Journal of the Persian Gulf (Marine Science)/Vol. 5/No. 17/September 2014/14/1-14

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1 Journal of the Persian Gulf (Marine Science)/Vol. 5/No. 17/September 2014/14/1-14 Dietary Indispensable Amino Acid Concentrarions based on Amino Acid Profiles of Broodstock, Eggs and Larvae in Yellow fin Seabream, Acanthopagrus latus Zakeri, Mohammad 1* ; Marammazi, Jasem G 2 ; Haghi, Mahsa 3 1- Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, IR Iran. 2- South Iranian Aquaculture Research Center, Ahwaz, IR Iran. 3- Department of Marine Biology, Faculty of Marine Science, Khorramshahr University of Marine Science and Technology, IR Iran. Received: October 2013 Accepted: June Journal of the Persian Gulf. All rights reserved. Abstract A study was undertaken to establish the optimum dietary indispensable amino acid (IAA) profile for yellow fin seabream broodstock based on the amino acid (AA) profile of broodstock, eggs and larvae of Acanthopagrus latus. Three isonitrogenous (40%) and isolipid (20%), named diets 1, 2 and 3 were formulated with different ratios of total essential amino acids per total non-essential amino acid (TEAA/TNEAA) (0.611, or 0.822, respectively) for 138 days. Amino acid profiles of carcass, eggs, hatchling and 3 days posthatching larvae were affected by the corresponding dietary treatments. In general, added concentration of IAA resulted in added contents of crude protein, total EAA and TEAA/TNEAA ratio of eggs and larvae. Optimal balance and increase in concentration of EAA in diet influenced biochemical composition of yellow fin seabream. Results indicated the indispensable amino acid profile in broodstock diet had a considerable effect on the quality of amino acid profile of eggs and larvae in A. latus. Keywords: Acanthopagrus latus; Essential amino acids; Broodstock; Egg and larval quality. 1. Introduction Information on gross protein requirement is of limited value without the knowledge of indispensable amino acid (IAA) requirements values of concerned species. Thus, precise information of the dietary IAA requirements of a fish is very important in evaluating * zakeri.mhd@gmail.com the nutritive value of a protein source and generally in formulating balanced, cost-effective fish feeds through optimization of the protein utilization for better production, as quality protein and dietary amino acid composition of the diet are two major factors that affect fish production (Luo et al., 2005). Therefore, determining the essential amino acids requirement of fish is of extreme importance due to 1

2 Zakeri et al. / Dietary Indispensable Amino Acid Concentrarions based on Amino its major effects on muscle deposition, feed costs and nitrogen wastes (Small and Soares, 1998; Nguyen and Davis, 2009). During pre-feeding developmental stages of marine fish, all nutrients needed for development, growth and homeostasis come from the yolk, and very small amounts of exogenous nutrients are ingested (Zhu et al., 2003). The nutrient material required by the oocyte and their processing during growth and maturation of the oocytes, are key factors affecting egg quality (Craik and Harvey, 1984; Kjorsvik et al., 1990; Bromage et al., 1992; Brooks et al., 1997). All the contents of an egg, genetic and nutritive, that will determine its quality, must be incorporated into an egg, when it is an oocyte within the ovary. If an egg does not contain a particular compound or contains an inappropriate amount of a compound, it will not be able to sustain development of a viable embryo (Brooks et al., 1997). Dietary proteins and amino acids appear to influence egg quality (Rodríguez- González et al., 2006; El-Sayed et al., 2008), but these components have received little attention, than lipid and fatty acids. The present study was carried out to examine the effects of IAA content in broodstock diets on egg and larval quality, and to determine a suitable concentration of EAA for A. latus broodstock diets. By comparing the essential amino acid composition in the diets, broodstock carcass, eggs, hatchling and 3 days posthatching (DPH) larvae, the possible mechanisms by which dietary IAA concentrations in broodstock diets affect reproductive performance are discussed. 2. Materials and Methods 2.1. Experimental Fish and Culture Facility Yellow fin seabream brooders weighing 209 ± 17 g and 433 ± 29 g for males and females respectively, were procured from the Mariculture Research Station of the South Iranian Aquaculture Research Center, Mahshahr, Iran and randomly distributed in groups of 10 in 9 cylindric polyethylene tanks (1139 L tank 1 ) at a sex ratio of 1:1. Feeding period lasted for 121 days and fish were fed to satiation twice daily at 09:00 and 17:00 h (from late October to late February) prior to first spawning. Broodstock were weighed and checked in each tank at the end of the spawning period to determine weight gain and the number of spawned females. The experiment lasted 138 days (including feeding, spawning and development periods). The tanks, equipments, photoperiod, temperature controls and water supply system were as reported previously (Zakeri et al., 2009). Water temperature, salinity, dissolved oxygen and ph were recorded daily. Salinity ranged between 39.5 and 44 (41.7±0.6) and temperature between 14 and 23 C (16±0.4) during the experimental period. Average values for dissolved oxygen and ph were 7.5 and 7.8 mg.l 1, respectively Experimental Diets The formulation and proximate composition of the experimental diets are presented in Table 1. Three diets were formulated to be isonitrogenous (40% dry-weight basis) and isolipidic (20% dryweight basis). Fish meal was the major source of protein. In three experimental diets, protein was partially substituted by defatted soybean to obtain dietary IAA of 15.4%, 16.9% and 18.1% in diets 1, 2 and 3, respectively. The total essential amino acid to total non-essential amino acid (TEAA/TNEAA) ratio in diets 1, 2 and 3 were 0.61, 0.72 and 0.82, respectively (Table 2). All ingredients were mixed for 45 min and mechanically extruded to obtain pellets of the desired size (4 mm). The pellets were dried in a convection oven at 25 C to obtain a moisture level of approximately 100 g kg 1 and stored in airtight plastic bags at 20 C until use. 2

3 Journal of the Persian Gulf (Marine Science)/Vol.5/No. 17/September 2014/14/1-14 Table 1: Ingredients, formulation and proximate composition of the experimental diets (g/100 g dry diet) Ingredients (g 100 g dry diet -1 ) Diets Fish meal a Defatted soybean meal a Gelatin a Fish oil Wheat bran a Vitamin premix b Mineral premix b Cellulose Proximate composition c (%) Crude protein Crude lipid Ash Crude fibre NFE d GE e (MJ/Kg) Indispensable amino acids (%) f a Proximate composition as % Dry-weight basis [Fish meal (64% crude protein, 15% crude lipid); Gelatin (97% crude protein); Defatted soybean meal (51% crude protein); Wheat bran (16% crude protein, 3.4% crude lipid)]. b According to Zakeri et al. (2009). c Value are means of three replicate samples per diet. d Nitrogen-free extract (calculated by difference). e Gross energy, calculated based on 0.017, and MJ/g for carbohydrate, lipid and protein, respectively. f Calculated from protein content Total essential amino acids. Table 2: Amino acids profile (g/100 g total amino acids) of experimental diets (n=3). Amino acid Diets Essential amino acid (EAA) Histidine (His) Arginine (Arg) Threonine (Thr) Valine (Val) Methionine (Met) Leucine (Leu) Isoleucine (Ile) Phenylalanine (Phe) Lysine (Lys) Non-essential amino acid (NEAA) Aspartic acid (Asp) Glutamic acid (Glu) Serine (Ser) Glycine (Gly) Alanine (Ala) Proline (Pro) Tyrosine (Tyr) Cysteine (Cys) Total EAA Total NEAA TEAA/TNEAA

4 Zakeri et al. / Dietary Indispensable Amino Acid Concentrarions based on Amino 2.3. Spawning, Egg and Larval Collection The brooders were checked daily for determining the time of spawning. Spawning started in late February and took place at night (between 12:00 and 05:00) and the eggs were collected the next morning from the tanks and transferred to a 1-L beaker. Samples of eggs, hatchlings and 3 days post-hatch (DPH) larvae were labeled and immediately frozen at 80 C for biochemical analyses. In order to make direct comparisons between fish, the amino acid (AA) data were expressed as the A/E ratios [each EAA content (total NEAA content including cysteine and tyrosine) ] (Arai, 1981; Akiyama et al., 1997). It is possible to compare the AA requirements of different fish and to detect dietary AA imbalances irrespectively of the protein content of the food, since this index uses the relative balance among the EAA, instead of their absolute values. A dietary deficiency for a given IAA was assumed to occur when, through the analysis of this plot, the A/E ratio in the fish is above the A/E ratio in the food item Proximate Analysis, Protein and Amino Acid Analysis Initial body composition of fish was analyzed from 10 samples of fish (5 females and 5 males) frozen prior to the commencement of the trial. After spawning, four broodfish (two females and two males from each tank (12 fish per treatment) were individually weighed, gutted and frozen at 80 C for chemical analyses. Proximate composition of feed ingredients, experimental diets, broodfish carcasses, eggs and larvae were analyzed using standard method (AOAC, 1997). Each analysis was conducted in triplicate. Crude protein (N 6.25) was measured by an auto Kjeldahl unit (Buchi, German; model B-414, K-438, K-371 and K-370). The samples for amino acids (AA) analysis were hydrolyzed for 23 h with 6N hydrochloric acid at 112 C under N2 atmosphere. Samples were then derivatized with phenylisothiocyanate (PITC) reagent before separation by gradient exchange chromatography using a Waters HPLC (Waters auto sample model 717 plus; Waters binary pump model 1525; Waters dual absorbance detector model 2487), according to the Pico-Tag method (P ) as described by Cohen et al. (1989). Resulting peaks were identified, integrated and quantified with a Waters Breeze software package by comparing to a known amino acid standard (Pierce NC10180). The protein-bound tryptophan was not determined, since it is partially destroyed by acid hydrolysis. Glutamine is converted to glutamate and asparagine to aspartate during acid hydrolysis, so the sum of the respective amino acid in the proteins is reported Statistical Analysis Data were expressed as mean ± standard error. They were checked for normal distribution with the Kolmogrov-Smirnoff test. Simple linear and nonlinear regressions were performed to correlate the results obtained. A one-way analysis of variance (ANOVA) was used to determine differences among EAA concentrations, using SPSS 11.5 statistical software. Differences were considered significant at an alpha of 0.05 (P<0.05). Turkey s HSD multiple comparison test was used to determine significant differences among means. 3. Results 3.1. Amino Acid Profile of Carcass Protein content of broodstock carcasses seemed independent of dietary IAA concentrations, because the highest (P<0.05) protein content of carcass was found in the fish fed diet 2 (Table 3). The amino acid profile of carcass was generally affected by dietary amino acid composition. Histidine, Arginine, 4

5 Journal of the Persian Gulf (Marine Science)/Vol.5/No. 17/September 2014/14/1-14 Threonine and Valine content were significantly higher in carcass of broodfish fed diet with 16.88% essential amino acid concentration. Leucine was significantly higher (P<0.05) in fish fed diet 2. There was no significant difference in leucine value between diet 2 and 3. Significant increase was found in isoleucine content with the increasing of dietary EAA concentration. In general, lysine decreased with increase of plant protein supply whereas, no significant difference in lysine content was observed between diets 2 and 3 or diets 1 and 2. Methionine and phenylalanine contents of carcass showed no significant difference among treatments. The total EAA content of carcass was significantly higher (P<0.05) in broodstock fed diet 2. The concentration of total NEAA of carcass was the maximum (P<0.05) in diet 1. No significant changes were observed in aspartic acid, tyrosine and proline concentrations. Glutamic acid, serine, glycine and alanine contents were highest (P<0.05) in fish feed diet 1. Cysteine was significantly higher (P<0.05) in fish fed diet 3. There was no significant difference in cysteine content between diet 3 and 2. The TEAA/ TNEAA ratio of carcass was significantly highest in diet 2. Table 3: Protein content and amino acid profile (g/100 g total amino acids) of total protein of carcass from broodstock fed experimental diets (Mean ± SE values (n=3) with different superscript in each row are significantly different (P<0.05)). Amino acids Carcass Initial Diet 1 Diet 2 Diet 3 Essential amino acid (EAA) His. 1.7± 0.04 b 1.5±0.04 b 2.1±0.1 a 1.8±0.07 b Arg. 5.3± 0.4b c 4.4±0.2 c 6.3±0.8 a 5.7±0.4 b Thr. 4.8± 0.6 b 3.9±0.4 c 5.7±0.5 a 3.8±0.2 c Val. 2.2± 0.1 c 2.1±0.2 c 4.6±0.3 a 3.5±0.3 b Met. 1.6± ± ± ±0.04 Leu. 6.7± 0.8 ab 5.4±0.4 b 7.1±0.2 a 6.5±0.7 ab Ile. 1.8± 0.09 d 2.8±0.2 c 3.5±0.3 b 4.2±0.2 a Phe. 2.7± ± ± ±0.1 Lys. 7.9± 0.6 a 6.5±0.1 c 7.1±0.8 b 7.3±0.4 b Non-essential amino acid (NEAA) Asp. 15.2± ± ± ±1.2 Glu. 19.9±2.1 b 21.3±2.2 a 17.4±2.1 c 17.9±2.3 c Ser. 5.3±0.4 b 6.3±0.4 a 4.7±0.6 c 5.3±0.4 b Gly. 8.5±0.7 a 8.2±0.6 a 5.8±0.8 c 7.8±0.6 b Ala. 8.9±0.7 ab 9.5±0.6 a 8.7±0.4 ab 8.2±0.4 b Pro. 2.9±0.3 b 3.4±0.8 a 3.9±0.7 a 3.5±0.8 a Tyr. 2.7± ± ± ±0.7 Cys. 1.4±0.09 b 1.5±0.04 b 2.5±0.4 a 2.6±0.2 a Total EAA 34.7±2.1 bc 31.7±1.8 c 41.5 ± 1.7 a 37.4±1.4 b Total NEAA 64.8±1.5 a 68.5±1.4 a 59.1±1.4 b 62.9±1.7 ab TEAA/TNEAA 0.54±0.01 bc 0.46±0.02 c 0.70±0.02 a 0.59±0.05 b Proteins (%) ±2.6 c 56.8±1.7 c 63.5±2.1 a 59.1±2.1 b 1 Dry weight basis. 5

6 Zakeri et al. / Dietary Indispensable Amino Acid Concentrarions based on Amino The comparison of the A/E ratios off carcass of A. latus broodstock and their diets suggestt that diet 1 is deficient in threonine, phenylalanine (including tyrosine) and lysine (Figure 1A), diet 2 in threonine (Figure 1B) and diet 3 in phenylalanine (including tyrosine) and lysine (Figure 1C). Phenylalanine (included tyrosine), lysine and threonine are probably the first limiting EAA for the broodfish feed experimental diets. (P<0.05) in fish fed diet 3. There was no significant difference in crude protein value between diets 3 and 2. Histidine, threonine, valine and phenylalaninee concentrations inn eggs increased proportionally with an increase in dietary EAA. No significant difference was observed in threonine and valine contentsts between diets 1 and 2. Conversely, isoleucine content in eggs decreasedd with an increasee in dietary EAA. Lysine and serinee contents in eggs were the highest (P<0.05) in broodfish fed diet 2. Arginine and alanine contents weree higher in brood fish fed diet 2, whereas, no significant differences were observed in their contents between diets 1 and 2. Methionine and leucine contents showed noo significantt differences among treatments. Variations in NEAA did not follow any uniform pattern. Glutamic acid concentration of egg was the highest (P<0.05) in fish fed diet 3. Proline concentration did not varyy significantly between diets 3 and 1. Glycine G and tyrosine concentrations weree generally higher in fish fed diet 1. Aspartic acid and cysteine were the NEAA which did not differ significantly among the experimental diets Amino Acid Profile P of Hatchling and 3DPH Larvae Fig. 1: Comparison of the A/E ratios of carcass from Acanthopagrus latus broodstock fed diet 1 (A), diet 2 (B) and diet 3 (C) Amino Acid Profile of Eggs Increasing dietary IAA resulted in ncreasing total EAA and TEAA/TNEAA ratio in eggs (Table 4). Variations in NEAA did not follow any uniform pattern. Crude protein content was significantly higher The amino acid profile of hatchling and 3DPH larvae is shown in Table 5.. The protein content of hatchling and larvae at 3DPHH increased proportionally with increasing dietary d EAA.. However, there t was no significant difference in protein content of hatchling and larvae at 3DPH between diets 3 and 2. The concentration of most amino acids was influenced by the dietary EAA concentrations and displayedd significant differences among diets. Histidine, threonine and valine contentss of hatchling increased with ncreasing dietary EAA concentration. Isoleucine and lysine contents of hatchling weree significantly higher in fish fed diet 3. Phenylalanine e concentration of hatchling was higher (P<0.05) in 6

7 Journal of the Persian Gulf (Marine Science)/Vol.5/No. 17/September 2014/14/1-14 groups fed diet 2. No significant difference was observed in arginine and leucine concentrations of hatchling produced by broodfish fed diets 2 and 1. These amino acids were proportionally higher in diet 2. Methionine content of hatchling was the only NEAA that did not change significantly among the experimental diets. Among the EAA of 3DPH larvae, leucine, isoleucine and lysine contents were found to be the highest at a dietary EAA concentration of 18.14%. In general, valine content decreased with the increasing dietary EAA concentration. Arginine was higher in fish fed diet 3. There was no significant difference in arginine content between diets 3 and 2. Histidine, threonine, methionine and phenylalanine content of 3DPH larvae did not show significant differences among treatments. The highest total NEAA of hatchling and 3DPH larvae was found in the diet 1. Total EAA and TEAA/TNEAA ratio of hatchling and 3DPH larvae significantly increased with increasing dietary EAA concentration from 15.4% to 18.14%, but no significant difference in total EAA of hatchling was observed between diets 3 and 2. The protein content in eggs, hatchling and 3DPH larvae of all treatments were much lower than the respective protein content in carcass. Both the TEAA content and TEAA/TNEAA ratio in hatchling and 3DPH larvae of all treatments were much lower than the respective contents in egg (Tables 4, 5). Thus, there is a clear decreasing trend in EAA content from broodfish to eggs and larvae and from eggs to hatchling/3dph larvae. Table 4: Protein content and amino acid profile (g/100 g total amino acids) of total protein of eggs from broodstock fed experimental diets (Mean ± SE values (n=3) with different superscript in each row are significantly different (P<0.05)). Egg Amino acids Diet 1 Diet 2 Diet 3 Essential amino acid (EAA) His 1.6±0.4 c 2.2±0.1 b 2.9±0.1 a Arg 8.3±0.7 a 8.4±0.8 a 7.8±0.4 b Thr 5.0±0.4 b 5.1±0.4 b 6.5±0.5 a Val 4.0±0.4 b 4.3±0.5 b 6.5±0.4 a Met 3.6± ± ±0.1 Leu 10.0± ± ±1.1 Ile 8.8±0.7 a 6.5±0.4 b 5.4±0.4 c Phe 2.5±0.4 c 4.8±0.2 b 6.4±0.5 a Lys 7.7±0.8 c 9.7±0.9 a 9.3±0.7 b Non-essential amino acid (NEAA) Asp 8.1± ± ±0.4 Glu 10.6±1.0 b 9.8±1.0 c 11.3±1.2 a Ser 5.6±0.5 b 6.5±0.4 a 3.4±0.2 c Gly 7.8±0.9 a 7.4±0.9 a 6.7±0.5 b Ala 7.3±0.6 a 7.6±0.8 a 6.3±0.7 b Pro 5.3±0.4 a 4.4±0.6 b 5.4±0.4 a Tyr 3.5±0.4 a 2.4±0.2 b 3.1±0.4 a Cys 0.4± ± ±0.02 Total EAA 51.5±1.2 c 54.0±0.9 b 57.2±1.4 a Total NEAA 48.6±2.1 a 46.3±2.1 b 44.3±2.5 c TEAA/TNEAA 1.06±0.07 c 1.17±0.04 b 1.29±0.09 a Protein (%) ±2.2 b 56.8±2.0 a 57.1±2.5 a 1 Dry weight basis. 7

8 Zakeri et al. / Dietary Indispensable Amino Acid Concentrarions based on Amino Table 5: Protein content and amino acid profile (g/100 g total amino acids) of total protein of hatchling and 3DPH larvae from broodstock fed experimental diets. Hatchling 3DPH larvae Amino acids Diet 1 Diet 2 Diet 3 Diet 1 Diet 2 Diet 3 Essential amino acid (EAA) His. 1.5±0.04 c 2.1±0.1 c 2.8±0.1 a 2.2± ± ±0.07 Arg. 7.4±0.4 a 7.9±0.4 a 6.6±0.2 b 5.3±0.4 b 7.1±0.9 a 8.1±0.7 a Thr. 4.6±0.4 b 4.8±0.3 b 6.9±0.3 a 5.5± ± ±0.4 Val. 1.7±0.04 c 4.1±0.2 b 5.4±0.2 a 4.3±0.5 a 1.7±0.08 b 1.8±0.07 b Met. 3.3± ± ± ± ± ±0.3 Leu. 9.1±0.8 a 9.2±0.9 a 5.9±0.5 b 4.7±0.5 c 8.7±0.8 b 9.8±0.8 a Ile. 7.9±0.4 b 6.2±0.5 c 8.7±0.7 a 6.9±0.5 c 7.6±0.8 b 8.6±0.7 a Phe. 2.3±0.2 c 4.6±0.5 a 3.1±0.2 b 2.5± ± ±0.2 Lys. 9.6±0.8 b 9.3±0.8 b 10.8±1.0 a 8.6±0.7 c 9.4±0.8 b 10.4±1.1 a Non-essential amino acid (NEAA) Asp. 8.9±0.9 a 8.1±0.7 b 7.7±0.8 c 9.3± ± ±0.7 Glu. 11.7±1.2 a 10.3±1.1 b 10.5±1.1 b 12.6±1.2 a 12.2±1.2 a 10.8±1.2 b Ser. 6.1±0.4 a 6.8±0.4 a 4.3±0.4 b 5.2± ± ±0.4 Gly. 8.5±0.7 a 7.7±0.6 b 7.0±0.2 c 9.4±0.8 a 8.8±0.7 a 7.9±0.8 b Ala. 8.0± ± ± ±1.1 a 8.4±0.6 b 7.4±0.9 b Pro. 5.8± ± ± ± ± ±0.6 Tyr. 3.8± ± ± ± ± ±0.4 Cys. 0.3± ± ± ±0.07 a 0.3±0.01 b 0.3±0.02 b Total EAA 47.4±1.2 b 51.4±1.5 a 53.1±1.1 a 42.4±1.7 c 46.2±2.1 b 51.1±1.8 a Total NEAA 53.1±1.5 a 48.6±1.1 b 46.9±1.7 b 57.5±2.2 a 54.2±1.6 b 49.3±2.1 c TEAA/TNEAA 0.89±0.04 c 1.06±0.07 b 1.13±0.05 a 0.74±0.07 c 0.85±0.09 b 1.04±0.07 a Protein (%) ±2.1 b 44.3±1.7 a 46.5±2.0 a 41.2±2.2 b 42.8±1.5 a 44.6±1.9 a 1 (Mean ± SE values (n=3) with different superscript in each row are significantly different (P<0.05)). 2 Days post-hatching. 3 Dry weight bases. 4. Discussion The biochemical constitution including amino acid profile of eggs and larvae, reflect the nutritional status of broodstock. Newly spawned marine fish eggs have a total amino acid content of 40 60% of their dry mass (Fyhn, 1989; Rønnestad and Fyhn, 1993; Thorsen et al., 1993; Finn, 1994; Rønnestad et al., 1996, 1999). The relative composition of the IAA pool also differs significantly between pelagic and demersal eggs (Rønnestad et al., 1996). In contrast to demersal eggs, the IAA pool in pelagic eggs are dominated by neutral amino acids such as leucine, valine, isoleucine, alanine and serine. In the pelagic eggs free amino acids play important role as osmotic active compounds, necessary for protein synthesis and regulation of egg buoyancy (Thorsen and Fyhn, 1996, Lahnsteiner et al., 2009). The present results provide the first proof of the pivotal role played by dietary EAA in egg and larval quality in A. latus. Our results showed that the amino acid profile of yellow fin seabream carcass was generally affected by amino acid profile of the diet. Lysine, leucine, arginine and threonine were the most abundant essential amino acids in carcass of A. latus. Similar results were 8

9 Journal of the Persian Gulf (Marine Science)/Vol.5/No. 17/September 2014/14/1-14 reported by Limin et al., 2006 (Lateolabrax japonicas). In this study, concentrations of crude protein and EAA such as histidine, arginine, threonine, valine, leucine, total EAA and TEAA/TNEAA ratio seemed independent of increasing dietary EAA concentrations, because their highest concentrations were found in fish fed diet 2 containing intermediate EAA content. Protein synthesis and deposition are known to be most efficient, when all the required amino acids are present simultaneously at the synthesis sites (Cho and Kaushik, 1990; Ng et al., 1996). An imbalanced dietary amino acid profile may induce amino acids released from protein digestion to be oxidized, instead of being channeled for body protein and total EAA gain. Amino acid profiles have been suggested as indices of amino acid requirements for fish (Wilson and Poe, 1985). Mambrini and Kaushik (1995) concluded that the whole-body EAA profile better reflects the amino acid requirements of fish, compared to the EAA profile of different tissues. In this study, significant correlation was obtained between essential amino acid profile of diet 2 and the EAA requirement of broodfish (Figure 1B). In many fish species, reduced food intake and subsequent utilization of muscle and visceral reserves as sources of nutrients for ovarian growth during the reproductive season, have been reported (Lal and Singh, 1987; Lie et al., 1993; Ling et al., 2006). Broodstock nutrition has been shown to improve larval quality in fish (Izquierdo et al., 2001). According to the obtained results, dietary amino acid profile regarded to broodstock amino acids requirement, may have affected quality of A. latus larvae. The biochemical compositions of eggs are often examined to evaluate egg quality, as the egg must satisfy nutritional needs for embryonic and larval development (Furuita et al., 2002). However, the relationship between biochemical composition and egg quality is difficult to interpret (Morehead et al., 2001). Our results showed that the total protein content in eggs was determined by the broodstock diet which is in agreement with the reports on Oreochromis niloticus (El-Sayed and Kawanna, 2008; El-Sayed et al., 2003), Labeo rohita (Afzal Khan et al., 2005) and Plectorhynchus cinctus (Li et al., 2005). Amino acid profiles in eggs reflect the respective dietary amino acid composition of the broodstock. However, the effect of protein supplementation of broodstock diets on egg quality was not found with changes in amino acid profile (Tandler et al., 1995). Tandler et al. (1995) suggested that reduction in egg quality from broodstock fed an imbalanced EAA diet resulted from a change of the concentration at the vitellogenin binding site. Consequently, for improvement of egg and larval quality, the protein in the diet should have an EAA composition, similar to that of the egg protein. In the present study, the main essential amino acids of egg were leucine, isoleucine, arginine and lysine, comprising >55% of TEAA (Table 5). This observation was in agreement with the amino acid profile reported for marine species, such as Lutjanus campechanus egg (Hastey et al., 2010), Dicentrarchus labrax (Rønnestad et al. 1998), Hippoglossus hippoglossus (Finn et al. 2002) and Solea senegalensis (Parra et al. 1999). Comprising to the previous study was shown that lysine (in eggs) was the only EAA that was positively correlated with survival rate of larvae at 3DPH and negatively correlated with abnormal larvae percentage (Zakeri et al., 2013). Lysine probably was the major EAA that affect larval survival rate and quality. It should be noted that correlation does not validate but only implies causation. The EAA content is of great importance to the hatching and survival of marine teleost larvae (Rønnestad et al., 1999; Finn, 2007; Moran et al., 2007). According to the results of our study, concentrations of histidine, threonine, valine and phenylalanine in eggs showed an increasing trend with increase of dietary EAA concentrations in broodstock diet in A. latus. In the present study, total EAA concentration and TEAA/TNEAA ratio in eggs of fish fed diet 3 (high EAA content) were higher than 9

10 Zakeri et al. / Dietary Indispensable Amino Acid Concentrarions based on Amino in diets 1 and 2. Free amino acids of yolk proteins have been cited as a resource for energy production (Finn et al., 1996), protein synthesis (Ohkubo and Matsubara, 2002), and active osmotic compounds (Finn, 2007). However, fish species vary widely in how they partition dietary protein between growth and reproduction (De Silva and Anderson, 1995; Sink et al., 2010). It is likely that lipids are more readily available for use as metabolic fuel by yellow fin seabream (Zakeri et al., 2011) resulting in repartitioning of essential amino acids from metabolic energy pathways to deposition in eggs. This could be the main reason for the high fertilization and hatching rate percentage observed in broodstock fed the diet 3. We found the effects of dietary EAA concentrations on the amino acid profiles in both hatchling and 3DPH larvae. The concentration of most amino acids in hatchling and 3DPH larvae displayed positive correlation with the dietary EAA content broodstock diet suggesting that the effect of dietary EAA content on amino acid profiles of egg, could be extended to larvae. No marked difference was found in the protein contents of hatchling and 3DPH larvae of broodfish fed diets 2 and 3. Based on the concentration in hatchling and 3DPH larvae, it could be concluded that lysine has an important role in larval development in yellow fin seabream (Table 5). The content of most EAA decreased following the progression of development whereas, the contents of most NEAA showed an increase. Similar observations were made in Lutjanus campechanus (Hastey et al., 2010). There seems to be an apparent preference in the utilization of TEAA versus TNEAA during the development from egg stage to 3DPH larvae of A. latus. Free amino acid contributes to protein synthesis as well as providing a source of energy for developing embryos and larvae (Watanabe and Kiron 1994; Rønnestad et al. 1999; Hastey et al., 2010). In comparison with levels in egg, total EAA concentration and TEAA/NTEAA ratio in hatchling were slightly lower. Furthermore, total EAA concentration and TEAA/NTEAA ratio of 3DPH larvae were also lower than that of hatchling and egg. Similar results on decreasing EAA content from egg to larval stages have been reported in Asian seabass (Dayal et al., 2003). Brown et al. (2005) reported total free amino acid concentration of 16.3 μg/egg (dry wet) and 3.4 μg/larvae at 1 day post-hatch of Latris lineata. Information on the dietary indispensable amino acid requirements of broodstock was limited to a few species. The present study demonstrated that the nutritional value of indispensable amino acids in broodstock diet has a significant effect on the quality and amino acid composition of egg and larvae of A. latus. Acknowledgments The authors are thankful to the director of the South Iranian Aquaculture Research Center, Ahwaz, Iran, and the director and staff at the Mariculture Research Station, Mahshahr, Iran for providing the necessary facilities for the experiment. We would like to thank Khorramshahr University of Marine Science and Technology for supporting this work under research grant contract No 55. References Afzal Khan, M., Jafri, A.K. and Chadha, N.K., Effects of varying dietary protein levels on growth, reproductive performance, body and egg composition of rohu, Labeo rohita (Hamilton). Aquaculture Nutrition. 11: Akiyama, T., Oohara, I. and Yamamoto, T., Comparison of essential amino acid requirements with A/E ratio among fish species (review paper). Fisheries Science. 63: AOAC, Official Methods of Analysis of Association of Official Analytical Chemists, 16th ed. AOAC, Arlington, VA, 1298P. Aragão, C., Conceicão, L.E.C., Fyhn, H.J. and Dinis, M.T., Estimated amino acid requirements 10

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