Bass, Micropterus salmoides:
A Comparison of Methods of Analysis
of Dose-Response Trials Data
Jony Koji Dairiki Carlos Tadeu dos Santos Dias
José Eurico Possebon Cyrino
ABSTRACT. Lysine is a strictly essential amino acid, the reference for dose-response trials to determine dietary amino acids requirements of fish. This study compares estimation of amino acids require-ments of largemouth bass, Micropterus salmoides, from data of lysine dose-response trials, analyzed through different statistical methods: Polynomial regression analysis, broken-line regression analysis, and specific mathematical modeling. Amino acids requirements were esti-mated through the A/E relationship [A/E = (essential amino acid total essential amino acids⫹ cystine ⫹ tyrosine) ⫻ 1.000]. Groups of 25 feed-conditioned largemouth bass fingerlings (1.29 ± 0.03 g; 4.35 ± 0.17 cm) were stocked in 60-L cages (5 mm mesh) housed in 1,000-L plastic,
Jony Koji Dairiki, Doctoral Student, Departamento de Zootecnia, Escola Superior de Agricultura “Luiz de Queiroz,” Universidade de São Paulo, P.O. Box 9, 13418-900, Piracicaba, SP, Brazil.
Carlos Tadeu dos Santos Dias, Associate Professor, Departamento de Ciências Exatas, Escola Superior de Agricultura “Luiz de Queiroz,” Universidade de São Paulo, P.O. Box 9, 13418-900, Piracicaba, SP, Brazil.
José Eurico Possebon Cyrino, Associate Professor, Departamento de Zootecnia, Escola Superior de Agricultura “Luiz de Queiroz,” Universidade de São Paulo, P.O. Box 9, 13418-900, Piracicaba, SP, Brazil.
Address correspondence to: José Eurico Possebon Cyrino at the above address (E-mail: [email protected]).
Journal of Applied Aquaculture, Vol. 19(4) 2007 Available online at http://jaa.haworthpress.com © 2007 by The Haworth Press, Inc. All rights reserved.
indoor tanks, closed circulation system, and fed diets containing 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5% lysine, in a totally randomized experimental design trial (n = 4). The broken-line analysis method yielded more reliable and precise estimations of lysine requirements–2.1% of diet or 4.9% dietary protein–for final weight, weight gain, and specific growth rate. Best-feed conversion ratio was attained with 1.69% lysine in the diet or 3.9% lysine in dietary protein. Body amino acids profile was an adequate reference for estimation of largemouth bass amino acids requirements.
doi:10.1300/J028v19n04_01 [Article copies available for a fee from The
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KEYWORDS. Lysine, requirement, largemouth bass, Micropterus
sal-moides, polynomial regression, broken-line regression, dose-response trial
INTRODUCTION
Proteins are the main organic constituent of fish body and represent 65 to 75% of its dry mass. Dietary protein sources differ nutritionally and biologically. The biological value of a given protein varies with the composition and availability of amino acids. The deficiency of or low essential amino acid availability leads to a poor use of the protein, and con-sequently hampers growth and reduces fish feeding efficiency (Anderson et al. 1995; Masumoto et al. 1996).
Lysine is the most important of all essential amino acids. It can be used as dietary amino acid requirement reference because it is strictly essential and solely related to body protein deposition, the most limiting amino acid in fish feeds (Griffin et al. 1992; Schuhmacher et al. 1997). For instance, low lysine content in the diet of rainbow trout lowers the species’ collagen synthesis and deposition (Steffens 1989). Adequate dietary lysine contents improve survival and growth rate and prevent erosion and deformities of fish dorsal, pectoral, and ventral fins (Halver 1989; Keembiyehetty and Gatlin III 1992).
Lysine requirements of fish range from 5.0 to 6.8% of dietary protein, the highest values ordinarily related to nutritional requirements of carnivorous fish (NRC 1993). As a matter of fact, Coyle et al. (2000) reported that lysine requirement of largemouth is 2.8% of the diet or 6.0% of the dietary protein.
The carnivorous largemouth bass, introduced in Brazil in 1920 (Godoy 1954), is now considered not only as an important sport fish all through the country’s South and Southeastern regions, but also an excellent ex-perimental model. However, research on feeding and nutritional require-ments of the species in sub-tropical environrequire-ments is scarce and recent (Portz and Cyrino 2003, 2004; Portz et al. 2001). In addition, defining and validating suitable methodology for data analysis of dose-response trials aligned with nitrogen and amino acids utilization trials still concern re-search groups (Liebert et al. 2000; Portz et al. 2000; Robbins 1986). This work is targeted at these two research needs.
MATERIALS AND METHODS Experimental Design, Data Collection, and Analysis
Feed-conditioned largemouth bass fingerlings (1.29 ± 0.03 g; 4.35 ± 0.17 cm) were stocked in 60-L plastic cages (5 mm mesh; 25 fish per cage) installed in 1,000-L, indoor plastic tanks, set in a closed recircu-lation, continuous water flow and aeration system (temperature 24.7 ± 1.7⬚C; pH = 7.5 ± 0.3; dissolved oxygen 7.02 ± 0.2 mg/L), and fed ad libitum for 62 days with semi-purified diets (Table 1) (NRC 1993) con-taining 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5% lysine, in a totally randomized experimental design (n = 4). The following performance parameters were recorded: Initial weight (Wi); final weight (Wf); weight gain (WG = Wf⫺ Wi); feed consumption (FC); feed conversion ratio (FCR = FC⫼ WG); specific growth rate [SGR = (ln Wf⫺ ln Wi)⫼ number of days ⫻ 100]; hepato-somatic index (%) [HSI = (weight of liver⫼ body weight) ⫻ 100]; and survival rate (%).
All measurements were taken in the metric system (g; cm), except where otherwise noted. Data were submitted to ANOVA by the PROC GLM, SAS statistical package (SAS Institute 2000). Regression (PROC NLIN; SAS) and broken-line analysis (Robbins 1986; Portz et al. 2000) were used for decomposition of the ANOVA results, and further com-parison with mathematical modeling of nitrogen and amino acids utili-zation trials (Liebert et al. 2000).
Preparation of Experimental Diets
Diets were formulated to contain 43.6% crude protein (Portz et al. 2001; Ruchimat et al. 1997), observed as the ideal protein concept (Ogino 1980). Powdered, lyophilized Nile tilapia (Oreochromis niloticus) fillets
were used as intact protein source (Table 2). Amino acids profiles of largemouth bass roe and fillets (Portz 2001) were used as reference dietary amino acids profile. Based on recommendations of De Silva and Anderson (1995), synthetic amino acids were added to equalize amino acids contents of the intact protein source to the reference amino acids profile (Table 3). Synthetic lysine was added to set the different treat-ments in replacement of the cellulose in the basal diet.
Feed ingredients were mixed, added with 20% warm water, pelleted in industrial mincer, oven-dried (force air circulation; 45⬚C; 22 hours), broken down to 1-2 mm pellets, sized, seal-bagged, and frozen-stored (⫺20⬚C) before use. Chemical composition of diets (Tables 4 and 5) was determined at Institute of Animal Physiology and Animal Nutrition, Col-lege of Agriculture, George-August University, Götingen, Germany.
Routine Management and Sampling Procedures
Laboratory light conditions were kept at 14 h light and 8 h dark (Cyrino et al. 2000; Heinen 1998; Portz et al. 2001). Fishes received two daily
TABLE 1. Experimental, semi-purified diet.
Ingredients Quantity (%)
Nile tilapia fillet 15.77
Amino acids mixture 22.91
Dextrin 30.66 Fish oil 7.50 Mineral mixture1 4.00 Vitamin mixture2 3.00 Carboximethylcellulose 2.00 Bicalcium phosphate 1.00 Cellulose 8.24 Asp/Glu3 4.92 L-lysine HCl 0.00 Total 100.00
1Mineral mixture: Ca, 11.42%; P, 9.57%; K, 10.78%; Mg, 1.30%; S, 1.83%; Na, 3.30%; Cl, 14.62%; Fe,
3,000 ppm; Cu, 424.89 ppm; Zn, 3,750.04 ppm; I, 22.49 ppm; Mn, 746.52 ppm; Se, 15 ppm; Co, 5.01 ppm and Cr, 1.98 ppm.
2Vitamin mixture: vit. A, 290.000 UI; vit. D
3, 143.500 UI; vit. E, 5.700 mg; vit. K3, 143.82 mg; thiamine (B1),
334.42 mg; riboflavin (B2), 667.20 mg; niacin (B3), 1,333.53 mg; pyridoxine (B6), 666.63 mg; pantothenic acid
(B5), 1.334 mg; biotin, 34 mg; folic acid, 67 mg; cobalamine, 1.000μg and ascorbic acid (vit. C) 999.85 mg.
meals (0700 and 1600). Feed for immediate use was kept in plastic containers under refrigeration. Apparent feed consumption was esti-mated by weighing feed containers every three days.
Cages were screened daily for casualties and visual signals of nutri-tional deficiencies. At the end of the feeding trial, fishes were randomly sampled (12 fish per treatment), individually weighed, sacrificed by anesthetic overdose, and laparotomized for excision of hepatic tissue. Livers (whole) were weighed fresh for calculation of HSI. Samples of liver tissue were quick-frozen and stored in liquid nitrogen for chemical analysis.
TABLE 2. Chemical composition of Nile tilapia muscle.
Nutrient Value
Moisture 1.10%
Gross energy 5,472.00 (cal/g)
Crude protein 78.01% Lipids 11.00% Ash 4.96% Calcium 0.21% Total phosphorus 1.12% Amino Acids Arginine 4.36% Histidine 1.55% Isoleucine 3.22% Leucine 5.89% Lysine 6.34% Methionine 2.18% Cystine 0.60% Phenylalanine 3.32% Tyrosine 2.65% Threonine 3.25% Tryptophan 0.44% Valine 3.50% Alanine 4.42% Glycine 3.70% Proline 7.02% Serine 2.78% Aspartic acid 7.51% Glutamic acid 11.02%
The use of the whole-body amino acids profile as standard for dietary amino acids requirements relationship was validated through the use of the relationship A/E = essential amino acids⫼ (total essential amino acids⫹ cystine ⫹ tyrosine) ⫻ 1000 (Fagbenro 2000; Ngamsnae et al. 1999). Once A/E was deemed valid, the dietary requirement of essential amino acids was estimated against the reference amino acid–lysine.
RESULTS AND DISCUSSION
The energy-to-protein ratio (DE:CP) of the experimental diets was 10.4 kcal/g, considered elastic in comparison with recommendations of TABLE 3. Composition of the synthetic amino acids mixture and Nile tilapia’s muscle tissue. Synthetic amino acids Reference for 43% CP on the diet Amino acids profile1 15.77% of Nile tilapia muscle tissue Amino acids mixture (%) Total (%) Arginine 3.863 4.36 0.6876 3.17 3.86 Glycine 1.962 3.70 0.5835 1.38 1.96 Histidine 1.012 1.55 0.2444 0.77 1.01 Isoleucine 1.702 3.22 0.5078 1.19 1.70 Leucine 3.783 5.89 0.9289 2.85 3.78 Lysine 4.022 6.34 1.0000 0.00 1.00 Methionine 1.352 2.18 0.3438 1.01 1.35 Cystine 0.483 0.60 0.0946 0.39 0.48 Phenylalanine 1.812 3.32 0.5236 1.29 1.81 Tyrosine 1.593 2.65 0.4179 1.17 1.59 Serine 2.603 2.78 0.4384 2.19 2.60 Threonine 2.143 3.25 0.5125 1.63 2.14 Tryptophan 0.443 0.44 0.0694 0.37 0.44 Valine 2.633 3.50 0.5520 2.08 2.63 Proline 2.143 7.02 1.1071 1.03 2.14 Alanine 3.093 4.42 0.6970 2.39 3.09 Mixture 22.91 Asp/Glu 4.92 Fillet 15.77 Total CP 43.60 1From Table 2.
2Largemouth bass muscle tissue.
TABLE 4. Chemical composition of experimental diets. Treatment with lysine (%) Dry matter (%) Mineral matter (%) Crude protein1(%) Crude lipid1 (%) Crude fiber (%) Energy2 (MJ kg⫺1) 1.0 95.48 6.42 43.22 9.18 4.21 18.88 1.5 95.79 6.41 43.16 9.34 4.16 19.07 2.0 95.89 6.39 43.29 9.37 4.36 18.91 2.5 95.61 6.40 43.06 9.14 4.11 19.02 3.0 95.67 6.39 43.04 9.27 4.16 19.08 3.5 95.53 6.41 43.46 9.12 4.15 19.02 1Dry matter. 2Original matter.
TABLE 5. Amino acids composition of experimental diets.
Diets 1 2 3 4 5 6 DM 95.48 95.79 95.89 95.61 95.67 95.53 Amino acids1 Arg 3.48 3.46 3.47 3.48 3.51 3.50 His 1.00 0.98 0.99 0.98 0.99 1.02 Ile 1.74 1.69 1.70 1.71 1.71 1.73 Leu 3.76 3.79 3.80 3.81 3.81 3.78 Lys 1.16 1.65 1.99 2.46 3.05 3.49 Met 1.40 1.40 1.43 1.39 1.41 1.38 Phe 1.70 1.68 1.67 1.68 1.70 1.69 Thr 2.16 2.15 2.18 2.17 2.15 2.15 Trp 0.45 0.48 0.43 0.45 0.45 0.46 Val 2.56 2.61 2.58 2.57 2.58 2.58 Asp 2.46 2.45 2.41 2.45 2.44 2.44 Glu 2.58 2.56 2.57 2.58 2.57 2.59 Ala 3.11 3.15 3.13 3.11 3.13 3.13 Cys 0.51 0.54 0.52 0.52 0.53 0.54 Gly 1.90 1.92 1.91 1.90 1.94 1.91 Ser 2.72 2.74 2.75 2.74 2.76 2.74 Pro 2.16 2.18 2.18 2.20 2.17 2.15 Tyr 1.58 1.56 1.59 1.60 1.58 1.59 Total 36.43 36.99 37.30 37.80 38.48 38.87
Portz (1999), who determined DE:CP = 7.78-8.83 kcal/g as ideal for nu-trition of juvenile largemouth bass (14.46±0.81 g). However, no problems related to high dietary DE:CP were observed, since values registered for both weight gain (WG) and specific growth rate (SGR) were considered satisfactory and compatible with literature data.
Animal growth is understood and defined as increase of mass of the structural tissues–that is, bone and muscle–and organs; protein is the primary constituent of structural tissues (Millward 1989; Young 1974; 1985). Fish fed with energy-deficient diets usually present reduced growth rate, for part of the ingested protein will be spared as energy source. On the other hand, disproportionately higher dietary energy can halt voluntary feed intake before enough protein is ingested, impairing the use of other nutrients and increasing body fat deposition (NRC 1993; Cho and Kaushik 1990; De Silva and Anderson 1995).
Performance of Fish
Performance data were submitted to exploratory analysis by outlier data test, variance homogeneity, sample size, range of the response vari-able, and Box-Cox optimal potency test (SAS 2000). Data on feed con-version ratio (FCR) of a single replication of treatment 3.5% dietary lysine were detected as outlier and disconsidered for analysis. Treatment means differed (P < 0.05) regarding final weight (Wf), absolute weight gain (WGa), relative weight gain (WGr), feed consumption (FC), spe-cific growth rate (SGR) and FCR.
Foster and Ogata (1998) reported that juvenile Japanese flounder Paralichthys olivaceus fed lysine-deficient diets presented abnormal color pattern. Also, caudal fin erosion was reported by Ketola (1983) for rainbow trout Onchorhynchus mykiss (1.1 g) fed with lysine-deficient diets. No external deficiency signs or body deformities were registered for largemouth bass fingerlings in this study, independent of dietary lysine level.
Polynomial Regression Analysis
If quantitative factors interact at more than two levels, data analysis will establish functional correlations between factors’ levels and the studied variable (Gomes and Garcia 2002), for example the polynomial regression analysis herein used. Solving polynomial regression equa-tions allows not only estimating nutrients’ requirement levels (Tibaldi and Tulli 1999), but also representing it graphically.
Before regression curves were graphically represented, exploratory analysis data with regard to outlier’s tests, curvature, homogeneity of the variance, influential observations, and scale of variable response were extracted (SAS 2000). The quadratic polynomial regression determina-tion coefficient for the variable Wfwas considerably high (r2= 0.87), but
graphical determination of lysine requirement level for optimal Wfwas in the interval between 2.5 and 3.5% (Figure 1). Only deriving the qua-dratic regression equation enabled determining precisely that 3.1% dietary lysine elicits better Wf.
From regression curves for WGa and WGr, it was inferred that di-etary lysine ranging from 3.0 to 3.5% yields best performance (Figures 2 and 3). Once again, only deriving the quadratic regression equations enabled one to determine that 3.19 and 3.21% dietary lysine yielded best WGa and WGr, respectively. Feed consumption tended to increase linearly with increasing dietary lysine levels (Figure 4); this relation-ship registered the smallest r2= 0.79; that is, only 79% of variations on FC result from variations on dietary lysine contents (Vanni 1998).
Even though fish were fed ad libitum, careful feeding management elicited very low feed loss. The best FCR was registered within 2.0 to 3.0% dietary lysine levels (Figure 5). Once again, graphic determina-tion of dietary lysine level for best FCR was not possible. Deriving the cubic regression equation revealed that 2.27 (minimum) and 2.99% (maximum) dietary lysine result in the best FCR for fingerling large-mouth bass. 6.0 5.0 4.0 3.0 2.0 W f (g ) 1.0 0.0 0 0.5 1 1.5 2 y = −0.2857x2 + 1.7643x + 1.9643 r2 = 0.87 2.5 3 3.5 4 Lysine rates (%)
Best SGR was estimated for fish fed diets containing 2.5 to 3.5% lysine (Figure 6); deriving the regression equation showed that 3.0% dietary lysine enables best SGR. Recorded SGR values–1.57%/day (1.0% dietary lysine) and 2.08%/day (3.5% dietary lysine)–are considered high. However, working in comparable conditions, Almeida (2003)
4.0 3.0 2.0 WGa ( g ) 1.0 0.0 0.5 1 y = –0.2466x2+ 1.5743x + 0.8937 r2= 0.86 1.5 2 2.5 Lysine rates (%) 3 3.5 4
FIGURE 2. Quadratic regression curve adjusted for the absolute weight gain variable (WGa). 300.0 250.0 200.0 150.0 WGr (%) Lysine rates (%) 100.0 50.0 0.0 0.5 1 1.5 2 2.5 3 3.5 4 y = –19.448x2+ 125.11x + 62.996 r2= 0.87
FIGURE 3. Quadratic regression curve adjusted for the relative weight gain variable (WGr).
recorded SGR = 2.72%/day for juvenile pacus, Piaractus mesopotamicus, fed diets containing 32.0% crude protein and increasing levels of vitamin C. Therefore, SGR recorded for fingerling largemouth bass will not be considered surprising.
Working with fingerling Indian major carp, Cirrhinus mrigala (4.30 ± 0.25 cm; 0.63 ± 0.02 g), and using polynomials analysis techniques, Ahmed and Khan (2004) estimated the species lysine requirement as
Lysine rates (%) FC ( g ) 0.5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 1 1.5 2 2.5 3 3.5 4 y = 0.3442x + 3.4771 r2= 0.79
FIGURE 4. Linear regression curve adjusted for the feed consumption variable (FC). Lysine rates (%) 2.0 1.8 FCR 1.5 1.3 1.0 0.5 1 1.5 2 2.5 3 3.5 4 y = –0.119x3+ 0.939x2– 2.423x + 3.376 r2= 0.89
FIGURE 5. Cubic regression curve adjusted for the feed conversion ratio vari-able (FCR).
being 2.30% of the diet or 5.75% lysine in the dietary protein (LDP). These values are closer to those resulting from broken-line regression analysis, than to those resulting from graphic and algebraic analysis of polynomial regressions, in this experiment. Similar results were reported for fingerling grass carp, Ctenopharyngodon idella (3.15 ± 0.01 g), by Wang et al. (2005) who, using the polynomial regression analysis method, determined the species’ dietary lysine requirement to be 2.24% (5.89% dietary protein).
The graphic analysis of the regression curves reveals that lowest di-etary lysine levels–1.0 and 1.5%–resulted in low Wf, WG, FC, SGR, and poor FCR. Therefore, using polynomial regression analysis to evaluate results of nutrient requirement dose-response trials was effective only to a certain extent, once observation of the tendency lines elicited drawing preliminary conclusions. However, this analysis method does not allow precise graphic determination of the best requirement levels; only alge-braic solution elicited estimation of optimal dietary lysine requirements, and from there on, determination of the best performance responses.
Broken-Line Analysis Method
Analyzing dose-response trials data of nutritional requirements through the broken-line regression analysis method allows determining accurately the minimum level of a given nutrient that guarantees the maximum performance of a certain species. This result/response is con-sidered an important determinant of the cost-benefit relationship for for-mulation of fish feeds. Using this method through the SAS PROC NLIN procedure is a simple, fast, and efficient method of determination of
Lysine rates (%) SGR (%) 0.5 0 0.0 0.5 1.0 1.5 2.0 2.5 1 1.5 2 2.5 3 3.5 4 y = –0.125x2+ 0.7508x + 0.9623 r2= 0.84
FIGURE 6. Quadratic regression curve adjusted for the specific growth rate (SGR).
nutritional requirements. However, results can underestimate values de-termined by models traditionally used, which may not be necessarily more precise (Portz et al. 2000; Robbins 1986; Zeitoun et al. 1976).
According to Robbins (1986), the broken-line regression curve con-sists of an ascending or descending line, followed by a horizontal line, and their intersection points will determine the break (optimal) point. This inclination model is better fitted to estimate growth parameters. The utilized regression model (1) used was:
Yi= +L U(R⫺XLRi)+e , ii =1, 2...n n1, 1+1, ... n, (1) where (R⫺XLRi) = 0, for iⱖ n1+1, n1being the number of observations be-fore the break point and n the number of pairs of observations; L = coordinate of the ordinates axis; R = coordinate of the abscissas axis of a given break point; and U = line inclination coefficient when X < R.
Using the broken-line method yielded an estimated, optimum level (break point) of lysine for maximum Wf(4.6 g) equal to 2.12% dietary lysine, or 4.9% lysine in the dietary protein (LDP) (Figure 7). This value is not within (below) the interval determined through the graphic obser-vation of the quadratic polynomial regression–2.5 to 3.5% dietary lysine (Figure 1)–and much smaller than the value yielded by the algebraic solution of the adjusted quadratic equation (3.1% dietary lysine). In other words, determining dietary lysine requirements by the quadratic polynomial regression method alone overestimated actual dietary lysine requirement.
Optimum, calculated dietary lysine levels for WGa (3.34 g) and WGr (259%) were 2.17 and 2.26% dietary lysine, respectively (5.0 or 5.2% LDP, respectively); results do not differ (P > 0.05) (Figures 8 and 9) and, once again, are out in the lower side of both the graphic range determined by the polynomial regression (3.0 and 3.5% of the diet; Figures 2 and 3), and of the algebraic analysis solution for the quadratic equation (3.19 and 3.21% of lysine in the diet, respectively). The broken-line analysis con-cept observed that an average 2.2% dietary lysine would yield the same weight gain yielded by either higher dietary lysine requirement deter-mined by the polynomial regression method.
Keembiyehetty and Gatlin III (1992) determined that the sunshine bass, Morone chrysopsE × white Morone saxatilis F striped bass hybrid requires 1.41% dietary lysine. Considering and analyzing weight gain, plasmatic lysine concentration, and alimentary efficiency data through the broken-line method in two experiments with sunshine bass, Griffin et al. (1992) also determined dietary lysine requirements ranging from
1.2 to 1.4%. Both researches report dietary lysine requirement of sunshine bass to be smaller than that of largemouth bass. However, Small and Soares Jr. (1998) determined that the striped bass requires 2.2% dietary lysine, a value close to that of largemouth bass. Apparently, hybrid fish have differentiated dietary requirement.
Lysine rates (%) Wf ( g ) 0.5 0 0 2 1 3 4 5 6 1 1.5 2 2.5 3 3.5 4 2.12% (A) Wf = 2.384 + 1.05* level r2 = 0.99 (B) Wf = –0.2857x2+ 1.7643x + 1.9643 r2= 0.87
FIGURE 7. Regressions for the means of final weight (Wf) in function of lysine
level on the diet: (A) Broken-line (ⵧ); (B) Polynomial (䉬)
Lysine rates (%) WGa ( g ) 0.5 0 0 2 3.5 2.5 0.5 1 1.5 3 4 1 1.5 2 2.5 3 3.5 4 (A) WGa = 1.1266 +1.02* level r2 = 0.99 2.17% (B) WGa = −0.2466x2+ 1.5743x + 0.8937 r2 = 0.86
FIGURE 8. Regressions for the means of absolute gain weight (WGa) in
Small and Soares Jr. (2000) reported that 2.01% dietary lysine increased weight gain of fingerling striped bass (1.5 g). The authors con-sidered that the smaller lysine requirement of hybrid basses was corre-lated to smaller dietary energy requirements. Brougher et al. (2004) ratify the statement of Small and Soares Jr. (2000), but warn that farming hy-brid striped bass is justified solely because of their high weight gain performance–15.6 g versus 3.4 g of pure-bred striped bass in a 12-week trial–once hybrids accumulate more body fat, an undesirable carcass trait. Dietary lysine requirements smaller than those reported herein for the largemouth bass have been seen for the yellowtail, Seriola quinqu-eradiata (1.78% dietary lysine or 4.13% LDP), and for the milkfish, Chanos chanos (1.8-2.0% dietary lysine or 4% LDP) (Borlongan and Coloso 1993; Borlongan and Benitez 1990).
As far as FCR is concerned, the break point (FCR = 1.35) was registered for 1.69% dietary lysine (3.9% LDP) (Figure 10). The break point was in the lower side of either the dietary lysine requirement interval determined through polynomial regression (2.0~3.0% dietary lysine) (Figure 5) or by solving the cubic equation (2.27~2.99% dietary lysine). Recorded FCR values for largemouth bass can be considered just reasonable, and ex-plained by occurrences of late ejection of ingested pellets, as already ob-served by Kubitza and Lovshin (1997).
A calculated 2.17% dietary lysine (5.0% LDP) yielded SGR = 2.06%/ day (Figure 11). Once again, lysine requirement for optimized SGR was
Lysine rates (%) 0.5 0 0 50 100 150 200 WGr (%) 250 300 1 1.5 2 2.5 3 3.5 4 (A) (WGr = 98.2236 + 71.14* level r2 = 0.99 2.26% (B) WGr = –19.448x2+ 125.11x + 62.996 r2 = 0.87
FIGURE 9. Regressions for the means of relative gain weight (WGr) in function
in the lower side of both the intervals obtained by the graphic analysis of the regression curve (2.5~3.5% dietary lysine; Figure 6), and the algebraic solving of the quadratic regression equation (3.0% dietary lysine). Working with fingerlings striped bass (1.5 g), Small and Soares Jr. (2000) registered SGR = 1.55%/day for 2.01% dietary lysine. For this same dietary lysine level, fingerling largemouth bass would present larger SGR (2.06%/day).
Dietary lysine requirements reported by several authors for several fish (Rollin et al. 2003; Rodehutscord et al. 2000a, b; Rollin et al. 2003; Ruchimat et al. 1997; Tibaldi and Lanari 1991) were very close to those registered in this work for the largemouth bass. Notwithstanding, Coyle et al. (2000) determined lysine requirement of largemouth bass (2.8% dietary lysine; 6.0% LDP) as considerably higher than those determined in this study. However, fish used by Coyle et al. (2000) were also consid-erably older and larger (36.0 ± 0.5 g).
Comparing the graphic analysis polynomial regression curves with re-sults yielded by the broken-line analysis method, fingerling largemouth bass fed diets containing lysine levels estimated by polynomial regres-sion curves (1.0 and 1.5% dietary lysine) would have Wf, WG, and SGR significantly smaller, and FCR significantly worse. In addition, because
Lysine rates (%) 0.5 0 0 0.5 1 1.5 2 2.5 3.5 3 FCR 1 1.5 2 2.5 3 3.5 4 (A) FCR = 2.43 – 0.64* level r2 = 0.99 (B) FCR = –0.119x3+ 0.939x2 – 2.423x + 3.376 r2 = 0.89 1.69%
FIGURE 10. Regressions for the means of feed conversion ratio (FCR) in
determination coefficients registered for the broken-line regression curves (r2= 0.99) are considerably higher than those registered for
poly-nomial regression curves (r2 = 0.84~0.89), the broken-line analysis
method can be considered not only more accurate and precise, but also an elicitor of economical efficiency in the carnivorous fish feed formulation process.
The Mathematical Model
The mathematical model proposed by Gebhardt (1966) and redefined by Liebert et al. (2000) measures utilization efficiency and nutritional re-quirement of amino acids starting from the ideal protein concept (Ogino 1980), which recommends fish feeds to contain a full range of amino-acids, both essential and non-essential. Considering this principle, Liebert et al. (2000) stated that excess dietary amino acids are eliminated and pro-tein deposition efficiency is maximized.
Each particular animal species, fish included, has a definite genetic ca-pacity for (maximum) nitrogen deposition (Mohamed 2002). Therefore, N deposition capacity plus the amount of N required for maintenance represent the maximum capacity of N retention. The model is thus based on a mathematical description of N-balance pattern–or maximum N retention capacity–in growing animals, depending upon the amount of
Lysine rates (%) 0.5 0 0 0.5 1 1.5 SGR (%) 2 2.5 1 1.5 2 2.5 3 3.5 4 (A) SGR = 1.192 + 0.4* level r2 = 0.99 (B) SGR = –0.1252x2+ 0.7508x + 0.9623 r2 = 0.84 2.17%
FIGURE 11. Regressions for the means of specific growth rate (SGR) in
ingested nitrogen and quality of the food/feed protein, represented by equation (2):
y=PDmaxT (1 e− −bx) (2)
where y = actual daily N-balance⫹ NMR1/LW2kg0.67(mg), PD
maxT =
maximum theoretical capacity for daily N-balance⫹ NMR/LW kg0.67
(mg), x = daily N-intake/LW0.67(mg), b = slope of the curve, and e = basic
number of natural logarithm (1nitrogen maintenance requirement and 2live weight).
The value of b is calculated through equation (3) (Mohamed 2002):
b =ln a ln (a y)
x
− −
(3) where a = 500 (fixed; Mohamed 2002); x and y same as equation (2).
In this model, b is the quality of the protein ingested as feed, which is linearly related to contents and availability of essential amino acids. Therefore, b depends solely on the utilization of dietary, essential amino acids, as regulated by dietary protein’s digestibility, absorption, and me-tabolism. Nitrogen balance trials in growing animals thus allow deter-mining maximum values for essential amino acids utilization.
Average N retention (%/day) did not differ among treatments (P > 0.05). It was thus impossible to calculate levels of dietary lysine that would favor best N-retention efficiency. The studied model was origi-nally developed and successfully used for swine and poultry, which are homoeothermic and have nutritional requirements dissimilar to fish. Adjustment and adaptation of the model’s principles are necessary to elicit its use in fish nutrition research (Cho and Bureau 1998), especially with regard to neotropical, fresh-water fish.
A/E Relationship and Amino Acids Requirements
Tables 6 and 7 present and compare data on amino acids requirements of largemouth bass estimated through the A/E relationship, and amino acids requirements determined for other fish species; results are homoge-neous and similar. In exception of NRC (1993), data on amino acids requirements were determined with the aid of the A/E relationship and amino acids profiles of selected fish tissues.
Kim and Lall (2000) postulate that using the A/E rate enables deter-mining similarities in essential amino acids requirements of different
species at different ages (sizes). Comparing amino acids composition of body tissues of the Atlantic halibut, Hippoglossus hippoglossus; yellowtail flounder, Pleuronectes ferruginea; and Japanese flounder, Paralichthys ferruginea, Kim and Lall (2000) detected high similarity among essential amino acids contents expressed by the A/E rate. Similar observations were reported by Campos et al. (2006) for suburim, Pseudoplatystoma corruscans.
In addition, Borlongan and Coloso (1993) demonstrated that nutri-tional requirements in arginine, leucine, lysine, tryptophan, and valine determined experimentally for the milkfish, were comparatively lesser than the contents of those amino acids determined in proteic tissues of the species. But requirements of other essential amino acids were similar to their contents in the species proteic tissue.
In a dose-response trial, Berge et al. (1997) determined that the Atlantic salmon (383 ± 62 g) requires 2.02% dietary arginine (4.8% of the dietary protein). Alam et al. (2002) determined that dietary arginine requirement TABLE 6. A/E relationship and estimated nutritional requirements of essential amino acids.
Amino acid Reference for 43% of protein g/100 g Essential amino acids of body tissues Rate of essential amino acids (reference lysine) Estimated nutritional require-ment g/100 g Arginine 3.862 156 96 2.0 Histidine 1.011 41 25 0.5 Isoleucine 1.701 69 42 0.9 Leucine 3.782 152 94 2.0 Lysine 4.021 162 100 2.1* Methionine 1.351 54 34 0.7 Cystine† 0.482 19 12 0.3 Phenylalanine 1.811 73 45 0.9 Tyrosine† 1.592 64 40 0.8 Threonine 2.142 86 53 1.1 Tryptophan 0.442 18 11 0.2 Valine 2.632 106 65 1.4 Total 24.81 1,000
1Largemouth bass fillet.
2Largemouth bass roe.
of fingerling Japanese flounder (1.85±0.05g) is 2.05% (4.14% of the dietary protein). These values are similar to the arginine requirement estimated for fingerling largemouth bass in this study, in spite of behavioral and phylo-genetic differences between these species.
Tibaldi et al. (1994) determined the dietary arginine requirement of fingerling sea bass (2.1±0.05 g) is 1.81% (3.9% of the protein). Tibaldi and Tulli (1999) determined, utilizing the broken-line regression method, that juvenile sea bass (7.5±0.15 g) requires 1.26% dietary threonine. These re-sults are similar to those registered for the largemouth bass in this study. However, Thebault et al. (1985) determined that dietary methionine require-ment of juvenile sea bass (35 ± 5 g) is 1.0%, therefore slightly superior to values registered in this study for the largemouth bass.
Utilizing the broken-line method, Luo et al. (2005) determined that ju-venile grouper, Epinephelus coioides (13.25±0.19 g), require 1.31% dietary methionine. Dietary methionine requirement for optimal specific growth rate, feed efficiency, and conversion and protein efficiency ratio of the yel-low croaker, Pseuosciaena croacea, is 1.41% (Mai et al. 2005). Ahmed et al. (2004) and Ahmed and Khan (2005) determined that fingerling Indian major carp (0.52 ± 0.21 g and 0.62 ± 0.02 g, respectively) require 1.8% dietary TABLE 7. Comparative dietary amino acids requirements of varied species.
Amino acid Chum
salmon1 Stripedbass2 Red sea
bream3
Atlantic
salmon4 Largemouthbass5
Arginine 2.60 1.25 1.71 1.82 2.00 Histidine 0.70 0.51 0.68 0.67 0.50 Isoleucine 1.00 0.80 1.07 ND 0.90 Leucine 1.50 1.71 2.05 ND 2.00 Lysine 1.90 2.02 2.15 2.39 2.10 Methionine⫹ cystine 1.20 0.92 1.07 1.54 1.00 Phenylalanine⫹ tyrosine 2.50 1.60 2.00 2.51 1.70 Threonine 1.20 0.98 0.88 1.21 1.10 Tryptophan 0.30 0.19 0.29 0.33 0.20 Valine 1.20 0.91 1.22 1.41 1.40 ND = Non-determined. 1NRC (1993).
2Small and Soares Jr (1998).
3Foster and Ogata (1999).
4Rollin et al. (2003).
threonine and 0.38% dietary tryptophan. All aforementioned amino ac-ids requirements were superior to those registered here for largemouth bass. Notwithstanding, estimated essential amino acids requirements of largemouth bass through the A/E ratio are closely similar to the values registered for salmonids and other basses (Table 6).
The relationship A/E is a reliable and useful tool to estimate essential amino acids requirements of species, but species-specific variations should be considered. Using body amino acids profile to set base for dietary amino acids profile and requirements is a viable technique and may bring additional benefits of formulating diets eliciting higher feed-ing efficiency and reduced nutrient loss and waste and metabolites excretion.
Survival Rate and Hepato-Somatic Index (HSI)
Dietary essential amino acids deficiency may affect the survival rate of farmed fish. Working on dietary lysine requirement of juvenile hybrid striped (8.0 g), Keembiyehetty and Gatlin III (1992) registered 100% sur-vival, even for fish fed lysine-deficient diets. Moon and Gatlin III (1991), working with red drum, Sciaenops ocellatus (0.9 g), observed that groups of fish fed methionine-deficient diets presented smaller survival rate.
Over 50% mortality was registered by Rodehutscord et al. (1997) for trout fed lysine-deficient diets–0.45, 0.55, and 0.70% dietary lysine; fish fed diet containing 0.85 and 1.0% lysine presented improved survival rate; when dietary lysine exceeded 1.0%, survival rate was 100%. In the present study, survival rate (54.00~71.00%; Table 8) did not differ among treatments (P > 0.01).
The occurrence of metabolism disturbances was screened through the study of the HSI variation. HSI did not differ (P > 0.01) in the treatments (Table 8); values herein recorded were close to those reported by Tibaldi et al. (1994) for the sea bass (HSI = 2.39~3.28%), and by Cyrino et al. (2000) for fingerling largemouth bass (HSI = 3.62~4.39%) fed diets with varying protein contents. Seemingly, either the lysine-poor or the lysine-rich diets did not induce severe physiologic disturbances in the largemouth fingerlings (possibly as a result of the somewhat short exper-imental period). Finally, Berge et al. (2002) report that the main clinical sign of dietary arginine to lysine imbalance is reduced growth. Growth rate of fingerling largemouth bass fed the highest dietary lysine rate was either equal or superior to growth rate of fish fed diets containing the
TABLE 8. Performance parameters of largemouth bass fingerlings fed with diets containing increasing levels of lysine.* Treat- ments Performance variables W i (g) W f (g) WGa (g) WGr (%) FC (g) FCR (g) SGR (%) S §(%) HSI §(%) 11 .2 7 ⫾ 0.05 3.38 ⫾ 0.17 2.10 ⫾ 0.24 166.16 ⫾ 17.18 3.75 ⫾ 0.25 1.79 ⫾ 0.05 1.57 ⫾ 0.14 71.00 ⫾ 16.12 a3.74 ⫾ 0.35 2 1 .30 4.05 ⫾ 0.06 2.75 ⫾ 0.09 210.61 ⫾ 12.67 4.04 ⫾ 0.06 1.47 ⫾ 0.06 1.83 ⫾ 0.06 70.00 ⫾ 13.27 a3.57 ⫾ 0.36 3 1 .30 4.43 ⫾ 0.15 3.12 ⫾ 0.09 237.30 ⫾ 7.18 4.22 ⫾ 0.09 1.35 ⫾ 0.05 1.97 ⫾ 0.05 80.00 ⫾ 5.66 a 3.30 ⫾ 0.40 41 .2 7 ⫾ 0.05 4.55 ⫾ 0.10 3.26 ⫾ 0.06 251.41 ⫾ 7.78 4.40 ⫾ 0.15 1.35 ⫾ 0.06 2.05 ⫾ 0.05 69.00 ⫾ 20.75 a3.42 ⫾ 1.02 51 .2 7 ⫾ 0.05 4.55 ⫾ 0.17 3.30 ⫾ 0.22 261.02 ⫾ 17.34 4.36 ⫾ 0.13 1.32 ⫾ 0.05 2.05 ⫾ 0.13 61.00 ⫾ 19.97 a 3.25 ⫾ 0.80 6 1 .30 4.73 ⫾ 0.30 3.45 ⫾ 0.31 264.68 ⫾ 22.62 4.73 ⫾ 0.12 1.38 ⫾ 0.17 2.08 ⫾ 0.12 54.00 ⫾ 4.00 a 3.51 ⫾ 0.32 W i : initial weight; W f : final weight; WGa: absolute weight gain; WGr: relative weight gain; FC: feed consumption; FCR: feed conversion rate; SGR: specific growth rate; S: survival rate; HSI: hepato-somatic index. *Means (n = 4 ) ± standard deviation; §Tukey’s test (α = 0 .01). 22
estimated amino acids requirement. Therefore, the hypothesis of amino acid antagonism should be definitely ruled out.
ACKNOWLEDGMENTS
The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and to Dr. Leandro Portz from Departamento de Zootecnia, Escola de Agronomia, Universidade Federal da Bahia, Cruz das Almas, BA, Brazil.
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