Growth performance, oxidative stress indices and ...

Aquaculture Research, 2013, 1–14

doi:10.1111/are.12153

Growth performance, oxidative stress indices and hepatic carbohydrate metabolic enzymes activities of juvenile Nile tilapia, Oreochromis niloticus L., in response to dietary starch to protein ratios Mohamed Salah Azaza1, Noura Khiari1, Mohamed Naceur Dhraief1, Ne´ji Aloui1, Mohamed Mejdeddine Kraϊem1 & Abdelfattah Elfeki2 1

National Institute of Marine Sciences and Technologies,2025 Salammb^ o, Tunisia

2

Laboratory of Animal Ecophysiology, Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia

Correspondence: M S Azaza, National Institute of Marine Sciences and Technologies. 28, Rue 2 mars 1934, Salammb^ o 2025, Tunisia. E-mail: [email protected]

Abstract

Introduction

The effect of various dietary starch to proteins ratios (STA/P) on growth performance, oxidative status and liver enzyme activities involved in intermediary metabolism in juvenile Nile tilapia was evaluated. Four isocaloric-practical diets (12.73 MJ kg1 digestible energy) with increasing STA/CP ratios were formulated. These were designated D0 (344 g crude protein (CP) and 163.5 g starch (STA) kg1), D1 (310 g CP and 243 g STA kg1), D2 (258 g CP and 322 g STA kg1) and D3 (214 g CP and 401 g STA kg1). Each diet was fed to triplicate groups of 60 fish (2.7 g) for 45 days. Compared with the control diet (D0), significantly (P < 0.05) depressed growth and feed efficiency were observed only in the groups fed on diet D3. The activities of hepatic enzymes involved in glycolysis and lipogenesis pathways were significantly enhanced in groups fed on diet D3 compared with other diets. A significant (P < 0.05) increase in catalase activity was detected only in groups fed on diet D3. Similarly, a significant (P < 0.05) enhancement in superoxyde dismutase, glutathione S-transferases and glutathione peroxidise was observed in groups fed on diets D2 and D3 compared with other diets. Results demonstrate the ability of juvenile Nile tilapia to spare protein by dietary carbohydrate.

Whatever the cultured fish species, it is crucial that aqua feed be adequate and sustainable, as feeds represent the main contribution to production costs (40–60% of total variable expenses). Proteins are the most expensive components of formulated feed and are the limiting components in diets, both in terms of cost and supply and thus, considered as the most critical input in aqua feed. The use of other alternative sources that reduce feed costs is the most requisite to produce fish cost effectively. Carbohydrates are the lowest cost energy source in practical diet ingredients and efficiently used by omnivorous and/or herbivorous warm water fish such as common carp, Cyprinus carpio L. (Gao, Liu, Tian, Mai, Liang, Yang, Huai & Luo 2010), Nile tilapia (Zhao, Cao, Liu, Zhu, Chen, Lan & Wang 2011; Fortes-Silva & S anchez-V azquez 2012) and bagrid catfish, Mystus nemurus (Cuv & Val) (Hamid, Mahayat & Hashim 2011). The use of carbohydrate-rich diets have the advantage of being economical as fish would utilize the inexpensive carbohydrate as a source of energy instead of proteins, thus sparing the absorbed protein for growth (e.g. Mohanta, Mohanty & Jena 2007; Azaza, Mensi, Wassim, Abdelmouleh, Brini & Kraϊem 2009; Webb, Rawlinson & Holt 2010; Zhao et al. 2011 etc.). Nevertheless, increased dietary carbohydrate content, beyond optimal levels, can negatively impact fish health through metabolic disturbances and clinical signs such as hyperglycemia (Hemre, Mommsen & Krogdahl

Keywords: carbohydrate utilization, protein sparing, metabolic enzyme, Reactive oxygen species, oxidative status, Oreochromis niloticus © 2013 Blackwell Publishing Ltd

1

Proteins: starch ratios in tilapia diets M S Azaza et al.

2002), increased glycogen deposition (Rawles & Gatlin 1998; Rawles, Smith & Gatlin 2008), liver hypertrophy (Azaza, Mensi et al. 2009) and histopathological (Russell, Davies, Gouveia & Tekinay 2001). Physiological and biochemical characteristics can serve as an important tool in the evaluation of the physiological functions, welfare and health status of aquatic organisms. Hence, knowledge of the fish’s response to the dietary carbohydrate (i.e. metabolic and physiological responses) is of utmost importance to improve the nutritional profile of the diet and hence to provide an adequate and healthy diet for fish (Saidi, Azaza, Abdelmouleh, Pelt, Kra€ıem & El-feki 2010; Zhou, Chen, Qiu, Zhao & Jin 2011; Zuo, Ai, Mai, Xu, Wang, Xu, Liufu & Zhang 2012; Fjelldal, Hansen & Albrektsen 2012; Shao, Liu, Lu, Xu, Zhang, Wang & Zhu 2012; Yang, Liu & Liou 2012 etc.). As with other animals, under normal physiological conditions, fish cells produce reactive oxygen species (ROS) which are removed by antioxidant defences. When this equilibrium between the generation and removal of ROS is disturbed, oxidative stress is triggered. There is little known about how the level of dietary carbohydrate affects the oxida~o, Dias, tive status of fish (e.g. Rueda-Jasso, Conceicßa De Coen, Gomes, Rees, Soares, Dinis & Sorgeloos 2004). It is obvious that the nutrition influences oxidation and antioxidation defence mechanisms (Azambuja, Mattiazzi, Riffel, Finamor, Garcia, Heldwein, Heinzmann, Baldisserotto, Pavanato & Llesuy 2011) and thus the composition of the diet should be evaluated in terms of its effect on the balance between ROS generation and breakdown. However, thorough information and integrated studies combining enzyme regulation and oxidative status are very scant in fish species. For Nile tilapia, the second group of species in world aquaculture production nowadays and a key species for freshwater aquaculture in tropical and subtropical regions around the world, so far, no studies have examined these aspects. Therefore, the objective of this work was to determine the effect of dietary starch to protein ratio on growth performance, carbohydrate enzyme metabolic regulation and oxidative status of juveniles Nile tilapia. On the applied side, results of the present study will allow tailoring the composition (in term of protein and carbohydrate contents) of tilapia diets in better adequacy with fish health (oxidative status) which allows reducing stress and its harmful effects on growth and production.

2


Materials and methods Experimental diets: formulation and preparation Formulation and proximate analysis (AOAC 1990) of the diets are given in Table 1. Fish meal and soybean meal (SBM) were used as protein sources. Soybean oil and corn starch meal (CSM) were used as lipid and carbohydrate sources respectively. Table 1 Formulation and proximate composition of the experimental diets. Diets Components

D0

Ingredients (g kg1diet) Fish meal 180 Soybean meal 480 Maize meal 250 Corn starch meal 0 Soybean oil 70 10 Vit-mineral mixa CMCb (binder) 10 Proximate analysis (g kg1diet) Dry matter 911.6 (in original matter) Crude protein 343.9 Crude lipid 98.9 Crude fibre 71.1 Ash 62.3 Starch (STA) 163.5 0.48 STA/Pc 17.38 Gross Energy (MJ kg1 DM)d 13.51 Digestible Energy (MJ kg1 DM)e Anti-nutritional factors (g kg1) 0.13 Trypsin inhibitor activityf Tanninsg 0.20 Phytic acid 3.22

D1

D2

D3

180 380 300 50 70 10 10

180 280 350 100 70 10 10

180 180 400 150 70 10 10

898.7

897.3

910.4

309.6 96.0 56.8 60.1 242.8 0.78 17.62

257.9 95.7 60.2 60.7 322.0 1.25 17.79

214.2 94.8 53.0 57.5 401.3 1.87 18.58

13.03

12.33

12.05

0.10 0.63 3.63

ND 1.59 2.97

ND 2.48 2.64

a

Vitamin premix and mineral premix were described in Azaza, Mensi et al. (2008), Azaza, Mensi et al. (2009), Azaza, Wassim et al. (2009). b CMC = carboxymethylcellulose. c Starch-to-protein ratios on mass basis (g g1). d Calculated using the factors: carbohydrates, 17.2 kJ g1; protein, 23.6 kJ g1 and, lipids, 39.5 kJ g1 (New 1987). e DE = (Crude protein content 9 23.6 9 0.9) + (Crude lipid content 9 39.5 9 0.85) + Starch content 9 17.2 9 0.5) (Morais et al., 2001). f Milligrams of pure trypsin inhibited per gram of sample; trypsin units are defined as a decrease in absorption of 0.01 at 400 nm. g As tannic acid equivalents. ND, not detected

© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–14


Prior to use, all feed ingredients were analysed for their proximate composition. Based on the nutrient composition of the ingredients, four isoenergetic diets differing in starch and protein levels were made by balancing the inclusion of SBM and CSM (Table 1). CSM was included at 50, 100 and 150 g kg1, as a partial substitution for SBM, and diets were designated as diets D1, D2 and D3 respectively. A control diet (D0) (350 g kg1 crude protein and 13.51 MJ kg1 digestible energy) which had previously been demonstrated to support good growth performance was formulated according to Azaza, Mensi, Abdelmouleh and Kraiem (2005). Before its use in the diets, SBM was autoclaved at 110°C for 30 min according to Azaza, Kammoun, Abdelmouleh and Kra€ıem (2009). The test diets were prepared as described by Azaza, Mensi, Ksouri, Dhra€ıef, Abdelmouleh, Brini and Kra€ıem (2008), Azaza, Mensi et al. 2009, Azaza, Wassim, Mensi, Abdelmouleh, Brini and Kraϊem (2009). All the dietary ingredients were ground (Ultra Centrifugal Mill ZM 200 Retsch GmbH, Haan, Germany), sieved to pass through a 0.25 mm sieve and mixed with a vitamin-mineral premix and soybean oil in a feed mixer (model: CAM A30, Retsch GmbH, Haan, Germany). All feeds were supplemented with 10 g kg1 carboxymethylcellulose as a binder. Water was added gradually until a desirable paste-like consistency was reached and then the mix was pelleted through 2.5 mm holes in a kitchen meat grinder (model: amb TC22SL, Oni2, Bruxelles, Belgium) and sun-dried. The dry pieces of feed were then stored in a freezer at 20°C, in polythene bags, until required. Before feeding, the dried diets were crushed and graded to yield suitable particle sizes according to fish size throughout the experiment following the model (The SGR-to-feed particle size relationship) developed by Azaza, Dhra€ıef, Kra€ıem and Baras (2010). The proximate biochemical analysis of the experimental diets (Table 1) was carried out for moisture, ash, crude protein, crude fat (ether extract) using standard AOAC (1990) methodology. Starch was determined by the glucose-amylase-glucoseoxidase method (Thivend, Mercier & Guilbot 1972). Carbohydrate (as NFE) was calculated as follows: Nitrogen-free extract: 1000 – (lipid g kg1 + moisture g kg1 + protein g kg1 + fibre g kg1 + ash g kg1). Digestible energy content was calculated using average conversion values of gross energy of 23.6 MJ kg1 for protein, © 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–14


39.5 MJ kg1 for lipid and 17.2 MJ kg1 for carbohydrate and average digestibility values of 90% for protein, 85% for fat and 50% for carbohydrate (Morais et al., 2001). Gross Energy was calculated based on the conversion factors: carbohydrate (as NFE): 17.2 MJ kg1, protein: 23.6 MJ kg1 and fat: 39.5 MJ kg1 (New 1987). The total saponin content was determined using the method of Hiai, Oura and Nakajima (1976). Tannins were determined using the spectrophotometric methods described by Aganda and Mosase (2001) after extraction with organic solvent. Phytic acid was determined by a spectrophotometric method described by March, Villacampa and Grases (1995) after acid extraction (3% H2SO4) and enzymatic hydrolysis (phytase of Aspergillus ficum, Sigma Aldrich P-9792, 1.1 unit mg1). Trypsin inhibitor activity (TIA) was measured by the enzymatic method of Bergmeyer (1965) with N-benzoylarginine-p-nitroanilide (BAPNA) as substrate. TIA was expressed as trypsin units inhibited per mg of dry sample. All analyses were performed in triplicate. Fish and experimental conditions The nutrition trial was carried out at the National Institute of Marine Sciences and Technologies (INSTM), Fish-culture Research Station, BechimaGabes, Tunisia. 600 six-week-old uniform-sized fish (2.7 0.1 g; SE, n = 600) were sampled. For sampling procedure, fish were sedated with tricaine methanesulphonate (50 mg L1), weighed individually to produce 12 groups in which mean size and size heterogeneity were as similar as possible. Thereafter, the groups of fish were randomly allocated to 12 cylindro-conical fibreglass tanks (120 L) with 50 fish per tank. Each tank was part of an open circulated system previously described by Azaza, Dhraief and Kraiem (2008), Azaza, Mensi et al. (2008) and by Azaza, Mensi et al. (2009), Azaza, Wassim et al. (2009). The tanks were supplied with water coming from a geothermal source after undergoing a cooling process in a large tank. In all 12 tanks, water was constantly equally replaced by continuous flow at a rate of 3–5 L min1 tank1. Fish were acclimated to experimental conditions for one week prior to the feeding trial. During this period, they received a mixture of the different experimental diets using automated belt feeders (12 h day1). During the acclimation period, all

3


dead or apparently stressed fish were replaced with individuals of similar sizes. At the start of the feeding trial, each experimental diet was assigned randomly to three tanks. Fish were hand-fed to apparent satiety four times daily during the week (08:00, 11:00, 14:00 and 17:00 hours) and twice daily on weekends (09:00, 12:00 hours). Pellets were distributed slowly, allowing all fish to eat without feed wastage. Apparent satiation was considered achieved when the fish would no longer accept the offered feed after a period of active feeding. Daily feed consumption, for each tank, was measured by weighing the feed at the start and at the end of each day. Tanks were searched for dead fish twice a day, before and after the period of feed distribution. Dead fish (if any) were weighed to calculate feed conversion ratio (FCR). The tanks were syphoned daily, before the first feeding, to remove faecal material and they were thoroughly scrubbed and completely flushed fortnightly, when fish were removed for weighing to monitoring growth. As indicated above, to monitoring the growth and general health condition, all fish in each tank were individually weighed fortnightly during the 45-days trial. Fish were captured with a dipnet, sedated with 50 mg L1 tricaine methanesulfonate, individually weighed (nearest 0.01 g) and returned to their tank. On the weighing days, feed distribution was suspended during the morning, and resumed in the early afternoon, at least 3 h after the last fish were measured, to enable all fish to recover from handling stress and to minimize the effects of handling on feed intake. For water quality monitoring, water temperature, dissolved oxygen and pH levels in each tank were recorded automatically (every hour) with a digital thermo-oxymeter surveillance system (WTW, MIQ/C184, www.memecosales.com; accuracy of 0.1°C and 0.1 mg O2 L1). Total ammonium and nitrite were measured twice weekly using standard methods (APHA 1995). Water temperature was maintained at 28 1°C throughout the experiment, which corresponds to the thermal optimum for growth (T°opt) of Nile tilapia Maryut strain (Azaza, Mensi et al. 2008, Azaza, Dhraief et al. 2008). No critical values were detected for dissolved oxygen (>4.02 mg L1). Nitrite (NO2-N: 0.05) difference in feed intake among all treatments. Compared with the control diet, only the growth performance (SGR and TGC) of groups fed the lowest protein and highest starch diet (D3; STA/ P = 1.87) was significantly (P < 0.05) lower than of those fed the other diets. In fact, dietary protein can be reduced from 344 to 258 g kg1 diet by increasing starch from 163.5 to 401.3 g kg1 diet with satisfactory growth performance. FCR tended to increase with the increase in STA/P ratios. However, these tendencies were not statistically significant for diets D0, D1 and D2. Protein efficiency ratio (PER) was dependent on dietary starch to protein ratios level, and increased with the increase in STA/P ratios. Indeed, Fish fed the diets D2 (258 g kg1 crude protein and 322 g kg1 of starch; STA/P = 1.25) and D3 (214 g kg1 crude protein and 401 g kg1 of

6

starch; STA/P = 1.87) exhibited higher PER (P < 0.05) comparedwith fish fed the feeds containing D0 (344 g kg1 crude protein and 163.5 g kg1 of starch; STA/P = 0.48) and D1 (309 g kg1 crude protein and 242.8 g kg1 of starch; STA/P = 0.78) (Table 2). The HSI value observed in fish fed diet D3 (containing the higher starch and the lower protein) was significantly higher than that found in fish fed any other diet (P < 0.05) (Table 3). Likewise, VSI increased significantly in groups fed on diets D2 and D3 compared with other diets. Intraperitoneal fat ratios (IPFRs) of fish fed on diet D3 (starch/P ratio of 1.87) were significantly higher (P < 0.05) than those fed the other experimental diets (2.4% versus 1.6, 1.4 and 1.9 for diets D0, D1 and D2 respectively (Table 3). Carbohydrate metabolic enzymes The activities of hepatic enzymes are presented in Table 4. There was a significant positive correlation between the activities of glycolysis (Pyruvate kinase; PK and 6-phosphofructokinase; PFK-1) and lipogenesis enzymes (Glucose-6-phosphate dehydrogenase; G6PD and 6-phosphogluconate dehydrogenase; 6PG-DH) and dietary starch levels. In fact, compared with the control diet, the activities of these enzymes in the liver were significantly increased in fish fed diet D3 for the G6PD and PFK-1 enzymes. Similarly, for the 6PG-DH and PK in fish fed on diets D2 and D3. For the gluconeogenesis enzyme analysed (i.e. Fructose 1, © 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–14



Table 3 Slaughter variables of juvenile Nile tilapia fed the experimental diets at the end of the growth trial Experimental diets Variables

D0

D1

HSI (% BM) VSI (% BM) IPFR (% BM)

2.7 0.16 6.9 0.52a 1.6 0.65a

D2

2.6 0.14 6.8 0.94a 1.4 0.14a

a

D3

3.0 0.15 7.9 0.48b 1.9 0.48a

a

3.2 0.12b 7.8 0.71b 2.4 0.62b

a

Values are means standard error (n = 30 fish per treatment; 10 from each replicate) in the same row with different superscripts are significantly different (Fisher’s LSD test, P < 0.05). IPFR = Intraperitoneal fat ratios; HSI = Hepatosomatic index; VSI = Viscerosomatic index.

Table 4 Hepatic enzyme activities (G6PD, 6PG-DH, PFK-1 and PK) of juvenile Nile tilapia fed experimental diets for 45 days and regression equations for the relationships between dietary starch content (g kg1) and enzyme activity Diets Enzyme

D1

D0

D2

D3

Regression equation

rs

P-value

Glucose-6-phosphate dehydrogenase (G6PD) 0.7 0.10a 0.6 0.08a IU mg protein1 IU g liver1 36.5 3.06a 44.7 2.17b 69.1 8.35a 86.7 6.92b IU 100 g fish1

0.7 0.09a 63.7 4.02c 92.4 6.06c

1.1 0.09b 88.8 3.18d 143.5 9.87d

Y = 0.30x + 0.44 Y = 38.11x + 16.67 Y = 50.43x + 42.67

0.715 0.995 0.921