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Lysine demand and essential aminoacids estimate for tambaqui, Colossoma macropomum (Cuvier, 1818) / Exigência de lisina e estimativa dos aminoácidos essenciais para tambaqui, Colossoma macropomum (CUVIER, 1818)

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Braz. J. of Develop.,Curitiba, v. 6, n. 3, p. 10781-10804 mar. 2020. ISSN 2525-8761

Lysine demand and essential aminoacids estimate for tambaqui,

Colossoma macropomum (Cuvier, 1818)

Exigência de lisina e estimativa dos aminoácidos essenciais para

tambaqui, Colossoma macropomum (CUVIER, 1818)

DOI:10.34117/bjdv6n3-086

Recebimento dos originais: 29/02/2020 Aceitação para publicação: 06/03/2020

Edimar Lopes da Costa Mestrado em Biologia de Água Doce

Instituto Federal de Educação, Ciência e Tecnologia do Amazonas-IFAM, Campus Humaitá

Br 230, km 07, Zona rural, Humaitá – AM, Brasil E-mail: edimar.lopes@ifam.edu.br

Paulo Adelino de Medeiros Mestrado em Biologia de Água Doce Instituto Federal do Amazonas – IFAM

Rua Ferreira Pena, 1109, Centro, CEP 69025-010, Manaus, AM, Brasil E-mail: paulo.adelino@ifam.edu.br

Elenice Martins Brasil Doutorado em Aquicultura

Universidade Federal de Santa Catarina – UFSC

Rua Admar Gonzaga, Itacorubi, CEP 88034001, Florianópolis, SC, Brasil E-mail: nicebrasil@hotmail.com

Eduardo Akifumi Ono Mestrado em Aquicultura

Nova Acqua, Rua Belo Horizonte, 93, Adrianópolis, CEP 69057-060, Manaus, AM, Brasil

E-mail: onoedu@yahoo.com

Elizabeth Gusmão Affonso Doutorado em Ecologia e Recursos Naturais

Instituto Nacional de Pesquisas da Amazônia – INPA, Endereço completo, Aleixo, CEP 69055-010, Manaus, AM, Brasil

E-mail: pgusmao@inpa.gov.br

ABSTRACT

Four hundred-fifty (450) 7.7±0.06 g juvenile tambaqui were distributed in 500L boxes, fed a casein, gelatin and free amino acids-based semipurified diet containing six lysine levels (L-lysine HCL 0.9; 1.2; 1.5; 1.8; 2,1; 2.4% of the diet), in a completely randomized design, so that their lysine requirements could be determined. Findings demonstrated that diets with rising lysine levels did not influence the assessed zootechnical parameters.

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Tambaqui presented reduced consumption, low weight gains and high feed conversion with the use of the semipurified diet. This diet affected the hematological parameters significantly with no changes on total proteins and plasmatic glucose. The reduction on the plasmatic cortisol with the increase of lysine in the diet suggests the lower lysine levels to have had an influence on plasmatic cortisol level values. The reduced levels of triglycerides, cholesterol and body lipids with increasing lysine levels suggest a relationship of this amino acid with the accumulation of body lipids. Essencial amino acids demand estimated from tambaqui body amino acids content, showed to be similar to that found for other species and the lysine was estimated at dietary protein 6.0 and 6.2%, through the two utilized methods. The zootechnical performance exhibited by tambaqui, using semipurified diet for the lysine 0.9 to 2.4% interval in the diet did not allow its demand to be determined through dose-response method.

Keywords: aminoacids, physiology, nutrition, ideal protein RESUMO

Com objetivo de determinar a exigência de lisina para o tambaqui, 450 juvenis (7,7±0,06 g) foram distribuídos em caixas de 500 litros, alimentados com dieta semipurificada à base de caseína, gelatina e aminoácidos livres, contendo seis níveis de lisina (L- lisina HCL 0,9; 1,2; 1,5; 1,8; 2,1; 2,4% da dieta), em delineamento inteiramente ao acaso. Os resultados demonstraram que as dietas com níveis crescentes de lisina não influenciaram os parâmetros zootécnicos avaliados. O tambaqui apresentou consumo reduzido, baixo ganho de peso e alta conversão alimentar com o uso de dieta semipurificada. Essa dieta afetou significativamente os parâmetros hematológicos e sem alterações nas proteínas totais e glicose plasmática. A diminuição no cortisol plasmático com o aumento de lisina na dieta sugere que níveis mais baixos de lisina influenciaram nos valores de cortisol plasmático. Os níveis reduzidos de triglicerídeos, colesterol e lipídios corporais com o aumento dos níveis de lisina sugerem uma relação deste aminoácido com o acúmulo de lipídios corporais. As exigências dos aminoácidos essenciais, estimados a partir do conteúdo de aminoácidos corporais do tambaqui, foram similares ao encontrados para outras espécies e a lisina foi estimada em 6,0 e 6,2% da proteína dietética pelos dois métodos utilizados. O desempenho zootécnico, apresentado pelo tambaqui, com uso de dieta semipurificada para o intervalo de 0,9 a 2,4% de lisina na dieta, não permitiu determinar a exigência para a espécie por meio do método dose-resposta.

Palavras-chave: aminoácidos, fisiologia, nutrição, proteína ideal

1 INTRODUCTION

Efficient diets, with high nutritional quality, contribute significantly, for the growth and feeding efficiency in fish, optimizing protein utilization and lowering production costs and effluents inflow into the aquatic environment, in addition to strengthening the immunological system, reducing diseases incidence and mortality rates (Crab et al., 2007; NRC, 2011; Dairiki et al., 2013b; Pohlenz and Gatlin III, 2014). Currently, Currently, the nutritional requirement for amino acids for fish, has been the

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focus of several studies related to balancing rations being developed for different species of interest in aquaculture (Dairiki et al., 2007; Grisdale-Helland et al., 2011; Dairiki et

al., 2013b; Riche, 2014).

Aminoacids demand can be estimated by utilizing the species’ body profile and an amino acid as refference. Generally, a reference amino acid is attained through dose-response type treatment, just as it happens with lysine, which is amongst the most studied amino acids in fish (Dairiki et al., 2007; Abimorad et al., 2010; Salze et. al., 2011). Lysine is an essential amino acid for fish, limiting in many feed ingredients utilized in diets, presenting no endogenous synthesis and is found in higher concentrations in fish carcass and muscle. It is considered an amino acid exclusively oriented for body protein deposition and, its limitation in the diet can lead to growth and weight gain losses, thus deserving special attention in feed formulations for different speciess (Rollin et al., 2003; Dairiki et al., 2013; Ovie e Eze, 2013).

In Brazil, recent studies on aminoacids nutritional requirement in fish have utilized lysine as reference and focused their their research on some species, such as tilapia Oreochromis niloticus, dorado Salminus Brasiliensis, pacu Piaractus

mesopotamicus and surubim Pseudoplatystoma spp (Dairiki et al., 2007; Bicudo et al.,

2009; Abimorad et al., 2010; Bomfim et al., 2010; Prado, 2011; Furuya et al., 2012; Dairiki et al., 2013; Ovie and Eze, 2013). In this context, tambaqui Colossoma

macropomum, the second most cultivated species in Bazil and the first amongst the native

ones (44,56% in 2011) (MPA, 2013), still has few studies being addressed to its aminocid nutritional requirement. Thus, the present study assesses the tambaqui lysine and the other essential aminoacids nutritional demand estimate, based on the ideal protein concept.

2 MATERIAL AND METHODS Fishes and experimental conditions

The experiment was carried out at Laboratório de Fisiologia Aplicada à Piscicultura – LAFAP, da Coordenação de Tecnologia da Inovação – COTI do Instituto Nacional de Pesquisas da Amazônia – INPA. It was authorized by the Ethics on animal use Committee– CEUA/INPA, under number 010/2013.

Tambaqui juveniles were acquired from commercial pisciculture and acclimated to experimental conditions for two weeks up to the beginning of the experiment. Four

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hundred and fifty tambaqui juveniles, weighed (7.7±0.06 g) and grouped into batches of 25 fish, were kept in 500L PVC tanks with partial water renewal and forced blower aeration system comprising six treatments and three replicates. The experimental design was completely randomized and lasted for 90 days. Fish were fed three daily meals (9:00, 13:00 and 17:00 h), until visual apparent satiety throughout the trial period.

Experimental diets

Six semipurified and isonitrogenous (30 % crude protein-CP) diets were formulated containing rising lysine levels at 0.3 (L-lysine HCL 0.9, 1.2, 1.5, 1.8, 2, 1 and 2.4 % of the diet intervals) using casein, gelatin and amino acids as crystalline protein ingredients (Tables 1 and 2). The profile of the aminoacids of the diets simulated that of the body aminoacids of tambaqui, except for lysine. To maintain the diets isonitrogenated, the addition of L-Lysine HCL was substituded in the same ratio (1:1) by the aspartic acid and glutamic acid mixture according to Bicudo et al., (2009) (Table 2). The diets gross energy (kcal/100g) was obtained through bomb calorimetry. The ingredients were mixed, homogenized and moistened with water and sodium hydroxide solution (6N NaOH) in the (1:1.5) ratio to neutralize the acidity of the feed. Subsequenty, the mixture was pelletized in a (model CAF n° 22)3mm-die meat grinder and dried in a ventilated oven at 45 °C for 24 h. The feed was ground and homogenized in 2-2.5 mm granulometry-sized sives and stored in freezer at -20 oC for further experimental use.

Table 1. Ingredients and chemical composition of experimental diets.

Ingredients Experimental Diets (%)

1 2 3 4 5 6 Casein 11.60 11.60 11.60 11.60 11.60 11.60 Gelatin 2.30 2.30 2.30 2.30 2.30 2.30 Premix of Aminoacids¹ 13.72 13.72 13.72 13.72 13.72 13.72 Dextrin 31.61 31.61 31.61 31.61 31.61 31.61 Microfine Cellulose 15.67 15.67 15.67 15.67 15.67 15.67 carboxymethylcellulose 5.00 5.00 5.00 5.00 5.00 5.00 Canola oil 6.50 6.50 6.50 6.50 6.50 6.50 Bicalcium phosphate 3.50 3.50 3.50 3.50 3.50 3.50 Vitamin-Mineral3 supplement 3.50 3.50 3.50 3.50 3.50 3.50 DL-α-tocopherol 0.20 0.20 0.20 0.20 0.20 0.20 BHT 0.02 0.02 0.02 0.02 0.02 0.02 L-Lysine HCL 0.00 0.45 090 1.35 1.80 2.25

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Asp:Glu (1:1) 6.38 5.93 5.48 5.03 4.58 4.13

Proximate composition (dry matter %)

Dry Matter 92.05 91.75 91.75 90.80 90.60 91.95

Crude Protein 30.4 30.7 31.4 31.1 30.5 31.1

Ether extract 4.70 3.55 3.85 4.2 4.65 4.05

Mineral Matter 6.70 6.35 7.10 8.10 8.30 7.30

Gross Energy (kcal/100g) 398.7 406.1 405.4 403.9 406.3 410.0

1(%) of aminoacids: arg-1.66; hist- 0.53; isol- 0.59; leu-1.22; met-0.50; fen-0.62; tre-0.93; tri-0.24; val-0.69; tir-0.32; ser-0.66; ala-1.65; glu-2.06; pro-0.43; cis-0.27

3 Enrichment of mMicrominerals and vitamins in mg/kg of feed: manganese (26); zinc (140); ferro (100); cobre (14); cobalto (0,2); iodo (0,6); selênio (0,6), Vit. A (10.000 UI); Vit D3 (4.000 UI); Vit E (100); Vit K (5); Vit B1 (25); Vit B2 (25); Vit B6 (25); Vit B12 (30); niacin (100); pholic acid (5); panthotechnic acid (50); biotin (0,8); choline (2000); inositol (50); Vit C (350).

Table 2.Ingredients aminoacids composition and mixture (% DM).

EAA Casein (11,60%) Gelatin (2,30%) AA Mixture (%) Total Tambaqui body profile (30%) Arg 0.37 0.19 1.64 2.21 2.21 His 0.27 0.02 0.53 0.81 0.81 Iso 0.46 0.03 0.59 1.08 1.08 Leu 0.95 0.07 1.21 2.23 2.23 Met 0.29 0.02 0.50 0.80 0.80 Fen 0.49 0.04 0.62 1.16 1.16 Tre 0.44 0.04 0.93 1.41 1.41 Tri 0.11 0.00 0.24 0.35 0.35 Val 0.66 0.05 0.69 1.40 1.40 NEAA Cis 0.18 0.02 0.27 0.47 0.47 Tir 0.57 0.01 0.32 0.90 0.90 Ser 0.57 0.08 0.65 1.30 1.30 Gli 0.20 0.56 2.05 2.80 2.80 Ala 0.32 0.21 1.65 2.17 2.17 Pro 1.04 0.33 0.43 1.79 1.79

Tambaqui body tissue and diet composition

Tambaqui diets and body aminoacids profiles were attained through high performance liquid chromatography analysis performed by the Laboratory “CBO

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Análises”, Campinas-SP (Tables 2 and 3). The corporal profile was obtained from five whole ungutted tambaqui specimens (248±26.67 g), originating from the experimental station at INPA, euthanized, ground, lyophilized and, then submitted for analysis. Fish and diet chemical composition was determined from the bromatologic analysis, according to AOAC (2000), analyzed at the INPA Fish Nutrition Laboratory. Twelve (12) juveniles from the same population, in the beginning of the experiment, and fifteen specimens from each treatment at the end, totalizing 90 individuals, were euthanized so as to ascertain their body composition.

Table 3. Essential and non-essential aminoacids composition in the experimental diets.

Dry matter %

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6

EAA Arg 1.5 1.6 1.6 1.6 1.6 1.6 His 0.7 0.7 0.7 0.7 0.7 0.7 Iso 1.2 1.2 1.2 1.2 1.2 1.2 Leu 2.2 2.2 2.2 2.3 2.2 2.2 Lis 1.0 1.2 1.5 1.9 2.0 2.5 Met 0.7 0.7 0.8 0.7 0.6 0.8 Fen 1.1 1.2 1.2 1.2 1.2 1.2 Tre 1.4 1.4 1.4 1.3 1.4 1.4 Tri 0.2 0.3 0.3 0.3 0.3 0.3 Val 1.5 1.4 1.4 1.4 1.5 1.4 NEAA Cis 0.5 0.4 0.5 0.4 0.5 0.5 Tir 0.8 0.9 0.8 0.9 0.9 0.9 Ac. Asp 4.3 4.3 4.4 3.8 3.8 3.6 Ser 1.3 1.3 1.3 1.3 1.3 1.3 Ac. Glu 5.0 5.0 4.7 4.8 4.6 4.4 Gli 2.8 2.8 3.0 2.9 2.8 2.9 Ala 2.2 2.1 2.2 2.3 2.1 2.2 Pro 1.9 1.9 2.1 2.1 2.0 2.1

Moisture content (MC) was determined by the Weende / Germany method with pre- drying, by sample lyophilisation and, dry matter determination through sample weight difference after drying in an oven at 105 ° C, with constant moisture, temperature and weight. Crude protein (CP) was determined by the Kjeldahl method, through the

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sample digestion (digestor Block /Modelo TE-040-G40/25), destilation (Distillator/Model NT 415) and titration process determining the total nitrogen converted by the 6.25 conversion factor. Ether extract (EE) was determined by extracting (Extrator SOXLETH/Model TE 044-8/25 micro) with petroleum ether solvent. Ashes (A) were obtained through incineration of the sample (2 g) in a muffle oven at 550 ºC for 3 h. Gross Energy (GE - kcal / 100g), estimated on the basis of energy values for protein (5.64 kcal / g ) , ether extract ( 9.44 kcal / g ) and carbohydrates (4.11 kcal / g) (NRC , 1993). Crude Fiber (CF), obtained through basal acid digestion waste, following the Weende method (Agricultural Experimental Station Weende/ Germany), and the non-nitrogenated extract (NNE) content, by calculating the difference between the total weight of each sample according to NNE % = 100 – (M% + CP% +EE %+ CF% + A%) calculation.

Zootechnical parameters

For assessing the diets effects on the fish performance, three biometries (initial, monthly and final) were performed so as to enable us to calculate the following zootechnical indexes: Mean weight gain (WG; g) = Mean weight final (g) – Mean initial weight (g); Protein efficiency rate: (PER; %) = [Weight gain (g) / Ingested crude protein (g)]*100; Mean individual feed intake: (MIFi) = Amount of feed provided (kg) / Number of fishes; Apparent Food Conversion: (AFC) = MIFi / [(mean final weight (g) – mean initial weight (g)]; Specific Growth Rate: (SGR%) = 100 x (mean final weight In– mean initial weight In) / time; Survival Rate: (SR%) = 100% x (final number of fish /initial number of fish ); Food Efficiency Index (FEI) = WG/CT (CT – total feed intake); Hepatosomatic Relation: HSR (%) = [(liver weight /carcass weight) x 100]; Viceral-Somatic Relation: VSR (%) = [(visceral fat weight /carcass weight) x 100]; Protein Productive Value (PPV):PPV = 100x(CPfinal-WGfinal)/(CPinitial-WGinitial)/CPIngery.

Essential amino acids concentration

The requirements for other essential amino acids were determined on the basis of the whole body essential amino acid profile of tambaqui, utilizing the equation proposed by Meyer and Fracalossi (2005), adapted by Abimorad and Castellani (2011), where: Demand on EAA = [(content of an EAA in the carcass or muscle of the fish) × (average of the sum of the requirements among channel catfish, Nile tilapia, common carp, jundiá

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and pacu)] / (sum of the EAAs content in the carcass and muscle of the fish). Another method proposed by Arai (1981) was used to compare the values of the amino acids found in the first método, where: AA demand= essential aminoacids E/A ratio × (lysine demand (%) ÷ Lysine in the muscle E/A ration). The E/A ratio was calculated according to the following proposal: E/A ratio = [(essential amino acid /total of essential aminoacids +cystine+tyrosine) x 1000].

Water quality monitoring

The water quality, in the tanks, was monitored through daily measurements of dissolved oxygen concentrations, (mg/L), temperature (ºC), pH and electric conductivity (EC - µ.S/cm), determined in the morning, through a digital oximeter (YSI model 85 ) and digital pH meter (YSI 60 model),so as to ascertain the experimental conditions Total ammonia (NH3+ NH4+) and nitrite (NO2), concentrations were measured weekly according to Verdouw (1978) and Boyd & Tucker (1992) respectively, utilizing visible/UV spectrophotometer (BIOPLUS/model 2000). Findings showed there to have been no significant difference on the assessed water physicochemical variable values, which were considered satisfactory fot the species (Aride et al., 2007).

Physiological Parameters

Blood samples were collected, at the end of 90 days, by caudal vein puncture from five fish in each experimental unit, which had been previously anesthyzed with 0.25 mg/L of eugenol. The following hematological parameters were determined: hematocrit (Ht) by microhematocrit method; hemoglobin concentration ([Hb]) by the method of cyanmethaemoglobin with wavelength of 540 nm (BIOPLUS / model 2000); erythrocyte count (RBC) using Natt and Herrick solution (1952) and determined in a Neubauer chamber. From the values of RBC, Ht and [Hb], of each individual, RBC indices mean corpuscular Volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), were calculated with the following formula: MCV (fL) = Ht*10/RBC and MCHC (%) = [g/dL] *100/Ht (Wintrobe, 1934).

From the blood plasma were determined: glucose by enzymatic- colorimetric method (glucose oxidase), cholesterol and triglyceride levels by colorimetric system using a commercial kit and reading on a 490-510 nm wavelength spectrophotometer,

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respectively. Total proteins were obtained through the modified biuret method using a commercial kit and reading the absorbance in a 550-nm wavelength spectrophotometer. Cortisol was obtained by enzyme immunoassay technique by competing with measured absorbance microplate reader (Direct Elisa kit cortisol - USA Diagnostica®). Analyses were carried out at Laboratório de Fisiologia Aplicada à Piscicultura - LAFAP/INPA.

Statistical analysis

The experiment was conducted in a completely randomized design, consisting of five treatments (0.9; 1.2; 1.5; 1.8; 2.1; 2,4% lysine in the diet) and three replicates (a tank holding 25 fish was considered as an experimental unit). To ensure homogeneity in the beginning of the experiment, fish were sampled by weight and subjected to Cochran's test, with 5% level of significance. Zootechnical performance and physiological parameters data from each treatment, were subjected to analysis of variance (ANOVA), using the statistical package Statistical Analysis System - SAS (2008). When significant differences (p<0.05) were observed, Tukey test with a significance level of 5 %, was applied for comparison between means.

3 RESULTS

The survival index during the experimental period showed to be 100%. The results of zootechnical performance parameters of juvenile tambaqui fed diets with increasing levels of lysine, are shown in Table 5. With use of experimental diet, the fish had low food consumption and there was no significant effect (p> 0.05) of the treatments on the zootechnical parameters evaluated. Even with no significant difference among treatments, the fish treated with 1.8 % dietary lysine showed higher WG, PER, SGR%, PRE, FEI and MIFi values relative to the other treatments, besides presenting lower AAC value (Figure 3). Fish fed diets containing 2.1 and 2.4 % lysine presented the lowest values for weight gain and mean feed intake among the levels tested (Figure 1). Feed intake influenced weight gain in every treatment (Figures 1, 2 and 3).

The evaluated physiological parameters are shown in tables 6 and 7. There was no effect (p> 0.05) from the treatments on the plasma protein, glucose and MCV. Even without significant effect, the glycemic index showed to be higher (117.6 mg / dL) in the diet with 1.8 % lysine. CBR, Ht and [ Hb] values, presented cubic behavior when

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subjected to regression analysis. There was significant effect (p <0.05) on serum Cholesterol and triglycerides parameters, which tended to decrease as the lysine level increased. Fish fed on diet with 0.9 % lysine showed the highest cholesterol and triglycerides values, in addition to having higher ether extract content in their body composition (Tables 7 and 8). The hematimetric indexes MCH and MCHC showed the highest values for the diet containing 2.4 % lysine and MCHC showed quadratic behavior. The diet with 1.2 % lysine showed the highest plasma cortisol value among treatments, showing a tendency to decrease as the lysine level in the diet increased (Figure 4).

The proximal, tambaqui carcass composition values are shown in table 8. There was significant effect (p<0.05) for protein and ether extract values. There was no effect (p>0.05) on moisture contents.

Table 4. Zootechnical parameters of juvenile tambaqui, Colossoma macropomum subjeted to diet with differen lysine levels (%).

Different letters in the same line indicate significant diferences (p<0.05) by the Tukey test. Values expressed as means ± standard deviation. 1Protein efficiency rate; 2 Mean individual feed intake; 3 Dietary intake index; 4 Specific growth rate; 5 Dietary efficiency index; 6 Protein productive value; 7Hepatosomatic ratio; 8 Vicerosomatic ratio.

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Table 5. Hematological Parameters: hematocrit (Ht), red blood cell count (RBC), hemoglobin concentration ([Hb]), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH) of juvenile tambaqui, Colossoma macropomum, subjected to different diatary lysine levels.

Values expressed in means ± standard deviation; diferente letters in the same column indicate significant differences (p<0.05) by the Tukey test.

4 DISCUSSION

The survival of 100% of the fish and, there not having been observed any signs of external deficiency and erosion of the fins, corroborate other studies utilizing lysine in the diet (Dairiki et al., 2007; Abimorad et al., 2010; Dairiki et al., 2013; Ovie e Eze, 2013). According to Keembiyehetty and Gatlin (1992), lysine helps to produce anticodies and to prevent fins from eroding.

In this study, juvenile tambaqui, fed semipurified diets, exhibited reduced feed intake, low weight gains performance and high food conversion values. We found no pulished data pertaining to the use of this kind of diet for this fish species so as to base the comparison with the results herein obtained, however. In general, tambaqui displays excellent culture characteristics, when fed practical diets in coventional culture systems (Gomes et al., 2006; Rodrigues, 2014). Pacu, an omnivorous species with ecologic and morphometric characteristics similar to those of tambaqui, when fed semipurified diet, presented specific growth rate of 2.54 to 2.81% (Bicudo et al., 2009), higher than the one found in the present study (Table 5). On the other hand, the omnivorous jundiá, fed purified diet with 18% of free aminoacids, exhibited low weight gain and high dietary conversion (Montes-Girao and Fracalossi, 2006) similar to the findings attained in this study (Table 5).

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Table 6. Plasma biochemical parameters: total protein (TP), triglycerides (TR), total cholesterol (TC), plasma glucose (PG) and plasma cortisol of juvenile tambaqui, Colossoma macropomum, subjected to different lysine dietary levels.

Values expressed as means ± standard deviation; Different letters in the same line indicate significant differences (p<0.05) by the Tukey test.

In the present study, the use of free aminoacids corresponded to 18% of the diet, being within what is recommended by NRC (2011), which is, at most, 20% in diets for fish. Nevertheless, some studies indicate that the use of free aminoacids as a protein source in the diet does not promote the same performance obtained with intact proteins (Bureau e Encarnação, 2006), corroborating the findings presented for tambaqui. Some factors may be related to the low performace displayed by juvenile tambaqui, such as, the use of free aminoacids in the diet, low palatability of the purified ingredients, diet pH, feeding frequency and antagonism between lysine and argenine (Montes-Girao and Fracalossi, 2006; Bicudo et al., 2009; Dairiki et al., 2013; Ovie and Eze, 2013). According to Bureau and Encarnação (2006) and NRC, (2011), the use of either purified or semipurified diets provides, for some species, a lower weight gains mainly related to the use of free aminoacids and the low palatability of the ingredients utilized in those diets.

Some Authors suggest that the arginina: lisina ratio imbalace may affect animal performace (Furuya et al., 2012; Dairiki et al., 2013). The same can be observed in the study of Furuya et al. (2013) who utilized 1.43:1 arginina: lisina ratio in the diet for tilápia (O. niloticus) and found that ratio to have influenced the lysine requirement when compared to earlier studies undertaken with the same species, suggesting that ratio to be able to affect the animal’s nutritional need. In the present study, the highest and lowest arginine: lysine ratios showed to be 1.45:1 and 0.65:1 respectively, yet, the best zootechnical parameter values for tambaqui were observed with the 0.87:1 ratio, near to values (0.98:1 and 0.91:1) observed by Furuya et al. (2006) for the best weight gain with tilápia. In literature, that arginine: lysine ratio shows a wide variation for some species in

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different studies, with different nutritional requirement findings (Montes-Girao and Fracalossi, 2006; Bicudo et al., 2009; Abimorad et al., 2010; Gridale-Helland et al., 2011; Ovie Eze, 2013). The deficiency or escess of any one of the essential aminoacids may limit protein systhesis and, consequently, reduce weight gain, bringing about an imbalance in the relationship between them, and metabolic symptoms of toxicity, antagonism, antagonism or imbalacing, in addition to affecting intake rate, nutrients transport and catabolism (Portz; Furuya 2011).

Studies on the requirements of aminoacids (AA) in fish, are usually undertaken through dose-response experiments. However, these studies present variations on the type of diest, experimental design and methods being applied to attain nutritional demand values (Encarnação et al., 2004; Bureau and Encarnação, 2006; Abimorad et al., 2010; Grisdale-Helland et al., 2011; Dairiki et al., 2013; Ovie and Eze, 2013). According to Riche (2014), studies on fish aminoacids requirement should first observe a test diet that has the same performance as that observed in practical diets for the species, and that may serve as a standard for determining the AA demand. That Author, when developing a semipurified test diet for determining the aminoacids demand for Trachinotus carolinus, obtained good results using 200 g/kg of casein in the diet, supplemented with free aminoacids, to form the body profile of the species.

In the presente work, the diets with increasing levels of lysine did not influence the assessed zootechnical parameters, which would allow to estimate their lysine requirement through the dose-response curve. Nevertheless, the zootechnical performance of tambaqui was remarkably below what was expected with the use of semipurified diets for the species. One way of corroborating zootechnial performance data from fish fed with diets deficient in a particular nutrient is to assess its physiological coditions for better understanding the effects of the diet on the wellfare and health of the animal (Tavares Dias e Moraes, 2004; Affonso et al., 2007).

The physiological parameters Ht, RBC and [Hb], evaluated for tambaqui, presented significant diferences (p<0,05), yet, when subjected to regression analysis, they displayed a cubic effect on the lysine levels in the diet, not exhibing a specific standard on the lysine levels in the diet. These parameters are related to the carrying capacity of the blood oxygen and, under stress conditions undergo changes that can lead to anemia

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in fish, or blood hemodilution or hemoconcentration (Tavares-Dias and Moraes, 2004; Affonso et al., 2012).

Some studies suggest the lysine influence on the hematocrit, as decribed by Bicudo et al. (2009), who found high Ht values with the increase of the lysine level in pacu (P. mesopotamicus), however, the values attained for tambaqui do not indicate this trend. According to Tavares Dias and Moraes (2004), these parameters are good indicators of fish oxygen-carrying capacity, however, the changes observed in this study are likely related to the low diet intake and nutritional deficiency. For hemoglobin concentrations, they were similar to those observed for this species in other studies, suggesting that tambaqui maintained their hemoglobin production capacity, even with low production of red cells, pointed out by the low, observed values of hematocrit and RBC.

MCH and MCHC parameters presented cubic effect telative to the lysine levels in the diet, demonstrating a trend to increase with higer lysine levels. Mean MCH and MCHC values presented a trend to increase as the lysine level in the diet increase occurred, with the highest levels being observed in the diets with higher lysine level, which has not been described in literature as of yet. In general, the hematimetric indexes are directly related to the volume of erythrocytes, as well as the quantity of hemoglobin in the blood, and, thus, the values found in the present study demonstrate that the semiporified diet, in the initial phase of tambaqui, affected these hematologic indexes, with no specific standard with the tested diet, however.

Another way to evaluate the wellfare of the animal is by analyzing some biochemical parameters such as glucose and cortisol, which are important, fish stress indicators (Inoue et al., 2011). In the present study, the values of glucose show no diferences (p>0.05) among treatments, with the highest glicemic index (117.9 mg/dL) being found with the 1.8% lysine diet. These findings indicate that animal stress level, observed by glucose values, corresponds to the ability of the animal to adapt to the conditions employed in the different experiments. Cortisol, also known as stress hormone, is generally related to cronic exposures. In that study the plasma cortisol values showed a trend to reduce as the lysine in the diet increased, with the highest values being found for diet 2 and 1 respectively (Figura 6). The first three lysine levels had the highest cortisol levels and showed to be close to the values (182.1 and 333.8 ng/ml ) observed by Tavares

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- Dias et al. (2001 ) before and after capture and handling stress, respectively. Findings suggest lysine lower levels or deficiency to have influenced plasmatic cortisol values in fishes, indicating stress increase for the lowest levels.

The increase of protein in the carcass, with the inclusion of the lysine levels was not observed for tambaqui as described by Furuya et al. (2006) for tilapia, Bicudo et al. (2009) in pacu (P. mesopotamicus) and Prado, (2011) for surubim (Pseudoplatystoma spp). The highest body protein content was observed for the diet with 2.1 % lysine content and the lowest for the lowest level of lysine (Table 8). Yet the carcass lipid content tended to decrease as the lysine content in the diet increased, with the same effect being observed by Berge et al. (1998) and Bicudo et al. (2009), with rainbow trout (Oncorhynchus

mykiss), pacu (P. mesopotamicus) and Atlantic salmon (Salmo salar) respectively.

Nevertheless, Ovie and Eze (2013), assessing the lysine requirement for tilapia (O.

niloticus), observed the increase of body lipids with the increase of the lysine in the diet.

Dairiki et al. (2013) observed no significant differences for the body composition in juvenile dory (Salminus brasiliense) fed with rising lysine levels. Some sodies have reported the increase of protein and a reduction of body lipids in diets with rising lysine contents (Rodehutscord et al., 2000; Mai et al., 2006). This standard was not observed tambaqui, however. The values observed for the hepatosomatic index (Table 5), showed the same effect found for lipids, with a trend to come down with the rising lysine levels, being corroborated by the findings found by Bicudo et al. (2009) for pacu. According to Abimorad et al. (2010) a balanced aminoacids diet reduces its catabolism, and, consequently, increases the protein synthesis and diminishes the lipid reserves accumulation. According to these Authors, the reduction of lipids may be related to L-carnitine, wich is a substance synthesized from lysine and methionine, performing a major role in body fat burning. These findings corroborate the cholesterol and triglyceride values obtained from blood plasma, with higher values observed for the diet with the lowest level of lysine (Figures 7 and 8), suggesting that the lowest level of this amino acid implies higher body fat accumulation.

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Figure 1. Relationhip between dietary lysine levels (%) and weight gain of juvenile tambaqui Colossoma

macropomum.

Figure 2. Relationship between dietary lysine levels and mean individual feed intake of juvenile tambaqui

Colossoma macropomum. 35,00 39,00 43,00 47,00 51,00 55,00 59,00 0,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 2,3 2,5

M

IF

i

(g)

Dietary Lysine levels (% diet)

8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 0,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 2,3 2,5

WG

(g)

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Figure 3. Relationship between dietary lysine levels (%) and apparent feed conversion rate of juvenile tambaqui, Colossoma macropomum.

Figure 4. Relationship between dietary lysine levels and plasma cortisol of juvenile tambaqui, Colossoma

macropomum. 2,00 2,50 3,00 3,50 4,00 4,50 5,00 0,50 0,70 0,90 1,10 1,30 1,50 1,70 1,90 2,10 2,30 2,50

F

CR

Dietary Lysine levels (% diet)

y=-0.407x²+1.73x+228.48 R²= 0.65 0 50 100 150 200 250 0,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 2,3 2,5 2,7 P la sm a C o rt is o l (u g/d L)

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Even there being no significant diffrerences on zootechnical performance parameters among treatments, the diet with 1.8% lysine, displayed the highest values among them. According to the NRC (2011), the lysine nutritional requirements interval ranges from 1.2 to 3.3% lysine in the diet. Howsoever, just based on 1.8% diet and tambaqui body profile we estimated the lysine requiremeny fot the species, utilizing the methods proposed by Arai, (1981) and Abimorad & Castellani (2011), in which they found 6.0 and 6.2% lysine in protein values (Table 8) respectively.

Table 7. Proximate composition of the whole carcass of juvenile tambaqui subjected to rising dietary lysine levels.

Values expressed in means ± standard deviation.

Oliveira et al. (2011), utilizizing only the tambaqui body profile and the methodology proposed by Meyer and Fracalossi, (2005), estimated the aminoacids profile for tambaqui and obtained 7.2% lysine in the protein. Azevedo et al. (2012), also utilizing only the species body profile based on the ideal protein concept, estimated the lysine requirement for tambaqui to be 5.3% lysine in the protein. Bicudo et al. (2009), utilizing segmented regression model, determined the requirement for pacu (P. mesopotamicus) at 1.4-1.5% dietary lysine or 4.34-4.68% lysine in the protein. Abimorad et al. (2010) estimated the requirement at 1.64% dietary lysine or 7.3% dietary proteín for the same species, utilizing the same regression model. The values estimated by Bicudo et al. (2009) and Abimorad et al. (2010) are near those obtained for tambaqui in the present study, taking the phylogenetic proximity between the two species and the lysine requirement values from 1.2 to 3.3% dietary lysine reccomended by NRC, (2011), into account. Montes-Girao and Fracalossi (2006) estimated the lysine requirement for jundiá (R.

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quelem) at 1.36% dietary lysine or 4.5% dietary protein. Bomfim et al. (2010) estimated

the requirement for the Nile tilapia at 1.8% dietary lysine or 6.18% lysine in the protein, utilizing the diet supplemented with free amoniacids and 29,12% crude protein. Dairikiet

al. (2013) obtained the 5% lysine in the protein requirement for dory (S. brasilienses).

Dairiki et al. (2007) estimated the lysine requirement for the carnivorous “blackbass” (Micropterussalmoides) at 1,69% dietary lysine or 3.9% of the protein and Furuya et al. (2013) determined the digestible lysine requirement for the Nile tilapia at 1.31% dietary lysine, maintaining the arginine: lysine ratio constant.

Table 8. Essential amino acids requirement for tambaqui, estimated through the methods proposed by Arai (1981) and Abimorad & Castellani (2011), based on tambaqui body amino acids profile and the nutritional requirement of other species.

¹ Values obtained by Abimorad and Castellani. (2011); Oliveira et al. 2011; Abimorad et al. (2010); Montes-Girao and Fracalossi (2006); NRC (1993).

²Adaptad by Meyer and Fracalossi (2005).

The essential aminoacids profile estimated for tambaqui (Figure 9) showed to be similar to that observed for other species, just as the one found in literature for tambaqui (Montes-Girao and Fracalossi, 2006; Bicudo et al., 2009; Oliveira et al., 2011; Azevedo

et al., 2012). Nevertheless, further sudies on lysine and other aminoacids are necessary

so as to consolidate nutritional requirement data for tambaqui.

In general, lysine estimates suggested here are close to the values found for different species described in the literature and can be used as a reference for further studies on tambaqui nutrition. Yet, this is one of the first studies on tambaqui aminoacids requirement by using the semipurified diet, it being necessary to deepen the knowledge on that subjet, considering the diferent diets and lysine levels, for a higher procision of

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the data on the essential aminoacids requirement for tambaqui. In general, more than one tests are to be conducted so as to attain the nutritional requirement estimate.

5 CONCLUSION

With the findings obtained under the conditions employed in this study, we were unable to determine the lysine requirement, through the dose-response method, between the 0.9-2.4% dietary lysine interval with the use of a semipurified diet. Lysine estimates through the proposed methods, utilizing the ideal protein concept, showed to be similar to those found for other species. Tambaqui exhibited low feed intake with the use of semipurified diet. The semipurified diet with rising lysine levels, changed the hmatological parameters, yet these showed no specific pattern related to lysine. Body lipids accumulation found for lysine lower levels, suggests there being relationship between lysine and lipids, probably related to the production of L-carnitine, responsible for burning the fat on the tissues. This study will serve as a reference for the adjustment and formulation of a diet for providing better performance and enabling the determination of nutritional aminoacids requirements for tambaqui.

ACKNOWLEDGEMENTS

Thanks DARPA / FINEP and INCT- ADAPT / CNPq / FAPEAM project for thanked for its financial support and the National Council for Scientific and Technological Development - CNPq for the scholarship. We thank our coworkers in this study: Technicians, Scholarship students and Doctors who collaborated with the development of this project.

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Imagem

Table 1. Ingredients and chemical composition of experimental diets.
Table 2.Ingredients aminoacids composition and mixture (% DM).
Table 3. Essential and non-essential aminoacids composition in the experimental diets
Table 4. Zootechnical parameters of juvenile tambaqui, Colossoma macropomum subjeted to diet with  differen lysine levels (%)
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