Effect of Dietary Inositol on Growth, Feed Utilization and Blood Biochemical Parameters for Juvenile Barramundi (Lates calcarifer Bloch)

Problem statement: The utilization of inositol was detected for growth performance and erum biochemical parameters of juvenile barramundi (Lates calcarifer ). Approach: A 56 day feeding trial was conducted to evaluate the effect of inosi tol on growth, feed utilization and serum biochemic al parameters for juvenile barramundi Lates calcarifer (initial size 5.51±0.07g). Six experimental fish meal-based isonitrogenous (42% crude protein) and i solip dic (10% crude lipid) diets containing levels of inositol (350, 364, 458, 507, 720, 1050 mg kg −1 diet) were formulated. Results: Fish fed diet containing inositol 507 mg kg diet had the significantly highest weight gain (WG , %) among all the groups (p<0.05) and had significantly lower Feed Conversio n Ratio (FCR) than fish fed the diet containing inositol 458 mg kg diet (p<0.05). Survival and Hepatosomatic Index (HS I) were not significantly affected by graded levels of dietary inositol (p>0. 05) However, significantly lower Viscerasomatic In dex (VSI) was found in fish fed diet without supplement al inositol (p<0.05). Dietary inositol levels did n ot affect whole body moisture, crude protein and lipid contents (p>0.05). Total protein and triacylglycer ol in serum increased with increasing dietary inositol le vels up to 507 mg kg −1 diet (p<0.05). The significantly lowest blood urea nitrogen was found in fish fed di etary inositol 458 mg kg −1 diet among all groups except for 720 mg kg −1 diet. Total Cholesterol (TC) of fish fed dietary i nositol 507-1050 mg kg −1 diet was higher than those of fish fed dietary inositol 350458 mg kg diet. Conclusion: Results of the present investigation demonstrated significant improvement of growth and feed utilization of juvenile barramun di can be achieved by inositol supplementation at 507 mg kg diet.


INTRODUCTION
Barramundi (Lates calcarifer) is native to the Indo-Pacific region (Glencross, 2006) and an economically important species in Southeast Asian countries (Tantikittia et al., 2005). It is a carnivorous species which can be reared in marine water, brackish water, or freshwater (Harpaz et al., 2005). Glencross (2006) reviewed barramundi nutrition research included the requirements for most nutrients, energy demand and ingredient utilization. Recent research indicated that lupin protein (Katersky et al., 2009) and defatted soybean protein (Tantikittia et al., 2005) were suitable protein sources for barramundi feeds. Williams et al. (2006) also indicated that a dietary n-3 HUFA was required for rapid growth and efficient feed conversion of juvenile barramundi. In our laboratory, research has focused on determining effects of dietary Bacillus licheniformis  and Chinese herbal medicines

on blood biochemical indices in cultured Lates calarifer.
Inositol is widely distributed in plants and animals (Peres et al., 2004), which is classified as a vitamin-like nutrient (Shiau and Su, 2005) and is an essential dietary ingredient for most aquatic animals (Michael and Koshio, 2008). The requirement of dietary inositol has been reported by Li et al. (2001), Shiau and Su (2005) and Wen et al. (2007) in various aquatic animals. Dietary inositol also affected blood chemistry in fish (Waagbo et al., 1998;Wen et al. 2007). Certain dietary vitamins have been identified as essential for barramundi; however, it is difficult to define the requirement for all essential vitamins because of poor acceptance of a purified diet by barramundi (Glencross, 2006).
The purpose of this study was to evaluate the influence of the dietary levels of inositol on growth performance, feed utilization and serum biochemical parameters in juvenile barramundi.

MATERIALS AND METHODS
Experimental diets: Diets were formulated to be grossly isonitrogenous (crude protein, 43%) and isolipidic (crude lipid, 12%). The experimental diet formulation and proximate are shown in Table 1. Inositol (Sigma) was added to the test diets at a concentration of 0, 50, 100, 200, 400 and 800 g kg −1 diet. All ingredients were mechanically mixed, pressure-pelleted using a laboratory pelleting machine through a 2.5 mm die. The pellets were air-dried at room temperature to a moisture content of about 10% and stored in a freezer at -20°C until used.
Experimental fish and feeding: The trial was conducted at an experimental station of South China Sea Fisheries Research Institute of CAFS (Sanya, Hainan). Barramundi were obtained from a private commercial hatchery and acclimated to the laboratory conditions for 2 weeks. At the end of the acclimation period, a total of 540 fish (mean initial weight 5.51±0.07 g) were randomly distributed into each of 18 500 L cylindrical plastic tanks. Each experimental diet was randomly assigned to triplicate tanks. Tanks were supplied with filtered seawater with a flow rate of approximately 2 L min −1 in a flow-through system with aeration. The fish were fed with the respective diet to apparent satiation twice a day at 0800 and 1700 h. During the trial, the water temperature was maintained ant 29.0±2.0°C and salinity was 28±0.3 g L −1 . Dissolved oxygen ranged from 5.10-6.93 mg L −1 . The experimental units were under a natural light and dark cycle. The experiment lasted for 56 days.
Sampling: At the conclusion of the 8-week period, the fish were deprived for 1 day prior to sampling, then counted and batch weighed to determine survival, Final Body Weight (FBW), Weight Gain (WG), Feed Conversion Ratio (FCR). Five fish from each tank were randomly collected, killed for the determination of the Hepatosomatic Index (HSI) and Viscerasomatic Index (VSI). Blood was taken by puncturing the caudal veins immediately after catching.
Chemical analysis: Crude protein was determined by the Kjeldahl method using a Kjeltec 2200 Auto Distillation Unit (Foss Tecator AB, Switzerland). Crude lipid was determined by the ether-extraction method using a Soxtec Avanti 2050 (Foss Tecator AB, Switzerland). Moisture and ash were determined using the standard methods (AOAC, 1984). The inositol concentration in the diets was determined using an enzymatic assay as described by Ashizawa et al. (2000).
The standard methods on human were used to determinate biochemical parameters (Rehulka, 2000). The serum was collected by centrifuging blood samples at 3000 rpm for 10 min. The biochemical indices of the serum were determined within 24 h of storage at 4°C. A Beckman LX20 automatic biochemical analyzer (Harbor Blvd., Fullerton, USA) was used for the determinations. These included Total Protein (TP), Blood Urea Nitrogen (BUN), Total Cholesterol (TC) and Triacylglycerol (TG).

Statistical analysis:
The data were subjected to oneway Analysis Of Variance (ANOVA). If significant (p<0.05) differences were found, Duncan's multiple range test was used to rank the groups using the SPSS program Version 13.0 for Windows (SPSS Inc., Michigan Avenue, Chicago, IL, USA).

Survival and growth parameters:
The fish growth performance of fish fed various dietary inositol was shown in Table 2. Weight gain was the significantly highest for barramundi fed the diet containing inositol 507 mg kg −1 diet among all the groups (p<0.05). FCR of fish fed the diet containing inositol 507 mg kg −1 diet was significantly lower than the fish fed the diet containing inositol 458 mg kg −1 diet; however, no significant difference was found compared to the other groups. Dietary inositol did not significantly affect survival (p>0.05). Hepatosomatic index was not significantly affected by graded levels of dietary inositol (p>0.05). However, significantly lower viscerasomatic index was found in the fish fed diet without supplemental inositol (p<0.05).

The proximate composition of the fish whole body:
The whole-body composition of fish fed diets containing graded levels of inositol is presented in Table 3. The moisture content of the whole body averaged 71.0%.; the crude protein and lipid contents averaged 17.5 and 7.0%, respectively. The moisture, crude protein and lipid contents of the fish whole body was not affected by inositol levels (p>0.05).

DISCUSSION
The importance of dietary inositol for normal growth of juvenile Lates calcarifer maybe demonstrated in the present study. Although dietary inositol did not significantly affect survival (p>0.05), weight gain was the significantly highest for barramundi fed the diet containing inositol 507 mg kg −1 diet among all the groups (p<0.05). Glencross (2006) reviewed that no differences in growth, feed conversion or survival were observed when barramundi were fed practical diets added inositol. On the other hand, inositol was required for its normal growth using semipurified diets. Weight gain increased for fish fed dietary inositol up to the requirement level. The optimal dietary inositol requirement for the maximum growth of juvenile Jian carp (Cyprinus carpio) was estimated to be 518.0 mg kg −1 diet (Jiang et al., 2009); juvenile tilapia (Oreochromis niloticus × Oreochromis aureus) required 400 mg kg −1 diet (Shiau and Su, 2005); juvenile flounder Paralichthys olivaceus required 800-1200 mg kg −1 diet (Li et al., 2001) and Lateclabrax japonicus reqired 500 mg kg −1 diet (Zhong and Zhang, 2001); however, the suitable requirement for dietary inositol of growing grass carp (Ctrnopharyngodon idella) was approximately 166-214 mg kg −1 diet (Wen et al., 2007). Waagbo et al. (1998) reported that no real effect of dietary inositol supplementation on growth and mortality in Atlantic salmon (Salmo salar) feeding practical diets. Moreover, juveniles Nile tilapia (Oreochromis niloticus) (Peres et al., 2004) and sunshine bass (Morone chrysops♀ × Morone saxatilis♂) (Deng et al., 2002) fed puried diets did not require an exogenous source of inositol for normal growth and feed utilization. Dietary inositol did not significantly affect survival (p>0.05). This maybe practical diet contain sufficient levels of inositol to meet various metabolic needs of fish because this vitamin is widely distributed in common feed ingredients (Peres et al., 2004).
Hepatosomatic weight were higher at high inositol levels compared with the unsupplemented diet maybe due to inositol increased the promotion of liver development (Jiang et al., 2009). In the present study, the results showed that HSI of barramundi was not affected by dietary levels of inositol. This result demonstrated that high inositol levels had no potential to affect the health of fish. It was also found that dietary inositol did not affect HSI of in Atlantic salmon (Waagbo et al., 1998), sunshine bass (Deng et al., 2002) and Nile tilapia (Peres et al., 2004).
The significantly higher accumulation of lipid in liver and muscle of fish could be an indication of inositol deficiency (Peres et al., 2004). In the present study, no significant difference was found in the moisture, crude protein and lipid contents of the fish whole body. This is also consistent with studies on Atlantic salmon (Waagbo et al., 1998), grass carp (Wen et al., 2007) and Kuruma shrimp (Marsupenaeus japonicus) (Michael and Koshio, 2008). However, in Jian carp (Jiang et al., 2009), whole body moisture was not affected by inositol levels (p>0.05), but body protein increased significantly with increasing dietary inositol levels up to 384.2 mg kg −1 diet (p<0.05) and body fat was the highest for fish fed diets containing 838.8 and 990.3 mg kg −1 diet and the lowest for fish fed the unsupplemented diet (p<0.05).
Good state of health for fish is the basic precondition for successful fish culture. Monitoring the physiological state of fish has become an integral part of the routine examination of fish health (Rehulka, 2000). Apparently any adverse effects were not found on the state of the fish health in this study. However, differences were observed in serum parameters of cultured barramundi among dietary groups. Dietary herbs  and B. licheniformis ) could change some blood hematological and blood biochemical indices of the fish. Serum TP levels can be used as a diagnostic tool and a valuable test for evaluating the general physiological state in fish (Pedro et al., 2005). In the present study, TP increased with increasing dietary inositol levels up to 507 mg kg −1 diet (p<0.05). Significantly low levels of total plasma protein were reported for some infected fish (Benli and Yildiz, 2004;Yildiz and Aydin, 2006;Rehulka and Minarik, 2007); meanwhile, clinical chemistry analyses in blood plasma indicated the decreased levels of total protein (Rehulka and Minarik, 2007). The significantly lowest Blood Urea Nitrogen (BUN) was found in the fish fed dietary inositol 458 mg kg −1 diet among all the groups except for the 720 mg kg −1 diet. The increase of BUN may reflect kidney dysfunction  and its increased levels generally signal a higher rate of conversion of nitrogen compounds (Rehulka and Minarik, 2003). The TC of fish fed dietary inositol 507-1050 mg kg −1 diet was higher than those of the fish fed dietary inositol 350-458 mg kg −1 diet, but no significant difference was observed (p>0.05). In grass carp (Wen et al., 2007), TC was significantly lowest for fish fed the unsupplemented diet (p<0.05). However, the total protein and cholesterol concentrations in plasma were not significantly affected by dietary inositol (Waagbo et al., 1998). TG increased with increasing dietary inositol levels up to 507-720 mg kg −1 diet (p<0.05) in this study. This result did not agree with Waagbo et al. (1998) for Atlantic salmon and Wen et al. (2007) for Ctrnopharyngodon idella, which plasma TG was negatively correlated to dietary inositol supplementation.

CONCLUSION
Results of the present investigation demonstrate significant improvement of growth and feed utilization of juvenile barramundi can be achieved by inositol supplementation at 507 mg kg -1 diet.