**4. Discussion**

In order to determine whether the addition of curcumin in the diet has an effect on the resistance to external environmental stress of greater amberjack, or whether it effectively protects the in vivo multiple functions of fish and enhances the capacity of recovery from stress, we analyzed and confirmed the results from multiple perspectives, such as nutrition, organ protection and the immune system, etc.

The role of dietary curcumin supplementation in animal nutrition is our first concern, on account of the digestion and absorption ability of fish having direct and important links with final growth performance [50–52]. Digestive enzymes including amylase, lipase, and trypsin were determined after two phases of the experiment. Comparisons of digestive enzyme activity after acute ammonia nitrogen stress and recovery provided the evidence of the promotion of recovery of digestive functions owing to the dietary curcumin supplementation. In particular, the activity of amylase and lipase of greater amberjack fed with 0.02% curcumin additive was not only maintained at the previous level but also increased. In contrast, the counterparts of the control and the 0.01% additive amount group decreased together and showed no change in their relative relationship, indicating that the high content of the curcumin additive maintained and even strengthened the activity of amylase and trypsin during the recovery following stress. Similar findings occurred in other fish. For instance, in crucian carp (*Carassius auratus*), significant increases were found in trypsin and lipase activities in the intestine after supplemental doses of dietary curcumin at 5 g/kg [35]. In addition, Ref. [53] demonstrated the ascent of these two digestion enzymes in another fish, *Ctenopharyngodon idella*, along with a rise in amylase activity. However, in the present study, the same positive assistance was not seen in trypsin activity in the fish fed at both dosage rates of curcumin additive, which is inconsistent with previous studies. This contradiction may be due to an insufficient dose, or there may be another possibility that should be further investigation by experiment. As for absorption capacity, in our experiment the enhanced effect of curcumin supplementation on Na+/K+–ATPase, γ-GT and CK activity was more obvious. The recovery mechanism was started earlier, compared with the control, regardless of the additive amount; in other words, the stress recovery process was accelerated. The studies mentioned above on crucian carp and grass carp also witnessed a positive increase in these three enzymes related to nutrition absorption with additional curcumin supplementation in the fish diet. Furthermore, a great deal of research has provided more direct enhanced-growth data more intuitively (increases in WG, SGR, DWG, FCR and FE, etc.) [54–58]. Collectively, it can be concluded that dietary curcumin supplementation can not only increase growth in daily culture by improving digestion and absorption, but can also raise the performance in a stress environment.

The role of curcumin as an immunostimulant that stimulates the activity of the nonspecific immune system in fish is also worthy of attention. The activity of lysozyme is an important marker for representing in vivo non-specific immunity of fish [59,60]. In this study, when there was no significant change in the control group, lysozyme activity increased in the fish fortified with both curcumin diets. The improving effect of curcumin on lysozyme activity was also reported by Leya et al. [61], who fed *Labeo rohita* fingerlings

a diet with 0.5%, 1%, 1.5% and 2% curcumin for 60 days; a significant rise was observed in lysozyme activity from 15 days to the end. Furthermore, another study also showed an increase in the lysozyme activity of *Oreochromis niloticus* fed the 50 mg and 100 mg/kg curcumin diet, but not in all the groups [38]. That indicated that the benefits of the additive amount of curcumin did not improve with higher amounts; however, the proportion of 0.01% and 0.02% in our present study remained positive for lysozyme activity. Albumin (ALB) should be another important component of a non-specific immune system [62,63]. Ref. [64] studied the effect of a curcumin-supplemented diet in *Cirrhinus mrigala* and found that fishes fed a curcumin-enriched diet (1–1.5%) showed significantly higher serum albumin. Similarly, research into the effects of two kinds of Supplementary Materials on *Nile tilapia* immunity also witnessed a remarkable increase in the curcumin-addition group [65]. However, the ALB of the two treatment groups showed no obvious change, while the ALB of the control pulled away from the state of no significant difference among the three groups. The results of the ALB were inconsistent with previous studies, and whether it was related to the amount of curcumin addition should be further explored.

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are produced and released by hepatocytes, and they are considered biomarkers of liver damage injury [66–69]. As for the protective effect of curcumin on the liver, the involvement of AST and ALT has been reported in some research. A significantly lower level of ALT and AST activities were observed in *Megalobrama amblycephala* from the 60 mg/kg curcumin diet group [70]. In addition, a study on grass carp showed that ALT and AST activities decreased significantly on account of curcumin addition [71]. The protective effect of curcumin on the liver was also found in this study, and a greater reduction in serum AST or a quicker reduction in serum AST were observed. However, no similar trend occurred in ALT activity. This phenomenon illustrated that, at least at the curcumin level of this experiment, a protective effect of restricting ALT activity was not activated.

Curcumin has been proved to have lipid-lowering and anti-obesity effects on mammals [72–76]. Therefore, its function of improving lipid metabolism in fish was also what we wanted to understand, and triglyceride (TG) and total cholesterol (TC) were tested in this study for verification. The present study showed that TG and TC content were basically maintained at the original level or even showed a slight decrease when the control group increased significantly after recovery. Based on that, facilitation of lipid metabolism on greater amberjack by curcumin has been very clear. These results are consistent with the results of a study [36] in which TC, TG and LDL-c were observed in the curcumin treatment group.

Heat-shock proteins (HSPs) are a highly conserved protein family; commonly analyzed HSP70 and HSP90 are included. These can be induced by a variety of stressors and perform their protective roles. Many heat shock proteins have molecular chaperone activity to preserve the tertiary structure of other proteins. Therefore, the proteins are an important part of the cellular stress response and considered excellent markers of stress response [77–81]. The relative expressions of HSP70 and HSP90 in four tissues (liver, kidney, spleen and gill) were determined in this study. Regarding HSP70, through the comparison of stress state and recovery state, curcumin obtained an outstanding performance in the relative expressions of HSP70 in fish, but its effect on liver cannot be clarified from the data. Regarding HSP90, its situation was very different from that of HSP70. HSP90 in all tissues except gill remained in the state of stimulated stress and did not fully recover. Nonetheless, the positive effect of curcumin also can be found in liver, kidney and spleen tissues. It should be noted, however, that different additive amounts of curcumin resulted in great differences.

Cytokine is a class of small molecular proteins with a wide range of biological activities. The class mainly includes interleukins (ILs), tumor necrosis factors (TNFs), transforming growth factor-β family (TGF-β family) and interferon (IFN), etc. [82]. Cytokine also can be roughly divided into two types: pro-inflammatory cytokines and anti-inflammatory cytokines. Pro-inflammatory cytokines determined in this study were IL8, IL1β, TNF-α and IFN-γ, and an anti-inflammatory cytokine was TGF-β1. The function related to the

anti-inflammation role of curcumin has been widely reported [83,84]. The study in rats reported that the expression of TNF-α and IL-6 in hypercholesterolemic rats decreased after a 200 mg/kg curcumin treatment. In fish, Ref. [85] investigated the protective effects of curcumin on liver-damaged *Cyprinus carpio*; the results indicated that 0.5% and 1.0% curcumin reduced the CCl4-induced damage in liver by inhibiting NF-kB, IL-1β, TNF-α, and IL-12 expression. Regarding pro-inflammatory cytokines in the present study, curcumin showed only its inhibition capacity on IL1β and IFN-γ for their sharper declines during recovery. In addition, the sole anti-inflammatory cytokine TGF-β1 we determined to be obviously improved in the high curcumin-dosage group. Transcription factor nuclear factor kappa B (NF-κB) has a central role in promoting the process of inflammation and cancer, and cytokine expressions can be activated via this signaling pathway [86,87]. Curcumin inhibits the expression of cytokine via blocking the NF-κB pathway. Ref. [88] provided the evidence with the study on *Oncorhynchus mykiss*; NF-κB expression declined dramatically after curcumin was added to the diet. Another test on *Cyprinus carpio* showed an attenuation effect on the expression of NF-κB caused by Pb exposure [89]. The result of this study was similar with theirs. The expression of NF-κB1 decreased to a higher degree in fish fed with 0.02% dietary curcumin, thus avoiding the continuation of inflammation response more quickly.

Some other immune-related genes were determined in this study; both effector molecules related to the innate immune system and the adaptive immune system were included (C3, C4, Hepc, Mx and lgT). The complement system comprises more than 35 proteins and plays an important role in the innate immune system of fish. It can exert positive effects on phagocytosis, antigen-antibody aggregate clearance, inflammatory reactions, and antibody production [90–93]. Antimicrobial peptides (AMPs) are also a vital component of the innate immune system which cannot be ignored. Hepcidin (Hepc), as a member of AMPs which are rich in cysteine, also performs well in antimicrobial activity. Coupled with its specific iron regulation function, it is of great importance to organism [94–96]. In addition, proteins encoded by the Mx gene are high-molecular-weight GTPases induced by interferon; they have strong intrinsic GTP hydrolysis, broad-spectrum antiviral activity and can inhibit a variety of negative-strand RNA viruses [97,98]. Immunoglobulins (Igs) play a key role in the adaptive immune system to help the organism against pathogens. Immunoglobulin T (lgT) was the last found among all immunoglobulins in teleost fish. It has been proved to have a more specific focus on gut mucosal immunity compared to other immunoglobulins (IgM, IgD, IgG, IgE, and IgA) [99–101]. The expression of these genes was sufficient to reflect the status of many important parts of the fish immune system, which enabled us to glimpse the truth of immune changes. In the present study, no striking enhancement was found in the restorative ability of C4, Hepc and Mx under both proportions of dietary curcumin treatments. However, 0.01% dietary accelerated the recovery pace of C3 and lgT after stress. Although positive results were not acquired for all the immune-related genes, the possibility that curcumin improves the immune functions of greater amberjack is assured, and more exciting results can be obtained on more indexes after more investigations into additive schemes.

Recent research into dietary curcumin supplementation on more fish species than our present study covers further broadens its application. For example, dietary curcumin proved to promote gilthead seabream larvae digestive capacity and modulate the oxidative status [102], and it was also found to work at the post-larval stage [103]. Dietary curcumin was also found to relieve the lipopolysaccharide (LPS)-induced and deltamethrin (DEL) induced stress response (oxidative stress, inflammation and cell apoptosis) in *Channa argus* via Nrf2 and NF-κB signaling pathways [104,105]. With more related studies combined with the results of our research, it can be predicted that dietary curcumin supplementation will play an important role in the healthcare and maintenance of robustness of fish.
