Impact of flavonoids as functional food additives on the health of Nile tilapia, Oreochromis niloticus

1 Introduction

Phytochemicals are gaining significant attention as functional feed additives due to their diverse bioactive properties, including antioxidant and antibacterial effects. Flavonoids are a group of natural substances that possess varying phenolic structures and exhibit strong antioxidant properties, along with a range of other biological activities (Dias et al., 2021; Zahra et al., 2024). These activities include the ability to interact with protein phosphorylation and chelate metal ions (Boyle et al., 2000; Panche et al., 2016). The antioxidant capacity of flavonoids is mainly attributed to the numerous hydroxyl groups present in their molecules, which enhance their ability to donate hydrogen atoms and scavenge free radicals (Pietta, 2000; Burda and Oleszek, 2001). Additionally, flavonoids can regenerate other antioxidants. For example, they can regenerate tocopherol by donating a hydrogen atom to the tocopheroxyl radical (Boyle et al., 2000). Flavonoids are a diverse group of more than 4,000 natural phenolic compounds found in plants (Ren et al., 2003). They are primarily classified into six categories based on their saturation level and the status of the central pyran ring. These categories are flavanones, flavones, flavonols, isoflavonoids, anthocyanins, and flavans. Flavanones are most commonly found in citrus fruits, flavones are present in various herbs, isoflavonoids are predominantly in legumes, while anthocyanins and catechins can be found in fruits. Flavonols are widely distributed across all fruits and vegetables (Peterson and Dwyer, 1998).

Quercetin is a type of flavonoid that falls under the flavonol subclass. Quercetinsupplementation has been shown to improve feed utilization, increase laying rates, and enhance egg quality in hens during late laying periods (Liu et al., 2014). In tilapia, quercetin supplementation has been found to improve antioxidant parameters (Liu and Zhai, 2014) and to reduce the pro-oxidant effects induced by antibiotics in silver catfish (Pês et al., 2018). In olive flounder, a quercetin-supplemented diet boosted immune function and helped maintain physiological homeostasis (Shin et al., 2010). Quercetin has been proposed as an antioxidant agent in the storage of fish oil (Nieto et al., 1993).

Hesperidin, one of the most abundant flavonoids found in citrus fruits, acts as an antioxidant by inhibiting lipid peroxidation and reducing oxidative stress. It belongs to flavanone subclass of flavonoids (Jain and Somani, 2015). Additionally, hesperidin enhances both mucosal and humoral immunity by increasing the number of intestinal intraepithelial lymphocytes, improving the indices of lymphoid organs (thymus, spleen, and bursa), and boosting antibody titers against avian influenza and Newcastle disease in poultry (Yatao et al., 2018). Furthermore, Abuelsaad et al. (2014) demonstrated that hesperidin may help suppress the inflammatory response induced by Aeromonas hydrophila toxins. It does this by downregulating reactive oxygen species (ROS) production and the expression of CD14 and adhesion molecules, while also increasing the ratio of CD4+ to CD8+ cells (Abuelsaad et al., 2014).

Catechin belongs to the flavanols subcategory of flavonoids and is primarily found in tea, cacao, and pome fruits (Huang et al., 2010). It is well-known for its strong antioxidant properties that protect against metal chelation and lipid peroxidation by functioning as a free radical scavenger. Research has demonstrated that plants or extracts rich in catechins offer a range of beneficial effects, including antioxidant, antimicrobial, antifungal, anticarcinogenic, hypoallergenic, and hypoglycemic properties (Yang et al., 2003; Koo and Cho, 2004). Supplementing quail diets with catechin has been shown to lower malondialdehyde (MDA) concentrations in the liver and blood (Tuzcu et al., 2008; Sahin et al., 2010) and in eggs (Kara et al., 2016). This supplementation also enhances reproductive performance, leading to improved fertility, and the hatchability of fertile eggs, while extending the shelf life of animal products (Kara et al., 2016).

Extensive research has demonstrated that crowding stress resulting from aquaculture intensification increases the risk of pathogen transmission and impairs fish immune systems, thereby enhancing their vulnerability to various diseases (Lin et al., 2018). Several phytogenic bioactive compounds have been proved remarkably efficient in promoting mucosal fitness in fish (Firmino et al., 2021). Flavonoids are now recognized as important components in a variety of nutraceutical, pharmaceutical, and cosmetic applications due to their antioxidant, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties (Panche et al., 2016). Research has shown that dietary supplementation with flavonoids, such as quercetin and tea polyphenols, at concentrations below 1% can enhance fish performance (Zhai and Liu, 2013; Shin et al., 2010; Yang et al., 2025). Although a few studies have explored the effects of plant extracts that are rich in these flavonoids, such as green tea and citrus peel on fish, since pure flavonoids offer precise, dose-dependent, targeted, and generally higher bioactivity than plant extracts that contain multiple bioactive compounds (Vaou et al., 2022), supplementation of a single flavonoid in the fish diet might affect fish immunity and disease resistance, as well as fish growth performance differently. Additionally, further investigation is needed to clarify the safety of using these substances as high-level feed supplements in the fish feed industry. Thus, this study aims to evaluate and compare the effects of three specific flavonoids—catechin, quercetin, and hesperidin—at two different concentrations—0.1% and 1%—on the growth, feed utilization, hematological parameters, immune responses, and resistance of Nile tilapia, Oreochromis niloticus, against Streptococcus iniae challenges.

2 Materials and methods

2.1 Flavonoid antioxidant and antimicrobial properties

2.1.1 2,2’-azino-bis 3-ethylbenzthiazoline-6- sulphonic acid assay

The flavonoids hesperidin (from Citrus aurantium citrus peel), green tea leaf extract (from Camellia sinensis) containing 80% catechins, and quercetin dihydrate were purchased from Bulk Supplements in Henderson, Nevada, USA. The radical scavenging activity was assessed using the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation discoloration assay, following a slightly modified procedure outlined by Re et al. (1999). A 7 mM ABTS stock solution was prepared by mixing it with potassium persulfate to a final concentration of 2.45 mM, and the mixture was allowed to react in the dark for 16 hours before use, then diluted with distilled water to create a working solution with an absorbance of 0.700 ± 0.02 at 734 nm. Flavonoids were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 µg/ml. For the assay, triplicate 10 µl of flavonoid samples at concentrations ranging from 1.95 to 62.5 µg/ml for quercetin and catechin, and from 0.25 to 8 mg/ml for hesperidin, were combined with 200 µl of the ABTS working solution. For the assay, various concentrations of flavonoids dissolved in dimethyl sulfoxide (DMSO) were combined with the ABTS working solution. After the reaction, the absorbance was measured at 734 nm using a spectrophotometer (Epoch/2, BioTec Instrument Inc.). The results were expressed as Trolox equivalent % inhibition.

2.1.2 2,2-Diphenyl-1-picrylhydrazyl assay

The DPPH assay is based on the reduction of DPPH by antioxidants, as described by Rahman et al. (2015) and Rubab et al. (2022). 1M DPPH (α, α-diphenyl-β-picrylhydrazyl) was prepared in methanol. A 1M DPPH (α, α-diphenyl-β-picrylhydrazyl) was prepared in methanol. For the assay, 30 µl of flavonoid samples at concentrations ranging from 1.95 to 62.5 µg/ml for quercetin and catechin, and from 0.25 to 8 mg/ml for hesperidin, were placed in a set of triplicate tubes. Then the DPPH solution (90 ml) was added to each sample. The mixtures were allowed to react for 30 min in the dark, and the absorbance was measured spectrophotometrically at 517 nm using an Epoch/2 (BioTek Instrument Inc.). The median effective concentration (EC₅₀) for each sample was determined by the following formula:

[Abs(control) – (Abs(sample)-Abs (blank))]/Abs(control) x 100.

2.1.3 Antibacterial activities of flavonoids

Paper disks impregnated with flavonoid solutions were placed on agar plates inoculated to create a lawn of S. iniae. The plates were then incubated to allow bacterial growth and the flavonoids to diffuse into the agar. After incubation, clear zones of inhibition were photographed to demonstrate the effectiveness of each flavonoid.

To evaluate the bactericidal activity of flavonoids, S. iniae was inoculated into TSB containing 0, 0.05, 0.1, or 0.2 mg/ml of flavonoids. After 24 hours of incubation at 28 °C, the S. iniae colonies were streaked onto plain TSA plates to observe bacterial growth. Each concentration was tested in duplicate.

2.2 Experimental diets, feeding, and sampling

The commercial diet used in this study contained 36.7% protein, 3.25% lipids, and 7.5% fiber. It was obtained from SouthFresh Feeds in Demopolis, AL. The diet ingredients consisted of plant protein products, processed grain byproducts, grain products, animal protein products, and fish oil. The diet was supplemented with various minerals, including dicalcium phosphate, calcium carbonate, zinc sulfate, ferrous sulfate, sodium selenite, copper sulfate, manganese sulfate, and ethylenediamine dihydriodide. Additionally, vitamins (vitamin A, vitamin D3, vitamin E, calcium pantothenate, niacin, ascorbic acid (vitamin C), menadione dimethylpyrimidinol disulfite, pyridoxine hydrochloride, riboflavin, thiamine mononitrate, vitamin B12, and folic acid) were included. No other additives aimed at promoting fish health and immune response were included in the diet. Diet was finely ground using a hammer mill and supplemented with three types of flavonoids (quercetin, hesperidin, and catechin) at two levels, 0.1% and 1%, and a control diet containing no additives. The moist mixture was extruded with a Hobart meat grinder. The pellets were air-dried at room temperature, then ground to the desired size and stored at -20 °C until used.

2.3 Experimental fish

Nile tilapia, Oreochromis niloticus, fingerlings were obtained from Aquasafra Inc. (Bradenton, FL) and maintained at the USDA-ARS, Aquatic Animal Health Research Laboratory (AAHRU; Auburn, AL, USA) on commercial fry and fingerling diets. Fish were acclimated to the experimental basal diet for 1 week before stocking. At the end of the acclimation period, fish (average weight of 11.5 ± 0.077 g) were randomly stocked into 21, 110 L aquaria at a density of 50 fish per aquarium. Aquaria were supplied with flow-through, dechlorinated, heated (28 °C) city water at a flow rate of about 0.7 L/min. During the trial, water temperature averaged 27.3 ± 0.40 °C, and dissolved oxygen averaged 6.77 ± 0.48 mg/L. Fish were hand-fed multiple times during each feeding session until they reached satiation for 8 weeks. Daily feed consumption was recorded by calculating the difference in diet weight between the first and last feedings each day. Once a week, the aquaria were cleaned, and accumulated waste was siphoned. Every two weeks, fish in each aquarium were group-weighed and counted to determine the average weight gain and survival. The feed consumption and feed efficiency ratio (FER) were calculated at the end of the feeding trial.

2.4 Hematological assays

At the end of the experiment, blood samples (3 fish/tank) were collected using dried heparinized (100 IU) tuberculin syringes after the fish were anesthetized with tricaine methanesulfonate (MS-222) at a concentration of 150 mg/L. Hematological assays were performed according to the method described by Yildirim-Aksoy et al. (2020). Blood cell counts were performed by diluting whole blood (1:10,000) and enumerating in a Spencer Bright Line hemacytometer. The hematocrit of each fish was determined using microhematocrit capillaries. Hemoglobin was determined using a cyanomethemoglobin method (Point Scientific, St. Louis, MO, USA).

2.5 Catechin effect on erythrocytes

Erythrocyte osmotic fragility was assessed using gradient saline solutions as described by Yildirim-Aksoy et al. (2009). Serial dilutions (1:10) of catechin (20 mg/ml) in phosphate-buffered saline (PBS) were prepared to yield concentrations of 200 µg/ml, 20 µg/ml, and 2 µg/ml. Each of these concentrations was mixed with blood in a 1:10 ratio and incubated overnight at 4 °C. Then, 50 µl of the catechin/blood mixture was added to 1 ml of various NaCl solutions (0%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, and 0.85%) using 24-well plates. After incubating at room temperature for 30 minutes, the plates were centrifuged, and the absorbance of the supernatant was measured at 415 nm using an Epoch 2 microplate reader (BioTek Instruments, Inc.). The percentage of hemolysis was calculated.

To observe the effect of catechin on erythrocytes, a suspension of blood and catechin was mixed with Yokoyama solution. This mixture was then placed on clean microscope slides and swiftly spread evenly using a second slide. After allowing the slides to air dry, they were examined under a BX41 microscope (Olympus, Tokyo, Japan) using a 100× oil-immersion lens.

2.6 Liver indexes

2.6.1 Hepatosomatic index and liver lipid content

At the end of the growth trial, livers from three fish per tank that had been bled for serological assays were removed and individually weighed for determination of hepatosomatic index (HSI).

Livers from each tank were pooled and lyophilized using Lyovapor L-200 (Buchi Crop., New Castle, DE). Lyophilized liver samples were ground into fine powder with a mortar and pestle set. Lipid content of liver samples was determined by petroleum ether extraction using a Soxtec System (2055 Soxtec Avanti; Foss Tecator, Höganäs, Sweden).

2.6.2 Total polyphenolic content of liver

The lyophilized liver samples in duplicate were extracted using 80% methanol. The total phenolic content (TPC) of the liver extracts was estimated using the Folin-Ciocalteu reagent, following the method of Soleas et al. (2001). In a glass test tube, 100 μl of the liver extract (diluted 20 times) was mixed with 500 μl of the 10-fold diluted Folin-Ciocalteu reagent. The mixture was vortexed and left at room temperature for three minutes. Afterward, 500 μl of a 7.5% sodium carbonate (Na₂CO₃) solution was added, and the sample was vortexed again, covered, and incubated at 30 °C for 1 hour. The resulting solution was measured at 760 nm using an Epoch/2 spectrophotometer (BioTek Instrument Inc.). The total phenolic content in liver samples was measured using gallic acid as the standard reference.

2.7 Serum biochemical and nonspecific immune responses

Serum biochemistry indexes, including serum total protein, immunoglobulins, albumin, alkaline phosphatase, alanine aminotransferase, amylase, glucose, calcium, phosphorus, and potassium, were measured using Abaxis, VetScan VS2 analyzer (Union City, CA). The serum chemistry profiles were analyzed by using comprehensive diagnostic reagent rotors (Abaxis Inc., Union City, CA).

Serum natural hemolytic complement activity, which is based on hemolysis of red blood cells (Remel Inc., Lenexa, KS, USA) by complements present in fish serum, was performed as described in Yildirim-Aksoy et al. (2008). Briefly, sheep erythrocytes were washed and standardized to 5×10⁷ cells/mL in PBS3⁺ before use. Sheep erythrocyte cell suspension was added to serially diluted (1:2) serum in PBS3⁺ in a round-bottom 96-well microtiter plate. Positive (100% lysis) and negative (spontaneous lysis) controls were also processed on each plate by replacing serum with distilled water and buffers, respectively. Complement hemolytic activity is quantified as the ACH₅₀ value, which indicates the volume of serum required to lyse 50% of target cells under standardized conditions. The results are presented in units/ml.

2.8 Bacterial challenge

One week before the bacterial challenge, 35 of the remaining fish in each aquarium were tagged with Visible Implant Elastomer (VIE) tags purchased from Northwest Marine Technology, Inc. (Shaw Island, WA, USA), to identify their dietary treatment group. Fish were anaesthetized with tricaine methanesulfonate (MS-222) at 100–200 mg/L for 2–5 minutes before tagging. Tagged fish were distributed to give 5 fish from each treatment per aquarium (35 fish per aquarium) across 9 aquaria (5 fish x 9 aquaria = 45 fish/treatment). A frozen glycerol stock of a S. iniae (ARS 98-60) was grown in tryptic soy broth (TSB) at 28 °C until the optical density reached 1.0, measured spectrophotometrically at 540 nm to give a S. iniae concentration of 1.3×10⁹ colony-forming units (cfu)/ml. To determine the LC₅₀ (concentration lethal to 50% of exposed fish), groups of 10 fish were intraperitoneally (IP) injected with 0.1 mL of 0, 1.3x10³ 1.3x10⁴ 1.3x10⁵ 1.3x10⁶ S. iniae cfu/ml using a tuberculin syringe. Fish were monitored twice daily, and moribund and dead fish were recorded and removed for 7 days. The calculated LC₅₀ (concentration lethal to 50% of exposed fish) was 1x10⁴ cells/ml, and the concentration used to challenge tagged fish (5 fish/trt/tank, 35 fish/treatment) as described above.

2.9 Statistical analysis

Statistical analysis was performed with analysis of variance (ANOVA) using GraphPad Prism 10 (San Jose, CA, USA). The Data from each group were compared to control using Dunnett’s multiple comparisons test (one-way ANOVA). Two-way ANOVA was also used to test the effects of three dietary flavonoids (quercetin, hesperidin, and catechin) at two inclusion levels (0.1% and 1%), and their interactions excluding the control diet. Survival data from the bacterial challenge test were analyzed using Graphpad Prism 10 by Kaplan-Meier Log Rank Survival Analysis using the Mantel-Cox test (used for pairwise comparisons) and/or LogRank test for trend (for the comparison of all survival curves simultaneously). Differences were considered significant at or below the 0.05 probability level.

3 Results

Figure 1 and 2 show the antioxidant and antibacterial activities, respectively, of quercetin, hesperidin, and catechin. Quercetin and catechin exhibited considerably greater antioxidant activity, as measured by the ABTS and DPPH assays, compared to hesperidin, with catechin demonstrating the highest activity. Similarly, quercetin and catechin exhibited greater antibacterial activity against S. iniae compared to hesperidin (Figure 2). Both compounds (quercetin and catechin) at a concentration of 0.2 mg/ml demonstrated bactericidal effects against S. iniae.

Dietary supplementation with quercetin, hesperidin, or catechin at 0.1% did not significantly affect weight gain (Table 1). However, at a 1% level, both quercetin and catechin supplementation led to a significant reduction in weight gain in fish compared to the control group. In contrast, hesperidin supplementation at 1% resulted in the highest weight gain, though this difference was not significant. Feed intake mirrored weight gain trends (Table 1). Fish fed diets supplemented with high (1%) levels of quercetin or catechin showed significantly lower feed consumption (P<0.0001). However, feed efficiency was significantly decreased (P = 0.0006). only in fish fed a diet supplemented with 1% catechin (Table 1).

Table 1. Mean final weight gain, feed intake, FER, and survival of Nile tilapia, Oreochromis niloticus, fed diets supplemented with three different types of flavonoids (quercetin, hesperidin, and catechin) at two levels (0.1% and 1%) of each flavonoid and plus a control group (fed basal diet, without flavonoid supplementation) for 8 weeks¹.

Values are presented as mean from three replicate tanks. Asterisks indicate a significant difference between the control and flavonoid supplement-fed groups. The number of asterisks represents the degree of statistically significant difference from control; *p ≤.001; **p ≤.0001. Control is not included in two-way ANOVA.

²FER (feed efficiency ratio) = weight gain (g)/dry feed fed (g).

Hepatosomatic index, liver lipid content, and liver total phenolic content are presented in Figure 3. The hepatosomatic index (Figure 3a) (P = 0.0471) and lipid content (Figure 3b) (P = 0.0021) of fish that were fed a diet supplemented with 1% quercetin were significantly lower (P = 0.0003) than those of the control group. No significant differences were observed among the other treatment groups. The livers of all fish that received a 1% flavonoid supplementation, regardless of the type used, exhibited a significantly (P<0.0001) higher accumulation of phenolic compounds (Figure 3c). Interestingly, the liver of fish fed 0.1% quercetin showed significantly (P = 0.0003) lower phenolic compound levels than the control group.

Hematological values, including red blood cell (RBC) count (P = 0.0003), hemoglobin (P<0.0001), and hematocrit (P<0.0001), were significantly reduced in fish that were fed a high (1%) catechin diet (Table 2). At this level, both mean corpuscular volume (MCV) (P = 0.0058) and mean corpuscular hemoglobin (MCH) (P = 0.0134) were significantly increased, while mean corpuscular hemoglobin concentration (MCHC) (P = 0.0002) was decreased. Additionally, catechin supplementation at 0.1% also led to reduced hemoglobin and MCHC values. However, the RBC count and hematocrit were not significantly affected in fish fed the 0.1% catechin diet. The RBC count was also lower in fish fed a high (1%) quercetin diet, but this difference was not significant compared to the control group. Supplementary Figure 1 illustrates the impact of catechin on fish blood cells in vitro. Higher concentrations of catechin lead to increased damage to the blood cells (Supplementary Figure 1c). Fish erythrocytes treated in vitro with 2 µg/ml catechin showedincreased fragility in hypotonic saline (Supplementary Figure 2). Salt concentrations in saline solutions that caused 50% erythrocyte hemolysis were 47.7% and 24.4% for cells treated with 2 µg/ml catechin and untreated, respectively.

Table 2. Hematological values of Nile tilapia, Oreochromis niloticus, fed diets supplemented with three different types of flavonoids (quercetin, hesperidin, and catechin) at two levels (0.1% and 1%) of each flavonoid and plus a control group (fed basal diet, without flavonoid supplementation) for 8 weeks¹.

RBC , red blood cell count, WBC , white blood cell count, Hb , hemoglobin, Ht , hematocrit, MCV , mean corpuscular volume, MCH , mean corpuscular hemoglobin, and MCHC , mean corpuscular hemoglobin concentration.

Values are means of one determination per fish, three fish per tank and three tanks per treatment. The number of asterisks represents the degree of statistically significant difference from control; *p <.05; p <.01; *p <.001; ****p <.0001. Control is not included in two-way ANOVA.

²MCV = Ht (%)/RBC (x10⁶) x10; MCH = Hb (g)/RBC (x10⁶) x10; MCHC = Hb (g)/Ht (%) x100.

The innate immune values, including natural complement activity and immunoglobulins, along with serum biochemical values, are presented in Table 3. Natural complement activity in fish fed a diet supplemented with 1% hesperidin, as well as immunoglobulin levels in fish fed a diet with 1% catechin, significantly (P = 0.001) increased compared to the control group. Catechin at the same concentration significantly decreased serum phosphorus levels (P = 0.006) while increasing serum potassium levels (P = 0.001). Hesperidin, at both concentrations, did not have a significant effect on serum biochemical values. Additionally, lower levels of these flavonoids did not significantly influence serum biochemical values.

Table 3. Innate immune responses and serum biochemistry of Nile tilapia, Oreochromis niloticus, fed diets supplemented with three different types of flavonoids (quercetin, hesperidin, and catechin) at two levels (0.1% and 1%) of each flavonoid and plus a control group (fed basal diet, without flavonoid supplementation) for 8 weeks¹.

NCA , Natural complement activity, TP , total protein, GLOB , globulin, ALB , albumin, ALP , alkaline phosphatase, ALT , alanine aminotransferase, AMY , amylase, CA , calcium, PHOS , phosphorus, GL , glucose, and K , potassium. Data were presented as mean of three fish per tank and three tanks per treatment. Asterisks indicate a significant difference between the control and flavonoid-supplemented groups. The number of asterisks represents the degree of statistically significant difference from control; *p <.05; p <.01; *p <.001; ****p <.0001.

Hesperidin and catechin, at both concentrations (0.1% and 1%), significantly increased tilapia survival when challenged with S. iniae (Figure 4). In contrast, although higher than the control, quercetin did not significantly improve tilapia survival against the S. iniae challenge at either concentration compared to the control group. There was no difference in survival rate among fish receiving a flavonoid-supplemented diet.

4 Discussion

Flavonoids are associated with many health benefits. Almost all flavonoid groups exhibit antioxidant activity (Panche et al., 2016). In the present study, catechin and quercetin exhibited significantly greater antioxidant activity than hesperidin, with catechin being the most potent. Using the DPPH assay, the concentration of antioxidants needed to scavenge 50% of free radicals is approximately 150 times higher for hesperidin compared to quercetin, and about 250 times higher when compared to catechin. Research indicates that flavones and catechins are among the most potent flavonoids (Panche et al., 2016).

Similarly, quercetin and catechin exhibited greater antibacterial activity against S. iniae compared to hesperidin. Both compounds at 0.2 mg/ml demonstrated bactericidal activity in vitro against S. iniae. Many isolated flavonoids have shown antifungal, antiviral, and antibacterial properties. Their mode of action involves disrupting bacterial cell membranes and wall integrity and synthesis (Liu et al., 2025; Zhang et al., 2025), inhibiting nucleic acid synthesis and energy metabolism (Tan et al., 2022), reducing adhesion and biofilm formation (Shamsudin et al., 2022), and diminishing virulence factors.

Supplementing the diet with quercetin has been shown to enhance tilapia growth at levels ranging from 200 to 1600 mg/kg (Zhai and Liu, 2013) and flounder growth at 0.25% or 0.50% (Shin et al., 2010). Similarly, quercetin supplementation significantly improved the growth performance of Labeo rohita when fed a diet containing up to 8 mg/kg, with 4 mg/kg being the optimal concentration. Another study found that quercetin is non-toxic to rainbow trout, Salmo gairdneri, when fed at high levels (1% to 8%) over 8 months (Plakas et al., 1985). However, in the present study, supplementation with quercetin at 0.1% did not significantly affect tilapia growth, but at higher concentrations (1%), it significantly reduced growth of tilapia. Fish that received elevated quercetin doses showed reduced feed intake and even refused to eat a quercetin-rich diet. At either concentration, quercetin supplementation did not significantly enhance tilapia survival against the S. iniae challenge compared to the control.

The hepatosomatic index and liver lipid content of fish that were fed 1% quercetin diet were significantly lowered. Reduced lipid levels have also been reported in both serum and whole body of tilapia fed a quercetin-supplemented diet (Zhai and Liu, 2013). Although the mechanisms behind these effects are still unclear, quercetin appears to influence lipid metabolism by promoting the breakdown of fats (specifically fatty acid beta-oxidation), decreasing fat accumulation, and improving blood lipid profiles in animal studies.

Flavonoids are primarily metabolized in the liver by hepatic enzymes after being absorbed by enterocytes and transported to the liver (Hu et al., 2025). The livers of all fish that received a 1% flavonoid supplementation, regardless of the type used, exhibited a significantly higher accumulation of phenolic compounds. Total phenolic compound (TPC) depositions were highest in fish fed a high quercetin diet. Even at high concentrations, quercetin is generally regarded as safe when consumed through food, and it may help protect against liver injury, according to the National Institutes of Health (NIH). Dietary flavonoid antioxidants, including quercetin and tea polyphenols (catechin), protected tilapia from mycotoxin induced adverse effects, and improved toxin-induced liver injury levels by enhancing liver GST activity (Deng et al., 2019). However, taking high doses of pure quercetin supplements may lead to accumulation in the liver and other organs (Skibola and Smith, 2000; Mennen et al., 2005).

The differing effects of flavonoids on liver phenolic deposition may be attributed to several factors, including their solubility, bioavailability, metabolism, and elimination rates (Manach et al., 1999; Scalbert and Williamson, 2000). Manach et al. (1999) compared the bioavailability of quercetin and catechin in rats and observed significant differences in their metabolism during the post-absorptive period. They found that catechin has a higher elimination rate compared to quercetin. Plasma concentrations of quercetin metabolites either increased or remained stable over the entire 24-hour period, while those of catechin metabolites dropped dramatically between 12 and 24 hours after the experimental meal. The researchers suggested that the delayed elimination of quercetin is due to its metabolites being more strongly bound to albumin than those of catechin. Another notable difference is that, unlike quercetin and hesperidin, catechin is primarily water-soluble and is mainly eliminated through urine, whereas quercetin is excreted in bile (Manach et al., 1996, Manach et al., 1999).

Studies regarding dietary hesperidin in aquatic animals are quite limited. Supplementation with hesperidin at doses of 100–300 mg/kg has been shown to enhance nonspecific immunity, antioxidant capacity, immune-related gene expression, and growth performance in Pacific shrimp, Litopenaeus vannamei (Arshadi et al., 2025). Similarly, an optimal dosage of 50–150 mg/kg of hesperidin improved nonspecific immunity, antioxidant capacity, and growth performance in the red swamp crayfish, Procambarus clarkii (Liu et al., 2020). In the present study, hesperidin-supplemented diets serve as effective immunostimulants, enhancing natural complement activity and providing improved protection against S. iniae in tilapia. Hesperidin showed no adverse effects, even at high concentrations. At both dietary hesperidin levels (0.1% and 1%), the survival rate of tilapia challenged with S. iniae significantly increased. This increase in survival may be due to hesperidin’s ability to interfere with bacterial pathogenesis, rather than having a direct bactericidal effect, as only slight or no antibacterial activity was observed in vitro tests against S. iniae.

Similar to quercetin diets, supplementation at 0.1%, catechin did not significantly affect tilapia growth, but at higher concentrations (1%), it significantly reduced weight gain and feed efficiency. A high intake of dietary catechin also significantly reduced hematological values, including RBC count, hemoglobin levels, and hematocrit. At this intake level, elevated MCV and MCH suggest macrocytic anemia. This condition arises when red blood cells develop improperly, leading to an increased cell size and higher hemoglobin content. Although not statistically different, the RBC count was also lower in fish fed a high (1%) quercetin diet. While antioxidants are beneficial for health, prolonged intake or high concentration can disrupt the normal balance of redox reactions in the body. This imbalance may lead to a pro-oxidant state (Daraghmeh and Karaman, 2024), potentially increasing hemolysis —the destruction of blood cells. An imbalance between antioxidants and oxidants creates oxidative stress, contributing to hemolysis and the release of hemoglobin into the bloodstream. When red blood cells break down, their intracellular contents, such as hemoglobin and potassium, are released into the bloodstream, leading to elevated serum potassium levels (Farnsworth et al., 2025). The increased serum potassium levels in fish fed a high-catechin diet were possibly related to intracellular components released from blood cells during breakdown. The lower impact on hematological values in fish fed high levels of quercetin is likely due to its low solubility in biological fluids (Chen et al., 2016; Pool et al., 2013), which makes it difficult for the substance to be effectively absorbed.

Catechin at both concentrations significantly improved tilapia survival when exposed to S. iniae. However, a high-catechin diet resulted in significantly reduced weight gain and reduced hematological parameters. Abdel-Tawwab et al. (2010) reported that dietary supplementation with green tea leaves, rich in catechins, at levels ranging from 0.125% to 25 g/kg of diet significantly improved tilapia survival against an Aeromonas hydrophila challenge, without negatively affecting fish performance. A lower level (200 mg/kg) of dietary green tea extracts (tea polyphenols) was shown to help hybrid crucian carp cope with overcrowding by strengthening internal defenses against stress damage and maintaining a healthier gut microbiota (Yang et al., 2025). In another study, incorporating 400–500 mg/kg of tea polyphenols into the diet was found to improve the liver’s antioxidant function without causing noticeable inflammation. However, this addition led to reduced growth (Daniel et al., 2024). Similarly, research on tea residue, which is rich in polyphenols, especially catechins, has shown potential as an effective functional food additive for fish. For example, Hu et al. (2023) evaluated fermented tea residues as a feed additive for juvenile largemouth bass, Micropterus salmoides, at levels ranging from 2% to 6%. They found that a 2% inclusion level was the most effective. In tilapia, an 8-week feeding trial showed increased weight gain with increasing levels of green tea waste inclusion up to 1.6%, after which weight gain decreased. Moreover, fish fed diets containing 0.8% and 1.6% green tea waste had a lower feed conversion ratio and better protein deposition than other treatments (Zheng et al., 2017). The levels of green tea extract (80% catechin) used in this study are clearly too high to be beneficial as a feed additive for tilapia.

In conclusion, the inclusion of hesperidin in tilapia diets showed promise for enhancing immunity and providing better protection against S. iniae. Evaluations of growth, feed utilization, and blood parameters indicate that hesperidin can be safely included in tilapia diets at concentrations of up to 1%. While catechin may offer some benefits in bacterial resistance, high levels of supplementation can lead to significant adverse effects on hematological health. On the other hand, dietary supplementation with quercetin showed no benefits and appears to be unfavorable for growth and liver-related indices at a concentration of 1%. Therefore, the development of sources rich in hesperidin or catechin at optimum doses as eco-friendly functional feed additives for aquatic animals could reduce the need for antibiotics in their diets, specifically under stressful conditions. This change may enhance the safety of animal products and promote sustainable aquaculture practices.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/faquc.2026.1814106/full#supplementary-material

Image of blood cells mixed with various concentrations of catechin. Control (a), 0.02 mg/ml catechin (b), and 0.2 mg/ml catechin (c). Supplementary material at the source

Erythrocyte osmotic fragility of tilapia mixed with different concentrations of catechin. Values are means of three replications. Supplementary material at the source

Footnotes

Edited by: Eric Amenyogbe, University of Environment and Sustainable Development, Ghana

Reviewed by: Enric Gisbert, Institute of Agrifood Research and Technology (IRTA), Spain

Mohamed Aboraya, Shanghai Ocean University, China