Aquaculture 296 (2009) 81–86
Contents lists available at ScienceDirect
Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
The optimum dietary essential amino acid profile for gilthead seabream (Sparus aurata) juveniles Helena Peres ⁎, Aires Oliva-Teles CIMAR/CIIMAR — Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal Departamento de Zoologia e Antropologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Edifício FC4, 4169-007 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 4 March 2009 Received in revised form 27 April 2009 Accepted 30 April 2009 Keywords: Ideal protein Essential amino acid Nitrogen retention Gilthead seabream
a b s t r a c t A study was undertaken to establish the optimum dietary essential amino acid (EAA) profile for gilthead seabream juveniles based on the amino acid (AA) deletion method. For that purpose 11 diets were formulated to be isonitrogenous (6.72% N) and isolipidic (18%). In the control diet half of the nitrogen was provided by protein bound-AAs (fish meal) and the other half by a mixture of crystalline L-AAs. The overall AA profile of this diet was made similar to that of fish meal protein. Ten other diets were formulated identical to the control except for the deletion of 45% of a single EAA in each diet and by adjusting the nitrogen level with a non-essential AA mixture. Each diet was assigned to four groups of 22 fish (initial body weight of 4.6 g) and the trial lasted 43 days at a water temperature of 25 °C. Fish were fed by hand, twice daily, using a pair-feeding scheme. At the end of the trial the relationship between nitrogen gain and AA intake of the test and control diets was determined. Based on these data, and assuming that nitrogen retention responds linearly to dietary EAA content when a given AA is limiting, the quantity of each EAA that can be removed from the control diet without affecting nitrogen retention was computed and the ideal dietary EAA profile for gilthead seabream juveniles was estimated. Expressed relative to lysine (= 100) A/E ratios were estimated to be: arginine, 108.3; threonine, 58.1; histidine, 36.8; isoleucine, 49.7; leucine, 92.7; methionine, 50.8; phenylalanine + tyrosine, 112.3; valine, 62.6; and tryptophan, 14.6. This EAA profile correlates tightly to the whole-body EAA composition of gilthead seabream (R2 = 0.99; p N 0.001). © 2009 Published by Elsevier B.V.
1. Introduction Formulation of diets with an optimum essential amino acid (EAA) profile and adequate protein content is a prerequisite for improving amino acid (AA) utilization for growth, and thereby reducing nitrogen (N) excretion. This is particularly important in carnivorous fish, like gilthead seabream, which uses protein preferentially to lipids or carbohydrates as an energy source (Peres and Oliva-Teles, 1999). The classic approach for estimating EAA requirements of a given species implies performing several dose–response experiments for establishing the requirements of each EAA for growth and maintenance and available data are limited (Mambrini and Kaushik, 1995a). As an alternative, estimation of the EAA requirements based on the ideal protein concept is as a highly versatile approach that is being used extensively in pigs (Boisen, 2003) and poultry (Baker, 2003). Indeed, this approach allows that modifications in requirements for all EAA related to animal age, environmental conditions or ⁎ Departamento de Zoologia e Antropologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Edifício FC4, 4169-007 Porto, Portugal. Corresponding author. Tel.: +351 220 402 736; fax: +351 22 339 06 08. E-mail address:
[email protected] (H. Peres). 0044-8486/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2009.04.046
different diet protein to energy ratios can be easily corrected by evaluating changes in the requirement of a single amino acid. The optimum dietary EAA profile that meets maintenance and production needs and maximizes N retention is often called “ideal protein” (Boisen, 2003). Knowing the ideal protein, each EAA can be computed as a proportion of the total EAA amount (A/E ratios) and its quantitative requirement estimated based on the determined requirement of a sole EAA. For consistent results with this approach it is required that reliable information on the optimum EAA profile for the animal is obtained and that the requirement of the EAA used as reference (usually lysine) is accurately established (Boisen, 2003). In fish, EAA profiles of whole-egg protein, ovarian tissue, eggs, muscle and whole-fish carcass were considered by different authors as ideal proteins (Wilson, 2003). Among them, carcass composition seems to best fit the dietary EAA requirements profile and is generally used as the starting point for establishing an ideal protein, although it does not take into account the dynamics of the living animal such as maintenance costs (Mambrini and Kaushik, 1995a). For gilthead seabream juveniles, an estimation of the dietary EAA requirements was already advanced by Kaushik (1998) based on the A/E ratio of wholebody composition and the lysine requirement estimated by Luquet and Sabaut (1974).
82
H. Peres, A. Oliva-Teles / Aquaculture 296 (2009) 81–86
The deletion method is a rapid and accurate technique to assess the ideal EAA profile, which was initially developed for pigs, and has recently been applied in salmonids (Green and Hardy, 2002; Rollin et al., 2003). This technique is based on the concept that each EAA is equally limiting to protein accretion (Wang and Fuller, 1989) and therefore changes in N gain due to partial removal of one EAA in turn in an otherwise balanced diet will provide an estimation of the optimum EAA profile. Compared to salmonids, the protein requirement of gilthead seabream is higher (Oliva-Teles, 2000) while protein productive value is lower (Tibaldi and Kaushik, 2005). This may be species-specific or may have a nutritional basis; thus, it is expected that a better estimation of the amino acid (AA) requirements will improve the efficiency of N utilization (Tibaldi and Kaushik, 2005). Therefore, the main objective of this study is to establish the optimum dietary EAA profile for gilthead seabream juveniles by the deletion method approach. 2. Material and methods 2.1. Experimental diets Eleven experimental diets were formulated to be isonitrogenous (6.72% N, DM basis) and isolipidic (18%, DM basis). Dietary N was fixed just below the estimated optimum requirement for growth of gilthead seabream (Oliva-Teles, 2000) to ensure maximum utilization of the limiting EAA, and a relatively high lipid level was chosen to ensure no energy limitation. The control diet was formulated to contain half of its N provided by protein bound-AAs (fish meal) and the other half provided by a crystalline L-AAs mixture. The overall AA profile of this diet was made similar to that of fish meal protein, according to Peres and Oliva-Teles (2007). Ten other diets were formulated identical to the control but with the deletion of 45% of a single EAA. N level in
these diets was adjusted by adjusting the non essential amino acid (NEAA) mixture level. Before mixing with the other ingredients crystalline-AAs were coated with agar, to avoid leaching and to delay absorption from the digestive tract. Dietary ingredients were thoroughly mixed and dry- pelleted in a laboratory pellet mill (CPM, California Pellet Mill), through a 2-mm die. Pellets were air dried at room temperature and stored in sealed containers in the refrigerator until used. Data on ingredients and proximate composition of the experimental diets is presented in Table 1 and that of AA composition in Table 2. 2.2. Experimental fish The experiment was performed at the Marine Zoology Station, at University of Porto, with gilthead seabream (Sparus aurata) juveniles obtained from a commercial hatchery. The growth trial lasted 43 days and was performed using two thermoregulated recirculation water systems. Each system contained a battery of 22 fibreglass tanks of 100-l capacity each, supplied with a continuous flow of filtered seawater (2–3.0 l/min). During the trial, water temperature averaged 25 ± 0.1 °C, salinity averaged 34 ± 1‰, and dissolved oxygen averaged 95% of saturation. Photoperiod was the natural one for spring. Thirteen hundred fish were randomly stocked in both systems and acclimatized for 15 days to the tanks and water temperature. Thereafter, 44 groups of 22 fish with an average body weight of 4.6 g were randomly distributed to each tank. Each experimental diet was randomly assigned to quadruplicate groups in a randomized complete block design, i.e., two groups in each water system. During the first 4 days of the experimental period, all groups were fed to visual satiation and the lowest feed intake value was determined. This value was used to subsequently pair-feeding all the
Table 1 Ingredient composition and proximate analysis of the experimental diets. Diets C
ARG
LYS
THR
HIS
ILE
LEU
MET
PHE
TRP
VAL
Ingredients (% dry weight) Waxy corn starcha Fish mealb Soluble fish protein concentratec Cod liver oil Constant componentsd Dibasic calcium phosphate L-arginine L-lysine-HCl L-threonine L-histidine-HCl L-isoleucine L-leucine L-methionine L-phenylalanine L-tryptophan L-valine NEAA mixtureg
25.0 29.0 1.0 14.6 4.5 4.67 1.99 1.23 0.84 0.67 0.73 1.17 0.69 1.94 0.21 0.78 11.0
21.4 26.7 1.0 14.9 4.5 4.95 – 1.38 0.92 0.72 0.82 1.32 0.76 2.07 0.23 0.87 17.5
24.6 26.7 1.0 14.9 4.5 4.95 2.13 – 0.92 0.72 0.82 1.32 0.76 2.07 0.23 0.87 13.6
24.7 26.7 1.0 14.9 4.5 4.95 2.13 1.38 – 0.72 0.82 1.32 0.76 2.07 0.23 0.87 13.0
23.8 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 – 0.82 1.32 0.76 2.07 0.23 0.87 13.7
24.8 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 0.72 1.32 0.76 2.07 0.23 0.87 12.8
24.9 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 0.72 0.82 – 0.76 2.07 0.23 0.87 13.2
24.9 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 0.72 0.82 1.32 – 2.07 0.23 0.87 12.7
25.4 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 0.72 0.82 1.32 0.76 – 0.23 0.87 13.5
24.7 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 0.72 0.82 1.32 0.76 2.07 – 0.87 12.4
24.7 26.7 1.0 14.9 4.5 4.95 2.13 1.38 0.92 0.72 0.82 1.32 0.76 2.07 0.23 – 12.9
Proximate analyses (% dry weight) Dry matter (%) Total nitrogen Crude fat Ash
92.8 6.80 18.5 10.8
92.7 6.76 19.1 11.4
91.8 6.70 18.9 10.9
90.9 6.80 17.7 11.0
92.6 6.80 18.1 11.0
92.9 6.85 17.7 10.9
97.9 6.71 17.6 10.9
94.6 6.75 17.7 10.9
94.4 6.89 17.8 10.9
92.9 7.01 17.7 10.8
97.9 6.74 17.9 10.8
a
Cerestar, France (≈ 99% amylopectin). Prime Quality Fish Meal, Triple Nine, Denmark (CP: 77.2% DM; GL: 11.1% DM). c Sopropêche, France (CP: 75.8% DM; GL: 18.8% DM). d Constant components (% of diet): vitamin mixe, 1.0; mineral mixf, 1.0; choline chloride (50%), 0.5; carboxy methil cellulose, 1.0; and agar, 1.0. e Vitamins (mg kg− 1 diet): retinol, 18000 (IU kg− 1 diet); calciferol, 2000 (IU kg− 1 diet); alpha tocopherol, 35; menadion sodium bis., 10; thiamin, 15; riboflavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbyl monophosphate, 50; and inositol, 400. f Minerals (mg kg− 1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; potassium chloride, 1.15 (g kg− 1 diet); sodium chloride, 0.40 (g kg− 1 diet); dibasic calcium phosphate, and 5.9 (g kg− 1 diet). g Non-essential amino acids mixture (% mixture): L-alanine: 13.37; L-aspartic acid: 19.68; sodium glutamate: 32.29; L-glycine: 14.83; L-serine: 9.66; and L-proline: 10.17. b
H. Peres, A. Oliva-Teles / Aquaculture 296 (2009) 81–86
83
Table 2 Amino acid composition (g 16 g− 1 N) of the experimental diets. Diets Arginine Lysine Histidine Isoleucine Leucine Valine Methionine Phenylalanine Threonine Tyrosine Aspartic acid Glutamic acid Serine Glycine Alanine Proline
C
ARG
LYS
THR
HIS
ILE
LEU
MET
PHE
TRP
VAL
7.56 6.73 2.16 4.19 7.47 4.86 2.96 6.21 3.01 1.60 7.90 12.9 5.04 9.41 8.29 4.13
4.29 6.08 2.56 4.20 7.73 4.94 3.44 6.14 3.33 1.64 10.05 13.1 5.39 9.32 8.02 5.32
8.12 3.47 2.57 4.35 7.56 4.81 3.33 6.24 3.49 1.60 9.42 13.1 5.00 8.03 7.69 4.64
8.50 6.54 2.64 4.37 7.58 4.56 3.16 6.16 1.99 1.67 8.83 13.6 4.93 7.82 7.83 4.45
8.22 6.22 1.48 4.10 7.63 4.40 3.23 6.00 3.40 1.64 8.11 14.2 4.95 7.90 7.88 4.72
8.54 6.26 2.59 2.10 7.35 4.56 3.26 6.28 3.49 1.66 8.81 13.3 5.25 8.04 8.05 4.69
8.68 6.50 2.76 4.33 3.84 4.59 3.39 6.13 3.55 1.67 9.74 14.0 5.77 7.14 7.90 4.84
8.47 6.26 2.69 4.12 7.74 4.53 1.89 6.32 3.53 1.69 8.76 13.0 5.20 8.57 7.67 4.33
8.74 6.69 2.87 4.22 7.38 4.43 3.19 2.33 3.56 1.66 9.73 14.0 4.65 9.48 8.03 4.60
8.79 6.14 2.85 4.59 7.54 4.67 3.45 6.58 3.49 1.62 9.17 12.7 4.50 7.30 7.40 5.06
8.79 6.37 3.23 4.07 7.70 2.40 3.42 6.17 3.46 1.58 9.05 13.1 4.65 7.59 7.54 4.90
groups during the rest of the experimental periods. During that period, fish were fed by hand twice a day, 6 days a week. Feed amount was adjusted so that all tanks received the same amount of N as per cent of tank biomass. Utmost care was taken to avoid feed waste and to assure that all feed supplied was consumed. Fish were group weighed at the start, after 2 weeks and at the end of the trial, following 1 day of feed deprivation. On day 14, feed supply was readjusted according to tank biomass.
package by comparing to a known amino acid standard (Pierce NC10180). Tryptophan and cystine were not measured in this study. 2.4. Data analysis Based on whole-body composition analysis, daily N retention (mg N kg− 1 MBW or % N intake) was estimated as follows: −1 −1 Daily N retention mg N kg MBW day = ðFBW × FBN − IBW × IBNÞ 0:75 0:75 = IBW + FBW = 2 × days
2.3. Diets and body composition analysis Chemical analysis of the experimental diets and whole-fish were made in triplicate according to the following procedures: dry matter after drying in an oven at 105 °C until constant weight; ash by incineration in a muffle furnace at 450 °C for 16 h; protein (N × 6.25) by the Kjeldahl method after acid digestion using a Kjeltec system and lipid by petroleum ether extraction in a Soxtec System HT apparatus. The samples for 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 (Waters auto sample model 717 plus; Waters binary pump model 1525; Waters dual absorbance detector model 2487), according to the Pico-Tag method as described by Cohen et al. (1989). Chromatographic peaks were identified, integrated and quantified with a Waters Breeze software
N retention ðkN Þ = ðFBW × FBN − IBW × IBNÞ = ðNIÞÞ × 100 where MBW is average metabolic body weight; IBW and FBW are initial and final body weights; IBN and FBN are initial and final body N content; NI is nitrogen intake. Estimation of the optimum dietary EAA profile was done according to Wang and Fuller (1989). It was assumed that: 1) removal of the first limiting AA in the diet will reduce N retention to the greatest extent; 2) if removal of a dietary AA does not reduce N retention, then the quantity removed is in excess relatively to the first limiting AA; 3) if removal of a dietary AA results in an N retention intermediate between 1) and 2) then the proportion that could have been removed without affecting N retention can be interpolated proportionally.
Table 3 Effect of deleting single amino acids from the diet on growth performance, feed utilization efficiency and mortality of gilthead seabream juveniles.a Diets Initial body weight (g) Final body weight (g) Weight gain (g kg ABW⁎− 1 day− 1) Specific growth rate (%)b Feed efficiency ratioc Protein efficiency ratiod Mortality (%) N intake (g kg MBW†− 1 day− 1) N retention (g kg MBW†− 1 day− 1) N retention (% N intake) a
C
ARG
LYS
THR
HIS
ILE
LEU
MET
PHE
TRP
VAL
SEM
4.64 16.1f 25.6d 2.88f 0.84f 1.97f 0.0 3.85bc 1.21e 31.3d
4.63 14.3e 23.8cd 2.62e 0.77ef 1.82ef 0.0 3.77ab 1.01bcd 26.7c
4.63 13.7cde 22.9bc 2.51cde 0.74de 1.76de 0.0 3.71a 0.91abc 24.5abc
4.63 11.2a 19.3a 2.05a 0.58a 1.35a 2.27 3.89bc 0.81a 20.9a
4.64 12.9bc 21.9abc 2.37bc 0.65bc 1.54abc 2.27 4.02d 0.98bcd 24.4abc
4.64 14.2de 23.6c 2.60de 0.75de 1.76de 0.0 3.85bc 1.08de 28.0cd
4.64 13.5cde 22.8bc 2.49cde 0.70cde 1.67cde 2.27 3.88bc 1.06cde 27.3cd
4.63 12.1ab 20.7ab 2.22ab 0.62ab 1.46ab 0.0 3.96cd 0.80ab 22.3ab
4.63 12.9bc 22.0bc 2.39bcd 0.68bcd 1.57bc 0.0 3.93cd 0.95abcd 24.2abc
4.64 13.2bcde 22.3bc 2.43bcde 0.68bcd 1.56bcd 1.14 3.95cd 0.98bcd 24.9abc
4.64 13.1bcd 22.1bc 2.40bcd 0.67bcd 1.60bcd 1.14 3.93cd 1.01bcd 25.7bc
0.00 0.36 0.45 0.06 0.02 0.05 1.01 0.03 0.03 0.88
Means in the same row with different superscript letters are significantly different (P b 0.05). SEM: pooled standard error of the mean; N: nitrogen. SGR: ((ln(final body weight) − ln(initial body weight)) / (time in days)) × 100. FE: (wet weight gain/dry feed intake). d PER: (wet weight gain/crude protein intake). ⁎ABW: Average body weight: (initial body weight + final body weight) / 2. † MBW: Metabolic body weight: (initial body weight^0.75 + final body weight^0.75) / 2. b c
84
H. Peres, A. Oliva-Teles / Aquaculture 296 (2009) 81–86
Following these assumptions, the quantity of each EAA that can be removed from the control diet without affecting N retention was calculated as: NRTest −1 requirement g 16g N = ðEAAÞControl × 2 − DEL − NRControl where (EAA)Control is the EAA content in the control diet; DEL is the measured deletion rate of the EAA in the test diets; NRTest is N retention (mg N kg− 1 MBW day− 1) in the test diet and NRControl is N retention in the control diet (mg N kg− 1 MBW day− 1). As tryptophan was not measured in this study, the tryptophan requirement value was computed based on the estimated tryptophan content of control and TYP diets. Statistical evaluation of the data was done by analysis of variance according to a randomized complete block design, using as a block the two recirculation water systems. The probability level for rejection of the null hypothesis was 0.05. Significant differences among means were determined by the Tukey's multiple range test. Statistical analysis was done using the SPSS 17.0 for Windows software package. 3. Results All experimental diets were well accepted by the fish. No pathological signs were observed during the trial and mortality was very low and unaffected by dietary treatment (Table 3). As fish were pair-fed, feed intake was similar in all groups and averaged 42.4 g kg BW− 1day− 1. Results of the single dietary EAA deletion on growth performance and feed utilization efficiency are presented in Table 3. Overall, high growth rates and feed efficiency were observed in all groups during the trial. Deletion of EAA significantly reduced growth performance, but the extent of growth depression depended on the EAA removed. Deletion of threonine led to the highest growth reduction while deletion of arginine, lysine, isoleucine, leucine and tryptophan led to the lowest growth reduction. Except for arginine, feed efficiency and protein efficiency ratio were also significantly reduced by EAA deletion. EAA deletion significantly reduced daily N retention (g kg MBW− 1 and % N intake) except for isoleucine and leucine. Similar to growth performance, deletion of threonine promoted the greatest reduction in N retention, followed by methionine. At the end of the trial there were no differences in whole-body composition of the experimental animals (Table 4) except for protein, which was significantly lower in fish fed the diet with lysine deletion than the control or diets with leucine or valine deletion, and for ash which was lower in fish fed the diet with arginine deletion compared to diets with threonine or methionine deletion. The relationship between N retention and AA intake for the control and the experimental diets is presented in Fig. 1. Based on these data and assuming a linear response between N retention and EAA intake when a given AA is limiting, the quantity of each EAA that can be removed from the control diet without affecting N retention was computed. From these data, and assuming that each EAA is equally limiting, the ideal dietary EAA profile expressed as A/E ratios relative to lysine
Fig. 1. Effect of deleting 45% of each essential amino acid from the control diet on nitrogen retention of gilthead seabream juveniles.
(=100) was estimated to be: arginine, 108.3 ± 18.96; threonine, 58.1 ± 1.96; histidine, 36.8 ± 1.95; isoleucine, 49.7 ± 2.47; leucine, 92.7± 3.96; methionine, 50.8 ± 1.52; phenylalanine + tyrosine, 112.3 ± 4.82; valine, 62.6 ± 4.37; tryptophan, and 14.6± 1.08. Expressed as g/16 g N, the optimal balance was estimated as: arginine, 5.55± 0.97; lysine, 5.13 ± 0.73; threonine, 2.98 ± 0.1; histidine, 1.89 ± 0.1; isoleucine, 2.55± 0.13; leucine, 4.75 ± 0.2; methionine, 2.60± 0.08; phenylalanine+ tyrosine, 5.76 ± 0.25; valine, 3.21 ± 0.22; and tryptophan, 0.75± 0.06. 4. Discussion The deletion method is generally accepted as an efficient and rapid tool to estimate the ideal EAA profile of a given species (Boisen, 2003; Baker, 2004). This method was initially outlined by Wang and Fuller (1989) in pigs and is based on the concept that each EAA is equally limiting to protein accretion. In fish, estimation of the ideal dietary EAA profile by the deletion method was already applied in salmonids (Green and Hardy, 2002; Rollin et al., 2003). Comparing these EAA profiles to the one estimated in the present study denotes only small differences among them (Fig. 2): rainbow trout/seabream: R2 = 0.93; p b 0.001 (Green and Hardy, 2002) and salmon/seabream: R2 = 0.87; p b 0.001 (Rollin et al., 2003). The highest difference among species is in arginine followed by histidine and threonine, which are higher in gilthead seabream than in salmon, and in methionine which is higher in salmon than in gilthead seabream. Using the ideal protein concept, Kaushik (1998) estimated the EAA profile for gilthead seabream and values obtained (Fig. 2) are very consistent to those estimated in the present study (R2 = 0.99; p b 0.001). In fact, different proteins have been proposed as best indicators of the optimum dietary EAA profile in fish, and among them the EAA pattern of whole fish carcass seems to best fit the requirements profile (Wilson and Cowey, 1985; Mambrini and Kaushik, 1995a; Rollin et al., 2003). A good correlation between the ideal dietary EAA profile and that of whole-body EAA profile is indeed to be expected in fast-growing animals in which a great proportion of EAA are used for protein deposition, while only a
Table 4 Whole-body composition (wet-weight basis) of gilthead seabream fed the different experimental diets.a Diets Dry matter (%) Protein (%) Lipid (%) Ash (%) a
Initial
C
ARG
LYS
THR
HIS
ILE
LEU
MET
PHE
TRP
VAL
SEM
26.1 14.6 7.63 4.46
29.3 15.6b 10.5 4.33ab
28.4 14.6ab 10.2 4.17a
27.8 14.1a 10.2 4.36ab
28.3 15.1ab 9.88 4.66b
28.0 15.5ab 9.36 4.54ab
29.2 15.4ab 10.2 4.39ab
29.0 15.8b 9.54 4.50ab
29.1 15.1ab 10.5 4.66b
28.9 15.1ab 10.2 4.47ab
28.8 15.2ab 10.5 4.52ab
29.6 15.6b 9.90 4.54ab
0.648 0.28 0.36 0.08
Means in the same row with different superscript letters are significantly different (P b 0.05). SEM: pooled standard error of the mean.
H. Peres, A. Oliva-Teles / Aquaculture 296 (2009) 81–86
85
Fig. 2. EAA profile of gilthead seabream juveniles determined in this study by the deletion method and by Kaushik (1998) based on whole-body EAA composition, and those determined for rainbow trout (Green and Hardy, 2002) and Atlantic salmon (Rollin et al., 2003) by the deletion method. EAA profile presented relatively to lysine level (= 100).
residual AA proportion is used for meeting maintenance requirements. Even though, some differences in EAA requirements for growth and for maintenance are to be expected, as it was already shown for arginine in gilthead seabream, European sea bass, turbot and rainbow trout (Fournier et al., 2002). In this study, the magnitude of the effects resulting from the individual EAA deletion diet was different according to the EAA tested. Deletion of threonine led to the highest reduction of growth and N retention, clearly indicating that threonine was the first limiting EAA in the control diet and methionine was the second limiting EAA. Deletion of isoleucine and leucine significantly reduced growth but not N retention. Partial deletion of each EAA from the control diet reduced N retention between 11 and 33%. This proportion is clearly lower than the dietary deletion of each EAA (45%). The deletion method relies on different assumptions which may limit its application; however, consolidation of the discrete scientific findings on AA nutrition in fish is needed to better clarify the potentialities of this methodology. First, the deletion method relies on the assumption that N retention is a linear function of the dietary EAA content when a particular EAA is limiting. Therefore, a deviation from this linearity may induce errors in the estimation of the ideal EAA profile. However, even though it was suggested that the utilization of a limiting EAA best-fits a non-linear model (Mercer et al., 1989; Shearer, 2000), a linear model generally gives adequate fitting and provides good estimation of the EAA requirements (Rodehutscord and Pack, 1999). Indeed, Wilson (2003) reviewing previously conducted studies concluded that the broken-line model was the most used in dose– response studies of EAA requirements, as linear increases in weight gain or N retention are usually observed with increasing EAA intake up to a break point. The deletion method also assumes that the efficiency of EAA utilization is constant irrespectively of its dietary level. In terrestrial animals, such assumption is generally accepted up to the point where the requirement is met (Baker, 2004). In fish, this subject has been much less studied. Recent studies suggest that efficiency of EAA utilization may be different depending on the EAA considered and its level in the diet, being higher for the most limiting EAA (Rodehutscord et al., 1995; Ronnestad et al., 2001, Rollin et al., 2003; Peres and OlivaTeles, 2008). Another assumption of the deletion method is that maintenance requirement is identical for each EAA. Very little is known regarding EAA requirements for maintenance in fish. Though, as overall N requirement for maintenance are very small compared to growth requirement, overall EAA requirement for maintenance must also be small. However, evidence suggests that some EAA may be needed in greater quantities for maintenance than for growth while the opposite may be true for other EAA (Cho et al., 1992; Mambrini and Kaushik, 1995b; Rodehutscord et al., 1997; Fournier et al., 2002). This will imply different EAA profiles for growth and for maintenance. In the present study fish growth was high therefore it is expected that the effect of maintenance on total EAA profile would be minimal.
Application of the deletion method implies formulating diets using crystalline-AAs to precisely modify the EAA composition of the experimental diets. However, the lower utilization efficiency of crystalline-AAs compared to protein bounded-AAs suggested by many authors (Dabrowski and Guderley, 2002), may bias the estimation of EAA pattern. Taking this into consideration, Green and Hardy (2002) on applying this method to rainbow trout applied a correction factor on N retention in order to obtain a more realistic EAA pattern. Nonetheless, several studies also suggested that utilization of crystallineAAs may be improved, becoming similar to that of protein boundedAAs, by coating the crystalline-AAs (Schuhmacher et al., 1997; Fournier et al., 2002; Segovia-Quintero and Reigh, 2004), not exceeding a safe dietary inclusion level (Peres and Oliva-Teles, 2005); adjusting the EAA to NEAA ratio (Green and Hardy, 2002; Peres and Oliva-Teles, 2006) and the composition of NEAA mixture (Mambrini and Kaushik, 1994; Schuhmacher et al., 1995); and adjusting the frequency of feeding (Zarate et al., 1997; Barroso et al., 1999). In the present study, besides coating crystalline AA with agar, the experimental diets were formulated with an N level just below the optimum for growth of gilthead seabream (Oliva-Teles, 2000) and a high non-protein energy level to assure maximum efficiency of AA utilization for synthetic proposes. Overall, the good feed acceptance and growth performance observed for the control diet confirmed the good utilization of the dietary crystalline-AAs. Results of this study support that the deletion method can be used to estimate the optimum EAA pattern for gilthead seabream juveniles. Although, this methodology relies on some presumptions that need to be further clarified, fast growth of animals and rigorous dietary formulation are expected to improve consistency of results. Based on the present results, the optimal balance of essential amino acids in the diets for gilthead sea bream juveniles is (g/16 g N): arginine, 5.55; lysine, 5.13; threonine, 2.98; histidine, 1.89; isoleucine, 2.55; leucine, 4.75; methionine, 2.60; phenylalanine + tyrosine, 5.76; valine, 3.21; and tryptophan, 0.75. References Baker, D.H., 2003. Ideal dietary amino acids patterns for broiler chicks. In: D'Mello, J.P.F.D. (Ed.), Amino Acids in Animal Nutrition. CABI Publishing, UK, pp. 223–235. Baker, D.H., 2004. Animal models of human amino acid responses. J. Nutr. 134, 1646S–1650S. Barroso, J.B., Peragon, J., Garcia-Salguero, L., de la Higuera, M., Lupianez, J.A., 1999. Variations in the kinetic behaviour of the NADPH-production systems in different tissues of the trout when fed on an amino-acid-based diet at different frequencies. Int. J. Biochem. Cell Biol. 31, 277–290. Boisen, S., 2003. Ideal dietary amino acid profiles for pigs. In: D'Mello, J.P.F.D. (Ed.), Amino Acids in Animal Nutrition. CABI Publishing, UK, pp. 157–168. Cho, C.Y., Kaushik, S., Woodward, B., 1992. Dietary arginine requirement of young rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 102, 211–216. Cohen, S.A., Meys, M., Tarvin, T., 1989. The pico-tag method. A Manual of Advance Techniques for Amino Acid Analysis. Waters Chromatography Division, Milford, MA. 124 pp. Dabrowski, K., Guderley, H., 2002. Intermediary metabolism, In: Halver, J.E. (Ed.), Fish Nutrition, 3rd Edition. Academic press, San Diego, pp. 309–365.
86
H. Peres, A. Oliva-Teles / Aquaculture 296 (2009) 81–86
Fournier, V., Gouillou-Coustans, M.F., Metailler, R., Vachot, C., Guedes, M.J., Tulli, F., Oliva-Teles, A., Tibaldi, E., Kaushik, S.J., 2002. Protein and arginine requirements for maintenance and nitrogen gain in four teleosts. Br. J. Nutr. 87, 459–469. Green, J.A., Hardy, R.W., 2002. The optimum dietary essential amino acid pattern for rainbow trout (Oncorhynchus mykiss), to maximize nitrogen retention and minimize nitrogen excretion. Fish Physiol. Biochem. 27, 97–108. Kaushik, S.J., 1998. Whole body amino acid composition of European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata) and turbot (Psetta maxima) with an estimation of their IAA requirement profiles. Aquat. Living Resour. 11, 355–358. Luquet, P., Sabaut, J.J., 1974. Nutrition azotée et croissance chez la daurade et la truite, Colloque sur l'aquaculture. Actes des colloques. CNEXO, Brest, pp. 243–253. Mambrini, M., Kaushik, S.J., 1994. Partial replacement of dietary protein nitrogen with dispensable amino acids in diets of Nile tilapia, Oreochromis niloticus. Comp. Biochem. Physiol. 109, 469–477. Mambrini, M., Kaushik, S.J., 1995a. Indispensable amino acid requirements of fish — correspondence between quantitative data and amino acid profiles of tissue proteins. J. Appl. Ichthyol. 11, 240–247. Mambrini, M., Kaushik, S., 1995b. Effect of temperature on sulfur amino acid requirements for maintenance and growth of juvenile rainbow trout. In: Nunes, A.F., Portugal, J.P., Costa, J.P., Ribeiro, J.R. (Eds.), Proc. 7 Int. Symp. on Protein Metabolism and Nutrition. EAAP Publ., pp. 117–122. n°81. Mercer, L.P., May, H.E., Dodds, S.J., 1989. The determination of nutritional-requirements in rats — mathematical-modeling of sigmoidal, inhibited nutrient-response curves. J. Nutr. 119, 1465–1471. Oliva-Teles, A., 2000. Recent advances in European sea bass and gilthead sea bream nutrition. Aquac. Int. 8, 477–492. Peres, M.H., Oliva-Teles, A., 1999. Effect of dietary lipid level on growth performance and feed utilization by European sea bass juveniles (Dicentrarchus labrax). Aquaculture 179, 325–334. Peres, H., Oliva-Teles, A., 2005. The effect of dietary protein replacement by crystalline amino acid on growth and nitrogen utilization of turbot Scophthalmus maximus juveniles. Aquaculture 250, 755–764. Peres, H., Oliva-Teles, A., 2006. Effect of the dietary essential to non-essential amino acid ratio on growth, feed utilization and nitrogen metabolism of European sea bass (Dicentrarchus labrax). Aquaculture 256, 395–402. Peres, H., Oliva-Teles, A., 2007. Effect of the dietary essential amino acid pattern on growth, feed utilization and nitrogen metabolism of European sea bass (Dicentrarchus labrax). Aquaculture 267, 119–128. Peres, H., Oliva-Teles, A., 2008. Lysine requirement and efficiency of lysine utilization in turbot (Scophthalmus maximus) juveniles. Aquaculture 275, 283–290.
Rodehutscord, M., Pack, M., 1999. Estimates of essential amino acid requirements from dose–response studies with rainbow trout and broiler chicken: effect of mathematical model. Arch. Tieärernhr. 52, 223–244. Rodehutscord, M., Mandel, S., Pack, M., Jacobs, S., Pfeffer, E., 1995. Free amino acids can replace protein-bound amino acids in test diets for studies in rainbow trout (Oncorhynchus mykiss). J. Nutr. 125, 956–963. Rodehutscord, M., Becker, A., Pack, M., Pfeffer, E., 1997. Response of rainbow trout (Oncorhynchus mykiss) to supplements of individual essential amino acids in a semipurified diet, including an estimate of the maintenance requirement for essential amino acids. J. Nutr. 126, 1166–1175. Rollin, X., Mambrini, M., Abboudi, T., Larondelle, Y., Kaushik, S.J., 2003. The optimum dietary indispensable amino acid pattern for growing Atlantic salmon (Salmo salar L.) fry. Br. J. Nutr. 90, 865–876. Ronnestad, I., Conceicao, L.E.C., Aragao, C., Dinis, M.T., 2001. Assimilation and catabolism of dispensable and indispensable free amino acids in post-larval Senegal sole (Solea senegalensis). Comp. Biochem. Physiol. 130, 461–466. Schuhmacher, A., Munch, M., Gropp, J.M., 1995. Non-essential amino acid sources in crystalline amino acid diets for trout (Oncorhynchus mykiss). J. Appl. Ichthyol. 11, 317–321. Schuhmacher, A., Wax, C., Gropp, J.M., 1997. Plasma amino acids in rainbow trout (Oncorhynchus mykiss) fed intact protein or a crystalline amino acid diet. Aquaculture 151, 15–28. Segovia-Quintero, M.A., Reigh, R.C., 2004. Coating crystalline methionine with tripalmitin–polyvinyl alcohol slows its absorption in the intestine of Nile tilapia, Oreochromis niloticus. Aquaculture 238, 355–367. Shearer, K.D., 2000. Experimental design, statistical analysis and modelling of dietary nutrient requirement studies for fish: a critical review. Aquac. Nutr. 6, 91–102. Tibaldi, E., Kaushik, S., 2005. Amino acid requirements of Mediterranean fish species. In: Montero, D., Basurco, B., Nengas, I., Alexis, M., Izquierdo, M. (Eds.), Proceedings of the Workshop on Mediterranean Fish Nutrition, Rhodes (Greece), 1–2 June, 2002: Cahier Options Méditerranéennes, vol. 63, pp. 59–65. Wang, T.C., Fuller, M.F., 1989. The optimum dietary amino acid pattern for growing pigs. 1. Experiments by amino acid deletion. Br. J. Nutr. 62, 77–89. Wilson, R.P., 2003. Amino acid requirement in fish and crustaceans. In: D'Mello, J.P.F.D. (Ed.), Amino acids in animal nutrition. CABI Publishing, UK, pp. 427–447. Wilson, R.P., Cowey, C.B., 1985. Amino acid composition of whole body tissue of rainbow trout and Atlantic salmon. Aquaculture 48, 373–376. Zarate, D.D., Lovell, R.T., Payne, M., 1999. Effects of feeding frequency and rate of stomach evacuation on utilization of dietary free and protein-bound lysine for growth by channel catfish Ictalurus punctatus. Aquac. Nutr. 5, 17–22.
