Lemon extract in the diet of Amazon tambaqui (Colossoma macropomum) improves growth, health, and survival rate after infection with Aeromonas hydrophila

Abstract

This study aimed to explore the effects of diets with different concentrations of lemon (Citrus limon) extract (CLE) on the growth, survival, hematological, and plasmatic variables of the Amazon tambaqui (Colossoma macropomum) infected by Aeromonas hydrophila. Six diets (in triplicate) with graded concentrations of CLE (0.0, 0.2, 0.4, 0.8, 1.6, and 3.2 g kg^-1) were supplied to the fish for 60 days. Afterward, the fish were infected with A. hydrophila for 10 days. Flavonoids and polysaccharides were the main constituents of CLE. Quadratic models indicated optimal feed conversion ratio and growth performance at 1.62 and 1.90 g CLE kg^-1 of diet (P < 0.05). The plasma aspartate aminotransferase activity was higher in the control group than in the other groups (P < 0.05). As the CLE inclusion level increased, hematological parameters increased proportionally (P < 0.05). After the bacterial challenge, no mortality or skin lesions were observed in tambaquis fed diets containing more than 0.8 g CLE kg^-1 (P < 0.05). Therefore, adding 1.9 g CLE kg^-1 to Amazon tambaqui juveniles’ diet is indicated, as it improves growth performance, feed conversion ratio, and overall health, and enhances resistance to diseases caused by A. hydrophila.

Key words
Citrus limon; feed conversion ratio; fish diseases; fish health

INTRODUCTION

In modern aquaculture, where the search to maximize production without compromising fish welfare and avoiding infection by pathogenic agents has gained prominence, the nutrition of these organisms becomes crucial to meeting these goals. Therefore, fish growth, health, and resistance against pathogens must be widely investigated in the most diverse aquaculture centers worldwide. In South America, especially in Brazil, the most prominent native fish species for aquaculture is the tambaqui (Colossoma macropomum), a species native to the Amazon Basin (St. Louis et al. 2022). It has received increasing attention from consumers due to the quality of its fillet (taste, texture, and presence of essential fatty acids, omega fatty acids, protein, and lipids) (Cavali et al. 2022), resistance to handling and transport stress (Castro Neto et al. 2025), and ability to adapt to intensive production systems (Ananias et al. 2024). Despite this, inadequate aquacultural practices make it highly susceptible to infectious diseases caused by pathogenic bacteria such as Aeromonas hydrophila (Paz et al. 2019, Pellin et al. 2023).

Good nutrition contributes to healthy development and disease resistance in fish. Several studies have shown that supplementing diets with natural plant products has benefited the growth, health, and immunity of tambaqui (Paz et al. 2019, Costa et al. 2020, Lopes et al. 2020, Chung et al. 2021, Monteiro et al. 2021, Souza et al. 2023, Pereira et al. 2024, 2025, Pereira Júnior et al. 2025). These studies highlight that plant-derived supplements are biodegradable and rich in bioactive compounds (e.g., polysaccharides, phenolic compounds, tannins, flavonoids, and organic acids). These compounds are essential for stimulating appetite and improving metabolism and physiology, and they have the potential to replace synthetic antibiotics in treating pathogenic infections.

Plants of the genus Citrus are cultivated in about 150 countries, mainly in tropical and subtropical regions, which together produce about 58.8 million tons of citrus fruits annually (Ramírez-Sucre et al. 2024, Sardoei et al. 2024). Lemon (Citrus limon ) extract (CLE) is rich in bioactive compounds, such as polysaccharides, phenolic compounds, essential oils, dietary fibers, vitamins, minerals, and ascorbic acid, which have been linked to improved weight gain and feed efficiency and strengthened immunity in fish (Laein et al. 2018, Macedo et al. 2023, Akrami et al. 2024, Oliveira e Silva et al. 2024). The wide availability of CLE and its bioactive compounds makes it an excellent alternative for dietary supplementation in fish. Although the effects of supplementary CLE on tambaqui have not been reported, this extract has already demonstrated beneficial effects on the growth, health, and defense against infections in striped catfish (Pangasius hypophthalmus) (Macedo et al. 2023), rainbow trout (Oncorhynchus mykiss) (Akrami et al. 2024), Nile tilapia (Oreochromis niloticus) (Oliveira e Silva et al. 2024), and African catfish (Clarias gariepinus) (Wei et al. 2024).

Infection with A. hydrophila impairs immune function and causes hemorrhagic septicemia, leading to high fish mortality and economic losses for fish farmers (Harikrishnan & Balasundaram 2005, Gallani et al. 2020, Felix e Silva et al. 2022). In aquaculture, bacterial disease prevention and control are typically achieved through vaccines and antibiotics (Wei et al. 2024). However, A. hydrophila has numerous strains, making it difficult to develop specific vaccines, antibiotics, and complex prophylactic methods, and often necessitating the use of other medications to control the pathogen (Souza et al. 2018, Gallani et al. 2020, Macedo et al. 2023). Furthermore, the use of antibiotics in aquaculture warrants caution, as indiscriminate use can select for resistant pathogens and negatively impact the aquatic environment (Souza et al. 2018, Paz et al. 2019).

Therefore, alternative dietary supplements should be investigated to improve fish growth and health. Given the proven benefits and positive effects of CLE in fish species, this study aimed to evaluate tambaqui juveniles fed diets with different CLE concentrations by assessing zootechnical performance, hemato-biochemical parameters, and resistance following A. hydrophila infection.

MATERIALS AND METHODS

Location, fish, and experimental conditions

The experiments were conducted in the Aquaculture Laboratory of the Federal University of San Francisco Valley (UNIVASF), Petrolina, PE, Brazil. A local fish farm supplied the tambaqui juveniles. During the 10-day acclimatization period, the fish were fed commercial feed (extruded; 36% crude protein; 3,200 kcal kg−1 digestible energy; Nutripiscis TR, Uberlândia, MG, Brazil) four times a day (08:00, 11:00, 14:00, and 17:00 h) at the rate of 5% of fish weight. The Committee on Ethics from UNIVASF approved this study (Protocol Number 2/260819).

A randomized experimental design included six treatments and three replicates in 18 500-L fiberglass tanks (usable volume of 450 L per tank) with 10 fish (22.30 ± 0.55 g apiece) per tank. A recirculating aquaculture system (continuous aeration, physical and biological filters) with a 60 L h–1 flow rate and a 12 L/12 D photoperiod was used. Growth, hematological, and metabolic parameters were collected 60 days after the fish began feeding on the specific diet. The fish were then infected with A. hydrophila for 10 days, during which survival and skin lesions in the fish were assessed.

The water quality parameters were measured every two days. The dissolved oxygen (5.90 ± 0.32 mg O₂ L−1) and temperature (29.10 ± 0.31 °C) were monitored with the aid of an oximeter (Pol-60, Politerm, São Paulo, Brazil), and the pH (6.76 ± 0.28) was monitored with a pH meter (HI 98130, Hanna, Barueri, Brazil). Alkalinity (55.00 ± 0.01 mg CaCO₃ L−1) and non-ionized ammonia (< 0.01 mg NH₃ L−1) were monitored by a kit from Alfatecnoquímica (Florianópolis, Brazil).

Experimental diets

Six diets with graded levels of CLE were formulated with 0.0 (control), 0.2, 0.4, 0.8, 1.6, and 3.2 g kg^-1 (Table I). The CLE levels were based on previous studies of Macedo et al. (2023) and Oliveira e Silva et al. (2024), who added CLE to diets of striped catfish and Nile tilapia, respectively. Before the experiments began, the fish had a 7-day adjustment period on the experimental diets. During acclimatization, food was offered four times daily (08:00, 11:00, 14:00, and 17:00 h) at 5% of fish weight.

Table I
Composition of experimental diets supplied to Amazon tambaqui containing different Citrus limon extract.

Ingredients (g kg-1)	CLE (g kg-1)
	0.0	0.2	0.4	0.8	1.6	3.2
Corn	340.90	340.90	340.90	340.90	340.90	340.90
Soybean meal (45%)	227.26	227.26	227.26	227.26	227.26	227.26
Corn gluten meal (60%)	172.54	172.54	172.54	172.54	172.54	172.54
Fish meal (60%)	142.20	142.20	142.20	142.20	142.20	142.20
Dicalcium phosphate	30.00	30.00	30.00	30.00	30.00	30.00
Soybean oil	28.30	28.30	28.30	28.30	28.30	28.30
Citrus limon extract	0.00	0.20	0.40	0.80	1.60	3.20
Inert	3.20	3.00	2.80	2.40	1.60	0.00
Premix1	50.00	50.00	50.00	50.00	50.00	50.00
Salt	5.00	5.00	5.00	5.00	5.00	5.00
Vitamin C	0.500	0.500	0.500	0.500	0.500	0.500
Butylated hydroxytoluene	0.10	0.10	0.10	0.10	0.10	0.10
	Analyzed proximate composition
Crude protein	32.90	32.95	32.91	32.89	32.93	32.92
Crude energy (kcal kg-1)	4011.05	4011.30	4011.50	4011.80	4011.97	4011.70
Crude lipid	5.57	5.62	5.60	5.65	5.62	5.67
Crude fiber	1.99	1.92	1.93	1.97	1.93	1.95

• ¹Fish premix (per kg of product; Neovia, São Lourenço da Mata, Brazil): Vit A - 1,210.00 UI; Vit B1 - 4.85 mg; Vit B12 - 4.80 mg; Vit B2 - 4.85 mg; Vit B6 - 4.85 mg; Vit C - 48,000.00 mg; Vit D3 - 210.00 UI; Vit E - 1.20 mg; Vit K3 - 2.30 mg; folic acid - 1.25 mg; biotin - 48.00 mg; D-calcium pantothenate - 12.00 mg; choline chloride - 108,000.00 mg; niacin - 24.00 mg; Se - 110.00 mg; I - 100.00 mg; Co - 10.00 mg; Cu - 3.00 mg; Fe - 50.00 mg; Mn - 20.00 mg; Zn - 30.00 mg; antioxidant - 25,000.00 mg.

The ingredients were mixed, macerated (hammer-milled through 1.00 mm sieves), remixed, moistened (30% water), and extruded. The extrusion was done in a temperature-controlled laboratory extruder (Inbramaq, São Paulo, SP, Brazil) using a 5.0-mm die plate, with a temperature of 90 °C for 2 sec. Then, the diets were dehydrated in a forced-air, recycling-system oven at 55°C for 24 h. The pellets were refrigerated at 4 °C in glass containers with hermetically sealed caps until use.

Diet samples were analyzed in triplicate for crude protein, crude lipid, gross energy, and crude fiber, according to AOAC (2016). The Kjeldahl method was used to determine crude body protein (% N × 6.25). Lipid content was determined by ether extraction in a Soxhlet extractor (Luca-202, Lucadema, São José do Rio Preto, Brazil). Gross energy was determined in a Parr bomb calorimeter. Crude fiber was extracted by acid hydrolysis, as described by Silva & Queiroz (2002).

The chemical characterization of CLE (Nor-Spice AB, Nor-Feed, organic production of fruit of lemon, certificate CE 834/2007, Beaucozé, France) was done by nuclear magnetic resonance (NMR) analysis for mass spectrometry (MS) and MS/MS analyses as described by Macedo et al. (2023) and Oliveira e Silva et al. (2024). The NMR spectroscopic technique identifies metabolites in complex mixtures, such as plant extracts, by detecting chemical shifts characteristic of primary and secondary metabolites. Macedo et al. (2023) and Oliveira e Silva et al. (2024) found that the 1H NMR spectrum showed high-intensity signals for polysaccharides (3.00 and 5.50 ppm) and aromatic regions (5.50–7.50 ppm) in CLE. Carbohydrates (primarily maltose and glucose) and phenolic compounds (highlighting flavonoids) were present as the main components, in addition to the presence of pectin and essential oils (Figure 1).

Figure 1
1H nuclear magnetic resonance (NMR) verified to Citrus limon extract (CLE) (400 MHz, DMSO-d6).

Growth performance

On days 1, 16, 31, 46, and 61, all juveniles were anesthetized with 50 mg L−1 of eugenol and weighed to estimate the amount of feed to be offered (feeding rate of 5% of fish weight). The quantified zootechnical variables were:

$$\mathrm{Weight} \mathrm{gain} \left(\mathrm{WG}\right) \left(g\right) = \mathrm{Fbw} - \mathrm{Ibw}$$

where Fbw = final (day 61) body weight (g) and Ibw = initial (day 1) body weight (g).

$$\mathrm{Specific} \mathrm{growth} \mathrm{rate} (\mathrm{SGR}, \% \mathrm{per} \mathrm{day}) =100 \times \frac{\mathrm{Ln\; Fbw} -\mathrm{Ln\; Ibw}}{\mathrm{time} \left(\mathrm{days}\right)}$$

$$\mathrm{Feed\; intake}(\mathrm{g\; fish}-1)=\frac{\mathrm{Total\; feed\; intake}\left(\mathrm{g}\right)}{\mathrm{Number\; of\; fish}}$$

$$\mathrm{Feed} \mathrm{conversion} \mathrm{ratio} \left(\mathrm{FCR}\right) =\frac{\mathrm{Total\; feed\; intake}}{\mathrm{Total\; weight\; gain}}$$

​​​​$$\mathrm{Survival\; rate}(\%)=100\times \frac{\mathrm{Final\; fish\; number}}{\mathrm{Initial\; fish\; number}}$$

Blood collection and hematological and plasmatic analysis

On day 61, blood was collected from anesthetized fish (n = 2 per tank) via a venocaudal puncture with a syringe containing ethylenediaminetetraacetic acid (anticoagulant) for hematological (1.0 mL) and plasmatic (1.0 mL) analysis. The hematological analysis was performed according to Blaxhall & Daisley (1973) to determine the hematocrit (%), number of erythrocytes (×106 µL–1), hemoglobin concentration (g dL–1), mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin (MCH, pg), and mean corpuscular hemoglobin concentration (MCHC, g dL–1). A heparinized capillary centrifuge (Benfer, São Paulo, Brazil) was used for hematocrit measurements. Natt and Herrick solution, Neubauer chamber, and a microscope were used for counting erythrocytes. A colorimetric kit (Bioclin, Belo Horizonte, Brazil) and a spectrophotometer (Biospectro 2,000 UV, Curitiba, Brazil) were used for measuring hemoglobin concentration.

For plasmatic analysis, the collected blood was centrifuged at 3,000 x g at 4 °C for 10 min (UniCen M, Helolab, Wiesloch, Germany) to separate the plasma. The plasma samples were stored at –80 °C. The variables measured were glucose (mg dL−1), albumin (g dL−1), cholesterol (mg dL−1), total protein (g dL−1), and aspartate aminotransferase (AST) (U L−1). The colorimetric enzymatic method used commercial kits (Labtest, Lagoa Santa, MG, Brazil) and a spectrophotometer (Biospectro 2,000 UV) for these analyses.

Aeromonas hydrophila challenge

The bacterial collection at UNIVASF provided the A. hydrophila strain (code A8B9761; www.ncbi.nlm.nih.gov/biosample/SAMN13514045). The stock material was obtained from the kidneys and skin of diseased tambaqui juveniles found in the region’s rivers (9°23’19.9” S, 40°30’10.8” W). The lethal dose (LD₅₀) was determined by testing bacterial concentrations (106, 107, and 108) in colony-forming units (CFU) mL-1. Then, the bacterial strain was diluted in sterile 0.85% saline to an experimental concentration of 1 × 108 CFU mL–1. On day 61, 0.2 mL of the A. hydrophila solution was injected intraperitoneally into anesthetized fish with no disease symptoms. Eight fish were used per tank (n = 24 per treatment in triplicate), as the fish from which blood was collected were not used.

Mortality and cutaneous lesions (reddish coloration and hemorrhaging) per fish were recorded daily for 10 days. A bacterial smear was prepared from the skin lesions and stained using the Gram method. Bacterial infection was confirmed by Gram staining of tank water and fish caudal kidney samples.

Statistical analysis

The data showed normality (Shapiro-Wilk test) and homogeneity of variance (Levene test) (both with P = 0.05). The treatments were compared using orthogonal polynomial contrasts (significant P < 0.05). The P and R2 values determined the best model. In addition, the means were compared using a one-way analysis of variance, followed by a post hoc Tukey’s test (significant at P < 0.05). The statistical analyses were performed using InfoStat software (2010).

RESULTS

Growth performance

No mortality or signs of disease occurred during the first 60 days of fish growth on the various treatment diets. Juveniles that were not fed CLE had significantly lower (P < 0.05) growth performance (Fbw, SGR, and weight gain) than juveniles fed with CLE > 0.4 g kg^-1 of diet. Similarly, the FCR in the control group was significantly higher (P < 0.05) than that of the groups fed CLE at 0.4–1.6 g kg^-1 of diet. Fed intake was not significantly different among treatments (P > 0.05) (Table II).

Table II
Growth performance of Amazon tambaqui fed Citrus limon extract (CLE) diets for 60 days.

Variables	CLE (g kg-¹)								p-Regression
	0.0	0.2	0.4	0.8	1.6	3.2	SEM	*P>F	Linear	Quadr	R2
Initial weight (g)	22.56	22.07	22.03	22.17	22.45	22.49	0.12	0.739	>0.050	>0.050	NR
Final weight |(g)	87.56b	94.99a	97.48a	101.04a	102.29a	98.36a	1.29	<0.001	<0.001	<0.001	0.71
Weight gain (g)	65.00b	72.92ab	75.45a	78.86a	79.48a	75.87a	1.32	<0.001	<0.001	<0.001	0.68
Feed intake (g fish–1)	103.81	104.96	97.79	99.65	99.68	108.28	1.97	0.698	>0.050	>0.050	NR
SGR (% per day–1)	3.02b	3.24ab	3.31a	3.37a	3.36a	3.28a	0.03	0.006	0.003	0.002	0.52
FCR	1.50a	1.42ab	1.29b	1.31b	1.33b	1.43ab	0.02	0.011	0.099	0.001	0.41

• SGR = specific growth rate. FCR = feed conversion ratio. SEM = standard error of the mean. NR = no regression. *ANOVA P-value. Different letters represent statistical differences by the Tukey test (P < 0.05) between treatments. N = 3 tanks per treatment.

Quadratic regression analyses revealed that tambaqui fed with CLE at 0.8 and 1.6 g kg^-1 of diet had increased growth performance for Fbw (y = – 3.929x2 + 14.919x + 90.470), weight gain (y = – 3.975x2 + 14.979x + 68.216), and SGR (y = –0.096x2 + 0.353x + 3.116) (all P < 0.05). For FCR, the best results occurred with CLE between 0.4 and 1.6 g kg^-1 of diet (y = 0.062x2 – 0.201x + 1.446) (P < 0.05) (Table II). Therefore, the optimal supplementation rate of CLE to obtain the highest Fbw, SGR, and WG and lowest FCR were 1.90, 1.88, 1.84, and 1.62 g kg^-1 of diet, respectively (Figure 2).

Figure 2
Graphical representation of weight gain (A), final weight (B), specific growth rate (SGR) (C), and feed conversion ratio (FCR) (D) as a function of different Citrus limon extract (CLE) concentrations in the Amazon tambaqui (Colossoma macropomum) diet.

Hematological and plasmatic analyses

For plasma AST activity, there was a quadratic effect (y = 7.273x2 – 20.822x + 96.327), where the lowest enzymatic activity occurred in the juveniles supplemented with 0.4 or 0.8 g of CLE kg^-1 of diet (P < 0.05). In the control group, plasma AST activity was significantly higher than in groups fed a diet supplemented with 0.2–1.6 g CLE kg^-1 (P < 0.05). Plasmatic albumin was significantly higher (P < 0.05) in fish fed with 1.6 g CLE kg^-1 of diet than in fish fed with 0.0 or 3.2 g CLE kg^-1 of diet (P < 0.05). Plasma glucose, total protein, and cholesterol were not significantly different among treatments (P > 0.05) (Table III).

Table III
Plasma metabolic intermediates of Amazon tambaqui fed Citrus limon extract (CLE) diets for 60 days.

Variables	CLE (g kg-1)								P-Regression
	0.0	0.2	0.4	0.8	1.6	3.2	SEM	*P>F	Linear	Quadr	R2
Glucose (mg dL–1)	85.54	87.18	84.81	82.76	86.15	85.05	0.91	0.842	>0.050	>0.050	NR
Albumin (g dL–1)	0.84b	1.11ab	1.04ab	1.10ab	1.14a	0.85b	0.11	0.301	>0.050	>0.050	NR
Total protein (g dL–1)	5.03	4.90	5.69	5.35	5.53	5.57	0.47	0.352	>0.050	>0.050	NR
Cholesterol (mg dL–1)	124.23	108.10	122.54	119.44	121.78	127.04	2.69	0.570	>0.050	>0.050	NR
AST (U L–1)	107.37a	91.79b	76.20c	79.04c	91.43b	102.26ab	2.14	<0.001	0.280	<0.001	0.35

• AST = aspartate aminotransferase. SEM = standard error of the mean. NR = no regression. *ANOVA P-value. Different letters represent statistical differences by the Tukey test (P < 0.05) between treatments. N = 6 fish per treatment.

As the level of CLE in the diet increased, there was a proportional increase in the hematocrit (y = 0.916x + 30.943), number of erythrocytes (y = 0.076x + 3.450), hemoglobin concentration (y = 0.882x + 10.708), MCH (y = 1.750x + 31.243), and MCHC (y = 1.669x + 34.673) (P < 0.05). Fish fed 3.2 g of CLE kg^-1 of diet generally showed significantly higher hematocrit and hemoglobin concentrations (P < 0.05) than those in the other treatments. In addition, the control group had a significantly lower hemoglobin concentration than fish fed diets with CLE > 0.4 g kg^-1 (P < 0.05). The MCH and MCHC levels in the control group were significantly lower (P < 0.05) than those in the treatment group given 3.2 g of CLE kg^-1 of diet. MCV was not significantly different among treatments (P > 0.05) (Table IV).

Table IV
Hematological parameters of Amazon tambaqui fed different Citrus limon extract (CLE) diets for 60 days.

Variables	CLE (g kg-1)								P-Regression
	0.0	0.2	0.4	0.8	1.6	3.2	SEM	*P>F	Linear	Quadr	R2
Hematocrit (%)	30.50b	31.67b	31.67 b	31.33b	32.17ab	34.00a	0.26	0.001	<0.001	0.001	0.43
Erythrocyte (×106 µL–1)	3.53	3.44	3.47	3.40	3.66	3.69	0.06	0.048	0.047	0.158	0.11
Hemoglobin (g dL–1)	9.96c	11.20bc	11.67b	11.42b	11.93b	13.53a	0.24	<0.001	<0.001	0.001	0.52
MCV (fL)	87.11	92.32	91.68	92.56	88.09	92.71	1.65	0.061	>0.050	>0.050	NR
MCH (pg)	28.37b	32.63ab	33.81ab	33.87ab	32.72ab	36.90a	0.87	0.015	0.005	0.043	0.21
MCHC (g dL–1)	32.66b	35.41ab	36.87ab	36.54ab	37.15ab	39.76a	0.62	0.001	<0.001	0.012	0.29

• MCHC = mean corpuscular hemoglobin concentration. MCV = mean corpuscular volume. MCH = mean corpuscular hemoglobin. SEM = standard error of the mean. NR = no regression. *ANOVA P-value. Different letters represent statistical differences by the Tukey test (P < 0.05) between treatments. N = 6 fish per treatment.

Aeromonas hydrophila challenge

Significantly reduced mortality (y = 14.053x2 – 58.519x + 45.651) was observed in tambaquis fed diets containing 0.4 g of CLE kg^-1 or more (P < 0.05) (Table V). All fish that received diets with 0.8 g of CLE kg^-1 or more survived. Significantly fewer skin lesions (y = 19.403x2 – 80.002x + 60.781) were observed in tambaquis fed diets containing CLE (P < 0.05) (Table V). All fish that received diets with 0.8 g of CLE kg^-1 or more had no skin lesions.

Table V
Mortality and skin lesions of Amazon tambaqui fed different Citrus limon extract (CLE) diets for 60 days, followed by 10 days of challenge with Aeromonas hydrophila.

Variables	CLE (g kg-1)
	0.0	0.2	0.4	0.8	1.6	3.2			P-Regression
							SEM	*P>F	Linear	Quadr	R2
Mortality (%)	58.33a	33.33ab	11.11b	0.00c	0.00c	0.00c	5.57	0.009	0.005	<0.001	0.90
Skin lesions (%)	83.33a	38.89b	11.11 c	0.00d	0.00d	0.00d	7.62	0.001	0.008	<0.001	0.92

• SEM = standard error of the mean. *ANOVA P-value. Different letters represent statistical differences by the Tukey test (P < 0.05) between treatments. N = 18 fish per treatment.

DISCUSSION

The use of plant extracts as additives in fish feed has been gaining popularity as a natural and safe alternative for nutrition and protection against microbial infections (Yilmaz 2019). Our research determined that the highest weight gain and the best FCR would be obtained with CLE levels of 1.62 and 1.90 g kg^-1 of diet, respectively. The properties of CLE, especially the presence of flavonoids and polysaccharides (the main active components), pectin, and essential oils (González-Molina et al. 2010), likely contributed to the promising results in growth performance and feed efficiency. Flavonoids and polysaccharides can exert prebiotic-like effects, as they contribute to beneficial changes in the morphology of intestinal villi, which can improve nutrient absorption (Pu et al. 2017) and stimulate the appetite of fish (Macedo et al. 2023). These components reinforce immune function and antioxidant activity, and boost protein and growth factor synthesis (Mohamed et al. 2021). CLE’s maltose and glucose (polysaccharides) may also have contributed to fish body mass (Ighwela et al. 2015). The pectin in Citrus promotes nutrient absorption and supports protein synthesis and growth factor production (Wei et al. 2024).

Similar effects on zootechnical performance to those observed in our study were described by Macedo et al. (2023) and Oliveira e Silva et al. (2024), who verified an improvement in the growth of striped catfish and Nile tilapia that were given diets enriched with 0.4 and 1.6 g of CLE kg^-1 of diet, respectively. Orange (Citrus sinensis) peel waste (10 g kg−1 of diet) improved the growth performance and antioxidant response of black rockfish (Sebastes schlegelii) (Lee et al. 2024). Furthermore, dehydrated lemon supplementation improved the growth, antioxidant status, and immune response of gilthead seabream (Sparus aurata) (1.5–3.0% peel) (Beltrán et al. 2017), common carp (Cyprinus carpio) (1.5–5.0 g of pomace kg^-1) (Laein et al. 2018), and rohu (Labeo rohita) (0.5–2.5% peel) (Harikrishnan et al. 2020).

The hemato-biochemical parameters are also essential tools for better understanding fish health and nutrition because they reflect physiological responses that affect development (Copatti et al. 2019, Michael et al. 2019). In our study, the metabolic parameters found (plasma glucose, cholesterol, albumin, and total protein) in tambaquis were similar among treatments, indicating that the fish maintained metabolic homeostasis with or without CLE. On the other hand, the addition of CLE to the diet affected hematological parameters and plasma AST activity.

In our study, diets with CLE enhanced the tambaquis physiological status. As the CLE concentration was increased, there was an increase in hematocrit, hemoglobin, erythrocytes, MCH, and MCHC. These results can be interpreted alongside the findings on growth performance. The improvement in erythrocyte count and oxygen transport capacity tends to enhance the tissue’s ability to obtain energy from the breakdown of organic molecules (Macedo et al. 2023, Oliveira e Silva et al. 2024), contributing to increased tambaqui growth in diets supplemented with CLE, as verified in this study. Erythrocytes are the cells that transport oxygen, and hemoglobin is the protein present inside erythrocytes and is directly related to the supply of oxygen to cells and the transfer of toxic metabolites to the external environment (Mohamed et al. 2021). The hematocrit is a percentage based on the number and size of erythrocytes in the blood volume, and it represents protection against dehydration and anemia (Lemos et al. 2025). An augmentation of hematimetric parameters reflects increased hemoglobin in erythrocytes (Michael et al. 2019).

Another key result of our study was that fish fed diets containing CLE at 0.4 and 0.8 g kg−1 showed reduced plasma AST activity. AST is an enzyme found in the liver, muscles, gills, and other tissues and can indicate liver, heart, and muscle injuries in animals (Copatti et al. 2019, Castro Neto et al. 2025). It is critical to predict amino acid energy use, thereby reducing deamination and improving protein use in tissues (Souza et al. 2023; Lemos et al. 2025). This was expected, as the properties present in plants of the genus Citrus have been associated with improvements in liver health and reductions in transaminase levels in this organ in fish (Akrami et al. 2024, Oliveira e Silva et al. 2024).

The improvement in the tambaqui’s physiological status and plasma AST levels in our study indicates better health, which was likely reflected in higher survivorship and fewer skin lesions when they were infected with A. hydrophila. This bacterium is an opportunistic pathogen that infects aquatic organisms, causing skin lesions, abdominal edema, exophthalmos, septicemia, and mortality (Harikrishnan & Balasundaram 2005, Felix e Silva et al. 2022). Its replication rate is influenced by abiotic factors (e.g., temperature and oxygenation), which can increase or reduce its ability to affect fish survival rate (Pereira et al. 2024). Under the conditions of our study, the highest mortality rate and the greatest number of skin lesions occurred among juveniles in the control group (diet without CLE) after bacterial challenge. Diets with 0.8 g of CLE kg−1 demonstrated a therapeutic effect against the bacterium and ensured survival without signs of skin lesions in these fish.

Protecting fish against aeromoniasis improves survival and growth, boosts feed efficiency, reduces physiological stress, and stabilizes fish production, thereby lowering treatment costs (Paz et al. 2019, Harikrishnan et al. 2020, Monteiro et al. 2021, Oliveira e Silva et al. 2024, Pereira et al. 2024, Pereira Junior et al. 2025). It is vital in the aquaculture sector because Motile Aeromonas septicemia can cause up to 100% mortality in outbreaks and is a major constraint on its productivity and profitability (Kari et al. 2022, Semwal et al. 2023). In addition, non‑antibiotic approaches reduce reliance on antibiotics and the spread of multidrug‑resistant A. hydrophila, supporting long‑term farm sustainability and market access (Souza et al. 2018, Felix e Silva et al. 2022).

The flavonoids and polysaccharides of CLE not only act positively on growth and nutrient absorption (Mohamed et al. 2021, Oliveira e Silva et al. 2024), but they also strengthen resistance to diseases triggered by microorganisms, as they play an immunological and antioxidant role (Harikrishnan et al. 2020), for example, by regulating proinflammatory cytokines and increasing activities of the mitochondrial respiratory complexes and antioxidative enzymes (Liu et al. 2021). Our results corroborate previous research. Adding 0.80–3.20 g of CLE kg−1 to the diet of Nile tilapia reduced mortality when the fish were challenged with A. hydrophila (Oliveira e Silva et al. 2024). Adding orange essential oil (400 and 800 mg L−1) improved tambaqui health and enhanced resistance to A. hydrophila (Pereira Júnior et al. 2025). Reduced mortality in Nile tilapia and African catfish after A. hydrophila infection was associated with dietary supplementation with dried lemon peel (0.1–0.2 g kg−1) (Rahman et al. 2019). Thus, based on the beneficial properties of Citrus and research reporting its advantages as a dietary additive for fish, CLE has the potential to enrich fish diets, thereby improving growth, welfare, and resistance to pathogen-caused diseases.

CONCLUSIONS

CLE showed benefits for the growth and health of Amazon tambaqui farming at concentrations above 0.8 g kg−1 of diet. Dietary supplementation with 1.9 g of CLE kg−1 is recommended for formulating practical tambaqui diets. At this concentration, greater zootechnical performance, optimal physiological status, reduced plasma AST activity, lower mortality, and fewer lesions after A. hydrophila infection are expected.

Acknowledgements

The Almeida, J.R.G.S. (# PQ 313235/2021-0) and Copatti, C.E. (# PQ 303337/2025-7) are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the productivity research grant awarded.

• Data availability
  The data used in this study are available upon request from the corresponding author.

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Edited by

• Handling editor
  Marília Rodrigues

Data availability

The data used in this study are available upon request from the corresponding author.

Publication Dates

• Publication in this collection
  18 Sept 2026
• Date of issue
  2026

History

• Received
  11 Aug 2025
• Accepted
  24 Mar 2026