Dietary Supplementation with Ginger (Zingiber officinale) Residue from Juice Extraction Improves Juvenile Black Rockfish (Sebastes schlegelii) Growth Performance, Antioxidant Enzyme Activity, and Resistance to Streptococcus iniae Infection

Simple Summary Plant-derived feed additives are gaining interest as environmentally friendly and practical alternatives to antibiotics in fish aquaculture. In this study, we evaluated the efficacy of a ginger by-product generated from juice extraction as a feed additive for fish. We compared the effects of varying dietary levels of ginger residue from juice extraction (GRJE) on the growth performance and health status of black rockfish. GRJE diet supplementation had a positive influence on growth, feed utilization, non-specific immunity, and disease resistance and produced no adverse effects. Dietary supplementation of 0.75% GRJE is recommended for improving juvenile black rockfish performance. Abstract Plant-derived feed additives provide cost effective and environmentally friendly alternatives to antibiotics for improving fish performance in aquaculture. An 8-week feeding trial was conducted to evaluate the effects of dietary ginger residue from juice extraction (GRJE) on juvenile black rockfish (Sebastes schlegelii) growth performance, antioxidant enzyme activities, and resistance to Streptococcus iniae infection. Juvenile rockfish (n = 450; initial weight = 2.2 ± 0.01 g) were randomly distributed into 30 L rectangular tanks (30 fish per tank). Five experimental diets with GRJE concentrations of 0% (control), 0.25%, 0.5%, 0.75% and 1% were prepared in triplicate. Three groups of fish were randomly assigned to each diet and fed to apparent satiation twice daily. After the feeding trial, fish were challenged with S. iniae, and cumulative survival was observed for six days. Growth parameters, feed efficiency, and the protein efficiency ratio showed a quadratic correlation with the GRJE concentration in the fish diet. Proximate composition and plasma chemistry were not significantly affected. Plasma lysozyme, superoxide dismutase, glutathione, and catalase activities linearly increased with increasing GRJE supplementation levels. Moreover, survival in the S. iniae challenge test was significantly higher in fish fed diets supplemented with 0.75–1% GRJE. Our findings demonstrated that 0.75% GRJE dietary supplementation enhanced the growth performance, antioxidant activity, and disease resistance of juvenile black rockfish with no adverse effects.


Introduction
Fish culture is subject to considerable constraints on fish production due to frequent disease occurrence and low survival rates [1]. Fish farmers use synthetic antibiotics or

Experimental Diet Preparation
Ingredients and nutritional information for the five formulated diets are shown in Table 1. The protein sources in the control diet were 50% pollock meal and 11.5% fermented soybean meal. Fish oil (4.5%) and soybean oil (4.5%) were used as lipid sources, whereas wheat flour (27%) was used as a carbohydrates source. The control (GRJE0) diet did not include GRJE. For the experimental diets, graded levels of GRJE supplementation at 0.25% (GRJE0.25), 0.5% (GRJE0.5), 0.75% (GRJE0.75), and 1% (GRJE1) were included at the expense of an equal amount of wheat flour. The feed ingredients were physically combined with the oils and distilled water in a mixer, and the dough was extruded into 3-5 mm diameter pellets using a laboratory pellet extruder (SL Machinery, Incheon, Korea). The experimental pellets were dried at a temperature of 20 • C for 48 h in a dryer (KED-M07D1, Kiturami Co. Ltd., Seoul, Korea) and then frozen at a temperature of −20 • C until use.

Experimental Fish and Conditions
Juvenile rockfish was obtained from a local fish farm (Namhae-gun, Gyeongsangnamdo, Korea) and transferred to the Marine Bio-Education and Research Center (Tongyeong, Gyeongsangnam-do, Korea) in 5-ton circular tanks equipped with sufficient aeration and seawater. For two weeks, fish were adapted to the experimental settings by being fed a commercial feed (Jeil Feed Co., Gyeongsangnam-do, Korea). Juvenile rockfish (n = 450; mean ± SD body weight = 2.2 ± 0.01 g) were randomly distributed into 15 30 L flowthrough tanks (water capacity, 25 L; 30 fish per tank) with seawater flow (1.2 L/min) and aeration. Three tanks were assigned to each experimental group. For 8 weeks, triplicate groups were fed the experimental diets twice daily (09:00 h and 17:00 h) until visible satiation. Feed consumption was recorded daily for each tank. The mean water temperature, salinity, and dissolved oxygen during the experimental period were 21.2 ± 0.22 • C, 30.13 ± 0.12 psu, and 7.0 ± 0.06 mg/L, respectively. The photoperiod followed natural conditions.

Bacterial Challenge Test
After the feeding trial, 10 fish were randomly selected from each tank and redistributed into 15 30 L tanks for the challenge test. The Korean Culture Collection of Aquatic Microorganisms, National Institute of Fisheries Science (Busan, Korea) provided the pathogenic bacteria, S. iniae. Then, 0.1 mL of S. iniae culture suspension (5.0 × 10 6 CFU/mL) was intraperitoneally injected into each fish. The water temperature and dissolved oxygen were maintained at 20.5 ± 0.15 • C and 6.8 ± 0.25 mg/L, respectively. Cumulative mortalities were recorded daily for six days.

Sample Collection
After the feeding trial, the surviving fish were counted and starved for 24 h before the total biomass was recorded to calculate growth performance. Ten fish in each tank were randomly selected and anesthetized using 100 ppm tricaine methanesulfonate (MS-222). Using heparinized syringes, blood samples were obtained from the caudal veins of fish. Plasma was collected and kept in a freezer at −80 • C until analysis following centrifugation (7500 rpm for 10 min). After blood sampling, the remaining fish (≥5 fish) in each tank were homogenized and used for whole-body proximate composition analysis.

Chemical Analyses
The diets and whole-body of fish were analyzed in accordance with the method of Association of Official Agricultural Chemists methods [29]. The crude protein content was evaluated using the Kjeldahl method with a KD310-A-1015 KjelROC Analyzer (OPSIS Liquid LINE, Sweden). The Soxtec extractor (ST 243 Soxtec™; FOSS, Hillerod, Denmark) was used to determine the crude lipid content. The moisture content was assessed by oven drying at 105 • C for 24 h, and the ash content was determined using a muffle furnace at 550 • C for 4 h.

Lysozyme Activity Analysis
The activity of lysozyme in plasma was measured using a commercial kit (EnzChek™ Lysozyme Assay Kit, E22013, Thermo Fisher Scientific, Waltham, MA, USA). This kit measures lysozyme activity using fluorophore fluorescein incorporated in Micrococcus lysodeikticus cell walls, which quenches the fluorescent signal. Plasma was diluted with 25 mL of 1X reaction buffer, which was made up of 0.01 M NaCl and 0.01 mg NaCl. It was then mixed in with 50 mL of fluorescein isothiocynate-labeled Micrococcus lysodeikticus (50 mg/mL) in a 96-well plate at 37 • C for 30 min. Fluorescence intensity was measured with a fluorescence reader (1420 Multilabel Counter Victor3, Perkin Elmer, CT, USA) at 485/535 nm. Each value was divided by the amount of fluorescence in the background, which was measured for a no-enzyme control. The lysozyme activity of the samples that were used in the study was determined from a standard curve made with lysozyme from chicken egg whites.

Superoxide Dismutase Activity Analysis
The activity of superoxide dismutase (SOD) was determined according to the manufacturer's instructions using a Cayman's Superoxide Dismutase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). To determine the activity, 10 µL of plasma was introduced to 200 µL of the radical detector, and then they were mixed together. The xanthine oxidase was added to 20 L of the mixture, and it was incubated on a shaker for 20 min to initiate. The spectrophotometer (Thermo Scientific MULTISKAN GO, Vantaa, Finland) was used to measure the absorbance at 440 nm.

Catalase Activity Analysis
The catalase (CAT) activity was analyzed following the manufacturer's instructions using a Cayman's Catalase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). To analze the activity, 20 µL of plasma was mixed with 30 µL of methanol and 100 µL of assay buffer, and then mixed again. The 20 µL of H 2 O 2 was used to initiate the reaction. The mixture was incubated at room temperature for 20 min. Potassium hydroxide and purpald chromagen were added to the mixture to terminate it, and the mixture was kept at room temperature for 10 min. Finally, 10 µL of potassium periodate was added and shaken for 5 min at room temperature. At 540 nm, the solution's absorbance was measured using a spectrophotometer (Thermo Scientific MULTISKAN GO, Vantaa, Finland).

Calculations and Statistical Analyses
The following parameters were used to calculate the grow performance of fish:

•
Fish at the end of the feeding trial/number of fish at the initial of feeding trial) × 100; • Weight gain (WG) = (final body weight-initial body weight)/initial body weight; • Specific growth rate (SGR, %/day) = (ln final weight of fish-ln initial weight of fish)/days of feeding) × 100; • Feed consumption (g/fish) = total feed intake/number of surviving fish; • Feed efficiency (FE) = WG/feed consumed; • Protein efficiency ratio (PER) = WG/protein consumed; • Protein retention (PR) = Protein gain × 100/protein consumed.
All percentage values were arcsine transformed before statistical analysis. Variable data were checked for normality and homogeneity of variance using the Kolmogorov-Smirnoff and Levene's tests, respectively [30]. ANOVA was conducted to determine where dietary GRJE concentrations significantly affected the observed response (p < 0.05); then, orthogonal polynomial contrasts (linear, quadratic, and cubic) were used to evaluate the response for all dependent variables [31]. The Kaplan-Meier and log-rank and Wilcoxon tests were used to plot the fish survival curves during the S. iniae challenge test. SPSS version 25.0 software was used for all statistical analyses (SPSS Inc., Chicago, IL, USA).

Growth Pergormance
After the 8-week feeding trial, the effects of GRJE supplementation on juvenile rockfish growth performance and feed utilization were determined and are presented in Table 2. Dietary GRJE had no significant effect on SR and FC. However, increasing dietary GRJE levels affected the FBW, WG, SGR, FE, and PER, presenting quadratic trends (p < 0.05). The FBW, WG, SGR, FE, and PER were significantly higher in the GRJE0.25, GRJE0.5, GRJE0.75, and GRJE1 treatments than in the GRJE0 treatment (p < 0.05).

Whole-Body Chemical Composition
The moisture content of whole-body of fish varied between 72.0% and 72.4%, the crude protein content between 16.6% and 16.9%, the crude lipid content between 5.7% and 5.9%, and the ash content between 4.1% and 4.3%. Graded levels of dietary GRJE exhibited no significant effect on whole-body proximate composition (Table 3).   Table 4 shows the effect of GRJE supplementation on juvenile rockfish plasma biochemical parameters. The AST, ALT, T-CHO, TP, and GLU were significantly different among diet groups.

Plasma Lysozyme Activity and Antioxidant Parameters
The effects of GRJE supplementation on plasma lysozyme activity and antioxidant parameters are shown in Table 5. Lysozyme, SOD, and CAT activities and GSH content showed a linearly increasing trend with the increasing dietary GRJE level; lysozyme (p < 0.05), SOD, and CAT activities and GSH content were significantly higher in the GRJE0.75 and GRJE1 groups than in the GRJE0, GRJE0.25, and GRJE0.5 groups (p < 0.05).

Discussion
Supplementing fish diets with various types (powder, extract, oil, by-product, etc.) of ginger (Zingiber officinale) has been demonstrated to enhance fish performance [2,16,[32][33][34]. In our previous study, we showed that dietary GRJE supplementation significantly improved juvenile black rockfish growth performance, innate immunity, and disease resistance against Gram-negative bacteria V. harveyi [34]. However, further investigation was needed to establish the stimulatory dose of GRJE in fish diets. Here, we evaluated the effects of graded levels of dietary GRJE (0%, 0.25%, 0.5%, 0.75%, and 1%) on juvenile black rockfish growth performance, non-specific immune response, and survival in a bacterial challenge test with Gram-positive S. iniae.
Dietary GRJE supplementation significantly improved rockfish growth (final weight, weight gain, and SGR) and feed utilization (FE and PER), even at the lowest dose (0.25% GRJE). Numerous studies have shown that dietary supplementation with various types

Discussion
Supplementing fish diets with various types (powder, extract, oil, by-product, etc.) of ginger (Zingiber officinale) has been demonstrated to enhance fish performance [2,16,[32][33][34]. In our previous study, we showed that dietary GRJE supplementation significantly improved juvenile black rockfish growth performance, innate immunity, and disease resistance against Gram-negative bacteria V. harveyi [34]. However, further investigation was needed to establish the stimulatory dose of GRJE in fish diets. Here, we evaluated the effects of graded levels of dietary GRJE (0%, 0.25%, 0.5%, 0.75% and 1%) on juvenile black rockfish growth performance, non-specific immune response, and survival in a bacterial challenge test with Gram-positive S. iniae.
Dietary GRJE supplementation significantly improved rockfish growth (final weight, weight gain, and SGR) and feed utilization (FE and PER), even at the lowest dose (0.25% GRJE). Numerous studies have shown that dietary supplementation with various types and concentrations of ginger significantly affects growth and feed utilization in fish [1,20,21,[34][35][36]. Similar to our study, Naliato et al. [19] showed that growth performance parameters of Oreochromis niloticus including final body weight, WG, SGR, feed intake and feed conversion ratio, and PER were responsive to the increase on dietary ginger powder supplementation, showing a dose-dependent response and a linear correlation. These authors also described that zingiberene is the most concentrated compound in the dehydrated ginger powder, terepene is known for its smell and flavor [37], and this substance improved the feed intake and feed conversion ratio, resulting in enhanced growth and PER. Ginger oils are known to enhance palatability, which may reflect improved FE and fish growth, as reported for O. niloticus [20]. Ginger extracts also stimulate digestive enzyme secretion in Oncorhynchus mykiss and Mesopotamichthys sharpeyi, improving growth performance through better feed utilization [38,39]. Ahmadifar et al. [40] reported an increase in enzyme activity (amylase) in fish fed 0.1-0.3% dietary ginger powder, but no effect on growth (SGR). In contrast, Cardoso et al. [20] observed improvements in O. niloticus growth with ginger supplementation but no differences in digestive enzyme activity (protease, lipase, and amylase).
Rockfish whole-body chemical composition was not significantly affected by the dietary GRJE concentration. This finding is consistent with our previous study, in which the proximate composition of juvenile S. schlegelii was not significantly affected by diet supplementation with 1% GRJE [34]. In contrast, Kim et al. [33] reported significantly higher crude protein, crude lipid, and moisture content in juvenile S. schlegelii fed 1% dried ginger for 8 weeks. Additionally, juvenile Cyprinus carpio fed varying doses of ginger extract showed significant differences in carcass composition [6]. Ginger composition is affected by the type (dry powdered plant, extract, residue), variety, agronomics, drying, and storage conditions, which may contribute to the varied effects of dietary ginger on fish [40].
Blood biochemical measurements are important aquaculture parameters that reflect fish performance and health [41,42]. In this study, plasma parameters of S. schlegelii were not significantly affected by dietary GRJE supplementation. Chung et al. [21] reported that increasing concentrations of ginger essential oil in O. niloticus diet had a negative linear effect on plasma cholesterol level but did not significantly relate to plasma glucose, triglyceride, TPs, albumin, ALT, and AST. However, Cyprinus carpio fed ginger extract showed increases in serum TP, albumin, and globulin content [6]. The advantageous effects of ginger supplementation on biochemical parameters indicative of promoted health have also been reported in Lates calcarifer [16], Danio rerio [40], Huso huso [43], and O. mykiss [35].
Lysozyme is compositionally expressed, synthesized, and secreted by neutrophils, monocytes, and macrophages and is considered one of the most important bacterial enzymes involved in fish immunity [44]. Lysozyme is an indispensable defense against infectious agents [45]. SOD, CAT, and GSH, which are responsible for detoxifying harmful reactive oxygen species generated during normal metabolism, are also important aspects of innate immunity in fish [45]. The SOD and CAT activity and GSH content trends observed in this study corroborate the lysozyme activity results; there was a linear increase in antioxidant enzyme activity with increasing GJRE levels. Stoilova et al. [46] demonstrated that ginger acts as an antioxidant by scavenging DPPH (1,1-diphenyl-2-picrylhydrazyl) and inhibiting the formation of secondary products from fat auto-oxidation. Additionally, ginger polyphenols have a high chelatoforming capacity with Fe 3+ , which prevents hydroxyl radical formation and lipids [46]. Masuda et al. [47] emphasized that the antioxidant activity of ginger might be due not only to radical scavenging activity, but also to its affinity with the substrate. Ahn et al. [24] reported that the ginger by-product of medicinal extraction contains polyphenol and flavonoids, which also have DPPH and ABTS (3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging abilities. These findings demonstrate the benefits of GRJE dietary supplementation at levels of 0.75% and higher for sustaining and improving the oxidative status of juvenile rockfish.
Ginger-induced enhancement of the non-specific immune response was previously demonstrated for O. niloticus fed 1% and 1.5% ginger powder [48,49]. Similarly,Şahan et al. [50] observed that 0.5% and 1% ginger supplementation enhanced SOD and CAT activities in the liver, gills, and gut of O. niloticus. Significantly higher activities of SOD and CAT were observed in ginger-fed groups, notably in the 2% ginger extract supplementation group [6]. Moreover, ginger is a potent antioxidant [9], and the beneficial effects of ginger on fish antioxidant systems have been reported [1,40,51]. These effects are attributed to bioactive compounds in ginger, especially polyphenols, flavonoids, tannins, and saponins [16,52,53].
Immunostimulants increase immunocompetency and disease resistance by augmenting non-specific and specific defense mechanisms in fish [54][55][56]. In this study, dietary GRJE supplementation at 0.75-1% effectively improved disease resistance to S. iniae, which was also reflected in linearly positive changes in non-specific immune parameters. Similarly, dietary ginger powder administration to different fish species increased bactericidal activity against A. hydrophila [35], V. harveyi [1,16], and A. salmonicida [1]. Moreover, ginger powder administration significantly improved O. niloticus resistance against S. agalactiae infection [17]. Dietary ginger powder also positively impacted the treatment of Grampositive and/or Gram-negative bacterial infections in S. schlegelii [33,34,57]. Lee et al. [34] reported that 1% GRJE-supplemented S. schlegelii groups attained a higher probability of survival against Gram-negative bacteria (V. harveyi) compared with groups that did not receive GRJE supplementation. These findings support the use of GRJE feed additives to reduce mortality caused by Gram-negative and Gram-positive bacterial infections.
Thus, we can conclude that, along with other ginger feed supplements, GRJE acts as a growth promoter and immunostimulant [1,6,16,21], enhancing antioxidant capacity, innate immune functions, and the overall health status of fish.

Conclusions
Dietary 0.75% GRJE supplementation is recommended as an effective and safe immunostimulatory agent for protection against bacterial diseases and the improvement of juvenile black rockfish health.

Conflicts of Interest:
The authors declare no conflict of interest.