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Lysine Requirements of Largemouth
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: jepcyrin@esalq.usp.br).
Journal of Applied Aquaculture, Vol. 19(4) 2007
Available online at http://jaa.haworthpress.com
© 2007 by The Haworth Press, Inc. All rights reserved.
doi:10.1300/J028v19n04_01 1
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
Haworth Document Delivery Service: 1-800-HAWORTH. E-mail address:
<docdelivery@haworthpress.com> Website: <http://www.HaworthPress.com>
© 2007 by The Haworth Press, Inc. All rights reserved.]
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 thatlysine requirementof largemouthis 2.8%of thediet or 6.0%
of the dietary protein.
2 JOURNAL OF APPLIED AQUACULTURE
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 environments is scarce and recent
(Portz and Cyrino2003, 2004;Portz etal. 2001).In addition,definingand
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 (W
i
); final weight (W
f
); weight gain (WG =
W
f
⫺ W
i
); feed consumption (FC); feed conversion ratio (FCR = FC ⫼
WG); specific growth rate [SGR = (ln W
f
⫺ ln W
i
) ⫼ 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 etal. 1997),observed asthe idealprotein concept(Ogino
1980). Powdered, lyophilized Nile tilapia (Oreochromis niloticus) fillets
Dairiki et al. 3
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 atInstitute of Animal Physiologyand Animal Nutrition, Col
-
lege of Agriculture, George-August University, Götingen, Germany.
Routine Management and Sampling Procedures
Laboratory light conditions were kept at 14 hlight and8 hdark (Cyrino
et al. 2000; Heinen 1998; Portz et al. 2001). Fishes received two daily
4 JOURNAL OF APPLIED AQUACULTURE
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 mixture
1
4.00
Vitamin mixture
2
3.00
Carboximethylcellulose 2.00
Bicalcium phosphate 1.00
Cellulose 8.24
Asp/Glu
3
4.92
L-lysine HCl 0.00
Total 100.00
1
Mineral 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.
2
Vitamin mixture: vit. A, 290.000 UI; vit. D
3
, 143.500 UI; vit. E, 5.700 mg; vit. K
3
, 143.82 mg; thiamine (B
1
),
334.42 mg; riboflavin (B
2
), 667.20 mg; niacin (B
3
), 1,333.53 mg; pyridoxine (B
6
), 666.63 mg; pantothenic acid
(B
5
), 1.334 mg; biotin, 34 mg; folic acid, 67 mg; cobalamine, 1.000 μg and ascorbic acid (vit. C) 999.85 mg.
3
Mixture of aspartic and glutamic acid to adjust the quantity of nitrogen (protein) in all diets.
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.
Dairiki et al. 5
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
6 JOURNAL OF APPLIED AQUACULTURE
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 profile
1
15.77% of Nile
tilapia muscle
tissue
Amino acids
mixture
(%)
Total
(%)
Arginine 3.86
3
4.36 0.6876 3.17 3.86
Glycine 1.96
2
3.70 0.5835 1.38 1.96
Histidine 1.01
2
1.55 0.2444 0.77 1.01
Isoleucine 1.70
2
3.22 0.5078 1.19 1.70
Leucine 3.78
3
5.89 0.9289 2.85 3.78
Lysine 4.02
2
6.34 1.0000 0.00 1.00
Methionine 1.35
2
2.18 0.3438 1.01 1.35
Cystine 0.48
3
0.60 0.0946 0.39 0.48
Phenylalanine 1.81
2
3.32 0.5236 1.29 1.81
Tyrosine 1.59
3
2.65 0.4179 1.17 1.59
Serine 2.60
3
2.78 0.4384 2.19 2.60
Threonine 2.14
3
3.25 0.5125 1.63 2.14
Tryptophan 0.44
3
0.44 0.0694 0.37 0.44
Valine 2.63
3
3.50 0.5520 2.08 2.63
Proline 2.14
3
7.02 1.1071 1.03 2.14
Alanine 3.09
3
4.42 0.6970 2.39 3.09
Mixture 22.91
Asp/Glu 4.92
Fillet 15.77
Total CP 43.60
1
From Table 2.
2
Largemouth bass muscle tissue.
3
Largemouth bass roe.
Dairiki et al. 7
TABLE 4. Chemical composition of experimental diets.
Treatment
with lysine (%)
Dry matter
(%)
Mineral
matter (%)
Crude
protein
1
(%)
Crude lipid
1
(%)
Crude fiber
(%)
Energy
2
(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
1
Dry matter.
2
Original matter.
TABLE 5. Amino acids composition of experimental diets.
Diets
123456
DM 95.48 95.79 95.89 95.61 95.67 95.53
Amino acids
1
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
1
Percentage of original matter.
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 theingested 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 (W
f
), 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.
8 JOURNAL OF APPLIED AQUACULTURE
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 W
f
was considerably high (r
2
= 0.87), but
graphical determination of lysine requirement level for optimal W
f
was
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 W
f
.
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 r
2
= 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.
Dairiki et al. 9
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 = −0.2857x
2
+ 1.7643x + 1.9643
r
2
= 0.87
2.5 3 3.5 4
Lysine rates (%)
FIGURE 1. Quadratic regression curve adjusted for the final weight variable (W
f
).
Best SGR was estimatedforfish feddiets containing2.5 to3.5% 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)
10 JOURNAL OF APPLIED AQUACULTURE
4.0
3.0
2.0
WGa (g)
1.0
0.0
0.5 1
y = –0.2466x
2
+ 1.5743x + 0.8937
r
2
= 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 = –19.448x
2
+ 125.11x + 62.996
r
2
= 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
Dairiki et al. 11
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
r
2
= 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 = –0.119x
3
+ 0.939x
2
– 2.423x + 3.376
r
2
= 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 W
f
, 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
12 JOURNAL OF APPLIED AQUACULTURE
Lysine rates (%)
SGR (%)
0.50
0.0
0.5
1.0
1.5
2.0
2.5
1 1.5 2 2.5 3 3.5 4
y = –0.125x
2
+ 0.7508x + 0.9623
r
2
= 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 X e , i 1, 2...n n , ... n
LRi i 1 1+1
=+ + =⫺ ),,
(1)
where (R⫺X
LRi
)=0,foriⱖ n
1+1
,n
1
being 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 W
f
(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 forthe 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 chrysops E × 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
Dairiki et al. 13
1.2 to 1.4%. Both researches report dietary lysine requirement of
sunshine bass tobe smaller thanthat of largemouthbass. 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.
14 JOURNAL OF APPLIED AQUACULTURE
Lysine rates (%)
W
f
(g)
0.50
0
2
1
3
4
5
6
1 1.5 2 2.5 3 3.5 4
2.12%
(A)
W
f
= 2.384 + 1.05* level
r
2
= 0.99
(B)
W
f
= –0.2857x
2
+ 1.7643x + 1.9643
r
2
= 0.87
FIGURE 7. Regressions for the means of final weight (W
f
) in function of lysine
level on the diet: (A) Broken-line (ⵧ); (B) Polynomial (䉬)
Lysine rates (%)
WGa (g)
0.50
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
r
2
= 0.99
2.17%
(B)
WGa = −0.2466x
2
+ 1.5743x + 0.8937
r
2
= 0.86
FIGURE 8. Regressions for the means of absolute gain weight (WGa) in func-
tion of lysine level on the diet: (A) Broken-line (ⵧ); (B) Polynomial (䉬).
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 energyrequirements. Brougheret 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
Dairiki et al. 15
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
r
2
= 0.99
2.26%
(B)
WGr = –19.448x
2
+ 125.11x + 62.996
r
2
= 0.87
FIGURE 9. Regressions for the means of relative gain weight (WGr) in function
of lysine level on the diet: (A) Broken-line (ⵧ); (B) Polynomial (䉬).
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 thegraphic analysispolynomial 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 W
f
, WG, and SGR
significantly smaller, and FCR significantly worse. In addition, because
16 JOURNAL OF APPLIED AQUACULTURE
Lysine rates (%)
0.50
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
r
2
= 0.99
(B)
FCR = –0.119x
3
+ 0.939x
2
– 2.423x + 3.376
r
2
= 0.89
1.69%
FIGURE 10. Regressions for the means of feed conversion ratio (FCR) in func
-
tion of lysine level on the diet: (A) Broken-line (ⵧ); (B) Polynomial (䉬).
determination coefficients registered for the broken-line regression
curves (r
2
= 0.99) are considerably higher than those registered for poly-
nomial regression curves (r
2
= 0.84~0.89), the broken-line analysis
method can be considered not only more accurate and precise, but also an
elicitor of economicalefficiency 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) statedthat excessdietary aminoacids areeliminated and pro
-
tein deposition efficiency is maximized.
Each particularanimal species, fish included, has adefinite 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
Dairiki et al. 17
Lysine rates (%)
0.50
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
r
2
= 0.99
(B)
SGR = –0.1252x
2
+ 0.7508x + 0.9623
r
2
= 0.84
2.17%
FIGURE 11. Regressions for the means of specific growth rate (SGR) in func
-
tion of lysine level on the diet: (A) Broken-line (ⵧ); (B) Polynomial (䉬).
ingested nitrogen and quality of the food/feed protein, represented by
equation (2):
yPD T(1e )
max
bx
=−
−
(2)
where y = actual daily N-balance ⫹ NMR
1
/LW
2
kg
0.67
(mg), PD
max
T=
maximum theoretical capacity for daily N-balance ⫹ NMR/LW kg
0.67
(mg), x = daily N-intake/LW
0.67
(mg), b = slope of the curve, and e= basic
number of natural logarithm (
1
nitrogen maintenance requirement and
2
live 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 requirementsdeterminedfor other fishspecies; resultsare 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
18 JOURNAL OF APPLIED AQUACULTURE
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 highsimilarity amongessential
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 contentsof 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
Dairiki et al. 19
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
3.86
2
156 96 2.0
Histidine
1.01
1
41 25 0.5
Isoleucine
1.70
1
69 42 0.9
Leucine
3.78
2
152 94 2.0
Lysine
4.02
1
162 100 2.1*
Methionine
1.35
1
54 34 0.7
Cystine
†
0.48
2
19 12 0.3
Phenylalanine
1.81
1
73 45 0.9
Tyrosine
†
1.59
2
64 40 0.8
Threonine
2.14
2
86 53 1.1
Tryptophan
0.44
2
18 11 0.2
Valine
2.63
2
106 65 1.4
Total
24.81 1,000
1
Largemouth bass fillet.
2
Largemouth bass roe.
*Determined value for dose-response trial;
†
Nonessential amino acid.
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. Dietarymethionine requirement for optimal specific growth
rate, feed efficiency, and conversion and protein efficiency ratio of the yel
-
low croaker, Pseuosciaena croacea, is1.41% (Mai et al. 2005). Ahmed etal.
(2004) andAhmed and Khan (2005) determinedthat fingerling Indian major
carp (0.52 ± 0.21 g and 0.62 ± 0.02 g, respectively) require 1.8% dietary
20 JOURNAL OF APPLIED AQUACULTURE
TABLE 7. Comparative dietary amino acids requirements of varied species.
Amino acid Chum
salmon
1
Striped
bass
2
Red sea
bream
3
Atlantic
salmon
4
Largemouth
bass
5
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.
1
NRC (1993).
2
Small and Soares Jr (1998).
3
Foster and Ogata (1999).
4
Rollin et al. (2003).
5
Original research’s data.
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), KeembiyehettyandGatlin III(1992) registered100% sur-
vival, even for fish fed lysine-deficient diets. Moon and Gatlin III (1991),
working withred drum,Sciaenops ocellatus(0.9 g),observed thatgroups
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
Dairiki et al. 21
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
§
(%)
11.27⫾ 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
a
3.74 ⫾ 0.35
a
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
a
3.57 ⫾ 0.36
a
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
a
41.27⫾ 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
a
3.42 ⫾ 1.02
a
51.27⫾ 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
a
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
a
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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