The omnivorous Nile tilapia (Oreochromis niloticus) is an important commercially farmed species worldwide due to characteristics such as strong disease resistance, fast growth and the ability to accept lower cost diets with terrestrial-based ingredients (Ng and Romano, 2013).
Fish production costs are increasing while fish price in the market is relatively stable (Tacon and Metian, 2015). Increased price of fish feed is one of the main reasons for increasing production cost as feed constitute 50%–70% of the operational cost in aquaculture. This creates pressure to develop new strategies to reduce the cost of feeding aquaculture-produced fish by improving efficiency and developing cheaper feeds. Formulating feeds with reduced digestible protein to digestible energy (DP:DE) ratios is a way to reduce feed costs (NRC, 2011), which has been practically demonstrated for tilapia (Kabir et al., 2019a; Kabir et al., 2019b). While reducing the protein to energy ratio, the non-protein energy in the diet increases. Non-protein energy can come either from lipid or carbohydrate. Energy sources including proteins, lipids (L) and carbohydrates (CHO) are the major expenditure in formulated feeds in aquaculture. In comparison with terrestrial animals, fish need more protein, which is the most expensive nutrient ingredient, to maintain normal growth. High protein content is usually associated with uneconomical feed cost. Therefore, reducing dietary protein supplementation and maximizing protein utilization in fish is an effective way to reduce formulated diet cost (NRC, 2011). Carbohydrate and lipid are the major non-protein energy sources in fish diets. To reduce dietary protein levels, much attention was given to analyze the feasibility of non-protein energy substitutes, and proved that providing adequate energy with dietary lipid and carbohydrate can minimize the use of costly protein and increase feed efficiency (Darias et al., 2015). However, excess dietary lipid or carbohydrate may impair lipid homeostasis, and lead to excessive lipid accumulation in the liver of farmed fish, accompanied by poor growth, low survival and weak resistance to pathogens (Zamora-Sillero et al., 2013; Li et al., 2016). Carbohydrates and lipids are two critical nutrients in fish nutrition, playing plenty of roles in fish growth, development, and immunity (Dong et al. 2018). Depending on the doses and the chemical composition, adequate dietary carbohydrate and lipid (CHO:L) are essential to play roles in sparing effect on protein (Gao etal. 2010). At present, most studies have shown that optimal feed CHO:L ratios can improve growth, feed efficiency, reduce the excretion of ammonia and nitrogen, and reduce pollution to the breeding environment ( Dias et al. 2004; Gao et al. 2010). The aim of this study was to evaluate the effect of four dietary inclusion levels of crude carbohydrate and lipids on growth performance, body composition and nutrient utilization of Nile tilapia (Oreochromis niloticus) fingerlings.
MATERIALS AND METHODS
Two hundred and forty Nile tilapia fingerlings with an average body weight (10g ±0.2g) were obtained from Fish Research Center, Faculty of Agriculture, Suez Canal University-Egypt. Fish were acclimated to laboratory conditions for 2 weeks before being randomly divided into four equal experimental groups (20 fish each treatment, three replicate/tanks,) representing four nutritional groups. The experimental fish were weighted every 15 days in order to adjust the daily feed rate which was 3 %of the total biomass at three times/ day (8.30, 12.30, and 4.30 pm) for 90 days.
The present study was conducted in the Fish Research Center, Faculty of Agriculture, Suez Canal University -Egypt. The experimental fish were stocked in 12 circle fiber glass tanks (380L) supplied with fresh water through a closed recycling system. Tank water was aerated continuously by using an air compressor. Water flow rate was maintained at approximately 1.5L/min. Photoperiod was 12h light/ 12h dark. Water temperature was maintained at (27 ±1oC) by using a 250- watt immersion heater with thermostat. Water temperature and dissolved oxygen were recorded daily (by metteler Toledo, model 128.s/No1242) where the average range of dissolved oxygen was above 5.8 mg/l. Other water quality parameters including pH and ammonia were measured every two days by pH meter (Orion model 720A, s/No 13062) and ammonia meter (Hanna ammonia meter), where the average range of total ammonia was 0.12 - 0.23 mg/l and pH was in range of 7.2 ± 0.5 during the experiment.
Four isonitrogenous (30% Crude protein) and Soybean oil was used as a source of lipid, four lipid levels (6, 8.10 and 12% lipid), the diets were formulated from practical ingredients (Table1). The experimental diets were formulated to contain almost 30% crude protein. The experimental diets were prepared by individually weighing of each component and by thoroughly mixing the mineral, vitamins and additives with corn. This mixture was added to the components together with oil. Water was added until the mixture became suitable for making granules. The wet mixture was passed through CBM granule machine with 2mm diameter. The produced pellets were dried at room temperature and kept frozen until experimental start.
At the end of the 10 weeks, 12 h after the last feeding, fish in each tank was anesthetized with eugenol (1:10,000), counted, and weighed to determine survival, weight gain rate (WG), specific growth rate (SGR), and feed conversion ratio (FCR). Five fish were randomly removed from each replication Tank; liver and viscera were dissected and weighed, respectively, to obtain hepatosomatic index (HSI) and viscerasomatic index (VSI). After then, the back muscles were separated and stored in the − 80 °C refrigerator for composition analysis and glycogen content. Then, seven fish were randomly taken from each Tank to collect blood in the fish tail vein. The collected blood samples were placed in a 1.5-mL anticoagulation tube, allowed to stand at room temperature for 4 h, and then centrifuged (3500×g, 10 min, 4 °C), and the collected plasma was stored in a − 80 °C refrigerator for plasma biochemical measurements. After that, fish was stored in the refrigerator at − 20 °C for whole-body composition analysis. Finally, three fishes were randomly selected again to weight and separate the liver, stomach, and proximal intestine tissues. Liver, stomach, and intestine tissues are stored in − 80 °C refrigerator for measuring enzyme activities and liver glycogen content.
The tested diets were analyzed for crude protein (CP %), ether extract (EE %), crude fiber (CF %), ash (%) and moisture while whole body composition of fish samples were also analyzed except crude fiber (CF %) and whole body according to the procedures described by standard AOAC methods (1995). The nitrogen free-extract (NFE %) was calculated by differences. Blood sample was collected using heparinized syringes from caudal vein of the experimental fish at the termination of the experiment. Blood was centrifuged at 3000rpm for 5 minutes to allow separation of plasma which was subjected to determination plasma biochemical indices were determined by an automatic biochemistry analyzer (Hitachi 7020, Tokyo, Japan).
The liver and muscle, glycogen content were determined by a commercial kit (provided by Nanjing Jiancheng Biological Engineering Institute, China), and the corresponding operation was conducted according to the manufacturer’s instructions.
Calculation formula and statistical analysis
Weight gain rate (WG, %) = 100 × (final body weight (g) − initial body weight (g))/initial body weight (g)
Specific growth rate (SGR, %) = 100 × [ln final body weight (g) − ln initial body weight (g)]/days of experiment
Feed conversion ratio (FCR) = feed intake (g)/(final body weight (g) − initial body weight (g))
Protein efficiency ratio (PER) = fish weight gain (g)/fish protein intake (g)
Survival (%) = 100 × final fish number/initial fish number
Condition factor (CF, %) = 100 × body weight (g)/body length3 (cm)
Hepatosomatic index (HSI, %) = 100 × hepatic weight (g)/body weight (g)
Viscerosomatic index (VSI, %) =100 × viscera weight (g)/final body weight (g)
All data were analyzed by one-way analysis of variance (ANOVA) using the general linear models procedure of statistical analysis system (SPSS) version 8.02.(2002) Duncan's multiple range test (Duncan, 1955) was used to resolve differences among treatment means at 5% significant level
RESULTS AND DISCUSSION
The growth performance parameters of Nile tilapia (Oreochromis niloticus) fingerlings which fed diets congaing four carbohydrate and lipid are shown in Table (2). Average of initial body weight of Nile tilapia fingerlings fed the experimental diets at the start did not differ, indicating that groups were homogenous. At the end of the experimental period (90 days),
7.64, 5.60, 4.04, 3.60
The growth performance were significantly affected by dietary CHO:L ratios (P < 0.05), and the fish fed with diets D2 had a significantly (P<0.05) highest BWG and SRG than those fed with diets D2, D3 and D4 group (P < 0.05) (Table 2). Similarly, better FCR were detected in the D2 had a significantly (P < 0.05) lowest FCR thanD1, D3 and D4 groups respectively (Table 2). The overall total feed consumption by Oreochromis niloticus was affected by the variability in CHO:L ratios. This indicated that both carbohydrates and lipids were equally acceptable by fish within the levels used on an energy equivalent basis in this experiment. As far as the carbohydrate and lipid levels in fish diets are concerned, our results were in close agreement with the findings of Teshima et al., (1985) and El-Sayed and Garling (1988). The increase in FCR values with the decrease in the CHO:L ratio indicates the poor utilization of diets when the dietary lipid level increases over 12%. Similarly, the efficiency of protein utilization decreased when the CHO:L ratio in the diets dropped to 4.04 and 3.60. These results indicate that O. niloticus is capable of best utilizing lipids up to a level of 8% with a minimum level of carbohydrates (47.10%) in their diets. Lowering the carbohydrate level beyond this limit with a simultaneous increase in lipid level, even on an energy equivalent basis, not only affected their growth performance but also the overall efficiency of energy and protein utilization. Fish fed low lipid high carbohydrate diets might metabolize less protein to meet their energy needs than fish fed high lipid low carbohydrate diets, resulting in higher dietary protein retention in tissues. Our results are also in line with the findings of Erfanullah and Jafri (1998) they reported that the highest weight gain, SGR, FCR, PER, and FE values were observed in fish fed 47.10% dietary carbohydrates and 8% lipids corresponding to a CHO:L ratio of 5.60. Tilapia fed low lipid high CHO:L ratio diets metabolized less protein to meet their energy needs than fish fed the diets with low CHO:L ratio in diets, resulting in higher dietary protein retention in tissues. In agreement with Meurer et al., (2002). Improvement of growth and PER with increasing dietary energy level at constant dietary protein level was found for rainbow trout (Beamish and Medland, 1986) and white sea bass (Lopez et al., 2009).
A number of studies have shown that most cultured fish can efficiently utilize both carbohydrates and lipids to achieve better growth (Li et al., 2010, 2012; Darias et al., 2015). In this study, high SGR values were presented in tilapia fed modest CHO: L ratio diets (CHO: L ratio ranged from 1.95 to 6.40). some studies in Nile tilapia, acceptable dietary CHO: L ratios were reported to range from 2.06 to 4.95 (the highest dietary CHO: L ratios of 4.95 was supplemented) (Ali and Al- Asgah, 2001). Similarly, an appropriate dietary CHO: L ratio can improve growth performance in other fish species, such as African catfish (Clarias gariepinus) (Ali and Jauncey, 2004), yellowfin seabream (Sparus latus) (Hu et al., 2007), yellow catfish (Pelteobagrus fulvidraco) (Wang et al., 2014), dourado (Salminus brasiliensis) (Moro et al., 2015), golden pompano (Trachinotus ovatus) (Zhou et al., 2015), large yellow croaker (Larmichthys crocea) (Zhou et al., 2016). While, excessive dietary carbohydrate or lipid (D3 diets and D4 diets) directly depressed the growth rate of fish (Moro et al., 2015; Zhou et al., 2016). Therefore, a balance between dietary carbohydrates and lipids must be met, in which carbohydrates are used as a cheaper energy source to enhances the diet's palatability and maximize growth, while lipids are used to satisfy the requirements for essential fatty acids (EFA) in fish (Ng and Romano, 2013). . Both HSI and VSI were significantly affected by dietary CHO: L ratios (P < 0.05). In particular, fish fed with diets D2 had a significantly (P<0.05) highest HSI and VSI than those fed with diets D1, D3 and D4 respectively (Table 4).In agreement with Hilton and Atkinson (1982) and Hanley (1991).
Proximate composition and glycogen content in tissue
No significant differences were observed in moisture, protein and ash content of whole body proximate composition among all treatments (Table 2). However, significant differences (P<0.05) were detected in crude lipid content between the fish fed with low-CHO: L ratios diets (D2 and D1).
Both HSI and VSI were significantly affected by dietary CHO: L ratios (P < 0.05). In particular, fish fed with diets D4 had significantly higher HSI and VSI than those fed with diets D1 and D2 (P < 0.05) (Table 3).In agreement with Hilton and Atkinson (1982) and Hanley (1991). Also no significant (P<0.05) differences were observed in moisture, protein and ash content of whole body proximate composition among all treatments (Table 3). However, significant differences were detected in crude lipid content between the fish fed with low-CHO: L ratios diets D1 and D2, D3 and D4 respectively. Li et al., (2010) recorded that, the moisture content, lipid content of whole body and carcass of blunt shout bream (Megablorama amblycephala) increased significantly (p<0.05).
Liver and muscle glycogen content of Nile tilapia increased gradually with the increase of dietary CHO: L ratios (Table 3). The liver and muscle glycogen content of D1 group was significantly highest than that of the other four groups (P < 0.05). The liver and muscles are the main parts where glucose is stored in the form of glycogen (NRC 2011). The dietary carbohydrate enters the liver and blood in the form of glucose after digestion and absorption (Kamalam et al., 2017). When the carbohydrate level of the diet is high, the glycogen synthesis of the liver will increase (Hu et al., 2018). In this experiment, the glycogen content in liver and muscle, HSI, and VSI in Nile tilapia increased gradually as the level of dietary CHO: L ratios increased. These results indicate that the high carbohydrate in the diet of Nile tilapia will be converted into glycogen and deposited in the liver. Also, some of these carbohydrates are converted to lipid, which is deposited in the viscera and muscles, according to crude lipid content in body and muscle. Similar results were observed in cobia (Rachycentron canadum) (Ren et al., 2011), sea cucumber (Xia et al., 2015), white sturgeon (Acipenser transmontanus) (Deng et al., 2001), sunshine bass (Morone chrysops ( Morone saxatilis )Keembiyehetty and Wilson 1998), grouper (Wang et al., 2016), Nile tilapia (Boonanuntanasarn et al., 2018),
Plasma biochemical parameters
Serum TG, CHO, and HDL levels were significantly (P < 0.05) affected by dietary CHO: L ratios (Table 4). TG, CHO, and HDL values of fish fed diets with CHO: L ratios higher than 4.04 were significantly lower than those of the other groups (P < 0.05). Whereas no significant difference (P<0.05) in plasma GLU and LDL levels was observed among treatments (P > 0.05). The ingredients of feed nutrition do not only affect the growth of fish but also the physiological conditions, such as plasma biochemical parameters, tissue physiology and biochemical indexes (Wang et al., 2014). However, the plasma cholesterol and triglyceride levels decreased with increased dietary CHO: L ratios, which were consistent with those reported in yellowfin seabream, yellow catfish, large yellow croaker and hybrid snakehead (Zhou et al., 2016; Zhang et al., 2017). Plasma profiles reflect general metabolism and physiological status in tissues, especially in the liver, which is involved in nutrient homeostasis. Consistently, the HIS, hepatic lipid content and droplet levels showed significant difference among the fourth groups, but all increased as the dietary CHO: L ratios rose. This conclusion is supported by the fact that lower plasma triglyceride, cholesterol and HDL levels were observed in fish fed diets with higher CHO: L ratios, as cholesterol and HDL are used to evaluate the activity of endogenous lipid transport (Wang et al., 2014; Zhou et al., 2016). Various studies also indicated that excess lipid accumulation is highly correlated with high dietary carbohydrate levels in fish (Azaza et al., 2015; Li et al., 2016). These results indicated that carbohydrate, like lipid, could be utilized as a promoter of hepatic lipid deposition in tilapia. Lipid deposition is a complex process involving lipid transport, uptake, synthesis and catabolism. Although lipids and carbohydrates are available for fish, lipid metabolism is reported to be highly modulated by dietary carbohydrates or glucose load (Moro et al., 2015; He et al., 2015; Zhou et al., 2016).
As shown in Table 4, different dietary CHO: L ratios could significantly affect plasma biochemical indexes (P < 0.05). Plasma glucose (GLU), cholesterol (CHOL), triglyceride (TG), and total protein (TP) increased with the increase of carbohydrate level. GLU and TG reached the maximum value in D1 group, while CHOL reached the maximum value in D1 group. Similar results obtained by Li et al,.( 2019). Plasma biochemical parameters are known to vary depending upon the nutritional status of fish (Tian et al., 2012). Blood glucose is the primary transport mode of carbohydrate in animals, generally in dynamic balance, but vulnerable to external stimuli and changes (Enes et al. 2009). TG and CHOL are the forms of lipids present and transported in the blood, and their content represents the metabolism of lipids in the body (Hu et al. 2018).
In the present study, the plasma GLU, CHOL, and TG level of Nile tilapia increased significantly as the dietary CHO:L ratios increased, indicating that glucose and lipid transport became more active in response to the higher dietary carbohydrate level. This is in agreement with the results reported for Nile tilapia (Boonanuntanasarn et al. 2018), European sea bass (Castro et al. 2015), and rainbow trout (Kamalam et al. 2013). These results indicated that carbohydrate, like lipid, could be utilized as a promoter of hepatic lipid deposition in tilapia.
It could be concluded that an optimal dietary CHO: L ratio of 5.60 is suitable for tilapia culture concerning the growth performance and health. The present results will be of great significance in improving the utilization of dietary lipid and carbohydrate to achieve better nutrition efficiency in tilapia.