https://orcid.org/0000-0002-7133-3633
Figures
Abstract
The present study evaluated the effects of curcumin on growth, immune and antioxidant response in tilapia (Oreochromis niloticus). An optimum dose of curcumin was investigated by feeding four different levels of this compound in combination with three different regimes of stocking density (12 treatments). Fish were reared at three densities; low density (LD = 1.50 kg/m3), medium density (MD = 3.00 kg/m3), and high density (HD = 4.50 kg/m3). Each treatment was fed with four different levels of dietary supplementation of curcumin (C0 = 0 mg/kg, C1 = 50 mg/kg, C2 = 100 mg/kg, and C3 = 150 mg/kg) for 60 days. Each treatment has three replicates (n = 50/replicate in LD, 100/replicate in MD, 150/ replicate in HD). Although better growth was observed in MD, however treatments at all densities fed with C1 diet showed improved growth as compared to other diets. Chemical composition of fish and activity of amylase, lipase and protease in all treatments were noted to be similar. Levels of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) and cortisol in MD and HD treatments were similar to those in LD treatment. However, fish fed with C1 diet in each density treatment showed the lowest values of antioxidant enzymes. Similarly, the levels of malondialdehyde were noted to be similar in MD and HD treatments as compared to that in LD. Its levels were lower in fish fed with C1 and C3 diets in all density treatments. Expression of pro-opiomelanocortin-α (POMC-α), Somatostatins-1 (SST-1) and Interleukin 1-β (IL-1β) did not increase in MD and HD treatments in response to high stocking density when compared with LD treatment. The lowest levels of these genes were noted in fish fed with C2 and C3 diets in all treatments. In conclusion, supplementation of curcumin in diet of tilapia improved growth and antioxidant response in tilapia. optimum dose of curcumin for tilapia culture is 50 mg/kg at the density of 3.00 kg/m3which might be further investigated for intensive culture.
Citation: Komal W, Fatima S, Minahal Q, Liaqat R (2024) Enhancing growth, antioxidant capacity, and immune response in tilapia (Oreochromis niloticus) through curcumin supplementation across varied stocking density paradigms. PLoS ONE 19(11): e0311146. https://doi.org/10.1371/journal.pone.0311146
Editor: Hamida Hamdi Mohammed Ismail, Cairo University, Faculty of Science, EGYPT
Received: April 7, 2024; Accepted: September 6, 2024; Published: November 20, 2024
Copyright: © 2024 Komal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data have been provided in MS.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
1 Introduction
Fish is widely recognized as a vital source of animal protein for human world widely [1]. There is a compelling demand to enhance aquaculture development to meet the rising global demand for human population. Global production of tilapia in year 2020 was seven million tonnes [2]. Nile tilapia (Oreochromis niloticus) stands out as a valuable species for aquaculture due to its rapid growth, minimal reliance on expensive animal protein in its diet and adaptability to high stocking densities in intensive production systems. The success and profitability of intensive fish culture systems depend on the growth rate of the fish and the stocking density employed [3–7].
High stocking density decrease the production by affects the physiology of fish by decreasing growth rate [8], increasing levels of cortisol and mortality and causing oxidative stress [9]. Elevating the number of fish in striking the space tends to harm their growth performance [10] as it deteriorates water quality, impacts social behavior, and uncontrolled metabolic rates due to the stress linked to crowding [11]. The fish stress responses are closely linked to hormonal reactions in the brain. The hypothalamic-pituitary interrenal (HPI) axis plays a key role in this process, triggering the production of corticotropin-releasing hormone (CRH) in the hypothalamus [12]. To assess how stocking density affects fish physiology, common parameters such as blood composition and alkaline phosphatase (ALP) levels are often examined [13]. Research indicates that high stocking density can have adverse effects on various blood parameters, including hematology and blood biochemistry [14] and can lead to chronic stress by increasing cortisol and glucose levels [15].
Crowding in production systems also causes oxidative stress, evident in the elevated production of reactive oxygen species (ROS) [16]. Accumulation of free radicals in the form of ROS occurs more rapidly leading to various forms of cellular damage i.e. mutation in nucleic acid [17]. Moreover, oxidative stress influences the expression of genes related to energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) [18], growth (Growth hormone, [19] Insulin-like growth factor-1 (IGF-1) [20], myostatin [21], immunological (nuclear factor-kβ) [22] and antioxidant enzymes (Nuclear factor erythroid 2-related factor 2 (Nrf2) [23], and other cellular defense proteins [22] in fishes. These biomolecules stimulate cascade of reactions to cope with damage caused by stress.
Fish experience changes in their innate and adaptive immune responses due to oxidative stress, starting with the activation of the HPA or HPI axis. This triggers the release of corticotropin-releasing factor (CRF), leading to the production of pituitary pro-opiomelanocortin (POMC) peptides, including adrenocorticotropic hormone (ACTH), which stimulates cortisol and other hormone synthesis to manage stress [24]. Somatostatins-1 (SST-1) are a diverse group of peptide hormones influencing growth, development and metabolism in vertebrates [25]. SST-1 which negatively affect growth hormone secretion, also play a role in regulating growth and metabolism [26]. Additionally, Interleukin 1-β (IL-1β), a proinflammatory cytokine, helps balance the immune system and alleviate stress by influencing the HPA axis [27].
This oxidative stress can be mitigated by supplementing with natural substances possessing antioxidant properties [28,29]. Recently researchers have found a positive connection between supplementing diets with antioxidants and reducing harmful effects such as health of fish and the activation of stress responses due to stocking density [30]. It also slows growth rates as in rainbow trout and grass carp (Ctenopharyngodon idella) juveniles [31,32], Nile tilapia (Oreochromis niloticus) [33,34], and largemouth bass (Micropterus salmoides) [35]. In this context, curcumin plays a role in repairing biomolecules, and membrane systems damaged by oxidative stress. in addition, it helps to maintain balance of normal physiological system and boosts the immune system [36]. Natural sources of antioxidants with higher phenolic compound levels have demonstrated the effectiveness in reducing oxidation, comparable to synthetic antioxidants [37–40]. When used as a preservative, curcumin can reduce oil oxidation and when included as a dietary supplement, it can enhance antioxidant capacity up to 80% [41]. The antioxidant effects of curcumin result from its ability to bind with free radicals, and provide a hydrogen atom [42].
Curcumin has the ability to decrease the generation of free radicals through the fenton reaction by binding to Fe2+/3+, Cu2+, [VO]2+ and Mn2+ [43]. It is crucial to acknowledge that curcumin is known to have low bioavailability (an average of 490 nmol/L in plasma) [44]. The antioxidant and cellular effects observed in vitro at high curcumin concentrations may not be replicated at physiological concentrations in vivo [40]. Additionally, in vivo curcumin is rapidly transformed into various metabolites through processes like glucuronidation and sulfation, either enzymatically or spontaneously [45]. Curcumin can also indirectly protect against free radicals by inhibiting reactive-oxygen-generating enzymes such as NADPH oxidase (NOX), lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase, and inducible nitric oxide synthase (iNOS) or by inducing antioxidant enzymes [46].
Present study conducted with different doses of curcumin against variant stocking density condition in tilapia. Study also determines the optimum dose of curcumin against different stocking density specifically at high stocking density. The objective of study was to assess the impact of a water-soluble curcumin formulation in the diet on the growth performance, stress physiology, antioxidant status, and gene expression of POMC-α, IL-1β and SST-1 in tilapia exposed to different stocking densities. This optimum dose could be used as dietary supplement in tilapia commercial culture to enhance growth and immune response at high stocking density.
2. Materials and methods
2.1. Diet preparation
In present study, commercial curcumin (C21H20O6: sigma aldrich, USA; purity ≥65%) was used as a feed supplement. Curcumin used in this study was sourced from Curcuma longa (Turmeric) powder with the formula weight of 368.38 g/mol. Its solubility color was from yellow to orange and with UV/VIS absorbance range of 420–430 nm. Treatment diets were prepared by mixing the finely ground ingredients with four levels of curcumin (C0 = 0 mg/kg, C1 = 50 mg/kg, C2 = 100 mg/kg, C3 = 150 mg/kg) and pellet of 1mm was prepared using mechanical pellet machine (Table 1). The pellets were air-dried at room temperature and stored at 4°C in sealed bags. Fish were fed at a proportion of 2% of their body mass on daily basis, twice a day. The recommended standard feeding ration for tilapia (Oreochromis niloticus) is 2% [47]. The feed utilized in this study contained 30% crude protein, aligning with the tilapia protein requirement range of 25–30%. The dietary requirement for tilapia in context to total energy content (calorie) is 2,500–3,000 kcal/kg and a fiber content ranging between 3% and 7% [48].
2.2. Experimental design
Fish (n = 3600, initial weight = 30.00±1.20g) were procured from a local fish hatchery (Lahore, Pakistan) and transferred to Aquaculture facility, Lahore College for Women University. There was no mortality of fish during the transfer. This study was commenced after approval of Animal Ethics Committee of Department of Zoology, Lahore College for Women University (Approval #: Zoo/LCWU/932). Fish were randomly distributed in 36 fiber glass tanks (water volume/tank = 1 m3) according to the details given in Fig 1. Each tank has water supplied from the same water sump, treated with UV filter and biofilters. All tanks had their own water supply. Fish were acclimatized for one week before commencement of trial. The duration of this trial was 60 days and all fish were healthy by end of this trial. Survival rate is mentioned in section 2.3. This time period for the trial was selected following [49], which investigated growth of tilapia over a period of 171 days, aiming to achieve a final stocking density of 57.81 kg/m3. Furthermore, 60 days is the most recommended study period to observe the effect of any dietary supplement on growth of fish. Duration of trial (60 days) was the only designated humane end point in this study to terminate experiment. Fish husbandry conditions and health were excellently maintained throughout the entire trial period to minimize the mortality as mentioned in section 2.3.
Three stocking density regimes were studied in this trial; low density (LD) (1.50 kg/m3), medium density (MD) (3.00 kg/m3), high density (HD) (4.50 kg/m3). The total number of fish stocked in LD, MD and HD treatment was 600, 1,200 and 1,800, respectively. Each density treatment had three replicates (Fig 1). Fish in all stocking density treatments (LD, MD, HD) were fed with four different levels of dietary supplementation of curcumin (C0, C1, C2, C3). Dose of curcumin in each dietary level is given in section 2.1. Each curcumin supplementation level was studied in further three replicates (Fig 1). These four different levels of curcumin were fed to low density treatment (LDC0, LDC1, LDC2, LDC3), medium density treatment (MDC0, MDC1, MDC2, MDC3), and high-density treatment (HDC0, HDC1, HDC2, HDC3). Fish were fed by hand with daily ration calculated at the rate of 2% of biomass in each replicate. Random weight check of fish was performed for each replicate after every 15 days to adjust the daily ration.
2.3 Water quality parameters and survival rate
Water quality was very well maintained in all tanks to ensure the welfare of fish. A total 20% of water was exchanged every day from each tank. Water quality parameters were measured twice a day to ensure its standard quality levels throughout the trial period. Aeration pumps (120V/60Hz, Airmax SilentAir LR25, USA) were used to deliver air via diffuser grids. There was one rectangular diffuser grid in each tank (L ×W: 1 × 0.5 ft). Each diffuser grid was built by using the anti-microbial tubing (outer diameter: 25.4 mm; inner diameter: 12.7 mm; airflow: 2.2 m3/h/meter) to generate microbubbles ensuring the good air saturation in water (>80%). All tanks were back washed on daily basis to drain solid waste settled at the bottom of tank. Water quality parameters including water temperature (25.86 ± 0.30–27.78 ± 0.23°C), dissolved oxygen (4.13 ± 0.31–4.88 ± 0.27 mg/L), and pH (7.52 ± 0.45–8.76 ± 0.01) were monitored twice a day by using portable meters (HI98494, Hanna, USA). Ammonia (0.07±0.03–0.28±0.12 ppm), and nitrite (0.11±0.01–0.21±0.10 mg/L) were monitored twice a week by using commercial kits (HI733, HI93708, Hanna, USA). Fish in every tank were checked for any sign of disease, abnormal behavior and mortality twice a day. Dead fish were removed immediately if found and carefully recorded. Survival rate observed in LD, MD and HD were 100%, 100% and 98.75%, respectively due to well-maintained husbandry conditions over the study period. Mortality of only 1.25% observed in HD treatment, was due to high density. However, it was much lower than the designated limit of 10%, approved by Animal Ethics Committee for Aquaculture trials.
2.4. Sample analysis
At end of the trial, five fish were randomly sampled from each replicate (Fig 1) of all density treatments (20 samples per treatments). A total of 180 fish were used for this terminal sampling out of 3600 fish used in the trial following the designated limit of 5% of population for euthanization by Animal Ethics Committee. Remaining 3420 fish were humanely released in nearby lake, administered by Department of Fisheries, Pakistan for stocking purpose (Release Approval #: DOF/27856/2022). Before sampling, fish were fasted for 24 h. On sampling day, they were euthanized using clove oil (0.8 ml/L of water, Sigma-Aldrich, USA). This dose of clove oil is standard to euthanize fish in a very humane way which took less than ten minutes to euthanize sampled fish.
Blood was collected from the caudal vein in two tubes. One tube was coated with ethylenediamine tetra acetic acid (EDTA: (for hematology) while second tube contained clot activator for plasma collection. Blood samples were centrifuged at 5,000 rpm for 15 min and plasma was collected in separate eppendorf tubes and stored at -20°C. The total body weight and total body length were measured before dissection. Fish were dissected, and gills were collected, rinsed in deionized water and preserved in 10% formaldehyde solution for 24 h for histological study. Fish muscle samples were collected and stored at– 20°C for whole body chemical composition. following the guidelines of Association of Official Analytical Chemists (AOAC) [50]. Muscle samples were dehydrated in an oven at 80°C until a consistent dry weight was reached. These dried samples were processed for further chemical analysis. The Kjeldahl apparatus (PCSIR Laboratories, Pakistan) was used to determine the crude protein, while crude lipids were identified using the Folch method [51] using the Soxhlet apparatus (PCSIR Laboratories, Pakistan). The ash content in muscles was determined using muffle furnace (PCSIR Laboratories, Pakistan). The intestinal samples from midgut were weighed, rinsed with deionized water and homogenized in 0.86% sterile normal saline solution (1:9). This mixture was centrifuged at 5000 rpm for 15 min. Supernatant was collected and stored at -20°C. Each sample was analyzed in three replicates for each laboratory analysis. Liver samples were collected and homogenized in liquid nitrogen at -80°C for genes expression analysis. Condition factor (K), specific growth rate (%) (SGR), hepatosomatic index (HSI), fish weight gain, survival rate, feed conversion rate (FCR) was measured by using the given formulae.
2.5. Hematological analysis
Hematological parameters such as hemoglobin (Hb) (g/dl), white blood cells (WBC) (103/μL) count such as Neutrophils (%), Eosinophils (%), Lymphocytes (%), Monocytes (%), red blood cell (RBC) (106/μL) count, platelets (103/μL) were determined by using auto-hematology blood analyzer (Sysmex-KX-21, Japan), calibrated for fish.
2.6. Biochemical analysis
Triglyceride (TG) (mg/dl) was estimated through a triglyceride colorimetric assay kit (Thermo Fisher Scientific, USA, CAT No. EEA028) following the protocol of the manufacturer. The level of albumin (Alb) (g/dl) was determined through the use of bromocresol green (BCG) dye binding technique, utilizing an albumin kit (LOT. DR379E249; ANMOL-LAB Pvt. Ltd, India). The quantification of alkaline phosphatase (ALP) (U/L) was carried out using commercial kit (Thermo Fisher Scientific, USA, CAT No. EEA002, E.C. 3. I. 3.1.). Aspartate aminotransferase (AST) (U/L) was estimated through commercial ELISA kit (Thermo Fisher Scientific, USA, CAT No. MAK055, E.C. 2.6. 1.1.). Activity of alanine aminotransferase (ALT) (U/L) was measured using commercial ELISA kit (Thermo Fisher Scientific, USA, CAT No. MAK052, E.C. 2.6. 1.2.). The concentration of glucose (GLU) (mg/dl) was measured by using laboratory blood glucose analyzer (Human, Germany).
2.7. Cortisol assay
The concentration of cortisol (ng/ml) in blood plasma was measured using ELISA (Calbiotech, USA, CAT No. CO368S, CID 5754) having a sensitivity of 1.16 ng/ml. The intra-assay and inter-assay precision were 3.8% and 8.65%, respectively. The detection limit was 0–800 ng/ml. The absorbance value was read on spectrophotometer at 450 nm.
2.8. Antioxidants assay
Plasma catalase (CAT) (U/ml) activity was determined using a commercial ELISA colorimetric activity kit (Thermo Fisher Scientific, USA, CAT No. EIACATC, EC 1.11.1.6) having an analytical sensitivity of 0.052 U/ml. The absorbance was read at 560 nm at 37°C. The activity of superoxide dismutase (SOD) (ng/ml) were measured by using ELISA kit (PARS BIOCHME, China, CAT No. PRS-02005hu, EC 1.15.1.1) with an assay range of 0.3 ng/ml– 10 ng/ml. Malondialdehyde (MDA) (nmol/ml) ELISA kit (PARS BIOCHME, China, CAT No. PRS-00991hu, CAS 542-78-9) with an assay range of 0.3 ng/ml– 7 nmol/ml. Activity of glutathione peroxidase (GPx) (IU/ml) were measured by using ELISA kit (PARS BIOCHME, China, CAT No. PRS-00680hu, EC 1.11. 1.9) with an assay range of 3 IU/ml– 200 IU/ml. The absorbance value of SOD, MDA, and GPx was read at 450 nm and 37°C.
2.9. Digestive enzymes assay
For digestive enzyme analyses, the supernatant of processed whole intestine samples was utilized. Activity of lipase (U/L) was assayed with a commercial ELISA kit (Sigma Aldrich, USA, CAT No. MAK046, EC 3.1.1.3) with a detection limit of 5 U/L to 250 U/L at 37°C and 570 nm of wavelength. Amylase (U/L) activity was measured using a commercial ELISA kit (Sigma Aldrich, USA, CAT No. MAK009A, EC 3.2. 1.1.) with a detection limit of 0.2439 U/L—2200 U/L at 37°C and 405 nm of wavelength. The activity of protease was determined following instructions [52]. Casein 1% w/v was used as substrate in 0.2 M phosphate buffer at pH 7.0. One unit of protease indicates the amount of enzyme that releases 1 μg/ml/min of tyrosine determined at 660 nm of wavelength.
2.10. Histological study
Preserved gill samples were dehydrated by passing through different grades of alcohol (70%, 90% and 100%) and xylene. For the infiltration of wax, gills were processed in paraffin wax. Microtome were used for sectioning and wax blocks were trimmed at 10 μ and then transverse sections of 4 μ thickness were cut. For dewaxing, xylene and alcohol were used and stained with haematoxyline and eosin. Stained section of gills was mounted with DPX (mixture of distyrene, plasticizer and xylene) (Merck, Germany). Microphotographs were taken at digital camera fitted optical microscope (Trinocular E-200, Nikon Japan Eil-12).
2.11. Gene expression analysis
Liver samples (50 mg/sample) were used to extract total RNA by using trizol (Catalog No. 15596026, Thermo, USA) method at 37°C. The quality and quantity of each sample was verified on Nanodrop 2000 spectrophotometer (Thermo, Waltham, MA, USA). The first strand cDNA was synthesized using super script III first strand cDNA synthesis kit (Cat No. 18080051, Life technologies). The 5.0 μg of total RNA was used for cDNA synthesis. cDNA synthesis was performed in the first step with poly-A tail primedoligodT in a total volume of 20 μl. The first reaction mixture was prepared having RNA 5 μg, 50 μMoligo (dT) 20 of 1 μL, 10 mMdNTP mix of 1 μL and then water was added upto 10 μL. The mixture was incubated at 65°C for 5 min. cDNA synthesis Mix-2 was prepared by adding 10 X RT buffer (2 μL), 25 mM MgCl2 (4 μL), 0.1 M DTT (2 μL), RNaseOUT™ (40 U/μL) (1 μL), SuperScript® III RT (200 U/μL) (1 μL), a total of 10 μL.
The prepared 10 μL of cDNA Synthesis mix was added to each RNA/primer mixture, mixed gently and collected by brief centrifugation. The tube was incubated for 50 min at 50°C. The reaction was terminated at 85°C for 5 min. The cDNA was store at -20°C. The PCR reaction was performed in a separate tube with gene specific primers (forward and reverse) using 2 μl cDNA templates. The following set of primer was used for real-time PCR which were designed by using software Primer Quest from integrated DNA technology (Table 2). Each set of primer 1 μl (10 μM) along with 12.5 μl SYBR green PCR master mix (Maxima SYBR Green/ROX qPCR Master Mix (2X)) were used. First denaturation step was carried out as 95°C for 2 min, followed by 95°C denaturation for 15 sec, annealing step was carried out at 55°C for 1 min and extension step was carried out at 72°C for 1 min. β-actin was used as the housekeeping gene for reference. The 2-fold induction was determined by ΔΔCT method (relative quantification).
2.12. Statistical analysis
For all the statistical analyses, SPSS v.29 software was used. Data were presented as Mean± SE for all the parameters. Levene test were performed to check the homogeneity of variance of data. The effect of density and curcumin supplementation dose on different parameters was determined by Two-Way ANOVA. To reject the null hypothesis, 0.05 probability level was used. Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters (red color) show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3. Results
3.1 Water quality parameters
The range of water quality parameters including water temperature (25.86 ± 0.30–27.78 ± 0.23°C), dissolved oxygen (4.13 ± 0.31–4.88 ± 0.27 mg/L), and pH (7.52 ± 0.45–8.76 ± 0.01), ammonia (0.07±0.03–0.28±0.12 ppm), and nitrite (0.11±0.01–0.21±0.10 mg/L) (Table 3).
3.2. Growth
In three different densities treatment (LDC, MDC, HDC), total body length (df2, F = 14.03, P = 0.0000), total body weight (df2, F = 6.88, P = 0.0024), condition factor (df2, F = 16.31, P = 0.0000), specific growth rate (df2, F = 7.39, P = 0.0016), hepatosomatic index (df2, F = 15.72, P = 0.0000) were significantly different (Table 4). A significant variation in total body length (df3, F = 4.25, P = 0.01), condition factor (df3, F = 3.54, P = 0.02), specific growth rate (df3, F = 2.79, P = 0.05) and hepatosomatic index (df3, F = 0.79, P = 0.50), except total body weight (df3, F = 2.62, P = 0.06) were observed between different levels of curcumin supplementation in all three-density treatment (levels of supplementation: 04 in each treatment). Other than this, effect of density*curcumin calculated by two-way ANOVA also showed a significant effect on total body length (df6, F = 2.67, P = 0.03), total body weight (df6, F = 2.58, P = 0.03), condition factor (df6, F = 2.47, P = 0.04), specific growth rate (df6, F = 2.85, P = 0.02) except hepatosomatic index on which the effect was insignificant (df6, F = 1.69, P = 0.14). The survival rate of fish both in LSD and MSD was 100% but in HSD the its rate was 98.22% - 99.56%.
Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3.3. Chemical composition of muscles
The moisture content (df2, F = 408.67), crude protein (df2, F = 756.29), crude ash (df2, F = 1641.52) and crude fat (df2, F = 2251.88) was significantly different (P = 0.0000) between three density treatment (Table 5). A significant difference (P = 0.0000) in the content of moisture (df3, F = 1746.03), crude protein (df3, F = 3392.86), crude ash (df3, F = 3549.21) and crude fat (df3, F = 3013.29) was observed between different levels of curcumin supplementation in all three-density treatment (levels of supplementation: 04 in each treatment). Effect of density*curcumin calculated by Two-way ANOVA also showed significant effect (P = 0.0000) on moisture (df6, F = 1924.60), crude protein (df6, F = 2440.48), crude ash (df6, F = 1596.83) and crude fat (df6, F = 3328.77).
Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3.4. Digestive enzymes activity
The activity of amylase (df2, F = 1256956.88), protease (df2, F = 417165.81) and lipase (df2, F = 447251.52) were significantly different (P = 0.0000) between three density treatment (LDC, MDC, HDC) (Fig 2). Different levels of curcumin supplementation in density treatment (four in each treatment) showed significant variations (P = 0.0000) in the activity of amylase (df3, F = 2119.64), lipase (df3, F = 21273.81) and protease (df3, F = 104146.83). In addition to this, effect of density*curcumin on profile of amylase (df6, F = 162860.12), lipase (df6, F = 170659.52) and protease (df6, F = 441651.60) calculated by Two-way ANOVA was also significant (P = 0.0000).
Levels of (A) amylase, (B) lipase and (C) protease (Mean ± SE) determined in three density treatment (LDC, MDC, HDC) having four curcumin supplementation levels (C0 = 0 mg/kg, C1 = 50 mg/kg, C2 = 100 mg/kg, C3 = 150 mg/kg). Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3.5. Profile of cortisol
The levels of cortisol differed significantly (df2, F = 560177080.92, P = 0.0000) between density treatment (LDC, MDC, HDC) (Fig 3). Effect of different levels of curcumin supplementation in all three-density treatment (four in each treatment) was also significant (df3, F = 39657792.65, P = 0.0000). Effect of density*curcumin on the level of cortisol calculated by Two-way ANOVA was also significant (df6, F = 29618622.42, P = 0.0000).
Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3.6. Blood biochemistry and hematology
A significant effect (P = 0.0000) was observed on the content of Hb (df2, F = 14772.95), platelets (df2, F = 102144144.38), WBC (df2, F = 78.05), RBC (df2, F = 3262.60), monocytes (df2, F = 2984.85), eosinophils (df2, F = 380.09), neutrophils (df2, F = 792620.57) and lymphocytes (df2, F = 872715.80) in three density treatment (Table 6). Content of triglycerides (df2, F = 5456572.95), ALT (df2, F = 6292715.80), AST (df2, F = 60185715.80), ALP (df2, F = 1251790858.66), albumin (df2, F = 47058.66), glucose (df2, F = 14722334.85), urea (df2, F = 14010430.09) showed a significant effect (P = 0.0000) in three density treatment except in creatinine (df2, F = 1.52) which was insignificantly different (P = 0.23) (Table 7).
Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
Effect of different levels of curcumin supplementation in all three-density treatment was also studied on hematology and biochemical parameters (four levels in each treatment). A significant effect of this supplementation was observed (P = 0.0000) on Hb (df3, F = 4503.96), WBC (df3, F = 12.81), RBC (df3, F = 1900.79), platelets (df3, F = 59515873.01), neutrophils (df3, F = 825396.82), lymphocytes (df3, F = 404761.90), monocytes (df3, F = 59.52), eosinophils (df3, F = 1646.82), triglycerides (df3, F = 15706349.20), glucose (df3, F = 21190476.19), ALT (df3, F = 1005952.38), AST (df3, F = 24609126.98), ALP (df3, F = 80325396.82), albumin (df3, F = 28015.87), urea (df3, F = 492063.49) and creatinine (df3, F = 1488.09).
A significant effect (P = 0.0000) of density*curcumin calculated by Two-way ANOVA were observed on the content of Hb (df6, F = 12539.68), WBC (df6, F = 17.64), RBC (df6, F = 606.15), platelets (df6, F = 12783730.15), neutrophils (df6, F = 1998015.87), lymphocytes (df6, F = 541666.66), monocytes (df6, F = 59.52), eosinophils (df6, F = 1051.58), triglycerides (df6, F = 7944444.44), glucose (df6, F = 8767857.14), ALT (df6, F = 4101190.47), AST (df6, F = 20484126.98), ALP (df6, F = 82182539.68), albumin (df6, F = 31825.39), urea (df6, F = 640873.01) and creatinine (df6, F = 2619.04).
3.7. Antioxidant assay
A significant difference (P = 0.0000) was observed in the levels of CAT (df2, F = 76.19), SOD (df2, F = 21.15), GPx (df2, F = 654.52) and MDA (df2, F = 719.06) between three different density treatment (Table 8). A significant variation in the levels of CAT (df3, F = 10.03, P = 0.0000), SOD (df3, F = 6.39, P = 0.0010), GPx (df3, F = 68.03, P = 0.0000) and MDA (df3, F = 13.42, P = 0.0000) were observed between different levels of curcumin supplementation in three density treatment (four in each treatment). Effect of density*curcumin calculated by Two-way ANOVA on the levels of CAT (df6, F = 11.02, P = 0.0000), SOD (df6, F = 2.84, P = 0.01), GPx (df6, F = 67.46, P = 0.0000) and MDA (df6, F = 13.42, P = 0.0000) was significant.
Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3.8. Gene expression
The expression of SST-1 gene (df2, F = 3.93, P = 0.03) and POMC-α (df2, F = 5.33, P = 0.008) was significantly different between three density treatment (Fig 4). However, the expression of interleukin 1-β (df2, F = 1.31, P = 0.28) was insignificantly different between three density treatment. Different levels of curcumin supplementation in three density (four in each density treatment) showed in significant effect on the levels of SST-1 (df3, F = 2.35, P = 0.08), interleukin 1-β (df3, F = 0.25, P = 0.86), and POMC-α (df3, F = 1.73, P = 0.17). Effect of density*curcumin calculated by Two-way ANOVA on the levels of SST-1 (df6, F = 1.65, P = 0.15), interleukin 1-β (df6, F = 0.36, P = 0.90) and POMC-α (df6, F = 1.54, P = 0.18) was insignificant.
Levels of gene expression of (A) Somatostatin 1, (B) interleukin 1-β and (C) POMC-α (Mean ± SE) determined in three density treatment (LDC, MDC, HDC) having four curcumin supplementation levels (C0 = 0 mg/kg, C1 = 50 mg/kg, C2 = 100 mg/kg, C3 = 150 mg/kg). Superscripts represented as upper case letters show the comparison (P < 0.05) between three density treatments (LDC, MDC, HDC). Superscripts represented as lower-case letters show the comparison (P < 0.05) between four different levels of curcumin supplementation within one density treatment.
3.9. Histological analysis
Histology of gills was done for all treatments (density*curcumin) (Fig 5). Low density treatment showed minute disruption in structure of lamellae (Fig 5B–5D). Medium and high-density treatment showed high alteration in gills structure indicated by the degeneration of primary and secondary lamella (Fig 5E–5L), and tissue debris (Fig 5J–5l), compared with low density treatment. In high density treatment lamellar fusion (Fig 5I), necrosis (Fig 5J), epithelial lifting (Fig 5K) which is the detachment of epithelial cells from secondary lamellae and congestion in blood (Fig 5L) were observed. Low density treatment showed normal structure of gills including primary lamella and secondary lamella with less or no structural alterations.
Light micrographs of a paraffin section stained with eosin (10x). A(LDCO), B(LDC1), C(LDC2), D(LDC3), E(MDC0), F(MDC1), G(MDC2), H(MDC3), I(HDCO), J(HDC1), K(HDC2), L(HDC3). PL: Primary lamellae; SL: Secondary lamellae; DPL: Degeneration of primary lamellae; DSL: Degeneration of secondary lamellae; TD: Tissue debris; BC: Blood congestion; N: Necrosis; EL: Epithelial lifting; LF: Lamellar fusion.
4. Discussion
Studies have pointed out the adverse effect of overcrowding fish in high density conditions on their health and general wellbeing without any dietary supplementation [53,54]. The present research identified that dietary supplementation of curcumin showed a notable increase in growth parameters among treatments with different stocking densities. Specifically, treatment supplemented with dietary supplementation of curcumin (C1 = 50mg/kg) led to improved growth in fish raised under high-density conditions (HD = 4.50 kg/m3). Present study coincides with previous research conducted on various fish species in response to curcumin [31,35,55–60]. Growth performance of fish also linked with somatostatin gene-1 (SST-I). Present study noted high expression of SST-I gene in both low as well high stocking density treatments. This aligns with previous findings indicating that sst1 was upregulated in tilapia at high density [61] as well as in cichlids [62,63]. After the dietary supplementation of curcumin in current study at high density the expression of SST-1 drops which indicates the stimulatory effect in terms of growth.
Along with growth parameters, chemical composition, specifically crude protein slightly increased with dietary supplementation of curcumin within different stocking densities in current study. Increase in the chemical composition against curcumin supplementation has also been previously observed in Nile tilapia [64]. Present study aligns with previous findings indicating that SST-I was upregulated in tilapia at high density [61] as well as in cichlids [62,65]. This increase in the growth parameters and crude protein is due to digestive enhancer properties of curcumin [66,67]. Present study showed that the utilization of dietary supplementation of curcumin demonstrated a notable improvement in the activity of lipase, protease and amylase in the intestine as in previous studies [67].
In addition to digestive activities, dietary supplementation of curcumin improved the immune response of fish with different stocking densities [56]. The present study observed an elevated level of RBC and hemoglobin in fish raised at high density after dietary supplemented with curcumin compared to C0 treatment. Red blood cells (RBC) and hemoglobin are pivotal for transporting oxygen to fish tissues and eliminating harmful substances through the gills into the environment [68]. This finding is consistent with similar study on rainbow trout [56,57] and common carp [69]. The levels of triglycerides were observed lower due to improved lipid metabolism in fish, possibly by enhancing lipolysis and less fat accumulation in fish raised at high density due to dietary supplementation of curcumin compared to the C0 treatment [70,71]. Similar results have been observed in response to curcumin supplementation in rainbow trout [57], and amberjack (Seriola dumerili) where triglyceride and total cholesterol remained stable or even slightly decreased [72]. White blood cell (WBC) count, acting as a frontline defense in fish increases in response to infections [73]. This study noted an increase in WBC with dietary supplementation of curcumin at high density aligning with previous research on rainbow trout where curcumin supplementation enhanced WBC content [56,57]. The highest level of ALT and AST levels in high-density conditions were observed with C0 treatment which indicates potential harm to liver cells, as these enzymes are usually contained within cells but are released into the bloodstream when cell integrity is compromised [74]. Whereas these enzymes decreased in the treatments supplemented with dietary supplementation of curcumin as previously observed in common carp [75] and tilapia [76] as well.
The present study showed an increase in cortisol levels in fish with high-density treatment fed with C0 diet which showed stress level, changes in catecholamine levels, fluctuations in corticosteroid hormone levels and changes in blood profiles of fish [77] as compared to lower density groups aligning with previous study [15,78–80]. Whereases, curcumin supplemented treatments showed reduced levels of cortisol. This may be due to inhibitory effects of cortisol induced by ACTH and suppressed transcription of genes such as steroidogenic acute regulatory protein (StAR) and Cytochrome P450 (CYP)11a1 mRNAs in response to both ACTH and cAMP stimulation [81,82]. Current study aligns with previous studied conducted on dietary supplementation of curcumin in different fish species such as snakehead fish [83], tilapia [84], common carp [85] and Pacific white shrimp [86].
ACTH stops the activation of StAR and Cytochrome P450 (CYP) genes, as well as the pro-opiomelanocortin (POMC) gene. ACTH regulates cortisol through negative feedback control [87]. Present study indicated that treatment without curcumin supplementation (C0) at high stocking density showed markedly increased POMC-α expression due to higher stress level which triggers the activation of the hypothalamic-pituitary-adrenal (HPA) axis [88,89]. This process involves the release of corticotropin-releasing factor (CRF), which prompts the synthesis of pituitary pro-opiomelanocortin (POMC). POMC is then processed into adrenocorticotropic hormone (ACTH), leading to the stimulation of cortisol release via the melanocortin 2 receptor (MC2R) [90–92]. Whereases, the treatments supplemented with dietary curcumin exhibited decreased level of POMC-α gene expression compared to treatment C0 treatment suggests a positive outcome by reducing stress among high density treatment [93]. Addition of curcumin in diet significantly influences the expression of appetite-regulating neuropeptides like POMC in tilapia [94]. The present study aligns with previous findings conducted on tilapia [61].
The present research highlights an enhanced response of oxidative enzymes (CAT, SOD, GPx) under high-density conditions in C0 treatment. While, a decrease in these enzymes is observed with dietary curcumin supplementation, particularly at the C1 dose, especially in high-density situations. Curcumin has the ability to attach to free radicals and deliver a hydrogen atom, preventing anti-inflammatory, antibacterial, anticancer, antidiabetic, antiviral, antifungal agent and aiding in wound healing and immunomodulation [95–97] which plays a crucial role in its antioxidant effects [42,98]. A few studies have suggested that incorporating curcumin into the diet has positive effects on the performance and oxidative stability of other animal species i.e. chick [99] and quail [100] exposed to stressors other than high density such as chromium [101]; Aeromonas salmonicida [56]; Aeromonas hydrophila [34].
The production of oxidative enzymes including CAT, SOD and GPx alleviates adverse effects of high density after dietary curcumin supplementation compared to the C0 treatment. Similar results have been noted in various fish species [31,34,57,58,66,75,102,103]. MDA is also a marker of oxidative stress linked to lipid peroxidation [104], which increases in oxidative stress. However, contrary to existing literature, the study found that curcumin supplementation in diet did not increase levels of MDA [34,55,69].
Both antioxidant system and immune response are positively corelated. Raising fish, particularly in high-density environments that create elevated stress levels which can benefit from strengthening the fish immune system through the inclusion of natural antioxidants in their diet. Interleukin-1 beta (IL-1β), a proinflammatory cytokines released from activated macrophages plays a crucial role in regulating innate immune functions and inflammatory responses [105,106]. The current study suggests that curcumin supplement did not show any difference in IL-1β level in all treatments nor in different doses of dietary curcumin supplementation. Contrary to present study, other research has demonstrated that dietary curcumin reduced the mRNA levels of IL-1β in vivo and in vitro studies [107,108], including in snakehead [109], grass carp [31], tilapia [110], common carp [75,111], and Nile tilapia [64] and some studies also reported an increase in IL-1β levels in tilapia [112].
5. Conclusion
The Present study concluded that dietary supplementation of curcumin in different stocking density treatment showed better growth, antioxidant response and significant regulation of stress related genes (POMC-α). Curcumin proved to be an effective dietary supplement in alleviating oxidative stress, as evidenced by improvements in both antioxidant enzyme activity and the regulation of the POMC-α gene. While all curcumin doses were effective in mitigating various stress parameters, the C3 dose (150 mg/kg) yielded the most favorable outcomes, particularly in terms of enhanced antioxidant enzyme levels and suppressed POMC-α gene expression against high density = 4.50 kg/m3. This study highlights curcumin potential to enhance tilapia health against high stocking density. Dietary supplementation of curcumin C3 = 150mg/kg would be used in intensive farming against high stocking density to gain high yield without compromising fish well-being.
Supporting information
S1 Table. Output of two-way ANOVA indicating the effect of stocking density, curcumin and interactive effect of stocking density and curcumin on different parameters measured in the study.
https://doi.org/10.1371/journal.pone.0311146.s001
(XLSX)
References
- 1. Yue GH, Lin HR, Li JL. Tilapia is the fish for next-generation aquaculture. Int J Mar Sci Ocean Technol. 2016;3(1):11–3.
- 2.
Agriculture Organization of the United Nations. Fisheries Department. The State of World Fisheries and Aquaculture, FAO.; 2020.
- 3. Andrade T, Afonso A, Pérez-Jiménez A, Oliva-Teles A, de las Heras V, Mancera JM, et al. Evaluation of different stocking densities in a Senegalese sole (Solea senegalensis) farm: implications for growth, humoral immune parameters and oxidative status. Aquac. 2015; 438:6–11.
- 4. Van Doan H, Lumsangkul C, Sringarm K, Hoseinifar SH, Dawood MA, El-Haroun E, et al. Impacts of Amla (Phyllanthus emblica) fruit extract on growth, skin mucosal and serum immunities, and disease resistance of Nile tilapia (Oreochromis niloticus) raised under biofloc system. Aquac Rep. 2022; 22:100953.
- 5. Van Doan H, Lumsangkul C, Hoseinifar SH, Harikrishnan R, Balasundaram C, Jaturasitha S. Effects of coffee silver skin on growth performance, immune response, and disease resistance of Nile tilapia culture under biofloc system. Aquacu. 2021; 543:736995.
- 6. Van Doan H, Hoseinifar SH, Harikrishnan R, Khamlor T, Punyatong M, Tapingkae W, et al. Impacts of pineapple peel powder on growth performance, innate immunity, disease resistance, and relative immune gene expression of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol. 2021; 114:311–9.
- 7. Van Doan H, Hoseinifar SH, Jaturasitha S, Dawood MA, Harikrishnan R. The effects of berberine powder supplementation on growth performance, skin mucus immune response, serum immunity, and disease resistance of Nile tilapia (Oreochromis niloticus) fingerlings. Aquacu. 2020; 520:734927.
- 8. Baldwin L. The effects of stocking density on fish welfare. 2011.
- 9. Diao W, Jia R, Hou Y, Dong Y, Li B, Zhu J. Effects of Stocking Density on the Growth Performance, Physiological Parameters, Antioxidant Status and Lipid Metabolism of Pelteobagrus fulvidraco in the Integrated Rice-Fish Farming System. Animals. 2023;13(11):1721.
- 10. Battisti EK, Rabaioli A, Uczay J, Sutili FJ, Lazzari R. Effect of stocking density on growth, hematological and biochemical parameters and antioxidant status of silver catfish (Rhamdia quelen) cultured in a biofloc system. Aquac. 2020; 524:735213.
- 11. Tolussi CE, Hilsdorf AW, Caneppele D, Moreira RG. The effects of stocking density in physiological parameters and growth of the endangered teleost species piabanha, Brycon insignis (Steindachner, 1877). Aquaculture. 2010;310(1–2):221–8.
- 12. Best J, Nijhout HF, Reed M. Serotonin synthesis, release and reuptake in terminals: a mathematical model. T Biol Med. 2010; 7:1–26. pmid:20723248
- 13. Barton BA. Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. Integr compar biol. 2002;42(3):517–25. pmid:21708747
- 14. Gang L, Hongxin T, Guozhi L, Chuan SD. Effect of density on Scortum barcoo (McCulloch & Waite) juvenile performance in circular tanks. Aquac Res. 2010;41(12):1898–904.
- 15. Odhiambo E, Angienda PO, Okoth P, Onyango D. Stocking density induced stress on plasma cortisol and whole blood glucose concentration in Nile tilapia fish (Oreochromis niloticus) of Lake Victoria, Kenya. Int J Zool. 2020; 2020:1–8.
- 16. Ahmad I, Hamid T, Fatima M, Chand HS, Jain SK, Athar M, et al. Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochim Biophys Acta (BBA)-Gen Subj. 2000;1523(1):37–48.
- 17. Evans MD, Cooke MS. Factors contributing to the outcome of oxidative damage to nucleic acids. Bioessays. 2004;26(5):533–42. pmid:15112233
- 18. Rius-Pérez S, Torres-Cuevas I, Millán I, Ortega ÁL, Pérez S. PGC-1α, inflammation, and oxidative stress: an integrative view in metabolism. Oxidative med cell longev. 2020;2020.
- 19. Saera-Vila A, Calduch-Giner JA, Prunet P, Pérez-Sánchez J. Dynamics of liver GH/IGF axis and selected stress markers in juvenile gilthead sea bream (Sparus aurata) exposed to acute confinement: differential stress response of growth hormone receptors. Comp Biochem Physiol Part A: Mol Integr Physiol. 2009;154(2):197–203.
- 20. Liu ZS, Zhang L, Chen WL, He CF, Qian XY, Liu WB, et al. Insights into the interaction between stocking density and feeding rate in fish Megalobrama ambylcephala based on growth performance, innate immunity, antioxidant activity, and the GH-IGF1 axis. Aquac. 2024; 580:740355.
- 21. Yuan X, Tao L, Hu X, Lin R, Yang J, Feng M, et al. Expression profile analysis of muscle growth regulation genes and effects of water flow stress on their expression levels in zebrafish. 2023.
- 22. Jomova K, Raptova R, Alomar SY, Alwasel SH, Nepovimova E, Kuca K, et al. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: Chronic diseases and aging. Arch toxicol. 2023;97(10):2499–574. pmid:37597078
- 23. Chen B, Lu Y, Chen Y, Cheng J. The role of Nrf2 in oxidative stress-induced endothelial injuries. J Endocrinol. 2015;225(3):R83–99. pmid:25918130
- 24. Shi C, Lu Y, Zhai G, Huang J, Shang G, Lou Q, et al. Hyperandrogenism in POMCa-deficient zebrafish enhances somatic growth without increasing adiposity. J Mol Cell Biol. 2020;12(4):291–304. pmid:31237951
- 25. Very NM, Sheridan MA. The role of somatostatins in the regulation of growth in fish. Fish Physiol Biochem. 2002; 27:217–26.
- 26. Feng P, Tian C, Lin X, Jiang D, Shi H, Chen H, et al. Identification, Expression, and Functions of the Somatostatin Gene Family in Spotted Scat (Scatophagus argus). Genes. 2020;11(2):194.
- 27. Zou J, Secombes CJ. The function of fish cytokines. Biol, 2016;5(2), 23. pmid:27231948
- 28. Lee KJ, Dabrowski K, Sandoval M, Miller MJ. Activity-guided fractionation of phytochemicals of maca meal, their antioxidant activities and effects on growth, feed utilization, and survival in rainbow trout (Oncorhynchus mykiss) juveniles. Aquac. 2005;244(1–4):293–301.
- 29. Alasmari WA, El-Shetry ES, Ibrahim D, ElSawy NA, Eldoumani H, Metwally AS, et al. Mesenchymal stem-cells’ exosomes are renoprotective in postmenopausal chronic kidney injury via reducing inflammation and degeneration. Free Radic Biol Med. 2022; 182:150–9. pmid:35218913
- 30. Küçükbay FZ, Yazlak H, Karaca I, Sahin N, Tuzcu M, Cakmak MN, et al. The effects of dietary organic or inorganic selenium in rainbow trout (Oncorhynchus mykiss) under crowding conditions. Aquac Nutr. 2009;15(6):569–76.
- 31. Ming J, Ye J, Zhang Y, Xu Q, Yang X, Shao X, et al. Optimal dietary curcumin improved growth performance, and modulated innate immunity, antioxidant capacity and related genes expression of NF-κB and Nrf2 signaling pathways in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila. Fish shellfish immunol. 2020; 97:540–53.
- 32. Mişe Yonar S, İspir Ü, Ural MŞ. Effects of curcumin on haematological values, immunity, antioxidant status and resistance of rainbow trout (Oncorhynchus mykiss) against Aeromonas salmonicida subsp. achromogenes. 2019.
- 33. Amer SA, El-Araby DA, Tartor H, Farahat M, Goda NI, Farag MF, et al. Long-term feeding with curcumin affects the growth, antioxidant capacity, immune status, tissue histoarchitecture, immune expression of proinflammatory cytokines, and apoptosis indicators in Nile tilapia, Oreochromis niloticus. Antioxid. 2022;11(5):937.
- 34. Mahmoud HK, Al-Sagheer AA, Reda FM, Mahgoub SA, Ayyat MS. Dietary curcumin supplement influence on growth, immunity, antioxidant status, and resistance to Aeromonas hydrophila in Oreochromis niloticus. Aquac. 2017;475:16–23.
- 35. Wang L, Yu A, Yu C, Ibrahim UB, Chen J, Wang Y. Curcumin Supplementation Enhances the Feeding and Growth of Largemouth Bass (Micropterus salmoides) Fed the Diet Containing 80 g/kg Fish Meal. Aquac Res. 2023;2023.
- 36. Alhawas B, Abd El-Hamid MI, Hassan Z, Ibrahim GA, Neamat-Allah AN, El-Ghareeb WR, et al. Curcumin loaded liposome formulation: Enhanced efficacy on performance, flesh quality, immune response with defense against Streptococcus agalactiae in Nile tilapia (Orechromis niloticus). Fish Shellfish Immunol. 2023;138:108776.
- 37. Ibrahim D, Arisha AH, Khater SI, Gad WM, Hassan Z, Abou-Khadra SH, et al. Impact of omega-3 fatty acids nano-formulation on growth, antioxidant potential, fillet quality, immunity, autophagy-related genes and Aeromonas hydrophila resistance in Nile tilapia (Oreochromis niloticus). Antioxid. 2022;11(8):1523.
- 38. Harikrishnan R, Rani MN, Balasundaram C. Hematological and biochemical parameters in common carp, Cyprinus carpio, following herbal treatment for Aeromonas hydrophila infection. Aquacu. 2003;221(1–4):41–50.
- 39. Harikrishnan R, Balasundaram C. In vitro and in vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish. J Aquatic Ani Health. 2008;20(3):165–76.
- 40. Harikrishnan R, Balasundaram C, Dharaneedharan S, Moon YG, Kim MC, Kim JS, et al. Effect of plant active compounds on immune response and disease resistance in Cirrhina mrigala infected with fungal fish pathogen, Aphanomyces invadans. Aquac Res. 2009;40(10):1170–81.
- 41. Jayaprakasha GK, Rao LJ, Sakariah KK. Antioxidant activities of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food chem 2006;98(4):720–4.
- 42. Wei QY, Chen WF, Zhou B, Yang L, Liu ZL. Inhibition of lipid peroxidation and protein oxidation in rat liver mitochondria by curcumin and its analogues. Biochim Biophys Acta (BBA)-Gen Subj. 2006;1760(1):70–7. pmid:16236451
- 43. Hatcher H, Planalp R, Cho J, Torti FM, Torti SV. Curcumin: from ancient medicine to current clinical trials. Cell mol life sci. 2008;65:1631–52. pmid:18324353
- 44. Shehzad A, Ha T, Subhan F, Lee YS. New mechanisms and the anti-inflammatory role of curcumin in obesity and obesity-related metabolic diseases. Eur j nutr. 2011; 50:151–61. pmid:21442412
- 45. Shen L, Ji HF. The pharmacology of curcumin: is it the degradation products? Trends mol med. 2012;18(3):138–44. pmid:22386732
- 46. Lin JK. Molecular targets of curcumin. The molecular targets and therapeutic uses of curcumin in health and disease. 2007:227–43. pmid:17569214
- 47. El‐Saidy DM, Gaber MM. Effect of dietary protein levels and feeding rates on growth performance, production traits and body composition of Nile tilapia, Oreochromis niloticus (L.) cultured in concrete tanks. Aquac res. 2005;36(2):163–71.
- 48. Abdel-Aziz MF, Hassan HU, Yones AM, Abdel-Tawwab YA, Metwalli AA. Assessing the effect of different feeding frequencies combined with stocking density, initial weight, and dietary protein ratio on the growth performance of tilapia, catfish and carp. Sci African. 2021;12: e00806.
- 49. Fatima S, Komal W, Manzoor F, Latif AA, Liaqat R, Ameen S, Janjua RS. Analysis of the growth performance, stress, profile of fatty acids and amino acids and cortisol in Tilapia (Oreochromis niloticus), cultured at high stocking density using in-pond raceway system. Saudi J Biol Sci. 2021;28(12):7422–31.
- 50.
AOAC. Official method of Analysis. 18th Edition, Association of Officiating Analytical Chemists, Washington DC, Method 935.14 and 992.24. 2005.
- 51. Folch J, Lees M, Sloane-Stanley GM. A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J Biol Chem. 1957; 226:497–509.
- 52.
Walter HE. Proteases and their inhibitors. 2. 15. 2 Method with hemoglobin, casein, and azocoll as substrate. Methods of enzymatic analysis, Netherland, Academic Press, 1984; 270–277.
- 53. Moradyan H, Karimi H, Gandomkar HA, Sahraeian MR, Ertefaat S, Sahafi HH. The effect of stocking density on growth parameters and survival rate of rainbow trout alevins (Oncorhynchus mykiss). W J F M S. 2012;4(5):480–5.
- 54. Ellis T, North B, Scott AP, Bromage NR, Porter M, Gadd D. The relationships between stocking density and welfare in farmed rainbow trout. J fish biol. 2002;61(3):493–531.
- 55. Akdemir F, Orhan C, Tuzcu M, Sahin N, Juturu V, Sahin K. The efficacy of dietary curcumin on growth performance, lipid peroxidation and hepatic transcription factors in rainbow trout Oncorhynchus mykiss (Walbaum) reared under different stocking densities. Aquac Res. 2017;48(8):4012–21.
- 56. Yonar ME, Yonar SM, İspir Ü, Ural MŞ. Effects of curcumin on haematological values, immunity, antioxidant status and resistance of rainbow trout (Oncorhynchus mykiss) against Aeromonas salmonicida subsp. achromogenes. Fish shellfish immunol. 2019; 89:83–90.
- 57. Aqmasjed SB, Sajjadi MM, Falahatkar B, Safari R. Effects of dietary ginger (Zingiber officinale) extract and curcumin on growth, hematology, immunity, and antioxidant status in rainbow trout (Oncorhynchus mykiss). Aquac Rep. 2023; 32:101714.
- 58. Giri SS, Sukumaran V, Park SC. Effects of bioactive substance from turmeric on growth, skin mucosal immunity and antioxidant factors in common carp, Cyprinus carpio. Fish shellfish immunol. 2019; 92:612–20.
- 59. Ashry AM, Hassan AM, Habiba MM, El-Zayat A, El-Sharnouby ME, Sewilam H, et al. The impact of dietary curcumin on the growth performance, intestinal antibacterial capacity, and haemato-biochemical parameters of gilthead seabream (Sparus aurata). Animals. 2021;11(6):1779.
- 60. Xavier MJ, Engrola S, Conceição LE, Manchado M, Carballo C, Gonçalves R, et al. Dietary antioxidant supplementation promotes growth in Senegalese sole postlarvae. Front physiol. 2020; 11:580600. pmid:33281617
- 61. Rodriguez‐Barreto D, Rey O, Uren‐Webster TM, Castaldo G, Consuegra S, Garcia de Leaniz C. Transcriptomic response to aquaculture intensification in Nile tilapia. Evol App. 2019;12(9):1757–71. pmid:31548855
- 62. Hofmann HA, Fernald RD. Social status controls somatostatin neuron size and growth. J Neurosci. 2000;20(12):4740–4. pmid:10844043
- 63. Trainor BC, Hofmann HA. Somatostatin and somatostatin receptor gene expression in dominant and subordinate males of an African cichlid fish. Behav brain res. 2007;179(2):314–20. pmid:17374406
- 64. Abd El-Hakim YM, El-Houseiny W, Abd Elhakeem EM, Ebraheim LL, Moustafa AA, Mohamed AA. Melamine and curcumin enriched diets modulate the haemato-immune response, growth performance, oxidative stress, disease resistance, and cytokine production in Oreochromis niloticus. Aquat Toxicol. 2020; 220:105406.
- 65. Trainor BC, Hofmann HA. Somatostatin regulates aggressive behavior in an African cichlid fish. Endocrinol. 2006;147(11):5119–25.
- 66. Jiang J, Wu XY, Zhou XQ, Feng L, Liu Y, Jiang WD, et al. Effects of dietary curcumin supplementation on growth performance, intestinal digestive enzyme activities and antioxidant capacity of crucian carp Carassius auratus. Aquac. 2016;463:174–80.
- 67. Ling J, Feng L, Liu Y, Jiang J, Jiang WD, Hu K, et al. Effect of dietary iron levels on growth, body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian). Aquac nutr. 2010;16(6):616–24.
- 68. Mohammadi G, Rashidian G, Hoseinifar SH, Naserabad SS, Van Doan H. Ginger (Zingiber officinale) extract affects growth performance, body composition, hematology, serum and mucosal immune parameters in common carp (Cyprinus carpio). Fish Shellfish Immunol. 2020; 99:267–73.
- 69. Yonar ME. Chlorpyrifos-induced biochemical changes in Cyprinus carpio: Ameliorative effect of curcumin. Ecotoxicol envir safety. 2018 30; 151:49–54.
- 70. Adineh H, Naderi M, Yousefi M, Khademi Hamidi M, Ahmadifar E, Hoseini SM. Dietary licorice (Glycyrrhiza glabra) improves growth, lipid metabolism, antioxidant and immune responses, and resistance to crowding stress in common carp, Cyprinus carpio. Aquac Nutr. 2021;27(2):417–26.
- 71. Ruiz A, Sanahuja I, Andree KB, Furones D, Holhorea PG, Calduch-Giner JA, et al. Supplementation of gilthead seabream (Sparus aurata) diets with spices as a functional strategy to control excess adiposity through lipid, cholesterol and bile acid metabolism, and to induce an immunomodulatory intestinal regulation. Aquac. 2024; 581:740378.
- 72. Hong J, Fu Z, Hu J, Zhou S, Yu G, Ma Z. Dietary Curcumin Supplementation Enhanced Ammonia Nitrogen Stress Tolerance in Greater Amberjack (Seriola dumerili): Growth, Serum Biochemistry and Expression of Stress-Related Genes. J Mar Sci Eng. 2022;10(11):1796.
- 73. Vahedi AH, Hasanpour M, Akrami R, Chitsaz H. Effect of dietary supplementation with ginger (Zingiber officinale) extract on growth, biochemical and hemato-immunological parameters in juvenile beluga (Huso huso). Sustainable Aquac Health Management J. 2017;3(1):26–46.
- 74. Liu G, Ye Z, Liu D, Zhao J, Sivaramasamy E, Deng Y, Zhu S. Influence of stocking density on growth, digestive enzyme activities, immune responses, antioxidant of Oreochromis niloticus fingerlings in biofloc systems. Fish shellfish immunol. 2018; 81:416–22.
- 75. Cao L, Ding W, Du J, Jia R, Liu Y, Zhao C, et al. Effects of curcumin on antioxidative activities and cytokine production in Jian carp (Cyprinus carpio var. Jian) with CCl4-induced liver damage. Fish Shellfish Immunol. 2015;43(1):150–7.
- 76. Eissa ES, Ezzo OH, Khalil HS, Tawfik WA, El‐Badawi AA, Abd Elghany NA, et al. The effect of dietary nanocurcumin on the growth performance, body composition, haemato‐biochemical parameters and histopathological scores of the Nile tilapia (Oreochromis niloticus) challenged with Aspergillus flavus. Aquac Res. 2022;53(17):6098–111.
- 77. Saurabh S, Sahoo PK. Lysozyme: an important defence molecule of fish innate immune system. Aquac res. 2008;39(3):223–39.
- 78. Varela JL, Ruiz-Jarabo I, Vargas-Chacoff L, Arijo S, León-Rubio JM, García-Millán I, et al. Dietary administration of probiotic Pdp11 promotes growth and improves stress tolerance to high stocking density in gilthead seabream Sparus auratus. Aquac. 2010309(1–4):265–71.
- 79. Li D, Liu Z, Xie C. Effect of stocking density on growth and serum concentrations of thyroid hormones and cortisol in Amur sturgeon, Acipenser schrenckii. Fish physiol biochem, 2012;38, 511–520.
- 80. Ruane NM, Komen H. Measuring cortisol in the water as an indicator of stress caused by increased loading density in common carp (Cyprinus carpio). Aquac. 2003;218(1–4):685–93.
- 81. Enyeart JA, Liu H, Enyeart JJ. Curcumin inhibits bTREK-1 K+ channels and stimulates cortisol secretion from adrenocortical cells. Biochem biophys res comm. 2008;370(4):623–8. pmid:18406348
- 82. Enyeart JA, Liu H, Enyeart JJ. Curcumin inhibits ACTH-and angiotensin II-stimulated cortisol secretion and Cav3. 2 currents. J nat prod. 2009;72(8):1533–7.
- 83. Kong Y, Li M, Guo G, Yu L, Sun L, Yin Z, et al. Effects of dietary curcumin inhibit deltamethrin-induced oxidative stress, inflammation and cell apoptosis in Channa argus via Nrf2 and NF-κB signaling pathways. Aquac. 2021; 540:736744.
- 84. Abdel-Ghany HM, El-Sisy DM, Salem ME. A comparative study of effects of curcumin and its nanoparticles on the growth, immunity and heat stress resistance of Nile tilapia (Oreochromis niloticus). Sci Rep. 2023;13(1):2523.
- 85. Rohmah MK, Salahdin OD, Gupta R, Muzammil K, Qasim MT, Al-Qaim ZH, et al. Modulatory role of dietary curcumin and resveratrol on growth performance, serum immunity responses, mucus enzymes activity, antioxidant capacity and serum and mucus biochemicals in the common carp, Cyprinus carpio exposed to abamectin. Fish Shellfish Immunol. 2022; 129:221–30.
- 86. Moghadam H, Sourinejad I, Johari SA. Dietary turmeric, curcumin and nanoencapsulated curcumin can differently fight against salinity stress in Pacific white shrimp Penaeus vannamei Boone, 1931. Aquac Res. 2022;53(8):3127–39.
- 87. Wunderink YS, de Vrieze E, Metz JR, Halm S, Martínez-Rodríguez G, Flik G, et al. Subfunctionalization of POMC paralogues in Senegalese sole (Solea senegalensis). Gen Comp Endocrinol. 2012;175(3):407–15.
- 88. Mateus A, A Costa R, Cardoso JC, Andree KB, Estevez A, Gisbert E, et al. Thermal imprinting modifies adult stress and innate immune responsiveness in the teleost sea bream. J Endocrinol. 2017;233(3):381–94. pmid:28420709
- 89. Pham LP, Jordal AE, Nguyen MV, Rønnestad I. Food intake, growth, and expression of neuropeptides regulating appetite in clown anemonefish (Amphiprion ocellaris) exposed to predicted climate changes. Gen Comp Endocrinol. 2021;304:113719.
- 90. Kalananthan T, Lai F, Gomes AS, Murashita K, Handeland S, Rønnestad I. The melanocortin system in Atlantic salmon (Salmo salar L.) and its role in appetite control. Front neuroanat. 2020; 14:48.
- 91. Chang HS, Won ES, Lee HY, Ham BJ, Kim YG, Lee MS. The association of proopiomelanocortin polymorphisms with the risk of major depressive disorder and the response to antidepressants via interactions with stressful life events. J Neural Transm. 2015; 122:59–68. pmid:25448875
- 92. de Freitas Souza C, Descovi S, Baldissera MD, Bertolin K, Bianchini AE, Mourão RH, et al. Involvement of HPI-axis in anesthesia with Lippia alba essential oil citral and linalool chemotypes: gene expression in the secondary responses in silver catfish. Fish physiol biochem. 2019; 45:155–66. pmid:30120603
- 93. Mv S, Lekha D. Curcumin in diet modulates fatty acid levels and mRNA expression of appetite‐regulating neuropeptides in brain and enhances growth via the GH‐GHR‐IGF axis in tilapia, Oreochromis mossambicus. J Fish Biol. 2023;103(1):22–31.
- 94. Delgado MJ, Cerdá-Reverter JM, Soengas JL. Hypothalamic integration of metabolic, endocrine, and circadian signals in fish: involvement in the control of food intake. Front neurosci. 2017; 11:354. pmid:28694769
- 95. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer res. 2003;23(1/A):363–98. pmid:12680238
- 96. Neerati P, Devde R, Gangi AK. Evaluation of the effect of curcumin capsules on glyburide therapy in patients with type‐2 diabetes mellitus. Phytother Res. 2014;28(12):1796–800. pmid:25044423
- 97. Perrone D, Ardito F, Giannatempo G, Dioguardi M, Troiano G, Lo Russo L, et al. Biological and therapeutic activities, and anticancer properties of curcumin. Exp ther med. 2015;10(5):1615–23. pmid:26640527
- 98. Trujillo J, Chirino YI, Molina-Jijón E, Andérica-Romero AC, Tapia E, Pedraza-Chaverrí J. Renoprotective effect of the antioxidant curcumin: Recent findings. Redox biol. 2013; 1(1):448–56. pmid:24191240
- 99. Durrani FR, Ismail M, Sultan A, Suhail SM, Chand N, Durrani Z. Effect of different levels of feed added turmeric (Curcuma longa) on the performance of broiler chicks. J Agric Biol Sci. 2006;1(2):9–11.
- 100. Sahin K, Orhan C, Tuzcu Z, Tuzcu M, Sahin N. Curcumin ameloriates heat stress via inhibition of oxidative stress and modulation of Nrf2/HO-1 pathway in quail. Food Chem Toxicol. 2012;50(11):4035–41. pmid:22939939
- 101. Mohamed AA, El-Houseiny W, Abd Elhakeem EM, Ebraheim LL, Ahmed AI, Abd El-Hakim YM. Effect of hexavalent chromium exposure on the liver and kidney tissues related to the expression of CYP450 and GST genes of Oreochromis niloticus fish: Role of curcumin supplemented diet. Ecotoxicol envir safety. 2020; 188:109890.
- 102. Hoseini SM, Gupta SK, Yousefi M, Kulikov EV, Drukovsky SG, Petrov AK, et al. Mitigation of transportation stress in common carp, Cyprinus carpio, by dietary administration of turmeric. Aquac. 2022; 546:737380.
- 103. Manju M, Akbarsha MA, Oommen OV. In vivo protective effect of dietary curcumin in fish Anabas testudineus (Bloch). Fish Physiol Biochem. 2012; 38:309–18.
- 104. Gu Y, Gao M, Zhang W, Yan L, Shao F, Zhou J. Exposure to phthalates DEHP and DINP May lead to oxidative damage and lipidomic disruptions in mouse kidney. Chemosphere. 2021; 271:129740. pmid:33736212
- 105. Yarahmadi P, Miandare HK, Hoseinifar SH, Gheysvandi N, Akbarzadeh A. The effects of stocking density on hemato-immunological and serum biochemical parameters of rainbow trout (Oncorhynchus mykiss). Aquac Int. 2015; 23:55–63.
- 106. Dinarello CA. Overview of the IL‐1 family in innate inflammation and acquired immunity. Immunol rev. 2018;281(1):8–27. pmid:29247995
- 107. Kurzrock R, Li L. Liposome-encapsulated curcumin: in vitro and in vivo effects on proliferation, apoptosis, signaling, and angiogenesis. J Clin Oncol. 2005;23(16_suppl):4091. pmid:16092118
- 108. Kalinski T, Sel S, Hütten H, Röpke M, Roessner A, Nass N. Curcumin blocks interleukin-1 signaling in chondrosarcoma cells. PLoS One. 2014;9(6): e99296. pmid:24901233
- 109. Li M, Kong Y, Wu X, Guo G, Sun L, Lai Y, et al. Effects of dietary curcumin on growth performance, lipopolysaccharide-induced immune responses, oxidative stress and cell apoptosis in snakehead fish (Channa argus). Aquac Rep. 2022; 22:100981.
- 110. Abdelkhalek N, El‐Adl M, El‐Ashram A, Othman M, Gadallah H, El‐Diasty M, et al. Immunological and antioxidant role of curcumin in ameliorating fipronil toxicity in Nile tilapia (Oreochromis niloticus). Aquac Res. 2021;52(6):2791–801.
- 111. Fazelan Z, Hoseini SM, Yousefi M, Khalili M, Hoseinifar SH, Van Doan H. Effects of dietary eucalyptol administration on antioxidant and inflammatory genes in common carp (Cyprinus carpio) exposed to ambient copper. Aquac, 2020; 520:734988.
- 112. Mahfouz ME. Ameliorative effect of curcumin on aflatoxin B1-induced changes in liver gene expression of Oreochromis niloticus. Mol Biol. 2015;49: 275–286.