1 Introducion

Aquariology is constantly expanding, and it is estimated that, in 2020, more than 4 million tons of live ornamental fish were traded worldwide (Food and Agriculture Organization of the United Nations - FAO 2022). This sector is gaining increasing importance as restrictions related to extractivism are being implemented (Ladisa et al., 2017). In addition, fish hold the top position in the global ranking of pet groups, and in Brazil, they are the fourth group, behind only dogs, birds, and cats, in 2023 (Brazilian Association of the Pet Products Industry - ABINPET 2024).

Among ornamental species, the common carp, Koi variety (Cyprinus carpio), stands out. It is a freshwater species native to Asia, belonging to the order Cypriniformes and the family Cyprinidae (Shi et al., 2024). According to data from the FAO (2024), the production of C. carpio accounts for 4,012.6 tons of the global freshwater aquaculture production. The species has a high market value due to its vibrant colors, body shape, and its easy adaptation to different farming systems (Laksono et al., 2021; Yanuhar et al., 2021).

Although ornamental fish production has developed significantly with advances in infrastructure and technologies to meet market demand (Goswami et al., 2023), this growth has also contributed to the frequent increase in health issues in farming environments (Saengsitthisak et al., 2020). These problems are triggered by a variety of factors, such as poor water quality, inadequate management practices, and the provision of imbalanced diets, which impact the development of these animals and, consequently, make them more vulnerable to disease outbreaks (Debnath et al., 2024).

In light of this situation, some tools are commonly employed in an attempt to mitigate these effects, such as the use of chemotherapeutics in the prophylaxis and treatment of bacterial infections (Cardoso et al., 2021). However, when applied inappropriately, this practice has consequences for the environment and for animals, because, as well as leaving chemical residues in the environment, it can promote the selection of resistant bacterial strains (Hossain and Heo, 2021). Considering this scenario, other tools are being developed with the intention of reducing the use of chemotherapeutics, among which are phytochemicals. These have been investigated as nutritional strategies for use as modulators of immune, physiological, and antioxidant responses, acting as health promoters in animal production (Xu et al., 2020). An example of phytotherapeutics with great potential for use in aquaculture is açaí (Euterpe oleracea), an Amazonian fruit.

Açaí (E. oleracea) is a species from the Arecaceae family, native to several countries in the Amazon region, such as Brazil, Venezuela, and Ecuador (Oliveira et al., 2020). This fruit stands out for its antioxidant, anti-inflammatory, and immunomodulatory properties, in addition to being rich in bioactive compounds such as polyphenols, essential fatty acids, and vitamins (Moura et al., 2022). Its main chemical constituents are anthocyanins, polyphenols, and flavonoids (Inácio et al., 2013). Furthermore, studies have shown that this fruit provides a wide range of therapeutic benefits and promotes health due to its nutritional value and phytochemical composition (Pacheco-Palencia et al., 2008). In consideration of the above, this research aimed to evaluate the impact of different levels of inclusion of freeze-dried açaí in the diet of koi carp on liver health through histological methods.

2 Materials and Methods

2.1 Biological materials

The juvenile koi carp (C. carpio) were acquired from the Girassol fish farm in Joinville, Brazil, with an average weight of (6.2 ± 1.2) g and an average length of (7.8 ± 1.4) cm. The açaí (E. oleracea) used was of commercial origin. The feeding trial was conducted at the Aquatic Organism Health Laboratory (AQUOS) at the Federal University of Santa Catarina (UFSC), and all procedures were approved by the Animal Ethics Committee (CEUA) of UFSC (CEUA/UFSC 5552130324).

2.2 Dietas experimentais

The procedure for preparing the feed was adapted from Heluy et al. (2023), using a commercial freeze-dried açaí product (Liomeal®), incorporated into a commercial feed that meets the nutritional requirements of the target species (Figure 1). For the formulation of the diets, 20 kg of feed was ground in a Willey knife mill. The feed was then divided into four treatments (5, 10, 15, and 20 g/kg) and a control group without açaí addition (0.0%), resulting in a total of five experimental diets.

The inclusion of açaí was achieved by adding distilled water at 55 °C to promote the gelatinization and agglomeration of the starch. After this process, the mixture was homogenized in a horizontal mixer until a semi-solid consistency was reached. The pelleting process was then carried out using a die with a 2.5 mm opening. The feed pellets were dried in an oven at 50 °C for 6 hours, broken, and sifted to separate the granules sized between 2 and 2.5 mm and stored at ~20 °C until use.

The proximal composition of the formulated diets (Table 1) was determined following the methods established by AOAC (2016). Crude protein (N × 6.25) was determined by the Kjeldahl method. The crude lipid content was evaluated using a Soxhlet extractor with solvent extraction (petroleum ether). Crude fiber was analyzed through acid and alkaline digestion of the samples, as described by Silva and Queiroz (2002).

2.3 Experimental design

A total of 240 juvenile koi carp were distributed in 20 tanks with 80 L of water at stocking density of 12 fish per tank and 4 replicates each treatment. The animals were acclimated for 10 days and were fed with the feed recommended by the producer during this period. The tanks, containing the fish, were connected to a recirculating aquaculture system (RAS) consisting of a clarifier, mechanical and biological filters, and a UV reactor, with a 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, 2000 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.

For the analysis of lesions, corresponding values were assigned to the histological alterations: 0 (no alteration), 1 (mild alteration, corresponding to up to 25% of the analyzed tissue area), 2 (moderate alteration, 26%~50% of the tissue area), and 3 (severe alteration, >50% of the tissue area). The intensity grades were used solely to measure the severity of the lesion and were subsequently converted into percentages (%) according to Cardoso et al. (2024).

2.6 Statistical analysis

The data was subjected to the Shapiro-Wilk and Levene tests to assess normal distribution and homoscedasticity, respectively. If the data was declared non-parametric, it was transformed. Subsequently, in compliance with the assumptions, the data was analyzed using ANOVA (analysis of variance) and, when appropriate, the means were separated using Tukey's post-hoc test. All tests were carried out at a 5% significance level using Statistica 12.0 software.

3 Results

The chemical analysis of the diets (Table 2) revealed that phenolic compounds were more pronounced (p > 0.05) in the diets with higher levels of açaí inclusion, DA1.5% and DA2.0% (30.50 ± 0.35 mg/g and 33.07 ± 0.07 mg/g, respectively), compared to the diet without inclusion, DC0.0% (23.40 ± 0.10 mg/g). A similar pattern was observed for flavonoids, with DA1.5% (14.90 ± 0.10 mg/g), DA2.0% (16.60 ± 0.10 mg/g), and DC0.0% (12.00 ± 0.10 mg/g); for tannin content, DA1.5% (1.53 ± 0.06 mg/g), DA2.0% (1.73 ± 0.06 mg/g), and DC0.0% (1.07 ± 0.06 mg/g); and for antioxidant potential concentration, DA1.5% (3.33 ± 0.07%), DA2.0% (3.77 ± 0.06%), and DC0.0% (1.13 ± 0.05%).

Regarding the histological alterations in the hepatic tissue of koi carp (Table 3), a significant difference (p < 0.05) was observed in the loss of cord-like appearance, which was more pronounced in DC0.0% (62.50 ± 13.06%) (Figure 1A) compared to the other treatments: DA0.5% (50.00 ± 10.66%), DA1.0% (45.45 ± 15.08%) (Figure 1B), DA1.5% (56.82 ± 11.68%), and DA2.0% (54.55 ± 15.08%). The diet DA2.0% resulted in greater congestion in the sinusoidal cords, with 54.17 ± 20.87% (Figure 2A) compared to DC0.0% (33.33 ± 11.68%) and DA1.5% (35.42 ± 12.87%), while DA0.5% and DA1.0% did not differ (p > 0.05) from the other diets, showing 46.88 ± 8.84% and 43.75 ± 15.54%, respectively. The control group exhibited a higher concentration (p < 0.05) of mononuclear inflammatory infiltrates, at 72.92 ± 7.54% (Figure 2B), compared to DA1.0% (54.17 ± 20.87%) and DA1.5% (50.00 ± 21.32%), but no significant difference (p > 0.05) was found concerning the other inclusion levels.

Additionally, in the control diet (DC0.0%) and the diet with the highest level of açaí inclusion (DA2.0%), a greater intensity (p < 0.05) of hepatic cytoplasmic vacuolization was observed, with concentrations of 72.92 ± 7.54% (Figure 3A) and 70.83 ± 9.73%, respectively, compared to diets DA1.0% (47.92 ± 22.51%) and DA1.5% (35.42 ± 16.17%), with the latter showing the lowest intensity of this alteration. Areas of necrotic tissue were more prevalent (p < 0.05) in the control group (64.58 ± 12.87%) (Figure 3B) and in the DA0.5% diet (62.50 ± 13.06%) compared to DA1.0% and DA1.5% (45.83 ± 20.87% and 43.75 ± 24.13%, respectively). The DA2.0% group (56.82 ± 11.68%) did not differ (p > 0.05) between the treatments and the control.

4 Discussion

The highest concentrations of phenolic compounds, tannins, flavonoids, and antioxidant potential in diets with higher inclusion of açaí are understandable, considering that this Amazonian fruit, according to Laurindo et al. (2023), is rich in various bioactive compounds found not only in the pulp but also in the leaves, seeds, and skin. According to Pacheco-Palencia et al. (2008) and Laurindo et al. (2023), the bioactives found in freeze-dried pulps range from phenolic acids, such as protocatechuic, p-hydroxybenzoic, vanillic, syringic, and ferulic acids, to flavonoids, including catechin, quercetin, homoorientin, orientin, taxifolin, deoxyhexose, isovitexin, and scopoletin.

In simplified terms, diets supplemented with açaí, due to the higher concentration of phytochemicals, contribute to the high antioxidant capacity observed in this study. According to Abbate et al. (2021) and Akbari et al. (2022), phenolic compounds and polyphenols, such as flavonoids, are effective in combating oxidative stress, promoting cell health, and increasing resistance to diseases and stress. The antioxidant process possibly involved is the neutralization of reactive molecules by non-enzymatic substances. Li et al. (2022) describe that these substances play an important role in reducing the activity of free radicals, leading to a decrease in the chain reactions mediated by these radicals and, consequently, protecting cells from oxidative damage. Among the non-enzymatic molecules present in açaí, flavonoids stand out, which may be associated with this antioxidant action, as evidenced by Rudenko et al. (2023) and Hu et al. (2025). The research by Carvalho et al. (2017) on the antioxidant capacity of genotypes of Euterpe oleracea pulp supports the results of this study, as it showed that açaí pulp exhibited high values in 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests, as well as an increase in Oxygen Radical Absorbance Capacity (ORAC) and Trolox Equivalent Antioxidant Capacity (TEAC), confirming the exceptional effectiveness of açaí pulp in eliminating free radicals.

The results obtained indicated attenuated histological changes as the inclusion of açaí in the fish diet increased. The hepatic tissue plays a key role in the metabolism process, including nutrient absorption and detoxification (Popović et al., 2023). Therefore, monitoring this tissue is crucial for assessing the animals' health status. The loss of the cord-like structure was more intense in the control group, possibly related to the larger area of necrotic tissue, which was also more pronounced in this group. As a result, tissue degeneration hindered the formation and organization of the sinusoidal cords.

These findings suggest that açaí supplementation exerts a protective action against hepatic tissue degeneration, possibly due to its antioxidant and anti-inflammatory compounds. The high antioxidant capacity of açaí neutralizes free radicals and reduces oxidative stress in hepatic tissues, as observed by Laurindo et al. (2023), which likely contributed to a hepatoprotective effect in the supplemented animals. These results are significant as they support observations made by Colombo et al. (2020), who identified antioxidant and hepatoprotective effects in Litopenaeus vannamei after the inclusion of açaí (10%) in the diet, and by Moura et al. (2022), who obtained promising results regarding the inclusion of açaí in the diet on mortality rates and batch uniformity, at concentrations of 2.48% for Pterophyllum scalare and 0.88% for Heros severus.

However, in the group that received 2.0% açaí in the diet, the highest index of sinusoidal congestion was observed compared to the other treatments. This condition may be associated with an increase in blood flow to the liver or a reduction in the organ’s ability to adequately drain blood, potentially affecting liver function and leading to more severe complications (Téllez et al., 2022). According to Pereira et al. (2016), an increase in sinusoidal congestion was also observed in rats fed fat-rich diets supplemented with açaí. Furthermore, the congestion observed may indicate an adverse effect of the high concentration of açaí, highlighting the need for more detailed analyses to determine the appropriate dosage of this fruit supplementation in fish diets.

It was observed that the animals receiving diets supplemented with açaí at concentrations of 1.0% and 1.5% had lower indices of Mononuclear Inflammatory Infiltrates (MI) compared to the control group and the other inclusion levels. The high presence of MI in the control group may be associated with a cellular inflammatory immune response in the hepatic tissue. The same effect may have occurred in the fish fed the 2.0% diet, suggesting that this concentration may be harmful to the organ's integrity. Our results support the findings of Leite et al. (2021), who reported in their trials with tilapia that açaí oil (3% and 6%) stimulates antioxidant effects in the liver, increasing the activity of glutathione peroxidase (GSH-Px) and Superoxide Dismutase (SOD). Meanwhile, in the study by Silva et al. (2023), juveniles of Colossoma macropomum that received 5% açaí in their diet showed positive results related to growth, metabolic biomarkers, and antioxidant capacity.

Additionally, cytoplasmic vacuolization was significant in the control group and the group with 2.0% açaí inclusion. This histological change is characterized by the presence of small fat accumulations within the hepatocytes. In the other diets, this effect was minimized, suggesting that açaí at lower concentrations may reduce the accumulation of vacuoles in hepatocytes, while higher concentrations may enhance the presence of these vacuoles. This effect was also observed by Zheng et al. (2017) in tilapia supplemented with rutin, one of the antioxidants found in açaí, at a dosage of 0.3 g/kg in the diet.

5 Conclusion

As far as we know, this is the first report on the impact of diets containing freeze-dried açaí on the histopathology of koi carp liver tissue. The results indicate that açaí has considerable potential as a dietary supplement for koi carp, provided it is administered in adequate quantities, taking advantage of its antioxidant benefits, and improving the fish's liver health. In this study, the inclusion of 1.5% açaí showed the best results. However, we stress the need for future studies that analyze oxidative stress in the liver, with the aim of determining the ideal dose of açaí and carrying out experiments with other fish species, to gain a comprehensive understanding of the effects of açaí in the diet of ornamental fish.

Authors’ contributions

Fernandes M.C. and Costa D.S. contributed to the conceptualization, methodology, software development, validation, formal analysis, investigation, data curation, and the writing of the original manuscript, as well as visualization. Silva A.V. was responsible for designing the experimental trial structure and methodology. Lopes E.M. participated in the investigation and contributed to the original writing. Ventura A.S. and Cardoso C.A.L. were involved in methodology development, conducting chemical analysis of the feed, and reviewing and editing the manuscript. Tedesco M. played a key role in methodology development and performed the histological analysis. Mouriño J.L.P. and Martins M.L. provided support in visualization, supervision, and project administration.

Acknowledgments

The authors thank the National Council for Scientific and Technological Development (CNPq) for research grants to J.L.P. Mouriño (CNPq 301524/2017-3) and M.L. Martins (CNPq 306635/2018-6, 409821/2021-7). This study was partially funded by CAPES (AUXPE 2722/2023, code 001).

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