EFFICACY OF DIFFERENT PROTEIN LEVELS ON GROWTH, IMMUNITY AND MEAT QUALITY OF NILE TILAPIA (Oreochromis niloticus) UNDER ZERO EXCHANGE HETEROTROPHIC CULTURE

INTRODUCTION

Nile tilapia (Oreochromis niloticus) has emerged as one of the most significant aquaculture species globally, representing approximately 8% of total finfish culture production worldwide. This species has been successfully introduced throughout tropical and subtropical regions due to its remarkable adaptability, fast growth rate, disease resistance, and ability to thrive in diverse farming systems (Abduljabbar et al., 2015; Teodósio et al., 2020). The global expansion of tilapia aquaculture has provided substantial benefits for livelihoods in developing countries,  serving as both a source of household income and family consumption(Mridha et al.,2017).Astheaquaculturesector continues to grow (accounting for nearly 47% of total fish production globally) the sustainability of Nile tilapia farming depends increasingly on the development of cost- effective and environmentally friendly feeding strategies (Prabu et al.,2019;Teodósio et al.,2020). Dietary protein constitutes the most expensive component of formulated fish feeds, typically representing over 50% of operational costs in intensive aquaculture systems (Abdel‐Tawwab & Ahmad, 2009; da Silva  et  al., 2018). Consequently, optimizing protein levels in tilapia diets has become a critical research focus, balancing economic viability with environmental sustainability.  Extensive research has demonstrated that protein requirements for Nile Tilapia vary significantly across different life stages. Fry weighing less than 1 g requires relatively high protein levels (35-50%), while fish in the 1- 5 g range need 30-40% protein, and those in the 5-25 g category perform optimally with 25-30% protein (Hafedh, 2001). Studies have shown that fry (approximately 0.5 g weight) requires 45% crude protein (CP) for optimal growth, whereas juveniles (approximately 40 g weight) achieve maximumperformance with35%CP diets(Abdel- Tawwab et al., 2010). These variations reflect the changing metabolic demands as fish develop, with younger fish requiring higher protein for tissue formation and growth,  while larger fish can utilize protein more efficiently for maintenance(Hafedh,2001). 

The environmental implications of protein management in aquaculture cannot be overstated. Excess dietary protein that exceeds the fish's metabolic capacity is catabolized and excreted as nitrogenous waste, contributing to water pollution and eutrophication (Teodósio  et  al., 2020; Zablon  et  al., 2022). In traditional aquaculture systems with water exchange, this waste is partially diluted, but in zero-exchange systems (which are increasingly adopted for environmental and biosecurity reasons) the accumulation of nitrogenous compounds becomes a critical concern. Biofloc technology (BFT), a form of zero-exchange heterotrophic culture, has gained prominence as a sustainable aquaculture approach that convertspotentialpollutantsinto microbialproteinthrough controlled heterotrophic processes. In such systems, the relationship between dietary protein levels and water quality parameters becomes particularly complex, as the microbial community plays a crucial role in nutrient recycling(Hisano et al.,2020;Long et al.,2015).

Recent research by Hisano  et  al.  (2020) has demonstrated that, in biofloc systems, Nile tilapia fingerlings (6-25g) can maintain growth performance with reduced dietary protein levels (28% CP) compared to  conventional recommendations (36% CP), resulting in significantcostsavingsandreducedenvironmentalimpact. These findings challenge traditional protein formulation standards and suggest that the microbial protein generated within BFT systems may partially compensate for reduced dietary protein (Debnath  et  al.,  2025). However, most  existing studies have focused primarily on growth performance and feed conversion efficiency, with limited attentiontoimmunefunctionand meatqualityparameters.

Theinteractionbetweendietaryproteinlevelsand immunefunctioninNiletilapiaremainspoorlyunderstood, despite evidence that protein nutrition significantly influences immune competence in fish. Adequate protein intake is essential for the synthesis of immune-related proteins, antibodies, and acute-phase proteins, whereas protein deficiency can compromise immune responses and increase disease susceptibility. Conversely, excessive  protein intake may induce metabolic stress that could negatively impact immune function (Gaye-Siessegger et al., 2006;Nguyen et al.,2021).Inzero-exchangesystems where water quality parameters fluctuate more dramatically than in flow- through systems, the relationship between protein nutritionand immune statusbecomeseven more critical,as suboptimal water conditions can exacerbate stress responses(Hisano et al.,2020;Wang et al.,2015).

Meatqualityrepresentsanothercrucialdimension affected by dietary protein levels. The proximate composition of fish muscle (including protein, lipid, moisture, and ash content) is directly influenced by dietary formulation. Studies have shown that variations in dietary  protein can alter the fatty acid profile, texture, color, and overall sensory characteristics of fish fillets (Hafedh, 2001). For example, Ogweny  et al. (2023) investigated the impactofproteinsourcesonwater quality,growthandfeed utilization ofNile tilapia fingerlings reared under BFT. Eid et al. (2020) focused on the impact of various protein levels in improving the growth and utilization of food under BFT system. Similarly, a study conducted by Ghaedi and Khanjani (2025) examined the impact of varying protein levels on water quality and reproductive performance of Niletilapia brood stockinbiofloc system. Inanother study, the meat quality of Nile tilapia flash was evaluated under BFT system (Kattakdad  et  al., 2026). In zero-exchange heterotrophic systems, where microbial communities contribute to nutrient cycling and potentially to the fish's nutritional status, the relationship between dietary protein  and final product quality may differ significantly from conventionalculturesystems(Hisano et al.,2020).

However few studies have investigated how varying dietary protein concentrations influence growth,  immunity and meat quality in tilapia cultured under zero- exchange heterotrophic conditions, where microbial biomass contributes significantly to nutrition and health. The current study addresses these critical knowledge gaps by investigating the efficacy of different protein levels on growthperformance, immune function, and meatqualityof Nile tilapia under zero-exchange heterotrophic culture conditions. This research builds upon previous findings that demonstrated the feasibility of protein reduction in biofloc systems (Hisano  et  al., 2020), while expanding the evaluation to include comprehensive immune parameters and detailed meat quality assessments. By establishing the optimal protein level that maximizes growth, maintains robust immune function, and ensures high- quality fillets whileminimizingenvironmentalimpact,thisresearchaims to provide practical guidance for sustainable Nile Tilapia production in zero-exchange systems, an increasingly important aquaculture approach in the context of water scarcity and environmental regulations. Therefore, the present study was designed to investigate the efficacy of different protein levels on growth, immunity and meat quality of Nile Tilapia (Oreochromis niloticus) under Zero exchangeheterotrophicculturesystem.

MATERIALSANDMETHODS

Ethics approval: The experiment was conducted in Fish Nutrition Laboratory, Department of Zoology, Wildlife and Fisheries, University of Agriculture Faisalabad, in compliance with the ethical committee of University of Agriculture, in the guideline of Fish Nutrition Laboratory, Department of Zoology, Wildlife and Fisheries of the same university at Faisalabad. All the ethical principles and standards have been followed during the dissection and handlingofspecimens.

Experimental design and ethical considerations: The experiment was carried out under indoor conditions in glass aquaria for 4 months. The volume of each aquarium was calculated to be 120 liters using the dimensions: 80cm (length) × 35cm (width) × 43cm (height). The effect of various protein levels 15, 20, 25 and 30% on growth, immunityandmeatqualityofNiletile underzeroexchange heterotrophic culture was conducted in Fish Nutrition Laboratory, Department of Zoology, Wildlife and Fisheries, University of Agriculture Faisalabad, in compliance with the ethical committee of University of Agriculture, in the guideline of Fish Nutrition Laboratory,

Department of Zoology, Wildlife and Fisheries of the same university at Faisalabad. All the ethical principles and standards have been followed during the dissection and handling of specimens. Acclimatization of Nile tilapia larvae was done for one week on basal diet before being distributed to respective glass aquarium of experimental units.

The experiment was conducted indoor conditions using glass aquarium over a period of 4-months. Four experimental groups were made with the same stocking density(50fishes/m3)andfed withvariouslevelsofprotein (T1 (15%), T2(20%), T3(25%) and T4(30%) with three replications of each aquarium (n=3),As aquarium were considered as experimental unit for statistical analysis and individual fish were tested as subsamples. Prior to the start of the trial, all aquarium were cleaned thoroughly, dried andfilled up with fresh water and inoculated ofbiofloc was done form a prepared cultured aquarium. Juveniles of Nile tilapia were fed daily at the rate of 6% of their body weight throughoutthestudyperiod.

Floc preparation: The clean aquarium was taken with 45 liters of water and continue vigorous aeration was done. Accordingtotheprotocol,for thepreparationof45litersof fresh water, 5 L of inoculum was required for the floc

development, So, 600g of pond soil, 0.3 g of Ammonium sulphate and 6 g of carbon source (starch) were added in water aquarium. Within 24-48h the inoculum was ready and then shifted to the main aquarium. The carbon source added daily in the inoculum for floc development. So, every 1kg of feed given, 600 g of carbon source was added tothesystemto maintainC:N(10:1).Oncetheflocvolume reaches 15-20ml further addition of carbon source was not required(Avnimelech,1999).

Diet formulation: Soybean meals, wheat flour, rice bran, wheat bran, fish meal, soy oil, vitamins, and minerals premixweremixedintofourdifferentdietformulatomake feed for fishes. All these ingredients were bought from the local market of Faisalabad. Ingredients were weighed and thoroughly mixed before feed diet formulation, and all the feed ingredients were properly grinded into fine particles by using mortar and pestle and the large sized particles were removed with the help of a sieve. Weight balance was usedtoweightheingredients. ASmallquantityofminerals and vitamins were added and mixed well. At the end soya oil was added and mixed well to ensure thorough mixing. The diets were prepared, packed and labeled for ease (Table1).

Table1:Formulationandcompositionofexperimentaldiet

Water quality parameters: Water quality parameters were monitored daily throughout the experimental time. Temperature in centigrade was measured by using dissolved oxygen meter (TP-101), Dissolved oxygen (DO) was measured by using Dissolved Oxygen Meter (HI- 9147). Total dissolved solids (TDS) were measured by using YL-TDS2-A meter, and pH of water was checked by using pH meter (HANNA) pH tester). Total Ammonia, Nitrite and Nitrate were measured by using APA freshwaterAmmoniakit.

Proximate analysis of fish meat and diet: The proximate composition (Protein, Lipid and Ash) of the biofloc material and fish meat was analyzed according to standard protocols AOAC (2006). Meat quality parameters include crudeprotein wascalculated byKjeldehal’s method.Crude Protein was calculated by applying this formula (Crude Protein = N2×6.25), and crude fat was calculated by

Soxhlet apparatus (Total fat (%) = (Weight of empty extraction cup-weight of extraction cup with fat after) / (weightofsample) and ash content was calculated byusing this formula(Totalash(%)=(Weightofash(g)/(weightof sample)×100).

Growthperformance:Growthparametersincludingbody weight in grams and length gain in cm were calculated on weekly basis. Survival percentage (%), Specific Growth Rate (SGR), feed conversion ratio (FCR) was also measured. At the end of experimental trial, the following variableswereevaluated: Weight gain wasassessedbythe givenformula:

Weightgain(g)=Finalweight(g)-Initialweight (g)……….(a)
Lengthgainwasmeasuredbyusingthisformula: Gaininlength(cm)=Finallength(cm) -Initiallength (cm)…(b)

TheformulaforthecalculationofSGRisgivenbelow:

Specificgrowthrate(SGR)=

……………(c)

FCRwasassessedbyfollowingformula:

Foodconversionratio(FCR)=

……………(d)

Survivalrateassessedbyfollowingformula: Survivalrate(%)= ….(e)

Innateimmunityandbloodprofile

Immunoglobulin levels were measured directly by subtracting the values of albumin from those of total proteinbyusing12%ofpolyethyleneglycolsolution.

WBCs(Whitebloodcells)werecalculatedbyusing Neubauerhemocytometer.

NoofWBCs(103/µL)=

……………….(f)

Lysozymeactivity:Theturbidmetricassaywasusedfor determininglysozymeactivity. Lysozymeactivity(µl/ml)=(Transmittance(530nm))/ (seruminmiused)x1000.(g)

RBCsThehemocytometer method used totakereadingsof red blood cells (RBCs) (Rusia et al., 1996) No. of RBCs=

Hemoglobin content, Cyanmethemoglobin methods were employedtoevaluatethehemoglobincontentoffishblood. Hemoglobintestkitwasused(Blaxhall&Daisley,1973).

Hemoglobinconcentration( )=

Hematocrit contents, A standard microhematocrit method was needed for the measurement of hematocrit. The capillaries tubes will be used for the collection of Heparinized, non-clot blood. The capillaries tubes will be sealed with wax and will be centrifuged at 12000 rpm for 5 min(Wintrobe,1967)

Hematocrit= ×100

Antioxidant enzyme activity: Assay of antioxidant enzyme activity were done to assess the antioxidant enzyme activity of Nile Tilapia (Asghar & Masood, 2008). Superoxide dismutase (SOD) was measured by using Tetrazolium salt (Maier & Chan, 2002). Glutathione peroxidase (GPx) activity was measured indirectly by the kinetic colorimetric assay (Paglia and Valentine, 1967). Catalase activity was determined by Catalase assay kit (Weydert&Cullen,2010).

Statistical Analysis: The data arranged in excel and mean± standard error was calculated. The experiment consisted of 4 different crude protein treatments, each

conducted in three replicate aquaria (n=3). The aquarium was considered as experimental unit. The statistical analysis was performed using Statistix version 8.1. The datawassubjectedtoANOVAundercompleterandomized design (CRD) and mean were further subject to post hoc test by Tukey HSD (using honest significant difference) (p≤0.05).

RESULTS

Water quality maintenance and fish survival: During the experimental period, the mean system temperature was 27 °C ± 2 (range 26–30 °C). Dissolved oxygen (DO) concentration averaged 5.2 mg/L (range 4–6 mg/L), and pH averaged 7.06 (range 7.06–8.003), generally within the optimal range for tilapia culture, except for occasional low pH values observed during the trials. Nitrite (NO₂⁻), nitrate (NO₃⁻), ammonia (NH₄⁺), total dissolved solids (TDS), and total suspended solids (TSS) exhibited fluctuations across tanks with different dietary crude protein (CP) levels (15, 20, 25, and 30%) (Fig. 1). No significant differences (p ≤ 0.05) were detected in nitrite, nitrate, or TDS levels amongtreatments.However,thehighestnitrate,nitrite,and TSS concentrations were observed in the 30% CP group (10.06 ± 1.07 mg/L and 694 ± 6.3 mg/L, respectively) (Fig. 1). Floc volume (FV, ml/L) increased significantly throughout the study, with maximum values of 105.9 ± 0.5 ml/L recorded at 20% and 30% CP, though these differences were not statistically significant. Overall, FV development did not differ significantly among treatments(p ≤ 0.05)(Fig. 1).

Proximate composition of experimental diets (%) and Niletilapia(after trialperiod):Proximate compositionof dietandmeatofNiletilapia(O.niloticus)fed withdifferent CP-levels under zero exchange heterotrophic culture system showed (Tab. 2). The nutritional values in the experimentaldiet(crudeprotein(CP),crudelipid (CL),ash (%) and dry matter (DM) content percentage) were calculated. The results indicated that highest CP, and DM values, and the lowest CL and ash values were recorded in the diet and fish meat fed with 30 % CP, followed by25, 20 and 15 % CP under zero exchange heterotrophic culture (TukeyHSD;p≤0.05).

The results showed that supplementation of CP in diet of Nile tilapia significantly enhanced the proximate composition of fish body (p≤0.05). The addition of 30 % CP in diet had more significant impact on Nile tilapia body ascomparedtoothertreatments(lowerlevelsofCP).Italso improves crude lipid, Ash and DM content of Nile tilapia body followed by 25, 20 and 15 % supplementation of crude protein in diet under zero exchange heterotrophic culture (Tukey HSD: p≤0.05) (Tab. 2). The proximate attributes of Nile tilapia body were recorded; CP (47.033±0.46 to 52.633±0.26 %), CL (20.900±0.34 to 26.800±0.40 %), ash (10.433±0.18 to 15.500±0.15%) and

Arshad et al.,   J. Anim. Plant Sci., 36 (4) 2026

DM content (91.877±0.04 to 92.123±0.03 %), after the (p≤0.05)(Tab.2). treatment of four different level of crude proteins in diet

Figure 1: Effect of different crude protein (CP) levels on dissolved inorganic nitrogen concentrations, TSS, TDS and

FV of rearing cages throughout the experimental period. Values showed the mean ±S.E of four different CP levelswithreplication. (i)Nitrate, (ii)Nitriteand(iii)Ammonia, and(iv) TSS= Totalsolublesolids,(v)TDS= Totaldissolvesolidsand(vi)FV=flocvolume.

Growth response: Throughout the trial period, the results showed that the survival rate (%) of Nile tilapia (O. niloticus) wasabove90%in aquaria fed withfourdifferent levels of crude protein under zero-exchange heterotrophic culture, and all treatments differed significantly from each other (p≤0.05) (Tab. 3). Growth parameters such as final body weight (FBW), weight gain (WG), and fork length (FL) were significantly higher in fish fed with 30% CP compared to the other treatments under zero-exchange heterotrophic culture (Tukey HSD; p≤0.05) (Tab. 3), followed by 25%, 20%, and 15% CP levels. The highest

weight gain (101.1 ± 1.01 g) was recorded in this group compared to the other treatments (Tab. 3). The specific growth rate (SGR) increased significantly with increasing CP levels from 15% to 30%, with each treatment differing significantly from the others (Tukey HSD; p≤0.05). Conversely, a significant decrease in feed conversion ratio (FCR) was observed with increasing protein levels from 15% to 30% (Tukey HSD; p≤0.05) under zero-exchange heterotrophic culture. There was no significant difference in FCR between the 15% and 20% CP levels, or between the25%and30%CPlevels(Tab.3).

Table 2: Proximate composition of diet and meat of Nile tilapia (O. niloticus) fed with different CP-levels under zero

exchangeheterotrophicculturesystem.

Proximatecompositionofdiet

Mean ± S.E with lower case letter showed significance of the treatments on growth parameters (Tukey HSD, p≤0.05). IBW= Initial body weight (g), FBW= (Final body weight (g), FL= Final length (cm), FCR=Feed conversion ratio, SGR= Specific growth rate

Innate immunity, blood profile and antioxidants activity: Supplementation with varying levels of crude protein (15, 20, 25, and 30%) under a zero-exchange heterotrophic culture system significantly enhanced innate immunity, hematological parameters, and antioxidant activityinNiletilapia(O. niloticus) (Tukey HSD; p≤0.05) (Tab. 4). Fish fed 30% crude protein exhibited the highest levels of immunoglobulin (IG), erythrocyte content (EC), hemoglobin (Hb), and hematocrit (Hct), followed by those fed 25%,20%,and 15%crude protein, respectively(Tukey HSD; p ≤ 0.05) (Tab. 4). No significant differences were observed in WBC counts between fish fed 20% and 25% crude protein (p ≤ 0.05); however, fish fed 30% crude

protein showed a lower WBC count (28.900 ± 0.17c %) (Tab.4).

The supplementation of a 30% crude protein level inthedietsignificantlyenhanced theantioxidantactivityof Nile tilapia under zero-exchange heterotrophic culture (p≤0.05), followed by the 25%, 20%, and 15% crude protein treatments (Tab. 4). Significantly higher lysozyme (LZ, mL⁻¹) and catalase (CAT, µmol/mg) activities were recorded in Nile tilapia fed a diet containing 30% crude protein under zero-exchange heterotrophic culture (Tab. 4). The SOD activity did not differ significantly between the 25% and 30% crude protein treatments, with values of 55.33±0.11 and 57.77±0.76 µmol/mg, respectively (Tukey HSD; p≤0.05). Similarly, GPx activity after

supplementation with 25% and 30% crude protein was not significantly different under the zero-exchange heterotrophic culture system (Tab. 4). The lowest antioxidant activities were recorded in fish fed the 15% crude protein diet: LZ (25.10±0.11 µmol/mg), CAT (27.37

± 0.53 µmol/mg), SOD (43.13±1.01 µmol/mg), and GPx (3.96±0.13 U/g), which were significantly different from all other treatments under the zero-exchange heterotrophic culturesystem.

Table.4:ImmunologicalparametersandantioxidantenzymeactivityofNiletilapia(O.niloticus)fedwithdifferent crudeprotein(CP)sourcesunderzeroexchangeheterotrophicculturesystem.

Mean ± S.E with lower case letter showed significance of the treatments on immunological parameters and antioxidants ability of Nile Tilapia (Tukey HSD, p≤0.05). IG (mg/ml) = Immunoglobulin, WBCs= White blood cells, LA= Lysozyme activity, EC= Erythrocyte content, Hb= Hemoglobin content, Hct= Hematocrit.

DISCUSSION

The present study aims to check the effect of different protein levels on the growth, immunity and meat quality of Nile Tilapia under zero exchange heterotrophic culture. The experiment consists of four different experimental groups (CP at 15, 20, 25 and 30% level) each replicated three times (n=3) each with stocking density of 50 fish/m3.  The juvenile of Nile tilapia fed daily basis and physicochemical parameters chemical parameters were checked regularly and maintained during the study period. The pH, temperature and DO were not statistically significant from each other among experimental treatments. The currentstudyshowed that flocvolume(FV)(ml/L) was significantly higher in treatment T4 (30% CP) and recorded as 105.9±0.5 ml/L. This may be due to degradation of carbon and nitrogen components of floc, which provide large surface area for bacterial and other microorganisms’ growth, hence enhanced biofloc volume. The present studyis inagreement with Zahra and Mateen (2025), who reported that the addition of 25% crude protein with a higher carbon-to- nitrogenratio significantlyimproved bacterialbiomass in biofloc technology (BFT) systems. Moreover, higher CP may contribute to increased nitrogenous substrate inthe culture water, which, inthe presence of carbon sources, can enhanced heterotrophic microbial growth and subsequent floc formation. These biofloc not serve as biological filters but also act as supplemental nutrients sources for the culture of Nile

tilapia (Li  et  al., 2025; Yadav  et  al., 2025). The current study agreement with Mansour and Esteban (2017) stated that higher carbon and different protein level supplementation enhanced biofloc particles as well as water quality parameters. The study also agreement with Ferreira  et  al.  (2015) who started that increase in surface area and substrate for microbial action enhanced the waterqualityas well as theavailabilityof food,whichalsoimpactthefishgrowth.

The results showed that a higher level of NH4 was recorded during 3rd month and a decline in the last month of the experimental period, while values of ammonia were not statistically significant among the treatments (15, 20, 25 and 30 % CP levels). The nitrate values increased steadily throughout the study period and higher NO-3 level were recorded in aquarium fed with30%crudeprotein(Fig1).Theraisingofdissolve inorganic nitrogen by bacterial activity during recycling defecate a substantial amount of nitrogenous ammonia by fish (Burford et al., 2004; Crab et al., 2010) and bacteria are usually associated with the suspended biofloc particles, providing extra nutrients and exogenous digestive enzymes (Moreno‐Arias  et  al., 2017; Wang  et  al., 2015) and stimulating growth and survival(Abduljabbar et al.,2015;Ekasari et al.,2015).

The TSS and TDS were significantly enhanced in all the aquarium fed with different crude proteins and all the treatments were significantly different from each other’s except 20 % CP level whereas the TSS value remained constant throughout experimental period (Fig 1). In the current study the

TDS value remains between 626 ±6.8 to 694.5± 2.16 mg/L. The increase in TSS and TDS consistent with recent findings Abd-El Azeem  et  al.  (2025) who proposed that diet composition directly influences water quality parameters. Elevated protein diets have been associated with greater dissolved nitrogenous compounds and solids in culture system due to increased wasteproduction and mineralsreleased from feed breakdown (Wani  et  al., 2025). The study agreement with Long  et  al. (2015) who proposed that total dissolve solid amount reached to 1000 mg/L during the study period. The da Silva  et  al.  (2018) reported all treatments of proteins significantly impact the development and formation of biofloc in water aquariums, and gradual increase in TSS and BFV were recorded during the study periods. Furthermore, he stated that the amount of BFV in the treatment T4 was not statistically different from the amount of TSS after 15 days of experiment. The constant aeration and varied conditions keep thelevelofbioflocconstantand stableinthewatercolumn(Xu&Pan,2012).

The current study showed that the survival rate (%) of O. niloticus was above 90% in aquariums fed with four different levels of crude protein under zero exchange heterotrophic culture, and all the treatments were significantly different (p≤0.05) from each other. The highest survival % Nile tilapia was recorded (99.123±3.74a) inan aquarium fed with 30 %

crude protein (CP). Similar to the current study Mansour and Esteban (2017) stated that the survival percentage of Nile tilapia was recorded 90 to 100 % throughout study period, fed with 20 and 30 % crude protein or carbon sources (Zahra & Mateen, 2025). Azim  and  Little  (2008) also reported that the Nile tilapia survival rate was recorded 100% when BFT fed adietof24and35%crudeprotein(CP).Inanaquarium fed with 30 % CP showed a higher growth rate and lower FCR than the other treatments under zero exchange heterotrophic system (Table 3). Similar findings were reported in recent studies on biofloc- based and heterotrophic systems, where moderate protein-based diets supplementation results in superior growth, survival and lower FCR ration due to improved nutrient recycling and enhanced microbial activity. For example, Ediwarman  et  al.  (2025) who proposed that the feed containing 30 % protein yielded the best production performance, including higher growth rate and the lowest Feed Conversion Ratio (FCR) in a zero-water exchange culture system (Adineh et al.,2025;Yılmaz et al.,2024).

For optimal growth of fish, high quality of feed hasbeen utilized inrecent aquacultureapproaches which is not just impact growth but also maintain antioxidant status, physiology and immunological parameters of cultured fish (Kiron, 2012). The current study showed that higher value of immunoglobulin

(IG), erythrocyte content (EC), hemoglobin (Hb) and hematocrit (Hct) content were significantly enhanced by the supplementation of 30% crude protein level, followed by 25, 20, and 15 % of crude protein levels, respectively, under zero heterotrophic culture system (Tukey HSD, p≤0.05). Like current study Wang  et  al. (2024) proposed that increasing dietary protein supplementation evaluated the key immune function (i.e. immunoglobulin concentrations, lysozyme activity and innate immune defense) in Juvenile Hybrid Sturgeon. Long  et  al.  (2015) stated that Nile tilapiashowedimprovementinimmunehealthafterthe supplement of carbon in form of glucose. However, he also stated that there was no significant difference in WBCs,redbloodcell,hematocritorhemoglobinlevels as compared to fish reared in clear water. While Mansour and Esteban (2017) stated that fish reared under BFT showed higher WBC counts than fish maintained in clear water. Furthermore, the increased number of WBCs was due to the increase in both neutrophils and lymphocytes, while monocyte numbers remained unchanged. The number of leucocytes in blood indicates the health status of fish and they play a vital role in innate immunity during inflammation and stress (Klak et al., 2024; Philominal et al.,2024;Secombes,1996;Soliman&Barreda,2023).

Theincreaseinlysozyme activity(LA), CAT, SOD and GPx activity were recorded after the supplementation of different CP levels under zero exchange heterotrophic culture system and the maximum activity of CAT, SOD and GPx was recorded inNile tilapia fed with 30 % CP. This showed that higher CP level promotes antioxidant defense in Nile Tilapia. Abd-El Azeem et al. (2025) also reported that 30 and 35% protein in diet significantly enhanced the antioxidant activity and immune parameters (lysozyme activityand IgM) in Nile tilapia (Sherif et al., 2024).Similar tothecurrentstudy, the Rind et al. (2023) stated that different protein levels and carbon sources together significantly improved the SOD and CAT activity in O. niloticus ((Mansour & Esteban, 2017).The maximum activity of both enzymes was noticed in fish fed with 30% protein. At protein levels 20 and 30% in integration with carbon sources showed compensating effect of biofloc was manifest in case of

catalase activity. He further stated that maximum performance of antioxidant enzymes was noticed in fishes fed with 30 % of protein as compared to control, only contradiction is that the supplementation sources and condition were different from present study(Table 4). CAT and SOD are enzymes linked with the lipid peroxidationinhibition(Gulcin,2025).Thesuperoxide dismutase involves inthe catalysis ofsuperoxide anion to produce H2O2 (Tao  et  al., 2013), furthermore the catalyze enzyme acted and converted it into oxygen andwaterandinhibittheinitiationoflipidperoxidation

(Tavares-Sánchez et al.,2004).Thedietaryprotein level (greater than 26%) significantly enhanced the hepatic SOD and CAT activity and gene expression of other enzymes (Kou  et  al., 2025). Faisal  et  al.  (2025) also proposed that dietary purslane extract enhanced the CAT andSODperformance in Labeo catlafingerlings. These studies showed that high CP levels influence CAT and SOD activities in fish, reduce oxidative stress, and may improve fish welfare. These studies showed that high CP levels influence CAT and SOD activities in fish, reduce oxidative stress, and may improve fish welfare (Parrilla-Taylor  et  al., 2013; Salamanca  et  al., 2025). Verma  et  al.  (2016) proposed thatBFT treatment had astrongimpactonfish stressas it decreases physiological stress (glucose and cortisol level) in Labeo rohita. Similarly, another study lined with current results the shrimps feed on low amount of protein exhibited minimum antioxidant capacity as compared to those reared under zero exchange heterotrophicculturesystem(Xu&Pan,2014).

Conclusion: In conclusion study proposed that Nile Tilapia fed with 30% Crude protein had excellent growth performance, antioxidant abilities and efficient immune system. This optimum protein level was essential for maximizing biofloc volume and developmentofmicro faunaon whichNiletilapiafeed. The lower level of FCR at 30 % CP level means that feed is efficiently converted into fish weight. Specifically, diets with 25 and 30% crude protein content yielded the best growth, immune response and antioxidant activity, demonstrating the importance of crude protein supplementation in zero heterotrophic exchange system. Furthermore, it is suggested that a cost-effective product can be formulated through industrial linkages, and stakeholders should play their roleinthisregard.