Study of various curcumin-dietary feeding regimens on growth performance, non-specific immunity, and <i>Aeromonas hydrophila</i> resistance in juvenile common carp (<i>Cyprinus carpio</i>)

Yuanyuan Zhang; Liping Song; Guohong Ma; Shuquan Mao; Yanhua Zhang; Bingli Wang; Peng Xu; Jun Wu; Shuren Zhu; Wuxiao Zhang; Hong Lu; Han Ke

doi:10.46989/001c.154189

Introduction

The intensification and high-density practices of modern aquaculture have led to chronic oxidative stress, immune suppression, and reduced growth performance in farmed fish, posing significant challenges to industry sustainability. To address these issues, feed additives have gained prominence as a strategy to enhance aquatic animal health and production efficiency. However, concerns persist regarding their safety and efficacy, particularly due to the overuse of antibiotics and synthetic compounds, which contribute to drug residues, pathogen resistance, and environmental contamination. These challenges underscore the demand for sustainable alternatives, driving research toward eco-friendly phytogenic feed additives.

Extensive studies have established that the efficacy of immunostimulants follows a clear dose- and duration-dependent relationship.¹ While short-term, high-dose administration can rapidly boost immune competence, sustained low-dose exposure is generally necessary to achieve immunomodulatory effects. Notably, supraphysiological concentrations of some immunostimulants may paradoxically suppress immune responses.²

Growing evidence highlights concerns about immunostimulant-induced fatigue in aquaculture species.³ Intermittent feeding strategies have emerged as a promising approach to counteract this effect. For instance, a 7-day-on/7-day-off peptidoglycan regimen significantly improved growth performance while preventing immune exhaustion in kuruma shrimp (Marsupenaeus japonicus).⁴ Similarly, a 4-day-on/3-day-off schedule enhanced both growth and immune parameters in Pacific white shrimp (Litopenaeus vannamei).⁵ In finfish, cyclical β-glucan administration (7-day intervals) demonstrated superior efficacy, boosting weight gain, phenoloxidase activity, and stress resistance compared to continuous feeding protocols.⁶

Curcumin, a polyphenolic compound derived from Curcuma longa stands out for its broad-spectrum bioactivities, including antioxidant, anti-inflammatory, and immunomodulatory properties. While its benefits in human medicine and livestock are well-established, emerging evidence highlights its potential to improve nutrition and health outcomes in aquatic species. Studies demonstrate that curcumin enhances antioxidant capacity by modulating the Nrf2-Keap1 pathway and reduces inflammation through TLR/NF-κB regulation.⁷ However, current research on dietary curcumin in fish has primarily examined continuous feeding, with intermittent administration remaining largely unexplored. This pulsed approach could prevent metabolic adaptation to bioactive compounds while lowering production costs, offering a potentially more sustainable aquaculture strategy. Common carp (Cyprinus carpio), a commercially important freshwater species in China, shows increased vulnerability to environmental stressors and pathogens under intensive farming conditions, often leading to growth impairment and immune dysfunction. Systematic evaluation of curcumin’s effects on growth, antioxidant status, and immune function under intermittent feeding regimes is therefore essential for optimizing its use in carp culture.

This study utilized common carp to comprehensively assess how intermittent curcumin supplementation affects growth performance, antioxidant capacity, and immune responses. The results provide both a theoretical basis for optimizing curcumin use in aquafeeds and practical insights for developing sustainable aquaculture health management strategies.

Materials and methods

Experimental diets. The basal diet formulation and proximate composition are presented in Table 1. The control diet contained fish meal, soybean meal, fish oil and soybean oil as protein and lipid sources (32.29% protein, 7.57% fat, 8.17% ash). The experimental diet was identical except for supplementation with 60 mg kg^-1 curcumin (Xi’an Feida Bio-Tech Co., Ltd, China). All feed ingredients were ground through a 60-mesh sieve before processing. After sequential addition of supplements, ingredients were homogenized with soybean oil and water to form a dough. This mixture was pelleted using a laboratory extruder, air-dried at room temperature, then crumbled and sieved to obtain uniform 2-mm pellets. All diets were stored at -20℃ until use.

Table 1.Formulation and nutrient contents of the experimental diets.

Ingredients	Percentage dry weight
Fish meal	15
Soybean meal	40
Beer yeast	6
Wheat middling	28
Soybean oil	2.5
Fish oil	2.5
Choline chloride (50%)	2
Vitamin premix^a	1
Mineral premix^b	1
Calcium dihydrogen phosphate	2
Proximate composition (%)
Crude protein	32.29
Crude lipid	7.57
Crude ash	8.17
Gross energy (kJ g^-1)^c	16.32

Note: ^a Vitamin premix supplied the following vitamins (IU or mg/kg): Vitamin A, 900000IU; Vitamin D,200000IU; Vitamin E, 4500 mg; Vitamin K₃ , 220 mg; Vitamin B₁,320 mg; Vitamin B₂, 1090 mg; Niacin, 2800 mg; Vitamin B₅, 2000 mg; Vitamin B₆, 500 mg; Vitamin B₁₂, 1.6 mg; Vitamin C, 5000 mg; Pantothenate, 1000 mg; Folic acid, 165 mg; Choline, 60000 mg.
^b Mineral premix supplied the following minerals (g/kg): FeSO₄·7H₂O, 25 g; CuSO₄·5H₂O, 2.0 g; ZnSO₄·7H₂O, 22 g; Na₂SeO₃, 0.04 g; KI, 0.026 g; MnSO₄·4H₂O, 7 g; CoCl₂·6H₂O, 0.1 g.
^c Energy, calculated by using standard physiological fuel values of 17.15, 23.64 and 39.54 kJ/g for carbohydrate, protein and lipid, respectively.

Fish and Experiment design. Juvenile common carp were obtained from the Freshwater Fisheries Research Institute of Shandong Province and acclimated for 15 days while fed the basal diet. Following acclimation, 450 fish (6.35 ± 0.022 g) were randomly allocated to 15 circular fiberglass tanks (300 L water/ tank, 3 tanks/group, 30 fish/tank) across five treatment groups: one control group (Regimen 1, R1) receiving the basal diet, the three intermittent feeding groups were fed a trial diet of 60 mg curcumin kg^-1 for one week (Regimen 2, R2), two weeks (Regimen 3, R3), and four weeks (Regimen 4, R4), respectively, followed by feeding with the basal diet for equivalent durations, repeating this cycle for the 8-week experimental duration, and one continuous feeding group (Regimen 5, R5) receiving the trial diet. This was an eight-week experiment. Water flow was maintained at 2 L min^-1 with the following parameters: 26±1 ℃, pH 7.4-7.8, dissolved oxygen≥6 mg L^-1, and NH-N 0.08-0.09 mg L^-1. Fish were hand-fed to satiation three times daily (07:30, 12:00, 16:30 h) under natural photoperiod for 8 weeks.

Sample collection. Following the 8-week feeding trial, fish were fasted for 24 h and anesthetized with 100 mg L^-1 MS-222 (tricaine methanesulfonate; Sigma-Aldrich). Total weight and number of fish per tank were recorded for growth analysis. From each tank, three randomly selected fish were sampled for blood collection via caudal venipuncture using heparinized syringes. Immediately after collection, 50 µL of whole blood was aliquoted for respiratory burst assays. The remaining blood was centrifuged (2,000×g, 4 ℃, 10 min) to separate leukocytes and plasma, with plasma stored at -80 ℃ for subsequent analyses.

Growth performance. Individual body weights were recorded at the start (Wi) and end (Wf) of the 8-week experiment. Growth performance was evaluated using the following metrics for each treatment group: weight gain percentage (WG), specific growth rate (SGR), feed conversion ratio (FCR), viscerosomatic index (VSI), and hepatosomatic index (HSI). The calculations were performed as follows:

Weight gain (WG)=100×(Wf -Wi)/Wi

Specific growth rate (SGR, %/d) =100×(Ln Wf-Ln Wi)/t.

Feed conversion ratio (FCR)=Feed consumed (g)/(Wf-Wi)

Viscerosomatic index (VSI, %)=100×viscera weight/body weight.

Hepatosomatic index (HSI, %)=100×hepatopancreas weight/body weight.

where Wf was final body weight (g), Wi was initial body weight (g), t was experimental duration in days.

Blood biochemical measurements. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured colorimetrically using commercial kits (Mindray Bio-Medical, China) with an automated biochemical analyzer (BS-400, Mindray).

White blood cell count (WBC). The white blood cell count (WBC) of obtained blood samples was directly measured using an Auto Hematology Analyzer (BC-5300Vet, Mindray, P.R. China) with a test kit from Shenzhen Mindray Medical International Co. Ltd. (P.R. China).

Respiratory burst activity. Phagocyte respiratory burst activity was assessed via nitroblue tetrazolium (NBT; Sigma-Aldrich) reduction assay following established protocols.^4,8 Absorbance was measured at 630 nm using a Multiskan spectrophotometer (Thermo Scientific) with KOH/DMSO as blank. Results were expressed as NBT reduction per 100 μL cell suspension.

Plasma tumor necrosis factor-α (TNF-α) assay. The activity of plasma tumor necrosis factor*-α (TNF-*α) was measured by the double antibody sandwich method using a TNF-α ELISA detection kit (IBL, Germany). Optical density was measured at 450 nm.

Total myeloperoxidase (MPO) assay. Peripheral blood leukocytes were isolated using density gradient centrifugation with modifications from Misra et al.⁹ Myeloperoxidase activity was determined following established methods.^10,11 Briefly, leukocyte suspensions (1×10⁶ cells/well, in triplicate) were incubated with 0.02% CTAB in Hank’s balanced salt solution (Ca²/Mg²-free, phenol red-free) for 20 min. The reaction was initiated by adding 20 mM TMB and 5 mM H₂O₂, terminated after 2 min with 2 M H₂SO₄, then centrifuged (600×g, 15 min). Supernatant absorbance was measured at 450 nm using a microplate reader (Bio-Rad).

Plasma antioxidant indicators measurement. The supernatant was collected for analysis. The activities of superoxide dismutase (SOD) was measured using xanthine oxidase method.¹² The reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured using monitoring the oxidation of 2’,7’-dichlorofluorescin by spectrofluorometry,¹³ and thiobarbituric acid colorimetry,¹⁴ respectively.

Challenge test. Gram negative Aeromonas hydrophila was originally isolated from the infected fingerling C. carpio. The seven day LC₅₀ was determined by intraperitoneal injection of 48 ﬁsh with graded concentrations of A. hydrophila (10^6^{; 4}7^{; 4}8^{; 4}9^ and 10¹⁰ CFU/ml) at 24℃, yielding a day 7 LC₅₀ of 5×10⁶ CFU/ml.

For the challenge test, fish were divided into triplicate groups (10 fish/group) and injected intraperitoneally with 0.5 ml of bacterial suspension (5×10⁶ CFU ml^-1 in sterile saline) per 50 g body weight. Fish were fasted during the 4-day observation period, with daily mortality recorded.

Statistical analysis. Data are expressed as mean±SEM. After logarithmic transformation, one-way ANOVA was performed using SPSS 23.0 (IBM). Significant differences among treatments (P < 0.05) were further analyzed by Duncan’s multiple range test to compare curcumin feeding regimens.

Results

Growth. After 8 weeks feeding, dietary curcumin significantly affected common carp growth performance across feeding regimes (Table 2). Initial body weight, FCR and survival rate did not differ among groups (P > 0.05). Compared to controls, final body weight was significantly lower in R2 and R3 (P < 0.05) but higher in R4 (P < 0.05), while R5 showed no difference. WGR was significantly greater in R4 and R5 versus other groups (P < 0.05), with R4 outperforming R5 (P < 0.05). No differences occurred in VSI or CF. However, HSI was significantly reduced in R2 and R4 (P < 0.05), suggesting curcumin may modulate hepatic metabolism.

Table 2.Growth performance of C. carpio in different feeding regimens of curcumin¹

Modes	Initial body weight (g)	Final body weight (g)	WG (%)²	SGR³(% d^-1)	FCR⁴	Survival rate (%)⁵
R1	6.35 ± 0.01	18.75 ± 1.14^a	195.28 ± 9.38^a	2.12 ± 0.02^a	1.23 ± 0.06	100
R2	6.33 ± 0.05	19.54±1.16^b	213.46±5.64^a	2.29 ± 0.03^a	1.34±0.14	98.67±1.33
R3	6.32 ± 0.01	21.46±1.07^b	239.41±5.98^ab	2.54 ± 0.07^ab	1.31±0.06	97.33±2.67
R4	6.35 ± 0.02	24.31±0.91^c	269.87±3.17^c	2.89 ± 0.05^c	1.42 ± 0.12	96±2.31
R5	6.4 ± 0.05	22.59 ± 1.2^b	252.97 ± 11.11^b	2.74 ± 0.08^b	1.22±0.04	100

¹ Values are means ± S.E. (n = 3×3). Values with different superscript letters in the same column are significantly different in Duncan multiple range test (P<0.05).
² Weight gain (WG) = (Final body weight-initial body weight) × 100/ initial body weight.
³ Speciﬁc growth rate (SGR) = (LnW_t - LnW₀) × 100/T, where W₀ and W_t are the initial and ﬁnal body weights, and T is the culture period in days.
⁴Feed conversion ratio (FCR) = total diet fed (kg) / total wet weight gain (kg).
⁵ Survival rate = (initial fish number-final fish number) × 100/ initial fish number

Blood biochemical measurements and haematological analyses. Dietary curcumin significantly influenced physiological parameters in common carp across feeding regimes (Table 3). Plasma ALT and AST activities were significantly lower in R2 and R4 compared to controls (P < 0.05), but did not differ among treatment groups. TP levels increased significantly in R4 and R5 versus controls (P < 0.05), while AKP activity peaked in R4, exceeding control, R2 and R3 values (P < 0.05) but matching R5. Blood glucose levels remained unaffected by curcumin supplementation across all groups (P > 0.05).

Table 3.Blood biochemical measurements of C. carpio in different feeding regimens of curcumin

Modes	R1	R2	R3	R4	R5
Aspartate aminotransferase (AST) （U/L）	26.86±39.62^a	17.6±3.22^b	21.59±3.01^ab	18.74±2.9^b	22.34±2.56^ab
Alanine aminotransferase (ALT) （U/L）	24.5±2.31^a	16.88±2.87^b	20.31±3.27^ab	16.03±3.31^b	20.21±2.05^ab
Total protein (TP)（mg/L）	22.94±2.47^a	28.5±2.16^ab	29.3±2.1^ab	32.57±1.31^b	31.91±1.25^b
Alkaline phosphatase (AKP)（U/L）	35.05±0.77^a	32.6±0.35^a	33.96±0.92^a	50.28±1.03^b	44.23±0.69^ab
Glucose (Glu)（mmol/L）	3.7±0.32	3.13±0.14	2.82±0.33	3.03±0.46	3.46±0.41

Note: Values are means ± S.E. (n =3×3). Values with different superscript letters in the same column are significantly different in Duncan multiple range test (P<0.05).

Immune response. Respiratory burst activity peaked in R4, significantly exceeding all other groups (P < 0.05; Fig. 1). R5 showed intermediate activity, higher than R3 and controls (R1) but lower than R4. TNF-α levels followed a similar pattern, with R4 values significantly surpassing R2 and controls (Fig. 2).