In recent years, many attentions have been paid to the use of algal resources and their derivatives as food additives in aquaculture (Fleurence et al., 2012; Rajauria, 2015; Thanigaiv el et al., 2016). The immunostimulatory properties of seaweeds in fish have been reported in various studies (Thépot et al., 2020). However, providing information about algae species, especially endemic species could be valuable. In the present study, the immunostimulatory and antioxidant effects of an endemic macroalgae, Gracilaria corticata extracts was examined in the goldfish by adding it in the diet. Based on our results, the growth performance the fish were not affected by dietary GCE. In general, the use of dietary Gracilaria sp. has resulted various results related to growth performance of fish. In Persian sturgeon, Acipenser persicus, dietary Gracilaria sp. (Adel et al., 2020) had no effects on growth performance. Similar results were observed in barramundi, Lates calcarifer and Meager, Argyrosomus regius supplemented with Gracilaria pulvinata and Gracilaria sp. respectively (Morshedi et al., 2018). In Siganus canaliculatus, use of Gracilaria lemaneiformis in the diet improved the fish growth (Xu et al., 2011). Contrary to these results, Younis et al. (2018) reported the reducing effects of dietary Gracilaria arcuata extracts in Nile tilapia, Oreochromis niloticus, which it was attributed to the high Nitrogen-free content of the extracts and its negative effects on digestibility and also to lower protein content of Gracilaria meal compared to fish meal. These results conclude that the growth response of fish to dietary Gracilaria sp. may be different depending on fish species, algal species and its chemical composition.
In this study, dietary GCE had enhancing effects on immune components and immune-related gene expressions in goldfish, as reported previously in other studies with Gracilaria sp. Hoseinifar et al. (2018) showed that dietary Gracilaria gracilis powder enhances the skin mucus immune components including total Ig and total protein, however the expression of immune (TNF-α, LYZ and IL) and antioxidant enzyme-related (SOD and CAT) genes showed no changes. Silva-Brito et al. (2020) reported a dose-dependent pattern for the effects of dietary Gracilaria sp. on antioxidant and immune responses of gilthead seabream, Sparus aurata, where a dietary level of 2.5 % Gracilaria sp. improved the plasma peroxidase activity. Use of Gracilariopsis persica in the diet of rainbow trout enhanced serum lysozyme, SOD, peroxidase activities and also the expression of immune-related gens such as LyzII, TNFα and IL-1β (Vazirzadeh et al., 2020). The immune enhancing effects of Gracilaria sp. have been widely reported in shrimps (Hou and Chen, 2005; Yeh and Chen, 2009; Yeh et al., 2010; Lin et al., 2011; Sirirustananun et al., 2011; Chen et al., 2012). Nevertheless, some studies have also reported the reducing impacts of dietary Gracilaria sp. on fish immunity. In European sea bass, Dicentrarchus labrax, Gracilaria-supplemented fish showed lower ACH50 activity compared to non-supplemented fish (Peixoto et al., 2016). Silva-Brito et al. (2020) reported a decrease in GPx activity in the gilthead seabream fed 2.5% and 5% Gracilaria sp. In the present study, GCE at dietary levels of 1 % increased the activity of mucosal lysozyme, alkaline phosphatase and protease. Mucosal proteases play an essential role in the breakdown of proteins involved in inflammatory, coagulation, apoptotic and tissue regeneration processes (Ahmad et al., 2021). Skin mucus alkaline phosphatase is known as part of fish mucosal immune system, though its actual mode of action is still unknown. However, some antibacterial properties have been reported for skin mucus alkaline against water pathogens (Esteban et al., 2015).
Based on above results, it is recognized that the effects of dietary Gracilaria sp. on fish immune system may be different depending on fish species, algal species, the dose administrated and condition of experiment.
MDA levels are known as biomarker of lipid peroxidation in organisms including fish (Avci et al., 2005; Grotto et al., 2009; Mendes et al., 2009; Rafieepour et al., 2018; Hajirezaee et al., 2019). In this study, the MDA levels did not show significant changes between the experimental groups, which could indicate that the GCE did not induce oxidative stress. However, GCE at high concentrations (1-1.5 %) enhanced the activity of liver antioxidant enzymes (CAT, SOD, GPx), which clearly shows the enhancing effects of GCE on antioxidant defense system of the fish.