*2.2. Experimental Diet*

The addition amounts of curcumin in the three experimental diets were 0, 100 and 200 mg/kg, respectively. All diets were designed and produced by the Tropical Fisheries Research and Development Center, South China Sea Fisheries Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Science. The added curcumin (purity > 95%) was provided by Xi'an Feida biotechnology Co., Ltd. (Xi'an, China). The formulation of experimental diet is shown in Table 1.


**Table 1.** Formulation and proximate composition of the experimental diet.

<sup>1</sup> 0.01% CUR and 0.02% CUR indicate the treatment groups in which the proportion of curcumin supplementation in the diet are 0.01% and 0.02%. <sup>2</sup> Vitamin premix (mg/kg diet): vitamin A—9,000,000 (IU/kg diet), vitamin K3—600 (IU/kg diet), vitamin D—2,500,000 (IU/kg diet), vitamin E—500 (IU/kg diet), vitamin B—(B1 3200 (IU/kg diet), B2—10,900 (IU/kg diet), B5—20,000 (IU/kg diet), B6—5000 (IU/kg diet), B12— 1160 (IU/kg diet), vitamin C—50,000 (IU/kg diet), phaseomannite—1500 mg/kg diet, calcium pantothenate— 200 mg/kg diet, niacin—400 mg/kg diet, folic acid—50 mg/kg diet, biotin—2 mg/kg diet. <sup>3</sup> Mineral premix (mg/kg diet): KCl—70; KI—1.5; MgSO·7H2O—300; MnSO4·4H2O—3; CuCl2—5; ZnSO4·7H2O—14; CoCl2·6H2O— 0.5; FeSO4·7H2O—15; CaCl2—2.8 (g/kg diet); KH2 PO4·H2O—4.5 (g/kg diet). The dietary energy was calculated as carbohydrate: 17.15 MJ/kg, protein: 23.64 MJ/kg, lipid: 39.54 MJ/kg.

#### *2.3. Ammonia Nitrogen Challenge Assay*

After being fed with experimental diets for eight weeks, a challenge assay was conducted on the fish. Ammonium chloride (NH4Cl) was added into the aquaculture water at the concentration of 1 g/L. At six minutes, all the fish showed either manic agitation or lack of apparent consciousness; based on our pre-experiment, two fish from each tank were selected. One was sampled as the challenge assay sample, and the other was placed in water with no ammonia poison to recover. It was sampled after recovery from lack of apparent consciousness. Recovery time was about 5 min.
