Dietary Supplementation of Sophora flavescens Root Extract Improved the Growth Performance, Antioxidant Capacity, Innate Immunity, and Disease Resistance against Edwardsiella tarda Challenge in Turbot (Scophthalmus maximus)

The impacts of dietary supplementation with graded levels of Sophora flavescens root extract (SFE) on growth performance, antioxidant capacity, immune status, and resistance against Edwardsiella tarda challenge in Scophthalus maximus were investigated in this study. In all, 600 turbot (initial body weight: 8.38 ± 0.07 g) were randomly distributed in 12 tanks with 50 fish per tank and fed four experimental diets supplemented with 0, 0.05%, 0.1%, or 0.2% SFE (named as: SFE0, SFE0.05, SFE0.1, and SFE0.2, respectively), for 56 days. The results showed that 0.1% and 0.2% SFE supplementation have significantly increased the FBW, WGR, SGR, and PER of turbot, while decreased the FCR of turbot (p < 0.05). Dietary SFE supplementations have significantly increased the activities of plasma SOD, CAT, GPx, T-AOC, GST and LZM, decreased plasma MDA contents in turbot under normal or challenge condition (p < 0.05). Meanwhile, SFE addition dramatically enhanced the hepatic mRNA expression of antioxidant parameters (including Nrf2, Keap1, SOD, CAT, Trx2, GST and GR) during the normal condition. mRNA levels of NF-κB p65, IκBα, TNF-α, TGF-β, and IL-10 in the liver of fish were notably up-regulated by SFE treatment during normal condition (p < 0.05), while the transcription of IL-1β was down-regulated by SFE whenever under normal or challenge condition. 0.1% and 0.2% SFE administration have significantly increased the survival rate of turbot against E. tarda challenge (p < 0.05). In conclusion, dietary SFE supplementation improved the growth performance, antioxidant activity and disease resistance of turbot, and SFE could be a potential feed additive for turbot.


Introduction
Nowadays, aquaculture is faced with great challenges due to intensive culture modes, and fish are more susceptible to oxidative stress and infectious diseases [1]. Turbot, Scophthalmus maximus L., is an important industrial mariculture species in Northern China, with an annual output of about 60,000 tons, and has a good culture prospect. However, the high-density intensive culture usually led to disease out-breaking in turbot culture. Antibiotics are used to prevent bacterial pathogens and reduce the economic loss of aquaculture [2]. The indiscriminate use of antibiotics can be hazardous to the environment and aid in the evolution of antimicrobial-resistant genes [3][4][5], which has also become a experimental diets used white fish meal and chicken meal as primary protein sources, fish oil as chief lipid sources, α-starch as the main carbohydrate source.
The feed ingredients were mixed and ground sufficiently through a 178 µm mesh sieve. Then, the fish oil was added to the powder and mix the ingredients. All ingredients were supplemented the distilled water and mixed adequately to form into 2 mm pellets by extruded pelletizer (EL-260, Youyi Machinery Factory, Weihai, Shandong, China), which were placed in a ventilated place to dry at room temperature for 30 min. The prepared diets were stored at −20 • C for further use. The dietary formulation and proximate compositions are shown in Table 1.

Fish and Growth Trial
Turbots were obtained from a commercial aqua-farm (Tianjin, China). All fish are acclimated to the laboratory conditions and fed the prepared basal diet for 2 weeks. After acclimatization period, 600 fish (average weight: 8.38 ± 0.07 g) were assigned to twelve tanks (capacity: 560 L) with three replicates per treatment and 50 fish per tank. Fish were hand-fed the corresponding diets twice a day (8:00 and 18:00) to apparent satiation. The feeding trial was conducted for 56 days. During the feeding trial, water temperature was maintained at 16-18 • C, salinity at 15-20‰, ammonia nitrogen lower than 0.05 mg/L and dissolved oxygen higher than 6.0 mg/L.

Sample Collection
At the end of feeding trial, all fish were anesthetized with 100 mg/L MS-222 solutions (Sigma-Aldrich, St. Louis, MO, USA) and batch weighed. Four fish per tank were dissected and stripped to calculate VSI and HSI. Two fish per tank were used for chemical analysis and gene expression. The blood of fish was collected from the tail vein of the syringe moistened by heparin sodium and centrifuged with 3000× g 15 min to measure the plasma non-specific immune and antioxidant indexes. The liver was stripped to analyze the mRNA expression. All the samples were immediately frozen in liquid nitrogen and stored in the refrigerator at −80 • C until use.
After feeding trial, 20 fish per tank were exposed to bacteria challenge test. Edwardsiella tarda was provided from Fish Disease Laboratory of Hebei Agricultural University. The bacteria were inoculated into TSA solid medium and cultured at 30 • C for 12 h. Then the single bacteria were selected in the aseptic beef soup liquid medium, cultured in 200 rpm shaker at 30 • C for 24 h, 4000 rpm centrifugation 10 min, remove the supernatant, rinse with 3.5% aseptic salt water and dilute the precipitated bacteria. The fish in 12 tanks were injected intraperitoneally with 3 × 10 8 CFU/mL E. tarda solution 0.3 mL (the injection concentration and dose were determined by the pre-experiment). Mortality was observed for 7 days, the dead fish was counted every 6 h and the survival rate was calculated. The plasma and liver of turbot were sampled for further analysis after 72 h challenge as the process described above.

Chemical Analysis
The proximate compositions of diets were analyzed according to standard methods, as previously described [29]. Moisture was determined by oven drying at 105 • C to constant weight. Crude protein (N × 6.25) was measured by Kjeldahl method using a 4800 Kjeltec Analyzer Unit (FOSS Tecator, Haganas, Sweden). Crude lipids were detected by petroleum ether extraction using the Soxhlet method. Crude ash was determined by combustion in a muffle furnace at 550 • C for 12 h. Gross energy was evaluated by an oxygen calorimeter (Parr 6300, Parr Instrument Company, Houston, TX, USA).
Plasma parameters, including lysozyme (LZM), complement 3 (C3), immunoglobulin (IgM), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC), glutathione-S-transferase (GST) activities, glutathione reductase (GR) and malondialdehyde (MDA)content were measured using commercial kits (Jiancheng Bioengineering Ltd., Nanjing, China). The determination method and calculation follow its instructions. The LZM, complement 3 and IgM activity were measured by turbidimetric assay. The activity of CAT was measured according to visible light spectrophotometry. The activity of SOD activity was evaluated according to hydroxylamine method. The T-AOC present in plasma was measured according to ABTS method. The MDA activity in the plasma was measured by means of a TBA method. These parameters were analyzed using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) to determine the contents. Activities of GPx and GR were determined by recording the colorimetry through TU-1810 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China).

Statistical Analysis
All data were presented as mean values and standard deviation (S.D.). Statistical analyses were performed using the STATISTICA 10.0 (StatSoft Inc., Tulsa, OK, USA) software, the normality and variance homogeneity of data were tested. The parameters of growth (including FBW, FR, WGR, SGR, FCR, PER, SR) and morphology (containing CF, VSI, HSI) were analyzed by one-way ANOVA. Two-way ANOVA was employed in plasma biochemistry indexes (including LZM, Complement C3, IgM, CAT, SOD, T-AOC, GPx, GST, GR, and MDA) and gene expression parameters. Duncan multiple comparisons were used when there was a significant difference in ANOVA analysis, and p < 0.05 was considered statistically significant.

Plasma Immune Indexes
Dietary supplementation with SFE positively influenced the plasma LZM (p < 0.05). The LZM was highest in the SFE 0.1 group compared with that in other groups. However, no significant difference was observed in plasma complement C3 and IgM levels among all groups (p > 0.05) ( Table 5).

Antioxidant-Related Gene Expression
As seen from Figure 1, SFE treatment has significantly affected the Nrf2 signaling pathway and its downstream genes expression. In the normal condition, SFE has dramatically enhanced the mRNA levels of Nrf2 and Keap1 expect for Nrf2 in SFE 0.1 group (p < 0.05), the Nrf2 gene expression increased with the increasing of dietary SFE levels after pathogen challenge, it is significantly higher in SFE 0.2 than in SFE 0 group (p < 0.05). The SFE additions have significantly increased the mRNA levels of CAT and SOD during the normal condition. When exposed to pathogen challenge, the gene expressions of CAT, SOD in SFE 0.05 group were significantly lower than that in other treatments (p < 0.05). est transcription levels of Trx2 and TrxR2 was in SFE 0.1 group, the highest mRNA expressions of GST and GR were observed in SFE 0.2 group. When exposed to pathogen challenge, the gene expressions of TrxR2, Trx2, GST and GR in SFE 0.05 group were significantly lower than that in other treatments (p < 0.05).  The transcription levels of the thioredoxin regulatory system and glutathione antioxidant regulatory system related genes are presented in Figure 2. The gene expression of GPx was not affected by SFE treatment (p > 0.05), while SFE additions have significantly increased the mRNA levels of Trx2, GST and GR during the normal condition, the highest transcription levels of Trx2 and TrxR2 was in SFE 0.1 group, the highest mRNA expressions of GST and GR were observed in SFE 0.2 group. When exposed to pathogen challenge, the gene expressions of TrxR2, Trx2, GST and GR in SFE 0.05 group were significantly lower than that in other treatments (p < 0.05).

Inflammation-Related Gene Expression
SFE treatment significantly regulated the key regulators NF-κB p65 and IκBα in the NF-κB signaling pathway, and their downstream pro-inflammatory factors TNF-α and IL-1β, anti-inflammatory factors TGF-β and IL-10 (p < 0.05) (Figure 3). Under normal condition, mRNA levels of NF-κB p65, IκBα, TNF-α, TGF-β and IL-10 in the liver of fish were notably increased by SFE treatment (p < 0.05), NF-κB p65, IκBα, TNF-α, and IL-10 increased first and then decreased, and reached the highest value in the SFE 0.1 group (p < 0.05). Anti-inflammatory cytokine TGF-β expression was the highest in SFE 0.2 group. Dietary SFE significantly decreased the transcription levels of pro-inflammatory cytokines IL-1β (p > 0.05) whenever under normal or challenge condition. After pathogen challenge, the mRNA levels of NF-κB p65 were significantly increased in SFE 0.1 and SFE 0.2 groups. Pro-inflammatory cytokines TNF-α was down-regulated in the SFE 0.05 and SFE 0.2 groups. Anti-inflammatory cytokine IL-10 were down-regulated in all SFE treatment groups (p < 0.05).

Disease Resistance
After pathogen challenge, dead of turbot was observed firstly in SFE 0 group, and then the mortality was dramatically increased during 2-6 days post challenge. Dietary supplementation with SFE increased the survival rate of turbot exposed to pathogen challenge ( Figure 4). The survival rates in SFE 0 and SFE 0.05 were significantly lower than that in SFE 0.1 and SFE 0.2 groups (p < 0.05).

Disease Resistance
After pathogen challenge, dead of turbot was observed firstly in SFE 0 group, and then the mortality was dramatically increased during 2-6 days post challenge. Dietary supplementation with SFE increased the survival rate of turbot exposed to pathogen challenge ( Figure 4). The survival rates in SFE 0 and SFE 0.05 were significantly lower than that in SFE 0.1 and SFE 0.2 groups (p < 0.05).

Discussion
Many herbs or their compounds have been used as supplements and additive in animals as well as human diets because of their numerous beneficial properties such as growth promotion, anti-cancer, anti-pathogenic, anti-inflammatory, immunomodulatory, and antioxidant properties [13]. In recent years, there has been increasing interest in using natural plant extract as functional compounds to enhance the immunity of fish in aquaculture. The root extracts of S. flavescens, a kind of traditional Chinese herbal medicine, has a wide range of medicinal values, such as antibacterial and anti-inflammatory [16]. However, little information was available about the functions and regulating mechanism of S. flavescens in fish. This study demonstrated that S. flavescens root extract enhanced the growth performance, antioxidant capacity, immunity, and disease resistance of turbot.

SFE improved the Growth Performance and Feed Utilization of Fish
In this study, 0.1% and 0.2% dietary SFE supplementation significantly increased the growth performance and feed utilization of fish, which is similar with previous studies of plant herbs extract. It is reported that Radix glycyrrhizae extract, Panaxnotoginseng extract and tea tree oil have increased the WGR, SGR of yellow catfish (Pelteobagrus fulvidraco), grouper (Epinephelussp) and prawn (Macrobrachium rosenbergii) [31][32][33], respectively. This is probably related to the bioactive compounds isolated from these herbs. It is proved that the bioactive substances contained in Chinese herbal medicine are the main factors to promote the rapid growth of aquatic animals [15]. Many studies have shown that alkaloids (such as berberine), flavonoids, triterpenes (such as hesperidin) and polysaccharides (such as Astragalus polysacharin) extracted from Chinese herbal medicine can significantly increase fish FBW, WGR and SGR, and reduce FCR [34][35][36][37]. The composition of S. flavescens extract is complex, more than 200 compounds are isolated from S. flavescens root, and the main bioactive components are alkaloids and flavonoids [16]. It might be

Discussion
Many herbs or their compounds have been used as supplements and additive in animals as well as human diets because of their numerous beneficial properties such as growth promotion, anti-cancer, anti-pathogenic, anti-inflammatory, immunomodulatory, and antioxidant properties [13]. In recent years, there has been increasing interest in using natural plant extract as functional compounds to enhance the immunity of fish in aquaculture. The root extracts of S. flavescens, a kind of traditional Chinese herbal medicine, has a wide range of medicinal values, such as antibacterial and anti-inflammatory [16]. However, little information was available about the functions and regulating mechanism of S. flavescens in fish. This study demonstrated that S. flavescens root extract enhanced the growth performance, antioxidant capacity, immunity, and disease resistance of turbot.

SFE improved the Growth Performance and Feed Utilization of Fish
In this study, 0.1% and 0.2% dietary SFE supplementation significantly increased the growth performance and feed utilization of fish, which is similar with previous studies of plant herbs extract. It is reported that Radix glycyrrhizae extract, Panaxnotoginseng extract and tea tree oil have increased the WGR, SGR of yellow catfish (Pelteobagrus fulvidraco), grouper (Epinephelussp) and prawn (Macrobrachium rosenbergii) [31][32][33], respectively. This is probably related to the bioactive compounds isolated from these herbs. It is proved that the bioactive substances contained in Chinese herbal medicine are the main factors to promote the rapid growth of aquatic animals [15]. Many studies have shown that alkaloids (such as berberine), flavonoids, triterpenes (such as hesperidin) and polysaccharides (such as Astragalus polysacharin) extracted from Chinese herbal medicine can significantly increase fish FBW, WGR and SGR, and reduce FCR [34][35][36][37]. The composition of S. flavescens extract is complex, more than 200 compounds are isolated from S. flavescens root, and the main bioactive components are alkaloids and flavonoids [16]. It might be ascribed to these bioactive components from SFE improved the antioxidant capacity and non-specific immune responses, which in turn boosts turbot growth performance.

SFE Enhanced the Antioxidant Capacity of Fish
Antioxidant enzymes are indicative biomarkers of health and the reaction in oxidative stress response when the reactive oxygen species (ROS) and free radicals production were uncontrolled [38], and MDA is regarded as a bio-indicator of oxidative stress [39]. In the current study, the activities of plasma CAT, SOD, T-AOC, GPx, GST, and GR were significantly increased, and plasma MDA contents were decreased in fish fed S. flavescens diets. The results of this study indicated that S. flavescens extract could improve the antioxidant capacity of juvenile turbot. Previous research has confirmed that S. flavescens can trigger the antioxidant defense system of mammals [40]. Similarly, antioxidant enzymes activity increased significantly in fish with dietary administration of grape seed proanthocyanidin extract [41] or curcumin [42], and decreased the MDA levels in the fish. The current results proved that S. flavescens extract improved the antioxidant system of turbot and reduced oxidative stress. This may be because the bioactive components in S. flavescens extract can activate the antioxidant related pathway Nrf2 and thus lead to the change of enzyme activity level. As we know, the increased enzyme activity is caused by the increased synthesis of enzyme protein, which largely relies on its gene transcription and translation [43]. Therefore, in order to further confirm whether the change of antioxidant enzyme activity is caused by the change of mRNA level of related genes in Nrf2 signaling pathway, this study determined the influence of S. flavescens extract on the expression level of antioxidation-related genes.
A number of reports have demonstrated the importance of the Nrf2-Keap1 signaling pathway against oxidative stress in the body. The nuclear transcription factor Nrf2 is widely expressed in an extensive range of cell and tissue types and regulates antioxidant defense [44,45]. Nrf2 transcriptional activity can be inhibited by Keap1-controlled ubiquitination-proteasomal degradation [46]. Nrf2 translocates into the nucleus and binds ARE to regulate and code are involved in redox regulation (CAT, SOD and Trx), and enzymes related to glutathione synthesis metabolism, such as GST, GR, GPx, etc., to regulate oxidation in the body [47]. In this study, S. flavescens extract supplementation activated Nrf2/Keap1 signaling pathway under normal condition, thus increased the expression of downstream related antioxidant genes, the mRNA expression results were consistent with the results of their enzyme activities under normal condition, which revealed that dietary SFE could strengthen antioxidant activity associated with decreased MDA content of turbot. Similar to our research, dietary supplementations of P. tectorius in C. carpio [48], emodin in M. amblycephala [49], curcumin in C. argus [42] regulated the expression of Nrf2, Keap1 and its downstream regulated genes such as SOD, CAT, GPx and GST to improve the antioxidant activity in fish. To date, no reports have investigated the potential effects of S. flavescens on the thioredoxin regulatory system of fish. The thioredoxin system is one of the major redox systems in cells and is an important regulator of eliminating ROS accumulation, the main components are thioredoxin (Trx) and thioredoxin reductase (TrxR) [50,51]. In the current study, the expressions of hepatic Trx2 and TrxR2 were significantly up-regulated in turbot fed an S. flavescens extract diet. S. flavescens extract increases the expression of thioredoxin and its related reductases by activating the Nrf2/Keap1 signaling pathway, and further improves the antioxidant capacity of turbot, so that it can quickly eliminate excessive ROS to protect the body. Furthermore, it is interestingly found in this study that the expression of SOD was down-regulated in SFE groups compared to control after 72 h pathogen challenge. There probably exists the negative feedback suppression. The activities and mRNA expression of SOD in SFE treatments were all at higher levels during normal condition, which showed there are enough SOD activities in SFE treatments to defense the bacterial invasion to maintain the body homeostasis, no need to activate the genes to produce more enzymes. Hence, in this study, the transcriptional levels of SOD in SFE groups were not significantly increased by E. tarda challenge, while the level in SFE 0 significantly increased, which revealed that dietary SFE supplementation could protect turbot from pathogen persecution. Based on the results of antioxidant parameters determined in this study, a dietary supplement of SFE improved the antioxidant capacity of turbot by activating the Nrf2 pathway and enhancing antioxidant systems including SOD, CAT, GPx, GST and Trx and protected lipids from peroxidation.

SFE Enhanced the Innate Immunity and Disease Resistance of Fish
Lysozyme, the first line of protection against pathogen invasion, plays a key role in nonspecific immune response in fish. It can cleave bacteria by hydrolyzing the β-1,4 glycosidic bond of peptidoglycan layer of the bacterial cell wall [52]. In this study, S. flavescens exerted its antibacterial effect by increasing the activity of plasma LZM and enhancing immunity in juvenile turbot. This is consistent with the results in tilapia and flounder [19,20]. Similarly, dietary Eucommia ulmoides Oliver and Astragalus polysaccharides increased LZM in turbot [37,53]. Allium mongolicum Regel significantly improved LZM in Channa argus [35]. This indicated that the bioactive substances in the extracts of Chinese herbal medicine could improve the activity of LZM, and then improve the immunity of fish.
Moreover, the transcription levels of immune-related genes were analyzed in this study to further evaluate the effects of SFE on immune responses of turbot. NF-κB pathway is involved in the initiation, amplification and regression of inflammation and is activated by different stimuli [54,55]. Cytokines play a vital role in the immune system by binding to specific receptors and setting off a cascade of immunological events [56]. Proinflammatory cytokines (IL-1β, TNF-α) regulate multiple aspects of the immune response in fish [57]. TGF-β and IL-10, anti-inflammatory cytokine, suppresses the production of pro-inflammatory cytokines [58]. In this study, SFE treatment activated the expression of NF-κB p65 and IκBα in the normal condition, then up-regulated the mRNA expression of anti-inflammatory cytokine (TGF-β and IL-10), and down-regulated the mRNA expression of pro-inflammatory cytokines (IL-1β). Similar results were found in other plant extract studies, such as turmeric in Cyprinus carpio [59], Allium mongolicum Regel in Channa argus [35]. However, in the present study, the transcription of TNF-α, another antiinflammatory cytokine, were up-regulated sharply in SFE 0.1 and SFE 0.2 groups. TNF-α is a pro-inflammatory cytokine secreted from activated macrophages, vital in regulating innate immune functions and inflammatory responses [60]. In previous studies, dietary P. tectorius extract and Agaricus bisporus polysaccharides up-regulated TNF-α expression in the fish [61,62], while dietary turmeric significantly decreased the expression of TNF-α in Cyprinus carpio [59]. The difference of gene expression results may be due to the difference of the type and dose of plant immune stimulants, feeding time and fish species. Under challenge condition, the gene expressions of NF-κB p65 and IκBα in SFE0 group were up-regulated compared with normal condition, and the mRNA levels of IL-1β and IL-10 increased sharply, which means that excessive inflammatory response occurred in turbot after E. tarda infection. Meanwhile, dietary SFE administration significantly decreased the mRNA levels of IL-1β and IL-10, which suggested that dietary supplementation of SFE could inhibit the inflammatory response of turbot infected by E. tarda to protect the body from excessive inflammatory reaction.
Due to the prevalence of industrialized intensive farming, fish usually face greater risks of antioxidant stress and pathogen attack. E. tarda, a causative infectious agent of disease in aquaculture, caused mass mortalities and considerable economic losses, particularly in turbot [63]. Bacterial challenge test is often used as an effective indicator of fish immunity and health status. In this study, after the challenge, turbot in SFE 0 treatment firstly displayed the clinical symptoms of E. tarda infection: red bleeding plaques in the head and muscles, dark red mucus in the gills, blood spots on the fins, anal congestion, and protrusion. Then, the first dead turbot was observed in the SFE 0 group on the second day, the mortality lasted to the sixth day after challenge. Further, 0.1% SFE administration significantly relieved the symptoms and increased the survival rate of turbot. The survival rates of SFE administration groups were higher than that of SFE 0 group. Therefore, the elevation of disease resistance against E. tarda is contributed to the regulation of immune response and antioxidant capacity by SFE administration. That is to say, SFE could affect mRNA levels of immune-related genes by activating Nrf2/Keap 1 and NF-κB signaling pathways, and then improved the antioxidant capacity and immunity of turbot, which also improved the ability of juvenile fish to resist E. tarda infection. It also suggests that antioxidant capacity, immunity, and disease resistance are inextricably linked.

Conclusions
Overall (Figure 5), the present study was apparently the first report that dietary SFE supplementation for 56 days promoted the immunity and antioxidant capacity through activating Nrf2/Keap1 and NF-κB signal pathways, thus improving the growth performance and disease resistance against E. tarda. SFE could be a potential feed additive for turbot. ministration significantly relieved the symptoms and increased the survival rate of turbot. The survival rates of SFE administration groups were higher than that of SFE 0 group. Therefore, the elevation of disease resistance against E. tarda is contributed to the regulation of immune response and antioxidant capacity by SFE administration. That is to say, SFE could affect mRNA levels of immune-related genes by activating Nrf2/Keap 1 and NF-κB signaling pathways, and then improved the antioxidant capacity and immunity of turbot, which also improved the ability of juvenile fish to resist E. tarda infection. It also suggests that antioxidant capacity, immunity, and disease resistance are inextricably linked.

Conclusions
Overall (Figure 5), the present study was apparently the first report that dietary SFE supplementation for 56 days promoted the immunity and antioxidant capacity through activating Nrf2/Keap1 and NF-κB signal pathways, thus improving the growth performance and disease resistance against E. tarda. SFE could be a potential feed additive for turbot.  Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee of Hebei Normal University (protocol code is 208007; approval date is 15 July 2020).
Informed Consent Statement: Not applicable. ministration significantly relieved the symptoms and increased the survival rate of turbot. The survival rates of SFE administration groups were higher than that of SFE 0 group. Therefore, the elevation of disease resistance against E. tarda is contributed to the regulation of immune response and antioxidant capacity by SFE administration. That is to say, SFE could affect mRNA levels of immune-related genes by activating Nrf2/Keap 1 and NF-κB signaling pathways, and then improved the antioxidant capacity and immunity of turbot, which also improved the ability of juvenile fish to resist E. tarda infection. It also suggests that antioxidant capacity, immunity, and disease resistance are inextricably linked.

Conclusions
Overall (Figure 5), the present study was apparently the first report that dietary SFE supplementation for 56 days promoted the immunity and antioxidant capacity through activating Nrf2/Keap1 and NF-κB signal pathways, thus improving the growth performance and disease resistance against E. tarda. SFE could be a potential feed additive for turbot.