarose gel electrophoresis and their concentration was determined using a Qubit fluorometer (Invitrogen, Carlsbad, CA). Based on the concentration, the DNA sample was diluted to 1 ng/μL using sterile water. The V3–V4 regions of the 16S ribosomal DNA genes were amplified by PCR based on the method of Sun et al. [17]. Sequencing libraries were generated using an Illumina MiSeq Reagent Kit (Illumina, San Diego, CA) on an Illumina MiSeq Sequencer. Single-end reads were assigned to samples based on their unique barcode in the adaptor sequence. Quality filtering of the raw reads was performed to obtain high-quality clean reads according to the Cutadapt quality controlling process [18]. The reads were compared with the reference database [19] using the UCHIME algorithm [20] to detect chimeric sequences [21], and clean reads were finally obtained using the Uparse software (Uparse v7.0.1001) [22]. Sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTU). For each representative OTU, the Silva Database was used to annotate taxonomic information based on the Mothur algorithm [19]. To study the phylogenetic relationships between different OTU, multiple sequence alignment was performed using the MUSCLE software (Version 3.8.31) [23]. Alpha diversity was applied to analyze the complexity of species diversity within groups, including the Observed-species, Chao1, ACE, and Shannon indices. Beta diversity analysis was used to evaluate differences between groups using non-metric multi-dimensional scaling. Two different complementary analyses, analysis of similarity and multiresponse permutation procedure, were used to determine significant differences between jejunum microbiota in their response to fermented diets with or without ginseng polysaccharide. All these indices were calculated using the quantitative insights into microbial ecology pipeline (version 1.7.0) and displayed using the R software (version 2.15.3).

Statistical analysis

The experimental design included a completely randomized design. All data, including villus height, crypt depth, and the ratio of villus height to crypt depth were analyzed using one-way ANOVA and Duncan’s test with SPSS version 17.0 (IBM Corp., Armonk, NY). The R software was used to perform MetaStat analysis to determine differences in the relative abundance of fecal microbiomes [24]. The P values indicated the significant differences at the levels of P < 0.05. The correlation analyses of microbiota composition with the intestinal morphology were tested by the cor function (x, y, use = “p”) (http://127.0.0.1:11153/library/stats/html/cor.html), and illustrated with a function-labelled Heatmap (Matrix, xLabels, yLabels) in the R package WGCNA (http://127.0.0.1:11153/library/stats/html/cor.html).

Results

Histomorphological measurements of the intestine

The effects of fermented feed and ginseng polysaccharide on the villus height, crypt depth, and the ratio of villus height to crypt depth of the jejunum of Xuefeng black-bone chicken were evaluated, as shown in Fig 1. The villus height was significantly increased in the Fe and FP groups (P < 0.05) but not in the Po group compared to that in the Cn group (P > 0.05). The crypt depth was significantly decreased (P < 0.05) in the Fe, Po, and FP groups compared with that in the Cn group. However, no significant differences in crypt depth were observed between the Fe, Po, and FP groups (P > 0.05). The ratio of villus height to crypt depth was significantly higher (P < 0.05) in the Fe, Po, and FP groups compared with that in the Cn group. However, no differences in villus height to crypt depth ratio were observed between the Po and FP groups (P > 0.05) while higher than those in the Fe group (P < 0.05).

Fig 1

Effects of dietary treatment on villus height, crypt depth, and the ratio of villus height to crypt depth in the jejunum of broiler chickens.

Treatments with different letters are significantly different at P < 0.05. Data were obtained from transmission electron microscopy, and were means of 10 birds (2 bird from each pen). Cn, 100% complete feed group; Fe, 100% microbial fermented feed group; Po, 100% complete feed and ginseng polysaccharide group; FP, 100% microbial fermented feed and ginseng polysaccharide group. VH, villus height; CD, crypt depth; V/C, the ratio of villus height to crypt depth.

16S rRNA gene sequencing and annotation analysis

After DNA extraction, the hypervariable V3–V4 regions of the 16S ribosomal DNA were enriched in each sample, and subsequent high-throughput analysis generated a total of 2,886,390 raw reads. On an average, each sample produced approximately 90,200 joined tags, which were assembled using PandaSeq v2.8 (min = 77,322, max = 99,325). Over 77.65% ± 4.33% of the total joined tags from each sample passed quality control and were processed for further analysis (S2 Table). OTU clustering of the high-quality tags yielded 3,318 unique OTU candidates at 97% sequence similarity, and 1,648 candidates that were shared across all samples were defined as core OTU. The core OTU comprised approximately 49.67% of the total candidates, while only 134,166,65 and 370 OTU were identified uniquely in the Cn, FP, Po, and Fe groups (Fig 2A and S3 Table). Additionally, the microbial diversity in the jejunum contents of broiler chickens was assessed using quantitative insights into microbial ecology pipeline based on the OTU annotation, which identified the top 10 phyla (Fig 2B and 2C). The most abundant phylum in the jejunum contents of Xuefeng black-bone chicken was Firmicutes, which accounted for approximately 46.75% of all sequences, followed by Proteobacteria (27.36%) and Bacteroidetes (12.20%) (S4 and S5 Tables). At the class level, a total of 59 classes were detected, and 9 classes, including Clostridia, Gammaproteobacteria, Bacteroidia, Epsilonproteobacteria, Bacilli, Alphaproteobacteria, unidentified_Actinobacteria, Deltaproteobacteria, and Melainabacteria had a relative abundance greater than 1.0%. The most abundant class in the jejunum contents of Xuefeng black-bone chicken was Clostridia (40.21%) (S6 and S7 Tables). At the order level, we detected 11 orders with a relative abundance greater than 1.0% (S8 and S9 Tables). Specifically, the most abundant order in the jejunum contents of Xuefeng black-bone chicken was Clostridiales, which accounted for approximately 39.73% of all sequences, followed by Bacteroidales (11.69%), Campylobacterales (7.12%), Enterobacteriales (6.17%), Lactobacillales (4.64%), Oceanospirillales (4.62%), Alteromonadales (1.87%), Rhizobiales (1.73%), Desulfovibrionales (1.53%), Bifidobacteriales (1.29%), and Caulobacterales (1.04%). At the family level, among the 189 families detected, 17 families, including Ruminococcaceae, Lachnospiraceae, Helicobacteraceae, Enterobacteriaceae, Peptostreptococcaceae, Halomonadaceae, Bacteroidaceae, Lachnospiraceae, Rikenellaceae, Christensenellaceae, Shewanellaceae, Prevotellaceae, Desulfovibrionaceae, Clostridiales_vadinBB60_group, Bifidobacteriaceae, Caulobacteraceae, and Enterococcaceae had a relative abundance greater than 1.0%. The most abundant family in the jejunum contents of Xuefeng black-bone chicken was Ruminococcaceae (19.23%) (S10 and S11 Tables). Among the 442 genera detected, 18 genera, including Helicobacter, Serratia, Romboutsia, Bacteroides, Halomonas, Lactobacillus, Ruminococcaceae_UCG-010, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Shewanella, Lachnoclostridium, Ruminococcaceae_UCG-005, Enterococcus, Faecalibacterium, Eubacterium_coprostanoligenes_group, Desulfovibrio, Ruminococcaceae_UCG-014, and Ruminococcaceae_NK4A214_group had a relative abundance greater than 1.0% (S12 and S13 Tables). At the species level, among the 216 species detected, 3 species, including Serratia_marcescens, Bacteroides_barnesiae, and Shewanella_algae had a relative abundance greater than 1.0%. The most abundant species in the jejunum contents of Xuefeng black-bone chicken was Serratia_marcescens (4.93%) (S14 and S15 Tables).

Fig 2

Venn Graph representation of the shared and exclusive OTUs at the 97% similarity level among the three groups of fecal microbiota (A). Bar plots analysis shows relative abundance of fecal microbiota at the phylum level in each group (B) and in each sample (C).

Microbial diversity in the jejunum contents of Xuefeng black-bone chicken

We compared the alpha-diversity (within-sample diversity or estimate of species richness and evenness) of each sample with differing sequence counts or sampling efforts. Rarefaction curve analysis indicated that the number of sequences and sequencing depth were sufficient for this study (S1 Fig). We further used observed species, Chao1, ACE, and Shannon indices to evaluate the diversity of jejunum content of Xuefeng black-bone chicken among the different groups (Fig 3). Specifically, observed species, Chao 1, ACE, and the Shannon diversity index indicated a significant decrease in the diversity of jejunum content of the Po group compared with that of the Cn, Fe, and FP groups (P = 0.007, P = 0.071, P = 0.003, and P = 0.003, respectively). For beta diversity analysis, the relationship between the microbial communities in the jejunum of chickens fed different diets was assessed by non-metric multi-dimensional scaling, as shown in Fig 4. There were overlaps between the Cn and Fe groups, but the microbial communities of the FP and Po groups formed a distinct cluster separated from those of the Cn group. In addition, pairwise analysis of similarity [25] suggested that there were highly significant differences between the Cn and Fe groups (global R = 0.139, P = 0.045), the Cn and FP groups (global R = 0.239, P = 0.004), the Cn and Po groups (global R = 0.253, P = 0.005), the Fe and FP groups (global R = 0.387, P = 0.003), the Fe and Po groups (global R = 0.436, P = 0.004), and the FP and Po groups (global R = 0.165, P = 0.05) (Table 1). We also performed a multiresponse permutation procedure [26] in within- and between-group populations. The differences for between-group homogeneity were higher than those for within-group with an A value > 0, and the change degree reached a significant level between the Cn and Fe groups (P = 0.031), the Cn and FP groups (P = 0.018), the Cn and Po groups (P = 0.01), the Fe and FP groups (P = 0.009), the Fe and Po groups (P = 0.004), and the FP and Po groups (P = 0.038) (Table 1).

Fig 3

Microbial diversity indices in the fecal microbiome.

Summary of four measures of α-diversity (observed species, Chao 1 index, ACE and Shannon).

Fig 4

First two dimensions from the (non-metric) multi-dimensional scaling (NMDS) of the Bray-Curtis dissimilarity matrix.

Each point in the figure represents a sample, the distance between points indicates the degree of difference, and the samples in the same group are represented by the same color.

Table 1

Significant differences in community structure in the jejunal microbiota of between different groups.

Group	Anosim		MRPP
	R value	P value	A value	Observed delta	Expected delta	P value
Cn-Fe	0.139	0.045	0.024	0.607	0.622	0.031
Cn-FP	0.239	0.004	0.033	0.618	0.639	0.018
Cn-Po	0.253	0.005	0.036	0.632	0.655	0.01
Fe-FP	0.387	0.003	0.057	0.584	0.619	0.009
Fe-Po	0.436	0.004	0.069	0.597	0.642	0.004
FP-Po	0.165	0.05	0.028	0.608	0.626	0.038

Anosim, analysis of similarity; MRPP, multiresponse permutation procedure. The R-value ranged from -1 to 1, and an R-value > 0 that showed the significant differences in the between-group population compared with those in the within-group population, as well as A-value (analysed by MRPP). The P values indicated the significant differences at the levels of P < 0.05 or P < 0.01.

Alternative diets changed the intestinal microbiota composition

Differences in the microbial composition (relative abundance) of the jejunum microbiomes between the Cn and FP groups and the Cn and Po groups were evaluated by the MetaStat method using Fisher’s exact test [24]. A total of 19 genera displayed a significant difference in relative abundance between the Cn and FP groups. Among them, the abundance of Ureaplasma, Synechococcus, Coprococcus_1, Fonticella, Butyricicoccus, Elusimicrobium, Sutterella, Anaerostipes, Faecalitalea, Thalassospira, and Bacteroides was increased, and that of Sporolactobacillus, Bacillus, Halomonas, Aquamicrobium, Devosia, Microbacterium, Asteroleplasma, and Shewanella was decreased in the FP group (S16 Table). Compared with that in the Cn group, the relative abundance of 4 genera, including Thermoplasmatales, Odoribacter, Erysipelotrichaceae_UCG-003, and Sutterella was significantly increased, and that of 7 genera, including Bacillus, Coprococcus_2, Sporolactobacillus, Alkaliphilus, Aequorivita, Asteroleplasma, and Sphingobacterium was significantly decreased in the Po group (S17 Table). At the species level, a total of 8 species displayed a significant difference in relative abundance between the Cn and FP groups. Among them, the abundance of Clostridiales_bacterium_NK3B98, Lactobacillus_phage_Sal3, Bacteroides_vulgatus, and Eubacterium_tortuosum was increased, and that of Mycoplasma_gallinarum, Acinetobacter_lwoffii, Gallibacterium_anatis, and Asteroleplasma_anaerobium was decreased (S18 Table). A total of 6 species displayed a significant difference in relative abundance between the Cn and Po groups. Among them, the abundance of Methanogenic_archaeon_CH1270, Clostridiales_bacterium_60-7e, and Lactobacillus_phage_Sal3 was increased, and that of Mycoplasma_gallinarum, Asteroleplasma_anaerobium, and Brevundimonas_diminuta was decreased (S19 Table).

Correlation between intestinal microbiota and histomorphology of jejunum

To further identify the genera that significantly correlated with the jejunum histomorphology, we performed Pearson’s correlation test. Results showed that the villus height of the jejunum of Xuefeng black-bone chicken displayed a strong positive correlation with the relative abundance of Coprococcus_1, Odoribacter, and Butyricicoccus (P = 0.02, P = 0.01, and P = 0.05, respectively) and a negative correlation with that of Coprococcus_2 (P = 0.008) (Fig 5). The crypt depth of the jejunum of Xuefeng black-bone chicken showed a positive correlation with the relative abundance of Asteroleplasma (P = 0.03), and a negative correlation with that of Coprococcus_1 (P = 0.03) and Faecalitalea (P = 0.03) (Fig 5). The relative abundance of Thalassospira, Sutterella, Coprococcus_1, Elusimicrobium, Odoribacter, Butyricicoccus, Synechococcus, and Faecalitalea was positively correlated with the ratio of villus height to crypt depth of the jejunum of Xuefeng black-bone chicken (P = 0.04, P = 0.01, P = 0.002, P = 0.04, P = 0.02, P = 0.03, P = 0.009, and P = 0.01, respectively). However, the ratio of villus height to crypt depth was negatively correlated with the relative abundance of Asteroleplasma (P = 0.04) and Coprococcus_2 (P = 0.04) (Fig 5). At the species level, the villus height of the jejunum of Xuefeng black-bone chicken showed a slight positive correlation with the relative abundance of Clostridiales_bacterium_60-7e (P = 0.07) and a negative correlation with that of Mycoplasma_gallinarum (P = 0.04) (S2 Fig). The crypt depth of the jejunum of Xuefeng black-bone chicken was positively correlated with the relative abundance of Asteroleplasma_anaerobium (P = 0.03), and negatively correlated with that of Bacteroides_vulgatus (P = 0.05) and Eubacterium_tortuosum (P = 0.03) (S2 Fig). The ratio of villus height to crypt depth showed a positive correlation with the relative abundance of Bacteroides_vulgatus (P = 0.01) and Eubacterium_tortuosum (P = 0.01), and a negative correlation with that of Asteroleplasma_anaerobium (P = 0.04) (S2 Fig).

Fig 5

Correlation analyses of genera taxa with the villus height, crypt depth, and the ratio of villus height to crypt depth in the jejunum of broiler chickens.

Each cell contains the corresponding correlation and P-value. The table is color-coded by correlation according to the color legend. V, the villus height; C, the crypt depth; V.C, the ratio of villus height to crypt depth.

Discussion

The primary function of the small intestine is the digestion and absorption of nutrients by the intestinal mucosa [27]. Its function is closely related to the villus height, crypt depth, and villus height to crypt depth ratio. A previous study reported that changes in the intestinal morphology, such as increased villus height and villus height to crypt depth ratio, and shallow crypts indicate enhanced digestion and absorption in the small intestine [28]. Consistent with our findings, several studies have shown that dietary supplementation of fermented feed with probiotics significantly improves intestinal morphology [29], and increases the villus height, crypt depth, and villus height to crypt depth ratio in the jejunum of broilers [30, 31]. In this study, we demonstrated that the Fe group displayed significantly increased villus height and villus height to crypt depth ratio, and decreased crypt depth in the jejunum (P < 0.05). This may be caused by fermentation of metabolites, including organic acids, amino acids, small peptides, and other substances, which improves the nutritional status of the intestine as well as intestinal morphology [32]. Additionally, we found that the Po group displayed slightly increased villus height, significantly increased villus height to crypt depth ratio, and significantly reduced crypt depth. This finding is in agreement with the results of Zahran et al. [32], who showed that dietary supplementation of astragalus polysaccharides caused a significant increase in the intestinal villus height in tilapia fish. Wang et al. [33] demonstrated that Sargassum fusiforme polysaccharides affect intestinal parameters, such as the ratio of villus height to crypt depth in mice. This may be caused by polysaccharides that are herbal plants with important biological activities to improve intestinal development [34]. Interestingly, in our study, compared to the Cn group, the FP group showed increased villus height, shallow crypt depth, and higher villus height to crypt depth ratio in the jejunum (P < 0.05). However, there is currently no information on the effect of fermented feed and ginseng polysaccharides on the intestinal structure of broiler chickens, and hence, further research is required.

Next, we characterized the microbial composition of the intestinal contents of 32 Xuefeng black-bone chickens. A total of 2,245,803 high quality valid tags were obtained across all samples, and the sequence size of each sample ranged from 49,767 to 80,545, which was greater than that reported in previous studies on broilers [35]. The Chao1 index indicated that this sequencing depth was sufficient for further analysis. Cluster analysis revealed that Firmicutes was the most dominant phyla among the total sequences. This finding is consistent with that of a study conducted by Wu et al. [36]. However, the functions of the phyla require further research. Additionally, in this study, the composition and structure of the gut microbial community was altered in the FP and Po groups. At the genus level, the FP and Po groups displayed significantly increased abundance of Sutterella and decreased abundance of Asteroleplasma. Interestingly, we found that the bacterial abundance of Sutterella was positively correlated to the villus height and villus height to crypt depth ratio, and negatively correlated to the crypt depth. In contrast, the bacterial abundance of Asteroleplasma was positively correlated to the crypt depth and negatively correlated to the villus height to crypt depth ratio. A previous study reported that the members of the genus Sutterella are widely prevalent in the human gastrointestinal tract, and their deficiency alters the colonic microbiota, resulting in disruption of the normal function of the colonic epithelium and inflammatory bowel disease [37, 38]. Min et al. [39] reported that an increase in the abundance of Asteroleplasma resulted in intestinal inflammation and other colonic epithelium diseases. At the species level, we discovered that the FP group showed significantly increased abundance of Bacteroides_vulgatus and Eubacterium_tortuosum and decreased abundance of Mycoplasma_gallinarum and Asteroleplasma_anaerobium. However, the abundance of Mycoplasma_gallinarum and Asteroleplasma_anaerobium was increased in the Po group, which may be because the combined effect of polysaccharides and fermented feeds is stronger than that of polysaccharides alone. Additionally, we found that the bacterial abundance of Mycoplasma_gallinarum was negatively correlated with the intestinal villus height. The crypt depth was positively correlated with the relative abundance of Asteroleplasma_anaerobium, and negatively correlated with that of Bacteroides_vulgatus and Eubacterium_tortuosum. However, the ratio of villus height to crypt depth was positively correlated with the relative abundance of Bacteroides_vulgatus and Eubacterium_tortuosum, and negatively correlated with that of Asteroleplasma_anaerobium. A previous study showed that an increase in the abundance of Asteroleplasma_anaerobium resulted in loss of pyruvate kinase activity, which slows glucose metabolism, thus hindering intestinal development, and an increase in the abundance of Mycoplasma_gallinarum resulted in intestinal inflammation and respiratory disease [40]. Previous studies have also reported that the relative abundance of Bacteroides_vulgatus is negatively correlated with inflammatory bowel disease [41]. Waidmann et al. [42] showed that an abundance of Bacteroides_vulgatus protected against Escherichia coli-induced colitis in gnotobiotic interleukin-2-deficient mice. Additionally, Tadayon et al. [43] reported that deficiency of Eubacterium_tortuosum caused suppurative inflammation of the intestine. Based on the above findings, we proposed a hypothesis that FP and Po might improve intestinal morphology by altering the microbial communities. However, further in-depth research is needed to confirm this hypothesis.

Conclusions

The results of the present study indicated that the Fe, FP, or Po groups showed improved intestinal morphology, including increased villus height and villus height to crypt depth ratio, and decreased crypt depth of the jejunum of Xuefeng black-bone chicken. Additionally, we also compared the intestinal microbial composition of Xuefeng black-bone chicken among the FP and Po groups. Moreover, they are closely related to the histomorphological characteristics of chicken jejunum, including villus height, crypt depth, and villus height to crypt depth ratio. Therefore, a combination of ginseng polysaccharides and microbial fermented feeds or ginseng polysaccharide alone can be used as a new, effective alternative to antibiotics for improving intestinal morphology. However, further in-depth research is required to reveal the underlying mechanisms.

Rarefaction curves.

Number of sequences (A) and number of sample (B) rarefaction curves for the sampled jejunum microbiotas. Number of detected OTUs on the y-axis; number of sequences (A) and of samples (B) on the x-axis.

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Correlation analyses of species taxa with the villus height, crypt depth, and the ratio of villus height to crypt depth in the jejunum of broiler chickens.

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Basic diet formulations and nutrient composition.

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Quality control and preprocessing of metagenomic datasets.

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Sequence composition of each sample at each level in the Greengenes database.

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The aligned counts that were annotated at the phylum level.

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The aligned percentages that were annotated at the phylum level.

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The aligned counts that were annotated at the class level.

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The aligned percentages that were annotated at the class level.

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The aligned counts that were annotated at the order level.

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The aligned percentages that were annotated at the order level.

(XLSX)

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The aligned counts that were annotated at the family level.

(XLSX)

Click here for additional data file.

The aligned percentages that were annotated at the family level.

(XLSX)

Click here for additional data file.

The aligned counts that were annotated at the genus level.

(XLSX)

Click here for additional data file.

The aligned percentages that were annotated at the genus level.

(XLSX)

Click here for additional data file.

The aligned counts that were annotated at the species level.

(XLSX)

Click here for additional data file.

The aligned percentages that were annotated at the species level.

(XLSX)

Click here for additional data file.

Significant differences between the Cn and FP groups identified at the genus-taxa level.

(XLSX)

Click here for additional data file.

Significant differences between the Cn and Po groups identified at the genus-taxa level.

(XLSX)

Click here for additional data file.

Significant differences between the Cn and FP groups identified at the species-taxa level.

(XLSX)

Click here for additional data file.

Significant differences between the Cn and Po groups identified at the species-taxa level.

(XLSX)

Click here for additional data file.

3 Jul 2020

PONE-D-20-12303

Effects of fermented feeds and ginseng polysaccharides on the intestinal morphology and microbiota composition of Xuefeng black-bone chicken

PLOS ONE

Dear Dr. SUN,

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The experiments seems well done and the data is important. However, the paper must have better description of Material and methods and stats.

The main topic, polisaccharides should be better addressed in the introduction, methods (describing better the product used) and discussion.

Also, the authors must to discuss better the data and correlate their findings with the literature, focusing on chickens.

Further comments are described in the file attached.

Reviewer #2: Abstract – group names were not defined before using abbreviations. The statement that basal diets were fermented by a variety of bacteria doesn’t make sense. The diet was actually fermented, or do you mean supplemented with these bacteria?

Introduction – Define microbially fermented feed, most readers will not know what this is.

Line 122 – was feed refreshed at these times and offered ad libitum or were birds fed a designated amount?

Was fermented feed the only source of nutrition for the birds, or was it combined with normal feed? If combined, at what ratio?

Was a nutrient composition analysis completed on the two different feeds? Perhaps differences noted in the experimental results were a result of changes in composition of the feed, and this would be interesting to note.

Line 116 describes 5 replicate pens per group, but line 130 suggests 4 replicate pens per group

In the discussion section, anywhere you refer to data shown in tables and figures, you should reference them.

The method of determining correlation between microbiome and intestinal morphometry is missing from the M&M section.

Mycoplasma gallinarum is a pathogen of chickens, they did not show clinical signs of disease despite the high abundance and presence in birds? This would explain the association with decreased villus height.

**********

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Reviewer #1: No

Reviewer #2: Yes: LR Bielke

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21 Jul 2020

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer: Many thanks for your kind help, and we have regulated to meet PLOS ONE's style requirements.

2. In your Methods, please state the method of anaesthesia used prior to exsanguination.

Answer: we have detailed the method of anaesthesia in main text with new line 131-133. The details as follow: On day 150, 2 broiler chickens were randomly selected from each pen (8 broiler chickens/treatment) and euthanized with an overdose of CO2 [12]. The intestinal tract was immediately removed.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was supported by grants from the Technical System of Modern Agricultural Industry in Guangdong Province [2018LM1121, 2018LM2158]."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"the Technical System of Modern Agricultural Industry in Guangdong Province [2018LM1121, 2018LM2158]."

Answer: Thanks. We have removed the Acknowledgments section of our manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Answer: Thanks.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Answer: Thanks.

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Answer: Thanks.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Answer: Thanks.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

The experiments seems well done and the data is important. However, the paper must have better description of Material and methods and stats. The main topic, polysaccharides should be better addressed in the introduction, methods (describing better the product used) and discussion. Also, the authors must to discuss better the data and correlate their findings with the literature, focusing on chickens. Further comments are described in the file attached.

Ethics Statement- Number of approve

ABSTRACT

Abreviation of the groups are not explained

Answer: Thanks so much. The informations of groups were showed in M&M section with new line 113-116. And we have explained the abreviation of the groups in our Abstract with red words.

INTRODUTION

Were all the diets fermented? No control diet? Distinguish Fe FP and Po groups

Answer: The related information was showed in M&M section with new lines 111-117. In our paper, a total of 400 Xuefeng black-bone chickens were randomly divided into 4 groups (5 replicates per group and 20 chickens in each replicate): 100% complete feed group (Cn group), 100% microbially fermented feed group (Fe group), 100% complete feed and ginseng polysaccharide (200 g/t) group (Po group), and 100% microbially fermented feed and ginseng polysaccharide group (FP group).

MATERIAL AND METHODS

Composition table of “basal diet” is missing- It is not complemental information

Need a better explanation of complete feed. Is that the basal diet without fermentation?

Describe phases of diet

Answer: Thanks so much for professional suggestions. In our paper, basic diet formulations and nutrient composition was showed in S1 Table. In our experiment, the basal diet was not fermented and termed as control group. Two phases of diets were described in our paper, and the information was also showed in S1 Table.

Describe how the animals were raised (installation, building, shavings, etc)

Answer: Thanks so much. And we have re-prepared the text with new lines 122-125. The details were also following as “Chickens were raised in floor pens, and placed into separate floor pens with 20 individuals per pen. Feed was provided twice a day at 8:00 am and 5:00 pm, and drinking water was provided ad libitum throughout the experimental period. Feed was removed from the pen 24 hours before sampling.”

“cetyltrimethylammonium bromide/sodium dodecyl sulfate method” – describe method or add reference

Answer: we have revised in the main text with lins 155-157. The details as follow: Total genomic DNA from the jejunum contents was extracted using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the specifcations of the manufacturers.

Composition of the ginseng polyssacaride- describe better the product. Is it from any kind of extraction? What its composition?

Answer: Thanks so much for your suggestion. Ginseng, one of the most well-known oriental medicine for several thousand years, has been widely used with mysterious powers as a tonic, prophylactic and restorative agent in china. In general, ginseng polysaccharides have a large content of polysaccharides, peptides, saponins, and other active substances. In our paper, the Ginseng polysaccharides were extracted from the roots of ginseng with hot water, precipitated by 80% ethanol and deproteinated based on our previous method. We have detailed in our text with lines 118-120.

Statistical Analysis- What P-value was considered significant?

-Was the data tested to normality? What was made with non normal data?

Answer: Thanks so much. We have added the related information in our main text with new lines 189-190. The details also showed as “The P values indicated the significant differences at the levels of P < 0.05.” Yes, the tested data was normal in our paper. Thanks again.

RESULTS

Figure 1. Describe the abbreviation of the graphics

Answer: Thanks so much. We have describe the abbreviation of the graphics. The details as bellow.

Fig 1. Effects of dietary treatment on villus height, crypt depth, and the ratio of villus height to crypt depth in the jejunum of broiler chickens. Treatments with different letters are significantly different at P < 0.05. Data were obtained from transmission electron microscopy, and were means of 10 birds (2 bird from each pen). Cn, 100% complete feed group; Fe, 100% microbial fermented feed group; Po, 100% complete feed and ginseng polysaccharide group; FP, 100% microbial fermented feed and ginseng polysaccharide group. VH, villus height; CD, crypt depth; V/C, the ratio of villus height to crypt depth.

Do you have the bromatological analysis of the feeds? Especially the fermented ones?

Would be interesting have the analysis of organic acids.

Answer: Thanks so much for your professional suggestion. In our paper, basic diet formulations and nutrient composition was analyzed and presented in S1 Tables, while the other characteristics of feeds, such as organic acids, were not tested. And we very interested to do these analysis, and thanks so much for your professional advices. Now, it is a pity that we have no more feed samples. In the future, we will follow your opinions to improve our research. Thanks.

Line 354- What kinds of polysaccharides?

You should explain better which kinds of polisaccharides are you talking. .

Line 355- 358- how polysaccharide have protein, minerals, …?

Answer: Thanks so much. In line 354 (new line 364), the reference 33 illustrated Sargassum fusiforme polysaccharides, and we have added related information in our paper in new line 363-364. In new line 365-366, we also revised “This may be caused by polysaccharides that are not only herbal plants with important biological activities but also contain abundant proteins, carbohydrates, vitamins, fats, and minerals, which play an important role in maintaining nutrient balance, thus improving intestinal development” to “This may be caused by polysaccharides that are herbal plants with important biological activities to improve intestinal development”.

Please describe correlations of villus height and crypt deep with chicken microbiota already find in the literature! Also, discuss data on microbiota and performance in chickens, how that can be associated with your data?

Answer: Many thanks for your kind helps. We have details the methods of correlation analysis in the M&M section with new lines 190-194. The details were also showed as bellow: The correlation analyses of microbiota composition with the intestinal morphology were tested by the cor function (x, y, use = “p”) (http://127.0.0.1:11153/library/stats/html/cor.html), and illustrated with a function-labelled Heatmap (Matrix, xLabels, yLabels) in the R package WGCNA (http://127.0.0.1:11153/library/stats/html/cor.html). And we also try our best to discuss our data between microbiota and performances. Thanks so much.

Figure and table subtitle should be better described.

Answer: Thanks. We have detailed the subtitle of our Figures and tables.

Reviewer #2:

Abstract – group names were not defined before using abbreviations. The statement that basal diets were fermented by a variety of bacteria doesn’t make sense. The diet was actually fermented, or do you mean supplemented with these bacteria?

Answer: Thanks so much. We have detailed the abreviation of the groups in our Abstract with red words. And the fermented methods were showed in M&M section with the lines 99-108.

Introduction – Define microbially fermented feed, most readers will not know what this is.

Answer: In the Introduction, we have added some description about fermented feed in new lines 71-73. The details were also showed as follow: “In general, the basal diet that fermented with probiotics has been extensively studied due to its benefits of increasing nutrient bioavailability and nutritional value [4]”.

Line 122 – was feed refreshed at these times and offered ad libitum or were birds fed a designated amount?

Was fermented feed the only source of nutrition for the birds, or was it combined with normal feed? If combined, at what ratio?

Was a nutrient composition analysis completed on the two different feeds? Perhaps differences noted in the experimental results were a result of changes in composition of the feed, and this would be interesting to note.

Answer: Thanks so much for your professional suggestion. In our paper, All birds received feed and fresh water ad libitum throughout the experiment. Feed was removed from the pen 24 hours before sampling. And we have also revised in our main text with new lines 123-125. The fermented feed was only for Fe (100% microbially fermented feed group) and FP (100% microbially fermented feed and ginseng polysaccharide group) groups. The information was described in new lines 111-117. In our paper, basic diet formulations and nutrient composition was analyzed and presented in S1 Tables, while the characteristics of fermented feed were not tested. And we very interested to do these analysis, but it is a pity that we have no more feed samples. In the future, we will follow your opinions to improve our research. Thanks.

Line 116 describes 5 replicate pens per group, but line 130 suggests 4 replicate pens per group

In the discussion section, anywhere you refer to data shown in tables and figures, you should reference them.

The method of determining correlation between microbiome and intestinal morphometry is missing from the M&M section.

Mycoplasma gallinarum is a pathogen of chickens, they did not show clinical signs of disease despite the high abundance and presence in birds? This would explain the association with decreased villus height.

Answer: Thanks so much for your kind help, and we have corrected in new line 113-114 as “The chickens were randomly divided into 4 groups (4 replicates per group and 25 chickens in each replicate)”. We have added the methods in new lines 189-194 as “The correlation analyses of microbiota composition with the intestinal morphology were tested by the cor function (x, y, use = “p”) (http://127.0.0.1:11153/library/stats/html/cor.html), and illustrated with a function-labelled Heatmap (Matrix, xLabels, yLabels) in the R package WGCNA (http://127.0.0.1:11153/library/stats/html/cor.html)”. Thanks so much for your professional suggestion about Mycoplasma gallinarum, and we have also updated our discussion.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: LR Bielke

Answer: Thanks.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

24 Jul 2020

Effects of fermented feeds and ginseng polysaccharides on the intestinal morphology and microbiota composition of Xuefeng black-bone chicken

PONE-D-20-12303R1

Dear Dr. SUN,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Michael H. Kogut, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

30 Jul 2020

PONE-D-20-12303R1

Effects of fermented feeds and ginseng polysaccharides on the intestinal morphology and microbiota composition of Xuefeng black-bone chicken

Dear Dr. SUN:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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