Egyptian Journal of Aquatic Biology & Fisheries Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt. ISSN 1110 – 6131 Vol. 25(3): 841 – 859 (2021) www.ejabf.journals.ekb.eg The effect of dietary protein level and amino acid supplementation on the Nile tilapia (Oreochromis niloticus) nursering performance under biofloc system conditions at cold suboptimal water temperature. Khaled H. Salama 1*, Shimaa Henish 2, Khadra A. Mohamed 4, Marian G. Nassif 2, Magdy T. Khalil 3, Ashraf Suloma5 1. 2. 3. 4. 5. Basic Sciences Dept., Institute of Environmental Studies and Research, Ain Shams University, Egypt. National Inst. of Oceanography and Fisheries (NIOF), Egypt. Zoology Dept, Fac. Sci, Ain Shams University, Egypt Aquatic Environment, National Center of water Research. Fish Nutrition Lab (FNL), Animal Production Dept, Faculty of Agriculture, Cairo University, Egypt. * Corresponding author: salamera61@hotmail.com _______________________________________________________________________________________ ARTICLE INFO Article History: Received: June 2, 2021 Accepted: June 24, 2021 Online: June 30, 2021 _______________ Keywords: Aquaculture, Biofloc, Nile Tilapia, Amino acids. ABSTRACT This study was conducted to evaluate the effect of dietary protein level and amino acid supplementation on tilapia nursering performance under biofloc system conditions at cold suboptimum temperature. Four experiment diets were examined; 30% CP diet as a positive control (30P), 22 % CP diet as negative control (22P), 22 % CP diet supplemented with 1% lysine (22PL) and 22 % CP diet supplemented with 0.5% threonine (22PT). Formulated diets were tested under biofloc system conditions in three replicates for each treatment. 12 experimental tanks 1000 L were used, each tank was filled with 400 L. each tank was stocked with 50 fries (6.20±0.01 gm. /fish). The experiment was extended for 97 days. Fish fed on a 22PT diet had the highest significantly the best values of weight gain (WG), feed intake (FI), feed convergent ratio (FCR), and specific growth rate (SGR) (11.10, 16.95, 1.55 and 1.40, respectively). Also, 22PT diet groups record the highest protein content of dried microbial floc and wet whole-body. The lowest total ammonia nitrite (TAN) values were recorded for the fish groups fed on a 22PT diet. It could be concluded that under biofloc system conditions, a low protein diet supplemented with threonine can be used as a tool to improve biofloc system performance during tilapia nursering at cold suboptimal water temperature. INTRODUCTION The World Aquaculture annually growth rate needs to be increase in order to mitigate the shortage in protein food supply, which is particularly situated in the developing countries (Subasinghe, 2005; Gutierrez et al., 2006; Matos et al., 2006). To determine the aquaculture potential of a species, studies should be undertaken to fine tuning the culture conditions for optimizing growth under different production systems including fish farming and Integrated Agri-Aquaculture Systems (Deacon, 1997; Suloma and Ogata, 2006; Kimera et al., 2021a and 2021b). Biofloc technology (BFT) is a technique of enhancing water quality through the addition of carbon source to the production unites which promoted nitrogen uptake by heterotrophic bacterial growth decreases the 842 Khaled H. Salama et al., 2021 ammonium concentration more rapidly than nitrifying bacteria (Widanarni et al., 2012 and Hargreaves, 2013). BFT minimize water usage in aquaculture units through maintaining adequate water quality within the culture unit, while producing low cost bioflocs rich in protein, which in turn can serve as a feed for aquatic organisms (Crab et al., 2007, 2009, 2010; El-Shafiey et al., 2018; Mabroke et al., 2021; Suloma et al., 2021). Avnimelech et al., (1999) discovered that expensive commercial protein sources in aquaculture feeds can partially replaced by single cell proteins produced using cheap carbon and nitrogen sources. Avnimelech (2011) reported that tilapia reared under BFT ponds and fed with 20% crud protein, had the highest growth performance and protein utilization rate compared to non-BFT ponds fed with 30% crude protein. Azim and Little, (2008) observed that the fish group fed on 24% CP under BFT condition had higher growth rate compere with fish group fed on 35% and reared on under clear-water conditions. Khalil et al. (2016) found that there was no significant difference in the growth of Keeled mullet (Liza carinata) fish fed on 25% CP, 30% CP and 35% CP diets under biofloc system. Tacon et al. (2002) reported that the growth performance of L. vannamei reared in unfiltered pond water and fed either on 25% CP or 35% CP showed no significant differences. Also, Hari et al., 2004 found no significant differences between the specific growth rates of P. monodon fed on 25% CP and 40% CP in extensive shrimp culture system and biofloc system. The perfect temperature range for the Nile tilapia is 26–30°C. There's a decrease in feed utilization, which leads to a critical decrease in development, when Nile tilapia are raised at a cold suboptimal temperature of 22°C, rather than at the ideal temperature of 28 °C Azaza et al. (2008). In general, the temperature extends at which feeding and voluntary development cease, as well as the lethal temperature, are significantly affected by hereditary qualities and nutrition Abdel-Ghany et al. (2019). Crab R et al., (2009) was investigated the effectiveness of BFT for maintaining good water quality in overwintering ponds for tilapia as a biological approach to overcome over-wintering problems, rapidly for nursing phase. Boyd, (1998) and Wilen et al., (2000) observed that temperature affected on dissolved oxygen in the water and both microbial community and the cultured species which affected on the fish development. Therefore, the aim of this study was to evaluate the interaction between reducing the dietary protein level and the supplementation of lysine and Threonine, on water quality and growth performance of Nile tilapia (Oreochromis niloticus) under the biofloc condition system at cold suboptimum temperature during the nursery phase. MATERIALS AND METHODS This study was conducted in Fish Nutrition Lab., Animal Production Department- Faculty of Agriculture, Cairo University. Experimental design and diets Nile tilapia (Oreochromis niloticus) were obtain from private Hatchery, Kafr El-Sheikh Governorate, Egypt, and transported lively in tanks to Fish Nutrition Lab. Fry was acclimated for two weeks to two 3m3 tanks. Four experiment diets were examined; 30% CP diet as positive control (30P), 22 % CP diet as negative control (22P), 22 % CP diet Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 843 supplemented with 1% lysine (22PL), 22 % CP diet supplemented with 0.5% threonine (22PT). Formulated diet were tested under biofloc system conditions in three replicates for each treatment. No artificial light was used in greenhouse. Fish in each replicate tank were weighted every 15 days, the amount of daily diet and carbon source (starch) readjusted according to the fish weight. The experiment was extended for 97 days. The experimental diets were formulated from Soyabean, fish meal, Gluten, yellow corn, and sunflower oil ingredients, and supplemented with amino acids (Lysine and Threonine), vitamins and minerals. The ingredients composition is (% dry matter bases) of ingredients and nutrients diets are presented in Table (1). All test diets were handled by mixing the dry fixings into a homogenous blend, at that point sun flower oil was included, a small mincer with little breadth was used to make the pellet diet were stored at 8ºC until delivered to tanks. Experimental conditions 12 experimental tanks 1000 L were used, each tank was filled with 400 L. each tank was stocked with 50 fries (6.20±0.01 gm. /fish). The tanks were aerated by air stone concentrated with 0.5HP ring blower to oxygen at (5-6 mg/l), and stored under a greenhouse enclosed structures covered by polycarbonate sheets without using heaters. Starch was used as a carbon source and added daily to maintain the C/N proportion at 1:10 activate the heterotrophic bacteria (Avnimelech, 1999). Starch was completely mixed with water cultured tank in a beaker before spread to tank surfaces at day time. Including carbohydrate beneath characteristic light and aeration conditions are the most reasonable circumstances that cause biofloc development and improvement (Azim and little, 2008). Fish were feed two times per day at 9am and 5pm. Biofloc volume measured two times a week (Monday and Thursday). Water quality Water temperature and pH were measured using Lovibond® Tintometer® water testing device and Milwaukee ph600 pocket pen. Total suspended solids, TAN, nitrogen (NH3N), nitrite (NO2-N) and nitrate (NO3-N) values were determined weekly using Lovibond® Multidirect device. Biofloc volume was measured weekly after 15-20 minutes of sedimentation using Imhoff cone (Avnimelech and Kochba, 2009). Alkalinity was measured by titration with sulphuric acid (0.02 N) to sample solution (50ml) till the pH value reaches 4.5 (Boyed and Tucker, 1992) Growth parameters SGR (%) = [ln(FW) – ln(IW)/ N] ×100, Fish (n = 50) of each replicate were weighed every fifteen day to estimate the growth parameters such as weight gain (%), feed conversion ratio (FCR), and specific growth rate (%) (SGR) as follows: weight gain (%) = (FW–IW), FCR=feed given (DW)/body weight gain (WW), Where FW=final weight, IW=initial weight, DW=dry weight, WW=wet weight, ln=natural log and N =number of culture days. 844 Khaled H. Salama et al., 2021 Table (1) Formulation and proximate composition (% dry weight basis) of the experimental diets Ingredient (%) 30P 22P 22PL 22PT a 36 22.8 22.8 22.8 Soyabean meal b 12 7.6 7.6 7.6 Fish meal c 6 3.8 3.8 3.8 Gluten d 36.9 56.7 55.7 56.2 Corn e 6 6 6 6 Vegetable oil f 2 2 2 2 Minerals &vitamins g 0.5 0.5 0.5 0.5 Salt i 0.05 0.05 0.05 0.05 Vitamin C j 0.05 0.05 0.05 0.05 PHT k 0.5 0.5 0.5 0.5 CMC l ------1 ---Lysine m ---------0.5% Threonine 100 100 100 100 Total % Diet composition 11.60 8.60 7.98 7.03 Moisture (%) 31.5 23.2 23.0 23.1 Protein (%) 9.59 5.22 5.44 5.82 Lipids (%) 6.95 2.31 2.42 3.80 Ash (%) n 40.36 59.17 55.86 61.25 Total carbohydrates 4360.58 4359.02 4442.28 4357.18 Gross energy o (kcal/kg) a; Soyabean meal, Food Technology Research Institute, Ministry of Agriculture, Giza, Egypt; b; Fish meal c; Gluten (60-63% P) Al-Ahram for food industries d; imported yellow corn from Argentina e; Vegetable oil commercial food-grade f; Minerals + vitamins, multimix, all essential vitamins + minerals for layer fatting feed g; Salt commercial food-grade I; Vitamin C L(+) ascorbic acid C6H5O6; M=176.13 gm/mol POCHSA- POLAND j; PHT Butylated Hydroxy Toluene 99% k; CMC carboxy methyl cellulose sodium sail (high viscosity)laboratory reagent Oxford Lab Chem l; Lysine m; Threonine, n; total carbohydrate content was determined by the difference: total carbohydrate=100−(% crude protein+% crude fat+% total ash+%moisture); o; dietary gross energy was calculated using the conversion factors of 5.6, 9.4, and 4.2 kcal/kg for protein, lipids and carbohydrates, respectively (Hepher et al., 1983). Proximate composition The proximate composition of fish, diets and floc meal samples generated from the experimental tanks were determined after completion of the experiment according to (AOAC, 1995). The moisture content was determined by drying the samples at 105°C (Binder oven, E series 28, Germany) to a constant weight, and the difference in weight of the sample indicated the moisture content. Ash content was determined by incinerating the samples in a muffle furnace at 600°C for 3 h. Total carbohydrate content was determined by the difference (total carbohydrate=100 − (% crude protein +% crude fat + Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 845 % total ash + % moisture). Crude lipid was determined by soxhlet extraction with ether (boiling point, 40–60°C) as a solvent. Crude protein were analyzed using Kjeldahl method (AOAC, 2016). Table (2): Essential amino acids content of the experimental diets. Amino acid 30P 1.85 Arginine 0.76 Histidine 1.30 Isoleucine 2.80 Leucine 1.76 Lysine Methionine 1.13 & Cystine 1.50 Phenylalanine 1.10 Threonine 1.70 Valine * Santiago & Lovell (1988) 22P 1.17 0.51 1.19 2.1 1.14 0.65 1.04 0.87 1.20 22PL 1,17 0.51 1.19 2.1 2.14 0.65 1.04 0.87 1.20 22PT 1.17 0.51 1.19 2.1 1.14 0.65 1.04 1.37 1.20 Tilapia requirement* 1.18 0.48 0.87 0.95 1.45 0.90 1.05 1.05 0.78 Zooplankton For zooplankton, five liters of water were filtered from subsurface layer of each site through standard plankton net 55μm mesh size. The collected samples were preserved immediately in plastic jars using 5% formalin solution. In the laboratory, a sub-sample of 1 ml was transferred to a counting cell (Rafter Sedwick Cell) and examined under a binocular compound Olympus microscope. This process was carried out 3 times for each sample and the average was calculated using the equation (APHA, 2005). Where: C = average number of organisms counted, V ′ = volume of the concentrated sample, mL, V ′′ = volume counted, mL, and V ′′′ = volume of the grab sample, m3. The total count of each group was expressed as individual/L. Organisms were identified to species level using Ruttner-Kolisko (1974), Koste (1978), Shiel and Koste (1992), Einsle (1996) and Smironov (1996). Statistical analysis All statistical analyses were performed using IPM SPSS Statistics 20.0 software. Data were analyzed by one-way ANOVA. Odd replicate value was omitted during statistical Khaled H. Salama et al., 2021 846 analysis to save data integrality. Duncan’s multiple range tests was used to identify differences among experimental groups at a significant difference of (P ≤0.05). RESULTS Water quality The results of water quality are showed in Table 3. The average water temperature during the experimental period ranged between 20 -30oC (Fig. 1). The average pH values in all the experiment tanks being 8.2 without significant difference among treatments all over the period of experiment (Fig. 2). Total nitrogen ammonia (TAN) values ranged from 0.1 to 0.2 mg/L and the lowest value was recorded on fish fed 22PT diet without stream changes all over the period of experiment (Fig. 3). The biofloc volume increased over the period of experiment and showed significant differences between treatments after the tenth week (Figure 4). At the end of culture period. The highest biofloc volume (16.79 ml/l) was recorded by fish group fed 22PT diet, whereas the lowest biofloc volume (11.81 ml/l) was recorded by fish group fed 22PL diet. Total suspended solids TSS values ranged from189.9 to 219.9 mg /l (Fig.4). The Alkalinity values ranged from 259.4 to 287.7 mg/l the highest value 287.7 mg/l was recorded for 22PT treatment (Fig. 5). Table (3): Water quality parameters of different experimental treatments. Variable 30P 24.4±2.8 (20.0-29.6) 8.2±0.4 (8.1-8.3) 0.2±0.1a (0.0-0.6) 0.1±0.2 (0.0-0.3) 15.2±12.9 (1.5-65.0) 22P 24.4±2.6 (20.0-29.1) 8.2±0.2 (7.9-8.6) 0.2±0.2a (0.0-0.8) 0.1±0.1 (0.0-0.3) 14.4±15.1 (0.5100.0) 22PL 24.3±2.6 (20.0-28.7) 8.2±0.2 (7.9-8.7) 0.2±0.1a (0.0-0.7) 0.1±0.1 (0.0-0.3) 11.8±8.7 (0.2-50.0) 22PT 24.5±2.9 (20.0-30.9) 8.3±0.2 (7.9-8.7) 0.1±0.1b (0.0-0.3) 0.1±0.1 (0.0-0.2) 16.8±18.1 (0.2-80.0) TSS (mg/l) 219.9±52.9 (135.0-355.0) 194.8±53.7 (115.0-355.0) 189.9±46.9 (96.0-276.0) 205.0±81.9 (75.0-392.0) ALK.(mg/l) 259.4±98.5 (118.8-486.2) 266.9±100.5(11 8.8-486.2) 275.0±100.5 (123.2-506.0) 287.7±96.1 (145.2-508.2) Temperature (ºC) pH TAN (mg/l) Nitrite (mg/l) Floc volume (mg/l) Values are mean 1 ±SD range Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 847 Fig (1). The pH values in all treatments over the course of the study. Temprature ˚C 30P 22P 22PL 22PT 30.0 29.0 28.0 27.0 26.0 25.0 24.0 23.0 22.0 21.0 20.0 19.0 week 1 2 3 4 5 6 7 8 9 10 11 12 13 Fig (2) Temperature values ˚C in all treatments over the course of the study. 14 848 Khaled H. Salama et al., 2021 Fig (3) TAN values in all treatments over the course of the study. Fig (4) BF volume in all treatments over the course of the study. Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 849 Fig (5) TSS values in all treatments over the course of the study. Fig (6) alkalinity values in all treatments over the course of the study. Growth performance and feed utilization Growth performance parameters of fish are represented in Table 4. 30P and 22PT treatment had higher values for WG (11.20 and 11.10, respectively); SGR (1.4 and 1.4, respectively). And 22PT treatment recorded the lowest value for FI and FCR. Khaled H. Salama et al., 2021 850 Table (4): Growth feed efficiency of Nile tilapia fed the experimental diets for 94 day. Variable Mean initial weight (g/fish) Mean final weight (g/fish) Weight gain (g/fish)1 Feed intake (g) FCR (feed: gain)2 SGR3 30P 22P 22PL 22PT 6.20±0.00 6.25±0.70 6.25±0.50 6.25±0.50 17.40±1.00 a 14.90±0.60 ab 13.30±0.20 b 17.35±1.45 a 11.20±1.00 a 20.85±0.35 a 1.90±0.28 ab 1.40±0.10 a 8.65±0.55 ab 18.40±0.50 b 2.15±0.05 ab 1.15±0.05 ab 7.05±0.07 b 17.50±0.20 b 2.50±0.00 a 1.00±1.00 b 11.10±1.40 a 16.95±0.63 b 1.55±0.25 b 1.40±0.10a Means in the same row with different superscripts are significantly different (P ≤0.05) by Duncan’s test. Weight gain (WG) = final body weight (g) − initial body weight (g) Feed conversion ratio (FCR) = feed intake (g)/body weight gain (g) 3 Specific growth rate (SGR) = (in final body wt. − in initial body wt.)/feeding days × 100 1 2 Fig (8): Weight with Time in all treatments over the course of the study. Whole fish Proximate Composition The whole fish chemical composition are reported in Table 5. The highest moisture value was recorded for fish fed 30P diet, while the lowest was recorded for fish fed 22PT treatment without significant difference among all the treatments. The highest protein value (17.5%) was recorded by fish fed 22PT treatment, while the lowest whole fish protein (11.7%) was recorded by fish group fed 22PL treatment without significant difference among all the treatments. The highest lipid percent was recorded for fish group fed 30P, 22P, and 22PT treatments and the lowest significant difference whole fish protein was recorded for fish group fed 22PL treatment. The highest ash percent 5.2% Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 851 was recorded by fish fed 22PT diet, while the lowest 3.3% was recorded with 22PL treatment without significant difference among the treatments. Table (5): Whole fish chemical composition as affected by experimental diets for 97day. Variable 30P 22P 22PL 22PT Moisture (%) 71.6±1.3 71.8±0.8 77.7±3.7 66.5±4.9 Protein (%) 15.2±0.7 15.3±0.3 11.7±3.0 17.5±3.4 Lipid (%) 8.7±0.7a 8.9±0.4a 6.6±0.8b 8.5±0.7ab Ash (%) 4.4±0.1 4.0±0.2 3.3±1.1 5.2±0.9 Means in lipid row with different superscripts are significantly different (P ≤ 0.05) by Duncan’s test. Biofloc Proximate Composition The biofloc chemical composition are presented in Table 6. The highest moisture value was recorded for fish fed 30P treatment, while the lowest value was recorded for fish group fed 22PL diets without significant difference among all the treatments. The highest protein values 30.5% and 29.3% were recorded by fish fed 22PT and 22PL treatment, respectively, without significant difference among all the treatments. While the lowest protein value 27.5% was recorded for 22P treatment without significant difference among all the treatments diet. The highest lipid percent was recorded for fish fed 30P diet while the lowest was recorded for fish fed 22P diets without significant difference among all the treatments. Fish fed diet 30P recorded the lowest ash percent without significant difference among all the treatments Table (6): chemical composition of BF as affected by experimental diets for 94 day. Variable 30P 22P 22PL 22PT 11.81±0.66a 9.77±0.01ab 8.51±0.63 b 9.00±1.13 ab Protein (%) 28.4±0.3 27.5±1 29.3±1 30.5±1.7 Lipid (%) 1.5±0.1 1.2±0.2 1.4±0.1 1.3±0.3 15.84±0.53b 24.80±1.26 a 23.94±2.04a 26.30±2.04a Moisture (%) Ash (%) Means in the lipid row with different superscripts are significantly different (P ≤ 0.05) by Duncan’s test Khaled H. Salama et al., 2021 852 Taxonomic composition of zooplankton The results of the zooplankton composition are presented in Table 7. In all the biofloc treatment tanks in this experiment, two phylum were identified in all treatment which are Rotifers and Ciliophora. Three genera were identified under phylum Rotifers which are Anuropsis fissa, Colurella sp., and Monostyla species, and one genera of Phylum Ciliophora, which is Tintinnidium pusillum. The lowest total count of Rotifera were recorded for 30P and 22PT treatments, and the highest total count for Phylum rotifers were recorded for 22P and 22PL treatments. The lowest total count for Phylum Ciliophora was found with 30P treatment, and the highest total count was recorded for 22PL treatment. Generally, the lowest total count of Rotifera and Ciliophora was recorded for 30P treatment, and the total count was increased with decreasing the dietary protein levels 22P, 22PL and 22PT. Zooplankton sp. Table (7) Taxonomic of zooplankton composition 30P 22P 22PL 22PT Rotifera/L Anuropsis fissa/L 106±34 108±93 180±0 25±5 9±9 99±72 54±18 60±60 Monostyla spp./L 487±26b 1033±123b 4239±125a 610±42b Total individual count 602 1,240 4,473 695 Colurella sp./L Ciliophora/L Tintinnidium pusillum/L 279±261 762±282 2511±2439 715±695 Total zooplankton/L 881 2002 6984 1410 Means in the same row with different superscripts are significantly different (P ≤ 0.05) by Duncan’s test DISCUSSION Water quality The result of this study indicated that all water quality parameters under the biofloc system conditions is suitable for the production of Nile Tilapia Emerenciano et al., (2017). The experimental diets did not affect significantly on water quality with expiation of TAN. The dietary protein levels didn’t effect on the TAN level and this agree with Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 853 García-Ríos et al. (2019) who reported that the protein levels have significant effect on water quality. The adding threonine to the low protein diets significantly decreased the TAN level. Zidan et al. (2017) reported that the optimal water temperature for fish culture ranged from 25.1–30.6 ºC. In the present study a descending decrease in water temperature along the period of the experiment were recorded and the average water temperature in all tanks was 24.4oC and ranged from 20.0 - 30.9 oC. De Almeida et al. (2021) reported that the tilapia suboptimum temperature20.8 ºC. In the present study water temperature gradually decreased within normal range tell the eight week and from the eight week the water temperature began decreased to the suboptimal temperature. The pH values show no significant or a stream change occurs all over the period of experiment (8.2 for all treatments). The pH values were in the normal range for growing tilapia under the biofloc system. The pH values were within normal range for growing tilapia under biofloc system this result agreed with (El-Sayed 2006; El-Sherif; El-Feky 2009) where they consider the ideal pH for tilapia ranges from7 to 8. The alkalinity values ranged from 259.4 to 287.7 mg/l with no significant differences among all the treatments which demonstrated the buffering capacity of the system and within the recommended range for aquaculture systems (Huet, 1986; Boyd and Tucker, 1998; Wurts, 2003). The alkalinity was high in all treatments especially 22PT treatment which recorded the highest value indicating that the amount needed of NaHCO3 will be decreased. This result disagree with (Azim and Little 2008) who observed oscillations in alkalinity values (80– 250 mg/l), which indicate a decrease in buffering capacity, and therefore require the frequent input of sodium bicarbonate (NaHCO3) to avoid pH oscillation in BFT. In this study the levels of TAN were distinctive and low, especially in the tanks delivered with 22PTdiet. TAN decreasing may attributed to the supplementation with threonine which agreed with Michelato et al. (2016), who reported that threonine addition increase protein and amino acid retention caused fast-growing of Nile tilapia. (Walton 1985; Abidi and Khan, 2008) recorded that threonine increase the metabolic rate and decrease ammonia excretion. The values of TSS in the present study (75- 392) were within the recommended limits for BFT stander conditions as recommended by Avnimelech (2011). However, (Azim and Little 2008 ; Silva et al. 2018) observed TSS reaching up to 1,000 mg/L in different studies with tilapia fed different CP concentrations. Growth performance and feed utilization Under the present study conditions, tilapia fed 22PT diets showed compensatory effect when subjected to suboptimal temperature. At the beginning of the treatment, there is a slight weight gain, and dramatically increase over time . This may attribute to the addition of threonine (fig.9). According to (Wohlfarth and Hulata 1983) the temperature below the suboptimum limit the adequate growth of the fish, Reproduction 854 Khaled H. Salama et al., 2021 stops at 22.8ºC and normal feed intake below 20.8ºC. De Almeida et al. (2021) tilapia feeding, swimming and vital physiological functions decrease at 20ºC while. Michelato et al. (2016) indicated that threonine is essential for growth, protein and amino acid retention of large Nile tilapia Lem-me (2003) reported that threonine is a critical essential amino acid on fish development serving as an antecedent of non-essential amino acids such as serine and glycine and the effects of dietary threonine were more expressive on protein and amino acids retention. Veldkamp et al. (2000) reported that for commercial male turkeys fed diets supplemented with amino acid threonine at low temperature caused significant reduce in FCR, while high temperature did not respond. This result agrees with Ferguson et al. (2003) who recorded that the environmental temperature effect on the response of growing pigs to Threonine. Avnimelech (2011) reported that feed rations in biofloc tilapia systems can be lowered to at least 20% compared to conventional non BFT. (Xu and Pan, 2014) in shrimp, BFT was found to be effective in offsetting a decrease in protein levels from 30 to 20 percent. Ogello et al. (2014), in bioflocs technology (BFT) lakes are potential food source for fish. In fact, the BFT can be considered as a self-sustaining biotechnology machine because it fabricated food concurrently, subsequently getting to be the ignored resource in aquaculture industry. (Megahed 2010 ; Kim et al. 2016) showed the possibility of reducing CP levels under biofloc system, and, this protein level reduction did not result in loss of performance of shrimp in BFT. Day et al. (2016) recorded the role of biofloc as a supplementary feed component of high nutritional quality, especially in terms of protein, has been demonstrated in several aquaculture species. In addition to the best FCR value recorded at tanks fed 22PT. Chemical composition of fish In the current study 22PT treatment recorded the highest in protein and ash content, and the lowest in lipid and moisture content which reflected in the dry matter level. The results indicate that supplementing threonine amino acid to fish diets may improve fish nutritive value. Helland et al. (2013) found a linear increase in crude protein and quadratic impact on whole-body humidity, unrefined lipid, and ash of Atlantic salmon bolstered expanding levels of threonine. Zhao et al. (2020) Dietary threonine improved the growth of hybrid catfish and improved muscle protein content. Increase ash values on wit basses may attributed to the addition of threonine to the experimental diet. Increase dry matter on wet basses may attributed to the addition of threonine to the experimental diet. Becerril et al. (2017) some fish species frequently observed in Biofloc can provide good protein, lipids and carbohydrates content. Biofloc proximate composition In the current study, the biofloc proximate are presented in Table 6. 22PT treatment had the highest nutritional value compared to the other treatments, due to the high percentage Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 855 of protein, ash and dry matter. (Tacon et al. 2002 ; Ballester et al. 2010) notes that biofloc quality in terms of fatty acid profile and protein content seems to be affected by the system input and microbial floc is a good source of vitamins and minerals and can supply the needs of these nutrients. In the present study the average ash content of floc meal were ranged between 15.8%–26.3% which within the range reported in previous studies (Azim et al., 2008 ; Emerenciano et al., 2013b). Increase protein values on wet basses may attributed to the supplementation by amino acids to the experimental diets. Mabroke et al. (2019) the amino acid content of different experimental diets seems to cover the requirement of tilapia even when the floc meal reached 50% from the total ingredients. CONCLUSION Adding of threonine amino acid to the low protein diets improved the growth performance, feed utilization, water and the tolerance to thermal stress during the nursery phase. REFERENCES Abdel-Ghany, H. M.; El-Sayed, A. F. M.; Ezzat, A. A.; Essa, M. A. and Helal A. M. (2019). Dietary lipid sources affect cold tolerance of Nile tilapia (Oreochromis niloticus). Journal of thermal biology, 79:50-55. Abidi Fatma, S. and Khan, M. (2008). Dietary threonine requirement of fingerling Indian major carp, Labeo rohita (Hamilton). Aquaculture Research, 39(14): 1498-1505. AOAC Official Method of analysis (1995). Official methods of analysis (Vol. 222). Washington, DC: Association of Official Analytical Chemists. AOAC Official Method of analysis (2016). 20th Kjeldahl method no.984.13-chapter 4p online. APHA (2005). Standard Methods for Examination of Water and Wastewater. 21sted. AOAC Official Method of analysis Avnimelech, Y. (1999). Carbon/nitrogen ratio as a control element in aquaculture systems. Aquaculture, 176(3-4): 227-235. Avnimelech Y. (2011) Tilapia Production Using Biofloc Technology Saving Water, Waste Recycling Improves Economics global aquaculture advocate May/June 2011. Azaza, M. S.; Dhraïef, M. N. and Kraïem, M. M. (2008). Effects of water temperature on growth and sex ratio of juvenile Nile tilapia Oreochromis niloticus (Linnaeus) reared in geothermal waters in southern Tunisia. Journal of thermal Biology, 33(2): 98-105. 856 Khaled H. Salama et al., 2021 Azim, M. E. and Little, D. C. (2008). The biofloc technology (BFT) in indoor tanks: water quality, biofloc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus). Aquaculture, 283(1-4): 29-35. Ballester, E. L. C.; Abreu, P. C.; Cavalli, R. O.; Emerenciano, M.; De Abreu, L. and Wasielesky Jr, W. (2010). Effect of practical diets with different protein levels on the performance of Farfantepenaeus paulensis juveniles nursed in a zero-exchange suspended microbial flocs intensive system. Aquaculture Nutrition, 16(2): 163-172. Becerril CD; Monroy DM; Emerenciano MG; Castro MG and Lara ARE. (2017) Nutritional importance for aquaculture and ecological function of microorganisms that make up Biofloc, a review. International Journal of Aquatic Science, 8(2):69-77. Boyd, C. E. and Tucker, C. S. (1992). Water quality and pond soil analyses for aquaculture. Water quality and pond soil analyses for aquaculture. Boyd, C. E. and Gross, A. (1998). Use of probiotics for improving soil and water quality in aquaculture ponds. Advances in shrimp biotechnology, 101-105. Crab, R.; Avnimelech, Y.; Defoirdt, T.; Bossier, P. and Verstraete, W. (2007). Nitrogen removal in aquaculture towards sustainable production. Aquaculture 270 (1-4): 1-14. Crab, R.; Kochva, M.; Verstraete, W. and Avnimelech, Y. (2009). Bio-flocs technology application in over-wintering of tilapia. Aquacultural Engineering, 40(3): 105-112. Crab, R.; Lambert, A.; Defoirdt, T.; Bossier, P. and Verstraete, W. (2010). The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi. Journal of applied microbiology, 109(5): 1643-1649. Day, S. B.; Salie, K. and Stander, H. B. (2016). A growth comparison among three commercial tilapia species in a biofloc system. Aquaculture International, 24(5): 13091322. Deacon, B. (1997). Global social policy: International organizations and the future of welfare. Sage. De Almeida, C. A. L.; de Almeida, C. K. L.; Martins, E. D. F. F.; Bessonart, M. ; Pereira, R. T. ; Paulino, R. R. and Fortes-Silva, R. (2021). Coping with suboptimal water temperature: modifications in blood parameters, body composition, and postingestive-driven diet selection in Nile tilapia fed two vegetable oil blends. Animal, 15(2): 100092. El-Sayed, A. F. M. (2006). Tilapia culture in salt water: environmental requirements, nutritional implications and economic potentials. Avances en Nutricion Acuicola. El-Sherif, M. S. and El-Feky, A. M. I. (2009). Performance of Nile tilapia (Oreochromis niloticus) fingerlings. I. Effect of pH. International Journal of Agriculture and Biology, 11(3): 297-300. Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 857 El-Shafiey, M. H. M.; Mabroke, R. S.; Mola, H. R. A.; Hassaan, M. S. and Suloma, A. (2018). Assessing the suitability of different carbon sources for Nile tilapia, Oreochromis niloticus culture in BFT system. AACL Bioflux, 11(3): 782-795. Emerenciano, M. G. C.; Martínez-Córdova, L. R.; Martínez-Porchas, M. and Miranda-Baeza, A. (2017). Biofloc technology (BFT): a tool for water quality management in aquaculture. Water quality, 5: 92-109. Ferguson, N. S.; Gous, R. M. and Iji, P. A. (2003). Determining the source of antinutritional factor (s) found in two species of lupin (L. albus and L. angustifolius) fed to growing pigs. Livestock Production Science, 84(1): 83-91. Gutierrez-Wing, M. T. and Malone, R. F. (2006). Biological filters in aquaculture: trends and research directions for freshwater and marine applications. Aquacultural Engineering, 34(3): 163-171. García-Ríos L.; Miranda-Baeza A.; Coelho-Emerenciano MG; Huerta-Rábago JA and Osuna-Amarillas P. (2019) Biofloc technology (BFT) applied to tilapia fingerlings production using different carbon sources: emphasis on commercial applications. Aquaculture 502: 26–31. Hargreaves, J. A. (2013). Biofloc production systems for aquaculture (Vol. 4503: pp. 111). Stoneville, MS: Southern Regional Aquaculture Center. Helland, S. and Helland, B.G. (2011) Dietary threonine requirement of Atlantic salmon smolts. Aquaculture, 321: 230–236. Helland, B.G. ; Lemme, A. and Helland, S. (2013) Threonine requirement for maintenance and efficiency of utilization for threonine accretion in Atlantic salmon smolts determined using increasing ration levels. Aquaculture, 372: 158–166. Khalil, M.; Ragaa, R.; Mohamed, R.; Abd-alatty, B.; Suloma, A. and Henish, S. (2016). Eco-friendly cultivation of Keeled mullet (Liza carinata) in biofloc system. Egyptian Journal of Aquatic Biology and Fisheries, 20(2): 23-35. Kim, K. W.; Moniruzzaman, M.; Kim, K. D.; Han, H. S.; Yun, H.; Lee, S. and Bai, S. C. (2016). Effects of dietary protein levels on growth performance and body composition of juvenile parrot fish, Oplegnathus fasciatus. International Aquatic Research, 8(3): 239-245. Kimera, F.; Sewilam, H.; Fouad, W. M. and Suloma, A. (2021a). Efficient utilization of aquaculture effluents to maximize plant growth, yield, and essential oils composition of Origanum majorana cultivation. Annals of Agricultural Sciences, 66(1): 1-7. Kimera, F.; Sewilam, H.; Fouad, W. M. and Suloma, A. (2021b). Sustainable production of Origanum syriacum L. using fish effluents improved plant growth, yield, and essential oil composition. Heliyon, 7(3): e06423. Lemme, A. (2003) Reassessing amino acid levels for Pekin ducks. Poult. Int., 42: 18–24. 858 Khaled H. Salama et al., 2021 Mabroke, R. S.; El-Husseiny, O. M.; Zidan, A. E. N. F.; Tahoun, A. A. and Suloma, A. (2019). Floc meal as potential substitute for soybean meal in tilapia diets under biofloc system conditions. Journal of Oceanology and Limnology, 37(1): 313-320. Matos, E., and Pires, D. (2006). Teorias administrativas e organização do trabalho: de Taylor aos dias atuais, influências no setor saúde e na enfermagem. Texto & ContextoEnfermagem, 15(3): 508-514. Megahed, M. E. (2010). The effect of microbial biofloc on water quality, survival and growth of the green tiger shrimp (Penaeus semisulcatus) fed with different crude protein levels. Journal of the Arabian Aquaculture Society, 5(2): 119-142. Michelato, M.; Vidal, L. V. O.; Xavier, T. O.; Graciano, T. S.; De Moura, L. B.; Furuya, V. R. B. and Furuya, W. M. (2016). Dietary threonine requirement to optimize protein retention and fillet production of fast‐growing Nile tilapia. Aquaculture Nutrition, 22(4): 759-766. Ogello, E. O.; Musa, S. M.; Aura, C. M.; Abwao, J. O. and Munguti, J. M. (2014). An appraisal of the feasibility of tilapia production in ponds using biofloc technology: A review. Santiago, C. B. and Lovell, R. T. (1988). Amino acid requirements for growth of Nile tilapia. The journal of nutrition, 118 (12): 1540-1546. Suloma, A., and Ogata, H. Y. (2006). Future of rice-fish culture, desert aquaculture and feed development in Africa: the case of Egypt as the leading country in Africa. Japan Agricultural Research Quarterly: JARQ, 40(4): 351-360. Suloma, A.; Gomaa, A. H.; Abo-Taleb, M. A.; Mola, H. R.; Khattab, M. S. and Mabroke, R. S. (2021). Heterotrophic biofloc as a promising system to enhance nutrients waste recycling, dry diet acceptance and intestinal health status of European eel (Anguilla anguilla). Aquaculture, Aquarium, Conservation & Legislation, 14(2): 1021-1035. Subasinghe, R. P.; Arthur, J. R.; Ogawa, K.; Chinabut, S.; Adlard, R. ... and Shariff, M. (2005). Disease and health management in Asian aquaculture. Veterinary parasitology, 132(3-4): 249-272. Tacon, A. G. J.; Cody, J. J.; Conquest, L. D.; Divakaran, S.; Forster, I. P. and Decamp, O. E. (2002). Effect of culture system on the nutrition and growth performance of Pacific white shrimp Litopenaeus vannamei (Boone) fed different diets. Aquaculture nutrition, 8(2): 121-137. Veldkamp, T.; Kwakkel, R. P.; Ferket, P. R.; Simons, P. C. M.; Noordhuizen, J. P. T. M. and Pijpers, A. (2000). Effects of ambient temperature, arginine-to-lysine ratio, and electrolyte balance on performance, carcass, and blood parameters in commercial male turkeys. Poultry Science, 79(11): 1608-1616. Effect of protein level on Oreochromis niloticus nursering performance under biofloc system ______________________________________________________________________________________ 859 Widanarni, W.; Wahjuningrum, D. and Puspita, F. (2012). Aplikasi Bakteri Probiotik melalui Pakan Buatan untuk Meningkatkan Kinerja Pertumbuhan Udang Windu (Penaeus monodon). Jurnal Sains Terapan, 2(1): 19-29. Wilén, B. M.; Nielsen, J. L.; Keiding, K. and Nielsen, P. H. (2000). Influence of microbial activity on the stability of activated sludge flocs. Colloids and Surfaces B: Biointerfaces, 18(2): 145-156. Wohlfarth, G. W.; Spataru P. and Hulata, G. (1983). Studies on the natural food of different fish species in intensively manured polyculture ponds. Aquaculture, 35: 283298. Wurts, W. A. (2003). Daily pH cycle and ammonia toxicity. World Aquaculture, 34(2), 20-21. Xu, W. J. and Pan, L. Q. (2014). Enhancement of immune response and antioxidant status of Litopenaeus vannamei juvenile in biofloc-based culture tanks manipulating high C/N ratio of feed input. Aquaculture, 412:117-124. Zhao, Y.; Jiang, Q.; Xiao-Qiu, Z.; Shang-Xiao, X.; Lin, F.; Liu, Y. ... and Jiang, J. (2020). Effect of dietary threonine on growth performance and muscle growth, protein synthesis and antioxidant-related signalling pathways of hybrid catfish Pelteobagrus vachelli♀× Leiocassis longirostris♂. The British journal of nutrition, 123(2): 121-134. Zidan, A. E. N. F.; Mola, H. R.; El-Husseiny, O.; Suloma, A. and Mabroke, R. S. (2017). Inclusion of biofloc meal in tilapia diets and its effect on the structure of zooplankton community under biofloc system condition. The Journal of Egyptian Academy Society for Environmental Development, 18(1): 47-57.