Xylanase has been widely applied in the broiler industry due to its ability to degrade arabinoxylans (AX) in poultry diets (Bautil et al., 2019). Corn and wheat are dominant feed grains for broilers diets; however, concentration and structure of AX in corn and wheat are different (Cowieson, 2010). At the present, most reports have focused on positive effects of xylanase on performance in broilers fed viscous wheat-based diets (Gonzalez-Ortiz et al., 2017; Liu and Kim, 2017; Wang et al., 2021). However, benefits of xylanase on growth performance of broilers supplemented non-viscous corn-based diets also were recorded (Khadem et al., 2016). Xylanase can reduce chyme viscosity, release nutrients from inside cell wall, and xylo-oligosaccharides (XOS) as a result of AX degradation in the gastrointestinal tract (GIT), which may then act like a prebiotic (Gonzalez-Ortiz et al., 2017; Petry et al., 2021). Previous studies have demonstrated that the dominant mode of action responsible for benefits of xylanase in corn- or wheat-based diets may be distinct and impacts of xylanase on growth performance, nutrient utilization, digesta characteristics, and cecal volatile fatty acid (VFA) concentrations of broilers fed corn- or wheat-based diets were compared (Kiarie et al., 2014; Munyaka et al., 2016; McCafferty et al., 2019a,b).

Cecal microbiota has the most complex microbiota in GIT which was associated with the health of broilers (Yadav and Jha, 2019). The GIT microbiota is shaped by both the environment and the broiler itself (Liu et al., 2021). Environmental changes, especially dietary factors, such as feed components and additives, have been widely demonstrated to regulate GIT microbiota (Shang et al., 2018). Cecal VFA concentrations are considered to be associated with bacterial fermentation in the large intestine (Ríos-Covián et al., 2016). Xylanase changed VFA concentrations in broilers cecum when the diets were corn- or wheat-based (McCafferty et al., 2019a,b). We hypothesized that supplemental xylanase might exert positive effects on cecal microbiota of broilers fed corn- or wheat-based diets by different gut bacterial communities.

16S rRNA gene sequencing analysis is a tool that has been widely applied to evaluate the diversity of GIT microbiota (Shang et al., 2018). To further elaborate the specificity of xylanase for feed ingredients, and the impacts of xylanase on cecal microbiota, we conducted the following experiment by feeding broilers corn- or wheat-based diets for a 6-week period and assessed the bacterial composition by 16S rRNA gene sequencing.

The present study was approved by the Laboratory Animal Welfare and Animal Experimental Ethical Inspection Committee of China Agricultural University (Beijing, China).

In the current study, broilers receiving corn-based diets showed higher ADG and ADFI than broilers supplemented wheat-based diets. Masey-O'Neill et al. (2014) also reported that feeding broilers with corn-based diets increased body weight gain and feed intake compared to broilers fed wheat-based diets from d 1 to 28. This observation might be related to the higher concentration of soluble NSP in viscous wheat. The soluble NSP is known to exhibit antinutritive effects by increasing intestinal digesta viscosity, affecting gut transit time, thus impair nutrient absorption and utilization of broilers (Kiarie et al., 2014). However, similar to our results, it has been reported that feeding broilers with wheat-based diets supplemented with xylanase could improve growth performance, the improvement might be associated with the alleviation of detrimental effects of soluble NSP on digesta viscosity (Gonzalez-Ortiz et al., 2017; Liu and Kim, 2017). Moreover, in this study, xylanase improved growth performance of broilers receiving corn-based diets. Benefits of xylanase on performance of broilers fed non-viscous corn-based diets also have been reported, indicating that there are other mechanisms besides this mode of viscosity reduction (Khadem et al., 2016). The primary mode of action may depend on the type of cereal grain, thus 16S rRNA gene high-throughput sequencing in this study was used to elaborate effects of xylanase on cecal microbiota of broilers supplemented corn- or wheat-based diets on d 14, 28, and 42.

The generated rarefaction curves have reached plateau with the Sobs index, suggesting that 16S rRNA sequencing libraries could represent most of OTUs present in all the samples sequenced in this study. As shown in Venn diagram, the number of OTUs in cecal microbiota among four dietary treatments was similar, and most of them shared. When comparing effects of xylanase on cecal microbiota alpha-diversity of broilers fed wheat- or corn-based diets, there was not a significant interaction between cereal type and xylanase supplementation in alpha-diversity, and xylanase main effect also did not affect alpha-diversity of broilers. However, we found that broilers receiving corn-based diets had richer and more diverse microbiota than broilers fed wheat-based diets on d 14 and 42, indicating that microbiota of broilers receiving corn-based diets exhibited higher richness. The increase in alpha-diversity has been reported to be beneficial to the health of broilers, the increased diversity might be able to promote the stability of microbiota (Díaz Carrasco et al., 2018; Dhillon et al., 2019). The analysis of PCoA based on dietary treatments did not exhibit distinct clustering patterns, the composition of GIT microbiota between WCON and WXY groups was similar, and beta diversity was also not different between CCON and CXY groups. However, the phylogenetic distances clearly revealed that type of cereal grain affected beta diversity, suggesting a distinction in microbial community structure of broilers receiving corn-based diets relative to broilers receiving wheat-based diets.

To further probe how xylanase modulates GIT microbial community of broilers receiving wheat- or corn-based diets, the variations in relative abundance of microbiota were analyzed. The development of GIT microbiota in broilers might be a continuous process of microbial community succession. In the current study, the development of microbiota community from an immature phase to a mature phase in four dietary treatments was clearly shown. As previously reported, cecal communities for all the broilers were dominated by members of Firmicutes and Bacteroidetes phylum (Ilaria et al., 2020). Firmicutes accounted for most of the cecal microbiota in young broilers, while Bacteroidetes were only enriched at the end of productive life. Butyrate-producing bacteria mostly belong to Firmicutes phylum (Chakraborti, 2015; Dai et al., 2021). Representatives of Bacteroidetes phylum mainly degrade polysaccharides producing propionate (Yang et al., 2013; Polansky et al., 2016). In quickly growing, young birds, butyrate is highly required to provide energy for the growth of intestinal cells, while adult birds have fully developed intestines and need less amount of butyrate (Petra et al., 2014; Onrust et al., 2015). Higher abundance of Bacteroidetes in adult broilers represented an effective balance between energy obtained from available nutrients and sustained growth (Ocejo et al., 2019). The alteration of the proportion of Firmicutes and Bacteroidetes indicates the change of produced butyrate in broilers cecum, thus can explain the results in this study that Bacteroidetes gradually increased throughout the production period.

When comparing cecal microbiota of broilers fed wheat-based diets, compared with WXY, higher abundance of family Ruminococcaceae on d 14 and phylum Bacteroidetes on d 28 was observed in WCON group. Ruminococcaceae has been reported to be related to cellulose-degrading capacity, and Ruminococcaceae is enriched in genes encoding for endo-1,4-beta-xylanase and cellulase (Biddle et al., 2013; Dai et al., 2020). Phylum Bacteroidetes also contain a high number of genes encoding for glycose hydrolases, such as β-xylosidase, endo-1,4-β-xylanase, and α-N-arabinofuranosidase, which makes Bacteroidetes become the primary phylum for the degradation of complex polysaccharides in GIT (Zhang et al., 2018a). Furthermore, it has been demonstrated that Bacteroides of the phylum Bacteroidetes was related to AX degradation (Zhang et al., 2018a,b). Dietary supplementation of xylanase to broilers could effectively hydrolyze AX in small intestines, thereby reducing the availability of polysaccharides to Ruminococcaceae and Bacteroidetes in the cecum. However, the abundance of microbiota associated with arabinoxylan-degrading capacity was not significantly different between broilers from CCON to CXY groups, we speculated that the difference might be related to the lower contents of arabinoxylans in corn.

Additionally, compared with WXY, the increased abundance of Ruminococcaceae in broilers from WCON group on d 14, and compared with CXY, higher abundance of order Mollicutes_RF9 in CCON group on d 14 was observed. Ruminococcaceae has been demonstrated to be sensitive to starch, increased abundance of Ruminococcaceae in broilers from WCON group might also be related to lower ileal starch digestibility, because the addition of xylanase has been reported to increase starch digestibility (Choct et al., 1999; McCafferty et al., 2019a). The metagenome-assembled genomes of Mollicutes_RF9 group also have the CAZyme related with starch degradation, thus lower concentration of undigested starch flowing into the cecum might limit the growth of Mollicutes_RF9 (Ma et al., 2019).

Compared to WCON, higher abundance of family Lachnospiraceae (containing norank_f_Lachnospiraceae, Shuttleworthia, Ruminococcus_torques_group, and Sellimonas) on d 14, phylum Firmicutes, genus Hydrogenoanaerobacterium, and Ruminococcaceae_V9D2013_group on d 28, genus unclassified_f_Ruminococcaceae, Anaerotruncus, Caproiciproducens, and Subdoligranulum on d 42 was observed in WXY group. Lachnospiraceae has been demonstrated to produce butyrate; in addition, genus norank_f_Lachnospiraceae, Shuttleworthia, and Ruminococcus_torques_group have been observed to be related to better performance (Torok et al., 2011; Lee et al., 2017; McCafferty et al., 2019a; Li et al., 2020). Genera (Hydrogenoanaerobacterium and Ruminococcaceae_V9D2013_group) from the phylum Firmicutes are also suggested to effectively produce butyrate (Fang et al., 2020; Ilaria et al., 2020; Sun et al., 2020). Genus unclassified_f_Ruminococcaceae, Anaerotruncus, Caproiciproducens, and Subdoligranulum (family Ruminococcaceae) are also known as short-chain fatty acid producers. Supplemental xylanase could modulate GIT microbiota by increasing XOS concentrations in the cecum, and XOS has been reported to increase the abundance of butyrate-producing bacteria (De Maesschalck et al., 2015; McCafferty et al., 2019a). Moreover, the present result showed that xylanase supplementation to wheat-based diets increased the abundance of Bifidobacterium (phylum Actinobacteria) on d 14, and the abundance of Lactobacillus on d 42. XOS has been demonstrated to result in better performance of broilers by the stimulation of Bifidobacterium and Lactobacillus (Oyeagu et al., 2019; González-Ortiz et al., 2020). Although some species of Actinobacteria can lead to diseases, some strains like Streptomyces spp. exhibited antibacterial activities (Jing et al., 2018). Bifidobacterium as lactate-producing bacteria benefits to the health of GIT (Jing et al., 2018; González-Ortiz et al., 2020). Members of Lactobacillus could stimulate butyrate-producing bacteria and inhibit the colonization of pathogenic bacteria (Munyaka et al., 2016; Proszkowiec-Weglarz et al., 2020). When comparing cecal microbiota of broilers fed corn-based diets, compared with CCON, higher abundance of order Clostridiales on d 28, genus Anaerofustis, Lachnospiraceae_UCG_010, and norank_f__Ruminococcaceae on d 42 was observed in broilers from CXY group. Clostridium-related bacteria within GIT have been demonstrated to be able to produce short-chain fatty acids and act as a barrier against invasion by other potentially pathogenic microbiota (Munyaka et al., 2016). The increase of these butyrate-producing bacteria might also be related to increased XOS concentrations in the cecum.

Furthermore, compared with CCON group, lower abundance of family Christensenellaceae, Desulfovibrionaceae, and genus Faecalitalea, Escherichia_Shigella on d 14, and higher abundance of family Erysipelotrichaceae on d 42 were observed in CXY group. Xylanase supplementation to wheat-based diets could also increase the abundance of genus Gordonibacter on d 14 and d 42. The family Christensenellaceae was negatively associated with overall GIT health (Richards et al., 2019). Faecalitalea which affiliates to Erysipelotrichaceae family was related to inflammatory disorders (Dai et al., 2020). In addition, the previous work has reported that the abundance of Desulfovibrio (family Desulfovibrionaceae) was increased with ulcerative colitis, indicating that Desulfovibrio was enriched in an inflammatory environment (Yan et al., 2019). Enterobacteriaceae family includes potential pathogens, such as Escherichia_Shigella, the abundances of Escherichia_Shigella were found to be negatively correlated with growth performance in broilers (Díaz Carrasco et al., 2018). Moreover, Erysipelotrichaceae showed a positive correlation with growth performance (Stanley et al., 2016). Gordonibacter was associated with a decrease risk or severity of different diseases, such as cancer and inflammatory bowel disease (Pérez-Burillo et al., 2018). Overall, the results indicated that xylanase supplementation could stimulate the colonization of beneficial bacteria or inhibit the colonization of harmful bacteria in broilers receiving wheat- or corn-based diets.

Additionally, we compared the cecal microbiota of broilers supplemented corn- or wheat-based diets. The abundance of Lactobacillus and Bifidobacterium increased in the cecal microbiota of broilers receiving wheat-based diets, and the increase may be associated with high contents of AX in wheat. The cecal fermentative ability of broilers depends on type and content of substrates flowing into cecum. Members of Lactobacillus contained a range of genes coding ford AX-degrading enzymes; therefore, they can be beneficial to the degradation of XOS and AX (Zhang et al., 2018b). It also has been reported that AX and XOS selectively increased the abundance of Bifidobacterium spp., a so-called bifidogenic effect exerted by these prebiotics. Also, Bifidobacterium spp. have been reported to contain large number of genes coding for AX-degrading enzymes (Rivière et al., 2014). Overall, AX and XOS are preferred substrates of Lactobacillus and Bifidobacterium, and the growth of Lactobacillus and Bifidobacterium was stimulated by wheat relative to corn.

In corn-based diets fed broilers, the abundance of butyrate-producing bacterium increased, such as Lachnospiraceae_UCG_010, Shuttleworthia, Anaerofilum, Ruminococcaceae_UCG_008, Ruminococcaceae_UCG_005, Ruminococcus_2, norank_f__Ruminococcaceae, Oscillibacter, and Ruminiclostridium_9 comparing to the broilers fed wheat-based diets. This difference may be attributed to the resistant starch content in corn and wheat. Corn contains the content of resistant starch 1.9-fold higher than that in wheat. Resistant starch is fermented by microbiota in large intestine and relates to increased abundance of butyrate-producing bacteria (Bednar et al., 2001; Yang et al., 2017b). Moreover, resistant starch-rich diet was associated with increased circulating propionate concentration in mice (Bai et al., 2021). In the current study, higher abundance of propionic acid-producing bacteria Propionibacteriaceae was likely to relate to the higher content of resistant starch in corn.

In wheat-based diets fed broilers, the abundance of Proteobacteria was higher than those fed corn-based diets, which may relate to proteinaceous substrates since wheat-based diets contain about 1% more protein than corn-based diets in this study. Proteobacteria are mainly responsible for utilization of amino acids and possess extensive gene coverage of amino acid responses (Dai et al., 2015; Diether and Willing, 2019). Moreover, the reason that higher abundance of Peptostreptococcaceae in the broilers fed corn-based diets was likely due to the different amino acid compositions of corn protein from wheat protein. Zein as the major fraction of corn protein is rich in leucine (Cowieson, 2005). Peptostreptococcus spp. was associated with leucine metabolism in hindgut (Ma and Ma, 2018).

Overall, AT-xynA supplementation to wheat- or corn-based diets modulated the relative abundance of specific bacteria without changing the overall microbial structure. We showed that bacterial community clustering was mainly due to cereal grain source rather than xylanase supplementation. The addition of xylanase to wheat-based diets increased the abundance of Lactobacillus, Bifidobacterium, and some butyrate-producing bacteria, the increase might be related to the release of XOS as a result of AX degradation in GIT. Moreover, the undigested polysaccharides flowing into the cecum resulted in higher abundance of (non-starch polysaccharides) NSP-degrading bacteria in broilers fed wheat-based diets without xylanase supplementation, such as Ruminococcaceae and Bacteroidetes. Xylanase supplementation to corn-based diets slightly affected the abundance of butyrate-producing bacteria and NSP-degrading bacterium, the difference might be related to AX concentration and composition in corn and wheat. However, xylanase still increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria in the cecum of broilers fed corn-based diets. Moreover, beneficial effects of xylanase on cecal microbiome improved growth performance.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA753623.

The animal study was reviewed and approved by the Laboratory Animal Welfare and Animal Experimental Ethical Inspection Committee of China Agricultural University.

YC and SL supervised and provided continuous guidance for the experiments. YC, BD, and CW conceived and designed the experiments. JW, HC, CB, and YL performed the animal experiments. JW, ZS, and BD analyzed the data and wrote the manuscript. All authors have discussed the results and reviewed the manuscript.

Our study was supported by the funds of National Natural Science Foundation of People’s Republic of China (no. 31760673) and the Project of introducing talents to Tibet (RXYJ-2019-06).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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