Introduction
W ith the current aquafeed formulations including high levels of plant-based ingredients, there is a higher carbohydrate inclusion , including non-starch polysaccharides (NSP), which are less digestible by fish (Kokou and Fountoulaki, 2018). To improve the NSP digestibility and thus feed efficiency, exogenous enzymes of microbial origin such as xylanases can be added (Maas et al., 2021 ). When such enzymes are included in the feeds, they can assist in the degradation of the NSP into less polymerised compounds, making them more available for fermentation by microbiota, resulting in the production of volatile fatty acids. Understanding how the dietary NSP levels and the enzyme addition may affect microbial composition and metabolite production in the gut, and how this relates to nutrient digestibility can assist in optimization of dietary formulations with aim to enhance fish performance and digestibility, especially when raw materials high in NSP are used. In this study, we explored the impact of two different non-starch polysaccharide and ezyme levels on the microbial composition and microbial metabolites in the gut of Nile tilapia ( Oreochromis niloticus ). Nile tilapia has omnivorous dietary habits and the potential for fibre fermentation (Amirkolaie et al., 2005), thus making it an interesting species to understand non-starch polysaccharide effects on the gut microbiota.
Materials and Methods
A 2 x 2 factorial design was used to test the effect of enzymes and dietary NSP levels. The enzyme supplementation included phytase (1000 FTU/kg, Danisco Animal Nutrition) and xylanase (6000 U/kg, Danisco Animal Nutrition). The NSP levels were included at 122 g NSP/kg DM diet (moderate) and at 311 g NSP/kg DM diet (high) . The feeding trial lasted 6 weeks, with four replicate tanks per diet. At the end of the trial, digesta samples were analyzed for microbiota composition , volatile fatty acid and NSP content. For the microbiota analysis, DNA was extracted with commercial kits and the V4 variable region of the 16S rRNA gene was sequenced , using Illumina MiSeq. Taxonomic composition was analyzed according to Kokou et al., 2020 . For diversity analysis, the ‘microeco’ R package was used. Volatile fatty acid concentrations were measured as described in Maas et al. (2021). The NSP content were measured as described by Blakeney et al. (1983). Network analysis using sparcc as correlation method was performed to explore associations between the different microbial taxa , including the environmental variables (nutrient digestibility, NSP content, VFA) as explanatory variables (Kurtz et al., 2015 ).
Results
The results showed that both NSP level and enzyme inclusion can affect the gut microbiota composition. An increased microbial richness and diversity was observed with high NSP or no enzyme addition compared to the enzyme addition in lower NSP levels. Such changes were attributes to a change of the Fusobacteria (Figure 1A), and specifically an increase in the abundance of Cetobacterium , in the gut of Nile tilapia, fed with the MEDENZ diets. This was in accordance with an increase in VFA content in the gut, which was higher in the specific diet (Figure 1B) . The digesta VFA content and the nutrient digestibility could explain the variation in the microbiota composition , as indicated by redundancy analysis. N etwork analysis indicated that NSP levels and enzyme addition may interfere with microbial associations , by altering the clustering co-efficiency. Our previous study showed that enzyme addition to high NSP diets may enhance the microbial associations in the distal gut of Nile tilapia , by increasing the network complexity (clustering co-efficiency and density), which was hypothesized to benefit the gut microbiota homeostasis (Maas et al., 2021).
Conclusion
These findings suggest that both NSP level and enzyme addition can have a significant effect on the gut microbio ta diversity and composition, with the latter being the major factor in this study. Volatile fatty acid content and composition were consistently altered by the enzyme addition in the diets. The interpretation of such results is important in order to better understand the microbial dynamics in the gut and how such dynamics may be altered by dietary formulations such as the presence of NSP or by enzyme addition , ensuring an optimal fish performance.
References
Amirkolaie , A.K., Leenhouwers, J.I., Verreth, J.A.J. and Schrama, J.W., 2005. Type of dietary fibre (soluble versus insoluble) influences digestion, faeces characteristics and faecal waste production in Nile tilapia ( Oreochromis niloticus L.). Aquac. Res. 36, 1157–1166.
Kokou, F. and Fountoulaki, E., 2018. Aquaculture waste production associated with antinutrient presence in common fish feed plant ingredients. Aquaculture, 495, 295-310.
Maas, R.M., Deng, Y., Dersjant-Li, Y., Petit, J., Verdegem, M.C., Schrama, J.W. and Kokou, F., 2021. Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia. Scientific reports, 11(1), pp.1-16.
Kokou, F., Sasson, G., Mizrahi, I. and Cnaani, A. 2020. Antibiotic effect and microbiome persistence vary along the European seabass gut. Sci. Rep. 10, 1–12.
Kurtz, Z.D., Müller, C.L., Miraldi , E.R., Littman, D.R., Blaser, M.J. and Bonneau, R.A., 2015. Sparse and compositionally robust inference of microbial ecological networks. PLoS computational biology, 11(5), p.e1004226.