The overpricing of aquafeed materials coincided with their severe shortage, prompted scientists to look for less expensive, readily available, and nontraditional feedstuff that can yield heathy fish with high productivity. A. pinnata is one of the most important macrophytes which provides appropriate growth levels, with a substantial protein content (20%-30%; dry weight) (Abou et al., 2007). Several studies were conducted to assess the nutritional value of A. pinnata as a replacer for several dietary components (Fasakin & Balogun 1998; Chareontesprasit & Jiwyam 2001; Gangadhar et al., 2017; and Magouz et al., 2020). As a nutritive and productive microphyte, A. pinnata was not practically examined as a fermented aquafeed. Our unique study was demonstrated to monitor the nutritive value of A. pinnata and assess its influence on intestinal digestive enzymes and morphometry in Nile tilapia, as well as its impact on immune response and antioxidative status in vivo. Furthermore, our results stated that up to 30% of BSFA could be incorporated into Nile tilapia diets without deleterious effects on health and growth status.
Interestingly, CP% elevated at a rate of 3.72%, and NDF% dropped at a rate of 11.59%. Moreover, amino acid profile of BSFA including arginine, methionine, threonine, and valine showed higher levels than raw Azolla. These reported results were also observed by shiu et al. (2015), who reported that fermentation of soybean meal with B. subtilis E20 (FSBM) enhanced its crude protein content and improved overall amino acids content compared to untreated one. In addition, Bacillus fermented physic nut seed meal (Jatropha curcas) was documented to increase crude protein content, while the fiber content was significantly diminished compared to nonfermented meal (Hassaan et al., 2017). This development in protein content may be attributed to the amino acid added during the fermentation process as a result of microbial protein formed (Belewu and Sam, 2010; Ismail et al., 2021). It was noted that protease enzymes released following fermentation shared in improving the amino acid profile (Hong et al., 2004; Lee et al., 2016). Additionally, in accordance with Hassaan et al. (2017), the diminishing in fiber content may be assigned to the fibrinolytic enzymes (cellulase, xylanase, amylases, hemicellulase, β-glycosidase, pectinases, and α-galactosidase) produced during the fermentation process.
Also, growth parameters including (FBW, BWG, FCR, SGR, and PER) revealed better values in fish fed BSFA at a rate of 30% than other experimental groups. Following Chareontesprasit and Jiwyam (2001), A. pinnata could be included in diets for Nile tilapia up to 15% without having detrimental effects on growth and feed utilization. Furthermore, dry Azolla was recommended at a level of 25% in Tilapia zilli diet without negative effects on growth and feed utilization (Abdel-Tawwab 2008). Interestingly, our progressive results could be explained by Liu et al. (2012) who reported that improving the growth of grouper fish (Epinephelu scoioides) fed B. subtilis-incorporated diet came back to the exoenzymes (proteases and lipases) secreted by B. subtilis which promote nutrient digestibility, thereby improving growth. In addition, certain essential nutrients were produced by many B. subtilis spp. as amino acids and vitamins (Sanders et al., 2003; Rosovitz et al., 1998), which, in turn, improves growth and feed intake. In contrast, a higher inclusion level of BSFA beyond 30% poorly affected on growth. This could be attributed to the climbing in fiber content and some deleterious substances like phytates and phenolics which, in turn, influence digestibility and feed acceptability (Shamna et al., 2015).
Hematological parameters were stated to reflect fish’s general health condition and physiological status (Maita, 2007). In the present study, BSFA did not show any specific trends in hematological (RBCs, Hb, WBCs, and hematoctit) and biochemical indices (AST, ALT, total protein, and creatinine). The same results were concluded in Japanese flounder (Paralichthys olivaceus) fed fermented soybean meal and squid by-product blend (Abdul Kader et al., 2012). In parallel with our data, hematological and biochemical assays including Hb, PCV, total protein, AST, ALT, TG, and glucose showed insignificant changes in juvenile rainbow trout fed fermented protein concentrates (Moniruzzaman et al., 2018). These results proved that fermentation has a remarkable effect on the antinutritional factors (ANFs) which associated with iron and the amine group of amino acids and diminished their accessibility in host blood (Soltan et al., 2008).
Lysozyme is a potent bactericidal enzyme (Saurabh and Sahoo 2008). It has a destructive effect on Gram-positive bacterial cell walls except opsonin which enhances a defense mechanism named phagocytosis. This mechanism is an efficient immunological response of the body against any infectious agent and can be assessed by determining the phagocytic activity and index (Harikrishnan et al., 2011). During this process, phagocytic cells produce reactive oxygen species (ROS) which are controlled by antioxidants to protect host cells. In this study, BSFA dietary inclusion dose showed a notable effect on nonspecific immune response and antioxidative status. Similarly, fermented vegetable products improved lysozyme and phagocytic activities in Japanese flounders (Ashida & Okimasu 2005). In addition, oral administration of B. subtilis in gilthead seabream (Sparus aurata) for two weeks significantly increased phagocytic activity (Dawood et al., 2016). On contrary, Nile tilapia fed Azolla meal did not show a significant effect on lysozyme activity, phagocytic activity, or phagocytic index (Magouz et al., 2020). Our results revealed that lysozyme activity, phagocytic activity and phagocytic index reached their peaks in BSFA30 group. SOD, GPx and MDA are oxidative stress indicators which illustrate the oxidative status of the host. SOD considered the first antioxidative line of defense, preventing cells from damage by catalyzing H2O2 and removing the reactive oxygen species (ROS) (Wan et al., 2016). In addition, GPx helps sustaining host cells’ health via disproportionation of the toxic ROS to inactive oxygen molecules and hydrogen peroxide (Dawood et al., 2020). Conversely, MDA is a final product of lipid peroxidation and can be used as an indicator of oxidative damage (Ding et al., 2015). The present study showed that BSFA had no observable effect on MDA, while SOD and GPx came to their climax in BSFA60 group. All these results suggest that BSFA modified the free oxygen radical scavenging capacity and developed the immune response of Nile tilapia. In line with our findings, juvenile black sea bream fed fermented soybean meal revealed a notable increase in GPx and SOD activity (Azarm et al., 2014). Furthermore, white shrimp fed a B. subtilis-supplemented diet recorded higher SOD activity (Liu et al., 2014).
Regarding whole body chemical composition, the experimental tilapia BSFA-fed had no observable effect compared to the control group. Similarly, Moniruzzaman, et al. (2018) stated that there were no significant changes in whole body chemical composition of juvenile rainbow trout fed fermented protein concentrates. Also, the same findings were noticed in O. niloticus fed graded levels of A. pinnata (Magouz et al., 2020). However, Abdel-Tawwab (2008) documented that the gradual increase of Azolla meal levels in Tilapia zilli-tested diets showed declining in crude protein and lipid, with increasing moisture and ash contents.
As a widely accepted indicator of feed utilization and digestibility of the host, intestinal digestive enzyme activity was determined to assess the nutritive value and optimized level of BSFA for our experimental Nile tilapia (Ueberschär, 1995). Curiously, amylase and protease enzymes are influenced by the level of BSFA included in diet and reached their summit in BSFA30 and BSFA15 groups, respectively, which agrees with the results found in the Nile tilapia group fed with a diet containing Bacillus subtilis spp. (Liu et al., 2017). As previously mentioned, wide range of Bacillus spp. were documented to produce exoenzymes which, in turn, participate in nutrients break down and digestion (Liu et al., 2009; Bandyopadhyay & Mohapatra 2009). On contrary, α-amylase, lipase, and trypsin activity in the intestine of juvenile black sea bream fish (Acanthopagrus schlegeli) were not influenced by dietary fermented soybean level (Azarm & Lee 2014). The activity of amylase and protease enzymes reduced with rising the levels of A. pinnata, which is compatible to findings of Magouz et al. (2020) in Nile tilapia. This can be assigned to the increase in oligosaccharides and nonstarch polysaccharides (NSPs), which, in turn, affect the viscosity and transit rate of digesta, and their binding action with bile salts leading to decreased bioavailability of nutrients and energy (Francis et al., 2001).
The histomorphometry in the current work supported the upgrading effect of BSFA, as previously mentioned. Intestinal morphometric analysis is a critical method for determining the impact of dietary supplements on the fish intestine’s absorption capacity, as well as local intestinal immunity, which reflects the fish’s overall immune condition (Abdel-Warith et al., 2021; Haygood, 2018; Dawood, 2020 c). In addition, the incorporation of immune cells within the tissues of the fish gut plays a role in immunity (Gewaily et al., 2021b). However, the general morphology of all investigated groups showed a normal appearance, the morphometric analysis clarified that the addition of BSFA to the Nile tilapia diet has a significant augmentation in the intestinal villi at the level of villus height, width, area, and goblet cell number. This may be due to the greater feed utilization in Nile tilapia BSFA-fed that was associated with increased intestinal villi surface area which is important for food absorption through the gut (Shukry et al., 2021; Zaki and Shatby 2015).
Disease resistance is the final indicator of host health status (Lim et al., 2009). A developed immune system can be proven by bacterial challenge test results. Our investigations revealed that dietary BSFA provided protection against A. septicemia infection in Nile tilapia. Similar results were reported in white leg shrimp (Litopaeneus vannamei) fed B. subtilis-fermented plant proteins, and challenged with Vibrio parahaemolyticus (Hamidoghli et al., 2020). In addition, administration of B. pumillus improved the survival rate in Nile tilapia challenged with A. hydrophyla (Aly et al., 2008). The same findings were also obtained in rockfish (Sebastes schlegeli) challenged with E. tarda (Lee et al., 2016), rainbow trout (Oncorhynchus mykiss) challenged with Aeromonas spp. (Newaj-Fyzul et al., 2007), and cobia (Rachycentron canadum) challenged with V. herveyi (Geng et al., 2011). Thus, the present study showed that Nile tilapia fed with a diet incorporated with 30% BSFA for 95 days exhibited the lowest cumulative mortality (%) when challenged with A. septicemia for 14 days, followed by the BSFA45 group.