Dietary digestible lysine requirement and essential amino acid to

Aquaculture Nutrition 2010 16; 370–377

doi: 10.1111/j.1365-2095.2009.00674.x

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1,2 1

1

1

1

UNESP – Universidade Estadual Paulista/CAUNESP – Centro de Aquïcultura, Jaboticabal, Saõ Paulo, Brazil; 2 APTA/SAASP – Ageˆncia Paulista de Tecnologia dos Agronegoćios/Po´lo Regional Noroeste Paulista, Votuporanga, Saõ Paulo, Brazil

To determine the digestible lysine requirement for pacu juveniles, a dose–response feeding trial was carried out. The fish (8.66 ± 1.13 g) were fed six diets containing the digestible lysine levels: 6.8, 9.1, 11.4, 13.2, 16.1 and 19.6 g kg)1 dry diet. The gradual increase of dietary digestible lysine levels from 6.8 to 13.2 g kg)1 did not influence the average values of the parameters evaluated (P > 0.05). The increase of dietary digestible lysine level to 16.1 g kg)1 significantly improved weight gain (WG), specific growth rate (SGR), protein productive value (PPV), protein efficiency rate (PER), and apparent feed conversion rate (FCR), but was not different from fish fed diets containing 19.6 g kg)1 lysine. Fish fed diets containing 16.1 and 19.6 g kg)1 digestible lysine showed lower body lipid contents than fish in the other treatments. The digestible lysine requirement as determined by the broken-line model, based on average WG values, was 16.4 g kg)1. The other essential amino acid requirements were estimated based on the ideal protein concept and the value determined for lysine. KEY WORDS: L-lysine,

body composition, digestibility, ideal protein, Piaractus mesopotamicus, white muscle

Received 9 October 2008, accepted 25 February 2009 Correspondence: APTA/SAA - SP - Ageˆncia Paulista de Tecnologia dos Agronegoćios/Po´lo Regional Noroeste Paulista, Votuporanga, Saõ Paulo, Brazil. E-mail: [email protected]

Pacu (Piaractus mesopotamicus) is a tropical climate migratory fish, native to the Basin that comprises the rivers Parana´, Paraguay, and Uruguay in South America (Saint-Paul 1986). Together with tambaqui (black-finned pacu) (Colossoma macropomum) and its hybrid tambacu (C. macropomum

$ · P. mesopotamicus #), production of these fish ranks third as the most cultivated species in Brazil, after tilapia and carp respectively (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renova´veis (IBAMA) 2005). Such importance stems from its rapid growth, omnivorous feeding behaviour, and excellent meat taste. Several researches on pacu nutrition have been conducted with regard to digestibility coefficients of energy and protein from several ingredients (Abimorad & Carneiro 2004), protein requirement (Merola 1988; Carneiro et al. 1994; Fernandes et al. 2000), non-protein energy levels and sources (Pezzato et al. 1992; Abimorad et al. 2007), and vitamin requirements (Martins 1995; Belo et al. 2005). However, few publications were found on its amino acid requirements (Munõz-Ramı´ rez & Carneiro 2002). The traditional methodology used to determine the amino acid requirements for fish is based on dose–response feeding experiments for each amino acid, which is costly and timedemanding (Small & Soares 1998). In 1964, with the ideal protein concept proposed by Mitchell for swine and poultry and, later discussed by Fuller et al. (1979), the all essential amino acids (EAA) requirements could be expressed as an ideal rate of a given amino acid in relationship to the EAA total in the animal tissue. Consequently, the amino acid profile of the skeletal muscle protein is the most used in researches to represent amino acid requirements, since that tissue is substantially formed during growth (Fuller et al. 1989; Small & Soares 1998; de la Higuera et al. 1999; Portz & Cyrino 2003; Abimorad et al. 2008). However, the muscle EAA profile only provides relative EAA values, and does not quantify the values to be used in the formulation of diets. A simpler alternative would be to determine the nutritional requirement of an essential amino acid, generally the most limiting one, to estimate the other amino acids requirements by of the ideal relationship between the EAA of muscle (Twibell et al. 2003; Wang et al. 2005). Researches have reached the conclusion that lysine is

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2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd

generally the first limiting amino acid in ingredients used for feed manufacturing, and is also the EAA found at the highest amounts in the carcasses of several fish species (Small & Soares 2000), including pacu (Munõz-Ramı´ rez & Carneiro 2002), and therefore it is the reference amino acid used in this type of study. Few studies have been conducted to determine digestible amino acid requirements in fish (Wang et al. 2005; Furuya et al. 2006). Knowledge about the availability of amino acids in diets is becoming increasingly important to formulate more efficient feeds. Therefore, the objective of this investigation was to determine the adequate dietary digestible lysine requirement for growth and protein utilization for pacu by the dose–response method, and estimate the other EAA requirements using the ideal protein concept.

This study was carried out at Aquatic Organisms Nutrition Laboratory from Aquaculture Center at Saõ Paulo State University (CAUNESP – Jaboticabal, SP, Brazil), over a 90-day period. A total of 108 pacu juveniles (8.66 ± 1.13 g) were distributed into 18 cubic fibre cement tanks (100 L), in a completely randomized design with six treatments (6.8, 9.1, 11.4, 13.2, 16.1 and 19.6 g kg)1 digestible lysine levels), three replicates, and six fish per experimental unit. The tanks were supplied with water from an artesian well, renovated at a rate of approximately 10 times a day. Each tank was supplied with aeration system as well as a system that allowed water to be drained directly from the bottom, to remove faeces and food residues; in addition, these were siphoned once a week. Mean water physicochemical parameter values, measured weekly, were as follows: 5.74 ± 0.38 mg L)1 dissolved oxygen, 202 ± 2.9 lS cm)1 electric conductivity, pH 8.04 ± 0.16, and temperature 29.6 ± 0.4 C. At the beginning of the study, 20 juveniles from the same population used in the experiment were sacrificed (benzocaine 0.2 g L)1) and stored in freezer for later determination of initial body composition. At the end of the study, six fish from each tank, after a 24-h fast, were weighed, sacrificed, and frozen to determine the body composition.

A basal diet (Table 1) was formulated to contain approximately 230 g kg)1 digestible protein and 14.3 MJ kg)1

digestible energy; maintaining the same concentration of non-protein digestible energy (Abimorad et al. 2007) and prioritizing a minimum digestible lysine level (6.8 g kg)1). The diets were either supplemented or not, with six levels of lysine: 0; 2.5; 5.0; 7.5; 10.0 and 12.5 g kg)1; the levels of all other EAA were maintained at the same proportion based on as in the muscle amino acid profile in relationship to the protein level of the basal diet (Tacon 1987). After finely ground, the ingredients in each diet were mixed manually for 10 min, adding distilled water (40%, v/w) little by little. The diets were processed in a meat grinder (CAF 22), forming 4–5 mm diameter granules, and dried in a forced air circulation oven at 45 C for 36 h. During the experimental period the fish were fed daily, twice a day (08:00 and 18:00 h), until apparent satiety.

A digestibility assay was carried out to determine the digestible protein, energy, amino acids values in each experimental diet. To accomplish that, 120 pacu juveniles (29.14 ± 4.97 g) were distributed into six feeding tanks (100 L) and fed experimental diets, added of 5.0 g kg)1 Cr2O3, for 5 days. GuelphÕs system (modified) was used to collect faeces. After the fish were transferred from the feeding tanks into the collection tanks, the faeces were collected repeatedly at 30-min intervals and were stored in a refrigerator. Such procedure was repeated three times at 1-day intervals, until the amount required for the analyses was achieved. ADC values were calculated by the following formula: ADC ¼ 100 100

% Cr2 O3 in diet % Cr2 O3 in faeces

% nutrient in faeces % nutrient in diet

The fish diet and carcass samples were analysed with duplicates for proximal composition. Determinations for dry matter, crude protein, lipids and ash were measured at CAUNESPÕs Aquatic Organisms Nutrition Laboratory, according to the Association of Official Analytical Chemists (AOAC) (2000) methodology. Gross energy was determined in Parr bomb calorimeter at UNESPÕs Animal Nutrition Laboratory, Jaboticabal, Brazil. Dietary and faecal total amino acids were measured by acid hydrolysis and ionic change chromatographic (HPLC) at ITALÕs Chemistry Center, Campinas, Brazil. The Cr2O3 concentrations in the diets and faeces were determined by nitric-perchloric digestion, according to Furukawa & Tsukahara (1966).

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2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 370–377

Table 1 Experimental diets

Digestible lysine levels (g kg)1) Diet Ingredients (g kg)1) Fish meal Soybean meal Corn gluten Yeast Corn Wheat bran Fish oil Dicalcium phosphate Limestone Salt (NaCl) DL-Methionine Threonine L-Lysine 78% Kaolin Vit/Min supplement* Analysed composition (g kg)1) Dry matter Crude protein Digestible protein Lipid Crude fiber Ash Nitrogen-free extract Gross energy (MJ kg)1) Digestible energy (MJ kg)1)

6.8

9.1

11.4

13.2

16.1

19.6

61.5 96.9 158.4 39.7 356.8 195.8 40.0 6.0 11.7 3.0 1.3 0.9 0.0 16.0 12.0

61.5 96.9 158.4 39.7 356.8 195.8 40.0 6.0 11.7 3.0 1.3 0.9 3.2 12.8 12.0

61.5 96.9 158.4 39.7 356.8 195.8 40.0 6.0 11.7 3.0 1.3 0.9 6.4 9.6 12.0

61.5 96.9 158.4 39.7 356.8 195.8 40.0 6.0 11.7 3.0 1.3 0.9 9.6 6.4 12.0

61.5 96.9 158.4 39.7 356.8 195.8 40.0 6.0 11.7 3.0 1.3 0.9 12.8 3.2 12.0

61.5 96.9 158.4 39.7 356.8 195.8 40.0 6.0 11.7 3.0 1.3 0.9 16.0 0.0 12.0

907 276 225 78 51 55 447 18.4 13.5

911 278 240 77 52 53 451 18.3 13.9

908 280 237 79 49 52 448 18.6 14.6

914 281 238 79 51 56 447 18.5 14.0

907 282 254 77 48 52 448 18.5 15.3

912 285 252 76 52 54 445 18.2 14.6

* (Ingredient kg)1 diet): Vitamins A = 600.000 IU; D3 = 24.000 IU; E = 600 IU; K3 = 120 mg; Thiamine = 180 mg; Riboflavin = 180 mg; Pyridoxine = 180 mg; B12 = 480 mcg; C = 1.800 mg; Folic Acid = 60 mg; Pantothenic acid = 480 mg; B.H.T. = 1.47 g; Biotin = 6.0 mg; Inositol = 120 mg; Nicotinamide = 840 mg; Choline = 4.8 g; Cobalt = 1.2 mg; Copper = 60 mg; Iron = 600 mg; Iodine = 6.0 mg; Manganese = 180 mg; Selenium = 1.2 mg; Zinc = 600 mg; Vehicle q.s. = 120 g.

The performance parameters were submitted to analysis of variance (one-way ANOVA), and differences between means were compared by DuncanÕs test (P < 0.05), using the Statistical Analysis System SAS v.9 software (SAS Institute Inc., Cary, NC, USA). The broken-line models were applied for the weight gain and apparent feed conversion to estimate the most adequate level of digestible lysine (Portz et al. 2000), using the PROC NLIN procedure in SAS.

No mortality or visible external pathological signs were observed in the fish during the experiment. After the determination of the ADC values, digestible protein, energy (Table 1) and amino acids values were calculated (Table 2). The performance results of pacu juveniles-fed different experimental diets are presented in Table 3. In general, the growth performance of this study is in line with other studies

with pacu, in similar experimental conditions. (Carneiro et al. 1994; Fernandes et al. 2000; Abimorad et al. 2007). There was no effect of the dietary treatments on feed intake (P > 0.05). The increasing in dietary digestible lysine level from 6.8 up to 13.2 g kg)1 did not significantly influence weight gain (WG), specific growth rate (SGR), and protein productive value (PPV). The increase in digestible lysine level to 16.1 g kg)1 significantly improved the means obtained for WG, SGR, PPV, PER and FCR, without statistical difference from fish fed-diets containing a higher dietary digestible lysine level (19.6 g kg)1). Fish fed diets containing 16.1 or 19.6 g kg)1 digestible lysine showed smaller body lipid and higher body moisture content relative to the other treatments. There was no effect of dietary treatments on the body ash and protein content (Table 4). The broken-line model was used to determine digestible lysine requirement, based on the mean values for weight gain and apparent feed conversion rate. The model estimated the optimal level for pacu juveniles as 16.4 g kg)1 to respond with greater weight gain, reaching a plateau at 51.15 g

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Table 2 Dietary crude and digestible essential (EAA) and non-essential (NEAA) amino acid values

Composition (g kg)1 dry matter) EAA Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine NEAA Aspartic acid Glutamic acid Alanine Cystine Glycine Proline Serine Tyrosine

Digestible lysine levels (g kg)1) 6.8

9.1

12.0 5.3 8.1 26.1 8.2 4.4 11.8 9.5 ND 9.4

(11.0) (4.7) (7.1) (24.3) (6.8) (4.0) (108) (8.4)

18.3 45.7 16.5 2.2 10.5 17.2 11.9 8.3

11.4

(11.0) (4.8) (7.2) (24.5) (9.1) (4.0) (10.9) (8.5)

(8.2)

11.9 5.3 8.1 26.1 10.4 4.4 11.8 9.5 ND 9.4

(16.1) (42.4) (148) (2.0) (9.1) (16.0) (10.7) (7.6)

18.2 45.6 16.5 2.2 10.5 17.2 11.9 8.2

13.2

(10.6) (4.4) (6.7) (23.3) (11.4) (3.7) (10.4) (8.1)

(8.4)

11.9 5.3 8.1 26.0 13.1 4.4 11.8 9.5 ND 9.4

(16.3) (42.8) (15.0) (2.0) (9.2) (16.2) (10.8) (7.6)

18.2 45.5 16.4 2.2 10.5 17.1 11.8 8.2

16.1

(10.9) (4.7) (7.1) (24.3) (13.2) (4.0) (10.8) (8.4)

(7.8)

11.9 5.2 8.1 26.0 14.5 4.4 11.8 9.4 ND 9.4

(15.6) (41.0) (14.0) (1.8) (8.7) (15.5) (10.3) (7.3)

18.1 45.4 16.4 2.2 10.5 17.1 11.8 8.2

19.6

(11.1) (4.8) (7.3) (24.7) (16.1) (4.0) (11.0) (8.6)

(8.3)

11.9 5.2 8.0 25.9 17.2 4.4 11.7 9.4 ND 9.3

(10.9) (4.7) (7.1) (24.4) (19.6) (4.0) (10.8) (8.3)

(8.5)

11.8 5.2 8.0 25.8 21.0 4.3 11.7 9.4 ND 9.3

(16.1) (42.4) (14.9) (2.1) (9.0) (16.3) (10.7) (7.6)

18.1 45.3 16.4 2.2 10.4 17.1 11.8 8.2

(16.5) (44.5) (15.2) (2.1) (9.4) (16.3) (10.9) (7.7)

18.1 45.1 16.3 2.2 10.4 17.0 11.7 8.1

(16.0) (42.4) (14.9) (2.0) (9.1) (16.1) (10.7) (7.5)

(8.2)

Crude AA (Digestible AA). ND, not determined.

Table 3 Performance of pacu juveniles-fed diets containing different digestible lysine levels Digestible lysine level (g kg)1) ANOVA

6.8 Mean initial weight (g) Mean final weight (g) Weight gain (g)1 Specific growth rate (% day)1)2 Feed intake (g/fish) Apparent feed conversion rate3 Protein efficiency rate4 Protein productive value (%)5

9.1 37.8 28.7 1.6

9.1 ± ± ± ±

0.8 7.5 b 7.8 b 0.3 b

8.1 40.8 32.7 1.8

11.4 ± ± ± ±

0.3 4.1 b 4.3 b 0.2 ab

8.6 45.38 36.8 1.9

13.2 ± ± ± ±

0.7 4.5 b 3.9 b 0.1 ab

8.8 49.5 40.7 1.9

16.1 ± ± ± ±

0.4 11.1 ab 11.0 ab 0.3 ab

9.0 62.1 53.1 2.2

P-values

19.6 ± ± ± ±

0.4 7.5 a 7.6 a 0.2 a

8.4 59.5 51.0 2.2

± ± ± ±

0.8 3.5 a 3.1 a 0.1 a

0.3747 0.0047 0.0048 0.0133

81.0 ± 6.4 3.1 ± 0.6 a

79.2 ± 4.0 2.7 ± 0.5 ab

77.6 ± 2.3 2.4 ± 0.7 ab

80.7 ± 3.1 2.5 ± 0.9 ab

79.8 ± 3.8 1.8 ± 0.5 b

71.7 ± 1.3 1.7 ± 0.3 b

0.0951 0.0355

1.3 ± 0.3 c 20.5 ± 3.9 c

1.5 ± 0.1 bc 23.3 ± 2.4 c

1.6 ± 0.1 bc 26.5 ± 3.6 c

1.8 ± 0.5 b 28.6 ± 7.2 bc

2.3 ± 0.2 a 36.2 ± 5.4 ab

2.5 ± 0.2 a 40.5 ± 2.1 a

0.0006 0.0008

Values are means of three replicates ± standard deviation. Means followed by different letters on the row are statistically different (Duncan P < 0.05). Homogeneity of variance for all parameters were >0.05 by Brown & ForsytheÕs test. 1 Weight gain (WG) = (final weight ) initial weight). 2 Specific growth rate (SGR) = (loge weight final ) loge weight initial) · 100/days. 3 Apparent feed conversion rate (FCR) = feed intake/weight gain. 4 Protein efficiency rate (PER) = weight gain/protein intake. 5 Protein productive value (PPV) = (Body proteinfinal · weight final) ) (Body proteininitial · weight initial) · 100/protein intake.

(Fig. 1), and 17.5 g kg)1 to respond with the best feed conversion rate, reaching a plateau at 1.68 (Fig. 2). Table 5 presents essential amino acid values in muscle of P pacu juveniles, EAA/ EAA ratio in muscle tissue (Arai 1981), and estimation of other digestible essential amino acids requirements by the ideal protein concept and the ADC of individual amino acids.

Most dose–response studies to determine amino acid requirements for fish use purified or semi-purified diets, which can be harmful for growth because they reduce intake (Berge et al. 2002), especially when they are deficient in essential amino acids, particularly lysine (Dabrowski et al.

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Table 4 Whole body composition of pacu juveniles-fed diets containing different digestible lysine levels Digestible lysine level (g kg)1) 6.8 9.1 11.4 13.2 16.1 19.6 P-value (ANOVA)

Composition (g 100 g)1 body weight)

Moisture 68.5 ± 0.5 67.3 ± 1.2 68.1 ± 0.3 67.5 ± 1.1 69.3 ± 1.4 70.0 ± 0.8 0.0061

bc c bc c ab a

Protein

Lipid

14.9 ± 0.4 15.3 ± 1.0 15.3 ± 1.0 15.1 ± 0.8 15.5 ± 0.8 16.0 ± 0.4 0.4203

12.8 ± 0.6 13.7 ± 1.0 13.5 ± 1.0 13.5 ± 1.4 12.0 ± 0.6 10.9 ± 0.5 0.0057

Ash ab a a a b b

3.8 ± 0.2 3.9 ± 0.2 3.8 ± 0.4 3.7 ± 0.1 3.6 ± 0.1 3.8 ± 0.2 0.5771

Values are means of three replicates ± standard deviation. Means followed by different letters in the column are different by DuncanÕs test (P < 0.05). Homogeneity of variance for all parameters were >0.05 by Brown & ForsytheÕs test.

65

Weight gain (g)

55

Y = 51.147 – 25.243 (16.4 -X) P = 0.0001 R2 = 0.96

45 35 25 15 5.0

X = 16.4

11.0 17.0 9.0 13.0 15.0 Digestible lysine levels (g kg–1)

7.0

19.0

21.0

Figure 1 Weight gain of pacu juveniles-fed diets containing different digestible lysine levels.

4.0

Food conversion rate

3.5

Y = 1.678 + 1.308 (17.5 –X) P = 0.0023 R2 = 0.95

3.0 2.5 2.0 1.5 1.0 5.0

X = 17.5 7.0

9.0 11.0 13.0 15.0 17.0 –1 Digestible lysine levels (g kg )

19.0

21.0

Figure 2 Apparent food conversion rate of pacu juveniles-fed diets containing different digestible lysine levels.

2007). In the present study, fish fed practical diets supplemented with synthetic amino acids and no effect on feed intake was observed. However, growth results indicated that lysine is indispensable for pacu juveniles, which were able to use L-lysine in practical diets. The digestible lysine requirement was determined by the broke-line model for weight gain and apparent feed conversion rate response, because it provided better model fitting to the data. Equation fitting was verified by the least sum of squared deviations, F test significance, and coefficient of determination (Shearer 2000; Murillo-Gurrea et al. 2001). The level determined for WG response (16.4 g kg)1) can be considered more adequate than the one determined for FCR response (17.5 g kg)1). The higher level determined for FCR response in relationship to the level determined for WG can be explained by the fact that a diet with higher concentration of a given nutrient, such as lysine, can provide better feed conversion rate, since no intake difference was observed (Maynard & Loosli 1974). On the other hand, to use more than 16.4 g kg)1 becomes unviable, due to the high cost of synthetic lysine and the low increment on WG. Therefore, it was considered that the most adequate dietary digestible lysine level for higher growth was 16.4 g kg)1, which corresponds to approximately 17.7 g kg)1 of crude lysine in the dry diet and 5.67 g 100 g)1 of dietary protein. Studies on amino acid requirements for fish are in general conducted using nutrients in their crude form. It is therefore difficult to make comparisons with the digestible lysine value determined in our study. Evaluation of optimal amino acids concentration in percent of the dietary protein is an additional way to make requirement data more comparable (Santiago & Lovell 1988; Wilson 2003; Liebert & Benkendorff 2007). Wang et al. (2005) evaluated the digestible lysine requirement of grass carp fry and estimated a 20.7 g kg)1 level for maximum growth, corresponding to 5.55 g 100 g)1 of dietary protein. Furuya et al. (2006) determined the digestible lysine requirement of Nile tilapia juveniles to be 14.4 g kg)1 for highest weight gain, which corresponded to 17.2 g kg)1 crude lysine in the dry diet and 5.23 g 100 g)1 in the protein fraction. The present study shows that the crude lysine requirement for pacu juveniles was 0.05% higher than for tilapia, but when the requirement is expressed as digestible values, pacu proved more efficient in utilizing dietary lysine. Lysine level in relation to dietary protein estimated for tilapia (5.23 g 100 g)1) by Furuya et al. (2006) is higher than the values found by Jackson & Capper (1982) and Santiago & Lovell (1988), who estimated requirements of 4.05 g 100 g)1 for Mozambique tilapia Oreochromis

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Table 5 Digestible essential amino acid requirements for pacu juveniles based on the ideal protein concept and the ADC of individual amino acids

g of EAA 100 g)1 pacu white muscle protein1 Arginine Phenylalanine Histidine Isoleucine Leucine Lysine Methionine Threonine Valine Cystine Tyrosine P EAA + Cys + Tyr

6.66 4.29 2.54 4.21 8.35 9.50 2.18 4.82 4.63 0.72 3.36 51.26

P EAA/ EAA in muscle · 10002

Ideal EAA profile3 (g kg)1)

Ideal digestible EAA profile4 (g kg)1)

129.92 83.69 49.55 82.13 162.90 185.33 42.53 94.03 90.32 14.05 65.55

12.4 8.0 4.7 7.8 15.6 17.7 4.1 9.0 8.6 1.3 6.3

11.6 7.4 4.4 7.2 14.6 16.4 3.8 8.2 7.9 1.2 5.8

1

Determined by Abimorad et al. (2008). Formula described by Arai (1981). 3 EAA requirements estimated by lysine requirement: (17.7/185.33) · result from AraiÕs 1981 formula. 4 Based on the ADC of individual amino acids. 2

mossambicus and 5.12 g 100 g)1 for Nile tilapia, respectively. In a study involving omnivorous catfish, Montes-Girao & Fracalossi (2006) estimated 4.50 g Lys 100 g)1 of dietary protein for jundia´ fry Rhamdia quelen. Tantikitti & Chimsung (2001) estimated 3.47 g Lys 100 g)1 of dietary protein for Asian Green catfish fry Mystus nemurus (Cuv. & Val.). Robinson et al. (1980) reevaluated the lysine requirement for Channel catfish Ictalurus punctatus, and recommended 5.0 g 100 g)1 of dietary protein. These differences maybe explained by several factors that interfere in this type of study, such as fish size, type of diet, dietary protein and energy concentrations, feeding frequency and quantity, and environmental conditions, among others. The dietary crude lysine level determined in this study (17.7 g kg)1) was near the value found for red sea bream juveniles Pagrus major by Forster & Ogata (1998), who estimated 17.3 g kg)1 lysine (3.6 g 100 g)1 of dietary protein). The low lysine proportion in relation to dietary protein content indicates a high protein content (480 g kg)1 CP), and consequently reveals the carnivorous feeding behaviour of the species under study. This suggests that comparisons should not be made between amino acid requirement values in species with distinct feeding behaviour, except when they are expressed as dietary protein. Otherwise, the result obtained in the present study was a little smaller than the result found for common carp by Zhou et al. (2008) (19.0 g kg)1 crude lysine in dry diet and 5.90 g 100 g)1 of dietary protein). This indicates that even among species considered to be omnivorous, the digestive processes (morpho-physiologic and biochemical) maybe distinct and lead to different results.

Several researches report smaller lipid accumulation and a slight increase of body protein content in fish fed diets supplemented with lysine (Zarate & Lovell 1997; Rodehutscord et al. 2000; Encarnaçaõ et al. 2004; Espe et al. 2007; Zhou et al. 2008). However, this effect was not observed by Anderson et al. (1992) in Atlantic salmon Salmo salar. Pacu has a tendency to accumulate lipid over its growth period due to its migratory behaviour; however, this depends on the feeding regime maintained during its life cycle. In another study on performance with pacu, Abimorad et al. (2007) evaluated the protein-sparring effect in juveniles fed dietary lipids and carbohydrates levels, and reported that fish that gained the most weight also accumulated more body lipid. In the present study, juveniles fed the highest levels of digestible lysine (16.1 and 19.6 g kg)1) showed higher PPV values and smaller body lipid content (Tables 3 and 4, respectively). This indicates that a better dietary amino acid balance probably prevents the selective catabolism of amino acids and consequently increases protein synthesis, while decreasing the accumulation of lipid reserves (Cowey & Sargent 1979; Benevenga et al. 1993; Tantikitti & Chimsung 2001; Ozo´rio et al. 2002; Conceiçaõ et al. 2003). This can occur due to lysine and methionine to be precursors to biosynthesis of the L-carnitine (Rebouche 1992). L-carnitine acts, theoretically, as a lipotropic factor, contributing to a reduction of body fat deposition (Dias et al. 2001). Another possible explanation is that a higher level of circulating lysine induces an anabolic process, by means of insulin release, increasing PPV and reducing body lipid by increasing lipolysis (Berne & Levy 1990).

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The ideal protein concept has been used as a method to estimate the other EAA requirements when only one EAA requirement is known, by the ideal rate of this amino acid relative to the EAA total in the fish tissues (Arai 1981). In the present study, in addition to estimating the other EAA requirements by the ideal protein concept, we also calculated the digestible EAA values by the ADC of individual amino acids. It was observed that the lysine requirement (17.7 g kg)1 dry diet, 5.67 g 100 g)1 dietary protein or 14,9 mg Lys fish)1 day)1), in pacu of 8–50 g body weight, was not substantially different from the requirements of other species with omnivorous feeding behaviour. The adequate dietary digestible lysine level (16.4 g kg)1) was determined based on lysine availability in the experimental diets. The other digestible EAA requirements estimated by ideal protein concept will allow the preparation of diets with an adequate amino acid balance into a minimum dietary digestible protein level to maximize growth, protein utilization efficiency, and carcass quality in pacu.

We would like to thank Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP) for granting a Doctoral Scholarship to the first author, proceeding no. 04/06060-6 and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (MCT/CNPq) for financial support, Universal Public Notice 019/2004 – proceeding no. 476281.

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