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Aquaculture Reports

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The impact of stocking density and dietary carbon sources on the growth,oxidative status and stress markers of Nile tilapia (Oreochromis niloticus)reared under biofloc conditionsMohamed A.A. Zakia, Ahmed N. Alabssawyb, Abd El-Aziz M. Noura, Mohammed F. El Basuinic,d,Mahmoud A.O. Dawoode,*, Saad Alkahtanif, Mohamed M. Abdel-Daimf,ga Department of Animal and Fish Production, Faculty of Agriculture, Alexandria University, EgyptbDepartment of Zoology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egyptc Department of Animal Production, Faculty of Agriculture, Tanta University, 31527, Tanta, Egyptd Education and Research Center for Marine Resources and Environment, Faculty of Fisheries, Kagoshima University, Japane Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516, Kafrelsheikh, EgyptfDepartment of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabiag Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt

A R T I C L E I N F O

Keywords:BioflocCarbon sourceGrowth performanceStress markersNile tilapiaStocking density

A B S T R A C T

The present study investigated the impact of stocking density and dietary carbon sources on the growth, oxi-dative status and stress markers of Nile tilapia (Oreochromis niloticus) reared under biofloc conditions. Six groupswere established at three levels of stocking densities [20, 40 and 60 fish (50.47± 0.05 g) per m3] and fed thebasal diet without carbon sources or with broken rice flour (BRF) or broken wheat grain flour (BWGF) in bioflocunits. Water quality [pH, biological oxygen demand (BOD), Total ammonia-nitrogen (TAN), and nitrite-nitrogen(NO2)] values were increased significantly (P<0.05) as the density increased and recorded the highest values ingroup (60 fish per m3) while dissolved oxygen decreased. Biofloc volume and bacterial counts were significantly(P<0.05) lower in 20 fish m−3 and the highest values were in 40 fish per m3. Significantly increased growthand feed utilization were recorded in 40 fish per m3 fed with BRF. The lipid content lowered significantly(P<0.05) in 60 fish m-3 group. Red blood cells count, hemoglobin and hematocrit values were reduced in fishstocked in high density while alanine transaminase (ALT) and aspartate transaminase (AST) increased in fishreared in low density. Glucose, cortisol, catalase and superoxide dismutase increased in fish reared at 20 fish m-3

and fed the basal diet. Thus, using of BRF for fish reared at 40 fish per m3 promotes growth and health status ofNile tilapia cultured in a biofloc system.

1. Introduction

In the last decades, aquaculture has become the best option forproviding sustainable seafood. Its high rate of return on investment hasattracted farmers and investors to the intensification systems with theapplication of modern technologies in order to increase profits (Dawoodet al., 2016; FAO, 2012; Towers, 2015). Intensive farming depends onhigh fish densities with the use of large quantities of high-protein levelsdiets (25–55%) aiming for increasing productivity in closed or semi-closed systems to overcome the limit of water and lands resources(Avnimelech et al., 2008; Dawood, 2016; Delong et al., 2009;Piedrahita, 2003; Pillay, 2005). High stocking densities combined withhighly nitrogenous diets in intensive fish culture negatively affect the

water quality especially the accumulation of inorganic nitrogen forms(NH3 and NO2) (Durborow et al., 1997; Hargreaves and Tucker, 2004).The common approaches to maintain water from deterioration andavoid nitrogen increases are through water exchange, using nitrifyingbiofilters and the microorganisms that grow while using a carbonsource (Avnimelech, 2006, 1999; Avnimelech and Kochba, 2009). Toovercome these bottlenecks, sustainable intensification by the adoptionof advanced culture systems and technologies becomes inevitable toimprove the production and productivity of the sector (Dawood et al.,2015, 2016; EL-Haroun et al., 2006).

Biofloc (BF) technology is an environmentally friendly aquaculturepractice considered as an efficient alternative system that promotescontinuous recycling and reused of nutrients. BF system approach

https://doi.org/10.1016/j.aqrep.2020.100282Received 9 November 2019; Received in revised form 25 January 2020; Accepted 27 January 2020

⁎ Corresponding author.E-mail address: mahmouddawood55@gmail.com (M.A.O. Dawood).

Aquaculture Reports 16 (2020) 100282

2352-5134/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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depends on growing microorganisms in the cultured media with noexchange of water or reduces exchange to the minimum (Avnimelech,2007; Kuhn et al., 2009). It was reported that the proportion of proteinin the diets can be reduced and the biofloc can be used as additionalprotein source (Avnimelech et al., 2015; Mansour and Esteban, 2017a,b). Kuhn et al. (2009) reported that microbial floc meal in tilapia andshrimp diets significantly boost the weight gain. Recently, BF techniquehave widely been used to maximize shrimp and tilapia production forits ability to support high densities cultivation, to improve water qualityand simultaneously recycling feed and protein production in the sameculture unit (Avnimelech, 2009; Azim and Little, 2008; Crab et al.,2007; Rakocy et al., 2004). In addition, BF units as zero-exchange in-tensive systems utilize different carbon inputs to enhance BF microbialcommunity’s growth (Burford et al., 2004; McIntosh, 2001). Moreover,BF microbial biomass can be used in aquafeed to reduce the com-plementary feed and production costs, as well as positively affects theimmunity and reducing the mortality (Azim and Little, 2008; Liu et al.,2018; Moss, 2002; Samocha et al., 2004; Tacon et al., 2002; Xu and Pan,2012). BF is considered as natural probiotics due to its role as an im-munostimulants that can increase the resistance of fish against in-fectious diseases (Crab, 2010).

The addition of organic carbon rich substrate to aquaculture systemto control the carbon-nitrogen ratio (C/N ratio) is considered to be oneof the prospect management measures to promote production, nutrientretention and in achieving low or zero-water exchange intensive culturesystems (Crab et al., 2007; Gao et al., 2012). But availability of cheapand locally available carbon source is a great issue to run this tech-nology throughout the world. Thus, cheap or easily available wastecarbon sources are required to further lower down the input cost and toincrease the net return. It was noted that the flour industries generatehuge quantity of unusable or unmarketable flours during machiningprocess (Deb et al., 2017; Mansour and Esteban, 2017a, b). Theydumped the materials in dumping yard or sometimes freely distribute touse as feed ingredients. Thus, these waste flours including, broken riceflour (BRF) or broken wheat grain flour (BWGF) were collected fromflour process industries (Deb et al., 2017).

Therefore, the present investigation examined the effects of dietarycarbon sources (BRF or BWGF) at different stocking densities on thegrowth, feed utilization, and stress markers of Nile tilapia (Oreochromisniloticus) cultured in a biofloc system.

2. Materials and methods

2.1. “Biofloc system

Two tanks were prepared as the BF source of inoculants 1 weekprior to the trial. 25 mg L−1 of N, 3.6 mg L−1 of PO4, 1 mg L−1 ofNaSiO3, and broken rice flour (BRF) or broken wheat grain flour(BWGF) were used as carbon sources to keep the C/N ratio equal to 15:1and added to the tanks. BRF and BWGF flours used in the trial were rawwaste products collected from flour mills collected from the localmarket. Proximate composition of the test carbohydrate sources (BRFand BWGF) are shown in Table 1. Biofloc volume (ml L-1) was regis-tered after 30 min sedimentation of 1000 ml of the tank water using anImhoff cones (Avnimelech and Kochba, 2009).

Production of biofloc was performed in the freshwater culture tanks(6 m3, 2 × 3 × 1 length × width × depth) and adopted at C:N ratio of15:1. The addition of carbon source to maintain the C:N ratio was fol-lowed using the method of (Avnimelech and Kochba, 2009) and(Menaga et al., 2019) for the transition of the heterotrophic system. Theaddition of carbon source promotes the heterotrophic bacteria to reducethe organic matter and assimilate the nitrogen waste into microbialprotein. The C:N ratio was maintained at 15:1 for the development ofbiofloc and addition of urea for nitrogen source.

Biofloc samples were collected from the rearing tanks and trans-ferred to the brain heart infusion (BHI) broth according to Venkat et al.

(2004). Biofloc samples were serially diluted with sterile saline [phos-phate-buffered saline (PBS, pH = 7.4)]. The viability of the bacterialcells into biofloc was assessed by spreading onto triplicate plates ofDeMan, Rogosa and Sharpe agar (MRS, MERCK, Darmastadt, Germany).The agar plate inoculated with each dilution was incubated for 27 °C for24 h. Colony forming unit (×106 CFU L−1) were determined for viablebacterial populations.

2.2. Fish and experimental design

Juveniles of Nile tilapia were purchased from a commercial fishfarm and then transferred to El-Kady fish farms group, Edkuo province,Alexandria Governorate, Egypt. Fish were kept in cement tanks for twoweeks for acclimatization and were fed with a commercial diet (30 %crude protein) before being distributed to their respective experimentalunits. Prior to the experiment, indoor rectangular concrete tanks (6 m3,2 × 3× 1 length × width × depth) were cleaned, dried and filled withfreshwater. Fish with an average initial body weight of 50.47± 0.05 gwere stocked in each of twelve indoor concrete tanks at different den-sities under natural light/dark regime.

This experiment was consisted of six treatment groups established atthree levels of stocking rates (20, 40 and 60 fish m−3) and were fed thebasal diet without additional carbon source or with additional carbonsources (BRF or BWGF) in biofloc units. The tanks were inoculated with100 ml of biofloc that previously prepared on the first day of the trial.Furthermore, the tanks were supplied daily with one of the testedcarbon sources, two hours after feeding to maintain the 15C: N ratio(Avnimelech et al., 1994).

The composition of the basal diet without additional carbon sources(BRF or BWGF) was shown in Table 2 (crude protein is 30 % and 22.12kJ g−1 gross energy). Ingredients thoroughly were grounded and mixedwith 3 % lipid source (sunflower oil) for 15 min then water at 30–35 %

Table 1Chemical analysis of carbohydrate sources used in the experimental treatments.

Carbohydrate sourcesProximate chemical(g kg−1, DM basis)

Broken rice flour (BRF) Broken wheat grain flour(BWGF)

Dry matter 773 906Crude protein 33 116Ether extract 4 20Ash 38 16Crude fiber 82 37Nitrogen free extract 807 811GE (kJ g−1)* 177 181

*Calculated using combustion values for protein, lipid and carbohydrate of23.6, 39.5 and 17.2 kJ g−1, respectively.

Table 2Formulation and proximate composition of the basal diet.

Ingredients (g kg−1) Proximate composition DM basis (g kg−1)

Fish meal 250 Crude protein 304.3Soybean meal 250 Ether extract 56.3Wheat bran 60 Ash 72.2Yellow corn 400 GE (kJ g−1)* 221.2Sunflower oil 30Vitamina 5Mineralsb 5

a Vitamin mixture (mg kg−1 premix): Vitamin A (3300 IU), Vitamin D3 (410IU), Vitamin E (2660 mg), Vitamin B1 (133 mg), Vitamin B2 (580 mg), VitaminB6 (410 mg), Vitamin B12 (50 mg), Biotin (9330 mg), Colin chloride (4000mg), Vitamin C (2660 mg), Inositol (330 mg), Para-amino benzoic acid (9330mg), Niacin (26.60 mg), Pantothenic acid (2000 mg).b Mineral mixture (mg kg−1 premix): Manganese (325 mg), Iron (200 mg),

Copper (25 mg), Iodine, Cobalt (5 mg). *Calculated using combustion values forprotein, lipid and carbohydrate of 23.6, 39.5 and 17.2 kJ g−1, respectively.

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was added and mixed for more 15 min. Feed was pelletized by a Cali-fornia pellet mill with 0.3 mm diameters and dried at 70 °C for 48 h.

2.3. Water characteristics

Water temperature (°C), pH, dissolved oxygen (DO, mg L−1), andbiological oxygen demand (BOD, mg L−1) were monitored weeklythroughout the experimental period, using thermometer, portable di-gital pH meter (Martini Instruments Model 201/digital) and WaterproofPortable Dissolved Oxygen and BOD Meter (model Hanna waterproofIP67). Total ammonia-nitrogen (TAN, mg L−1), nitrate-nitrogen (NO3,mg L−1) and nitrite-nitrogen (NO2, mg L−1) were measured color-imetrically.

2.4. The feeding trial and sample collection

Fish were hand-fed twice a day at 3 % of wet body weight for 84days experimental period. The left feed was collected and dried tocalculate the actual feed intake. Fish was weighed every 15 days to re-adjust the daily amounts of feeds. Prior to the final sampling, fish wasfasted for 24 h. Then, 10 fish per treatment were randomly collected,washed and kept for whole body proximate analysis at −20 °C. Five fishper tank was collected 24 h after weighing and anaesthetized (clove oil3 ml in 10 ml absolute ethanol). Blood samples were collected via thecaudal venous and the samples were placed in Eppendorf tubes withheparin-anticoagulant. Plasma was obtained by centrifugation at 3000rpm for 10 min under 4 °C. In addition, non-heparinized disposablesyringes were used to collect serum after whole blood was clotted andcentrifuged at 3000 rpm for 10 min. Plasma and serum samples werekept at −20 °C until the analysis. Livers of five fish of each tank werecollected, washed and kept at −60 °C for the catalase and superoxidedismutase (SOD) activities measurement.

2.5. Biochemical measurements

Chemical analysis of experimental basal diet, different carbohydratesources (BRF and BWGF), and fish whole body were performed in tri-plicate, using standard methods (AOAC, 1990) for moisture by drying at110 °C to constant weight, crude protein by the Kjeldahl method, crudelipid by solvent extraction method with a Soxhlet and ash by combus-tion at 550 °C for 4 h in Muffle furnace. Gross energy (GE) calculatedusing combustion values for protein, lipid and carbohydrate of 23.6,39.5 and 17.2 kJ g−1, respectively.

Hematocrit Ht was assessed in the partial heparinized whole bloodafter centrifugation at 13,000 rpm for 5 min. Total red blood cells(RBCs×1012 L−1) and total white blood cells (WBCs×109 L−1) werecounted according to (Dacie and Lewis, 1995). Hemoglobin con-centration (Hb) was determined spectrophotometrically based on

cyano-methemoglobin method (blood was diluted in a Drabkin solu-tion) then the concentration determined using standard curve (Noga,2010). The serum glucose levels (mg dL−1) were estimated calorime-trically using commercial kit (Transasia Bio-Medicals, India). Serumcortisol levels were measured using the Cortisol ELISA Kit (Caymanchemicals, Mumbai, India), and the values were expressed in ng mL−1.The activities of plasma alanine transaminase (ALT) and aspartatetransaminase (AST) were assayed by the method of Gella et al. (1985).

2.6. Statistical analysis

The results are presented as means± standard errors (n = 3). Thedata were analyzed by Two-way ANOVA using Statistical AnalysisSystem (SAS) version 8.02 for Windows at P<0.05 level to test theeffects of stocking densities and carbohydrate sources supplementation,as well as their interactions. Means were tested for significant differ-ences using Duncan’s multiple range test.”

3. Results

3.1. Water parameters

Means of the water quality parameters recorded during the trialwere: temperature (28.5± 0.06), pH (7.3± 0.03), DO (4.4± 0.1),BOD (23.5± 1.1), TAN (0.24± 0.02), NO2 (0.33± 0.02) and NO3(0.54±0.02) (Table 3). Only stocking density had significant effect onwater characteristics including pH,DO, BOD, TAN and NO2. pH, BOD,TAN and NO2 values increased significantly (P< 0.05) as the densityincreased and recorded the highest values in group (60 fish per m3)over the low density (20 fish per m3) while DO decreased following thesame trend. No significant (P> 0.05) effect was observed for thecarbohydrate sources or the interaction between the density and car-bohydrate sources.

3.2. Biofloc volume and bacterial counts

BF volume and bacterial counts were significantly (P< 0.05) lower(13.77 mg L−1 and 2.78 × 106 CFU L−1, respectively) at 20 fish m-3

and increase with increasing stocking level where the highest valueswere 26.58 mg L−1 and 3.83 × 106 CFU L−1, respectively at 40 fish m-3

(Table 4). Moreover, fish fed BWGF and BRF diets recorded sig-nificantly (P<0.05) higher values of BF volume and bacterial counts.Stocking density, carbohydrate sources and both factors interactionshowed significant (P< 0.05) effects on BF volume and total bacterialcounts.

Table 3Characteristics of water used for Nile tilapia culturing during the experimental period.

Items Temperature (ºC) pH DO1 (mg L−1) BOD2 (mg L−1) TAN3 (mg L−1) NO2 (mg L−1) NO3 (mg L−1)Stocking density (D)

20 28.47± 0.15 7.21± 0.04b 4.83± 0.04a 18.15± 0.11c 0.14±0.02b 0.89± 0.02b 8.12± 0.0240 28.40± 0.04 7.32± 0.04ab 4.45± 0.03b 24.45± 1.03b 0.29±0.03a 1.02± 0.01a 8.18± 0.0260 28.63± 0.05 7.44± 0.04a 3.89± 0.02c 27.88± 0.73a 0.31±0.01a 1.07± 0.01a 8.34± 0.01Carbohydrate sources (C)Basal diet 28.43± 0.15 7.43± 0.05a 4.32± 0.16 20.95± 1.92 0.25±0.09 1.37± 0.09 11.59±0.04BWGF 28.51± 0.07 7.31± 0.05a 4.39± 0.16 23.53± 1.90 0.23±0.03 1.33± 0.04 11.54±0.04BRF 28.56± 0.05 7.24± 0.04b 4.46± 0.18 26.00± 3.94 0.26±0.05 1.29± 0.04 11.50±0.04Two-way ANOVA P valuesD 0.405 0.008 0.000 0.000 0.000 0.000 0.274C 0.734 0.018 0.059 0.182 0.257 0.054 0.068D × C interaction 0.995 0.984 0.485 0.258 0.041 0.825 0.994

Means in the same column within each classification with different letters differ significantly (P<0.05). 1DO = Dissolved oxygen, 2BOD = Biological oxygendemand and 3TAN = Total ammonia nitrogen.

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3.3. Growth and nutrient utilization parameters

Values were reduced significantly (P<0.05) in fish reared at highstocking rate (60 fish m−3) when compared with other groups(Table 5). Significantly (P<0.05) higher growth performance and feedutilization (FW, WG, SGR, FI and PER) were recorded in fish reared at40 fish per m3 stocking rate. Fish reared at 20 and 40 fish per m3 re-ported significantly (P<0.05) higher protein productive value (PPV)and energy utilization (EU) and lower feed conversion ratio (FCR) thanthe intensive group without no differences among the two groups(Table 5). In addition, fish fed BRF as a carbon source showed sig-nificantly (P< 0.05) higher FW, WG, SGR, FI and PER and lower FCRcompared with other groups. Fish fed the basal diet showed the lowestPPV and EU among all the groups. Stocking density and carbohydratesources affected significantly (P< 0.05) on all the growth and nutrientutilization parameters while the interaction between the two factorswas reported only in case of FW, WG, SGR, FI and FCR.

3.4. Whole-body proximate analysis

The whole-body proximate compositions of tilapia showed nosignificant differences (P>0.05) except for the body crude lipid con-tent (Table 6). Whole body lipid changed significantly, yet slightly withstocking rate and the lowest value (22.41±0.13) recorded at 60 fish

per m3.

3.5. Blood parameters

RBC, Hb and Ht values were reduced within fish groups stocked athigh level (60 fish m−3) compared with other groups (P< 0.05)(Table 7). Meanwhile, the highest WBCs value was in fish reared at 40fish per m3. ALT and AST values were differ significantly among thetreatment groups (P<0.05) with the highest values in fish reared at 20fish per m3 and fed the basal diet and the lower values were recorded infish reared at 40 fish per m3 and fed BRF as a carbon source (Table 7).Generally, stocking density was a significant (P< 0.05) factor on themeasured blood parameters while the carbohydrate source was a sig-nificant factor only on ALT and AST. Significantly (P<0.05) interac-tion effects between stocking density and carbohydrate sources werereported in all blood parameters except for hemoglobin and hematocritparameters.

3.6. Oxidative status and stress resistance

Stocking density and carbohydrate sources and their interactionsignificantly (P< 0.05) affected catalase and superoxide dismutase(SOD) activities (Figs. 1A and 1B). Significantly (P< 0.05) highercatalase and SOD values were observed in the group reared at 20 fishper m3 and fed with basal diet, while the lower values were recorded inthe group reared at 40 fish per m3 and fed with BRF as a carbon source.

Similarly, stocking density, carbohydrate sources and their inter-action significantly (P<0.05) affected glucose and cortisol (Figs. 2Aand 2B). Serum glucose and cortisol levels varied significantly(P< 0.05) among the treatment groups with the highest values in fishreared at 20 fish per m3 and fed the basal diet, while lower values wererecorded in fish fed BRF as a carbon source and reared at 40 or 60 fishper m3.

4. Discussion

The farming system is one of the main factors which affect theperformance and the health status of aquatic organisms (Boyd, 1997;Boyd and Tucker, 1998; M’balaka et al., 2012). In the present study, thewater quality is within the suitable values for Nile tilapia rearing (Boyd,1990; Boyd and Tucker, 1998; El-Sayed, 2006) and a slight effect wasobserved for different stocking densities on water oxygen parameters(DO and BOD) and water nitrogen contents (NH4, NO2 and NO3).

Table 4Biofloc volume and total bacterial counts of water used during the trial period.

Items Biofloc volume (mlL−1)

Total bacterial counts (106

CFU L−1)

Stocking density (D)20 14±1.09b 2. 8± 0.21b

40 31±1.12a 3.5± 0.47a

60 27±1.13a 3.8± 0.35a

Carbohydrate sources (C)Basal diet 6± 1.19b 2.3± 0.04b

BWGF 29±1.36a 3.7± 0.30a

BRF 36±1.37a 4.1± 0.26a

Two-way ANOVA (P values)D 0.0001 0.0001C 0.0001 0.0001D × C 0.0001 0.002

Means in the same column within each classification bearing different super-script are significantly different at (P< 0.05).

Table 5Growth parameters and nutrient utilization of Nile tilapia cultured for 84 days.

Items FW2 WG3 SGR4 FI5 FCR6 PER7* PPV8 EU%9

Stocking density (D)20 115±1.15b 64.68± 1.17b 0.77±0.06b 130.24± 1.78b 1.95± 0.04b 1.56± 0.05b 28.40± 0.32a 18.01±0.34a

40 136±1.23a 85.97± 1.25a 1.02±0.07a 156.00± 1.80a 1.79± 0.02b 1.82± 0.09a 28.19± 0.25a 17.99±0.19a

60 98±1.76c 47.50± 1.78c 0.57±0.06c 114.96± 1.22c 2.47± 0.09a 1.35± 0.05b 24.92± 0.45b 15.61±0.34b

Carbohydrate sources (C)Basal diet 102±3.55c 51.17± 3.55c 0.61±0.07c 113.27± 3.74c 2.19± 0.18a 1.43± 0.08c 26.44± 0.76b 16.74±0.62b

BWGF 118±3.91b 67.17± 3.80b 0.80±0.08b 138.75± 4.93b 2.09± 0.11b 1.54± 0.06b 27.16± 0.84ab 17.30±0.58a

BRF 130±4.12a 79.81± 4.11a 0.95±0.09a 149.18± 3.34a 1.92± 0.09c 1.75± 0.12a 27.92± 0.60a 17.58±0.36a

Two-way ANOVA (P values)D 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001C 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.006 0.018D × C 0.0001 0.0001 0.0001 0.0001 0.0001 0.081 0.532 0.224

Means with different letters in the same column within each classification are significantly different (P<0.05). 1In wt.: initial mean weight (g), 2FW: final meanweight (g), 3WG: weight gain (g)= (FBW– IBW) ×100/IBW, 4SGR: specific growth rate (% day−1) = 100((LnFBW -LnIBW)/T), 5FI: feed intake (g dry diet fish−1 84days−1), 6FCR: feed conversion ratio = FI/WG, 7PER: protein efficiency ratio =WG (g)/dry protein intake (g), 8PPV: protein productive value= (protein gain in fish/protein intake in feed)× 100, 9EU: energy utilization=(energy gain in fish /energy intake in feed)× 100. Where FBW= body weight final (g), IBW = body weightinitial (g), T = duration of the trial in days, WG = wet weight gain (g) and FI = estimated feed intake (g).*PER does not include consideration of the protein contained in the carbohydrate sources added as the consumption of floc would complicate accurate calculation ofthe PER.

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Higher stocking densities normally resulted in poor water quality whichis the main stress factor in the aquaculture ponds (de Oliveira et al.,2012). Oxygen required for fish is generally affected by nitrite andammonia levels in the culture system (Remen et al., 2008; Tilak et al.,2007). Exchanging the water in the culture system is essential tomaintain the properties of water quality from deterioration. Frequently,using techniques with minimum or zero-water exchange increases ni-trogen levels in water (Iwama et al., 2000; Randall and Tsui, 2002). Inthe current study, the level of inorganic nitrogen concentrations wereincreased in BF tanks with increasing the stocking density (Labib, 2016;Xu and Pan, 2014). These findings agree with previous ones demon-strating that the in situ biofloc formation accelerates the nitrificationprocess in the water of tanks (Labib, 2016; Xu and Pan, 2014). Thebacteria formed in BF system is helping to keep the nitrogen at safelevels for fish rearing by utilizing nitrogen in situ microbial protein andincreasing the nitrification process to maintain the ammonia and nitriteat safe levels for Nile tilapia (Day et al., 2016a,b; Mansour and Esteban,2017a, b).

In the present study, higher values of BF volume and bacterialcounts were recorded in fish reared at 40 and 60 fish per m3 and fedBWGF or BRF diets as compared to the low density (20 fish per m3) fedthe basal diet. The surface of BF and its particle size can increase thesurface area required for bacterial growth to increase the produced BF(Mansour and Esteban, 2017a, b). Increasing the substrate surface areain BF tanks resulted in increased bacterial growth and improvement ofwater quality as well as food availability (Ferreira et al., 2016; NunesCaldini et al., 2015). The increased amounts of BF can be also attributedto the increased levels of nitrogen emissions as a result of increasing thestocking density. The differences may be due to the differences in fish

size, duration and experimental conditions.The growth parameters and feed utilization were better in BF units

than the control groups. These results are in consistence with thoseobtained with Nile tilapia reared in BF system (Mansour and Esteban,2017a, b). It has been reported by Azim and Little (2008) that the BFsystem can increase the fish total production by around 45 % over thenormal aquaculture systems. The increased growth performance in fishreared in BF with moderate stocking density (40 fish per m3) resultedfrom the optimum water quality values and availability of BF for thefish (Azim and Little, 2008; Ekasari et al., 2014; Emerenciano et al.,2012; Li et al., 2009; Qasem, 2016; Yoo and Lee, 2016). The groupsreared at high stocking level (60 fish per m3) performed less growthwhich is in consistent with the results obtained by (Bakeer et al., 2007;Sorphea et al., 2010). Tilapia can tolerate high densities; however, ti-lapia is a regional and aggressive fish and the adverse effect of densityon growth can be explained by its competition over territories, as wellas the constant stress resulting from overcrowding (Zhang et al., 2016).Carbon source affects the cultured species growth depending on theformatted biofloc characteristics, such as its “volume, chemical com-position, and ability to store bioactive compounds (e.g. polymers, car-otenoids, phytosterols and extracellular enzymes)” (Arnold et al., 2009;De Schryver and Verstraete, 2009; Wang et al., 2015; Zhao et al., 2016).Additionally, microbial flocs which are formed from different carbonsources act as a supplemental food source that constantly provide ad-ditional essential amino acids profile (microbial protein), poly-unsaturated fatty acids, minerals, vitamins, and an external source ofdigestive enzymes (Avnimelech, 2007; Azim and Little, 2008; Bakhshiet al., 2018; De Schryver and Verstraete, 2009; Luo et al., 2014). Theaddition of carbohydrate sources in the BF system may improving the

Table 6Whole-body proximate analysis (%) of Nile tilapia cultured for 84 days.

DM basisItems DM 1 CP2 EE3 Ash (kJ g−1)

Stocking density (D)20 27.52±0.9 56.69± 0.38 22.63± 0.13ab 20.67±0.27 223.2± 0.840 27.5± 0.4 56.34± 0.11 22.82± 0.7a 20.84±0.9 223.3± 0.360 27.69±0.7 57.16± 0.24 22.41± 0.13b 20.49±0.18 223.3± 0.3Carbohydrate sources (C)Basal diet 27.58±0.76 56.78± 0.37 22.58± 0.13 20.64±0.25 223.2± 0.2BWGF 27.53±0.74 56.4± 0.12 22.73± 0.8 20.87±0.7 222.9± 0.4BRF 27.6± 0.52 56.95± 0.3 22.95± 0.13 20.49±0.2 223.5± 0.3Two-way ANOVA P valuesD 0.057 0.080 0.027 0.093 0.922C 0.659 0.060 0.055 0.069 0.383D × C interaction 0.050 0.182 0.091 0.072 0.090

Means with different letters are significantly different (P<0.05). Means having same subscript letters were not significantly different. 1DM: dry matter, 2CP: crudeprotein, 3EE: ether extract.

Table 7Blood parameters of Nile tilapia cultured for 84 days.

Items RBC1

(109 L−1)WBC2

(1012 L−1)Hb3(U ml−1)

Ht4(U ml−1)

ALT(U ml−1)

AST(U ml−1)

Stocking density (D)20 4.14± 0.15a 241.00± 6.57b 98.00±0.08a 41.18± 0.84a 0.79± 0.06a 0.66± 0.06a

40 3.59± 0.14b 322.90± 6.62a 89.66±0.58b 39.00± 0.44a 0.51± 0.05c 0.34± 0.03c

60 2.91± 0.08c 256.00± 3.57b 68.33±0.84c 27.33± 0.75b 0.61± 0.06b 0.54± 0.03b

Carbohydrate sources (C)Basal diet 3.20± 0.20 288.10± 6.76 82.91±2.51 34.15± 2.68 0.79± 0.06a 0.63± 0.07a

BWGF 3.56± 0.18 274.15± 5.54 84.64±2.64 35.63± 2.75 0.64± 0.06b 0.49± 0.06b

BRF 3.87± 0.27 258.10± 5.57 86.58±2.44 37.37± 2.64 0.49± 0.05c 0.42± 0.04c

Two-way ANOVA (P values)D 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001C 0.158 0.434 0.897 0.704 0.0001 0.0001D × C 0.0001 0.026 0.783 0.770 0.028 0.002

Means in the same column within each classification bearing different superscript are significantly different at (P<0.05). 1RBC = Red blood cells, 2WBC = WhiteBlood Cells, 3Hb=Hemoglobin and 4Ht=Hematocrit.

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growth performance and feed utilization which depends on the use ofdifferent C: N ratios and different feed intake levels as well as the dif-ferences in the carbon sources (Arnold et al., 2009; De Schryver andVerstraete, 2009; Wang et al., 2015; Zhao et al., 2016). BRF and BWGFwere tested in the present study cheap and available carbon sources.

The proximate composition of the fish whole body may change as areflection of a number of factors, including water quality, stress factors,availability of nutrients, feed intake and utilization (Dawood et al.,2016; El Basuini et al., 2017). In the current study, the whole-bodyether extract showed a slight change with density levels and the lowestvalue recorded in case of fish reared at 60 fish per m3 may be due to thestress effect of increased density. The decreased ether extract can beattributed to the decreased feed intake that can result in low amounts ofaccumulated lipids in carcass composition.

Blood biochemistry indices are useful tools that aid in indicating thegeneral state of fish health which can differ with water characteristicand nutritional state (Dawood et al., 2015; El Basuini et al., 2017, 2016;Kuhn et al., 2009). Overall, blood parameter values recorded in thepresent study are within the acceptable limits of the Nile tilapia (Ayyatet al., 2017a, b; Mahmoud and El-Hais, 2017). In this study, over-stocking density (60 fish m−3) adversely affects RBCs, hemoglobin andhematocrit values of Nile tilapia. These findings are in accordance withMehrim (2009) and Kpundeh et al. (2013) who reported significantlydecreased hematological indices (RBCs, WBCs, Hb, PCV, Ht and bloodplatelets) by increasing stocking rate of Nile tilapia. Serum glucose andcortisol levels are signs of the fish stress state (Barreto and Volpato,2006; EL-Khaldi, 2010). In the current study, low glucose and cortisollevels observed in BF units with high rearing densities compared withfish reared in low density and fed the basal diet, which shows that usingthe BF system with high density levels showed moderate stress relatedfeatures to the fish stocking rate or/and carbon source. Low levels ofglucose and cortisol of Nile tilapia reared in high stocking density in thecurrent study can be attributed to the formed BF which can act as asupplemental food source and anti-stressor factor due to its high con-tent of essential amino acid profile (microbial protein), polyunsaturatedfatty acids, minerals, and vitamins (Avnimelech, 2007; Azim and Little,2008; Bakhshi et al., 2018; De Schryver and Verstraete, 2009; Luo et al.,2014).

The antioxidant defense system is highly linked with fish healthstatus and immune system (Dawood et al., 2016; Kuhn et al., 2009).Evaluation of superoxide dismutase and catalase as the important an-tioxidant enzymes can be considered as the biomarkers of oxidativestress besides indicating antioxidant capacity of aquatic organisms(Aruoma, 1998). The levels of catalase and superoxide dismutase ac-tivities were significantly altered among the treatment groups withlower recorded values in BF units particularly, at 40 fish per m3 groupand fed with BRF as a carbon source. These results may be due to theincreased stocking density which can be among the reasons of increasedoxidative stress in fish.

5. Conclusion

The present study demonstrated the effective application of brokenrice flour (BRF) or broken wheat grain flour (BWGF) as carbon sup-plement for biofloc. The addition of carbon sources particularly, BRF atstocking density 40 fish per m3 promotes growth performance, feedutilization and anti-stress markers of Nile tilapia cultured in BF systemas an effective environmentally friendly aquaculture practice.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgment

The first author would like to thank all staff member of Fish and

Fig. 1. Antioxidant enzymes: (A) catalase; (B) superoxide dismutase of Niletilapia cultured for 84 days. Values are expressed as mean± SE from triplicategroups. Bars with an asterisk are significantly different from those of controlgroup (P<0.05).

Fig. 2. Stress responses: (A) glucose; (B) cortisol of Nile tilapia cultured for 84days. Values are expressed as mean±SE from triplicate groups. Bars with anasterisk are significantly different from those of control group (P<0.05).

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Animal Department, Faculty of Agriculture, Alexandria University,Egypt for supporting this research. We express our sincere gratitude toEl-Kady fish farms group, Edkuo province, Alexandria Governorate,Egypt for the facilities, equipment and technical support. This work wasfunded by Researchers Supporting Project number (RSP-2019/121),King Saud University, Riyadh, Saudi Arabia.

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