International Journal of Aquaculture, 2025, Vol.15, No.1, 11-20 http://www.aquapublisher.com/index.php/ija 14 photoperiod of 12 hours. The experiment was conducted in a completely randomized design (CRD), with the animals being fed for 30 days with five distinct experimental diets and four repetitions, as follows: (1) Control diet without açaí inclusion: DC0.0%; (2) Diet with 0.5% açaí inclusion: DA0.5%; (3) Diet with 1.0% açaí inclusion: DA1.0%; (4) Diet with 1.5% açaí inclusion: DA1.5%; (5) Diet with 2.0% açaí inclusion: DA2.0%. During the experimental period, water quality variables, such as pH, dissolved oxygen (DO), and temperature, were measured daily using a multiparameter probe (HI9829 Hanna). Total ammonia, toxic ammonia, and nitrite were measured using colorimetric tests (Alfakit®). These variables remained within safe standards for the species, according to Qur’ania and Verananda (2017): pH (6.2 ± 0.1), DO (6.6 ± 1.4 mg/L), temperature (25.4 ± 0.9 ℃), total ammonia (0.13 ± 0.14 mg/L), and nitrite (0.12 ± 0.14 mg/L). 2.4 Chemical analysis of the diets 2.4.1 Phenolic compounds, flavonoids, tannin content and antioxidant potential A sample of 1 g of feed was used to determine the content of phenolic compounds and flavonoids. The compounds were extracted using 10 ml of ethanol for 28 minutes in an ultrasonic bath, followed by filtration and analysis. This procedure was performed for all experimental diets and for pure freeze-dried açaí. To quantify the phenolic compounds, 0.1 mL of the extract was added to 0.5 mL of Folin-Ciocalteu reagent and 1 mL of water, allowing for 1 minute of incubation. Then, 1.5 mL of sodium carbonate (20%) was added and analyzed using a spectrophotometer (Global Trade Technology, Brazil) at 430 nm (Djeridane et al., 2006). The quantification was performed using a standard curve of gallic acid, and the results were expressed in mg of gallic acid equivalent (GAE) per g of sample. For the determination of flavonoids, 1 mL of 2% aluminum chloride in methanol and 1 mL of the sample were added. After a 15 minute reaction, the reading was performed using a spectrophotometer at 430 nm (Djeridane et al., 2006). The quantification was carried out using a standard curve, expressing the results in mg of rutin equivalent (RE) per g of sample. The tannin content was determined in all experimental diets using the Folin-Denis spectrophotometric method (Pansera et al., 2003) with adaptations to the reagent volumes while maintaining the proportions. To this end, 0.5 mL of Folin-Denis reagent was added to 0.5 mL of the sample, shaken, and allowed to stand for 3 minutes. Then, 0.5 mL of sodium carbonate (0.75 M) was added, homogenized, and kept in the dark for 2 hours. Absorbance was measured at 725 nm. The concentration of tannins was calculated using a standard curve of tannic acid, with results expressed in mg of tannic acid equivalent (TAE) per g of sample. The antioxidant activity of the samples was evaluated using the DPPH free radical method (1.1-diphenyl-2-picrylhydrazyl), as described by Capanoglu et al. (2008). For each 100 µL of sample, 2 000 µL of 0.004% DPPH was added, and the reaction was allowed to proceed for 30 minutes in the dark before measuring the absorbance at 517 nm using a spectrophotometer. The percentage of inhibition was calculated using the equation: Q=(A0-Ac)/A0×100, where Q is the percentage of inhibition, A0 is the absorbance of the control, and Ac is the absorbance of the sample after the reaction. 2.5 Histological analysis For the histological analysis of the liver, 4 fish from each tank were anesthetized with eugenol (75 mg/L) and then euthanized by brain sectioning. Liver fragments were fixed for 24 hours in 10% buffered formalin and subsequently dehydrated through a graded series of ethanol, followed by embedding in paraffin. These tissues were then sectioned into 5 μm thick slices and stained with hematoxylin and eosin (HE), as described by Martins et al. (2018). The slides were analyzed using a light microscope and the Zeiss 12pro software to obtain microphotographs and identify any histological alterations.
RkJQdWJsaXNoZXIy MjQ4ODYzNA==