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Effect of dietary protein level, initial body weight, and their interaction on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.)

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Effect of dietary protein level, initial body weight, and their interaction on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.) Mohsen Abdel-Tawwab a, , Mohammad H. Ahmad b , Yassir A.E. Khattab b , Adel M.E. Shalaby c a Department of Fish Biology and Ecology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt b Department of Fish Nutrition, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt c Department of Fish Physiology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt abstract article info Article history: Received 11 August 2009 Received in revised form 26 October 2009 Accepted 27 October 2009 Keywords: Nile tilapia Dietary protein Fish weight Feed utilization Body composition Physiological status A 10-week feeding trial was conducted to assess the interaction between dietary protein level and sh weight on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.). Fish were categorized into three weights; 0.40.5 g (fry), 1722 g (ngerling), and 3743 g (advanced juvenile). Diets containing 25, 35, or 45% crude protein (CP) were fed by triplicate to each sh weight. Fish growth, feed utilization, and protein turn-over were signicantly affected by dietary protein level and sh weight, meanwhile their interaction signicantly affected specic growth rate and protein efciency ratio (PER) only. Unionized ammonia was signicantly affected by dietary protein level, sh weight, and their interaction. Moreover, protein and lipid contents in whole-body of sh were signicantly affected by dietary protein level and sh weight, while their interaction signicantly affected total lipids content only. Ash content signicantly differed with sh weight only. The optimum feed conversion ratio (FCR) was obtained with fry tilapia fed the 45%-CP diet; whereas, the poorest FCR was observed for advanced juveniles fed the 25%-CP diet. The lowest PER and protein productive value (PPV) values were obtained with the 45%-CP diet fed to advanced juveniles; whereas, the highest values were obtained with the 25%-CP diet fed to fry. The highest protein growth rate (PGR) was obtained with fry tilapia fed the 45%-CP diet, while the lowest one was obtained with advanced juvenile fed the 25%-CP diet. Hematological variables were signicantly affected by protein level, sh weight, and their interaction except for serum lipids which was not signicantly affected by the interaction. Activities of aspartate amninotransferase (AST) and alanine aminotransferase (ALT) in serum, liver, and muscles were signicantly affected by dietary protein level and sh weight. The interaction signicantly affected enzyme activities except for serum AST, which was not signicant. The optimum growth of fry tilapia was obtained at 45% CP, while ngerling and advanced juvenile showed optimum growth performance with the 35%-CP diet. Excess protein in ngerling and advanced juvenile might be deaminated and used as energy source resulting in increased blood glucose, protein, and lipids as well as increased unionized ammonia in the environment. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The feeding of prepared diets is a principal factor in aquaculture to increase growth and production of reared sh (Thankur et al., 2004; Liti et al., 2005; Abdel-Tawwab et al., 2007). Dietary protein is an important aspect in achieving efcient sh production and its needs should accommodate sh requirements due to age/weight. Protein is the most expensive ingredient in prepared feeds and thus it should be carefully formulated to meet the needs of the cultured organism. Understanding the sh's protein requirement during the growth period is fundamental in sh culture management leading to maximized feed conversion efciency, cost savings, and reduced nutrient loading into the aquatic ecosystem (Abdel-Tawwab and Ahmad, 2009). The physiological status of intensively farmed sh is an integral part of evaluating their health status. Diet composition, metabolic adaptations, and variations in sh activity are the main factors responsible for seasonal changes in physiological variables (Cnaani et al., 2004; Řehulka et al., 2004). Physiological alterations might be indicative of unsuitable environmental conditions or the presence of stressing factors such as toxic chemicals, excess organic compounds, and even usual procedures in aquaculture (Barton and Iwama, 1991; Wendelaar Bonga, 1997; Barcellos et al., 2004). Aquaculture 298 (2010) 267274 Corresponding author. Tel.: +20 55 2319821/+20 120570607; fax: +20 55 3400498. E-mail address: mohsentawwab@yahoo.com (M. Abdel-Tawwab). 0044-8486/$ see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.10.027 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Nile tilapia, Oreochromis niloticus (L.) accepts articial feeding from hatching and typically shows high survival rates and fast growth (El-Sayed, 2006). The effect of dietary protein on the intermediary metabolism of this species, however, remains scarcely known. Assessing the nutritional demands and the effect of dietary protein on the metabolism of this species at different weights is of particular interest. Therefore, this study was carried out to assess the effect of dietary protein level, initial body weight, and their interaction on growth, feed utilization, carcass composition, and physiological alterations of Nile tilapia. 2. Materials and methods 2.1. Fish culture technique The experiment design was factorial, including three weight classes and three dietary protein levels, by triplicate. Healthy Nile tilapia, O. niloticus (L.) of different weights were obtained from Abbassa sh hatchery and nursery ponds, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia, Egypt. Fish were acclimated in indoor tanks for 2 weeks by feeding a commercial diet containing 20% crude protein (CP). After that they were categorized according their weights into fry (0.40.5 g), ngerling (1722 g), and advanced juvenile (3743 g). Fish of each weight class were distributed into 100-L glass aquaria (75 × 60 × 50 cm) at a rate of 5 g/L. Each aquarium was supplied with compressed air via air-stones from air pumps. Well-aerated water was provided from a storage berglass tank. The temperature was adjusted at 27±1 °C by using thermostatically controlled heaters. Half of the aquarium's water with sh excreta was siphoned every day and replaced by an equal volume of well-aerated storage water. 2.2. Diet preparation and feeding regime Three experimental diets were formulated to contain 25, 35, or 45% crude protein (CP) (Table 1). The ingredients of each diet were blended together for 40 min to make a paste which was separately passed through a grinder, and cold-pelleted (1-mm diameter) in a paste extruder. The diets were dried in a forced-air drier at room temperature for 24 h and stored in plastic bags at -2 °C for further use. Each of the three diets was fed to sh in the three different size categories (fry, ngerling, and advanced juvenile of Nile tilapia) and three aquaria were assigned to each treatment. Fish were fed to satiation twice daily at 9:00 and 14:00 h for 6 days a week over 10 weeks. The amount of consumed feed for each aquarium was subsequently calculated as a summation of given diets during the experimental period. Fish in each aquarium were fortnightly group- weighed and dead sh were removed and recorded daily. 2.3. Water quality measurements Water samples were collected fortnightly at 15 cm depth from each aquarium. Dissolved oxygen was measured in situ with an oxygen meter (YSI model 58, Yellow Spring Instrument Co., Yellow Springs, OH, USA), unionized ammonia using DREL/2 HACH kits (HACH Co., Loveland, CO, USA), and pH with a pH meter (Digital Mini-pH Meter, model 55, Fisher Scientic, Denver, CO, USA). In all treatments, dissolved oxygen concentrations ranged from 6.9 to 7.2 mg/L and pH ranged from 7.8 to 8.1. All the water quality parameters were within the acceptable ranges for sh growth (Boyd, 1984). 2.4. Proximate chemical analysis of diets and sh The tested diets and whole-sh body from each treatment were analyzed according to the standard methods of AOAC (1990) for moisture, protein, fat, and ash. Moisture content was estimated by drying the samples to constant weight at 85 °C in a drying oven (GCA, model 18EM, Precision Scientic group, Chicago, Illinois, USA) and nitrogen content using a microKjeldahl apparatus (Labconco, Labconco corporation, Kansas, Missouri, USA). Crude protein was estimated by multiplying nitrogen content by 6.25. Lipid content was determined by ether extraction in a multi-unit Soxhlet extraction apparatus (Lab-Line Instruments, Inc., Melrose Park, Illinois, USA) for 16 h. Ash was determined by combusting dry samples in a mufe furnace (Thermo- lyne Corporation, Dubuque, Iowa, USA) at 550 °C for 6 h. 2.5. Fish performance Growth performance was determined and feed utilization was calculated as following: Specic growth rate (SGR; %/day) = 100(lnW 2 -lnW 1 )/ T; where W 1 and W 2 are the initial and nal weight, respectively, and T is the number of days in the feeding period; Feed conversion ratio (FCR) = feed intake (g)/weight gain (g); Protein efciency ratio (PER) = weight gain (g)/protein intake (g); Protein productive value (PPV; %) = 100 × (protein gain (g) / protein intake (g)); Protein growth rate (PGR; %/day) = 100 (Ln nal protein content -Ln initial protein content) / days of feeding; Hepatosomatic index (HIS; %) = 100 × [liver weight (g) / body weight (g)]. 2.6. Physiological measurements At the end of the feeding trial, sh were not fed during the 24 h immediately prior to blood sampling. Fish were anaesthetized with buffered tricaine methane sulfonate (20 mg/L) and blood was collected with a hypodermic syringe from the caudal vein. The extracted blood was divided in two sets of Eppendorf tubes. One set contained 500 U sodium heparinate/mL, used as an anticoagulant, for hematology (hemoglobin, haematocrit and red blood cell counting). The second set, without anticoagulant, was left to clot at 4 °C and centrifuged at 5000 rpm for 5 min at room temperature. The collected serum was Table 1 Ingredients and chemical composition of the experimental diets (on dry matter basis). Ingredients (g/100 g) Dietary protein levels 25% 35% 45% Fish meal 15.6 20.3 31.0 Soybean meal 20.0 40.0 50.0 Wheat bran 5.0 5.0 5.0 Ground corn 52.63 28.42 9.44 Fish oil + corn oil (1:1) 2.0 2.0 2.0 Vitamins and minerals premix a 1.5 1.5 1.5 Ascorbic acid 0.06 0.06 0.06 Starch 2.21 1.72 0.0 Carboxymethyl cellulose 1.0 1.0 1.0 Total 100 100 100 Chemical analysis (%) b Dry matter 92.48 ± 0.7 92.69 ± 0.6 93.09 ± 0.6 Crude protein 25.32 ± 0.24 35.41 ± 0.33 45.56 ± 0.46 Crude fat 5.87 ± 0.15 5.67 ± 0.25 5.99 ± 0.20 Ash 5.51 ± 0.23 6.31 ± 0.36 7.31 ± 0.37 Fiber 6.68 ± 0.15 5.50 ± 0.12 5.76 ± 0.13 NFE c 56.62 47.11 35.38 GE (Kcal/g) d 439.14 446.85 458.92 a Vitamin and minerals premix: each 2.5 kg contain vitamin A 12 MIU; D 3 2 MIU, E 10 g; K 2 g; B 1 1 g; B 2 4 g; B 6 1.5 g; B 12 10 mg; pantothenic acid 10 g; nicotinic acid 20 g; folic acid 1 g; biotin 50 mg; choline chloride 500 mg; copper 10 g; iodine 1 g; iron 30 g; manganese 55 g; zinc 55 g and selenium 0.1 g. b Means of ve replicates. c NFE (nitrogen free extract) = 100 -(protein% + lipid% + ash% +ber%). d GE (gross energy): calculated after NRC (1993) as 5.64, 9.44, and 4.11 kcal/g for protein, lipid, and NFE, respectively. 268 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267274
Aquaculture 298 (2010) 267–274 Contents lists available at ScienceDirect Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e Effect of dietary protein level, initial body weight, and their interaction on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.) Mohsen Abdel-Tawwab a,⁎, Mohammad H. Ahmad b, Yassir A.E. Khattab b, Adel M.E. Shalaby c a b c Department of Fish Biology and Ecology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt Department of Fish Nutrition, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt Department of Fish Physiology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt a r t i c l e i n f o Article history: Received 11 August 2009 Received in revised form 26 October 2009 Accepted 27 October 2009 Keywords: Nile tilapia Dietary protein Fish weight Feed utilization Body composition Physiological status a b s t r a c t A 10-week feeding trial was conducted to assess the interaction between dietary protein level and fish weight on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.). Fish were categorized into three weights; 0.4–0.5 g (fry), 17–22 g (fingerling), and 37–43 g (advanced juvenile). Diets containing 25, 35, or 45% crude protein (CP) were fed by triplicate to each fish weight. Fish growth, feed utilization, and protein turn-over were significantly affected by dietary protein level and fish weight, meanwhile their interaction significantly affected specific growth rate and protein efficiency ratio (PER) only. Unionized ammonia was significantly affected by dietary protein level, fish weight, and their interaction. Moreover, protein and lipid contents in whole-body of fish were significantly affected by dietary protein level and fish weight, while their interaction significantly affected total lipids content only. Ash content significantly differed with fish weight only. The optimum feed conversion ratio (FCR) was obtained with fry tilapia fed the 45%-CP diet; whereas, the poorest FCR was observed for advanced juveniles fed the 25%-CP diet. The lowest PER and protein productive value (PPV) values were obtained with the 45%-CP diet fed to advanced juveniles; whereas, the highest values were obtained with the 25%-CP diet fed to fry. The highest protein growth rate (PGR) was obtained with fry tilapia fed the 45%-CP diet, while the lowest one was obtained with advanced juvenile fed the 25%-CP diet. Hematological variables were significantly affected by protein level, fish weight, and their interaction except for serum lipids which was not significantly affected by the interaction. Activities of aspartate amninotransferase (AST) and alanine aminotransferase (ALT) in serum, liver, and muscles were significantly affected by dietary protein level and fish weight. The interaction significantly affected enzyme activities except for serum AST, which was not significant. The optimum growth of fry tilapia was obtained at 45% CP, while fingerling and advanced juvenile showed optimum growth performance with the 35%-CP diet. Excess protein in fingerling and advanced juvenile might be deaminated and used as energy source resulting in increased blood glucose, protein, and lipids as well as increased unionized ammonia in the environment. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The feeding of prepared diets is a principal factor in aquaculture to increase growth and production of reared fish (Thankur et al., 2004; Liti et al., 2005; Abdel-Tawwab et al., 2007). Dietary protein is an important aspect in achieving efficient fish production and its needs should accommodate fish requirements due to age/weight. Protein is the most expensive ingredient in prepared feeds and thus it should be carefully formulated to meet the needs of the cultured organism. Understanding ⁎ Corresponding author. Tel.: +20 55 2319821/+20 120570607; fax: +20 55 3400498. E-mail address: mohsentawwab@yahoo.com (M. Abdel-Tawwab). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.10.027 the fish's protein requirement during the growth period is fundamental in fish culture management leading to maximized feed conversion efficiency, cost savings, and reduced nutrient loading into the aquatic ecosystem (Abdel-Tawwab and Ahmad, 2009). The physiological status of intensively farmed fish is an integral part of evaluating their health status. Diet composition, metabolic adaptations, and variations in fish activity are the main factors responsible for seasonal changes in physiological variables (Cnaani et al., 2004; Řehulka et al., 2004). Physiological alterations might be indicative of unsuitable environmental conditions or the presence of stressing factors such as toxic chemicals, excess organic compounds, and even usual procedures in aquaculture (Barton and Iwama, 1991; Wendelaar Bonga, 1997; Barcellos et al., 2004). 268 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 Nile tilapia, Oreochromis niloticus (L.) accepts artificial feeding from hatching and typically shows high survival rates and fast growth (El-Sayed, 2006). The effect of dietary protein on the intermediary metabolism of this species, however, remains scarcely known. Assessing the nutritional demands and the effect of dietary protein on the metabolism of this species at different weights is of particular interest. Therefore, this study was carried out to assess the effect of dietary protein level, initial body weight, and their interaction on growth, feed utilization, carcass composition, and physiological alterations of Nile tilapia. paste extruder. The diets were dried in a forced-air drier at room temperature for 24 h and stored in plastic bags at −2 °C for further use. Each of the three diets was fed to fish in the three different size categories (fry, fingerling, and advanced juvenile of Nile tilapia) and three aquaria were assigned to each treatment. Fish were fed to satiation twice daily at 9:00 and 14:00 h for 6 days a week over 10 weeks. The amount of consumed feed for each aquarium was subsequently calculated as a summation of given diets during the experimental period. Fish in each aquarium were fortnightly groupweighed and dead fish were removed and recorded daily. 2. Materials and methods 2.3. Water quality measurements 2.1. Fish culture technique Water samples were collected fortnightly at 15 cm depth from each aquarium. Dissolved oxygen was measured in situ with an oxygen meter (YSI model 58, Yellow Spring Instrument Co., Yellow Springs, OH, USA), unionized ammonia using DREL/2 HACH kits (HACH Co., Loveland, CO, USA), and pH with a pH meter (Digital Mini-pH Meter, model 55, Fisher Scientific, Denver, CO, USA). In all treatments, dissolved oxygen concentrations ranged from 6.9 to 7.2 mg/L and pH ranged from 7.8 to 8.1. All the water quality parameters were within the acceptable ranges for fish growth (Boyd, 1984). The experiment design was factorial, including three weight classes and three dietary protein levels, by triplicate. Healthy Nile tilapia, O. niloticus (L.) of different weights were obtained from Abbassa fish hatchery and nursery ponds, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia, Egypt. Fish were acclimated in indoor tanks for 2 weeks by feeding a commercial diet containing 20% crude protein (CP). After that they were categorized according their weights into fry (0.4–0.5 g), fingerling (17–22 g), and advanced juvenile (37–43 g). Fish of each weight class were distributed into 100-L glass aquaria (75 × 60 × 50 cm) at a rate of 5 g/L. Each aquarium was supplied with compressed air via air-stones from air pumps. Well-aerated water was provided from a storage fiberglass tank. The temperature was adjusted at 27 ± 1 °C by using thermostatically controlled heaters. Half of the aquarium's water with fish excreta was siphoned every day and replaced by an equal volume of well-aerated storage water. 2.2. Diet preparation and feeding regime Three experimental diets were formulated to contain 25, 35, or 45% crude protein (CP) (Table 1). The ingredients of each diet were blended together for 40 min to make a paste which was separately passed through a grinder, and cold-pelleted (1-mm diameter) in a Table 1 Ingredients and chemical composition of the experimental diets (on dry matter basis). Ingredients (g/100 g) Dietary protein levels 25% 35% 45% Fish meal Soybean meal Wheat bran Ground corn Fish oil + corn oil (1:1) Vitamins and minerals premixa Ascorbic acid Starch Carboxymethyl cellulose Total 15.6 20.0 5.0 52.63 2.0 1.5 0.06 2.21 1.0 100 20.3 40.0 5.0 28.42 2.0 1.5 0.06 1.72 1.0 100 31.0 50.0 5.0 9.44 2.0 1.5 0.06 0.0 1.0 100 Chemical analysis (%)b Dry matter Crude protein Crude fat Ash Fiber NFEc GE (Kcal/g)d 92.48 ± 0.7 25.32 ± 0.24 5.87 ± 0.15 5.51 ± 0.23 6.68 ± 0.15 56.62 439.14 92.69 ± 0.6 35.41 ± 0.33 5.67 ± 0.25 6.31 ± 0.36 5.50 ± 0.12 47.11 446.85 93.09 ± 0.6 45.56 ± 0.46 5.99 ± 0.20 7.31 ± 0.37 5.76 ± 0.13 35.38 458.92 a Vitamin and minerals premix: each 2.5 kg contain vitamin A 12 MIU; D3 2 MIU, E 10 g; K 2 g; B1 1 g; B2 4 g; B6 1.5 g; B12 10 mg; pantothenic acid 10 g; nicotinic acid 20 g; folic acid 1 g; biotin 50 mg; choline chloride 500 mg; copper 10 g; iodine 1 g; iron 30 g; manganese 55 g; zinc 55 g and selenium 0.1 g. b Means of five replicates. c NFE (nitrogen free extract) = 100 − (protein% + lipid% + ash% + fiber%). d GE (gross energy): calculated after NRC (1993) as 5.64, 9.44, and 4.11 kcal/g for protein, lipid, and NFE, respectively. 2.4. Proximate chemical analysis of diets and fish The tested diets and whole-fish body from each treatment were analyzed according to the standard methods of AOAC (1990) for moisture, protein, fat, and ash. Moisture content was estimated by drying the samples to constant weight at 85 °C in a drying oven (GCA, model 18EM, Precision Scientific group, Chicago, Illinois, USA) and nitrogen content using a microKjeldahl apparatus (Labconco, Labconco corporation, Kansas, Missouri, USA). Crude protein was estimated by multiplying nitrogen content by 6.25. Lipid content was determined by ether extraction in a multi-unit Soxhlet extraction apparatus (Lab-Line Instruments, Inc., Melrose Park, Illinois, USA) for 16 h. Ash was determined by combusting dry samples in a muffle furnace (Thermolyne Corporation, Dubuque, Iowa, USA) at 550 °C for 6 h. 2.5. Fish performance Growth performance was determined and feed utilization was calculated as following: Specific growth rate (SGR; %/day) = 100(lnW2 − lnW1) / T; where W1 and W2 are the initial and final weight, respectively, and T is the number of days in the feeding period; Feed conversion ratio (FCR) = feed intake (g)/weight gain (g); Protein efficiency ratio (PER) = weight gain (g)/protein intake (g); Protein productive value (PPV; %) = 100 × (protein gain (g) / protein intake (g)); Protein growth rate (PGR; %/day) =100(Ln final protein content− Ln initial protein content) /days of feeding; Hepatosomatic index (HIS; %) = 100 × [liver weight (g) / body weight (g)]. 2.6. Physiological measurements At the end of the feeding trial, fish were not fed during the 24 h immediately prior to blood sampling. Fish were anaesthetized with buffered tricaine methane sulfonate (20 mg/L) and blood was collected with a hypodermic syringe from the caudal vein. The extracted blood was divided in two sets of Eppendorf tubes. One set contained 500 U sodium heparinate/mL, used as an anticoagulant, for hematology (hemoglobin, haematocrit and red blood cell counting). The second set, without anticoagulant, was left to clot at 4 °C and centrifuged at 5000 rpm for 5 min at room temperature. The collected serum was 269 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 stored at −20 °C for further assays. Red blood cells (RBCs) were counted under the light microscope using a Neubauer haemocytometer after blood dilution with phosphate-buffered saline (pH 7.2). Hemoglobin (Hb) level was determined colorimetrically by measuring the formation of cyanomethaemoglobin according to Van Kampen and Zijlstra (1961). Haematocrit values (Ht) were immediately determined after sampling by placing fresh blood in glass capillary tubes and centrifuging for 5 min in a microhematocrit centrifuge. Glucose was determined colorimetrically according to Trinder (1969). Total protein and total lipid contents in serum were determined colorimetrically according to Henry (1964) and Joseph et al. (1972), respectively. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, liver, and muscle were determined colorimetrically according to Reitman and Frankel (1957). 2.7. Statistical analysis Data were analyzed using a two-way ANOVA with protein levels and fish weights as factors. Statistical significance was set at the 5% probability level and means were separated using Duncan's new multiple range test. The software SPSS, version 12 (SPSS, Richmond, USA) was used as described by Dytham (1999). 3. Results 3.1. Growth performance Fish growth was significantly affected by protein level and initial weight, while their interaction significantly affected specific growth rate (SGR) only (P < 0.05; Table 2). The highest growth of fry tilapia was obtained at 45% CP, while fingerling and advanced juvenile showed optimum growth performance at 35% CP (Fig. 1). The poorest fish growth was obtained with the 25%-CP diet irrespective of fish weight. Feed intake (FI) and feed conversion ratio (FCR) were significantly affected by protein level and initial body weight and Fig. 1. Changes in live body weight (g) of Nile tilapia with different initial body weights and fed different protein levels for 10 weeks in glass aquaria. there was no significant effect due to their interaction (P < 0.05; Table 2). FI increased and FCR decreased significantly with increasing protein level up to 35% CP (P < 0.05), although FI and FCR of fingerling were not significantly different (P > 0.05). The optimum FCR was Table 2 Final weight, specific growth rate (SGR), feed intake, feed conversion ratio (FCR), hepatosomatic index (HSI), and survival of Nile tilapia as affected by dietary protein levels and different initial body weights. Variables Individual treatment means Fish size Fry Fingerling Advanced juvenile Protein level (%) Final weight (g)a SGR (%/day) Feed intake (g feed/g fish) FCR HSI (%) Survival (%) 25 35 45 25 35 45 25 35 45 5.1 7.7 10.3 41.1 45.2 44.3 58.6 64.7 62.9 0.421 3.289 3.900 4.287 1.007 1.143 1.107 0.524 0.672 0.635 0.022 8.3 11.8 14.5 46.2 47.9 47.3 50.2 55.7 55.5 0.409 1.81 1.65 1.49 2.22 1.92 1.98 2.79 2.29 2.45 0.020 3.29 2.47 1.79 2.01 1.91 1.69 2.30 1.82 1.57 0.202 96.7 100 100 100 100 100 100 100 100 0.258 7.7 43.5 62.1 34.9 39.2 39.2 3.825 1.086 0.61 1.607 1.905 2.010 11.5 47.1 53.8 34.9 38.5 39.1 1.65 2.04 2.51 2.27 1.95 1.97 2.52 p 1.87 q 1.90 q 2.53 2.07 1.68 98.9 100 100 98.9 100 100 b Pooled SE Means of main effectsc Fish size Fry Fingerling Advanced juvenile 25 35 45 ANOVA: P values Protein level Fish size Protein level × fish size a r q p y x x 0.001 0.0001 0.445 c b a e d d g f f 0.0001 0.0001 0.0001 q p p y x x 0.001 0.0001 0.426 q p p x y y 0.0001 0.0001 0.062 0.211 0.034 0.181 0.099 0.099 0.068 Initial weights for fry, fingerlings, and advanced juvenile were 0.51 ± 0.006, 20.33 ± 0.577, and 40.43 ± 1.528, respectively. Treatments means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different. c Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level. b 270 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 obtained when fry tilapia were fed the 45%-CP diet (1.49); whereas, the poorest one was obtained when advanced juvenile were fed the 25%-CP diet (2.79). The hepatosomatic index (HSI) was significantly affected by fish weight only (P < 0.05; Table 2) where it decreased significantly with increasing fish weight. The highest HSI was obtained by fry tilapia fed the 25%-CP diet; whereas, the lowest one was obtained by advanced juveniles fed the 45%-CP diet. No significant differences in fish survival were observed among fish groups and it was almost 100% except for that of fish fry fed the 25%-CP protein diet (96.7%; Table 2). 3.2. Protein utilization Protein utilization parameters, i.e., PER, PPV, and PGR were significantly affected by protein level and fish weight (P < 0.05; Table 3). The interaction of both factors significantly affected PER (P > 0.05). The dietary protein level inversely affected PER and PPV, while it positively affected PGR at different fish weights. The lowest PER and PPV were obtained with the 45%-CP diet for advanced juvenile (0.99 and 19.70%, respectively); whereas, the highest ones were obtained with the 25%-CP diet for fry tilapia (2.35 and 35.95%, respectively). PGR increased significantly with increasing fish weights; it was affected by dietary protein level for fingerling and advanced juvenile. The highest PGR was obtained with fry tilapia fed the 45%-CP diet (4.32%/day), while the lowest one was obtained with advanced juvenile fed the 25%-CP diet (0.69%/day). Unionized ammonia (UA) was significantly affected by protein level, fish weight, and their interaction (P < 0.05; Table 3). UA excreted in rearing water increased significantly with increasing protein level and fish weights. However, the highest UA value was obtained when advanced juvenile were fed the 45%-CP diet (1.622 mg/L); Table 3 Changes in protein efficiency ratio (PER), protein productive value (PPV), protein growth rate (PGR), and unionized ammonia (UA) of Nile tilapia fed different levels of dietary protein at different initial body weights. Variables Protein level (%) Individual treatment meansa Fish size Fry 25 35 45 Fingerling 25 35 45 Advanced juvenile 25 35 45 Pooled SE Means of main effectsb Fish size Fry Fingerling Advanced juvenile 25 35 45 ANOVA: P values Protein level Fish size Protein level × fish size PER 2.35 a 1.85 b 1.58 c 1.92 b 1.58 c 1.19 d 1.53 c 1.33 d 0.99 e 0.016 1.93 1.56 1.28 1.93 1.59 1.25 0.0001 0.0001 0.048 PPV (%) 35.95 28.97 25.18 26.80 24.17 19.73 27.72 21.92 19.70 0.254 30.03 23.57 23.11 30.16 25.02 21.54 p q q x y z 0.0001 0.0001 0.063 PGR (%/day) 3.98 3.94 4.32 1.00 1.21 1.24 0.69 0.81 0.89 0.024 4.08 1.15 0.80 1.89 1.99 2.15 p q r y y x 0.001 0.0001 0.179 UA (mg/L) 1.148 1.194 1.234 1.212 1.316 1.438 1.372 1.482 1.622 0.007 f ef e e d b c b a 1.192 1.322 1.492 1.244 1.331 1.431 0.0001 0.0001 0.009 a Treatment means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different. b Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level. whereas, the lowest one was obtained with fry tilapia fed the 25%-CP diet (1.148 mg/L). 3.3. Body composition No significant changes in moisture content were observed at the different treatments (Table 4). Protein and lipid contents in whole-body fish were significantly affected by dietary protein level and fish weight, while their interaction significantly affected lipids content only (P < 0.05; Table 4). Ash content significantly differed due to fish weight (P< 0.05). The highest protein content in whole body was obtained with the 45%-CP diet in all weight classes. The body lipid content decreased with increased dietary protein level within each fish weight; whereas, the highest lipid content was recorded for the 25%-CP diet at all fish weights. The lowest lipid content was obtained in fish fed the 35- and 45%-CP diets for fingerling and advanced juvenile. Ash content in whole body was unaffected by dietary protein levels at all fish weights, and the lowest contents were observed in fry (P< 0.05). 3.4. Physiological alterations RBCs, Hb, and Ht values were significantly affected by protein level, fish weight, and their interaction (P < 0.05; Table 5). Likewise, serum glucose, protein, and lipids were significantly affected by protein level and fish weight (P < 0.05; Table 5), while plasma lipids were not affected by their interaction. All physiological variables significantly increased with increasing dietary protein level in all fish weights. In some cases, these variables did not exhibit significant differences between fish fed the 35- or 45%-CP diets. AST and ALT activities in serum, liver, and muscles were significantly affected by dietary protein level and fish weight (P < 0.05; Table 6). The interaction significantly affected all Table 4 Proximate chemical analysis (%; on fresh-weight basis) of whole body of Nile tilapia fed different levels of dietary protein at different initial body weights. Variable Protein level (%) Individual treatment meansa Fish size Fry 25 35 45 Fingerling 25 35 45 Advanced juvenile 25 35 45 Pooled SE Means of main effectsb Fish size Fry Fingerling Advanced juvenile 25 35 45 ANOVA: P values Protein level Fish size Protein level × fish size Moisture Crude protein Total lipids Ash 71.6 72.0 72.5 75.2 74.9 74.2 73.1 74.7 73.5 0.709 15.4 15.6 16.0 14.0 14.7 15.4 14.6 14.3 15.5 0.145 9.7 a 8.9 b 7.8 c 5.8 d 5.1 e 5.1 e 6.5 e 5.3 de 5.1 e 0.065 3.3 3.5 3.7 5.0 5.3 5.3 5.8 5.7 5.9 0.047 72.0 74.8 73.8 73.3 73.9 73.4 15.7p 14.7q 14.8q 14.7y 14.9y 15.6x 8.8 5.3 5.6 7.3 6.4 6.0 3.5q 5.2 p 5.8 p 4.7 4.8 5.0 0.0001 0.0001 0.032 0.099 0.001 0.629 0.933 0.294 0.981 0.026 0.033 0.731 a Treatment means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different. b Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level. 271 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 Table 5 Changes in red blood corpuscles (RBCs), haemoglobin (Hb), haematocrit (Ht), serum glucose, protein, and lipids in Nile tilapia fed different levels of dietary protein at different initial weights. Variables Individual treatment meansa Fish size Fry Fingerling Advanced juvenile Protein level (%) RBCs (× 106/μL) Hb (g/L) Ht (%) Glucose (mg/L) Protein (g/L) Lipids (g/L) 25 35 45 25 35 45 25 35 45 1.262 1.298 1.866 1.856 1.972 2.186 1.728 2.166 2.412 0.041 75.4 c 84.4 c 119.4 a 83.7 c 100.6 b 102.2 b 79.6 c 99.8 b 109.1 b 1.274 21.8 f 23.8 e 26.8 d 29.0 c 31.0 ab 32.2 a 29.6 bc 31.6 a 32.2 a 0.206 648.9 c 1191.7 b 1282.0 b 641.2 c 653.0 c 1274.4 b 714.9 c 1425.5 a 1442.0 a 13.361 25.0 e 28.2 d 29.3 cd 30.3 cd 34.9 bc 37.7 ab 33.3 bc 41.0 a 43.1 a 0.709 15.0 15.4 16.2 15.8 16.8 19.6 16.8 19.7 20.5 0.174 93.1 95.5 96.2 79.6 94.9 110.2 24.1 30.7 31.1 26.8 28.8 30.4 1040.9 856.2 1194.1 668.3 1090.1 1332.8 27.5 34.3 39.1 29.5 34.7 36.7 15.5 17.4 19.0 15.9 17.3 18.8 Pooled SE Means of main effectsb Fish size Fry Fingerling Advanced juvenile 25 35 45 ANOVA: P values Protein level Fish size Protein level × fish size d d bc bc bc ab c ab a 1.475 2.005 2.102 1.6153 1.812 2.155 0.011 0.001 0.001 0.0001 0.015 0.001 0.0001 0.0001 0.046 0.0001 0.0001 0.0001 0.038 0.0001 0.015 r q p y xy x 0.0001 0.0001 0.596 a Treatment means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different. b Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level. enzyme activities except serum AST. In all fish weights, enzyme activities increased significantly with the increase of dietary protein level. The highest activities were observed in advanced juvenile fed the 45%-CP diet; whereas, the lowest values were obtained for fry fed 25%CP diet. 4. Discussion The present study shows that the dietary protein level markedly affects the growth, feed utilization, and physiological status of Nile tilapia in all weight classes. The optimum dietary protein required for Table 6 Changes in AST and ALT activities in serum (IU/L), liver (IU/g fresh weight), and muscles (IU/g fresh weight) of Nile tilapia fed different levels of dietary protein at different initial weights. Variables Individual treatment meansa Fish size Fry Fingerling Advanced juvenile Protein level (%) Serum AST Liver AST Muscle AST Serum ALT Liver ALT Muscle ALT 25 35 45 25 35 45 25 35 45 13.5 14.3 17.4 14.1 17.3 19.5 15.5 17.3 21.6 0.223 225.7 d 258.5 c 301.3 b 229.4 d 264.8 c 303.4 b 261.3 c 312.8 b 414.5 a 6.613 460.8 d 612.5 c 764.0 b 458.0 d 647.3 c 817.2 a 590.0 c 782.0 ab 819.8 a 7.785 14.7 c 18.5 b 20.3 ab 15.1 c 19.1 b 20.1 ab 19.0 b 20.7 ab 22.9 a 0.371 235.0 f 292.5 de 436.3 c 255.1 ef 363.7 cd 528.9 b 367.5 cd 535.5 b 675.3 a 9.223 337.3 d 427.1 bc 476.0 ab 340.3 d 411.2 bc 530.0 a 373.0 d 417.3 bc 548.1 a 7.541 15.1 17.0 18.1 14.4 16.3 19.5 261.8 265.9 329.5 238.8 278.7 339.7 612.4 640.8 730.6 502.9 680.6 800.3 17.8 18.1 20.9 16.3 19.4 21.1 321.3 382.6 526.1 285.9 397.2 546.8 413.5 427.2 446.1 350.2 418.5 518.0 Pooled SE Means of main effectsb Fish size Fry Fingerling Advanced juvenile 25 35 45 ANOVA: P values Protein level Fish size Protein level × fish size q p p z y x 0.0001 0.0001 0.096 0.0001 0.0001 0.043 0.0001 0.0001 0.006 0.0001 0.004 0.046 0.0001 0.0001 0.039 0.0001 0.028 0.002 a Treatment means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different. b Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level. 272 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 Nile tilapia is weight dependant; fry tilapia (~0.5 g) required the 45%CP diet for optimal growth; whereas, fingerling (~ 20 g) and advanced juvenile (~40 g) performed optimally with the 35%-CP diet. El-Sayed and Teshima (1991) found similar results in terms of the protein requirement of Nile tilapia; with values ranging between 20% and 56% CP. The studies of Balarin and Haller (1982) showed that fry of tilapia required a diet ranging between 35 and 50% CP, and 5–25 g fish between 25 and 35% CP. Tacon (1987) reported dietary protein levels varying from 42% for fry to 35% for growing adults of omnivorous fish species. Our results show a more accurate description of dietary CP needs for different life history changes, as we tested the possible variations within these ranges (25–45%). Variations in dietary CP needs related to fish weights may be attributed to different protein needs at different life history stages. El-Sayed and Teshima (1991) found that dietary protein requirements decreased with increasing fish weight and age. Both in fingerling and advanced juvenile of Nile tilapia, excess protein could not be utilized efficiently and might have been used for energy. Khattab et al. (2000) studied the optimum dietary protein level for three Nile tilapia strains of the same weight class (12 g) collected from different locations in Egypt and found that optimum dietary protein level ranged from 27 to 37%. Such variations in optimum dietary protein requirements for tilapia growth might be due to the variation in stocking density, hygiene, and/or environmental conditions in their natural environments. In cultured fish, such as those used in this study, the environmental variability was controlled and the effect of protein in the diet was not confounded by other factors. Feed utilization was significantly affected by protein level and fish weight but not their interaction. FCR increased with fish weight and coincided with previously published ranges for Nile tilapia (Siddiqui et al., 1988; Al-Hafedh, 1999; Abdelghany, 2000; Khattab et al., 2000). The hepatosomatic index was inversely affected by dietary CP in all fish weights except fingerlings due to the increased fish weight at higher CP, the liver to body weight ratio decreased. Likewise, Gallagher (1999) found that HSI was significantly higher in sunshine bass fed lower protein diets. The carcass proximate analysis of all weight classes was significantly influenced by dietary protein level and fish weight; the ash content was only significantly affected by fish weight. Gallagher (1999), however, did not find significant differences in moisture, protein, lipid, and ash in whole-body of sunshine bass fed different protein levels. Nile tilapia fed the 25%-CP diet had lower content of protein and higher lipid content than fish fed the 35%- or 45%-CP diet, for all weight classes. Due to the high feed intake, nutrient utilization, and the high nutrient digestibility, the deposited nutrients increased. Changes in protein and lipid contents in fish body could be linked with changes in their synthesis, deposition rate in muscle and/or different growth rates (Smith, 1981; Fauconneau, 1984; Soivio et al., 1989; Abdel-Tawwab et al., 2006). Similar results were obtained by Wee and Tuan (1988), Al-Hafedh (1999), and Khattab et al. (2000). All different measures of protein metabolism, including PER, PPV, and PGR, and ammonia production were affected by the treatments. PER, PPV, and PGR were significantly affected by protein level and fish weight. Protein utilization decreased with increasing dietary protein levels and with larger fish. These results may occur because the major part of weight gain was related to the deposition of protein, and protein accretion is a balance between protein anabolism and catabolism. Gastric emptying rate or solubility of the protein has been shown to affect the utilization of dietary protein (de la Higuera et al., 1998; Espe et al., 1999). In our study, fingerling and advanced juvenile of Nile tilapia did not use the excess protein over 35% suggesting that some dietary protein might be deaminated and produced ammonia. The increase of nitrogenous excretion is a consequence of using amino acids as energetic compounds (Hidalgo and Alliot, 1988; Kim et al., 1991). A direct relationship between protein intake and ammonia excretion has been found in fish (Li and Lowell, 1992; Chakraborty and Chakraborty, 1998). The increased protein breakdown in fish, resulting in increased plasma ammonia concentrations, was observed in Bidyanus bidyanus, Dicentrarchus labrax (Yang et al., 2002; Peres and Oliva-Teles, 2001), Anguilla australis australis (Engin and Carter, 2001) and Rhamdia quelen (Melo et al., 2006). Excess of ammonia is promptly excreted through the gills (VanWaarde et al., 1983). Therefore, the increase in unionized ammonia in the present study reflects an increased protein catabolism. Webb and Gatlin (2003) found similar results for red drum; when it was fed high-protein diet (45% CP); it excreted significantly more ammonia than those fed on low-protein diet (35% CP). Changes in the metabolic profile are proxies of fish performance and ability to cope with different dietary conditions (Bidinotto and Moraes, 2000; Moraes and Bidinotto, 2004; Lundstedt et al., 2004; Melo et al., 2006). Changes in RBCs, Hb, and Ht were significantly affected by dietary protein, fish weight, and their interaction. The increase in RBC count may have occurred because of its release from the storage pool in the spleen (Vijayan and Leatherland, 1989; Pulsford et al., 1994). Thus, it seems that spleen activity is different for different fish weight and that it is affected by dietary protein level. In addition, fish blood contains heterogeneous populations of erythrocytes where immature cells are generally smaller and contain less Hb than older and mature ones (Härdig and Høglund, 1983). These authors suggested that Hb synthesis occurs in the erythrocytes after their release into circulation. The knowledge herein gained on biochemical features may provide the basis for better understanding the handling and rearing of Nile tilapia. In serum, glucose, protein, and lipid tended to increase with increased diet protein content. Similar results were observed in Oncorhynchus mykiss (Lone et al., 1982), European eel (Suárez et al., 1995), and R. quelen (Melo et al., 2006). These increments may be because any excess of amino acids could be converted into carbohydrates or, in smaller amounts, to fat (Driedzic and Hochachka, 1978). In the present study, the surplus of amino acids in Nile tilapia was reflected in the increased amino acid concentration in the tissues due to the increased protein levels. This can be associated with increased absorption of amino acids from protein digestion (Yamamoto et al., 1999). Serum protein depends on many factors such as digestion efficiency, fish weight, composition of the diet, and temperature (Grove et al., 1981; Darcy, 1984). Although there was an increase in serum protein, the increased levels of ALT and AST suggests protein catabolism at high protein levels in the diet. The amino acids surplus from proteinrich diets cannot be directly stored in fish and they might be deaminated and converted into energetic compounds (Ballantyne, 2001; Stone et al., 2003). In this study, the rise of plasma protein with dietary protein could likely be due to the enhancement of digested protein (Lundstedt et al., 2002). Increased glucose in serum suggests gluconeogenesis as a consequence to increased dietary protein level. Serum lipids slightly increased due to the increase in protein level and it may be because the muscle is a pivotal compartment directly linked to amino acid turnover. This involves protein synthesis or breakdown of those molecules as energetic substrates. The expression of key enzymes of intermediary metabolism is modulated by nutritional status in fish (Metón et al., 1999, 2003). The levels of amino acid-metabolizing enzymes and nitrogen excretion are reliable indicators of dietary protein availability. Metabolism of amino acids involves deamination and transamination reactions. The activities of transaminases and deaminases are useful to evaluate the feeding status in some fish (Alexis and Papaparaskeva-Papoutsoglou, 1986; Moyano et al., 1991; Melo et al., 2006). The rise of ALT and AST activities observed in Nile tilapia fed the 45%-CP diets may reflect the use of excess hydrocarbons from amino acids to supply energetic demands. High protein/carbohydrate ratios in the feeding of Sparus aurata increased ALT and AST activity in the liver (Metón et al., 1999). Similar responses were observed in O. mykiss for ALT (Sánchez-Muros et al., 1998) and in R. quelen for AST and ALT (Melo et al., 2006). The rise in the hepatic activity of protein-metabolizing enzymes when fish were fed the 45%- M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 CP diet may denote use of excess dietary amino acids for growth as well as substrate for gluconeogenesis, particularly for AST and ALT activities. Physiological alterations were significantly affected by diet composition and weights of Nile tilapia and could be used as a metabolic tool for assessing the proper concentration of dietary protein in the feeding of Nile tilapia. Moreover, the proper ratio of protein/carbohydrate in the diet is fundamental to establish the optimal nitrogen content in the diet, to increase fish gain efficiency while preventing nitrogen waste and environmental damage. The ratio of protein/energy in the Nile tilapia diet seems to be specific and further studies concerning types and sources of carbohydrates and lipids to replace protein as energy sources are necessary. Acknowledgements The authors would like to thank Mamdouh A.A. Mousa, Department of Fish Biology and Ecology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia, Egypt, for his great help in doing the physiological assays and for his valuable comments and advises during the writing of this manuscript. References Abdelghany, A.E., 2000. Optimum dietary protein requirements for Oreochromis niloticus L. fry using formulated semi-purified diets. In: Fitzsimmons, K., Filho, J.C. (Eds.), Tilapia Aquaculture in the 21st Century. Proceedings from the 5th International Symposium on Tilapia Aquaculture, vol. 1, pp. 101–108. 3–7 Sept. 2000, Rio de Janeiro, Brazil. Abdel-Tawwab, M., Ahmad, M.H., 2009. Effect of dietary protein regime during the growing period on growth performance, feed utilization and whole-body chemical composition of Nile Tilapia, Oreochromis niloticus (L.). Aquacult. Res. 40, 1532–1537. Abdel-Tawwab, M., Khattab, Y.A.E., Ahmad, M.H., Shalaby, A.M.E., 2006. Compensatory growth, feed utilization, whole body composition and hematological changes in starved juvenile Nile tilapia, Oreochromis niloticus (L.). J. Appl. Aquac. 18, 17–36. Abdel-Tawwab, M., Abdelghany, A.E., Ahmad, M.H., 2007. Effect of feed supplementation on water properties, phytoplankton community structure and the growth of Nile tilapia, Oreochromis niloticus (L.), common carp, Cyprinus carpio L., and silver carp, Hypophthalmichthys molitrix V. polycultured in fertilized earthen ponds. J. Appl. Aquac. 19, 1–24. Alexis, M.N., Papaparaskeva-Papoutsoglou, E., 1986. Aminotransferase activity in the liver and white muscle of Mugil capito fed diets containing different levels of protein and carbohydrate. Comp. Biochem. Physiol., B 83, 245–249. Al-Hafedh, Y.S., 1999. Effects of dietary protein on growth and body composition of Nile tilapia, Oreochromis niloticus L. Aquac. Res. 30 (5), 385–393. AOAC, 1990. In: Helrich, K. (Ed.), Official Methods of Analyses, 15th edition. Association of Official Analytical Chemists Inc., Ar, VA, USA. Balarin, J.D., Haller, R.D., 1982. The intensive culture of tilapia in tanks, raceways and cages. In: Muir, J.F., Roberts, R.J. (Eds.), Recent Advances in Aquaculture. Crom Helm, London, United Kingdom, pp. 265–356. Ballantyne, J.S., 2001. Amino acid metabolism. Fish Physiol. 20, 77–107. Barcellos, L.J.G., Kreutz, L.C., de Souza, C., Rodrigues, L.B., Fioreze, I., Quevedo, R.M., Cericato, L., Soso, A.B., Fagundes, M., Conrad, J., Lacerda, L.A., Terra, S., 2004. Hematological changes in jundiá (Rhamida quelen Quoy and Gaimard Pimelodidae) after acute and chronic stress caused by usual aquacultural management, with emphasis on immunosuppressive effects. Aquaculture 237, 229–236. Barton, B.A., Iwama, G.K., 1991. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Annu. Rev. Fish Dis. 10, 3–26. Bidinotto, P.M., Moraes, G., 2000. Induced changes in the amylohydrolytic profile of the gut of Piaractus mesopotamicus (Holmberg, 1885) fed different contents of soluble carbohydrate; its correlation with metabolic aspects. Rev. Ictiol. Argent. 8, 47–51. Boyd, C.E., 1984. Water Quality in Warm water Fishponds. Auburn University Agriculture Experimental Station, Auburn, AL, USA. Chakraborty, S.C., Chakraborty, S., 1998. Effect of dietary protein level on excretion of ammonia in Indian major carp, Labeo rohita, fingerlings. Aquac. Nutr. 4, 47–51. Cnaani, A., Tinman, S., Avidar, Y., Ron, M., Hulata, G., 2004. Comparative study of biochemical parameters in response to stress in Oreochromis aureus, O. mossambicus and two strains of O. niloticus. Aquac. Res. 35, 1434–1440. Darcy, B., 1984. Availability of amino acids in monogastric animals. Diabetes Metab. 10, 121–133. de la Higuera, M., Garzon, A., Hidalgo, M.C., Peragon, J., Cardenete, G., Lupianez, J.A., 1998. Influence of temperature and dietary-protein supplementation either with free or coated lysine on the fractional protein-turnover rates in the white muscle of carp. Fish Physiol. Biochem. 18, 85–95. Driedzic, W.R., Hochachka, P.W., 1978. Metabolism in fish during exercise. Fish Physiol. 7, 503–543. Dytham, C., 1999. Choosing and Using Statistics: A Biologist's Guide. Blackwell Science Ltd., London, United Kingdom. 273 El-Sayed, A.-F.M., 2006. Tilapia Culture. CABI Publishing, CABI International, Willingford, Oxfordshire, United Kingdom. El-Sayed, A.-F.M., Teshima, S., 1991. Tilapia nutrition in aquaculture. Rev. Aquat. Sci. 5, 247–265. Engin, K., Carter, C.G., 2001. Ammonia and urea excretion rates of juvenile Australian Short-finned eel (Anguilla australis australis) as influenced by dietary protein level. Aquaculture 194, 123–136. Espe, M., Sveier, H., Hogoy, I., Lied, E., 1999. Absorption and growth in Atlantic salmon (Salmo salar): effects of fish protein concentrates in the diets. Aquaculture 174, 119–137. Fauconneau, B., 1984. The measurements of whole body protein synthesis in larval and juvenile carp (Cyprinus carpio L.). Comp. Biochem. Physiol. 78, 845–850. Gallagher, M.L., 1999. Growth response, tissue composition, and liver enzymes changes in juvenile sunshine bass Morone chrysops × M. saxatilis, associated with dietary protein and lipid level. J. Appl. Aquac. 9, 41–51. Grove, D.J., Loizides, L.G., Nott, J., 1981. Satiation amount, frequency of feeding and gastric emptying rate in Salmo gairdner. J. Fish Biol. 12, 507–516. Härdig, J., Høglund, L.B., 1983. On accuracy in estimating fish blood variables. Comp. Biochem. Physiol. 75A, 35–40. Henry, R.J., 1964. Colorimetric determination of total protein. Clinical Chemistry. Harper and Row Publ., New York, USA. Hidalgo, F., Alliot, E., 1988. Influence of water temperature on protein utilization in juvenile sea bass, Dicentrachus labrax. Aquaculture 72, 115–129. Joseph, A., Knight, M., Anderson, S., James, M., Rawie, H., 1972. Chemical basis of the sulfophospho-vanillin reaction for estimating total serum lipid. Clin. Chem. 18, 198–201. Khattab, Y.A.E., Ahmad, M.H., Shalaby, A.M.E., Abdel-Tawwab, M., 2000. Response of Nile tilapia (Oreochromis niloticus L.) from different locations to different dietary protein levels. Egypt. J. Aquat. Biol. Fish. 4, 295–311. Kim, K., Kyes, T.B., Amundson, C.H., 1991. Purified diet development and reevaluation of the dietary protein requirement of fingerling rainbow trout (Oncorhynchus mykiss). Aquaculture 96, 57–67. Li, M., Lowell, R.T., 1992. Effect of dietary protein concentration on nitrogenous waste in intensively fed catfish ponds. J. World Aquac. Soc. 23, 122–127. Liti, D., Cherop, L., Munguti, J., Chhorn, L., 2005. Growth and economic performance of Nile tilapia (Oreochromis niloticus L.) fed on two formulated diets and two locally available feeds in fertilized ponds. Aquac. Res. 36, 746–752. Lone, K.P., Ince, B.W., Matty, A.J., 1982. Changes in the blood chemistry of rainbow trout, Salmo gairdneri fish, in relation to dietary protein level, and an anabolic steroid hormone, ethylestrenol. J. Fish Biol. 20, 597–606. Lundstedt, L.M., Melo, J.F.B., Santos-Neto, C., Moraes, G., 2002. Diet influences proteolytic enzyme profile of the South American catfish Rhamdia quelen. Proceedings of International Congress on the Biology of Fish, Biochemistry and Physiology Advances in Finfish Aquaculture, Vancouver, Canada, pp. 65–71. Lundstedt, L.M., Melo, J.F.B., Moraes, G., 2004. Digestive enzymes and metabolic profile of Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition. Comp. Biochem. Physiol., B 137, 331–339. Melo, J.F.B., Lundstedt, L.M., Metón, I., Baanante, I.V., Moraes, G., 2006. Effects of dietary levels of protein on nitrogenous metabolism of Rhamdia quelen (Teleostei: Pimelodidae). Comp. Biochem. Physiol., A 145, 181–187. Metón, I., Mediavilla, D., Casearas, A., Cantó, E., Fernández, F., Baanante, I.V., 1999. Effect of diet composition and ration size on key enzyme activities of glycolysis– gluconeogenesis, the pentose phosphate pathway and amino acid metabolism in liver of gilthead sea bream (Sparus aurata). Br. J. Nutr. 82, 223–232. Metón, I., Egea, M., Baanante, I.V., 2003. New insights into regulation of hepatic glucose metabolism in fish. Recent Res. Dev. Biochem. 4, 125–149. Moraes, G., Bidinotto, P.M., 2004. Digestive proteases of pacu, Piaractus mesopotamicus fed on distinct protein-starch diets. J. Appl. Aquac. 15, 197–207. Moyano, F.J., Cardente, G., de la Higuera, M., 1991. Nutritive and metabolic utilization of proteins with glutamic acid content by the rainbow trout Oncorhynchus mykiss. Comp. Biochem. Physiol., A 100, 759–762. NRC (National Research Council), 1993. Nutrient requirements of fish. Committee on Animal Nutrition. Board on Agriculture. National Research Council. National Academy Press. Washington DC., USA. pp. 114. Peres, H., Oliva-Teles, A., 2001. Effect of dietary protein lipid level on metabolic utilization of diets by European sea bass (Dicentrarchus labrax) juveniles. Fish Physiol. Biochem. 25, 269–275. Pulsford, A.L., Lemaire-Gony, S., Tomlinson, M., Collingwood, N., Glynn, P.J., 1994. Effects of acute stress on the immune system of the dab, Limanda limanda. Comp. Biochem. Physiol. 109C, 129–139. Řehulka, J., Minařík, B., Řehulková, E., 2004. Red blood cell indices of rainbow trout Oncorhynchus mykiss (Walbaum) in aquaculture. Aquac. Res. 35, 529–546. Reitman, S., Frankel, S., 1957. Colorimetric determination of glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 53–56. Sánchez-Muros, M.J., Gárcia-Rejón, L., Gárcia-Salguero, L., de la Higuera, M., Lupiánes, J.A., 1998. Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout. Adaptive response to starvation and high-protein, carbohydrate-free diet on glutamate dehydrogenase and alanine aminotransferase kinetics. Biochem. Cell. Biol. 30, 55–63. Siddiqui, A.Q., Holder, M.S., Adam, A.A., 1988. Effects of dietary protein levels on growth, food conversion and protein utilization in fry and young Nile tilapia (Oreochromis niloticus). Aquaculture 70, 63–73. Smith, M.A.K., 1981. Estimation of growth potential by measurement of tissue protein synthetic rates in feeding and fasting rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 19, 213–220. Soivio, A., Niemisto, M., Backstrom, M., 1989. Fatty acid composition of Coregonus muksun Pallas: changes during incubation, hatching, feeding and starvation. Aquaculture 79, 163–168. 274 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 Stone, D.A., Allan, G.L., Anderson, A.J., 2003. Carbohydrate utilization by juvenile silver perch, Bidyanus bidyanus (Mitchell). III. The protein-sparing effect of wheat starchbased carbohydrates. Aquac. Res. 34, 123–134. Suárez, M.D., Hidalgo, M.C., García Gallego, M., Sanz, A., de la Higuera, M., 1995. Influence of relative proportions of energy yielding nutrients on liver intermediary metabolism of the European eel. Comp. Biochem. Physiol. 111A, 421–428. Tacon, A.G.I., 1987. The nutrition and feeding of farm fish and shrimp a training manual. 1. The essential nutrients. FAO Brasilia Brazil, GCP/RLA/075/ITA Field Document 2/E, p. 117. Thankur, D.P., Yi, Y., Diana, J.S., Lin, C.K., 2004. Effects of fertilization and feeding strategy on water quality, growth performance, nutrient utilization and economic return in Nile tilapia. In: Bolivar, R., Mair, G., Fitzsimmons, K. (Eds.), The 6th International Symposium of Tilapia in Aquaculture Pages 529–543. Philippine International Convention Center, Roxas Boulevard, Manila, Philippines. Trinder, P., 1969. Determination of glucose concentration in the blood. Ann. Clin. Biochem. 6, 24–27. Van Kampen, E.J., Zijlstra, N.C., 1961. Determination of haemoglobin. Clin. Chem. Acta 5, 719–720. VanWaarde, A., Van Den Thillart, G., Kesbeke, F., 1983. Anaerobic energy metabolism of the European eel, Anguilla anguilla L. J. Comp. Physiol. 149B, 469–475. Vijayan, M.M., Leatherland, J.F., 1989. Cortisol-induced changes in plasma glucose, protein, and thyroid hormone levels, and liver glycogen content of Coho salmon (Oncorhynchus kisutch Walbaum). Can. J. Zool. 67, 2746–2750. Webb, K.A., Gatlin III, D.M., 2003. Effects of dietary protein level and form on production characteristics and ammonia excretion of red drum Sciaenops ocellatus. Aquaculture 225, 17–26. Wee, K.L., Tuan, N.A., 1988. Effects of dietary protein level on growth and reproduction in Nile tilapia (Oreochromis niloticus). In: Pullin, R.S.V., Bhukasawan, T., Tonguthal, K., Maclean, J.L. (Eds.), The Second International Symposium on Tilapia in Aquaculture. ICLARM, Manila, Philippines, pp. 401–410. Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiol. Rev. 77, 591–625. Yamamoto, T., Unuma, T., Akiyama, T., 1999. The influence of dietary protein and fat levels on tissue free amino acid levels of fingerling rainbow trout (Oncorhynchus mykiss). Aquaculture 182, 353–372. Yang, S., Liou, C., Liu, F., 2002. Effects of dietary protein level on growth performance, carcass composition and ammonia excretion in juvenile silver perch (Bidyanus bidyanus). Aquaculture 213, 363–372.