Due to the antibiotic resistance of bacteria being on the rise (Guo et al., 2016) and the increasing consumer concern regarding poultry products, antibiotic-free flocks (Gadde et al., 2017), and environmental sustainability (Xing et al., 2015), a prohibition on antibiotics had been imposed in Europe (Stolker et al., 2007), South Korea (Kumar et al., 2012), and China (Hu and Cowling, 2020) since 2006, 2012, and 2020, respectively. Hence, replacing antibiotics with alternative products (Han et al., 2017) has become a hot research topic in recent years. Probiotics and enzymes are attracting much attention as important alternatives to antibiotics.

Bacillus is widely used in poultry diets as a type of probiotic, which improves the productive performance of poultry (Naumova et al., 2021) by producing naturally synthesized antimicrobial peptides, maintaining microbial flora balance in the intestie, accelerating the increase of beneficial microbiota along the gastrointestinal tract, and adjusting the immunological function and gut morphology (Sen et al., 2012; Grant et al., 2018). Many findings confirmed the significant improvement of growth performance, immune response, cecal microbial population, and intestinal morphology of weanling pig (Lee et al., 2014), Cherry Valley ducks (Guo et al., 2016), and Ross broiler chicks (Sen et al., 2012) with Bacillus-based diets. In addition, lacking enough enzymes to fully digest fiber, birds rely on exogenous enzymes in their diets to improve the fiber digestion process (Alagawany et al., 2018). As one of the most important exogenous enzymes, non-starch polysaccharidase (NSPase) is commonly used in poultry diets and plays many essential roles such as breaking down the non-starch polysaccharides, releasing nutrients encapsulated by the cell wall, reducing intestinal viscosity, improving animals’ utilization of nutrients, and affecting the composition and metabolic potential of bacterial populations (Kiarie et al., 2013; Ravindran, 2013; Amerah, 2015). Ao et al. (2010) and Coppedge et al. (2012) reported the supplementation of NSPase could improve the growth performance of market broilers and pigs and be used as a strategy to degrade antinutritional compounds so as to reduce dietary energy levels and costs.

Both Bacillus and NSPase can improve the productive performance of poultry, and are able to directly or indirectly affect the intestinal flora. However, it is not clear whether there is a synergistic effect between them. A few studies demonstrated that there was a certain synergistic effect on improving intestinal health, promoting nutrient digestion and growth in broiler chickens, when both Bacillus and NSPase were used as a supplement in the feed (Lin Q. et al., 2012; Wealleans et al., 2017). However, no such study has been conducted in ducks. In the present study, we aimed to study the effects of Bacillus and NSPase on the growth performance, serum antioxidant profiles, intestine morphology, and intestinal microbiota composition in ducks.

All the experimental procedures of this study were approved by the Animal Care and Use Committee of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences.

Several lines of studies demonstrated that Bacillus Probiotics could improve the growth performance of poultry (Jeong and Kim, 2014; Lee et al., 2015; Lin Y. et al., 2017). In the current study, we found that Bacillus probiotics supplementation could increase the average final weight and ADG by modulating antioxidative status and intestinal microflora. In fact, the antioxidant effect of Bacillus Probiotics has been verified in rats before (Paik et al., 2005). In our study, we found Bacillus probiotics have the efficacy of significantly decreasing the serum level of MDA and increasing the level of SOD and GSH. These results revealed that feeding ducks with Bacillus Probiotics can also improve the antioxidant status. Meanwhile, there were a higher relative proportion of the genera Ruminococcaceae, vadinBB60 group, and S24-7 group in the Bacillus-fed group in the cecal or ileal microbial community. Many species belonging to these genera are able to produce short-chain fatty acid (SCFA), especially butyrate (Hooda et al., 2012; Ridlon et al., 2015), which serves as a preferred energy source for enterocytes and a known regulator of cellular differentiation and proliferation within the intestinal mucosa (Sikandar et al., 2017; Bedford and Gong, 2018). This can contribute to improving the morphological development of the intestines and to reinforce the intestinal defense barrier, for instance, strengthening tight junctions, thereby promoting animal growth (Le Blay et al., 2000; Fukunaga et al., 2003; Bordin et al., 2004; Peng et al., 2007). Additionally, a significant increasing trend was also observed for Lactobacillus of the ileum in the Bacillus-treated group. This point deserves further investigation as it might be of interest, as Lactobacillus may provide nutrients to the host and help defend against opportunistic pathogens (Cross, 2002; LeBlanc et al., 2013). Interestingly, the Bacteroides genus was largely decreased with the presence of Bacillus, which conflicts with the findings of a very recent broiler study (Wang et al., 2017). However, studies using other species, such as swine, have shown that Bacillus subtilis supplementation decreased the copy numbers and percentage of Bacteroidetes while it increased the percentage of Firmicutes in the cecal contents (Cui et al., 2013). Similarly, a significant decrease trend was also revealed in Bacteroidetes of the ileum in our study. Given the fact that the decrease in Bacteroidetes and the increase in the Firmicutes/Bacteroidetes ratio are positively correlated with body mass index in humans (Koliada et al., 2017) and associated with an increase in ADG (Salaheen et al., 2017), our data revealed that Bacillus Probiotics promotes duck growth by improving the intestinal flora.

Nowadays, poultry feed with higher levels of NSP may reduce the digestibility of nutrients in their diets (Salim et al., 2010), which can lead to poorer growth and performance of birds (Annison and Choct, 1991). One solution to this issue accounting for feed cost and variability is to use exogenous enzymes (Woyengo et al., 2019), including NSPases. In our study, the addition of NSPases increased the average final weight and ADG of ducks, but insignificantly. A similar result was also obtained by Uthai et al. (2004). They found NSPase type mixture supplementation could improve the growth performances (average total weight gained, average daily gain, and average feed intake) of pigs also with non-statistically significant differences. Besides, Ao et al. (2010) reported that 2% NSPase supplementation to based diets for weaned piglets did not significantly improve performance. But interestingly, the average daily gain and gain/feed were increased 4.40 and 6.26% when fed with 1% NSPase supplementation. Variation in the results is likely associated with the content of substrates (Ao et al., 2010). However, a significant increase was found in the proportions of the S24-7 group, Lactobacillus, and Subgroup 2, while a significant decrease was found in the proportion of Bacteroides in the NSPase-treated group at the ileal level. Considering the results that the decrease in Bacteroidetes and the increase in S24-7 group, Lactobacillus, and Subgroup 2 are positively correlated with SOD activity. Thus, ducks fed with NSPases showed improved growth performance, and increased antioxidant capacity might be linked to the microbial changes. In many studies, the change of intestinal microflora was beneficial for the health and welfare of poultry. Naumova et al. (2021) found that the beneficial effect on the production of ducks was associated with the changes in gut microbiota due to Bacillus-feed probiotic supplementation. Dietary probiotics can enhance growth performance by the regulation of intestinal microbial composition, the immune system, and maintenance of intestinal integrity and barrier function, as described by Tuohy et al. (2003). In our study, the major changes were found within the foregut area and the predominant role of Lactobacillus was enhanced. Lactobacillus consists of gram-positive and facultative anaerobes that produce lactic acid, which can create a low pH environment and inhibit the growth of pathogens (Rodriguez-Cabezas et al., 2010). In addition, Lactobacillus was proven to be of antioxidant activity (Lin and Chang, 2000), and having the capacity of scavenging free radicals to alleviate damages induced by oxidative stress (Xin et al., 2014). It indicated that the increase of the genus Lactobacillus probably enhances the antioxidant activity of duck. However, other bacterial species and their interaction (significantly positively associated with SOD) were still unclear.

In the present study, Bacillus probiotics and NSPases supplementation have been proven to be a potential method to improve the average final weight and ADG of ducks. Our findings are consistent with the result reported by Lin Q. et al. (2012), who demonstrated that Bacillus probiotics and NSPases showed obvious synergistic growth-promoting effects on yellow-feathered broilers. Most noteworthily, compared to the basal diet group, the diversity index of ACE and Chao increased when the treatment of NSPase or Bacillus Probiotics alone or combined, especially, the significantly increased (P < 0.05) of ACE and Chao revealed in group IV. The PCoA and NMDS results further indicated that the group of diet NSPases + Bacillus was far from the control group, which was consistent with the above ACE and Chao analysis results. Our results were consistent with Klein et al. (2015) and Liu et al. (2018). They reported that the beneficial effects may be observed from the use of multiple enzymes or mixed-use of Bacillus subtilis and photosynthetic bacteria. Moreover, a significant increase was found in proportions of Ruminococcaceae, S24-7 group, Lactobacillus, and Subgroup 2, and a significant decrease was found in the proportion of Bacteroides in NSPase + Bacillus Probiotics-treated group at the cecal or ileal level. An obvious change in ACE or Chao index would suggest the deep changes in microbial diversity. On one hand, NSPase might affect the intestinal microbiota by reducing the undigested substrates and some short-chain oligosaccharides with potential prebiotic effects were created (in situ) from cell wall NSP (Kiarie et al., 2013). On the other hand, Bacillus probiotics could influence the distribution and colonization of the innate microflora along the gastrointestinal tract, reduce the competition for nutrients between microbes and the host, promote the growth and proliferation of other good symbiotic bacteria (Grant et al., 2018). To summarize, it has been suggested that Bacillus Probiotics and NSPases have a significant synergistic effect on improving the intestinal microflora of ducks.

Including Bacillus (25 mg/kg) and NSPases (150 mg/kg) in the diet could efficiently enhance growth performance via altering gut microbiota composition at the genera level and antioxidant indices of ducks. By comparison, the supplementation combining both Bacillus and NSPases showed the best effect for microbial abundances and diversities. Thus, using an additive of both Bacillus and NSPases could be recommended in diets during the early stage of Zhijiang ducks.

The data analyzed in this study is subject to the following licenses/restrictions: The data used to support the findings of this study are available from the corresponding author upon request. Requests to access these datasets should be directed to QL, linqian@caas.cn.

The animal study was reviewed and approved by the experimental procedures of this study were approved by the Institutional Animal Care and Use Committee of Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences.

HQ, QL, and QD conceived and designed the experiments. XW, YZW, TL, SP, and HZ prepared the samples. XW completed the serum indices measurement. YYW detected the growth performance index. YYW and QL performed the sequencing of the 16S rRNA gene. SP and SZ completed the data analysis. SP and QL wrote the manuscript. QL and JZ refined the article. All authors contributed to the article and approved the submitted version.

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.