The evaluation of growth performance, blood health, oxidative status and immune-related gene expression in Nile tilapia (Oreochromis niloticus) fed dietary nanoselenium spheres produced by lactic acid bacteria

Aquaculture 515 (2020) 734571

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The evaluation of growth performance, blood health, oxidative status and immune-related gene expression in Nile tilapia (Oreochromis niloticus) fed dietary nanoselenium spheres produced by lactic acid bacteria

T

Mahmoud A.O. Dawooda,∗, Mohsen Zommarab, Nabil M. Eweedaha, Azmy I. Helala a b

Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt Department of Dairy Science, Faculty of Agriculture, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt

ARTICLE INFO

ABSTRACT

Keywords: LAB-Fermented selenium Growth performance Immunity Nile tilapia Oxidative status

The present study was conducted to investigate the effects of lactic acid bacteria-produced nanoselenium spheres (LAB-Se) on the growth performance, blood health, oxidative status and immune-related gene expression in Nile tilapia (initial weight, 14.03 ± 0.04 g). LAB-Se was incorporated into the basal diet at 0.5, 1, 1.5 and 2 mg/kg of diet and fed to the fish for 8 weeks. The results revealed significantly improved final body weights, weight gain, specific growth rates and feed efficiency ratios in fish fed varying levels of LAB-Se (quadratic, P = 0.002, P = 0.002, P = 0.002, and P = 0.04, respectively). The blood haematology and biochemistry parameters of the fish fed varying levels of LAB-Se showed normal values with nonsignificant (P > 0.05) differences, indicating the non-toxic effect of LAB-Se. Additionally, dietary LAB-Se had significant influences on blood total protein and lysozyme and phagocytic activities in tilapia in a dose-dependent manner (quadratic, P = 0.04, P = 0.02, and P = 0.001, respectively). Dietary LAB-Se significantly increased SOD and CAT enzyme activities and decreased MDA levels (quadratic, P = 0.001, P = 0.03, and P = 0.007, respectively); GPX was not significantly affected by LAB-Se inclusion the diets (P > 0.05). Liver and spleen TNF-α expression was significantly upregulated in the fish fed LAB-Se (quadratic, P = 0.007 and P = 0.009, respectively). The expression of liver IL-1β was also upregulated significantly in the fish fed LAB-Se (quadratic, P = 0.005), while spleen IL-1β was not significantly affected by LAB-Se (P > 0.05). Thus, LAB-Se at 1 to 2 mg/kg can be effectively supplemented in tilapia diets to improve growth, oxidative status and immune-related gene expression.

1. Introduction Selenium (Se) is an indispensable microelement that is vital for the improved performance and wellbeing of aquatic animals (Pacitti et al., 2016). Se can protect animal cells from oxidation due to several stressors, including high-density populations, transportation, poor water quality, and infectious diseases (Hefnawy and Tórtora-Pérez, 2010; Pacitti et al., 2016). Se increases the activities of antioxidant-related enzymes and thyroid hormone metabolism and improves reproductive performance (Rider et al., 2009; Rotruck et al., 1973). Se is present in fishmeal and as a major ingredient in fish diets, but further inclusion of Se is highly recommended to achieve the requirements of some fish species (Le and Fotedar, 2013, 2014; Zhu et al., 2012). Aquatic animals require highly balanced diets that contain macro- and micro-ingredients to maintain high metabolic rates associated with increased growth performance (Dawood and Koshio, 2019; Sarkar et al., 2015). The

∗

addition of Se in fish diets can be affected by the form of Se, diet formulation, fish body size and species (Zhu et al., 2017). Normally, Se occurs in both organic and inorganic forms and has been included in fish diets (Saffari et al., 2017). Recently, Se in nanoparticle form (nanoSe) has been applied due to its high bioavailability and low harmfulness when fed to fish in adequate amounts (Dawood et al., 2019a,b; Godin et al., 2015; Izquierdo et al., 2017; Saffari et al., 2017, 2018; Terova et al., 2018). Additionally, nano-Se improved the growth, feed utilization and antioxidant defence capacity of numerous cultured fish (Ashouri et al., 2015; Naderi et al., 2017a,b,c; Saffari et al., 2017). Fermentation technology for the production of nanoselenium spheres by lactic acid bacteria (LAB-Se, Lactomicrosel®) is a new strategy in the production of Se. Lactomicrosel® is fermented by lactic acid bacteria (LAB) and has a similar role as natural nanoselenium spheres (Eszenyi et al., 2011). The new technology involved a manufacturing method that allows for suspension and powder formation with

Corresponding author. E-mail addresses: [email protected], [email protected] (M.A.O. Dawood).

https://doi.org/10.1016/j.aquaculture.2019.734571 Received 27 July 2019; Received in revised form 3 October 2019; Accepted 4 October 2019 Available online 13 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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distinctive features that contain precious nanoselenium spheres (Prokisch et al., 2008; Saleh, 2014). Materials prepared in this manner can be used as functional feed additives (Prokisch et al., 2008; Saleh, 2014). Nano minerals are known to be stable during feed preparation and fish feeding, without losing bioactive molecules, and allow the efficient acquisition of active molecules into biological systems at a lower cost than the current cost of adding minerals; they are associated with lower environmental impacts than the current impacts of minerals on the environment (El Basuini et al., 2016; Terova et al., 2018). The production method depends on the use of probiotic LAB, including Lactobacillus spp., Streptococcus spp. or yeast to form LAB-Se (Bai et al., 2019; Dalia et al., 2018; Pacitti et al., 2015, 2016). Bai et al. (2019) concluded that zebrafish fed bio-fermented Se exhibited improved growth performance and nonspecific immunity when Se was supplemented at the optimum level (3 mg/kg); however, excessive Se supplementation affected the health status of the fish. Furthermore, yeastSe resulted in increased stress resistance in gilthead seabream (Sparus aurata) (Izquierdo et al., 2017). LAB-Se seems to be more effective to fish, but its detailed effects on the aquatic animals remains to be studied. Accordingly, the aim of this work is to investigate the feasibility of using LAB-Se as a feed supplement for tilapia and to study its influence on growth performance, oxidative status and immune-related gene expression.

Table 1 Basal diet and proximate chemical composition (on dry matter basis). Ingredient Fish meal Soybean meal Wheat bran Yellow corn Rice bran Fish oil Dicalcium phosphate Vitamins and minerals mixturea

%

Chemical composition

%

10 44.4 10 18.6 10 5 1 1

Dry matter Crude protein Ether extract Total ash Gross energy (kcal/100 g)b

92.8 30.9 7.1 7.2 446

a Vitamin mixture (mg kg−1 premix): vitamin A (3300 IU), vitamin D3 (410 IU), vitamin E (2660 mg), vitamin B1 (133 mg), vitamin B2 (580 mg), vitamin B6 (410 mg), vitamin B12 (50 mg), biotin (9330 mg), colin chloride (4000 mg), vitamin C (2660 mg), inositol (330 mg), para-amino benzoic acid (9330 mg), niacin (26.60 mg), pantothenic acid (2000 mg), manganese (325 mg), iron (200 mg), copper (25 mg), iodine, cobalt (5 mg). b Gross energy was calculated as 5.65, 9.45, and 4.11 kcaL/g for protein, lipid, and carbohydrates, respectively.

Kafrelsheikh, Egypt. The fish were acclimated to the laboratory conditions in fibreglass aquaria for 14 days before being randomly distributed into fifteen 70-l capacity glass aquaria (15 fish/aquarium) in triplicate, with four dietary groups (0.5, 1, 1.5 and 2 mg/kg feed) and a control group (0 mg/kg feed). The fish in all the treatment groups were fed at a rate of 4% of their body weight for the first two weeks and 3% for the remainder of the experiment. Each diet was fed to the fish twice daily for a period of 8 weeks. The fish waste and half of the aquarium water were siphoned daily and replaced by well-aerated and dechlorinated water. All the fish were carefully weighed in bulk bi-weekly to determine growth, check their health condition and adjust their feed ration according to the mean fish weight. The fish were sedated using MS222 (Argent Laboratories, Redmond, Washington) to reduce handling stress. During the rearing period, there were no significant differences in water quality parameters due to LAB-Se supplementation. The water temperature was 26.1 ± 0.3 °C, and the dissolved oxygen was 6.2 ± 0.42 mg/L. The pH was 7.36 ± 0.12, and the unionized ammonia concentration was 0.22 ± 0.1 mg/L as measured by DREL/2 HACH kits (HACH Co., Loveland, Co.).

2. Materials and methods 2.1. Preparation of selenium nanoparticles and the experimental diets Nanoselenium spheres were produced by lactic acid bacteria (LABSe, Lactomicrosel®) as previously mentioned by Saleh (2014) and Prokisch et al. (2008). Selenium nanoparticles were prepared from pure yoghurt cultures of Streptococcus thermophilus (CNCM I-1670) and Lactobacillus delbrueckii subsp. bulgaricus (NCAIM B 02206), which was obtained from the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary. MRS medium was amended with 20 mg of filter-sterilized (SARTORIUS AG, Germany) selenium Se (IV) (sodium selenite, Na2SeO3.5H2O, SIGMA-ALDRICH, Switzerland) and incubated at 40 °C for up to 24 h. The media were centrifuged at 4500 g for 20 min at room temperature to spin down the bacteria cells. The cell pellets were washed 2 times with Tris-HCl buffer (50 mM, pH 7.5) and finally with ultra-pure water to obtain the Se nanoparticle fortified cell fraction. The Se content was analysed by inductively coupled plasma mass spectrometry (ICP-MS) (X series, THERMO FISHER SCIENTIFIC, Germany). The obtained selenium nanospheres were within the range of 100-500 nm, as suggested by Prokisch et al. (2008) and Eszenyi et al. (2011). Five experimental diets (31% crude protein and 7.1% lipids) were formulated to contain LAB-Se at a rate of 0 (control), 0.5, 1, 1.5 and 2 mg/kg of diet. The proximate chemical compositions of the main ingredients of the test diets are shown in Table 1. The dry ingredients of each diet were thoroughly mixed, and 100 ml of water per kg of diet was added. Afterwards, the mixture (ingredients and water) was blended to form a paste. Pelleting was carried out by passing the blended mixture through a laboratory pellet machine with a 1-mm diameter. The pellets were dried for 24 h at 57 °C in a drying oven and stored in plastic bags at -20 °C. The Se contents of the test diets were confirmed by the digestion of samples in nitric acid (AOAC , 1998). The concentration of Se in the diluted digested solution was determined using an Atomic Absorption Spectrophotometer-Graphite furnace (GBCAvanta E, Victoria, Australia). The actual Se content in the test diets was 0.08, 0.54, 1.11, 1.62 and 2.14 mg/kg of diet.

2.3. Blood and tissue sampling At the end of the 8-week feeding trial, all the fish were anaesthetized using 150 mg/l MS222 (Argent Laboratories, Redmond, Washington). The weight of each individual fish was measured. Blood samples were collected from the caudal blood vessels of 3 fish per aquaria using a syringe without an anticoagulant, and the serum was separated by centrifuging the clotted blood at 3000 rpm for 15 min at 4 °C and stored at -20 °C. After blood sampling, spleen and liver tissue samples were collected immediately from the same fish and kept in RNAlater (Bioshop, Germany) at -80 °C for RNA extraction and gene expression. 2.4. Blood haematology, biochemical and oxidative parameters Red blood cells (RBCs) and white blood cells (WBCs) were immediately counted with a haemocytometer after dilution with Natt and Herrick's solution (Houston, 1990) following the methods of Stoskopf (1993). For differential leucocytic counts, blood films were prepared and stained according to Lucky (1977), while cell counts were calculated by following Schalm (1986). For the haemoglobin assay, Drabkin's solution was added to blood samples, and the solution was centrifuged (3500 g for 6 min) to remove interferents. Afterwards, the blood haemoglobin concentration was determined with a spectrophotometer (Model RA 1000, Technicon Corporation, USA) at 540 nm using the method of Blaxhall and Daisley (1973).

2.2. Experimental conditions and experimental design Nile tilapia (O. niloticus), fingerlings, with an average initial weight of 14.03 ± 0.04 g, were obtained from a private farm located in 2

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The serum total protein, albumin, globulins, urea, uric acid, glucose, alanine aminotransferase, aspartate aminotransferase, and creatinine contents were determined with a RA-50 chemistry analyser (Bayer) using readymade chemicals (kits) supplied by Spinreact Co., Spain, according to the manufacturer's instructions. Serum superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT), and glutathione peroxidase (GPX) were measured using diagnostic reagent kits following the manufacturer's instructions (Cusabio Biotech Co., Ltd., China).

weights, respectively, and T is the experimental period (days). The obtained data were subjected to one-way ANOVA to evaluate the effect of dietary LAB-Se supplementation. Differences between the means were tested at the 5% probability level using Duncan's test as a post hoc test. Polynomial contrasts were used to detect linear and quadratic effects of various dietary LAB-Se levels on the observed response variables. The optimum LAB-Se level was determined using a polynomial regression analysis (Yossa and Verdegem, 2015). All the statistical analyses were performed using SPSS version 22 (SPSS Inc., IL, USA).

2.5. Phagocytic and lysozyme activities

3. Results

Following Kawahara et al. (1991), phagocytic activity was determined. The number of phagocytes was counted to calculate the phagocytic index using the following equations: phagocytic activity = macrophages containing yeast/total number of macrophages × 100; and phagocytic index = number of cells phagocytized/ number of phagocytic cells. Lysozyme activity was evaluated according to Parry et al. (1965).

3.1. Growth performance and feed utilization Table 3 represents the growth, feed efficiency and survival results. The survival rates were 91.7% and 98.3%, without significant alterations (P > 0.05). It was observed that dietary LAB-Se had a positive quadratic influence on fish FBW, WG, SGR and FCR in a dose-dependent manner (P = 0.002, P = 0.002, P = 0.002, and P = 0.04, respectively) (Table 3). The relationships between FBW (g), WG (%), SGR (%g/day), and FCR and dietary LAB-Se levels (Fig. 1) were expressed by the following secondorder polynomial regression equations (quadratic): FBW (y = 11.11 × 2 + 31.82x + 39.82, R2 = 0.9648, optimal dose = 1.43 mg/kg, Fig. 1A), WG (y = -76.897 × 2 + 219.62x + 184.1, R2 = 0.9666, optimal dose = 1.42 mg/kg, Fig. 1B), SGR (y = -0.3597 × 2 + 1.0191x + 1.7578, R2 = 0.9843, optimal dose = 1.41 mg/kg, Fig. 1C), FCR (y = 0.4173 × 2 1.0228x + 1.7145, R2 = 0.9373, optimal dose = 1.23 mg/kg, Fig. 1D).

2.6. Total RNA extraction, cDNA synthesis and real-time quantitative PCR assay Total RNA from the tested samples was extracted using easy-RED Total RNA Extraction kits (iNtRON Biotechnology, Inc., Korea) according to the manufacturer's instructions. RNA integrity was verified by agarose gel electrophoresis, while the concentrations and purities of the samples were examined with a NanoDrop spectrophotometer. Firststrand cDNA was synthesized using a HiSenScript cDNA Synthesis kit (iNtRON Biotechnology, Inc., Korea). Specific primers were used to amplify the selected genes, with; βactin was used as a housekeeping (internal standard) gene because it was stable among the test groups (Table 2). The mRNA abundance of tumour necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) was measured in liver and spleen samples using real-time quantitative PCR (qRT-PCR). The qRT-PCR assay was carried out using a Stratagene MX3005P real-time PCR system (Agilent Technologies, USA) and TOPreal™ PreMIX SYBR Green qPCR master mix (Enzynomics, cat. RT 500) following the manufacturer's recommendations. MxPro qPCR software was used for data collection. The relative gene expression levels were evaluated using the 2−ΔΔct method, as described by Pfaffl (2001). All the samples were analysed in triplicate, and no template controls or negative RT controls were included on the plates.

3.2. Blood haematology and biochemistry indices The blood haematology and biochemistry parameters of fish fed varying levels of LAB-Se showed normal values with nonsignificant differences (P > 0.05; Tables 4 and 5). 3.3. Blood immunity and phagocytosis Dietary LAB-Se has a significant quadratic influence on blood total protein and lysozyme and phagocytic activities in a dose-dependent manner (P = 0.04, P = 0.02, and P = 0.001, respectively) (Table 6). The relationships of blood total protein and lysozyme and phagocytic activities with dietary LAB-Se levels (Fig. 2) were expressed by the following second-order polynomial regression equations (quadratic): total protein (y = -0.6786 × 2 + 1.6595x + 3.5514, R2 = 0.8296, optimal dose = 1.22 mg/kg, Fig. 2C), lysozyme activity (y = 5.4148 × 2 + 12.611x + 20.425, R2 = 0.868, optimal dose = 1.16 mg/kg, Fig. 2A), and phagocytic activity (y = 0.1471 × 2 + 0.486x + 1.7098, R2 = 0.9014, optimal dose = 1.65 mg/kg, Fig. 2B).

2.7. Growth parameters and statistical analysis The following equations were used to calculate the growth performance parameters: Weight gain (WG) = [(FBW-IBW)/IBW] × 100; specific growth rate (SGR) = 100 (Ln FBW-Ln IBW)/T; feed conversion ratio (FCR) = 100 (feed intake/weight gain); where IBW and FBW are the initial and final

3.4. Oxidative status-related markers

Table 2 Primers used for qRT-PCR analysis. Gene

Primer sequence

GenBank Accession No.

Size (bp)

TNF-α

F:5’- GAGGCCAATAAAATCATCATCCC-3’ R: 5’- CTTCCCATAGACTCTGAGTAGCG-3’ F: 5’- AAGGATGACGACAAGCCAAC-3’ R: 5’- CGCTGTGCTGATGTACCAGT-3’ F:5’- GTGCCCATCTACGAGGGTTA-3’ R: 5’- CTCCTTAATGTCACGCACGA-3’

NM_001279533

161

KF747686.1

174

XM_003443127

156

IL-1β β-actin

The levels of SOD, CAT, GPX, and MDA in fish fed varying levels of LAB-Se are presented in Table 7. Dietary LAB-Se significantly increased SOD and CAT enzyme activities and decreased MDA levels (P = 0.001, P = 0.03, and P = 0.007, respectively), while GPX was not significantly affected by varying levels of LAB-Se (P > 0.05). The relationships between SOD, CAT, and MDA and dietary LAB-Se levels (Fig. 3) were expressed by the following second-order polynomial regression equations (quadratic): SOD (y = -6.0952 × 2 + 18.724x + 23.619, R2 = 0.9689, optimal dose = 1.54 mg/kg, Fig. 3A), CAT (y = 4.4762 × 2 + 11.419x + 34.229, R2 = 0.9244, optimal dose = 1.28 mg/ kg, Fig. 3B), and MDA (y = 9.1429 × 2 - 23.886x + 34.571, R2 = 0.8945, optimal dose = 1.31 mg/kg, Fig. 3C).

TNF-α= tumor necrosis factor alpha, IL-1β= interleukin 1 beta, β-actin= internal reference gene (house-keeping gene). 3

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Table 3 Growth performance and feed utilization of Nile tilapia fed different levels of LAB-Se for 8 weeks. Item

IBW FBW WG (%) SGR FCR Survival

LAB-Se (mg/kg)

Quadratic P value

0

0.5

1

1.5

2

14.03 ± 0.04 40.65 ± 0.61 189.8 ± 4.92 1.77 ± 0.03 1.75 ± 0.04 93.33 ± 1.67

14.12 ± 0.06 51.65 ± 2.59 265.68 ± 17.7 2.16 ± 0.08 1.21 ± 0.08 96.67 ± 1.67

14.16 ± 0.01 59.47 ± 2.5 319.96 ± 17.3 2.39 ± 0.07 1.17 ± 0.07 98.33 ± 1.67

14.23 ± 0.05 65.3 ± 3.27 358.69 ± 21.5 2.54 ± 0.08 1.14 ± 0.07 93.33 ± 4.41

14.18 ± 0.05 57.84 ± 4.34 307.82 ± 30.6 2.33 ± 0.13 1.32 ± 0.14 91.67 ± 3.33

NS 0.002 0.002 0.002 0.04 NS

*Values expressed as means ± SE (n = 3). NS means not significant (P > 0.05).

3.5. Relative immune-related gene expression levels

4. Discussion

Liver and spleen TNF-α expression levels were significantly upregulated in fish fed LAB-Se (P = 0.007 and P = 0.009, respectively) (Table 8). The expression of liver IL-1β was also upregulated significantly in fish fed varying levels of LAB-Se (P = 0.005), while spleen IL-1β was not significantly affected by varying levels of LAB-Se (P > 0.05) (Table 8). The relationships between liver and spleen TNF-α and liver IL-1β and dietary LAB-Se levels (Fig. 4) were expressed by the following secondorder polynomial regression equations (quadratic): liver TNF-α (y = 0.3448 × 2 + 0.8655x + 1.0323, R2 = 0.9383, optimal dose = 1.26 mg/ kg, Fig. 4A), spleen TNF-α (y = -0.2048 × 2 + 0.5675x + 1.0376, R2 = 0.8392, optimal dose = 1.39 mg/kg, Fig. 4C), and liver IL-1β (y = 0.0771 × 2 + 0.239x + 0.9901, R2 = 0.7591, optimal dose = 1.55 mg/ kg, Fig. 4B).

Micronutrients at adequate levels in formulated diets are highly recommended to meet the requirements of cultured fish to maintain standard growth and health conditions (Dawood and Koshio, 2018; Dawood et al., 2018, 2019c; El Basuini et al., 2017; Pacitti et al., 2016; Yan et al., 2017; Godin et al., 2015; Izquierdo et al., 2017; Terova et al., 2018). The obtained results revealed that the FBW, WG and SGR parameters of fish were increased at 1.23-1.42 mg LAB-Se per kg of diet. In general, our observations agree with previous trials conducted to assess the role of Se in different fish species, including grouper (Lin and Shiau, 2005), cobia (Lin et al., 2010), carp (Ashouri et al., 2015), Nile tilapia (Lee et al., 2016), gilthead seabream (Izquierdo et al., 2017), red sea bream (Dawood et al., 2019d), and rainbow trout (Wang et al., 2018). Specifically, a significant enhancement in growth performance was previously demonstrated in carp (Ashouri et al., 2015; Saffari et al.,

Fig. 1. Significant quadratic relationships and polynomial regressions analysis (P < 0.05) between final body weight (A), weight gain (B), specific growth rate (C), and feed conversion ratio (D) of Nile tilapia and dietary levels of LAB-Se. 4

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Table 4 Blood hematological parameters of Nile tilapia fed different levels of LAB-Se for 8 weeks. Item

Hb (g/100 ml) RBCs ( × 10/mm³) PCV (%) MCV (μm³/cell) MCH (pg/cell) MCHC (%) WBCs ( × 10³/mm³) Heterophils (%) Lymphocytes (%) Monocytes (%) Esinophils (%) Basophils (%)

LAB-Se (mg/kg)

P value

0

0.5

1

1.5

2

9.57 ± 0.37 3.01 ± 0.24 29.33 ± 2.40 97.44 ± 0.46 32.03 ± 1.53 32.88 ± 1.66 34.61 ± 3.18 10.00 ± 0.58 82.00 ± 1.00 7.33 ± 0.88 0.33 ± 0.33 0.33 ± 0.33

10.80 ± 0.89 3.54 ± 0.33 34.50 ± 3.18 97.48 ± 0.09 30.58 ± 0.32 31.38 ± 0.30 32.07 ± 1.71 9.00 ± 0.58 82.50 ± 0.87 7.00 ± 0.00 1.00 ± 0.00 0.50 ± 0.29

12.38 ± 0.36 4.01 ± 0.13 39.00 ± 1.73 97.27 ± 1.17 30.91 ± 0.10 31.80 ± 0.49 35.62 ± 2.52 9.00 ± 0.00 82.00 ± 0.00 8.00 ± 0.00 0.50 ± 0.29 0.50 ± 0.29

11.66 ± 1.15 3.82 ± 0.45 37.33 ± 4.06 97.98 ± 1.04 30.98 ± 0.65 31.62 ± 0.33 33.30 ± 2.91 10.33 ± 0.67 80.67 ± 0.67 7.33 ± 0.67 0.67 ± 0.33 1.00 ± 0.58

10.80 ± 0.53 3.27 ± 0.20 32.00 ± 2.08 97.80 ± 0.59 33.03 ± 0.34 33.81 ± 0.51 30.81 ± 0.23 9.67 ± 0.33 81.33 ± 0.33 7.33 ± 0.67 1.33 ± 0.33 0.33 ± 0.33

NS NS NS NS NS NS NS NS NS NS NS NS

*Values expressed as means ± SE (n = 3). NS means not significant (P > 0.05).

2017; Zhou et al., 2009) and tilapia (Lee et al., 2016) fed Se. Interestingly, Ashouri et al. (2015) reported improved growth performance at a similar inclusion level (1 mg/kg) for common carp using nanoforms of Se. Similarly, beneficial effects were achieved by increased levels (5.56 mg) in yellowtail kingfish (Le and Fotedar, 2013) and by decreased levels in grouper (0.77 mg) (Lin and Shiau, 2005). However, gilthead seabream fed diets supplemented with yeast-Se showed no significant effects in growth performance parameters (Izquierdo et al., 2017). The minimum Se level required to obtain the maximum growth performance may vary according to the form of Se, period of administration, and experimental procedure as well as fish species and fish size. Nonetheless, the results of this study confirm the hypothesis that identical growth performance in aquatic animal species can be achieved with relatively low levels of microminerals supplemented in nanoforms rather than in inorganic and organic forms (Lee et al., 2008). It is generally accepted that LAB-Se is more readily available to fish (Ashouri et al., 2015; Saffari et al., 2017) and probably displays wider and greater biological efficacy (Wang et al., 2007) than other Se sources. Over supplementation with Se can negatively affect fish growth performance (Kim and Kang, 2014, 2015; Pham and Fotedar, 2017). Excess Se supplementation may force Nile tilapia to use energy for the detoxification of Se rather than for growth and development; accordingly, growth is stunted. Regardless, our results indicated that supplementary Se could be provided to Nile tilapia at 1 to 2 mg LAB-Se per kg of diet, with no negative effects on fish growth and feed efficiency.

However, the decreased performance at 2 mg compared to 1 mg LAB-Se per kg suggests that a dietary LAB-Se up to 1 mg per kg diet may be beneficial for the growth of Nile tilapia under the conditions of this study. In addition, these results confirmed the crucial demand for dietary LAB-Se in Nile tilapia. The Se-induced improvements in growth and feed effectiveness are due to the roles of Se in the stimulation of growth hormone production, synthesis of selenoprotein, activation of intestinal protease enzymes, and increase in the intracellular protein content of intestinal cells. Se can increase the production of growth hormones and enhance growth performance in fish (Khan et al., 2017). In this study, dietary LAB-Se had a positive influence on the FCR in a dose-dependent manner. Wang et al. (2013) reported an increase in the intracellular protein in intestinal epithelial cells in the crucian carp, which may result in the better utilization of the fed diets, resulting in higher feed efficiency and growth performance (Saffari et al., 2017). In addition, the expression of selenoprotein genes were upregulated due to the role of Se in selenoprotein synthesis, which resulted in improved feed utilization and growth performance in rainbow trout (Wang et al., 2018). Micronutrients, including Se, act as co-enzymes in the production/activation of digestive enzymes (Shenkin, 2006). Previous observations have reported that dietary Se enhances protein digestibility and utilization by increasing the amount and activity of intestinal microbes and digestive proteases (Chaudhary et al., 2010; Shi et al., 2011). Thus, the reduced FCR values of tilapia fed LAB-Se may be explained by the high intestinal cell protein content that resulted in the efficient metabolism of the

Table 5 Blood biochemical parameters of Nile tilapia fed different levels of LAB-Se for 8 weeks. Item

Glucose (mg/dl) T-CHO (mg/dl) ALT (U/I) AST (U/I) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Urea (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl)

LAB-Se (mg/kg)

P value

0

0.5

1

1.5

2

12.32 ± 0.48 86.11 ± 4.10 2.39 ± 0.17 72.27 ± 2.39 3.06 ± 0.03 1.91 ± 0.07 1.99 ± 0.13 3.00 ± 0.08 0.23 ± 0.00 1.97 ± 0.09

10.56 ± 0.39 78.24 ± 0.71 2.84 ± 0.08 70.37 ± 2.84 3.90 ± 0.12 1.06 ± 0.04 2.00 ± 0.01 4.12 ± 0.01 0.24 ± 0.03 1.81 ± 0.11

10.50 ± 0.08 82.63 ± 3.86 2.94 ± 0.10 67.85 ± 2.02 4.79 ± 0.06 2.09 ± 0.01 2.20 ± 0.23 3.01 ± 0.08 0.25 ± 0.03 1.56 ± 0.00

11.56 ± 1.08 76.00 ± 3.60 2.52 ± 0.23 66.45 ± 5.06 3.53 ± 0.28 1.27 ± 0.21 1.92 ± 0.19 3.67 ± 0.34 0.20 ± 0.02 1.77 ± 0.13

11.17 ± 1.15 81.35 ± 5.00 3.14 ± 0.55 80.46 ± 15.3 4.21 ± 0.41 2.04 ± 0.11 2.50 ± 0.33 3.63 ± 0.63 0.28 ± 0.06 1.84 ± 0.24

*Values expressed as means ± SE (n = 3). NS means not significant (P > 0.05).

5

NS NS NS NS NS NS NS NS NS NS

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Table 6 Immune parameters of Nile tilapia fed different levels of LAB-Se for 8 weeks. Item

Lysozyme activity (unit/ml) Phagocytic activity (%) Total protein (g/dl)

LAB-Se (mg/kg)

Quadratic P value

0

0.5

1

1.5

2

19.76 ± 0.7 1.72 ± 0.1 3.59 ± 0.1

27.13 ± 1.1 1.91 ± 0.1 4.06 ± 0.03

26.39 ± 1.2 1.99 ± 0.1 4.79 ± 0.06

27.1 ± 1.3 2.19 ± 0.2 4.33 ± 0.2

24.24 ± 2.1 2.06 ± 0.04 4.21 ± 0.4

0.02 0.001 0.04

*Values expressed as means ± SE (n = 3).

absorbed nutrients. The blood parameter value found in this study were similar to those obtained from Nile tilapia (Dawood et al., 2019e,f). The blood haematology and biochemistry parameters of fish fed varying levels of LABSe showed normal values with nonsignificant differences among the fish, indicating the non-toxic effect of LAB-Se. Blood total protein is influenced by serum proteins (e.g., IgM) and serves as an indicator of the enhanced immune system in fish (Burgos Aceves et al., 2016; Dossou et al., 2018a,b; El Basuini et al., 2017; Uribe et al., 2011). The enhanced blood total protein in the current study is in line with the increased lysozyme and phagocytic activities, suggesting

immunomodulatory effects of LAB-Se on Nile tilapia. Thus, dietary LABSe can positively affect the welfare of cultured tilapia. Lysozymes are important defence components that are responsible for the lysis of pathogenic bacteria (Saurabh and Sahoo, 2008). In this study, fish fed LAB-Se demonstrated significantly enhanced lysozyme ac tivity. Fish neutrophils perform various phagocytic, bactericidal and respiratory burst activities (Lamas and Ellis, 1994; Vallejos-Vidal et al., 2016). In fish, as in other vertebrates, phagocytosis is a critical process of the immune system. Its role is to efficiently assist fish in avoiding pathogen attacks by recognizing the pathogens and limiting their spread and proliferation (Harikrishnan et al., 2011). Our study revealed

Fig. 2. Significant quadratic relationships and polynomial regressions analysis (P < 0.05) between lysozyme activity (A), phagocytic activity (B), and total protein (C) of Nile tilapia and dietary levels of LAB-Se.

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Table 7 Oxidative and antioxidative parameters of Nile tilapia fed different levels of LAB-Se for 8 weeks. Item

LAB-Se (mg/kg) 0 a

SOD (IU/L) CAT (IU/L)b GPX (IU/L)c MDA (IU/L)d

24 34 22 36

± ± ± ±

Quadratic P value 0.5

0.6 1.2 1.16 1.2

31 ± 39 ± 40 ± 21.67

2.1 0.6 0.58 ± 0.9

1

1.5

35.3 ± 1.45 42 ± 1.2 44.67 ± 1.16 21 ± 0.6

39.67 40 ± 36 ± 21 ±

± 1.5 1.2 1.16 0.6

2 36 ± 1.2 39.67 ± 1.2 36.33 ± 1.2 22.3 ± 0.67

0.001 0.03 NS 0.007

*Values expressed as means ± SE (n = 3). NS means not significant (P > 0.05). a SOD: serum superoxide dismutase. b CAT: catalase. c GPX: glutathione peroxidase. d MDA: malonaldehyde.

that LAB-Se promoted heightened immune responses and provided greater tolerance against infectious pathogens through an increase in phagocytosis. However, the current study lacked a challenge test against infection in Nile tilapia. Thus, further investigations are required to reveal the effects of LAB-Se on tilapia against infectious diseases.

The most vital role of Se is as an antioxidant because it forms “selenocysteine”, which is a part of the active centre of GPX (Köhrle et al., 2000; Terova et al., 2018). LAB-Se is an effective microelement for the prevention of oxidative stress (Ashouri et al., 2015; Saffari et al., 2017). The role of antioxidant enzymes is to remove damaging reactive oxygen species (ROS) from the cellular environment by catalysing the

Fig. 3. Significant quadratic relationships and polynomial regressions analysis (P < 0.05) between SOD (A), CAT (B), GPX (C), and MDA (D) of Nile tilapia and dietary levels of LAB-Se.

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Table 8 Relative gene expressions of liver and spleen TNF-α and IL-1β of Nile tilapia fed different levels of LAB-Se for 8 weeks. Item

LAB-Se (mg/kg) 0

Liver TNF-α Liver IL-1β Spleen TNF-α Spleen IL-1β

1 1 1 1

± ± ± ±

Quadratic P value 0.5

0 0 0 0

1.46 1.05 1.34 1.17

± ± ± ±

1 0.1 0.03 0.08 0.1

1.49 1.21 1.43 1.28

± ± ± ±

1.5 0.1 0.02 0.05 0.05

1.55 1.13 1.33 1.23

± ± ± ±

2 0.05 0.05 0.03 0.03

1.40 ± 0.1 1.17 ± 0.02 1.4 ± 0.08 1.09 ± 0.1

0.007 0.005 0.009 NS

*Values expressed as means ± SE (n = 3). NS means not significant (P > 0.05).

dismutation of two superoxide radicals to hydrogen peroxide and oxygen (Fattman et al., 2003; Lin et al., 2010). Consequently, antioxidant enzymes levels have been extensively used as an early warning indicator of toxicity and/or pollution (Lin et al., 2001). The main molecules of the antioxidant defence network are SOD, GPX and CAT, which inhibit ROS production by eliminating their precursors (Lee et al., 2000). Over-ROS creation can increase lipid peroxidation, which results in MDA production. The continuous presence of MDA can damage the cells by breaking down the DNA, protein and cytoplasm (Yao et al., 2010). Our results revealed lower MDA and higher SOD, CAT and

GPX activities in fish fed the LAB-Se diet, indicating reduced cell damage, than in fish fed a control diet. Similar results were reported by Bai et al. (2019) in zebrafish (Danio rerio) fed diets supplemented with bio-fermented Se. However, Terova et al. (2018) reported nonsignificant differences in SOD and GPX when Se nanoparticles were supplemented in the early weaning diets of gilthead seabream. The efficiency of Se may vary according to the period of administration, experimental procedures, fish species and fish size. Using these parameters, our study showed that fish fed diets supplemented with LAB-Se were more tolerant to oxidative stress than fish fed the control diet,

Fig. 4. Significant quadratic relationships and polynomial regressions analysis (P < 0.05) between of liver TNF-α (A), liver IL-1β (B), spleen TNF-α (C), and spleen IL1β (D), of Nile tilapia and dietary levels of LAB-Se.

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indicating an improved health status. The role of LAB-Se seems to be similar to that of immunostimulants, and LAB-Se supplementation mainly upregulates pro-inflammatory cytokines (Abarike et al., 2018). TNF-α and IL-1β are pro-inflammatory cytokines that demonstrate high transcription levels after immunostimulant supplementation in fish (Xia et al., 2018). In this study, the results showed the upregulation of immune-related gene (TNF-α and IL-1β) expression in Nile tilapia. TNF-α is a cytokine that is mostly secreted by activated macrophages, orchestrates immune defence mechanisms against pathogen invasion and colonization, and stimulates neutrophil base immunity (Bilen et al., 2019). IL-1β is a pro-inflammatory cytokine that activates lymphocytes and macrophages against disease-causing aetiologies (Low et al., 2003). In this study, the IL-1β expression increased in fish fed the LAB-Se diet compared to fish fed the control diet. TNF-α also stimulates macrophages to secrete other cytokines, including IL-6 and IL-1β (Hamdan et al., 2016; Striz et al., 2014). Activated phagocytic cells search for and engulf foreign invaders faster than inactivated cells.

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5. Conclusion In conclusion, the supplementation of LAB-Se at 1 to 2 mg/kg of feed has the potential to increase the growth, health and immunity of Nile tilapia. Dietary LAB-Se also stimulated the oxidation status of Nile tilapia in a particular way. LAB-Se activated blood immune responses and TNF-α and IL-1β gene expression, which significantly improves defence against stressors and infectious pathogens in Nile tilapia. Disease challenge studies are important to confirm the results obtained in the current study. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements This work was financially supported by the project “Biological production of nanoselenium spheres and its application in livestock production” by the National Strategy for Genetic Engineering and Biotechnology, Academy of Scientific Research and Technology, Egypt. References Abarike, E.D., Kuebutornye, F.K.A., Jian, J., Tang, J., Lu, Y., Cai, J., 2018. Influences of immunostimulants on phagocytes in cultured fish: a mini review. Rev. Aquac. 1–9. https://doi.org/10.1111/raq.12288. Ashouri, S., Keyvanshokooh, S., Salati, A.P., Johari, S.A., Pasha-Zanoosi, H., 2015. Effects of different levels of dietary selenium nanoparticles on growth performance, muscle composition, blood biochemical profiles and antioxidant status of common carp (Cyprinus carpio). Aquaculture 446, 25–29. AOAC (Association of Official Analytical Chemists), 1998. Official Methods of Analysis of Official Analytical Chemists International, sixteenth ed. AOAC, Washington, DC. Bai, Z., Ren, T., Han, Y., Hu, Y., Schohel, M.R., Jiang, Z., 2019. Effect of dietary Biofermented selenium on growth performance, nonspecific immune enzyme, proximate composition and bioaccumulation of zebrafish (Danio rerio). Aquaculture Reports 13, 100180. Bilen, S., Sirtiyah, A.M.A., Terzi, E., 2019. Therapeutic effects of beard lichen, Usnea barbata extract against Lactococcus garvieae infection in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 87, 401–409. Blaxhall, P.C., Daisley, K.W., 1973. Routine haematological methods for use with fish blood. J. Fish Biol. 5 (6), 771–781. Burgos-Aceves, M.A., Cohen, A., Smith, Y., Faggio, C., 2016. Estrogen regulation of gene expression in the teleost fish immune system. Fish Shellfish Immunol. 58, 42–49. Chaudhary, M., Garg, A.K., Mittal, G.K., Mudgal, V., 2010. Effect of organic selenium supplementation on growth, Se uptake, and nutrient utilization in Guinea pigs. Biol. Trace Elem. Res. 133 (2), 217–226. Dalia, A.M., Loh, T.C., Sazili, A.Q., Jahromi, M.F., Samsudin, A.A., 2018. Effects of vitamin E, inorganic selenium, bacterial organic selenium, and their combinations on immunity response in broiler chickens. BMC Vet. Res. 14 (1), 249. Dawood, M.A.O., Koshio, S., 2019. Application of fermentation strategy in aquafeed for sustainable aquaculture. Reviews in Aquaculture. https://doi.org/10.1111/raq. 12368.

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