## 2 Material and Methods
### Antioxidant Enzymes and Detoxification Activities in the Hepatopancreas  
At the end of the experiment, the hepatopancreases of 60 prawns (15 per experimental group; \(n\) = 6 replicates) were randomly collected to analyze the activity of enzymes related to the antioxidant defense system and lipid peroxidation. Prawns were euthanized by thermal shock (5 min in iced water) and the body surface was cleaned with ethanol 70% (\(v/v\)). The hepatopancreases were collected and immediately stored at \(-\)20 \({}^{\circ}\)C until further analysis. Samples were homogenized in 50 mM Tris/HCl buffer pH 7.4 using an electric homogenizer (IKA(r) T10 basic; IKA(r), Staufen, Germany). The entire procedure was carried out on ice to preserve the enzymatic activities. The homogenates were then centrifuged  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Ingredients (g kg\({}^{-1}\))** & \multicolumn{4}{c}{**Experimental Groups (g kg Diet\({}^{-1}\))**} \\
**Control** & **1.0** & **2.0** & **3.0** \\ \hline
**Proximate composition** & & & & \\ \hline Moisture (\%) & 8.57 & 8.92 & 8.69 & 9.04 \\ Dry matter (\%) & 91.43 & 91.08 & 91.31 & 90.96 \\ Crude protein (\%) & 39.5 & 39.12 & 38.22 & 38.54 \\ Ash (\%) & 9.17 & 8.99 & 9.46 & 8.65 \\ Ether extract (\%) & 7.07 & 7.15 & 6.9 & 7.01 \\ NDF (\%) & 29.42 & 29.45 & 28.27 & 28.6 \\ \hline \end{tabular} \({}^{\dagger}\) Vitamin guarantee levels kg\({}^{-1}\): 36 mg vitamin B2, 25.5 mg vitamin B6, 45 mg vitamin B12, 1200 mg vitamin C, 1.68 mg biotin, 11.2 mg folic acid, 265 mg inositol, 170 mg nicotinic acid, 67.5 mg pantothelic acid. Mineral guarantee levels kg\({}^{-1}\): 0.35 mg cobalt, 13.8 mg copper, 1.3 mg iodine, 65 mg iron, 85 mg manganese, 0.4 mg selenium, 150 mg zinc. EO-OC: essential oil of _Oximum gratisimum_. NDF: neutral detergent fiber.  
\end{table}
Table 1: _Cont._in a refriget centrifuge (Sigma, St. Louis, MO, USA, 3-16 KL) at 4 \({}^{\circ}\)C for 10 min at 12,000\(\times\)\(g\) and the resulting supernatant was collected for further analysis.  
The protein content was determined using the Bradford method [44], using bovine albumin as a standard and absorbance ratio at 595 nm. The protein content was used for subsequent calculations of enzymatic activities. The catalase (CAT) activity was determined based on the decomposition of H\({}_{2}\)O\({}_{2}\) into O\({}_{2}\) and H\({}_{2}\)O, with the consumption of 1 \(\mu\)mol H\({}_{2}\)O\({}_{2}\) min\({}^{-1}\), at an absorbance of 240 nm [45; 46]. The glutathione peroxidase (GPx) activity was determined by measuring NADPH oxidation at an absorbance of 340 nm, on the reduction of cumene hydroperoxide [47; 48]. The glutathione reductase (GR) activity was quantified by measuring the oxidation of NADPH at an absorbance of 340 nm, on the reduction of glutathione disulfide to reduced glutathione [49; 50]. Reduced glutathione (GSH) levels were determined by the non-protein thiol method using Ellman's reagent [50; 51]. Glutathione S-transferase (GST) activity was quantified by measuring the conjugation of GSH with 1-chloro-2,4-dinitrobenzene [52; 53]. Lipid peroxidation was measured based on the reaction of MDA and thiobarbituric acid reactive species (TBARS) [54; 55].