The Israeli Journal of Aquaculture - Bamidgeh, IJA_64.2012.868, 10 pages
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Effect of dietary eicosapentaenoic acid/docosahexaenoic acid and vitamin E on the reproductive performance and gonadal fatty acid composition of Betta splendens Sagar C. Mandal1*, S. Khogen Singh1, Pronob Das2, Mohd Ashraf Rather3* & Debtanu Barman4 1
College of Fisheries, Central Agricultural University (I), Lembucherra, Tripura (W)799210, India 2 Central Inland Fisheries Research Institute, Beltola, Guwahati-6, India 3 Central Institute of Fisheries Education, Versova Mumbai-61, India 4 Center for Aquaculture Research & Development, St. Xavier's Vocational Training Center, Don Bosco, Bishramganj-799103, Tripura, India
Key words: EPA/DHA, Vitamin E, fatty acids, Betta splendens, reproductive performance Abstract The present study elucidated the reproductive performance of Betta splendens fed with enriched eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) and vitamin E diets. Two hundred and forty Betta splendens fry (mean weight 0.141±0.02g) were equally distributed in four groups, each group with three replicates in 35 L glass aquaria. Fishes were fed twice daily for 126 days with the following different diets: Group 1formulated feed (FF); Group 2-FF+0.01% vitamin E; Group 3-FF+1% EFA/DHA and Group 4-FF+0.01% vitamin E+1% EPA/DHA. During the second spawning, the highest values were found in Group 2. After completion of both the spawning, highest (P0.05) were found after completion of both the spawning. No significant differences (P>0.05) were found in fry survival percentages. Fatty acid profile of diets did not reflect the profiles of the gonad, except n-6 PUFA levels. Positive correlations were found between n-6 PUFA levels in the diets with hatched larvae (y=10.02x-146.2; R²=0.819) and fry survival after two weeks rearing (y=5.581x-80.37; R²=0.823). Hence, considering all factors, it can be summarized that formulated feed enriched with vitamin E showed best reproductive performance and diets enriched with DHA/EPA did not show any betterment in the reproductive performance of Betta splendens. Introduction Fish nutrition plays an important role in maintaining good health and general behaviour of ornamental fishes and also enhancing the external appearance such as bright colour, enlarged fins, intact scales, skin and spawning performance. Feeding of * Corresponding author.
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broodstock in most of the ornamental fish farms still depends on live food such as blood worms, Tubifex that enhances the risks of introducing harmful pathogens (Radhika et al., 2007). Broodstock nutrition is still not properly understood due to difficulties in conducting studies involving proper feeding and reproduction of broodstocks (Chong et al., 2004). Some studies have reported that highly unsaturated fatty acid (HUFA) level of brood fish diet significantly affects the fecundity, fertility, hatching rate, viability of fish eggs and larval growth (Furuita et al., 2007). Different types of animal and plant lipid sources are used for aqua-feed formulation not only as a source of energy but also as a source of essential fatty acids (EFA), which are necessary for maintaining the impermeability barrier of skin, cholesterol transport and metabolism and also for normal growth and health status of fish. Significance of long-chain PUFA of the n-3 type, available in fish oils, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has increased considerably (Jaya-Ram et al., 2008). Vitamin E is a lipid-soluble vitamin and a naturally occurring antioxidant. Antioxidant function lies in its 6-hydroxychromane ring that donates a hydrogen atom to the chain propagating lipid peroxyl radical (Kagan et al., 1990). The form of vitamin E commonly added to fish feed is alpha-tocopheryl acetate. It becomes active as an antioxidant only after the hydrolysis of the acetate group in the fish body. It protects biological molecules from oxidation and requirements directly related to dietary HUFA level (Udilova et al., 2003), essential for the immune function and is only obtained through diet (Bekhit et al., 2009). If increasing dietary lipid concentrations increase the rate of lipid oxidation in fish, then vitamin E might be utilized for faster growth Chaiyapechara et al. (2003). Betta splendens is a fish with a maximum size of 5-6 cm for females and 8-10 cm for males, the market size of the males is at least 5-6 cm. For better understanding the requirements of fatty acids in B. splendens broodstock, present study was conducted to investigate and compare various combinations of dietary HUFA and vitamin E on the spawning performance of B. splendens. Materials and Methods Experimental design and fish Two hundred and forty Betta splendens fry (average body weight 0.141±0.02 g), produced in a laboratory were utilized for the experiment. They were equally distributed in four triplicate groups in 12 35-L aquariums. These were arranged in a Completely Randomized Design (CRD). Group 1 was fed formulated feed (FF); Group 2FF + 0.01% vitamin E; Group 3-FF + 1% EPA/DHA and Group 4-FF + 0.01% vitamin E + 1% EPA/DHA. Aeration was supplied by air blowers although B. splendens can intake atmospheric oxygen directly. The water quality parameters were measured weekly using standard methods (APHA, 1998) and all were found to be within the optimum range for the requirements of B. splendens. Formulation of the diets Four experimental diets having 35% crude protein (CP) were formulated (Table 1) based on the result of James and Sampath (2003). Dry ingredients were blended to make a homogenous mixture. The diets were mixed with boiled water to form dough. Butylated hydroxyl toluene (BHT) was dissolved in oil and mixed thoroughly in the dough and steam cooked for 15 minutes. Mineral-vitamin mixture and vitamin C were mixed after cooling the dough. Pellets of 1 mm size were prepared and dried in the oven at 60 0C to a moisture content of 7-8% and were packed in airtight polythene pack. Proximate composition of four diets (Table 1) was estimated according to AOAC (2005). Energy values were determined by Digital Bomb Calorimeter (Model no. RSB-3). Average temperature in the tanks was maintained at 29±1.0 0C using temperature controlled thermostat heaters. Initially, feed was given at 5% of the total biomass and was adjusted based on the biomass recorded at 21 days interval. Before
Effect of dietary eicosapentaenoic acid/docosahexaenoic acid and vitamin E on the reproductive performance and gonadal fatty acid composition of Betta splendens 3 feeding, pellets were broken into small pieces. The total feed was divided in to two instalments and given at 10:00 and 17:00 h. Spawning of Betta splendens The first breeding of B. splendens occurred on day 105 of the experiment, using 10 L capacity glass aquaria. One male from each aquarium was randomly selected and released in each of the spawning aquarium. One female from each of the aquarium was selected and introduced to the corresponding set of aquariums containing a male, for spawning. After completion of spawning, the female fish was removed, whereas, the male was left in the aquarium for parental care and guarding the eggs. Within 3-4 days when larvae become free swimming, the male was also removed. Similarly, second breeding was done on the 126th day of the experiment to ascertain the spawning performance of B. splendens with respect to their advanced age, using different spawning pairs. Table 1. Formulation of the four different experimental diets Ingredients (%) Fish meal1 Squid meal1 Beef liver meal1 Wheat bran1 Corn flour1 Soybean meal1 Cod liver oil2 Sunflower oil3 Mineral-Vitamin mix4 Vitamin C5 Carboxy methyl cellulose (CMC)6 Butylated hydroxyl toluene (BHT)6 EPA + DHA7 Vitamin E 8 Proximate composition Crude protein (%) Ether Extract (%) Ash (%) Total carbohydrate (%) Moisture (%) Gross energy (KJ g-1)
Group 1 12 10 10 22 20 16 4 4 1.5 0.01 0.5 0.01 34.05±0.20 9.51±0.22 10.57±0.07 45.88±0.06 6.46±0.09 19.78±0.05
Experimental diets Group 2 Group 3 12 12 10 10 10 10 22 21 20 20 16 16 4 4 4 4 1.5 1.5 0.01 0.01 0.5 0.5 0.01 0.01 1 0.01 33.30±0.15 10.33±0.20 10.49±0.01 45.88±0.04 7.26±0.05 19.93±0.04
32.42±0.06 14.04±0.12 10.67±0.30 42.87±0.36 7.48±0.03 20.69±0.03
Group 4 12 10 10 21 20 16 4 4 1.5 0.01 0.5 0.01 1 0.01 33.10±0.88 12.53±0.01 9.33±0.19 45.04±1.07 5.72±0.48 20.62±0.04
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Procured from local market Densa Pharma Private Limited, Mumbai 3 Ruchi Soya Pvt. Ltd., Raighad, India 4 Composition of vitamin mineral mix (EMIX PLUS) (quantity/ 2.5 kg): Vitamin A 55,00,000 IU; Vitamin D3 11,00,000 IU; Vitamin B2 2,000 mg; Vitamin E 750 mg; Vitamin K 1,000 mg; Vitamin B6 1,000 mg; Vitamin B12 6 mcg; Calcium Pantothenate 2,500 mg; Nicotinamide 10 g; Choline Chloride 150 g; Mn 27,000 mg; I 1,000 mg; Fe 7,500 mg; Zn 5,000 mg; Cu 2,000 mg; Co 450 mg; Ca 500 g; P 300 g; L- lysine 10 g; DL- Methionine 10 g; Selenium 50 ppm; Satwari 250 ppm; (Lactobacillus 120 million units and Yeast Culture 3000 crore units). 5 Sd Fines Chemicals Ltd., Mumbai, India 6 Himedia Laboratories Ltd., Mumbai, India 7 Maxepa (EPA 180 mg + DHA 120 mg), MERCK, Goa, India 8 Evion ® 200, MERCK, Goa, India. 2
Values are mean ± S.E. (n=3). Total carbohydrate was calculated by difference method given by the formula below: Total carbohydrate % = 100 - (CP% + EE% + Ash %) ; Gross energy was calculated based on 0.17, 0.40 and 0.24 kJ g-1 for carbohydrate, lipid and protein, respectively.
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Sampling and analyses of samples Hatching rate The number of hatched eggs in each spawning aquarium was counted carefully by deducting and removing the opaque, white and damaged eggs. Hatching rate was calculated according to Suquet et al. (2005) as follows: Hatching rate (%) = (Total number of hatched eggs/Total number of fertilized eggs) x 100 Total fry survival. The number of fry produced and their survival percentage was recorded after two weeks of rearing in each of the breeding aquarium as follows: Fry survival (%) = (Total number of fry on 14th day / Total number of hatched eggs) x 100 Fatty Acid Profile Extraction of lipid The experimental diets and female gonadal tissues were used for lipid extraction according to the method of Folch et al. (1957). The diets and tissues were homogenized with 20 volume of chloroform: methanol mixture (2:1) and transferred into a 25 ml graduated coppered measuring cylinder. The contents were allowed to stand for 6 h with occasional shaking. The mixture was then filtered and the residue was transferred into the cylinder and 10 volume of chloroform: methanol (2:1) was added. The filtrates were treated with 0.2 volume of chilled normal saline and the whole content was transferred to a separating funnel. Mixing was done gently and the separating funnels were held vertically till the two phases were clearly separated. The lower layer was transferred into a pre-weighed flat bottom flask and the solvent was evaporated off in a vacuum evaporator by a continuous flow of nitrogen to get the lipid extract. Preparation of fatty acid methyl esters (FAME) The AOAC (2005) method was followed to esterify the lipid extract. FAME was prepared by heating the lipid extract with methanolic sodium hydroxide first and then with boron tri-fluoride methanol for esterification. N-heptane (5 ml) was added to recover the methyl esters in organic phase. The mixture was washed with saturated sodium chloride solution and left undisturbed till the two phases separate out. The upper Nheptane phase was pipetted out and stored in 10 ml glass vials with glass lids in a refrigerator for GC-MS analysis. Gas Chromatography-Mass spectrometry (GC-MS) GC-MS measurements were performed in a Shimadzu QP2010 quadrupole mass spectrometer with ionization energy of 70 eV operating in positive electronic impact set to 100 µA, connected to a GC 8060 gas chromatograph (Shimadzu) equipped with a carbowax (25 m x 0.25 mm; 0.25 µm film thickness) column (Cromlab S.A.) with helium as a carrier gas. Injection was performed in split mode at 250 0C. FAMEs were separated at constant pressure (23.1kPa) with the oven program as (a) 50 0C for 2 min; (b) increase at a rate of 10 0C/min up to 230 0C. Mass spectrometer was tuned to get the relative abundances of m/z ranging from 40 to 550. Statistical analysis All the data were subjected to one-way ANOVA using the statistical software SPSS (Version 16.0) and the significance of difference between means was determined using Duncan’s multiple range test (DMRT). The results were expressed as the mean±S.E. and differences were considered significant at P0.05) were found for the values of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), EPA and the ratio of DHA/EPA among the different groups. Group 3 had the highest (P
