Aquaculture is rapidly expanding to meet the growing global demand for high-quality animal protein (El-dweny et al., 2025). The grass carp (Ctenopharyngodon idella) holds a position of paramount significance among the cultivated freshwater fish, as it contributes accounting for over 10% of global freshwater aquaculture production (FAO, 2018). However, this species is susceptible to bacterial diseases, which poses a challenge to the aquaculture industry (Qin et al., 2019). The bacterial enteritis caused by A. hydrophila is considered to be a highly prevalent ailment in the cultivation of grass carp (Song et al., 2014). In recent decades, there has been extensive utilization of antibiotics in the aquaculture industry to mitigate and control bacterial disease outbreaks. However, the excessive administration of antibiotics has led to a surge in bacterial resistance to these drugs, thereby causing detrimental effects on both food safety and the environment (Done et al., 2015). The possible alternative to antibiotic additives involves the utilization of additives as immune stimulators that enhance the ability of aquatic animals to resist diseases by improving innate defense mechanisms (Fuchs et al., 2015). Commercially available additives, such as probiotics, Bacillus subtilis and Lactobacillus spp., are live microorganisms that colonize the gut, inhibit pathogenic bacteria through competitive exclusion, and enhance immune responses and prebiotics, mannan oligosaccharides (MOS) and inulin, are non-digestible dietary fibers that selectively stimulate the growth and activity of beneficial gut microbes, thereby supporting mucosal immunity and gut integrity as well as, organic acidifiers, including malic, citric, and formic acids, lower the pH of the gastrointestinal tract, creating unfavorable conditions for harmful bacteria while improving nutrient digestibility and intestinal function (Lückstädt, 2008; Hoseinifar et al., 2020; Yilmaz et al., 2022).

In recent years, there has been a considerable amount of research dedicated to examining the utilization of medicinal herbs, owing to their various benefits including sustainability and reduced adverse reactions, valid alternatives to chemicals and antibiotics cost-benefit implications, health-promoting effects (Ahmadifar et al., 2021).

Ginger (Zingiber officinale) is a prime example, which encompasses the qualities of an edible plant, spice, and medicinal herb (Mohammadi et al., 2020). Bioactive components present in the rhizomes of ginger encompass terpenes, oleoresin, zingiberol, zingiberone, zingiberene, gingerol, shogaol, zingerone, and paradol (Jesudoss et al., 2017). In aquaculture, ginger extract and powder improve growth performance and health status of juvenile beluga, Huso huso, (Vahedi et al., 2017), juvenile rockfish, Sebastes chlegeli, (Kim et al., 2018), zebrafish, Danio rerio, (Ahmadifar et al., 2019), and common carp, Cyprinus carpio, (Mohammadi et al., 2020).

Malic acid (MA), a dicarboxylic acid consisting of four carbon atoms, is naturally present in various organic acids found in fruits (Sniffen et al., 2006) that playing a role in pH regulation (Hassaan et al., 2018). Additionally, malic acid serves as an effective means of microbial control, as it effectively inhibits the growth of pathogenic bacteria and fungi (Ricke, 2003). In the study by Hassaan et al. (2020) dietary supplementation with 5 g/kg MA for 84 days improved growth performance and immune response in Nile tilapia (Oreochromis niloticus). Similarly, the inclusion of 0.25% malic acid in the diet of rainbow trout (Oncorhynchus mykiss) for 8 weeks had a positive effect on their growth and health (Yousefi et al., 2023). Furthermore, in Carassius auratus gibelio, a combination of 0.2% malic acid and 0.2% citric acid for 8 weeks enhanced overall fish health (Zhang et al., 2020).

To the best of our understanding, there have been no previous investigations focusing on the combined impact of malic acid and ginger powder on fish growth performance and health. The malic acid is utilized as a flavor enhancer in food products (Hassaan et al., 2018) so using malic acid with ginger powder have a masking effect on the unpleasant taste of ginger and may preserve the bioactive compounds and antioxidant properties of ginger. Grass carp is economic importance in freshwater aquaculture across many regions, particularly in Asia. Despite its widespread use, grass carp are known to be susceptible to various stress-related and immune challenges under intensive farming conditions. However, nutritional strategies aimed at enhancing immunity and growth performance in this species are still limited compared to model fish like zebrafish or commonly studied species such as common carp. Therefore, the present study was designed to evaluate the individual and combined effects of dietary ginger powder and malic acid on growth performance, hematological parameters, innate immune responses, antioxidant enzyme activities, and resistance against Aeromonas hydrophila in grass carp. In addition, the study aimed to investigate the expression levels of key immune- and antioxidant-related genes in liver and head kidney tissues. This integrated approach seeks to provide novel insights into the potential of these natural dietary additives to enhance health status, immune competence, and disease resistance in aquaculture species.

A basal diet (Table 1) was prepared and enhanced with malic acid (MA) (DL-Malic acid; Merck Millipore, Darmstadt, Germany) and ginger powder (GP), leading to the formation of four distinct diets. In summary, the required quantities of ingredients were accurately measured and meticulously integrated and blended in the mixer (P310, Pars Electric, Tehran, Iran). Subsequently, MA and GP were introduced to a 1000 g mixture of dry ingredients in order to attain the desired concentrations of 0.25% MA, 0.2% GP, and a combination of both components (0.25% malic acid, 0.2% ginger powder) (referred to as T0, T1, T2, and T3). The determination of the quantities of malic acid and ginger powder used in the experiment was carried out by referring to the previous studies conducted by Mohammadi et al (2020) and Zhang et al. (2020). Subsequently, the dough that was prepared underwent the process of being transformed into pellets using a meat grinder (MG1400R, Pars Khazar, Tehran, Iran), followed by drying at ambient temperature for a duration of 24 hours. The resulting dried dough was then ground into particle sizes that were deemed desirable and subsequently stored at a temperature of -20 ˚C until it was ready to be used at a later time.

Healthy grass carp were obtained from a private breeding center (Golestan, Iran) and transported to the aquaculture laboratory located in technical and professional center of Gorgan (Golestan, Iran). All fish were accommodated in tanks (1000 L) and underwent a process of acclimation to the experimental conditions for a duration of two weeks, during which they were provided with a basic diet, 4% of their body weight twice daily. In this study, 240 individuals (4.4 ± 0.01 g) were randomly divided in twelve 150-L tanks with a density of 20 fish in each tank (four treatments with three replications). The fish were then given the experimental diets until they reached a state of apparent satiation, three times a day (at 7:30, 12:30, and 17:30), for 8 weeks (Qin et al., 2019). The tanks underwent a daily siphoning procedure with the objective of eliminating fecal matter, whereby 25% of the water volume was siphoned and replaced with freshwater every day before the first feeding time in the morning. The measurement of water physicochemical factors (dissolved oxygen, temperature, pH levels and water flow rate) in the experimental period were maintained at values of 7.13 ± 0.21 mg/l, 27.6 ± 1.2°C, 6.99 ± 0.05 mg l^− 1, and 0.8 L s ^− 1 respectively. A mercury thermometer (Zomorodazma Company, Iran) and Cyberscan Eutech instruments (DO 110, Singapore) were used to measure temperature and dissolved oxygen.

After 8 weeks, feeding was stopped for 24 hours and all fish were subjected to anaesthetization through the administration of 50 mg/L of MS-222. Subsequently, the fish were individually weighed and the growth factors were determined by following formula (Zhang et al., 2020; Yousefi et al., 2019; Chekani et al., 2021):

Weight gain (WG) = final weight (g) – initial weight (g)

Specific growth rate (SGR) = Ln final weight-Ln initial weight/ days ×100

Feed conversion ratio (FCR) = dry weight of feed given (g) / WG (g)

Survival rate (%) = (final number of fish/initial number of fish) ×100

After 8 weeks, a total of 6 fish per tank were chosen in a random manner and subjected to the process of anaesthetization by the administration of 50 mg/L of MS-222 (Zhang et al., 2019). Blood samples were procured from the caudal vein through the method of venipuncture. Subsequently, these samples were divided into tubes and centrifuged at 1600 g for 10 minutes. The serum was then stored at a temperature of -70°C for subsequent analysis. Following the collection of blood samples, the fish were dissected and the intestine from two fish per tank (six fish per treatment) was meticulously detached. This particular tissue was promptly frozen using liquid nitrogen and ultimately preserved at a temperature of -80°C for the purpose of conducting a gene expression assay.

Total levels of immunoglobulin (total Ig) were evaluated using the polyethylene glycol precipitation method for Ig and subsequent deduction of initial and final total protein, as outlined by Siwicki and Anderson (Siwicki, 1993). Lysozyme (LYZ) activity was quantified following the method described by Saurabh and Sahoo (Saurabh and Sahoo, 2008) using turbidimetric analysis. Immunoglobulin (IgM, Eastbiopharm) Glutathione peroxidase (GPx, ZelBio), superoxide dismutase (SOD, ZelBio), catalase (CAT, ZelBio), total protein (Bionik), urea (Pars Azmon), and triglyceride (Zistchimi) were analyzed by means of diagnostic reagent kits (Armobin et al., 2023).

At the end of the experiment, six fish (per treatment) were killed with a high dose of anesthetic and placed on ice. The fish's intestines form two fish per tank were removed and stored in a nitrogen tank and stored in a -80°C. First, RNA extraction was performed with the extraction EZ-10 spin column total RNA mini-prep kit (Takara, Japan). Then, the quality and quantity of extracted RNA was carried out by means of the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). After confirming the quality of RNA, cDNA synthesis was performed using Revert Aid first strand cDNA synthesis kit (Takara, Japan). The quantification of cDNA was evaluated using a spectrophotometer manufactured by Thermo Fisher Scientific (Wilmington, USA). Quantitative PCR (qPCR) was performed using RealQ Plus Master Mix Green (AMPLIQONIII) in accordance with the manufacturer’s protocol. Each reaction contained 10 µL of SYBR Green master mix, 1 µL of diluted cDNA, 0.5 µL of each primer, and deionized water (Millipore) to reach a final volume of 20 µL. The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 40 seconds, and extension at 72°C for 35 seconds. Gene expression levels were quantified using the 2^−ΔΔCT method described by Livak and Schmittgen (2001). All experiments were conducted in three independent biological replicates.

2–7. Challenging test with Aeromonas hydrophila

A. hydrophila (ATCC 7966) was procured from the Iranian Biological Resource Center (Tehran, Iran). The bacterial specimens were cultivated at a temperature of 25°C for the duration of one night and subsequently subjected to centrifugation (at 3000 g for 15 min) (Ahmadifar et al., 2022). The upper layer was removed and the cell pellet was cleaned with phosphate saline buffer (PBS, 0.1 M, pH7.2) three times by centrifugation at 3000xg for 15 min. Finally, the cell pellet was suspended in PBS. The bacteria concentration was measured with a spectrophotometer at the wavelength of 540 nm and adjusted to 10⁸ CFU. After the eight-week feeding trial, 10 fish per tank (30 fish per treatment) were taken and injected with 0.1 mL of bacterium suspension at 10⁸ CFU per fish (Yousefi et al., 2021). The dead fish were taken out and recorded every day and the mortality rate (%) was calculated for 14 days.

Statics

All the data were subjected to analysis using version 22 of the SPSS software with a confidence level of 95%. Initially, the normality of the data was assessed using the Kolmogorov-Smirnov test and the homogeneity of variance was assessed using Levene's test. One-way analysis of variance (ANOVA) was applied, and the assessment of distinctions between treatments was conducted using Duncan's multiple range test. In order to evaluate the survival rates in the challenge test, the Kaplan-Meier survival analyses were utilized along with a log-rank test at a significance level of 0.05.

A noticeable difference was observed in FW between various treatments, with the highest FW being noted in T3 which was not significantly different with T2 (Table 3, p < 0.05). WG and SGR were not affected by supplemented diets (Table 3, p > 0.05). FCR experienced a notable decrease in fish that were provided with supplemented diets specially in T2 and T3 (Table 3, p < 0.05). There was no mortality during feeding trial.

Grass carp fed the combined diet containing MA and GP (T3) exhibited a significantly higher total protein level compared to the control group (Fig. 1A; p < 0.05). Supplementation with MA and GP, either individually or in combination, led to a significant reduction in serum triglyceride levels (Fig. 1B; p < 0.05). However, no significant differences were observed in serum urea concentrations among the experimental groups (Fig. 1C; p > 0.05). Dietary inclusion of MA and GP also significantly enhanced non-specific immune responses. Specifically, lysozyme activity (Fig. 2A), total immunoglobulin (Fig. 2B), and IgM levels (Fig. 2C) were significantly elevated in all treatment groups compared to the control, with the highest values consistently recorded in the T3 group (p < 0.05).

The activities of CAT, GPx, and SOD were significantly elevated in fish receiving diets supplemented with MA and GP (Figs. 3A–C; p < 0.05), with the T3 group showing the greatest enhancement across all antioxidant enzymes.

The experimental groups, which were provided with diets containing MA and GP, displayed a noteworthy increase in the relative expression levels of CAT (Fig. 4A) and GPX (Fig. 4B) in comparison to the control group (P < 0.05). Remarkably enhanced levels of cytokines, including IL6 (Fig. 5A), and IL10 (Fig. 5B), were observed in the experimental groups that received diets supplemented with MA and GP, when compared to the control group (P < 0.05).

Fish challenged with A. hydrophila had variable survival rate and low mortality (Fig. 6) and high survival rate (Table 4) were evidenced in fish fed supplemented diets compared to the control. (P < 0.05).

In the aquaculture industry, the utilization of dietary medicinal herbs and acidifiers as immunostimulants and growth promoter has the potential to enhance growth performance and the innate defense mechanisms of fish in combating pathogens during times of stress (Haghighi and Sharif Rohani, 2013; Ahmadifar et al.,2021). In this study, the utilization of malic acid and ginger powder, whether used individually or in combination, has demonstrated significant enhancements on the various growth factors in the grass carp. Similarly, organic acids or salts improve growth efficiency in Nile tilapia (Hassaan et al., 2018, 2020; Reda et al., 2016) and rainbow trout (De Wet, 2005). Ahmadifaret al. 2020 found that dietary ginger (Zingiber officinale) alters biochemical and immunological parameters and gene expression related to growth, immunity and antioxidant system in zebrafish (Danio rerio). Jafarinejad, et al 2020 in investigation of dietary ginger growth performance, blood parameters, antioxidant capacity and gene expression in Cyprinus carpio reported a significant increase in growth parameters (final weight, weight gain, specific growth rate, food conversion ratio) in the treatment groups by 2% and 5% compared to the control group. In 2017, Vahedi et al. investigated the effects of ginger extract on farmed beluga (Huso huso) and found no significant difference in the growth factors of all treatment groups compared to the control group.

By investigating the effects of malic acid at levels of 0, 0.5, 1, 2% over a period of 60 days on common carp, Safari et al. (2021) found a significant increase in the growth performance, expression of the genes related to immunity and the genes related to antioxidant activity in the skin of the fish in the 2% malic acid treatment group (P < 0.05). In their study, there was no significant difference in genes related to growth among different groups. Yousefi et al. (2023) showed that dietary use of malic acid at the rate of 0.25% in O. mykiss diets improved the antioxidant status and immune responses of the fish without any negative effect on growth performance.

Also, increased growth in fish fed diets supplemented with ginger was observed in Labeo rohita (Sukumaran et al., 2016), common carp, Cyprinus carpio, (Mohammadi et al., 2020), and Nile tilapia (Brum et al., 2017). The increase in growth that occurs as a result of adding ginger to the diet can be attributed to the heightened release of intestinal proteases by the host, which in turn improves the process of digesting and absorbing protein components from the feed (Mohammadi et al., 2020). Additionally, the rhizomes of ginger serve as a plentiful source of proteinase, further enhancing protein digestion and the absorption of amino acids within the gastrointestinal tract (Hashim et al., 2011). Furthermore, ginger also has a positive impact on the probiotic bacterial population within the intestines and aids in the acquisition of even more nutrients (Ali et al., 2008). The mechanisms by which organic acids exert their effects seem to be distinct. Several hypotheses have been proposed to explain the impacts of organic acids on improving nutrient utilization in animals. These hypotheses encompass the following: the reduction of gastric pH, which in turn enhances pepsin activation; the decrease in intestinal pH, potentially leading to increased solubilization of minerals and subsequent enhanced absorption; or the inhibition of intestinal microbial activity, which would otherwise compete for nutrients that are now available for the host animal (De Wet, 2005; Hassaan et al., 2018). The utilization of malic acid as a flavor enhancer in food products has been documented by Hassaan et al. (2018). Consequently, the combination of malic acid with ginger powder may have a mitigating impact on the disagreeable taste of ginger, while simultaneously potentially maintaining the bioactive compounds and antioxidant properties of ginger.

The metabolic status of fish can be delineated through the analysis of serum biochemical indicators (Zhang et al., 2020). The present study, the concentrations of total protein displayed a noteworthy increase in fish fed-supplemented diets in comparison to the control group. Previous research conducted by other scientists has also demonstrated the beneficial impact of ginger supplementation on the levels of total protein in Zebra fish (Ferri-Lagneau et al., 2012), rainbow trout (Nya et al., 2009), and common carp (Mohammadi et al., 2020). Similar to the findings on malic acid supplementation have been found to significantly enhance total protein (Hassaan et al., 2017). The elevated levels of total protein is indicative of improved health and immune function in fish. Triglycerides, which are stored in adipose cells and between muscles and skin, are regarded as the primary forms of fat within the body (Banaee et al., 2011). In this study, the level of triglyceride reduced in fish fed-supplemented diets compared to the control group. Similar to our results, lower triglycerides level were reported in common carp fed herb extract (Raissy et al., 2022), and malic acid-fed Siberian sturgeon (Acipenser baerii) (Alizade et al., 2019). The notable reductions in blood triglycerides observed in these treatments can be ascribed to the impact of this extract on their accumulation in adipose tissues.

Dietary supplementation of malic acid and ginger extract has been demonstrated to enhance the immune response of grass carp, thereby bolstering their ability to resist diseases (Mohammadi et al., 2020; Hassaan et al., 2020). In this study, immune factors (total Ig, IgM, and LYZ) and their related genes (IL6 and IL10) increased significantly in fish fed malic acid and ginger powder. Similar to our results, higher LYZ, Ig levels were reported in rainbow trout fed with ginger powder (Nya et al., 2009; Haghighi and Rohani, 2013), ginger-fed common carp (Mohammadi et al., 2020), malic acid-fed rainbow trout (Yousefi et al., 2023), and the ferulic acid-fed hybrid grouper (Fu et al., 2022) than in fish fed with non-supplemented (the control) diets. Ahmadifar et al. (2019) observed a notable increase in serum Ig content following the administration of powdered ginger rhizomes at a supplementation rate of 3% in zebrafish. Correspondingly, Dawood et al. (2020) demonstrated that the inclusion of ferulic acid in the diet significantly enhanced serum LYZ in Nile tilapia exposed to heat stress. Mousavi et al., following the consumption of the grape seed extract (GSE) the expression of LYZ was upregulated in the rainbow trout (Mousavi et al., 2021). IL-6 is a pro-inflammatory cytokine involved in the early activation of the innate immune response, playing a key role in the acute phase reaction and defense against pathogens. In contrast, IL-10 is an anti-inflammatory cytokine that helps regulate immune homeostasis by suppressing excessive inflammatory responses (Fu et al., 2022). In this study, the gene expression of IL6 and IL10 significantly were up-regulated in fish fed supplemented diet. In line with our findings, increased expression of IL6 and IL10 genes has also been reported in Carassisu auratus gibelio fed with organic acid (Zhang et al., 2019), as well as in Labeo rohita (Sukumaran et al., 2016). The upregulation of IL-6 and IL-10 suggests a balanced immune response induced by dietary supplementation. The upregulation of IL-6 and IL-10 may be indicators of stronger immune power, which helps the fish to resist against pathogens (Mirghaed et al., 2020). This discovery implies that the intake of ginger and/or malic acid may promote the activation of immune cells, thereby enhancing the organism's ability to defend against harmful agents and stressors. The enhanced immune factors caused by malic acid can be explained by the role of acidifiers in increasing the permeability of the intestinal barrier in the gastrointestinal tract (GIT) to nutrients and folic acid. The heightened immune factors observed in the treatment involving malic acid and/or ginger powder indicate that the simultaneous use of malic acid and/or ginger powder leads to a more significant stimulation of innate immune responses compared to the individual use of either of these compounds in rainbow trout.

The evaluation of SOD, CAT, and GPx function and antioxidant related gene (CAT and GPx), being the primary enzymatic antioxidants, serves as a dependable indicator of the antioxidative potential of organisms (Ahmadifar et al., 2019). Our findings imply that the inclusion of malic acid and/or ginger extract in the diet can significantly enhance the antioxidative capacity in rainbow trout by augmenting the activities of SOD, CAT, and GPx and gene expression of CAT and GPx. The previous study documented that herbal extract (Mohammadi et al., 2020; Ahmadifar et al., 2021; Raissy et al., 2022) and organic acid (Sallah-Ud-Din et al., 2017; Asano et al., 2017; Zhang et al., 2020) were able to induce increases in antioxidant enzymes and their related gene. The antioxidant property of organic acid results from the presence of the hydroxyl group, which can easily create a resonance-stabilized phenoxy radical (Yu et al., 2017) and It is also capable of safeguarding biological membranes against lipid peroxidation while simultaneously counteracting peroxyl and alkyl radicals (Maurya and Devasagayam, 2010). Ginger possesses strong antioxidant properties (Si et al., 2018), and studies have indicated the positive impact of ginger on the antioxidant system of fish, as described by Fazelan et al. (2020), Sukumaran et al. (2016), and Ahmadifar et al. (2020). This effect can be attributed to the presence of bioactive compounds in ginger, particularly phenols, tocopherols, and flavonoids (Jelled et al., 2015). Therefore, it can be inferred that ginger extract, in conjunction with other additives that have demonstrated growth-promoting and immunostimulatory properties (Rashidian et al., 2020), can be utilized to enhance the antioxidant capacity, innate immune functions, and overall health of fish.

Aeromonas hydrophila, a prevalent pathogen in freshwater environments, is responsible for significant economic losses in the aquaculture industry (Anjur et al., 2021). One potential strategy to combat this disease is implementing fish immunization. Nevertheless, the advancement of commercial A. hydrophila vaccines is hindered by the existence of various strains (Mzula et al., 2019). Alternative approaches, such as feed additives, are recommended by different researchers (Anjur et al., 2021). The disease challenges are typically employed to assess the potential of utilizing medicinal herbs (Ahmadifar et al., 2019 a; Ahmadifar et al., 2019 b). The present study indicates that feeding malic acid and/or ginger powder increased the grass carp resistance against A. hydrophila challenge, which might be due to fish immune modulation, as suggested by the aforementioned immunological indices significantly upper in the treated grass carp. In agreement with such outcome dietary administration of herb extract improved the resistance against A. hydrophila in catfish (Pangasius bocourti) (Van Doan et al., 2016).

In conclusion, the administration of malic acid and/or ginger powder has the potential to significantly enhance the growth performance, antioxidant and immune indices of grass carp. By incorporating these substances into their diet, grass carp exhibit a stronger resistance to against A. hydrophila. There are no pharmacokinetic, synergistic, or sensory studies have yet confirmed interactions between these compounds and future studies need to address these mechanistic aspects. The strategic use of malic acid and ginger powder in aquaculture could serve as an effective approach to enhance the growth and health of grass carp, ultimately leading to more sustainable and profitable fish farming practices.