Growth performance, anti-oxidative status, innate immunity, and ammonia stress resistance of Siganus rivulatus fed diet supplemented with zinc and zinc nanoparticles

1. Introduction

Siganids (rabbitfishes) are a small family of algaevorous fish that inhabit the tropical and subtropical waters of the Middle East and Indo-Pacific region (Saoud et al., 2007). The marbled spinefoot rabbitfish, Siganus rivulatus, has a promising potential opportunity for aquaculture due to its herbivorous feeding habits and acceptability of artificial diet (Barakat et al., 2011; Abou-Daoud et al., 2014). Accordingly, the farming of siganids could be economically efficient with low environmental impact due to low dietary animal protein and nitrogenous wastes (Roumieh et al., 2013). Also, this fish has an acceptable growth (Bariche, 2005), with high salinity and temperature tolerances, and capability of rearing in intensive culture (Saoud et al., 2007, 2008). Meanwhile, the aquaculture situation of S. rivulatus is still restricted in some of the Mediterranean and Red Sea offshore cage fisheries due to the lack of proper aquaculture protocols and suitable diets (Stephanou, 2007).

Zinc (Zn) is one of the most abundant essential trace elements in the fish body, participating in several physiological and biochemical functions due to its catalytic and/or co-catalytic properties (Swain et al., 2016). Zinc is a cofactor with the cytosolic antioxidant enzyme Cu-Zn-superoxide dismutase (Miao, Clair, 2009). Zinc is involved in protein digestion as a component of pancreatic carboxypeptidase A and B (Chen et al., 2016). Also, it regulates the metabolism of macro and micronutrients in the body (Davis, Cousins, 2000).

In addition, Zn regulates the activity of the cell via binding to specific receptors on cell membranes, carriers, and channels (Miao, Clair, 2009; Swain et al., 2016). Also, Zn regulates proliferation, differentiation, apoptosis, and the gene expression of metallothionein (Colvin et al., 2003). Hence, the activity of hundreds of enzymes and thousands of transcription factors in different animals species is closely related to Zn (Wang and Wang, 2015; Swain et al., 2016). Therefore, Zn deficiency disrupts fish growth, survival, immune function, and causes poor appetite, frequent infections, cataracts, and fins and skin erosion (Zalewski et al., 2005; Wang, Wang, 2015).

Fish can obtain Zn from water and feed; nevertheless, the presence of some antinutritional factors in the diet (tricalcium phosphate, phytate, or phytic acid) may reduce the bioavailability of minerals (Hossain et al., 2003). Moreover, Zn is a non-stored mineral (Zalewski et al., 2005). Therefore, continuous Zn supplement in feedstuff is necessary to cover animal requirements and prevent Zn deficiency (Hossain et al., 2003; Swain et al., 2016). In nature, there are several Zn sources, including Zn carbonate, chloride, oxide, and sulfate. However, the primary mineral element content is higher in the Zn oxide form (ZnO; 780 mg kg⁻¹) than other sources (NRC, 2007). The additional fortification of ZnO improves growth, feed utilization, immune response, antioxidative status, thermal tolerance, reproductive performance of animals, and acts as an antimicrobial agent (Lin et al., 2013; Tsai et al., 2016; Kumar et al., 2017b, b; Kumar et al., 2020a). However, the bioavailability of Zn from inorganic sources has been reported to be quite low (Alishahi et al., 2011; Sahoo et al., 2014). Therefore, amino acid-chelated Zn is used as a diet supplementation for higher absorption and better incorporation in the animal tissues (Mansour et al., 2017; Khalil et al., 2019). Among different organic Zn forms (Zn-lysine, Zn-glycine, and Zn-methionine), Zn-methionine (Zn-Met) is the best source for improving growth and immunity of several fish species (Shahpar, Johari, 2019; Du et al., 2020).

In addition to organic and inorganic forms of minerals as feed supplements, nanotechnology has introduced a new atomic or molecular scale of Zn with better characteristics and availability (Gowda et al., 2013). The nanoparticles of minerals have novel characteristics, including high specific surface area, activity, catalytic efficiency, stronger adsorbing ability, and higher bioavailability (Albanese et al., 2012; Sheikh et al., 2016). Zinc oxide nanoparticles (ZnO-NPs) have showed new characteristics of transport, uptake, and have exhibited higher absorption efficiencies (Albanese et al., 2012).

The ZnO-NPs (60 mg kg⁻¹) improved survival, growth, and immunity of Macrobrachium rosenbergii (Muralisankar et al., 2014). The growth and haematological parameters of grass carp, Ctenopharyngodon idella, improved with 30 mg kg⁻¹ ZnO-NPs dietary supplementation (Faiz et al., 2015). Furthermore, the diets supplemented with ZnO-NPs enhanced thermal tolerance of Pangasius hypophthalmus reared under lead contamination and also protected this fish against oxidative and cellular metabolic stress(Kumar et al., 2017a, a). The microdiets supplemented with a mixture of selenium, magnesium, and Zn nano-particles improved growth, bone mineralization, and the stress response of early-weaned gilthead seabream, Sparus aurata (Izquierdo et al., 2017). Meanwhile, the ZnO-NPs in higher levels found to induces acute toxicity in fish. Whereas, the LC₅₀ of ZnO-NPs at 96 h was 21.89 mg L⁻¹ in Pangasianodon hypophthalmus and causes deleterious alteration on histopathology and cellular and metabolic activities (Kumar et al., 2020b). Also, fish cells line exposed to high levels of ZnO-NPs showed membrane function disturbance and free radicals generation (García-Gómez et al., 2020).

The improvement of aquaculture feed is a significant step towards the establishment of successful and economic aquaculture production of marbled spinefoot rabbitfish, as feed represents the highest operational expense in the aquaculture industry (Ghanawi et al., 2011). In addition, dietary supplementation with different antioxidants, especially under aquaculture conditions, could improve the antioxidant balance, general physiological status, growth performance, and economic revenue of the cultured fish (Mansour et al., 2017; Sallam et al., 2017; Abdallah Tageldien Mansour, 2018; Mansour et al., 2018; Fayed et al., 2019; Khalil et al., 2019). Therefore, the present study was designed to investigate the efficiency of dietary supplementation of different Zn sources, including ZnO, Zn-Met, and ZnO-NPs, on growth performance, feed utilization, body composition, anti-oxidative status, innate immune response, and ammonia stress resistance of the rabbitfish, S. rivulatus.

2. Materials and methods

2.1. Experimental fish and rearing conditions

A total of 480 marbled spinefoot rabbitfish, S. rivulatus, specimens (0.20 ± 0.01 g of average body weight and 2.6 ± 0.10 cm average body length) collected from the Mediterranean Sea (Abu Qir Bay, Alexandria, Egypt) was used in the present study. The fish were reared in cages (1 × 1 × 0.6 m, L × W×H) set in an outdoor concrete pond (8 × 3 × 1.2 m, L × W×H) for three weeks to acclimate to the artificial diets and experimental conditions by feeding them the control diet (38.03 % protein and 20.23 mg kg⁻¹ gross energy). The experiment was conducted at the Marine Fish Rearing Unit (El-Max Research Station), National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt. After acclimatization, fish were randomly allocated in each group (n = 40 fish cage^-1, 3 cages group^-1).

The daily water exchange rate was 30 % of the total concrete tank volume. During the experimental trial, the water temperature was recorded daily and ranged 27 ± 1 °C. The dissolved oxygen (DO) was monitored weekly and was above 6.00 mg L⁻¹ determined by using a portable DO meter (YSI, model Pro20, USA). Total ammonia-N was monitored once a week and was less than 0.16 ± 0.01 mg L^-1, salinity averaged 32.00 ± 2.00 g L^-1 as determined by refractometer, and pH was 7.70 ± 0.300 as measured by a digital multi-meter (Crison, model MM41, Spain). Natural light: dark regime was applied in the feeding trial. Water Zn levels were lower than 0.04 as measured according to Li et al. (2006).

2.2. Experimental design and diets

Four isonitrogenous (38.03% protein) and isocaloric (20.23 mg kg⁻¹ gross energy) experimental diets were formulated to contain three sources of Zn (Table 1). The first diet served as the control diet without Zn supplementation. The other three diets were supplemented with 30 mg kg^-1 diet of Zn oxide (ZnO), Zn-methionine (Zn-Met), and Zn oxide nanoparticles (ZnO-NPs). The supplementation level was selected to cover the fish recommended dose by considering the basal diet content of Zn (Zhao et al., 2011; Muralisankar et al., 2014; Tawfik et al., 2017; Shahpar, Johari, 2019). The fish were hand-fed to apparent satiation three times a day (09:00, 12:00, and 16:00) for eight weeks.

The experimental diets were prepared by grinding all the ingredients and mixing with vitamins and minerals, then the respective dose of the different Zn sources was added. Warm distilled water (35 °C) was added slowly until the diets began clumping, then pelleted by mill machine and dried using a forced air oven before storing in plastic containers at −20 °C. The resulting pellet size was 0.2 mm diameter x2 mm length. The ingredients and proximate chemical analysis of the different diets followed AOAC recommendation (AOAC, 1995) and are presented in Table 1.

The different Zn sources were ZnO (powder form provided by El-Gomhoria Co., for Chemicals and Pharmaceuticals, Alexandria, Egypt), Zn-Met (Mintrix® Zn was obtained from United BioMed for feed additives, Cairo, Egypt), and ZnO-NPs (PubChem Substance ID: 329763818) provided by Sigma-Aldrich (Saint Louis, USA). The specifications of ZnO-NPs were < 100 nm particle size, > 10.8 m² g⁻¹ sur-face area, purity > 97 % as described previously (Abdel-Daim et al., 2019).

2.3. Determination of zinc concentration in the diets and water

Zinc concentrations in the water and diets were analyzed according to the method described previously (Li et al., 2006). Briefly, approximately 0.15 g of dried and finely ground samples were digested with 15 mL 65 % nitric acid and 2 mL 70 % perchloric acid using Kjeldahl flasks. After digestion, samples were diluted with deionized water to 50 mL, and Zn contents were determined by atomic absorption spectroscopy equipped with a graphite furnace (Model AA-240Z, Varian, Australia).

Growth performance, anti-oxidative status, innate immunity, and ammonia stress resistance of Siganus rivulatus fed diet supplemented with zinc and zinc nanoparticles - Image 1

2.4. Measured parameters

2.4.1. Growth performance

Growth performance was determined as weight gain (WG), growth coefficient (GC), length gain (LG), and condition factor (K), and food utilization parameters, such as feed intake, feed conversion ratio (FCR), and protein efficiency ratio (PER). Also, the mortality percent, relative percentage of survival (RPS) (Amend, 1981), and viscera-somatic index (VSI) were calculated by the following equations:

Weight gain (WG; g) = final weight − initial weight

Growth coefficient (GC; %) = 100 × [final weight^1/3 – initial weight^1/³/Temperature (Cº)]

Length gain (LG; cm) = final length − initial length.

Condition factor (K value) = 100 × [body weight (g)/length (cm⁻³)].

Feed conversion ratio (FCR) = feed intake/body weight gain.

Protein efficiency ratio (PER) = weight gain (g)/protein intake (g).

Mortality (%) = 100× [(initial fish number-final fish number)/initial fish number].

Relative percentage of survival (RPS; %) = [1 − (% mortality/% control mortality)] × 100.

Viscera-somatic index (VSI; %) = 100 × [viscera weight (g)/body weight (g)]

2.4.2. Whole-body composition

Proximate analysis of experimental diets and whole-body of fish were analyzed in triplicate as described previously (AOAC, 1995). In particular, the moisture content was determined after samples were dried in an oven at 105 °C for 12 h. the protein content was determined by measuring the total nitrogen (N × 6.25) levels using the Kjeldahl method following acid digestion with an Auto Kjeldahl System (Model BUCHI K358, Flawil, Switzerland). The fat content was detected by ether extraction using a Soxhlet System (Model VELP Scientifica, SER 148, Italy) and ash content was determined by muffle furnace at 550 °C for 5 h.

2.4.3. The SDS-PAGE technique

A portion of the minced whole body (0.1 g fresh weight) was suspended in 1.0 mL lysing buffer (0.5 M Tris HCl, pH 6.8, 30 % glycerol, 10 % sodium dodecyl sulfate, 0.06 % bromophenol blue, and 5% 2-Mercaptoethanol), boiled for 5 min, cooled, and centrifuged (12,000 x g for 10 min at 4 °C). The 12 % slab gel was applied according to Laemmli (1970). The samples and protein marker (50 μL) were loaded, and electrophoresis was performed at 75 V through the stacking gel followed by 125 V for approximately 120 min. The gel was stained by 0.1% coomassie blue R- 250 for 120 min. Densitometric analysis of the protein bands was performed by using Totallab analysis software (Ver.1.0.1). Also, cluster analysis was done using Past Cluster analysis using Past Software version 3.14 based on protein profile (Hammer et al., 2001).

2.4.4. Anti-oxidative and immune parameters

2.4.4.1. Tissue homogenate preparation. At the end of the experiment, two fish from each replicate were killed with an overdose of clove oil (5 mg L⁻¹). The bodies were washed using sterile chilled saline, kept in an icebox, and then stored at −80 °C until homogenization. Frozen tissues (without the head and alimentary canal) were minced and homogenized (10 % w/v) in ice-cold sucrose buffer (0.25 M) in a Wise Tis® HG-15D Homogenizer (Daihan Scientific, Bangalore, India). The homogenate was centrifuged at 7063 x g for 20 min at 4 °C. The resulting supernatant was collected and stored at −20 °C and used for different enzyme determinations of anti-oxidative and immune parameters (Sallam et al., 2017), and the pellets of tissues were used for determination of phenoloxidase activity.

2.4.4.2. Malondialdehyde content (MDA). The measurement of the malondialdehyde content (MDA) content was done by a commercial chemical colourimetrical assay kit according to the manufacturer’s protocol (MDA assay kit; ZellBio GmbH). It used the MDA-TBA adduct formed by the reaction of MDA and thiobarbituric acid (TBA) under high temperature (90−100 °C) (Cao et al., 1995). MDA is measured in acidic media colourimetrically at 535 nm. This method can determine the MDA with 0.1 μM sensitivity. The intra- and inter-assay coefficient of variation claimed to be 5.8 % and 7.6 %, respectively.

2.4.4.3. Catalase activity (CAT). Catalase activity (U mg⁻¹ protein; EC1.11.1.6) was measured according to Luck (1974). Briefly, a 10 μL tissue homogenate sample was added to 1.25 mL of freshly prepared buffer containing 50 μL of H₂O₂ and 10 mL^-1Na-K-phosphate buffer (0.15 M, pH 7, El-Gomhoria Co., Egypt). The difference in absorbance was recorded after 20 s (A1) and 80 s (A2) of incubation at 240 nm against air. The CAT value was calculated as A1-A2/0.0008.

2.4.4.4. Superoxide dismutase (SOD). Superoxide dismutase (U mg⁻¹ protein; EC 1.15.1.1) activity was evaluated according to Misra, Fridovich (1972). Briefly, 20 μL of tissue homogenate was added to 940 μL sodium carbonate buffer (pH 10.2, 0.05 M, El-Gomhoria Co., Egypt) and 40 μL epinephrine (30 mmol L⁻¹ dissolved by adding 30 μL of HCL, Sigma, USA). The inhibition of epinephrine auto-oxidation in the alkaline medium to adrenochrome was recorded after 30 and 90 s at 480 nm. A control was prepared as 960 μL sodium carbonate buffer and 40 μL epinephrine.

The per cent of inhibition (%) = 100- [( A control − A sample/ A control) – 100]. SOD activity in tissue homogenate (U g⁻¹ protein) = % inhibition × 3.75.

2.4.4.5. Glutathione peroxidase (GSH-Px). Glutathione peroxidase (U mg⁻¹ protein; EC 1.11.1.9) activity was assayed using the method of Flohé, Günzler (1984) in tissue homogenate. Briefly, 25 μL of tissue homogenate was incubated for 5 min at 37 °C with 375 μL Tris HCl buffer (pH 7.6, 0.05 M), 50 μL reduced glutathione (pH 7.6), and 50 μL cumene hydroperoxide (pH 7.6). Then, 0.5 mL trichloroacetic acid (15 %) was added to stop the reaction, the sample was vortexed and incubated for 10 min and centrifuged (7063 x g for 10 min at 4 °C). The supernatant (250 μL) was transferred to 0.5 mL Tris HCl buffer (pH 8.9), and 25 μL of 5,5′-dithiobis (2-nitrobenzoic acid; DTNB) was added. The absorbance was read at 412 nm (Spectrophotometer PD-303 UV, APEL, Japan). A control tube was prepared for each sample by adding cumene after adding trichloroacetic acid.

2.4.4.6. Lysozyme activity. The lysozyme activity (U mg⁻¹ protein, EC3.2.1.17) in tissue homogenate was measured via a turbidimetric assay, according to Ellis (1990) with some modifications. Briefly, aliquots of 25 μL of tissue homogenate were added to 1 mL suspension of Micrococcus lysodeikticus (0.2 mg mL⁻¹ in a 0.05 M sodium phosphate buffer, pH 6.2), and the absorbance was measured at 670 nm after 30 s and 180 s by a spectrophotometer (Spectrophotometer PD-303 UV, APEL, Japan).

2.4.4.7. Phenoloxidase activity. The phenoloxidase activity (U mg⁻¹ protein, EC 1.10.3.1) was determined according to the method of Pérez-Jar et al. (2006) with some modifications (Huang et al., 2010). The resulting pellet from tissues homogenization and centrifugation was rinsed, re-suspended gently in a cacodylate-citrate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.10 M trisodium citrate, pH 7.0), and centrifuged again. The pellet was then re-suspended with 200 μL cacodylate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.01 M calcium chloride, 0.26 M magnesium chloride, pH 7.0), and a 100 μL aliquot was incubated with 50 μL trypsin (1 mg/mL), which served as an activator, for 10 min at 25–26 °C, 50 μL of DOPA was then added, followed by 800 μL of cacodylate buffer 5 min later. The optical density at 490 nm was measured using a spectrophotometer.

2.4.5. High ammonia challenge test

At the end of the feeding trial (8-weeks), twenty fish from each dietary group were selected randomly and transferred into two aquaria (35 × 30 × 40 cm; 35 L) at the same rearing conditions. Fish were starved for 24 h then challenged by dissolving requisite amounts of ammonium chloride (NH₄Cl; El-Nasr Chemicals Co., Egypt) to reach the total ammonia-N concentrations of 20 mg L⁻¹ according to Guo et al. (2018). The concentration was confirmed using a photometer (Milwaukee, model Mi 405, USA). The water temperature was 27 °C, pH 7.7, and salinity was 32 g L^-1. Aeration was provided continuously during the trial. Fish were carefully monitored, and the mortality was recorded at 1, 3, 5, and 7 h during the challenge experiment. The cumulative mortality (%) in different treatment groups was calculated by the following formula:

2.5. Statistical analysis

All data were statistically analyzed as a completely randomized design by ANOVA using SPSS (Standard version 17.0; SPSS, Chicago, IL, USA). Tukey test was used to compare the differences between means when significant F values were observed at the p ≤ 0.05 levels. All percentage data were arc-sign-transformed prior to analysis (Zar, 1984). However, data are presented in an untransformed form to facilitate treatment comparison. The results of the ammonia challenge test were presented in a Kaplan-Meier cumulative mortality curve (SigmaPlot® version 11).

3. Results

3.1. Growth performance

The growth performance in terms of FBW, WG, and GC of marbled spinefoot rabbitfish, S. rivulatus, increased significantly with Zn-Met and ZnO-NPs supplemented treatments compared to the control and ZnO treatments (Table 2). The final length, length gain, and condition factor tended to increase with all Zn supplemented treatments compared to the control group. The FCR and PER improved significantly with all Zn supplementation treatments, and the best results were obtained with ZnO-NPs. The mortality per cent ranged between 0.0–8.89% in all treatments without any pathological signs and the least mortality was recorded with Zn-Met treatments.

3.2. Whole-body composition

The whole-body proximate chemical composition of S. rivulatus showed a significant increase in the crude protein of fish fed ZnO-NPs supplemented diets (Table 3). Additionally, ether extracts decreased significantly with dietary Zn-Met compared to other treatments. However, the ash and moisture contents and VSI did not differ significantly among the studied treatments.

3.3. Body protein fractions

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a molecular biomarker technique for body protein bands of different dietary Zn source treatments is presented in Fig. 1 and Table 4. The values of protein fractions molecular weight (kDa) are presented in Tables 4. In all treatments, eight distinctive bands were detected, and the control bands started lower than the other bands for Zn supplemented treatments. The molecular weight of the control bands ranged from 90.78 to 32.19 kDa and represented the narrowest range. Meanwhile, the ZnO-NPs bands had the widest molecular weight range (108.25 to 20.09 kDa). The densitometric analysis (Fig. 1) and cluster analysis (Fig. 2) illustrated that fish fed diets supplemented with ZnO and ZnO-NPs were more similar than the control and Zn-Met groups.

3.4. Anti-oxidative and innate immunity parameters

The Zn supplemented treatments significantly decreased MDA levels and increased anti-oxidative enzyme activities (SOD, CAT, and GSH-Px), especially with the Zn-Met and ZnO-NPs forms (Fig. 3). The innate immune response of S. rivulatus fed different Zn forms illustrated significant improvements of lysozyme, and phenoloxidase activities compared to the control. The highest immune surveillance was recorded with ZnO-NPs, followed by Zn-Met treatment (Fig. 4).

3.5. Challenge test

The challenge with the acute ammonia test showed higher endurance of fish fed Zn (organic and nanoparticles forms) supplemented diets than fish fed the control diet. The lowest mortality (10 %) was recorded with ZnO-NPs and Zn-Met supplementation compared with 25% in the control group after 7 h of treatment. Moreover, increasing the exposure time increased the mortality rate of exposed fish in all treatments, but to a lesser degree in Zn-Met and ZnO-NPs compared with the control group (Fig. 5).

4. Discussion

Under the aquaculture system conditions, fish are exposed to several effects, from high intensity, improper handling, and being forcing to nourish by artificial diets (Kumar et al., 2014, 2016; Kumar et al., 2017a). Providing an artificial fish diet with essential elements is an important aquaculture practice for maintaining normal growth performance, proper physiological status, and maximize animal resistance against biotic and abiotic stressors (Kumar et al., 2017a; Khalil et al., 2019; Kumar et al., 2020a,b). Zinc represents the second microelements in the fish body and takes part in several enzymatic processes of animal metabolism (Imamoglu et al., 2005; Zalewski et al., 2005; Rajan et al., 2016; Tawfik et al., 2017; Uniyala et al., 2017). However, it cannot be stored in the body, so regular dietary Zn intake is, therefore, a necessity (Case, Carlson, 2002; Zalewski et al., 2005). Accordingly, the present study examined, for the first time, the efficacy of supplementation from different Zn sources (inorganic, organic, and nano-particle Zn forms) on growth performance, antioxidative status, immune response, and ammonia stress resistance of marbled spinefoot rabbitfish, S. rivulatus, the new emerging aquaculture species with promising characteristics.

The growth performance and feed utilization of fish fed Zn-supplemented diets surpassed the control fish, and Zn-Met and ZnO-NPs are greater growth stimulators than ZnO in bulk form in the current study. In line with the current findings, the dietary supplementation of 60 mg kg⁻¹, in both organic and inorganic forms, significantly improved growth performance and protein utilization of Nile tilapia, Oreochromis niloticus (Zhao et al., 2011; Tawfik et al., 2017). Also, dietary supplementation with the same ZnO-NPs level (60 mg kg^-1) has the ability to improve survival, feed intake, and growth of freshwater prawn, M. rosenbergii, and rainbow trout, Oncorhynchus mykiss (Shahpar, Johari, 2019; Thirunavukkarasu et al., 2019). Zn-Met supplementation improved growth performance and survival of Para-misgurnus dabryanus (Du et al., 2020). The growth performance and feed utilization of the grass carps, C. idella and P. hypophthalmus, were significantly improved with dietary supplementation of ZnO-NPs (Faiz et al., 2015; Kumar et al., 2018a).

The improvement of growth performance and feed utilization in all Zn supplemented groups could attribute to the role of Zn in a wide variety of physiological processes, including, sensing, digestion catalysis, and energy storage (Wang, 2004). Also, Zn supplementation revealed an improvement in the antioxidant balance and immune status of fish in the current study (Fig. 3 and 4). In addition, Zn supplementation in both organic and nanoparticles forms improved protein digestion by stimulating the secretion of Zn-containing endopeptidase (Rajan et al., 2016; Uniyala et al., 2017). Also, the improvement of growth associated with ZnO-NPs supplementation could be attributed to the stimulation of DNA and RNA synthesis, which leads to better regulation of cell function and division (Siklar et al., 2003) and improvement in growth hormone synthesis (Imamoglu et al., 2005; Tawfik et al., 2017). The high efficiency of both organic and ZnO-NPs when compared to ZnO in bulk form could be attributed to the higher intestinal absorption and bioavailability (Case, Carlson, 2002; Alishahi et al., 2011; Sahoo et al., 2014). Moreover, the low molecular size of ZnO-NPs increases the surface area to volume ratio, resulting in greater activity and a wider energy confinement level (Wang, 2004).

The interaction of Zn in carbohydrate and fatty acid metabolism via increasing glucose-6-phosphate dehydrogenase (G₆PD) activity (Murray et al., 2003), could also explain the significant increase in growth performance in the present study. Hence, it could reduce the catabolism of protein and increase glucose synthesis from carbohydrate (protein-sparing effect), accordingly protein retention increased and nitrogen wastes decreased (Zhao et al., 2011; Taheri et al., 2017). In the same sense, the current results revealed an increase in whole-body protein content and a decrease in the ether extract with Zn-Met and ZnO-NPs treatments as a suggested result of its protein-sparing effects.

The electrophoretic investigation of whole-body body homogenate in the present study showed different similarity protein band patterns among the control group and other Zn supplemented treatments depending on the bands molecular weight. However, the SDS-PAGE technique could only show differences in macro proteins according to their electrophoretic mobility, but it cannot identify the isolated proteins (Muhammad et al., 2018). The protein variation is an indicator of the physiological, nutritional, and health status of the animal (Knowles et al., 2006). The ZnO and ZnO-NPs treatments were electrophoretically more similar in the present findings, which could be due to both Zn sources have the same chemical formula (Zn to oxygen ratio) but it differ in the smaller size of ZnO-NPs which increases the energy confinement (Wang, 2004).

The dietary supplementation of different Zn forms in the current study improved the oxidant/antioxidant balance in favour of Zn-Met and ZnO-NPs. In accordance with the current findings, Zn supplementation reduced the MDA and protein carbonyls content, like lipids and protein peroxidation indicators, in the mitochondria and micro-somal membranes (Tapiero, Tew, 2003; Feng et al., 2011). The MDA value is an indicator for superoxide anion, hydroxyl, and hydrogen peroxide levels (Devasena et al., 2001). Therefore, reducing the MDA level, which is normally produced in the animal body during regular metabolism, phagocytosis process or during stress exposure, could participate in the protection of the cells from oxidative injuries (Tapiero, Tew, 2003; Miao, Clair, 2009). In addition, dietary Zn supplementation improved different antioxidant enzymes activity including, SOD, GSH-Px, and CAT in Cyprinus carpio (Feng et al., 2011). Similarly, dietary ZnO-NPs supplementation enhanced SOD, CAT activities, and total antioxidative status in O. niloticus (Awad et al., 2019) and marine medaka, Oryzias melastigma, (Wang et al., 2017) and im-proved erythrocytic and plasma SOD activity of aged laying hens (Tsai et al., 2016). ZnO-NPs increased the activity of enzymatic and non-enzymatic cellular antioxidant defences and reduced free radical levels (Prasad et al., 2017).

The positive effect of Zn on the antioxidant system could be attributed to its role as a cofactor for cytoplasm and extra-cellular SOD and the ability of Zn to maintain the activities of radical scavenging enzymes (Miao, Clair, 2009; Kumar et al., 2014, 2016). In addition, the organic and nanoparticles of Zn forms have higher availability, absorption, and incorporation in animal tissues than inorganic forms, which further facilitates SOD formation and activity (Albanese et al., 2012; Lin et al., 2013; Sheikh et al., 2016; Tsai et al., 2016). This suggests that the antioxidative effect of Zn may be attributed to the increase of antioxidant enzyme mRNA levels (Muthuraman et al., 2014). Moreover, increasing the ZnO-NPs supplementation level increased G₆PD activity (Taheri et al., 2017), which, in turn, increased NADPH production and led to the regulation of redox activity and coping with reactive oxygen species (Stanton, 2012).

The current findings revealed that the highest immune surveillance was recorded with both groups fed with supplemental Zn-Met and ZnO-NPs compared to ZnO and the control treatments. These results are similar to those found by Gopal et al. (1997), who found a sharp increase in the serum globulin level of Cyprinus carpio fed with a ZnO-NPs supplemented diet. In addition, the total protein, IgM titers, and antioxidant balance improved with increasing concentrations of ZnO-NPs in the diet of O. niloticus and P. hypophthalmus (Tawfik et al., 2017; Kumar et al., 2018a).

The phenoloxidase and lysozyme activities increased in the present study with Zn supplementation treatment. The phenoloxidase cascade is one of the important innate cell-mediated immunity mechanisms in animals (Travers et al., 2008). In accordance, phenoloxidase activity improved with increasing Zn levels in inorganic or organic form in the marine gastropod, Haliotis tuberculate and shrimp, L. vannamei, respectively (Lin et al., 2013). Also, the ZnO-NPs based β-glucan binding protein increased the lysozyme and myeloperoxidase activities in the serum of O. mossambicus (Anjugam et al., 2018).

Zn supplementation upregulated the expression of lysozyme mRNA in the gills of the L. vannamei and increased its activity in different tissues (Guo et al., 2011). Lysozyme is a non-specific humoral immune defence against both gram-positive and gram-negative bacteria and participates in the activation of complement cascade and phagocytosis (Saurabh, Sahoo, 2008). Therefore, the improvement of lysozyme and phenoloxidase activities in the current study resulted in an improvement of both cellular and humoral innate immune defences in fish fed Zn supplemented diets.

The improvement of immune status could also be attributed to the reduction of the Zn macromolecule to the nanoscale, which changed its properties and increased its efficacy (Rather et al., 2011). Hence, nanoparticles can stimulate the innate and adaptive immune response (Luo et al., 2015), by activating toll-like receptors (Lucarelli et al., 2004), and down-regulating pro-inflammatory cytokines (Tawfik et al., 2017). In addition, Zn has a strong positive antioxidant properties which could attenuate antioxidant-immune interaction (Miao, Clair, 2009; Kumar et al., 2014, 2016).

In addition, the challenge of S. revulatus with a total of ammonia nitrogen equivalent to unionized ammonia (NH³⁻; 0.70 mg L^-1) in the current study showed a mortality rate that ranged between 10–25 %. The ammonia level in the aquaculture system is one of the most detrimental water quality criteria for fish intensification (Foss et al., 2003; Ip et al., 2004). The increasing of total ammonia nitrogen above the toxic threshold could induce mass loss in the farmed fish (Randall, Tsui, 2002; Ip et al., 2004). The concomitant mortality to ammonia toxicity could be attributed to the disruption of osmoregulation (Roumieh et al., 2013) and the neuronal transmembrane potential, which in turn leads to hyper-excitability, hyperventilation, insufficient oxygen uptake, and convulsions (Ip et al., 2004). Moreover, high ammonia induces severe histological and ultrastructural alterations in gills (Roumieh et al., 2013) and liver of exposed S. rivulatus (Abdelmeguid et al., 2013). Meanwhile, the current findings and the results of Roumieh et al. (2013) revealed a high relative capability of S. rivulatus to tolerate ammonia exposure compared with other aquaculture species.

In addition, the dietary supplementation of Zn in different forms improved fish tolerance to ammonia exposure, especially with Zn-Met and ZnO-NPs treatments. The improvement associated with Zn supplementation in the present study could be attributed to the improvement of the anti-oxidative and immune status of treated fish as Aquaculture Reports 18 (2020) 100410 confirmed in the present findings (Fig. 1 and 2). Whereas ammonia exposure increased the level of free radicals (MDA and protein carbonyls contents) (Ching et al., 2009). In accordance, selenium supple-mentation alleviated the oxidative stress and maintained the normal liver ultrastructure against ammonia exposure, which could be due to the enhancement of antioxidative capacity (Guo et al., 2018). Also, ZnO-NPs improved the resistance of P. hypophthalmus, including antioxidant enzymes, immune parameters, and reduced stress indicators against biotic and abiotic multi-stressors (Kumar et al., 2018a).

5. Conclusion

The current findings revealed a significant improvement of growth performance, feed utilization, and antioxidant balance, in terms of high SOD, CAT, and GSH-Px activities and low MDA levels, with Zn-Met and ZnO-NPs forms dietary supplementation. Moreover, the innate immune status, in terms of lysozyme and phenoloxidase activities, and ammonia stress resistance were improved in S. rivulatus with both organic and nanoparticles of Zn forms. The continuous supply of Zn as non-stored essential mineral must be assured in the S. rivulatus diet to maintain proper physiological, nutritional, and growth performance and the recommended forms of Zn is Zn-Met and ZnO-NPs rather than ZnO in inorganic form.

Credit authorship contribution statement

Ahmed Elsayed Sallam: Conceptualization, Data curation, Formal analysis, Funding acquisition, Software, Supervision, Validation. Abdallah Tageldein Mansour: Conceptualization, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Ahmed Saud Alsaqufi: Funding acquisition, Visualization, Writing - original draft, Writing - review & editing. Mohamed El-Sayed Salem: Conceptualization, Investigation, Methodology, Project administration, Resources. Mohamed M.M. El-Feky: Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources.

Declaration of Competing Interest

The authors declare no conflict of interest among authors and organization.

Acknowledgements

The authors sincerely acknowledge the Fish Nutrition Laboratory staff members at the National Institute of Oceanography and Fisheries (NIOF) for their support and cooperation in the study. The authors would like to thank Aquaculture Division, NIOF, Egypt, for Providing partial financial support for this study. We gratefully acknowledge King Faisal University, Saudi Arabia and Alexandria University, Egypt for partially funding this study.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aqrep.2020.100410.

This article was originally published in Aquaculture Reports 18 (2020) 100410 https://doi.org/10.1016/j.aqrep.2020.100410. This is an Open Access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).