In aquaculture, growth promotion is a character of particular significance, because it directly connected with the productivity and profitability of enterprises (Serpil Mişe Yonar, 2019). Chitosan is a positive growth promoter and have been regarded as an essential prebiotic for the growth of aquatic animals, such as gibel carp (Carassius auratus gibelio) (Y Chen et al., 2014), cobia (Rachycentron canadum) (Geng et al., 2011b)and tiger shrimp (Penaeus monodon) (Niu et al., 2015). In this research, hybrid sturgeon fed with 1.00 g/kg, 3.00 g/kg and 5.00 g/kg dietary chitosan showed remarkable increase of growth performance evidenced by higher FBW, WG and SGR and lower FCR compared to the fish fed control diet. However, it is worth to note that when chitosan supplemented in diet reached 5.00 g/kg, the growth performance of hybrid sturgeon showed a comparatively decrease though the deference was not significant. The result is consistent with previous research which reported that the sea bass (Dicentrarchus labrax) feed with 0.5-4.0 g/kg chitosan have enhanced growth performance, and the optimal supplemental level was 1.0 g/kg (Zaki, Shatby, & Shatby, 2015). However, the significantly higher dietary concentration of chitosan was necessary in other fish species. For example, the growth character of grey mullet (Mugil cephalus) has a liner relation with the concentration of chitosan, and the fish fed on diets supplemented with dietary chitosan at concentrations of 10 and 15 g/kg showed significant growth performance compared with those fed with diets containing 5 g/kg chitosan (Akbary & Younesi, 2017). Similarly in European carp (Cyprinus carpio), the inclusion of chitosan at a high level of 20 g/kg enhanced the growth rates (Maqsood, Singh, Samoon, & Khansaheb Balange, 2010). The discrepancy in the optimal level of dietary chitosan partial due to differences among fish species or experimental conditions.
The quality of an intestine is determined by biological factors like values of muscle thickness and intestinal villus height as well as numbers of goblet cells (Banan Khojasteh, 2012). The absorption of nutrients by the intestine defines a host’s growth performance, which has a directly relation to its morphology (Pirarat et al., 2011). The increment in VL and VW induces the enhancement of intestinal absorption surface area for nutrients (Kuebutornye et al., 2020). Muscular layer mainly play a role in peristaltic motion and increased MT level commonly implies an improvement of intestinal function associated with digestion (F. Chen & Wang, 2013). The increase in crypt depth is coincidence with an improvement production rate of crypt-cell and an overall stimulation of cell renewal in the intestine that has universally been connected with a decreased digestive and absorptive activity (Jiménez et al., 2020; Pluske, Williams, & Aherne, 1996). Goblet cells produce and secrete biologically active substances, generally considered as mucus and glycoproteins components, which have been reported to exert an important role in gut immune systems (Blomberg, Henriksson, & Conway, 1993; Knoop & Newberry, 2018). Moreover, the produced substances cover on the surface of gastrointestinal epithelium where by intestine barrier function is enhanced (Deplancke & Gaskins, 2001). This study showed that dietary inclusion of chitosan increased the value of muscle thickness and amount of goblet cells in the mid intestine of fish, suggesting the improvement of nutrient utilization and immunity. In sturgeon, the supreme nutrient absorption occurred in spiral valve (Caimi et al., 2020), this segment was therefore considered. In the current research, no significant differences of VH, VW and CD were observed in the spiral valve of sturgeon among all experimental groups. However, evident increase of MT and the number of acid mucin producing goblet cells were recorded in chitosan dietary groups compared to the control group, indicating the positive effect of chitosan on spiral valve absorption and disease resistance.
The innate immune system, first line of defense for fish immunity, exerts a key role in preventing invaded pathogens and initiating adaptive immune responses (Magnadóttir, 2006). Lysozyme, referred to as N–acetylmuramide glycanohydrolase or muramidase, is an important bacteriolytic enzyme known to controls microorganism colonization and proliferation by destroying cell wall polysaccharides, resulting in cell wall lyse and bacterial death (Saurabh & Sahoo, 2008; Z.-H. Zhang et al., 2020). Previous studies revealed that chitosan supplementation increased the lysozyme capacity in juvenile loach (Misgurnus anguillicaudatus) (Yan et al., 2017), cobia (Rachycentron canadum) (Geng et al., 2011a), and gibel carp (Carassius auratus gibelio) (Y. Chen et al., 2014). In this study, the serum LZM activity was increased with the concentration of dietary chitosan and the highest value was observed in the CHI3 group which implied the dietary chitosan might improve the immunity of the hybrid sturgeon. ACP and AKP, two kinds of phosphatase capable of hydrolyzing organic phosphate esters, play pivotal roles in non-specific immune system of animals (M. Chen, Chen, Tian, Liu, & Niu, 2020; Tseng et al., 2009) and have been considered as a symbol of macrophage stimulation for the capacity to intracellularly digest antigens in the immune system of animals (Yin, Gong, Ke, & Li, 2015). In this research, as dietary chitosan increased, the activities of ACP and AKP exhibited the tendency of initially increased and then decreased. This was similar with the results in previous studies which demonstrated that the ACP and AKP activities have the similar tendency after fed diets supplement with prebiotics (Q.-Q. Chen et al., 2016; Jia et al., 2017). MPO is a particularly vital enzyme function in the elimination of microorganisms (X. Chen et al., 2020), which could utilize hydrogen peroxide to generate hypochlorous acid (Dalmo, Ingebrigtsen, & Bøgwald, 1997) and subsequently eliminate invading pathogens through destroying a variety of target substances (Johnston Jr, 1978). The current investigation displayed that the activities of MPO in serum showed an improvement as the concentration of chitosan increased from 1.00 to 3.00 g/kg, then declined when the supplement level reached 5.00 g/kg, indicating that dietary chitosan supplementation within a reasonable range could elevate the immune system and may be able to prevent sturgeon from harmful microorganisms. Various studies have demonstrated that prebiotics elevated the MPO activity in fish (Mohammadi, Rafiee, El Basuini, Abdel-Latif, & Dawood, 2020; G. Xu et al., 2018). In general, the increase of LZM ACP, AKP and MPO activities in the serum, suggesting that dietary chitosan could promote the innate immunity capacity of sturgeon.
The balance between generation and clearance of reactive oxygen species (ROS) is essential for sustaining the dynamics of normal metabolism in the fish body (Ibrahim et al., 2021). Under normal physiological conditions, cells generate ROS. Meanwhile, the body is enclosed by a complex network of antioxidant system that represent as SOD, CAT and GSH-Px to eliminate excessive ROS that may exert serious impairment of DNA and other macromolecules (Aliko, Qirjo, Sula, Morina, & Faggio, 2018; Regoli & Giuliani, 2014). The enzyme SOD, first line defense against oxidative stress, exerts the role in catalyzing toxic superoxide anions produced in the tissues through cellular metabolism or reactions to O2 and H2O2, which would be then transformed into H2O and O2 by CAT and GSH-Px to complete the detoxification process prior to attacking the cellular components (Jamalzad Falah, Rajabi Islami, & Shamsaie Mehrgan, 2020; Yu, 1994). Therefore, analysis of those enzyme activities can offer a reflection of the antioxidant function in animals, as also can serve as indicator of oxidative stress (C.-N. Zhang et al., 2013). The oxidative stress lead to the production of excessive ROS in cellular components which could induce lipid peroxidation and accumulation of lipid peroxides in cell (Ferreira, Moradas-Ferreira, & Reis-Henriques, 2005; S. M. Yonar, Sakin, Yonar, Ispir, & Kirici, 2011). MDA as a highly toxic substance produced by lipid peroxidation can be used to elevate the degree of lipid peroxidation and cell damage in fish (Buege & Aust, 1978). The present study illustrated that dietary chitosan could improve the antioxidant capacity where activities of SOD, CAT, and GSH-Px upregulated significantly, while values of MDA downregulated significantly. These results are indicative to the enhancement of antioxidant activity and the alleviation of oxidative stress. Similar results were observed in previous study which showed that chitosan supplementation induced an apparent increase of enzymatic (SOD, CAT and GSH-Px activities) antioxidant and a significant decrease in the MDA level (El-Naggar et al., 2021; Niu et al., 2015). The antioxidant activity of chitosan can be due to free radical-scavenging activities through the donation of hydrogen or one pair of electrons and its abilities of chelating metal ions (S. B. Lin, Chen, & Peng, 2009; Ngo & Kim, 2014).
Feeding fish with supplemented prebiotics and then challenging them with pathogenic microorganism is one of the most valuable way to investigate the potential function of the supplements in terms of resistance to pathogen infections (Xing, Xu, Tang, Sheng, & Zhan, 2019). The decrease of cumulative mortality rate or increase of relative percent survival reflected promotion of fish immunity and resistance to bacteria. In this research, it is mentioned that dietary supplement with chitosan relatively improved the resistant of hybrid sturgeon against A. hydrophila, where the dose of 5.00 g/kg diet recorded the highest protection. The increased protection was in line with the results previously reported in gibel carp (Carassius auratus gibelio) (Y. Chen et al., 2014), rainbow trout (Oncorhynchus mykiss) (Ahmed et al., 2021) and koi (Cyprinus carpio koi) (S. M. Lin et al., 2012), which revealed that fish administrated with prebiotics induced lower cumulative mortality rate or higher relative percent survival rate in the challenge experiment. The positive results may be explained by stimulation of antioxidant activity and non-specific immunity after administration of chitosan.