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Fishmeal and fish oil are becoming scarcer and more expensive due to the stagnant production growth of capture-based fisheries and the increasing demand for FM and FO resources. Furthermore, it can be argued that the use of FM and FO in industrially compounded aquafeeds are unsustainable especially for carnivorous marine finfish species based on the global average fish-in fish-out ratios which are generally greater than one for these high-value aquaculture species. Therefore, more affordable and available alternatives, such as plant-protein ingredients, are being readily incorporated into diets of fish. However, despite the presence of ANFs, such as NSP, most fish species do not possess the endogenous enzymes to optimally utilise plant ingredients.

The indigestible portion of aquafeeds, viz. NDOs, are regarded as functional feed additives and may exert positive effects on the growth and health of aquatic animals. Non- digestible oligosaccharides or immunosaccharies possess beneficial, immune stimulating properties and may enhance the innate immune response of fish in two ways: Firstly, immunosaccharides have shown to enhance the innate immunity through directly stimulating pattern recognition receptors and interacting with carbohydrate receptors on leucocytes in the GALT of fish. Secondly, immunosaccharides indirectly modulate the intestinal microbial community by stimulating the growth of specific, beneficial bacteria, such as lactic acid

48 bacteria, while also inhibiting the growth of harmful, pathogenic microbiota in the intestines of fish. Accompanying the enhanced growth of LAB is an increase in the production of beneficial metabolites, such as SCFAs, while harmful proteolytic metabolites are inhibited. Short-chain fatty acids are beneficial in that they may act as additional energy sources as well as having health-benefitting properties. Non-digestible oligosaccharides may be supplemented to fish feeds directly (via prebiotics) or indirectly (via NSP-degrading enzymes). The current study aimed to evaluate the effect of a prebiotic (AXOS) and an NSP-degrading enzyme (xylanase) on the production performance, innate immunity, hindgut SCFA production and hindgut microbial diversity of African catfish.

Dietary xylanase has shown to increase the nutrient digestibility and availability of aquafeeds through i) hydrolysing soluble NSP in the cell contents of plant cells, as well as ii) solubilising insoluble NSP in the cell wall fraction of plant cells. Soluble NSP in aquafeeds interferes with the interaction between nutrients and endogenous enzymes through increasing the intestinal digesta viscosity. On the other hand, insoluble NSP encapsulates potential nutrients inside the plant cell wall. Non-ruminant animals lack the endogenous enzymes that are capable of hydrolysing the glycosidic bonds of plant ingredients and are, therefore, unable to utilise plant feedstuffs optimally. Through hydrolysing the different NSP fractions of plant ingredients, xylanase increases the nutrient efficiency and digestibility of highly plant-based diets. Additionally, xylanase also produces NDOs that are fermented by carbohydrate fermenting bacteria in the large intestine of fish.

Information regarding the supplementation of dietary xylanase in aquaculture studies is lacking despite the positive results shown in the poultry and pig industries. From the literature survey, it is concluded that the effects of dietary xylanase in aquaculture studies vary according to the species, enzyme mixture, enzyme concentrations, and the diet constituents. It is, however, difficult to distinguish the effects of xylanase on fish performance as most studies involved the addition of xylanase in a multi-enzyme complex containing various other enzymes. Nevertheless, xylanase has shown to increase the nutrient efficiency of plant-based diets, especially wheat, but also maize and SBM-based diets. An increase in the growth performance, survival, body protein content, protein efficiency ratio and an improved feed conversion ratio, are among the most reported benefits regarding the supplementation of dietary xylanase in plant-based diets of fish.

Significant improvements have been reported regarding innate immunity parameters, endogenous enzyme activities and intestinal morphology. Additionally, studies have also shown that xylanase supplementation modulated the intestinal microbial communities of fish. The increase in growth performance caused by xylanase and other carbohydrase supplementation is most likely because of the increased nutrient utilisation efficiency caused by the increase in nutrient digestibility, intestinal morphology, physiological factors and a more

49 beneficial microbial community. These beneficial effects of xylanase seem to be more profound, but not limited to, omnivorous and herbivorous, freshwater species. This might be due to the greater inherent capability of these species to utilise plant ingredients compared to carnivorous, saltwater species.

Arabinoxylan-oligosaccharides are the hydrolysis products of arabinoxylan and have shown to possess potential prebiotic properties. Arabinoxylan-oligosaccharides can be produced through enzymatic (xylanase) hydrolysis or through thermochemical methods, such as steam explosion, of AX-containing raw materials, such as brewer’s spent grains. Depending on the method and raw material used, different AXOS preparations are produced. These different preparations of AXOS have different chemical structures and, therefore, different rates of fermentability. Dietary AXOS are indigestible to fish and thus passes through the GIT to the hindgut where it is fermented by facultative anaerobic bacteria. Positive microbial and immunomodulating effects seen in fish may be caused by the direct or indirect action of AXOS supplementation. Dietary AXOS may directly enhance the innate immune response by interacting with microbial-associated molecular patterns and pattern recognition receptors in the GALT of fish. However, most effects of AXOS on the immune system are indirect which involves the modulation of the intestinal microbiota.

Despite the reported benefits of AXOS supplementation in other animals and human studies, literature concerning the supplementation of dietary AXOS in aquafeeds are limited. From this literature study, it can be gathered that AXOS possesses prebiotic properties, including microbial and immunomodulating activities, that may enhance the growth and health of fish. The intricate structure of AXOS, compared to conventional prebiotics, may convolute its functional properties and, therefore, some discrepancies exist between studies. Dietary AXOS have shown to significantly increase innate immune response parameters and hindgut SCFA production of Siberian sturgeon and African catfish. Supplementation of AXOS has also shown to significantly modulate the hindgut microbial community of both these species. A significant decrease in the hindgut microbial diversity has been reported in Siberian sturgeon fed diets supplemented with dietary AXOS. Although the effects of AXOS are more profound on the intestinal microbial community and non-specific immunity parameters than on growth performance, one study did reveal that a combination of AXOS and a probiotic significantly increased the growth rate of juvenile Siberian sturgeon. These effects of AXOS on fish performance may be depended upon the AXOS preparation, fish species, supplementation dosage and the mixture of other prebiotics or probiotics used in combination with AXOS.

The African catfish species is a hardy, omnivorous, highly fecund, fast growing and airbreathing freshwater fish that is not only cultured in Africa but also in parts of Eastern Europe. The inherent capability of C. gariepinus to utilise plant ingredients relatively well may be attributed to the fact that its endogenous enzyme activities are similar to both herbivorous

50 and carnivorous fish species. However, literature has shown that in order to successfully rear African catfish the nutrient efficiency of highly plant-based diets needs to be improved while the presence of ANFs needs to be removed. Functional feed additives, such as xylanase and AXOS, have proved to be able to alleviate ANFs, such as NSP, as well as enhance the nutrient digestibility of plant-based diets whether by degradation of the NSP contents of plant products or by beneficially altering the microbial community of the host, ultimately leading to an improved health and growth of the host animal. However, information regarding the use of these feed additives in aquatic animals are scarce and, therefore, their effects on African catfish performance warrants more study.