*2.6. Statistical Analyses*

The relative quantity of gene expression was calculated according to the 2−ΔΔCt method [49]. The results were analyzed using the SPSS 19.0 statistical software packages. All data are expressed as the standard deviation (mean ± SD). Comparisons between different groups were conducted by one-way ANOVA and Tukey's test. Significant difference was considered at *p* < 0.05.

#### **3. Results**

Growth performance indicators (specific growth rate, weight gain percentage, feed coefficient and condition factor) after the feeding experiment are shown in Table 3. The condition factor of *Seriola dumerili* in the 0.01% treatment group was significantly higher than in the other two groups (*p* < 0.05), while there was no significant difference between the control group and the 0.02% treatment group (*p* > 0.05). The feed coefficient of *Seriola dumerili* in the control group was significantly higher than that in all treatment groups (*p* < 0.05), and there was no significant difference between treatment groups (*p* > 0.05). Moreover, different levels of dietary curcumin supplementation had no significant effects on weight gain percentage or specific growth rate of *Seriola dumerili* (*p* > 0.05).


**Table 3.** Growth performance of *Seriola dumerili* at different levels of curcumin in feed.

Data are presented as mean ± SD. In the same row, the same lowercase letters on the right side of the data indicated no significant difference (*p* > 0.05). Different lowercase letters indicated a significant difference (*p* < 0.05).

Digestive enzyme activities in the intestine of greater amberjack are shown in Figure 1. Only the 0.02% CUR group exhibited a significant difference with the control as to AMS activity (*p* < 0.05). Regarding LPS activity, two treatment groups had a similar difference with the control. There was no remarkable difference among all the groups regarding TPS activity (*p* > 0.05).

**Figure 1.** (**a**) Amylase (AMS), (**b**) Lipase (LPS), and (**c**) Trypsin (TPS) activities of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

The activity of Na+/K+–ATPase, γ-GT, and CK in intestinal tissue was also determined. The results are described in Figure 2. Na+/K+–ATPase activity in 0.02% CUR group is apparently lower than in the other two groups after the ammonia nitrogen challenge. An evident lower level was found in γ-GT activity in the 0.01% CUR group. The CK activity of both treatment groups was significantly higher than in the control.

LZM and ALB (Figure 3), AST and ALT (Figure 4) activity, as well as TG and TC (Figure 5) content of serum were determined after the ammonia nitrogen challenge. According to Figure 3, LZM activity of both two treatments groups was higher than that of the control, and there was no significant difference between them. In addition, ALB content of serum showed no difference among groups. The variations in AST and ALT activity were quite different. In particular, no sharp distinction was seen in AST activity among all groups. With respect to ALT activity, the 0.02% CUR group compared to other groups was lower after the ammonia nitrogen challenge. In addition, different change characteristics were also revealed in the TG and TC content of serum. The TG content of all treatment groups was obviously higher than that in the control. By contrast, the TC contents of both treatment groups were lower.

**Figure 2.** (**a**) Na+K+ adenosinetriphosphatase (Na+K+–ATPase), (**b**) γ-Glutamyl transferase (γ-GT), (**c**) creatine kinase (CK) activities of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

**Figure 3.** (**a**) Lysozyme (LZM) activity and (**b**) albumin (ALB) content of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

After recovery from apparent lack of consciousness, fish were sampled and the same in vivo indexes were determined as in the ammonia nitrogen challenge experiment. The activity of three digestive enzymes all showed a significant decline phenomenon compared with the control. The declines in AMS and TPS activity were seen in the 0.02% CUR group and the decline in LPS activity was found in both two curcumin-added groups. The levels of Na+/K+–ATPase and CK activity of the intestine in the two treatment groups after recovery were significantly higher than in the control. γ-GT activity in the two treatment groups was obviously lower than that of the control. Moreover, in serum, LZM activity in the two treatment groups was still maintained at a higher level than in the control. The ALB content of the control reached the highest level after recovery. Figure 4 provides the results of the AST and ALT activity. The lowest AST activity level appeared in the 0.01% CUR group, while in contrast the ALT activity in the 0.01% CUR group was the highest. In addition, the TG in 0.01% CUR and 0.02% CUR groups were both significantly lower than in the control

according to Figure 5. Regarding the TC content, the same significant difference was seen only in 0.01% CUR.

**Figure 4.** (**a**) Aspartate aminotransferase (AST) and (**b**) alanine aminotransferase (ALT) activities of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars indicate significant differences at the 0.05 level).

**Figure 5.** (**a**) Triglyceride (TG) and (**b**) total cholesterol (TC) content of greater amberjack fed with dietary curcumin supplementation after ammonia nitrogen challenge assay. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

Heat shock protein genes, containing HSP70 and HSP90, were quantified in liver (a), kidney (b), spleen (c) and gill (d) tissues of greater amberjack after the challenge assay (Figures 6 and 7). Regarding the HSP70 gene, the relative expression of the HSP70 gene of experimental groups in the liver was significantly lower than that of the control after the treatment challenge assay. In contrast, the relative expression of the HSP70 gene in the kidney and spleen of two curcumin-added groups were both higher than that of the control.

In the gill, only the relative expression of the HSP70 gene of the 0.02% CUR group was at the highest level.

**Figure 6.** The relative expression of heat shock protein (HSP70) genes in (**a**) liver, (**b**) kidney, (**c**) spleen and (**d**) gill tissues of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

The rules of the relative expression of HSP90 gene in each tissue were similar with those of HSP70. In kidney and spleen tissues, the relative expression of the HSP90 gene in both the two curcumin-added groups was significantly higher than that of the control, but the HSP90 expression level of the 0.02% CUR group was significantly lower than that of the 0.01% CUR group. The relative expressions of the HSP90 gene in liver were higher with the increase in the amount of added curcumin. Moreover, the change situation of the relative expressions in gills showed a huge difference between the two heat shock protein genes. The relative expression of the HSP90 gene in gills of the 0.02% CUR group was the highest; however, in HSP70, the relative expression of the HSP90 gene of the control was the highest.

**Figure 7.** The relative expression of heat shock protein (HSP90) genes in (**a**) liver, (**b**) kidney, (**c**) spleen and (**d**) gill tissue of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

There have been some changes in the relative expression of the HSP70 and HSP90 genes in four tissues after recovery compared to the state after the ammonia nitrogen challenge (Figures 6 and 7). As for the HSP70 gene, its relative expression in liver, kidney and spleen was obviously less than that of the control group, but in gill, its relative expression had no difference among all the groups. It was noted that the situation of the relative expression of HSP90 in gill was also quite similar with that of HSP70; experimental groups also showed no difference with the control group. Furthermore, the relative expression of HSP90 in liver was still kept at a lower level than the control group after recovery. However, the relative expression of HSP90 in kidney and spleen fell into a different situation. In spleen in particular, only the relative expression of HSP90 in the 0.01% CUR group different from other groups. In kidney, however, a higher value was found in the 0.01% CUR group and a lower value in the 0.02% CUR group compared with the control.

The relative expression of cytokine IL8, IL1β, TNF-α, IFN-γ and TGF-β1 genes in intestines of greater amberjack after the challenge assay are given in Figure 8. A lower level occurred both in the relative expression of the IL8 and TGF-β1 genes in two curcuminadded groups compared with the control. Regarding the TNF-α gene, only the high curcumin-added group showed the lower level. However, IL1β gene and IFN-γ gene

relative expressions of the curcumin-added group were all significantly higher than that of the control. The difference is that the highest level of the IL1β gene occurred in the 0.02% CUR group, and that of the IFN-γ gene occurred in the 0.01% CUR group.

**Figure 8.** The relative expression of (**a**) cytokine IL8, (**b**) IL1β, (**c**) TNF-α, (**d**) IFN-γ and (**e**) TGF-β1 genes in intestine of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

The relative expression of other genes (C3, C4, NF-κB1, Mx, Hepc and lgT) after challenge are analyzed in Figure 9. C3 and lgT genes were both found to have a higher relative expression in the 0.01% CUR group and a lower relative expression in the 0.02% CUR group. The relative expression of C4 and Hepc genes showed the same phenomenon; in both there appeared a distinct lower value in the 0.02% CUR group compared with other groups. In addition, NF-κB1 and Mx genes were on the opposite sides. Treatment groups in particular were significantly higher than the control regarding the NF-κB1gene and lower regarding the Mx gene after the ammonia nitrogen challenge compared with the control.

**Figure 9.** The relative expression of complements (**a**) (C3), (**b**) C4, (**c**) NF-κB1, (**d**) Mx, (**e**) Hepc and (**f**) lgT genes in intestine of greater amberjack fed with dietary curcumin supplementation. (Different letters above bars and asterisk sign (\*) indicate significant differences at the 0.05 level).

The results of the relative expression of some cytokine genes and other related genes after recovery are also provided in Figures 8 and 9. As for cytokine genes, the highest relative expression of IL8 and TGF-β1 genes was witnessed in the 0.02% CUR group and the highest relative expression of TNF-α and IFN-γ genes in the 0.01% CUR group instead. In addition, the IL1β gene was expressed the least in the 0.03% CUR group while the relative expression of IL1β in the 0.01% CUR group showed no significant difference to others.

The relative expression of the C3 gene in the 0.01% CUR group was remarkably lower than in other groups. On the contrary, the NF-κB1 gene's relative expression in the 0.01% CUR group was the highest among the groups. The relative expression of the C4 and Hepc genes had the same tendency: their level of expression in the 0.02% CUR group rose to a higher value compared with the control after recovery. Moreover, only the relative expression of the Mx gene in the 0.02% CUR group was significantly lower than in other groups, but both two curcumin-added groups witnessed a markedly lower value of the relative expression of the lgT gene than in the control.
