Aquaculture nutrition, tập 16, số 3, 2010

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Aquaculture Nutrition
2010 16; 223–230

..........................................................................................

doi: 10.1111/j.1365-2095.2009.00654.x

United States Department of Agriculture, Agricultural Research Service, Sustainable Marine Aquaculture Systems, Fort Pierce,
FL, USA

Two experiments were conducted with Florida pompano,
Trachinotus carolinus L. at 3 and 28 g L)1 salinity to determine apparent crude protein digestibility (ACPD), energy
digestibility (AED) and amino acid availability (AAAA)
from soybean meal (SBM), soy protein isolate (SPI) and corn
gluten meal (CGM). Mean AAAA was similar to ACPD. In
ﬁsh adapted to 3 g L)1 salinity, they were 81.2% and 81.9%
(CGM), 93.6% and 92.2% (SBM), 93.8% and 93.1% (SPI)
for AAAA and ACPD respectively. In ﬁsh adapted to
28 g L)1, they were 84.5% and 83.4% (CGM), 86.5% and
87.1% (SBM), and 83.4% and 85.0% (SPI) for AAAA and
ACPD respectively. The AED was highest for SPI and lowest
for SBM and inversely related to carbohydrate. The ACPD,
AED and AAAA of soy products appeared to be lower in
high salinity, whereas CGM was unaﬀected. The data suggest
that SBM, SPI and CGM should be further evaluated as
partial ﬁshmeal replacements in Florida pompano diets.
Application of the generated coeﬃcients can be used to
develop well-balanced, low-cost diets for Florida pompano
reared in low salinity or seawater.
KEY WORDS: amino acid availability, digestible protein, plantbased proteins, pompano

Received 8 October 2008, accepted 16 December 2008
Correspondence: Marty Riche, USDA, ARS, 5600 US Hwy. 1, North, Fort
Pierce, FL 39496, USA. E-mail:
Present address: Terhea N. Williams, Marine Bio-Resources, University of
Maine, Rogers Hall, Orono, ME 04469, USA.

Florida pompano, Trachinotus carolinus L. is a euryhaline
species representing a small marine ﬁshery in Florida with an

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Ó 2009 Blackwell Publishing Ltd
No claim to original US government works

estimated 227 000 kg total annual catch; however, because of
its highly prized taste and texture it maintains a high market
demand. Florida pompano tolerate a wide range of salinities,
stress, readily consume pelleted rations, successfully breed in
captivity, and are an excellent candidate for aquaculture
(McMaster et al. 2004). There is increased interest in rearing
euryhaline species such as Florida pompano in freshwater or
low-salinity conditions. However, nutritionally balanced
diets do not exist for Florida pompano presenting an
obstacle to development of large-scale commercial production in low salinity.
High quality ﬁsh meal is the best source of protein for ﬁsh,
particularly for carnivorous species. However, replacement
of ﬁsh meal with alternative protein sources will increase
sustainability and proﬁtability (Glencross et al. 2007). In
addition to palatability and anti-nutritional concerns, use of
ingredients as alternatives to ﬁshmeal is limited by unknown

availability of nutrients. Apparent digestibility coeﬃcients of
feed ingredients exist for only a few ﬁsh species, but not
Florida pompano. To develop low-cost, low-polluting diets
that achieve maximum eﬃciency, nutrient requirements and
nutrient availability from dietary ingredients must be determined to implement least-cost formulation of economical
and balanced diets.
There is also evidence that salinity aﬀects nutrient digestibility (Lall & Bishop 1976; MacLeod 1977; Dabrowski et al.
1986; Krogdahl et al. 2004). Digestibility in Golden-line
seabream, Sparus sarba (Forsskal) was higher in low salinity
relative to isosmotic or full-strength seawater (Woo & Kelly
1995). Similarly, protein digestibility in milkﬁsh, Chanos
chanos (Forsskal) was elevated in freshwater relative to
saltwater (Ferraris et al. 1986). Zeitoun et al. (1973) also
suggested that protein requirements of rainbow trout,
Oncorhynchus mykiss (Walbaum) were higher with increasing
salinity.

We hypothesized protein digestibility and amino acid (AA)
availability would be diﬀerent in low-salinity adapted Florida
pompano than saltwater adapted Florida pompano. Therefore, the objective was to determine apparent digestibility of
crude protein (CP), energy, and AA availability from soybean meal (SBM), soy protein isolate (SPI) and corn gluten
meal (CGM) at both 3 and 28 g L)1 salinity representing the
known range of salinity supporting normal growth of Florida
pompano.

Florida pompano broodstock were spawned at the USDA,
Agricultural Research ServiceÕs Center for Reproduction and
Larviculture, Fort Pierce, Florida, USA. Postlarval juveniles
were reared at 28 °C and 30 g L)1 salinity. Fish were fed a

commercial diet (EPAC-CW or IDL-CW; INVE Americas,
Salt Lake City, UT, USA) until they were approximately 3 g
in weight. Fish were subsequently transferred to a nursery
system where they were held at 28 °C and 7 g L)1 salinity
and fed a commercial trout diet (Silver Cup; Nelson & Sons,
Inc., Murray, UT, USA) until they were approximately 75 g
in weight at which time they were acclimated to 3 or 28 g L)1
salinity over a 1-week period.
Two simultaneous 4 · 4 Latin squares were set up to
evaluate the three feed ingredients at 3 and 28 g L)1 salinity.
Two 8750 L recirculating systems with sand, bead, cartridge
and carbon ﬁltration, and ultraviolet light sterilization were
used. Both systems were maintained at 28 °C. Four 100-L
tanks in each system with nominal ﬂow rates of 3 L min)1
served as experimental units. Fish were maintained under a
natural light cycle approximating 13 h light and 11 h dark.
A menhaden ﬁsh meal based formulation meeting the
known protein and energy requirements for pompano served
as the reference diet (Table 1). Solvent-extracted SBM
(Rangen, Inc., Buhl, ID, USA), SPI (Archer Daniels Midland, Decatur, IL, USA) and CGM (Rangen, Inc.) were
substituted at 300 g kg)1 for 300 g kg)1 of the reference diet
utilizing a modiﬁed diet replacement method. All diets
incorporated yttrium oxide (Y2O3) at 5 g kg)1 of the diet as
an inert marker. Feed ingredients were ground via hammermill (Prater Industries, Inc., Chicago, IL, USA) to pass a
250 micron screen. Dry feed ingredients were mixed in a
V-mixer (Patterson-Kelley, East Stroudsburg, PA, USA).
Following addition of water and oil, complete diets were cold
extruded and dried at 60 °C for 24 h. Pelleted diets were
stored at )20 °C until fed.
Twenty and 15 ﬁsh each, were stocked into 28 and 3 g L)1

salinity experimental units respectively. Fish were fed a

Table 1 Reference and test diets used to determine digestibility of
crude protein, energy and amino acid availability from soybean meal,
soy protein isolate, and corn gluten meal in Florida pompano
Trachinotus carolinus
Ingredient (g kg)1 dry diet)

Reference
diet

Test
diets

Test ingredient
Menhaden meal (low temperature)1
Soybean meal (solvent extracted)2
Corn gluten meal2
Porcine blood meal (spray dried)2
Fish solubles (dehydrated)3
Shrimp meal2
Dextrin (type-II from corn)4
Menhaden oil (stabilized)5
Sipernat 506
Mineral premix7
Vitamin premix8
Lecithin9
Ascorbyl-2-monophosphate8
a-Cellulose10
Carboxymethyl cellulose10

Yttrium oxide10

0.0
338.5
221.0
68.0
30.0
60.0
50.0
22.0
139.0
10.0
15.0
5.0
1.0
0.5
15.0
20.0
5.0

300.0
237.0
154.7
47.6
21.0
42.0
35.0
15.4
97.3
7.0

10.5
3.5
0.7
0.4
9.0
14.0
5.0

1

Special SelectÔ, Omega Protein, Inc., Houston, TX, USA.
Rangen Inc., Buhl, ID, USA.
3
International Proteins Corp., Minneapolis, MN, USA.
4
MP Biomedicals, Solon, OH, USA.
5
Alkali refined and stabilized with 500 ppm ethoxyquin, Omega
Protein, Inc., Hammond. LA, USA.
6
Degussa Corp., Parsippany, NJ, USA.
7
Mineral premix contained the following (g kg)1 premix): CaHPO4, 350.0; CaSO4Æ2H20, 100.0; KH2PO4, 200.0; MgSO4Æ7H20, 84.0;
FeSO4Æ7H2O, 16.0; ZnSO4Æ7H2O, 3.0; MnSO4ÆH2O, 2.0; CuCl2Æ2H20,
1.0; KF, 0.23; KI, 0.1; NaMoO4Æ2H2O, 0.05; CoCl2Æ6H2O, 0.02; Na2SeO3, 0.01.
8
Roche Vitamins Inc, Parsippany, NJ, USA.
9
USB, Cleveland, OH, USA.
10

Sigma-Aldrich, St. Louis, MO, USA.
2

commercial diet and allowed a 4-day acclimation to the new
environment. At initiation of the experiment, ﬁsh were
switched to their assigned experimental diet and fed 4.7%
body weight per day divided between a morning and afternoon feeding. Faecal samples were collected on day 5 and
day 7 of being fed the experimental diets. Faecal samples
were collected 3–4 h following the morning feeding on day of
collection.
Prior to faecal collection, ﬁsh were anaesthetized with
75 mg L)1 tricaine methanesulphonate (MS-222; Western
Chemical, Inc., Ferndale, WA, USA). Upon induction of
stage IV anaesthesia, the area around the anus was dried with
a towel and faecal samples collected by gentle expression of
the lower gastrointestinal tract (Austreng 1978). Immature
ﬁsh were used and care was taken not to contaminate samples with urine. Following collection, ﬁsh were resuscitated
and placed back into the experimental unit. Faeces collected

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Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230
No claim to original US government works

on both day 5 and 7 were pooled into one sample. Diets were
reassigned to the experimental units and procedures were
repeated until all four experimental units received each of the
four diets (4 weeks).
Feed and pooled faecal samples were analysed for yttrium

(Y), nitrogen (N), gross energy (GE) and AA. Proximate
composition of reference and test diets was determined and
test ingredients were analysed for each ingredientÕs contribution of nutrients to the test diet (Table 2). Coeﬃcients
were calculated as the ratio of nutrient and marker in feed
and faeces (Maynard & Loosli 1969) and adjusted for
nutrient concentration (Forster 1999).
Test ingredients, feed and faecal samples were lyophilized
to a constant weight and stored at )80 °C until analysis.
Nitrogen was determined following combustion (TruSpec
N-elemental analyser; Leco Corp., St. Joseph, MI, USA)
and CP calculated as N · 6.25. GE was determined by

Table 2 Analysed composition (g kg)1)
of test ingredients and experimental
diets fed to Florida pompano Trachinotus carolinus

adiabatic bomb calorimetry (Parr 1266; Parr Instruments
Co., Moline, IL, USA). Ash was determined following
incineration at 600 °C for 2 h (AOAC 2002). Crude lipid
was
determined
gravimetrically
following
chloroform : methanol extraction (Bligh & Dyer 1959) in a
Soxhlet apparatus. Crude ﬁbre was determined by a commercial laboratory (Barrow-Agee Laboratories, Memphis,
TN, USA).
Amino acids were analysed by a commercial laboratory
(Midwest Laboratories, Inc., Omaha, NE, USA). Brieﬂy,
samples were hydrolyzed with 6 N HCl at 110 °C for 24 h. A
separate aliquot was analysed for cysteine (Cys) and methionine (Met) following performic acid oxidation to cysteic

acid and methionine sulphone. Amino acids were separated
using a C-18 reverse phase HPLC column and quantiﬁed
with a photodiode array detector following postcolumn
derivatization with ninhydrin.

Test ingredient

International feed no.

CGM1
5-28-242

Proximate components
Dry matter
917.0
Crude protein
653.0
Crude lipid
22.0
Ash
26.0
Fibre
8.0
NFE4
214.0
Gross energy (kJ g)1)
20.9
Indispensable amino acids
Arginine
21.1

Histidine
19.8
Isoleucine
24.2
Leucine
84.8
Lysine
10.0
Methionine
24.4
Phenylalanine
45.5
Threonine
23.9
Valine
29.2
Dispensable amino acids
Alanine
66.6
Asx5
43.2
Cysteine
21.5
Glx6
170.3
Glycine
18.9
Proline
55.1
Serine

41.0
Tyrosine
35.8
1
2
3
4
5
6

SPI3
–

Reference
diet

CGM
diet

SBM
diet

SPI
diet

894.0
474.0
11.0
57.0
33.0

342.0
17.4

915.0
885.0
2.0
39.0
2.0
0.0
20.9

947.0
523.0
160.0
144.0
34.0
86.0
21.0

884.0
542.0
118.0
103.0
27.0
94.0
21.7

882.0
498.0
107.0

123.0
35.0
119.0
20.8

926.0
628.0
108.0
110.0
24.0
56.0
21.9

34.9
14.4
19.7
40.4
30.6
10.3
25.5
19.9
21.9

60.3
17.0
42.2
75.0
53.6
10.9
46.5

35.1
41.3

26.5
13.5
20.5
46.7
32.6
13.5
22.9
20.4
27.5

22.6
12.5
19.3
60.9
25.3
14.3
27.3
20.2
26.5

27.6
12.7
18.4
39.4
31.4
12.2
21.9

19.5
21.0

37.6
15.9
27.3
52.5
37.6
12.7
29.4
24.6
32.9

21.9
58.8
10.4
99.6
21.9
25.5
28.1
18.1

51.5
112.0
08.4
162.0
38.8
44.5
50.5
32.2

40.4
50.1
14.5
74.6
30.8
24.5
23.2
16.2

49.8
45.5
11.9
89.8
25.7
33.4
25.7
20.8

35.9
51.2
13.2
76.9
26.6
24.1
23.4
15.7

46.4
67.7

11.2
101.0
32.5
29.9
30.6
20.6

Corn gluten meal, Rangen, Inc., Buhl, ID, USA.
Dehulled, solvent-extracted soybean meal; Rangen, Inc., Buhl, ID, USA.
Soy protein isolate, Pro-FamÒ, Archer Daniels Midland, Decatur, IL, USA.
Nitrogen-free extract (100 ) moisture ) crude protein ) crude lipid ) ash ) fibre).
Aspartic acid + asparagine.
Glutamic acid + glutamine.

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Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230
No claim to original US government works

SBM2
5-04-612

Diﬀerences in apparent nutrient availability were analysed
using the model statement for a Latin square design:
Yijk ¼ l þ Ii þ columnj þ rowk þ eijk ;

Table 3 Mean (SEM, n = 4) apparent crude protein (ACPD) and
energy (AED) digestibility coeﬃcients (%) for soybean meal, soy
protein isolate and corn gluten meal fed to Florida pompano

Trachinotus carolinus adapted to 3 or 28 g L)1 salinity
ACPD

where I represents the main eﬀect of test ingredient, column
represents variation due to tank, and row represents variation due to week. Analysis was performed using the general
linear model procedure of SAS with software package version
9.1 (SAS Institute, Cary, NC, USA). Residuals were analysed
to evaluate normality of distribution and homogeneity of
variance. Where main eﬀect diﬀerences were detected pairwise contrasts between the three ingredients were evaluated.
Signiﬁcance was reported at P < 0.05 unless otherwise stated. Where analysis indicated row or column eﬀects in
3 g L)1 salinity (alanine) or 28 g L)1 (glutamic acid + glutamine) no further analysis was conducted as row and column both represent restrictions on randomization making
the F-test questionable. Regression analysis was performed
with test ingredient protein and energy as independent variables and apparent energy digestibility (AED) as the dependent variable.

Total ammonia-nitrogen ranged from 0.00 to 0.21 mg L)1
and 0.01 to 0.17 mg L)1 for the low-salinity and saltwater
systems, respectively. Nitrite-nitrogen was 0.04–5.01 and
0.04–0.56 mg L)1 for the low-salinity and saltwater systems,
respectively. The pH and alkalinity ranged from 6.92 to
8.08 mg L)1 and 138 to 190 mg L)1 as CaCO3 at 3 g L)1
salinity and from 6.62 to 7.95 mg L)1 and 86 to 139 mg L)1
as CaCO3 at 28 g L)1 salinity. Values were within acceptable
ranges for Florida pompano (Watanabe 1995; Weirich &
Riche 2006). No mortalities occurred during the experiment.
Apparent crude protein digestibility (ACPD) was signiﬁcantly higher in the soy products than CGM at low salinity,
but not in sea water where no diﬀerences were detected
(Table 3). The AED was higher from SPI than CGM and
SBM at low salinity. Despite a decrease in AED of the soy
products at 28 g L)1, the coeﬃcient for SPI remained higher
than SBM, but not CGM.

Insuﬃcient faeces necessitated reporting apparent Met and
Cys availability on either two or three samples. Therefore,
statistical analysis was not performed on these two AA.
Signiﬁcant diﬀerences in apparent amino acid availability
(AAAA) were detected for phenylalanine (Phe) and glutamic
acid + glutamine (Glx) at 3 g L)1 salinity (Table 4). No
other diﬀerences were detected at low salinity. Although not

AED

Test ingredient

3 g L)1

28 g L)1

3 g L)1

28 g L)1

Reference diet
Corn gluten meal
Soybean meal
Soy protein isolate

72.8
81.9
92.2
93.1

74.7
83.4
87.1
85.0

71.3
77.4
70.5
93.4

72.3
77.4
62.2
78.1

(0.5)
(4.2)b
(2.0)a
(1.9)a

(1.1)
(2.9)a
(3.6)a
(3.5)a

(1.2)
(4.2)b
(6.5)b
(2.5)a

(0.7)
(3.4)a
(4.0)b
(4.1)a

Mean values within a column having different superscripts were
significantly different (P < 0.05).

Table 4 Mean (SEM; n = 4) apparent amino acid availability
(AAAA) coeﬃcients (%) for soybean meal (SBM), soy protein isolate (SPI) and corn gluten meal (CGM) in Florida pompano
Trachinotus carolinus adapted to 3 g L)1 salinity

Amino acids
Indispensable
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine1
Phenylalanine
Threonine
Valine
Dispensable
Alanine
Asx2
Cysteine1
Glx3
Glycine
Proline

Serine
Tyrosine
Overall
mean AAAA

Reference
diet
CGM

SBM

83.3
80.3
80.4
86.1
81.9
83.0
84.0
75.3
82.2

(1.0)
(0.6)
(1.2)
(0.3)
(1.1)
(1.8)
(0.5)
(1.0)
(2.0)

73.5
76.8
68.1
88.1
76.2
100.0
83.2
81.0
81.6

(23.3)
(6.1)
(10.4)
(4.3)
(17.9)
(2.8)
(6.7)b
(9.4)
(7.9)

102.0
103.3
91.8
92.1
100.0
110.1
97.0
92.3
85.6

81.5
73.9
84.5
80.0
71.8
73.5
79.3
83.1
80.2

(1.3)
(0.3)
(0.5)
(0.6)
(1.4)
(0.7)
(1.3)
(1.0)
(1.0)

91.5
79.3
67.8
86.5
72.8
84.2
84.9
84.8
81.2

(5.0)
(7.1)
(10.9)
(3.7)b
(9.8)
(3.5)
(7.3)
(9.2)
(2.0)

89.7
87.7
91.5
94.0
71.7
88.9
94.5
99.4
93.6

SPI

(7.4)
95.3 (1.4)
(9.0)
92.5 (4.5)
(11.7) 96.4 (1.7)
(1.1)
94.7 (1.0)

(4.7)
94.1 (2.6)
(6.0) 105.7 (8.1)
(3.5)a 95.1 (2.7)a
(6.0)
89.9 (6.4)
(8.5)
98.6 (3.1)
(10.9)
(3.8)
(3.3)
(3.1)a
(12.3)
(2.5)
(4.4)
(7.1)
(2.1)

96.4
90.9
82.8
93.8
88.4
93.1
93.6
92.9
93.8

(2.0)
(4.1)

(5.2)
(3.1)a
(2.7)
(3.4)
(3.5)
(3.4)
(1.2)

Different superscripts across a row indicate significant differences
between ingredients tested (P < 0.05).
1
Not statistically evaluated due to insufficient material for suitable
replication (n = 2).
2
Aspartic acid + asparagine.
3
Glutamic acid + glutamine.

statistically diﬀerent, the overall pattern suggests that AAAA
appears higher from soy products than CGM at low salinity
in agreement with ACPD. The availability of Met
approached 100% for all ingredients. Overall mean AAAA
was similar to ACPD for all test ingredients, they were 81.2%
and 81.9% (CGM), 93.6% and 92.2% (SBM), 93.8% and
93.1% (SPI) for AAAA and ACPD respectively.

..............................................................................................

Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230
No claim to original US government works

Table 5 Mean (SEM; n = 4) apparent amino acid availability
(AAAA) coeﬃcients (%) for soybean meal (SBM), soy protein isolate (SPI) and corn gluten meal (CGM) in Florida pompano
Trachinotus carolinus adapted to 28 g L)1 salinity

Amino acids
Indispensable
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine1
Phenylalanine
Threonine
Valine
Dispensable
Alanine
Asx2
Cysteine1
Glx3
Glycine
Proline
Serine
Tyrosine
Overall
mean AAAA

Reference

diet
CGM

SBM

SPI

87.5
81.1
81.6
87.7
83.0
87.9
85.6
73.2
86.2

(0.6)
(1.1)
(1.6)
(0.7)
(0.4)
(0.3)
(0.4)
(3.5)
(0.6)

89.0
84.4
79.6

92.0
77.4
92.9
90.2
87.6
84.6

(3.3)
78.1 (10.2)
(5.5)
88.7 (7.7)
(5.4)
84.6 (17.9)
(1.4)
85.6 (7.1)
(7.9)b
95.6 (2.1)a
(1.8)
93.9 (6.0)
(0.9)
79.6 (11.8)
(5.3)
105.3 (4.8)
(0.8)ab 75.7 (5.3)b

79.4
83.6
91.9
85.4
83.8

90.2
85.2
87.7
88.1

(16.8)
(4.6)
(5.9)
(5.6)
(4.2)ab
(4.5)
(3.5)
(9.3)
(5.0)a

84.9
73.7
85.3
81.9
72.5
77.6
75.7
85.5
81.8

(0.7)
(2.4)
(1.0)
(0.4)
(1.5)

(0.4)
(3.8)
(0.5)
(1.3)

88.7
79.0
68.3
86.8
68.8
84.3
93.1
89.3
84.5

(1.9)
(6.2)
(1.7)
(2.8)
(9.6)
(3.1)
(5.5)
(1.2)
(1.8)

81.6
80.6
51.9
86.2
77.0

84.2
92.4
88.5
83.4

(5.3)
(5.6)
(8.1)
(3.5)
(7.3)
(4.1)
(6.8)
(4.2)
(2.2)

80.9
98.7
73.7
87.9
86.1
79.5
105.7
70.6
86.5

(10.9)
(15.4)
(2.4)
(3.0)
(16.4)

(8.2)
(4.8)
(19.2)
(2.5)

Different superscripts across a row indicate significant differences
between ingredients tested (P < 0.05).
1
Not statistically evaluated due to insufficient material for suitable
replication (CGM, n = 3; SBM, n = 3; SPI, n = 2).
2
Aspartic acid + asparagine.
3
Glutamic acid + glutamine.

Signiﬁcant diﬀerences in AAAA were detected for lysine
(Lys) and valine (Val) at 28 g L)1 (Table 5). Availability of
Lys was higher from SBM (95.6%) than CGM (77.4%), and
neither was diﬀerent from SPI (83.8%). Apparent availability
of Val was higher from SPI (88.1%) than SBM (75.7%), and
neither was diﬀerent from CGM (84.6%). No other diﬀerences were detected at 28 g L)1 salinity. As with low-salinity
treatments, overall mean AAAA was similar to ACPD for all
test ingredients. They were 84.5% and 83.4% (CGM), 86.5%
and 87.1% (SBM), and 83.4% and 85.0% (SPI) for AAAA
and ACPD respectively.

Apparent digestibility of CP was high for all test ingredients
regardless of salinity, particularly relative to the reference
diet. The high ACPDs suggest a potential for these plant
proteins as partial replacements for ﬁsh meal in Florida

pompano diets. Apparent digestibilities of CP and GE from
the reference diet were lower than reported for some marine
species fed compounded diets (Santinha et al. 1999; Peres &

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Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230
No claim to original US government works

Oliva-Teles 1999; Sa´ et al. 2006). The reason is unclear;
however, the values in this study are similar to previously
reported values (75.8% and 73.3% for ACPD and AED
respectively) for juvenile Florida pompano fed the same diet
formulation (Riche, new characters, 2009).
Poor digestibility is one reason attributed to low feed
eﬃciency (FE) in Florida pompano (Tatum 1973; McMaster
1988; Lazo et al. 1998; Weirich et al. 2006). However, SBM
digestibility and AAAA at 3 g L)1 salinity were similar to
that observed in yellowﬁn sea bream, Acanthopagrus latus
(Houttuyn) (Wu et al. 2006) and Atlantic cod, Gadus morhua
L. (Tibbetts et al. 2006). Also, ACPD for SBM at 28 g L)1
salinity was the same as reported for gilthead seabream,
Sparus aurata L. (Lupatsch et al. 1997). Although ACPD for
SBM was similar to that reported for haddock, Melanogrammus aegleﬁnus L. (92.2%) and Cobia, Rachycentron
canadum L. (92.8%), AED was approximately 18–20% lower
in Florida pompano than haddock or cobia (Tibbetts et al.
2004; Zhou et al. 2004). Low apparent digestible energy
values from SBM were also reported in European seabass,
Dicentrarchus labrax L. (da Silva & Oliva-Teles 1998) and red
drum, Sciaenops ocellatus L. (Gaylord & Gatlin 1996).

De Silva & Perera (1984) suggested that lower protein
digestibility occurs in diets with higher protein. However, in
this study no diﬀerence in protein digestibility between soy
products was detected at either salinity despite 130 g kg)1
higher protein in the SPI diet. Conversely, in this study AED
was directly proportional to dietary protein (r2 = 1.00) and
inversely proportional to dietary nitrogen free extract (NFE;
r2 = 0.99). Utilization of plant starch is limited in ﬁsh,
particularly carnivores. Digestible energy tends to be negatively correlated to dietary carbohydrate and positively correlated to dietary protein and lipid (Sullivan & Reigh 1995).
Carbohydrate digestibility in Florida pompano is about 50%
(Williams et al. 1985) underscoring its limited availability
and impact on energy digestibility.
Florida pompano have short digestive tracts. Intestinal
transit time for a ﬁsh meal/SBM diet was reported as 3 h in
seawater at 29–31 °C (Williams et al. 1985). This was later
conﬁrmed using the same dietary formulation serving as the
reference diet in this study (Riche, new characters, 2009). The
short transit may result in limited enzymatic contact time
attenuating digestion and absorption of nutrients, possibly
causing the poor FE reported for Florida pompano.
Faecal stripping was initiated 3 h postprandially. Consistent results with previous trials (Riche, new characters, 2009)
coupled with the small SEM of coeﬃcients in the reference
diet suggests that this was appropriate for the reference diet.
However, the high SEM of coeﬃcients associated with the

test ingredients, particularly AAAA coeﬃcients for SBM and
CGM suggests incomplete digestion or possible interactive
eﬀects. Composition, chemical, and physical characteristics
of feed can aﬀect both. Also, faecal collection method aﬀects

variability of availability values, with greater variability
observed using faecal stripping (Yamamoto et al. 1997).
Digestibility coeﬃcients are also generally lower using
intestinal stripping relative to other methods (Hajen et al.
1993; Yamamoto et al. 1997). However, Glencross et al.
(2005) demonstrated feed ingredients high in carbohydrates,
such as SBM and CGM, aﬀect faecal pellet integrity and
suggested that stripping is the preferred faecal collection
method for plant protein digestibility trials. Moreover, this
method obviates diluting nutrient concentrations by external
saltwater contamination of faeces.
Digestibility coeﬃcients for SBM and SPI reported for
Chinook salmon, Oncorhynchus tshawytscha (Walbaum)
were much lower than for Florida pompano (Hajen et al.
1993). Conversely, energy and N digestibility of SPI in
rainbow trout (Glencross et al. 2005) and Atlantic cod
(Tibbetts et al. 2004) were higher than for pompano, while N
digestibility for SBM was the same. The signiﬁcant diﬀerence
observed in AED between SBM and SPI was also observed in
rainbow trout and Atlantic salmon, Salmo salar L. (Glencross et al. 2004), again supporting the negative eﬀect of
carbohydrates on digestible energy in carnivorous species.
Protein digestibility of the soy products was higher than
CGM at low salinity, but not at 28 g L)1. Energy digestibility of CGM was similar in haddock, but ACPD in haddock was approximately 10% higher (Tibbetts et al. 2004).
Also, the energy digestibility coeﬃcient of non-extruded
CGM in rainbow trout was similar to that reported here, but
increased substantially following extrusion (Cheng & Hardy
2003). It is likely extrusion processing would increase ADE
of CGM in Florida pompano as well.
The AAAA from SBM in Florida pompano was similar to
yellowﬁn seabream, Sparus latus (Houttuyn) with the

exception of Lys and Phe availability being higher, and Val
lower in pompano (Wu et al. 2006). In cobia, AAAA from
SBM was similar to Florida pompano, but that from CGM
was higher ranging from 93.2% to 96.9% (Zhou et al. 2004).
Overall AAAA of SBM and SPI reﬂected CP digestibility as
reported elsewhere (Yamamoto et al. 1997; Allan et al. 2000;
Zhou et al. 2004).
The AAAA from CGM was 5.7–16.3% lower relative to
Australian silver perch, Bidyanus bidyanus (Mitchell) for all
indispensable AA except Met (Allan et al. 2000). They were
also substantially lower than in rainbow trout where all
AAAA were >95% (Yamamoto et al. 1997). Pompano fed a

CGM based diet supplemented with AA to match their whole
body AA proﬁle exhibited only 60% of the weight gain of
pompano fed a menhaden meal based diet with the same AA
proﬁle (Riche; unpublished data). Results from this study
suggest that poor weight gain previously observed was due in
part to lower AA availability from CGM.
Apparent availability of Met was high for all test ingredients, as it was in cobia (Zhou et al. 2004). The Met availability from test ingredients evaluated in low salinity was
100–110%, suggesting enhanced availability from the other
protein sources used in the test diets. However, caution
should be exercised in interpreting the Met values as insufﬁcient material in some cases limited the number of samples
for estimating means.
Signiﬁcantly, lower apparent Lys availability was observed
from CGM than SBM at the higher salinity (P < 0.05) and
appeared to be lower than both soy products at low salinity.
This is similar to that reported for Australian silver perch
(Allan et al. 2000), red sea bream, Pagrus major (Temminck
& Schlegel) (Yamamoto et al. 1998), and yellowtail, Seriola

quinqueradiata (Temminck & Schlegel) (Masumoto et al.
1996), but the opposite of cobia (Zhou et al. 2004) and
Atlantic salmon (Anderson et al. 1992). Lower Lys availability from CGM relative to the soy products may be an
artefact of lower Lys in CGM. Analysis of test ingredients
indicated Lys was 53.6, 30.6 and 10.0 g kg)1 dry matter for
SPI, SBM and CGM respectively. At low dietary Lys,
endogenous sources account for more of the recovered Lys
masking true availability and depressing apparent availability. The 10% increase in true Lys availability over apparent
Lys availability from CGM in red sea bream (Yamamoto
et al. 1998) and yellowtail (Masumoto et al. 1996) support
this hypothesis.
The low CGM coeﬃcients and high variability for Arg
(SEM of 23.3%) and Lys (SEM of 17.9%) in the low salinity
treatment are attributable to high recovery of these AA in
one faecal sample resulting in AAAA for that replicate of
6.5% and 29.1% for Arg and Lys respectively. Removal of
that sample from consideration would have resulted in
coeﬃcients of 95.8% and 91.9% for Arg and Lys, respectively, which are similar to the other ingredients. Although
residuals of the coeﬃcients tested as outliers (Snedecor &
Cochran 1967), the coeﬃcients were not removed from
analysis because row and column eﬀects could not be ruled
out. Moreover, it is possible the coeﬃcients could represent
true variability in AAAA for a marine species held at low
salinity.
Although the experimental design precludes statistical
analysis of test ingredients between the two salinities, the

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Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230

No claim to original US government works

trend was towards higher ACPD and AED from SBM and
SPI for pompano reared in low salinity water relative to
seawater. This could result in lower FE in saltwater and
suggests that dietary protein may need to be higher for
production in saltwater as reported for other species (Zeitoun
et al. 1973; Lall & Bishop 1976). The data suggest that further research is warranted to determine if digestibility values
are lower in a seawater environment.
In summary, the ACPD of SBM and SPI were >90% in
low salinity, and signiﬁcantly higher than CGM. However,
no diﬀerences in ACPD could be detected between the
three ingredients in seawater. As the ACPD coeﬃcient for
CGM was similar between the two salinities it appears
protein digestibility of the soy products may be lower in
seawater than freshwater, although this could not be tested.
The AED for the three test ingredients exhibited a parallel
response to salinity as the ACPD. The AED of SBM was
signiﬁcantly lower than SPI and was likely due to the CP/
NFE ratio as there was a positive linear relationship
(r2 = 1.00) with protein and inverse relationship
(r2 = 0.99) with NFE. The overall AAAA from the test
ingredients was similar to the ACPD coeﬃcients and suggests that SBM, SPI and CGM should be further evaluated
as partial ﬁshmeal replacements in Florida pompano diets.
Application of the protein, energy and AA coeﬃcients for
SBM, SPI, and CGM generated in this study can be used
to develop well-balanced, low-cost diets for Florida pompano reared in low salinity or in seawater addressing one of
the obstacles to large-scale commercial production of this
species.

The authors acknowledge David I. Haley and Patrick L.
Tracy for their skilful technical assistance in sample collection, preparation and processing. We would also like to
express gratitude to Dr T. Gibson Gaylord, Dr Jon Amberg
and Dr Hector Acosta-Salmon for critical review and advice
on preparation of this manuscript. The authors acknowledge
Rangen, Inc. Buhl, Idaho for generously donating the plantbased proteins. This work was funded in part by the Link
Foundation. Additional funding was provided by the
USDA/Agricultural Research Service Project No. 622563000-007-00D. Mention of trade names or commercial
products in this article is solely for the purpose of providing
speciﬁc information and does not imply recommendation or
endorsement by the US Department of Agriculture. All
programmes and services of the US Department of Agriculture are oﬀered on a non-discriminatory basis without

..............................................................................................

Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230
No claim to original US government works

regard to race, colour, national origin, religion, sex, marital
status, or handicap.

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Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230
No claim to original US government works

Aquaculture Nutrition
doi: 10.1111/j.1365-2095.2009.00655.x

2010 16; 231–236

..........................................................................................

1,2
1

1,3

1

1

1

1

1

The Key Laboratory of Mariculture (Education Ministry of China), Ocean University of China, Qingdao, China; 2 Center for
Bioengineering and Biotechnology, China University of Petroleum, Qingdao, China; 3 Guangdong Yuehai Feed Group Co. Ltd.,
Xiashan District, Zhanjiang, China

Correspondence: Kangsen Mai, The Key Laboratory of Mariculture
(Education Ministry of China), Ocean University of China, 5 Yushan
Road, Qingdao 266003, China. E-mail:

A 9-week feeding experiment was conducted to determine the
dietary biotin requirement of Japanese seabass, Lateolabrax
japonicus C. Six isonitrogenous and isoenergetic puriﬁed diets
(Diets 1–6) containing 0, 0.01, 0.049, 0.247, 1.238 and

6.222 mg biotin kg)1 diet were fed twice daily to triplicate
groups (30 ﬁsh per group) of ﬁsh (initial average weight
2.26 ± 0.03 g) in 18 ﬁbreglass tanks (300 L) ﬁlled with 250 L
of water in a ﬂow-through system. Water ﬂow rate through
each tank was 2 L min)1. Water temperature ranged from
25.0 to 28.0 °C, salinity from 28.0 to 29.5 g L)1, pH from 8.0
to 8.1 and dissolved oxygen content was approximately
7 mg L)1 during the experiment. After the feeding experiment, ﬁsh fed Diet 1 developed severe biotin deﬁciency syndromes characterized by anorexia, poor growth, dark skin
colour, atrophy and high mortality. Signiﬁcant lower survival
(73.3%) was observed in the treatment of deﬁcient biotin.
The ﬁnal weight and weight gain of ﬁsh signiﬁcantly
increased with increasing dietary biotin up to 0.049 mg kg)1
diet (P < 0.05), and then slightly decreased. Both feed eﬃciency ratio and protein eﬃciency ratio showed a very similar
change pattern to that of weight gain. Dietary treatments did
not signiﬁcantly aﬀect carcass crude protein, crude lipid,
moisture and ash content. However, liver biotin concentration (0–6.1 lg g)1) signiﬁcantly increased with the supplementation of dietary biotin (P < 0.05), and no tissue
saturation was found within the supplementation scope of
biotin. Broken-line regression analysis of weight gain showed
that juvenile Japanese seabass require a minimum of
0.046 mg kg)1 biotin for maximal growth.
KEY WORDS: biotin requirement, growth, Japanese seabass
(Lateolabrax japonicus)

Received 9 April 2008, accepted 7 January 2009

..............................................................................................

Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd

Biotin is a water-soluble vitamin generally included in vitamin B complex and functions in several speciﬁc carboxylation and decarboxylation reactions. It is part of the
coenzymes of several carboxylation enzymes ﬁxing CO2, such
as propionyl CoA in the formation of propionic acid, acetylCoA carboxylase and pyruvate carboxylase. Carboxylase
ﬁxation of CO2 to form methylmalonyl CoA is involved in
the carboxylation and decarboxylation of tricarboxylic acids.
Biotin is also involved in the syntheses of fatty acids, lipids
and citrulline.
As biotin is one of the most important and expensive
vitamins added to aquafeeds, it is necessary to quantify the
minimum requirement of biotin to manufacture cost-eﬀective
commercial feeds. However, several factors have been proved
to inﬂuence the need for dietary biotin in animals. For
example, high dietary lipid has been shown to obscure the
eﬀects of biotin in rats, chicks and brook trout (Jacobs et al.
1970; Marson & Donaldson 1972; Poston & McCarteney
1974). The quantitative requirement of biotin for optimal
growth has only been investigated in few species of ﬁsh. For
example, 0.1 mg kg)1 for lake trout (Poston 1976), 0.02–
0.03 mg kg)1 for common carp (Ogino et al. 1970), 0.05–
0.14 mg kg)1 for rainbow trout (Castledine et al. 1978;
Woodward & Frigg 1989), 2.0–2.5 mg kg)1 for mirror carp
(Gu¨nther & Meyer-Burgdorﬀ 1990), 0.06 mg kg)1 for hybrid
tilapia (Shiau & Chin 1999), 2.49 mg kg)1 for Asian catﬁsh
(Mohamed et al. 2000) and 0.25 mg kg)1 for Indian catﬁsh
(Mohamed 2001).
Japanese seabass (Lateolabrax japonicus) is an economically important food ﬁsh with fast growth and high market
value, and now has been widely cultured in China. However,

a main constraint to Japanese seabass culture is the limited

supply of trash ﬁsh that is presently the main feed source for
grow-out. Hence, there is an urgent need to develop a suitable practical diet for grow-out production of this ﬁsh. One
of the prerequisites for developing high eﬃcient diet for
Japanese seabass requires complete knowledge of its nutritional requirements. A few studies have been reported on the
nutrition of this seabass (Lin et al. 1994; Hu et al. 1995; Gao
et al. 1998; Hong et al. 1999; Pan et al. 2000; Ai et al.
2004a,b; Mai et al. 2006; Zhang et al. 2006). To our knowledge, no information is available on its dietary biotin
requirement. Hence, the present investigation was undertaken to determine the optimum dietary biotin requirement
on the basis of weight gain (WG) of juvenile Japanese seabass.

Six isonitrogenous and isoenergetic diets were formulated
with graded levels of biotin (Table 1). Vitamin-free casein
Table 1 Formulation and proximate composition of the basal diet
(g kg)1 dry matter)

Ingredients
Casein (vitamin free)
Gelatin
Dextrin
Menhaden fish oil
Soybean oil
Amino acid mixture1
Lecithin
Sodium alginate
a-Cellulose
Mineral premix2
Vitamin premix (biotin free)3
Proximate analysis (n = 3)
Protein
Lipid

Moisture

Content
(g kg)1)
360
90
280
70
40
40
20
10
30
40
20
432
125
95

Amino acid mixture (g kg)1 diet): aspartic acid, 12.5 g; glycine,
0.2 g; alanine, 6.7 g; arginine, 7.3 g; cystine, 0.4 g; valine, 1.3 g;
methionine, 2.9 g, isoleucine, 2.6 g; lysine, 6.1 g.
2
Mineral premix (g kg)1 permix): NaF, 0.2 g; KI, 0.08 g; CoCl2Æ6H2O
(1%), 5.0 g; CuSO4Æ5H2O, 1.0 g; FeSO4ÆH2O, 8.0 g; ZnSO4ÆH2O, 3.0 g;
MnSO4ÆH2O, 1.5 g; MgSO4Æ7H2O, 120.0 g; Ca (H2PO4)2ÆH2O, 750.0 g;
NaCl, 10.0 g; Zoelite, 101.22 g.
3
Vitamin premix (mg kg)1 diet): B1, 25 mg; B2, 45 mg; B6, 20 mg;
B12, 0.1 mg; pantothenic acid, 60 mg; niacin acid, 200 mg; folic

acid, 20 mg; A, 32 mg; D, 5 mg; E, 120 mg; K3, 10 mg; C, 2000 mg;
inositol, 800 mg; choline chloride, 2500 mg; antioxidant, 150 mg;
wheat middling, 14 013 mg.
1

(Sigma, Chemical, St Louis, MO, USA) and gelatin (Sigma,
Chemical) were used as protein source, dextrin (Shanghai
Chemical Co., Shanghai, China) as carbohydrate source, and
menhaden ﬁsh oil (Food grade) and soybean oil (Food grade)
as lipid sources. Amino acid (Shanghai Chemical Co.) mixture was added to simulate the whole body amino acid pattern of Japanese seabass ﬁngerling (Mai et al. 2006). Biotin
(Sigma) was added to the test diets at the expense of cellulose
to provide concentrations of 0, 0.01, 0.05, 0.25, 1.25 and
6.25 mg kg)1 diet. The biotin concentrations in the diets
determined by high performance liquid chromatography
(HP1100; Agilent, Palo Alto, CA, USA) (Lahely et al. 1999)
were 0, 0.01, 0.049, 0.247, 1.238 and 6.222 mg kg)1 diet,
respectively.
All the ingredients were ground into ﬁne powder through
220-lm mesh and thoroughly mixed with biotin, then with
menhaden ﬁsh oil and soybean oil. Finally, cold water was
added to produce stiﬀ dough, which was subsequently pelleted with an experimental diet mill [F-26 (II), South China
University of Technology, China] and dried for about 12 h in
a ventilated oven at 45 °C. After drying, the diets were
broken up and sieved into proper pellet size. The sizes of
pellets were 1.5 · 3.0 and 2.5 · 4.0 mm. All the diets were
sealed in bags and stored at )15 °C until used.

Experimental ﬁsh were obtained from a commercial farm in
Yantai, Shandong province, China. Prior to the feeding trial,
the ﬁsh were reared in a concrete pond (4.0 · 2.0 · 2.0 m),

and fed the control diet (Diet 1) for 2 weeks to acclimate to
the experimental diet and the rearing conditions. At the start
of the experiment, the ﬁsh were fasted for 24 h and weighed
after being anesthetized with eugenol (1 : 10 000) (Shanghai
Reagent Corp., Shanghai, China). Juvenile Japanese seabass
with similar size (2.26 ± 0.03 g) were randomly allotted into
18 ﬂow-through ﬁbreglass tanks ﬁlled with 250 L of water
(three tanks per treatment). Each tank was stocked with 30
ﬁsh and provided with continual aeration. The ﬁsh were fed
by hand twice daily at 08:00 and 17:00 hours respectively. To
prevent the waste of dietary pellets, ﬁsh were slowly hand-fed
little by little to apparent satiation on the basis of visual
observation of ﬁsh feeding behaviour. The feeding trial lasted
for 9 weeks, from weeks 1 to 4, 1.5-mm pellets were fed;
thereafter, 2.5-mm pellets were fed until the end of the
experiment. During the experimental period, feed consumption was recorded daily. The number and weight of dead ﬁsh
were recorded and a natural photoperiod was maintained.
Water ﬂow rate through each tank was 2 L min)1. Water

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 231–236

temperature ranged from 25.0 to 28.0 °C, salinity from 28.0
to 29.5 g L)1, pH from 8.0 to 8.1 and dissolved oxygen
content was approximately 7 mg L)1 during the experiment.
At the end of the experiment, the ﬁsh were fasted for 24 h
and ﬁsh in each tank were weighed and counted.

Fifty ﬁsh at the start and 15 ﬁsh per tank at the termination of the feeding trial were sampled and stored frozen
()20 °C) for the analysis of proximate carcass composition.
Livers of another ﬁve ﬁsh were removed and pooled for
liver biotin content assay. Proximate analyses on feedstuﬀs,
diets and ﬁsh were performed according to the standard
methods of AOAC (1995). Biotin contents of the diet and
ﬁsh liver were determined using the method of Lahely
et al. (1999).

The following variables were calculated:
Survival rate (%) = NI/NF · 100%
Weight gain (WG) (%) = (WF ) WI)/WI · 100%
Feed eﬃciency ratio (FER) = (WF ) WI)/FT
Protein eﬃciency ratio (PER) = (WF ) WI)/PT
Feed intake (FI) = FT/((WI + WF)/2 · T)
where NI and NF were initial and ﬁnal number of ﬁsh; WI
and WF were initial and ﬁnal weight of ﬁsh in g; FT was total
FI on dry basis in g; PT was protein intake on dry basis in g;
T was the experimental duration in day.
Values presented were treatment means with standard
error of the mean (SEM). All data were subjected to analysis

of variance and regression analysis where appropriate using
SPSS 10.0 for windows. Diﬀerences between the means were
tested by Tukey’s multiple range test. The level of signiﬁcance
was chosen at P < 0.05. After comparing the sum of squares
about regression (SSR) between broken-line regression
model and second-order polynomial regression model, the
broken-line model (Robbins et al., 1979), which gave the

least value of SSR and the highest estimation coeﬃcient (R2),
was used to estimate the optimal requirements of dietary
biotin for Japanese sea bass, on the basis of weight gain.

After 4 weeks of the experiment, ﬁsh fed the control diet
(Diet 1) began to show the biotin deﬁciency syndromes
characterized by heavy mortality, poor growth, anorexia
(Table 2), atrophy and dark skin colour were also observed.
However, the ﬁsh fed the other diets (Diets 2–6) did not show
any deﬁciency syndrome.

It can be seen from Table 2 that with the increase of dietary
biotin level, FI of ﬁsh was signiﬁcantly increased (Diets 1–4)
and then levelled oﬀ when the dietary biotin level reached
0.247 mg kg)1 (Diets 4–6). The lowest FI (21.42 g kg
BW day)1) was observed in the biotin-deﬁcient treatment
(Diet 1), while the highest FI (31.70 g kg BW day)1) in the
ﬁsh feeding on Diet 4 (0.247 mg biotin kg)1 diet). The survival rate of ﬁsh fed the biotin-deﬁcient diet (73.3%) was the
lowest among the six dietary treatments. The survivals in

Table 2 Initial weight (IW, g), ﬁnal weight (FW, g), survival rate (%), weight gain (WG, %), feed efﬁciency ratio (FER), protein efﬁciency ratio
(PER) and feed intake (FI, g kg)1 BW day)1) of Japanese seabass fed experimental diets with graded biotin levels for 9 weeks1
Diets
(biotin mg kg)1)

IW (g)

Diet
Diet
Diet

Diet
Diet
Diet

2.26
2.26
2.26
2.26
2.26
2.26

1
2
3
4
5
6

(0)
(0.010)
(0.049)
(0.247)
(1.238)
(6.222)

±
±
±
±
±

±

FW (g)2
0.03
0.03
0.03
0.03
0.03
0.03

4.46
7.36
14.59
13.58
13.66
12.83

Survival (%)
±
±
±
±
±
±

0.07d
0.17c
0.22a
0.32ab
0.44ab

0.13b

73.3
91.1
88.9
95.6
86.7
88.9

±
±
±
±
±
±

6.67b
2.22ab
2.22ab
2.22a
3.85ab
5.88ab

WG (%)
97.2
225.5
545.4
500.7
504.4
467.6

±
±
±
±
±
±

FER
2.99d
7.59c
9.73a
14.01ab
19.27ab
5.68b

0.41
0.70
0.85
0.79
0.76
0.75

FI (g kg)1
BW day)1)

PER
±
±
±

±
±
±

0.04b
0.04ab
0.05a
0.05a
0.10a
0.09a

1.10
1.50
1.97
2.00
1.93
2.01

±
±
±
±
±
±

0.04c
0.02b
0.03a
0.04a
0.02a

0.02a

21.42
25.46
28.22
31.70
30.83
30.89

ANOVA

F-value
P-value

265.367
0.000

3.127
0.049

265.367
0.000

one-way analysis of variance.
Values are means ± S.E.M of three replicate groups (n = 3).
Mean values with different superscript letter in the same column differ significantly (P < 0.05).

ANOVA,
1
2

5.673
0.007

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 231–236

141.688
0.000

4.588
0.014

±
±
±
±
±
±

1.41b
0.94a
1.11ab
2.33a
1.77a
2.81a

(141.9–146.9 g kg)1), crude lipid (49.9–56.3 g kg)1), moisture (761.3–776.8 g kg)1) and ash content (45.1–45.9 g kg)1).
Liver biotin concentration (0.0–6.1 lg g)1), however, significantly increased with increasing dietary biotin level
(P < 0.05), and no tissue saturation was observed within the
range of dietary biotin levels.

650

Weight gain (%)

500
Y = 522.1 8751 (0.046 X)
2
R = 0.9458

350

–1

X = 0.046 mg kg

200

50
–0.5

0.5

1.5
2.5
3.5

4.5
Dietary biotin level (mg kg–1)

5.5

6.5

Figure 1 Relationship between dietary biotin level and weight gain
(%) of Japanese seabass.

other dietary treatments (Diets 2–6) were from 86.7% to
95.6%, which were not signiﬁcantly diﬀerent from each
other. Fish WG signiﬁcantly increased with increasing dietary biotin level up to 0.049 mg biotin kg)1 diet (Diet 3)
(P < 0.05), and thereafter, it slightly decreased when dietary
biotin levels were between 0.049 and 1.238 mg kg)1 (Diets
3–5). When dietary biotin level reached 6.222 mg kg)1 (Diet
6), WG signiﬁcantly dropped compared with that of Diet 3
(P < 0.05). Broken-line regression analysis on the basis of
WG shows that juvenile Japanese seabass require a minimum
of 0.046 mg biotin kg)1 diet for maximal growth (Fig. 1).
Both FER and PER signiﬁcantly increased with increasing
dietary biotin level up to 0.049 mg biotin kg)1 diet (Diet 3),
and thereafter, remained nearly constant.

Carcass composition and liver biotin content of Japanese
seabass are presented in Table 3. Dietary biotin level
had no signiﬁcant inﬂuence on ﬁsh carcass crude protein

This study demonstrates that biotin is one of the essential
vitamins for Japanese seabass. Deﬁciency syndromes such as

high mortality, poor growth, anorexia, atrophy and dark
skin colour were observed in Japanese seabass fed on biotindeﬁcient diet (Diet 1) for 6 weeks (plus the acclimation period of 2 weeks). Similarly, Halver (1989) found that Paciﬁc
salmonids fed biotin-deﬁcient diets showed skin disorders,
atrophy, convulsions and loss of appetite.
It was also found that dietary biotin-deﬁciency resulted in
deﬁciency signs in Asian catﬁsh and Indian catﬁsh, which
were characterized by convulsions, heavy mortality, listlessness, anorexia, poor FI and feed conversion, dark skin colour, tetanus and weight loss after feeding on the control diet
(0 mg kg)1 biotin) for 6–7 weeks (Mohamed et al. 2000;
Mohamed 2001).
Dose–response experiments with increasing supply of
nutrients are accepted in principle as a method for determining dietary nutrient requirements. Nutrient requirements in ﬁsh can be estimated by either broken-line
regression analysis (Robbins et al. 1979) or polynomial
regression analysis (Zeitoun et al. 1976). Comparisons
between broken-line regression model and second-order
polynomial regression model have been made before the
analysis of optimal dietary biotin requirement on the basis
of WG. The sum of squares about regression and the
coeﬃcient of estimation (R2) have been calculated. The

Diets
(biotin mg kg)1)

Crude protein
(g kg)1)2

Crude lipid
(g kg)1)2

Moisture
(g kg)1)2

Ash
(g kg)1)2

Liver biotin
(lg g)1)

Diet
Diet
Diet
Diet
Diet
Diet

142
145
146
145
145
147

50
54
55
55
55
56

777
770

770
761
762
769

45
46
45
46
46
46

0
1
1
3
5
6

1
2
3
4
5
6

(0)
(0.010)
(0.049)
(0.247)

(1.238)
(6.222)

±
±
±
±
±
±

1
1
1
1
1
1

±
±
±
±
±
±

2
2
1
1
1
1

±
±
±
±
±
±

5
5
6
7
6
6

±
±
±
±
±
±

1
1
1
0
1
0

±

±
±
±
±
±

0f
1e
1d
1c
1b
2a

Table 3 Carcass composition and liver
biotin contents of Japanese seabass fed
experimental diets with graded biotin
levels for 9 weeks (wet weight)1

ANOVA

F-value
P-value

2.3
0.1

2.7
0.1

1.0

0.5

0.2
1.0

602.3
0.0

one-way analysis of variance.
Values are means ± SEM of three replicate groups (n = 3).
Mean values with different superscript letter in the same column differ significantly (P < 0.05).

ANOVA,
1
2

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 231–236

results indicated that the broken-line model is more suitable
to describe the relationship between dietary biotin level and
WG of Japanese seabass. Hence, the requirement of Japanese bass for dietary biotin was estimated to be
0.046 mg kg)1 using the broken-line regression model.
This result is comparable to the data of 0.02–0.03 mg kg)1
for common carp (Ogino et al. 1970), 0.05 mg kg)1 for
rainbow trout (Woodward & Frigg 1989) and 0.06 mg kg)1
for hybrid tilapia (Shiau & Chin 1999). However, it is lower

than those of 0.1 mg kg)1 for lake trout (Poston 1976), 2.0–
2.5 mg kg)1 for mirror carp (Gu¨nther & Meyer-Burgdorﬀ
1990), 2.49 mg kg)1 for Asian catﬁsh (Mohamed et al.
2000) and 0.25 mg kg)1 for Indian catﬁsh (Mohamed
2001). The variation observed in the requirements for biotin
among ﬁsh species may be due to the diet formulation, size
and age of the experimental ﬁsh and genetic diﬀerences.
The model used to analyse the dose–response relationship
also inﬂuences the estimate of requirements. Broken-line
model generally gives lower estimates of requirements
compared to nonlinear models (Baker 1986). It is also
possible that Japanese seabass may have a non-negligible
intestinal microﬂora contributing some biotin for the lower
requirement. Intestinal microorganisms are a signiﬁcant
source of water-soluble vitamins for some ﬁsh. Kashiwada
et al. (1971) isolated water-soluble vitamin-synthesizing
bacteria from the intestine of common carp. Robinson &
Lovell (1978) fed avidin in a biotin-free chemically deﬁned
diet to channel catﬁsh and found growth suppression,
suggesting that biotin synthesis by intestinal microﬂora
could take place in this species. However, in a later study
by Lovell & Buston (1984) no synthesis of biotin by the
intestinal microﬂora in channel catﬁsh could be detected.
Further research is necessary to identify whether the
intestinal microorganisms can synthesize biotin and whether
it is a signiﬁcant source of biotin for Japanese seabass.
Mohamed (2001) found that Indian catﬁsh fed the biotinfree diet had signiﬁcantly lower body protein and lipid
compared with the ﬁsh fed the biotin-supplemented diets. In
this study, dietary biotin level did not signiﬁcantly aﬀect the
carcass composition. However, both carcass crude protein

and crude lipid of Japanese seabass had the lowest values
when ﬁsh fed the biotin-free diet.
The liver biotin concentration is representative of the body
pool of biotin in Japanese seabass. It increased signiﬁcantly
as dietary biotin level increased and no tissue saturation was
found. The result indicates that the optimal level of dietary
biotin was not reached in term of maximizing the liver content of the vitamin. It is likely a higher requirement of dietary
biotin by Japanese seabass is needed to maximize liver biotin

concentration. The result agrees with Maeland et al. (1998),
Shiau & Chin (1999) and Mohamed et al. (2000). They
reported the body biotin concentration of Atlantic salmon,
hybrid tilapia and the liver biotin level of Asian catﬁsh
increased signiﬁcantly with the increasing of dietary biotin
level without tissue saturation. In this study, higher survival
rate and no biotin deﬁciency syndrome were observed in the
treatments when liver biotin concentrations were more than
0.5 lg g)1 (Diet 2).
In rats (Jacobs et al. 1970), chicks (Marson & Donaldson
1972) and trout (Poston & McCarteney 1974), the dietary
lipid content has been shown to obscure eﬀects of biotin
deﬁciency. However, in this study, the basal diet contained
432 g kg)1 crude protein and 125 g kg)1 crude lipid in
meeting the requirement of Japanese seabass (Ai et al.
2004a). Therefore, the data are adequate for the elucidation
of the biotin requirement of Japanese seabass fed the diet
with adequate amount of lipid.

This study was supported by National Key Technologies R &
D Program for the 10th Five-year Plan of China (Grant No.:

2004BA526B-06) and PCSIRT. We thank Tan, F.P. and
Xiao, L.D. in diet production. Thanks are also due to Liufu,
Z. G., Chen, J. H., Deng J. M., Cai, Y. H. and Liu, K. for all
their help during the experiment.

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young rainbow trout Salmo gairdneri determined by weight gain,
hepatic biotin concentration and maximal biotin-dependent
enzyme activities in liver and white muscle. J. Nutr., 119, 54–
60.
Zeitoun, I.H., Ullrey, D.E. & Mages, W.T. (1976) Quantifying
nutrient requirements of ﬁsh. J. Fish. Res. Board Can., 33, 167–
172.
Zhang, C., Mai, K., Ai, Q., Zhang, W., Duan, Q., Tan, B., Ma, H.,
Xu, W., Liufu, Z. & Wang, X. (2006) Dietary phosphorus
requirement of juvenile Japanese seabass, Lateolabrax japonicus.
Aquaculture, 255, 201–209.

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 231–236

Aquaculture Nutrition
doi: 10.1111/j.1365-2095.2009.00658.x

2010 16; 237–247

..........................................................................................

1,2
1

3

1

4

1

Unit of Research in Organismal Biology, University of Namur (FUNDP), Namur, Belgium; 2 Fish Culture Station of Rwasave,
National University of Rwanda, Butare, Rwanda; 3 Laboratoire de Chimie Biologique Industrielle, Faculte´ des Sciences
Agronomiques de Gembloux, Gembloux, Belgium; 4 Unite´ de Biochimie de la Nutrition, Faculte´ d ÕInge´nierie Biologique,
Agronomique et Environnementale, Louvain-la-Neuve, Belgium

The study was undertaken to evaluate the growth performance and feed utilization of African catﬁsh, Clarias
gariepinus, fed six diets (D) in which ﬁshmeal (FM) was
gradually replaced by a mixture of local plant by-products.
In diets 1 and 2, FM (250 g kg)1) was replaced by sunﬂower
oil cake (SFOC). In diets 3 and 4, FM (250 and 150 g kg)1,
respectively) was replaced by SFOC and bean meal (BM)
while FM was totally substituted by a mixture of groundnut
oil cake (GOC), BM and SFOC in diets 5 and 6. Sunﬂower
oil cake was cooked, soaked or dehulled in order to determine the appropriate processing techniques for improving
the SFOC nutritive value and to evaluate the apparent
digestibility coeﬃcient (ADC) values of the alternative diets.
No signiﬁcant diﬀerences were observed for daily feed intake,
weight gain, speciﬁc growth rate (SGR) and feed eﬃciency
(FE) among ﬁsh fed D1, D2, D3 (250 g kg)1 FM), D4

(150 g kg)1 FM) and D6 (0 g kg)1 FM). The highest SGR
(3.2% per day) and FE (1.2) were achieved in ﬁsh fed D3,
and the lowest in ﬁsh fed D5 (0% FM), suggesting a maximum acceptable dietary concentration of hulled SFOC below
250 g kg)1 in African catﬁsh juveniles. Protein eﬃciency
ratio ranged from 2.2 to 3.2 for all dietary treatments and
was positively inﬂuenced by FM inclusion. African catﬁsh
were able to digest plant protein very eﬃciently in all diets
tested. ADC of protein ranged from 88.6 to 89.5%, while
ADC of energy was relatively low for diets containing hulled
sunﬂower oilcake (71–74%) and high when sunﬂower oilcake
was dehulled (78.6–81.3%). Similarly, ADC of dry matter
was higher when sunﬂower was dehulled (72.1%) when
compared with crude SFOC (60.5%). Soaking increased

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd

ADC values for neutral detergent ﬁbre (NDF), dry matter,
energy, protein and amino acids (AA). There were no signiﬁcant diﬀerences in protein ADCs (88–90%) with increased
levels of dietary vegetable ingredients. Both soaking and
dehulling of sunﬂower before incorporation helped in the
reduction of NDF, antitrypsin and tannins. Digestibility of
all AA was generally high, greater than 90% for both
indispensable and non-indispensable AA. Based on the data
obtained, it was possible to totally replace menhaden ﬁsh
meal with a mixture of vegetable proteins (72% of total
dietary protein) when diets contained a relatively low percentage of animal protein (28% based on blood meal and
chicken viscera meal) without negative eﬀects.

KEY WORDS: anti-nutritional factors, apparent digestibility
coeﬃcient, Clarias gariepinus, feed utilization, growth performances, sunﬂower oilcake

Received 29 May 2007, accepted 3 February 2009
Correspondence: P. Kestemont, Unit of Research in Organismal Biology,
University of Namur, Rue de Bruxelles 61, B-5000 Namur, Belgium.
E-mail:

It has been shown that ﬁshmeal constitutes the most suitable
source of indispensable amino acids (IAA) for ﬁsh, given the
high correlation between whole body IAA proﬁle and the
IAA requirement pattern (Mambrini & Kaushik 1995).
However, in the absence of ﬁshmeal, it is important to
evaluate the nutritional value of alternative ingredients and
formulate diets based on a mixture of ingredients which can

collectively replace ﬁshmeal in the diet of ﬁsh. Among the
many protein sources available for animal feeds in many
African countries, plant proteins appear to be the most
appropriate alternatives to ﬁshmeal in ﬁsh diets, especially
those that are not suitable for human consumption. Partial
replacement of ﬁshmeal by plant proteins has been accomplished in many carnivorous cultured ﬁsh (Gomes et al. 1995;
Kaushik et al. 1995; Robaina et al. 1995; Masumoto et al.
1996; Hoﬀman et al. 1997; Fagbenro 1999), but total
replacement has met with success in only a few cases
(Kaushik et al. 1995; Regost et al. 1999). Some studies have
also stressed that a mixture of plant protein sources is more
appropriate than the incorporation of a single plant source
because of improved AA proﬁles (Regost et al. 1999; Fournier et al. 2004; Kaushik et al. 2004).

However, use of plant-derived materials as ﬁsh feed
ingredients is limited by the presence of a wide variety of
anti-nutritional factors (ANFs). Some ANFs inhibit speciﬁc
enzyme activities, e.g. inhibition of proteinase and amylase.
Haemagglutinins and lectins are proteins which can interact in
speciﬁc ways with certain carbohydrates (Hendricks 2002).
Saponins and glycosides, which are bitter, reduce the palatability of livestock feeds. Some saponins reduce feed intake
and growth rate of non-ruminant animals, while others are not
very harmful. Phytic acid can interfere with mineral element
absorption and utilization and react with proteins to form
complexes which have an inhibitory eﬀect on proteins digestion (Francis et al. 2001; Sugiura et al. 2001 in Sajjadi &
Carter 2004; Helland et al. 2006). The presence of tannins has
been associated with lower nutritive value and lower biological availability of macromolecules such as proteins and carbohydrates (Desphande & Cheryan 1985; Liener 1989 in
Francis et al. 2001). Plant meals also contain starch which
must be cooked to make it digestible to ﬁsh. In brief, according
to Lienner (1980), Huisman et al. (1989) and Krogdahl (1989),
insoluble ﬁbres (NDF), soluble ﬁbres (ADF), enzymes inhibitors, saponins, lectins, tannins, phytic acid and gossypol are
the most important anti-nutrients acting in the gut. They aﬀect
digestive functions and nutrient absorption by altering the
ﬂow of chyme, impairing interactions between nutrients and
digestive components, restricting diﬀusion, altering absorptive surfaces and changing microbial activity. For example,
insoluble ﬁbre appears to increase intestinal ﬂow rate, whereas
soluble ﬁbre decreases it (Meyer et al. 1988 in Krogdahl
1989). Increased rates tend to decrease nutrient absorption
(Krogdahl 1989). The consequences of such changes in the
intestines on nutrient absorption and general metabolism may
be large and eﬀect on growth and production of considerable
economic importance.

Attempts to increase utilization of plant protein by

improving digestibility and to partly reduce the presence of
ANFs include a wide range of processing techniques such as
cooking, dehulling, germination, roasting, extrusion, soaking
and recently extrusion cooking (Akpapunam & Sefa-Dedeh
1997; Alonso et al. 1998, 2000; Chong et al. 2002; Egounlety
& Aworth 2003; Garg et al. 2003; Nibedita & Sukumar 2003;
Koplik et al. 2004; Gill et al. 2006). As feed formulation
should be based on nutrient bioavailability, reliable data on
the digestibility of diﬀerent ingredients for each species might
well be considered as a necessary prerequisite. However,
potential interactions among ingredients should also be
considered.
Fish meal (FM), the conventional dietary protein source in
catﬁsh feed (40–60% of the total protein) (Van Weerd 1995)
is totally imported in Rwanda, soybean is scarce while sunﬂower oil cake is available and less expensive (Nyinawamwiza et al. 2007). Moreover, it has been demonstrated
that dietary incorporation of soybean meal, groundnut cake
and winged bean improved the growth performance, feed
intake and feed eﬃciency (FE) of Clarias gariepinus (Balogun
& Ologhobo 1989; Degani et al. 1989; Hoﬀman et al. 1997;
Fagbenro 1998, 1999). Our knowledge on anti-nutrient
eﬀects in African catﬁsh is very poor.
Based on the foregoing, several objectives were identiﬁed in
this study: to evaluate, in a ﬁrst experiment, the maximum level
of substitution of FM in diets for juvenile African catﬁsh when
a mixture of available by-products was used and to evaluate
the resulting inﬂuence on the growth response, protein utilization and FE of C. gariepinus ﬁngerlings. Among the tested
ingredients, sunﬂower oilcake was especially investigated by
applying diﬀerent processing methods such as soaking or
dehulling, and by combining it with other by-products. As
nutrients are not available to an animal before they are

absorbed in the digestive tract, in a second stage, apparent
digestibility coeﬃcients (ADCs) for dry matter, protein,
energy, ﬁbre and AA in experimental diets was studied.

Experiment 1: growth and feed utilization In the ﬁrst
experiment, ﬁsh were obtained by artiﬁcial reproduction
from broodstock cultured in earthen ponds at the Rwasave
Fish Culture Station of the National University of Rwanda
(Butare District). At 3–4 g body weight, ﬁsh were acclimatized to the experimental conditions for 3 weeks in plastic
tanks and received a mixture of the six experimental diets in

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

order to habituate them to locally formulated feed. Fish
actively ingested the food and feeding was interrupted when
ﬁsh stopped eating the delivered pellets (ﬁsh were fed to
appetite).
The experiment was conducted in a recirculating system
including eighteen 100-L rectangular tanks installed over a
4.5-m3 concrete tank for mechanical and biological water
ﬁltration. A total of 540 C. gariepinus ﬁngerlings, with initial
mean body weight of 7.49 ± 0.09 g, were randomly distributed as 30 ﬁsh of mixed sexes per tank. Three replicate
tanks per dietary treatment were used. In all 18 tanks, water
was equally aerated and exchanged at a ﬂow rate of
2–3 L min)1. Fish were subjected to natural photoperiod
(12-h light and 12-h dark). Water temperature, dissolved

oxygen and pH were checked daily. Water temperature was
maintained at 23 ± 1.5 °C, dissolved oxygen and pH ranged
from 3.1 to 6.0 mg L)1 and 6.3 to 7.8, respectively. Ammonia
and nitrites were monitored twice a week and varied between
0.00–0.417 and 0.002–0.134 mg L)1, respectively.
At the beginning of the experiment, 30 ﬁsh were sampled
for analysis of body composition, and at the end of the
experimental period, after 8 weeks, all ﬁsh were individually
weighed and measured (total length). Fish were hand-fed to
apparent satiation twice daily, at 9:00 and 16:00. Care was
taken to stop the feed as soon as the ﬁsh stopped eating. The
remaining pellets were weighed and the diﬀerence from the
initial weight was then recorded as the feed intake.
Experiment 2: digestibility measurements Apparent digestibility coeﬃcients for dry matter, protein, AA, ﬁbre and
energy of experimental diets were measured indirectly using
chromic oxide (Cr2O3) as an inert marker. Juveniles (initial
mean body weight: 20.0 ± 5.0 g) were obtained from the
Aquaculture Training and Research Centre in Tihange
(Belgium). The trial was conducted in the experimental
facilities at the Marcel Huet ﬁsh culture laboratory, Universite´ Catholique de Louvain (Belgium). Fish were reared in
165-L cylindroconical tanks (water ﬂow rate: 4 L min)1).
Two tanks were randomly allotted to each diet. Water
quality, temperature and photoperiod (LD 12:12) were in the
same range as in the ﬁrst experiment. The water was constantly replaced in the tank by continuous ﬂow at a rate of
4 L h)1. Fish were acclimated in experimental tanks and to
the experimental diets (Table 1) for 10 days before the start
of the experiment, followed by 3 weeks of faecal collection
from each tank, using an automatic faecal collector
(Choubert et al. 1982). During the trial, ﬁsh were fed by hand
to apparent satiation twice daily (09:00 and 17:00). About

30 min after each feeding, the tanks and the faecal collection

system were brushed out to remove feed residues and faeces
from the system. The faecal samples collected from each tank
were frozen daily. At the end of the digestibility trial, the
pooled faeces from each tank were freeze-dried prior to
analysis for chromic oxide, protein, AA, ﬁbre and energy.

Six diets were formulated containing graded levels of FM. A
ﬁrst diet with sunﬂower oilcake from hulled and unsoaked
seeds (SFOC) containing only 25% of FM was formulated as
reference. In the second diet, SFOC was soaked in water for
24 h before incorporation in the diets (SFOCS) in order to
diminish ANFs and to improve the feed intake (Amrish 2002).
In the third diet, SFOC level was reduced and it was mixed with
bean meal (BM), Phaseolus vulgaris (SFOC + BM), in order
to obtain a good balance in some essential AA, e.g. in lysine.
Indeed, the lysine content of sunﬂower (Helianthus annuus) is
low, whereas its content in methionine is high. On the contrary,
the lysine content of Phaseolus seeds is relatively high, the
amount ranging from 8 to 10 g per 16 g N (Abdel-El-Samei &
Lasztity 1984; Sen & Bhattacharyya 2000; Sauvant et al.
2002). This would favourably meet the Clarias requirement
for lysine estimated at 4.8% of protein for Clarias hybrids
(Unprasert 1994 in Wilson 2002). Webster & Lim (2002) found
lysine to be the main limiting AA in Channel catﬁsh Ictalurus
punctatus and perhaps in other warmwater ﬁsh as well
(Robinson et al. 1980 in Wilson 2002). Groundnut (Arachis
hypogea) oilcake (GOC) was used as a substitute for ﬁshmeal because of its high-crude protein content (480 g kg)1).
Because of the potentially higher digestibility of dehulled

sunﬂower meal/oilcake (SFOCD), and the food intake preferences (Gill et al. 2006), for the fourth diet, ﬁshmeal was
reduced to 150 g kg)1. Finally, ﬁshmeal was reduced to 0%
by using a mixture of local ingredients such as BM, GOC
and SFOC. Diet 5 = SFOCS + BM + GOC and diet
6 = SFOCD + BM + GOC.
Menhaden FM was obtained from Coppens International
bv, Helmond, The Netherlands. Other ingredients were
selected from local markets in Rwanda, partly based on their
potential as cheap and readily available protein sources.
All diets were analysed for proximate composition using
standard methods given in AOAC (1980) and results are
presented in Tables 1 and 2.
All collected ingredients were cooked in a pressure cooker
for 1–2 h at 100 °C with addition of a few volumes of water,
followed by sun drying. Before mixing, ingredients were
ground, mixed thoroughly with water, made into spaghetti
(2 mm diameter), and converted into pellets after sun drying.

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

Ingredients (g kg)1 diet)
Fish meal (menhaden)
Blood meal
Chicken viscera meal
Sunflower oilcake
Groundnut oilcake

Bean meal
Fish oil (menhaden)
Sunflower oil (local)
Mineral mixture1
Vitamin mixture2
Carboxymethylcellulose
Crude protein (g kg)1 dry matter)
theoretical
Chemical proximate composition
Dry matter (g kg)1)3
Crude protein (g kg)1 dry matter)3
Crude fat (g kg)1 dry matter)3
Gross energy
(kJ g)1 dry matter)3
NDF (% dry matter)3
ADF (% dry matter)3
Ash (% dry matter)3

25% Fish meal

15% Fish
meal
0% Fish meal

Diet 1 Diet 2 Diet 3

Diet 4

Diet 5

Diet 6

SFOC

SFOC + SFOCD + SFOCS +
SFOCD +
SFOCS BM
BM
BM + GOC GOC + BM

257
89
99
436
0
0
25
25
30
30
10
388

257
89
99
436
0
0
25

25
30
30
10
388

957
956
367
351
103
122
19.0
18.8
33.1
13.6
14.8

35.1
12.1
13.3

Table 1 Composition of the six experimental diets

257
89
99
248
0
197

25
25
25
25
10
384

149
99
99
426
0
137
20
20
20
20
10
378

0
99
99
217
396
99
20
20
20
20

10
378

0
99
99
296
317
99
20
20
20
20
10
379

961
384
82
17.8

929
378
63
17.4

952
350
51
17.5

934
381
84
18.0

36.9
10.6
11.1

22.6
4.7
5.0

23.6
10.5
11.7

17.3
5.1
5.6

NDF, neutral detergent fibre; ADF, acid detergent fibre.
Mineral mixture INRA Belgium, MLNP 763, (composition per kilogram: dibasic calcium phosphate: 500 g; calcium carbonate: 215 g; sodium chloride: 40 g; potassium chloride: 90 g; magnesium hydroxide: 124 g; iron sulphate: 20 g; zinc sulphate: 4 g; manganese sulphate: 3 g; cobalt
sulphate: 0.02 g; potassium iodide: 0.04 g; sodium selenite: 0.03 g and sodium fluoride: 1 g).
2
Vitamin mixture INVE Aquaculture, Belgium (composition per kilogram: Vit. A: 2 500 000 IU;
Vit. D3: 500 000 IU; Vit. E : 30 000 mg; Vit. K3 : 2000 mg; Vit. B1 : 2000 mg; Vit. B2 : 5000 mg;
Panthotenic acid: 10 000 mg; Niacin 5000 mg; Vit. B6: 4000 mg; Folic acid: 2000 mg; Vit. B12:4
mg; Vit. C: 20 000 mg; Biotin: 200 mg and Inositol: 80 000 mg).

3
Assayed.
1

For the digestibility experiment, 15 g kg)1 of chromic oxide
was added to the formulated diets (Table 1). The cooking
procedure for diets used for the digestibility test was similar
to that used for the growth experiment.

Diet and faecal samples were analysed in duplicate for
proximate composition (AOAC 1980) Dry matter was
calculated from weight loss after drying in an oven at 105 °C
for 24 h. Total lipids of ﬁsh carcass were extracted with
chloroform/methanol/water (10 : 10 : 9, vol/vol/vol) according to Folch et al. (1957), total nitrogen by the Kjeldahl
technique (protein = N · 6.25). Ash content was calculated
from weight loss after incineration of samples in a muﬄe
furnace for 24 h at 550 °C.

Gross energy of the diets and faeces was determined using
an adiabatic bomb calorimeter 1241, Parr Instrument Company, Moline-Illinois-USA). Neutral detergent ﬁbre (NDF)
and acid detergent ﬁbre (ADF) in diets and faeces were
measured by the method of Goering & van Soest (1970).
Chromic oxide was estimated spectophotometrically
following the method of Furukawa & Tsukahara (1966).
Total AA contents of diets (Table 2) and faecal samples
from each tank were measured by ion-exchange chromatography, Biochrom 20 Plus-Amino Acid Analyser, Biochrom
Ltd, Cambridge, UK. (Moore et al. 1958). For sulphur AA,
samples were ﬁrst oxidized by a performic acid-phenol to
oxidize methionine and cystine to methionine sulphone and
cysteic acid, respectively (Lewis 1966). These oxidized samples, as well as unoxidized samples, were hydrolysed in 6 N

HCl, for 24 h at 110 °C. Norleucine was used as an internal

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

Table 2 Proximate amino acids composition of the experimental diets (g per
16 g N)

25% Fish meal

15% Fish meal

0% Fish meal

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

Amino acid

(% dry matter)

SFOC

SFOCS

SFOC +
BM

SFOCD +
BM

SFOCS +
BM + GOC

SFOCD +
GOC + BM

Alanine
Arginine1
Aspartic acid/Asparagine
Cystein/Cystine
Glutamic acid/Glutamine
Glycine
Histidine1
Isoleucine1
Leucine1
Lysine1
Methionine1
Phenylalanine1

Proline
Serine
Threonine1
Tyrosine
Valine1

5.67
5.96
9.85
1.06
16.80
6.02
2.89
3.27
7.59
6.65
2.07
4.36
4.89
4.68
4.21
2.60
5.04

5.69
6.04
9.89
1.01
16.68
6.05

2.82
3.29
7.57
6.50
1.98
4.27
4.84
4.78
4.22
2.56
5.06

5.72
5.86
10.19
1.03
16.16
5.89
2.87
3.31
7.76
6.97
2.16
4.53
4.85
4.94
4.32
2.72
5.12

5.28
7.00
10.18
1.22
19.14
5.70
3.02
3.35
7.46
5.83
2.03
4.76
4.73
4.95
4.14
2.73
5.04

4.92
8.09
11.31
1.20
18.76
5.60
3.26
2.88
7.57
5.37
1.42
5.14

4.93
5.23
3.87
3.09
5.14

4.90
8.23
10.90
1.22
20.01
5.58
3.22
3.12
7.43
5.17
1.56
4.93
4.79
5.09
3.85
2.95
5.03

Tryptophan was not analysed.
Indispensable amino acid (IAA).

1

standard and sodium citrate (pH 2.2) as a buﬀer solution.

The AA were post-column derivatized with ninhydrin and
quantiﬁed at 570 nm for primary AA and 440 nm for secondary (imino acid, proline and hydroxy-proline). Tryptophan could not be analysed because of its destruction during
acid hydrolysis.
Among the multiple ANFs that can be found in the vegetable ingredients used in the experimental diets and in the
diets themselves, three were measured: antitrypsin, tannins
and phytic acid. These three factors were measured by
spectrophotometry.
The principle of the proportion of the antitrypsin is
based on the release of p-nitroaniline from N-benzoyl-DLarginine-p-nitroanilide (BAPNA), this being immediately
followed by an increase of extinction measured at 407 nm
during 10 min against a reagent blank. The protocol of
proportion has been established according to the method
of Bergmeyer (1965). Trypsin inhibition was expressed in
International Unit (IU), an antitrypsin unit being equal to
a diﬀerence of absorbance DDO of 0.001, in the experimental conditions.
Tannins present in the vegetable by-products were
quantiﬁed by measuring their absorbance at 550 nm against
a reagent blank after their extraction by means of organic
solvents in acid medium, and the reaction of these
polyphenols with hydrated ammonium ferric sulphate
NH4Fe(SO4)2Æ12H2O. The protocol of proportion used has
been modiﬁed from Aganda & Mosase (2001). Tannin

contents were expressed in gram of catechin equivalent per
kilogram of sample analysed, catechin being the standard
tannin used.
Phytic acid contents were determined according to the
method of March et al. (1995). The method of proportion
consists ﬁrstly in isolating phytates, after their extraction in
sulphuric acid, in the form of iron (III) phytate. Secondly,

NaOH and water were added to this solid iron (III) salt in
order to precipitate hydrated iron (III) oxide and liberate the
phytate. The absorbance was measured at 400 nm against a
reagent blank. Phytic acid contents were expressed as grams
of phytic acid per kilogram of sample analysed.

Fish performance was determined using the following formulae:
Weight gain ð%Þ ¼ 100 Â ðWf À Wi Þ=Wi
where Wi and Wf is the initial and ﬁnal body mass (g).
Specific growth rate ðSGR; % per dayÞ
¼ 100 Â ½lnðWf Þ À lnðWi Þ=Dt
where Wi and Wf is the initial and ﬁnal mean body mass (g)
and Dt is the duration of experiment.
Feed efficiency (FE) ¼ ðFB À IBÞ=TFI
where FB is the ﬁnal biomass per tank (g), IB is the initial
biomass per tank (g) and TFI is the total food intake (g).

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

and those fed other diets. In contrast, no signiﬁcant differences were obtained between ﬁsh fed diet 6 containing
dehulled SFOC (0% FM) and ﬁsh fed diets containing 15
or 25% FM. On the contrary, the best overall growth
response was obtained in ﬁsh fed diet 3 (25% ﬁshmeal when
SFOC was reduced to 25%). Similar results were observed
for FE and PER with a signiﬁcant reduction observed for
diet 5. No signiﬁcant diﬀerences were observed between ﬁsh

fed diets 1 and 2 containing unsoaked and soaked SFOC,
respectively.

Protein efficiency ratio (PER)
¼ Weight gain (g)=protein intake (g)

The ADC of dry matter were calculated according to
Maynard and Loosly (1969) in Burel et al. (2000) as follows:
ADC dry matter ð%Þ ¼ 100 Â ½1 À ðDiÞ=ðFiÞ

The ADCs of proteins, AA, ﬁbre and energy were calculated as follows (Cho & Slinger 1979):
ADC ¼ 100 Â ½1 À ðF =DÞ Â ðDi=FiÞ
where D is the dietary nutrient or energy content (%), F is the
faecal nutrient or energy content (%), Di is the dietary
marker content (%) and Fi is the faecal marker content (%).
All data were analysed by one-way analysis of variance
(ANOVA) followed by Fisher test LSD (least signiﬁcant difference) to determine if signiﬁcant diﬀerences occurred
among the dietary treatments. Variance homogeneity was
ﬁrst checked by Hartley test (Dagnelie 1975). Diﬀerences
were considered signiﬁcant at P < 0.05.

Apparent digestibility coeﬃcients for dry matter, protein,
energy, ﬁbre and AA in diets consumed by C. gariepinus
ﬁngerlings are shown in Table 4. ADCs of dry matter and
gross energy were signiﬁcantly aﬀected by experimental
diets (P < 0.05), generally high for the diets containing
dehulled SFOC and especially lowered by increased inclusion of hulled SFOC meal in the diet. Diet 3 gave intermediate results. Dry matter digestibility was highest in diet
4 followed by diet 6, whereas diet 1 gave the lowest ADC.
Dehulling increased ADC of gross energy and insoluble
ﬁbres (NDF) in diets 4 and 6, whereas it was lowest in diet

5. In comparing diets 1 and 2, soaking process increased
ADC values of NDF, dry matter, gross energy, protein
and AA ADCs. There were no signiﬁcant diﬀerences in
ADCs (88–90%) of protein with increased level of vegetable ingredients in diets (>0.05). Digestibilities of all AA
were generally high, over 90% for indispensable and nonindispensable AA little aﬀected by experimental diet.
Indeed, digestibilities of three IAA (isoleucine, methionine
and threonine) were higher in diets 1 and 2, but lower in
diet 5.

As shown in Table 3, daily voluntary feed intake decreased
with increase in dietary plant protein. This was signiﬁcantly
lowest in diet 5 (P < 0.05) when FM was totally replaced by
a mixture of plant by-products in a diet containing hulled
sunﬂower oil cake.
At the end of the experiment, a signiﬁcant decrease in
weight gain was observed between groups of ﬁsh fed diet 5

Table 3 Growth performance and feed efﬁciency of Clarias gariepinus ﬁngerlings fed experimental diets for 61 days
25% Fish meal

15% Fish meal

0% Fish meal

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

Parameters

SFOC

SFOCS

SFOC + BM

SFOCD + BM

SFOCS + BM + GOC

SFOCD + GOC + BM

Initial body weight (g)
Final body weight (g)
Weight gain (%)
SGR (%)
Daily feed intake
(g fish)1 day)1)
Feed efficiency
Protein efficiency ratio

7.54

42.8
468
2.80
0.51

7.56
53.3
604
3.17
0.62

7.44
38.7
419
2.70
0.47

7.43
26.0
250
2.04
0.40

7.45
35.6
377
2.55
0.48

±

±
±
±
±

0.08
13.1ab
173ab
0.50ab
0.12a

1.09 ± 0.16ab
3.11 ± 0.47a

7.51
42.1
460
2.79
0.51

±
±
±
±
±

0.02
9.7ab
129ab
0.39ab

0.10a

1.08 ± 0.12ab
3.22 ± 0.37a

±
±
±
±
±

0.04
11.8a
154a
0.38a
0.10a

1.16 ± 0.14a
3.16 ± 0.39a

±
±
±
±
±

0.09
2.3abc
259ab
0.08ab

0.02a

1.04 ± 0.03ab
2.97 ± 0.08ab

±
±
±
±
±

0.03
4.4c
637b
0.31b
0.04b

0.74 ± 0.12b
2.21 ± 0.37c

±
±
±
±
±

0.04
5.1bc
710ab
0.26ab

0.05a

0.87 ± 0.10ab
2.45 ± 0.27bc

Values are given as mean ± standard deviation. Values in the same row with common superscript letters are not significantly different
(P < 0.05).

..............................................................................................

Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

Table 4 Apparent digestibility coefﬁcients (%) for dry matter, crude protein, gross energy, NDF, ADF and amino acids in African catﬁsh fed
various local levels of vegetable protein in substitution of menhaden ﬁsh meal
25% Fish meal

15% Fish meal

0% Fish meal

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

Parameters

SFOC

SFOCS

SFOC + BM

SFOCD + BM

SFOCS + BM +
GOC

SFOCD + GOC +
BM

Digestibility of dry matter (%)
Digestibility of crude protein (%)
Digestibility of gross energy (%)
Digestibility of NDF (%)
Amino acids (%)
Alanine
Arginine1
Aspartic acid/Asparagine
Cystein/Cystine
Glutamic acid/Glutamine

Glycine
Histidine1
Isoleucine1
Leucine1
Lysine1
Methionine1
Phenylalanine1
Proline
Serine
Threonine1
Tyrosine
Valine1

60.5
87.7
72.4
44.3

±
±
±
±

1.3c
3.0
1.9bc
1.1e

64.2
88.8

74.4
52.2

±
±
±
±

1.1bc
0.6
0.6bc
0.7c

67.1
87.6
77.0
61.6

±
±
±
±

1.4ab
0.8
2.5ab
1.1b

72.1
88.5

81.3
65.9

±
±
±
±

1.9a
1.8
0.7a
0.1a

65.0
88.0
71.0
41.8

±
±
±
±

4.4bc
0.8
3.9c
1.4f

70.9
89.5

78.6
49.9

±
±
±
±

2.6ab
0.5
2.9a
1.2d

92.6
94.5
90.1
80.2
93.4
89.3
92.0
90.6
92.8
94.3
92.2
92.0
92.1
90.5
90.8
92.0
90.8

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.8
1.6
1.4
4.1
1.3
2.1
0.4
1.5a
1.0
1.0
1.1a

1.5
2.0
1.9
1.1a
2.8
1.4

93.0
95.4
90.9
79.8
94.0
90.2
92.8
91.0
93.0
94.5
91.8
92.8
92.8
91.4
91.3
92.8
91.5

±
±
±
±
±

±
±
±
±
±
±
±
±
±
±
±
±

0.3
0.4
0.5
2.3
0.5
1.2
0.2
1.0a
0.6
0.6
0.1a
0.5
0.7
0.8
0.5a
0.8
0.7

93.0
94.0
89.1
77.0
92.1
89.2
91.7
88.4
91.4
93.0
90.2
90.8
91.4
90.1
89.6
90.8
89.6

±
±
±
±
±
±
±
±
±
±
±

±
±
±
±
±
±

0.5
0.5
0.5
0.4
0.8
1.0
0.1
0.4ab
0.6
0.6
0.2ab
0.5
1.1
0.8
0.5ab
0.8
0.6

91.2
95.2
90.3
83.2
93.8

89.8
92.2
89.1
91.6
93.1
91.6
90.6
92.0
90.9
89.7
90.6
90.3

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.6
0.9
1.2
2.3
1.2
1.5
0.7
1.0ab
0.9
0.8
0.9a
1.2
1.1
1.0
0.8ab
1.2
0.9

91.2
95.6
90.7
82.6
93.2
86.2
91.0
86.8
90.1
91.1
87.6

92.1
90.7
89.6
86.9
92.1
88.7

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

1.8
0.4
1.9
1.2
1.7

2.7
0.6
0.6b
1.9
1.3
1.0b
1.8
1.4
2.2
2.1b
1.0
2.1

89.3
96.1
91.9
84.7
94.4
88.8
92.5
88.8
95.0
92.2
90.7
91.8
91.9
91.1
88.4
91.8
90.1

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

1.7
0.7
1.2
2.1
1.5
2.2
2.0
1.0ab
0.8
1.1
0.3ab

1.1
2.0
1.7
1.8ab
0.9
1.7

Values are given as mean ± standard deviation.
Indispensable amino acid (IAA).

1

Based on the present results, no signiﬁcant diﬀerences were
found between ﬁsh fed the higher inclusions of ﬁshmeal
(25.7% of total ingredients), ﬁsh fed 15% (diet 4) and 0%
ﬁshmeal (diet 6), respectively. SGR and PER were generally
high when compared with results obtained by Balogun &
Ologhobo (1989), Degani et al. (1989) and Hoﬀman et al.
(1997) with African catﬁsh of comparable size fed diets
containing various proportions of FM and plant products, as
well as with the results of Fagbenro (1999) who used 40%
menhaden FM, poultry by-product meal and maize meal to
ensure crude protein levels of 400 g kg)1 diet. This supports
the suggestion that the correct complementary mixture of
plant and animal by-products can partly or totally replace
the FM in Clarias diets. However, voluntary feed intake was
signiﬁcantly (P < 0.05) lower in Clarias fed diet 5 when
compared with other diets. Similarly, all nutritional indices
for ﬁsh fed diet 5 (0% FM) were signiﬁcantly (P < 0.05)
inferior to those of ﬁsh fed diet 3 (25% FM). This latter diet

was diﬀerent from the ﬁrst two (D1 and D2) in terms of plant
by-product content. While the 2 ﬁrst diets contained only
sunﬂower oilcake, a part of that oilcake was substituted by
BM in the third diet. It was apparent that Clarias ﬁngerlings
might be sensitive to a large (higher than 25.7%) inclusion of
hulled sunﬂower oil cake for several reasons. Firstly, because
of the high ﬁbre content in SFOC, and secondly because the
complementary nature of SFOC and BM leads to a better
essential AA balance. Diﬀerences between diets 5 and 6 can
only be explained by the dehulling of sunﬂower. Fishmeal
can thus be totally replaced by a combination of groundnut
oilcake, BM and sunﬂower oilcake, providing that sunﬂower
oilcake is dehulled before its incorporation into the diet.
Diets 1 and 2 provided similar results; the soaking of sunﬂower oilcake did not aﬀect these results, whereas dehulling
improved its nutritive value.

The results of the present study showed that soluble and
insoluble ﬁbre levels decreased appreciably in diets with
SFOCD when compared with SFOCS and SFOC. Dry

..............................................................................................

Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

matter digestibility coeﬃcients ranged from a minimum of
60.5% (D1) to a maximum of 72.1% (D4). All diets containing a high level of hulled SFOC meal were less digestible.
The low digestibility of dry matter and energy was probably

due to the high ﬁbre (ADF and NDF) content of the diet.
Pre-treatment of SFOC ingredients appeared to be relatively
important when considering the high digestibility coeﬃcients
recorded for all the diets evaluated. Soaking had little eﬀect
on ADC of dry matter and energy, whereas dehulling
appeared to be the most eﬀective method improving both dry
matter and energy ADCs. Both soaking and dehulling
enhanced starch digestibility by reduction of phytates and
tannins which inhibit activity of a-amylase. On the contrary,
rupture of starch granules in plant feedstuﬀs during heat
treatment makes substrates accessible and facilitates the
amylolysis (Deshpende & Cheryan 1984 in Alonso et al.
2000).
Protein digestibility coeﬃcients were very similar ranging
from 87.7 (D1, 25% FM, SFOC) to 89.5% (D6, 0% FM,
SFOCD). These results were consistent with the range of
protein digestibility values (75–95%) reported for other
freshwater ﬁsh fed practical selected diets (Kenan & Yasar
2005). Diets that contained a high level of animal protein and
those composed principally of plant-based ingredients were
all highly digestible. Improvement of protein digestibility
could be attributable to the reduction or elimination of different anti-nutrients during the pre-treatment process, especially phytic acid and tannins which are known to interact
with protein to form complexes. This can be also related to
higher eﬃciency of the thermal treatment, reducing trypsin
and chymotrypsin inhibitory activities (Alonso et al. 2000).
The present results are higher than the protein ADC of
soybean meal reported for channel catﬁsh, I. punctatus
(Brown et al. 1985), C. isheriensis (Fagbenro 1996) and
higher than the protein ADC reported for C. gariepinus fed
various dietary oilseed cakes (Fagbenro 1998). On the contrary, the present values were lower than the 92.8% for

menhaden FM reported for C. gariepinus (Fagbenro 1998).
Indispensable AA proﬁles in each diet were in agreement
with Clarias requirements and all IAA had globally high
ADCs (about 90%). The present results suggest that FM can
be replaced by plant feed stuﬀs in Clarias diets without AA
supplementation when an adequate mixture of plant feedstuﬀs is used. Highest AA ADCs were found for arginine and
lysine and this eﬀect is relevant given the high requirements
for these two AA in Clarias (Oellermann & Hecht 2000;
Wilson 2002).
Gross energy digestibility coeﬃcients ranged from 71 to
81%. The diﬀerence in gross energy ADCs in the present

study may be attributed to diﬀerences in ﬁbre content
(Table 1). These results were higher than the 68.9% for
cottonseed cake and similar to the 75.8 and 79% (except for
D1 and D5) for groundnut cake, sunﬂower cake and soybean
cake, respectively, reported for C. gariepinus (Fagbenro
1998). Bjo¨rck et al. (1984 in Cheng & Hardy 2003) suggested
that the increased soluble ﬁbre portion would improve ADCs
of ﬁbre and thus increase digestible energy, because
non-ruminant animals (such as pigs) could utilize the ﬁbre to
meet 30–50% of their energy needs via fermentation to
volatile fatty acids. Results of the present study suggest that
this is not true in African catﬁsh.

Anti-nutritional factors are present in sunﬂower oilcakes and
groundnut oilcake in similar proportions, whereas BM contained less phytic acid and displayed less antitryptic activity.
Both soaking and dehulling of sunﬂower before incorporation helped in the reduction of trypsin inhibitors and tannins
but not phytate. It was not possible to assay tannins in BM
because of pigment interference. According to Deshpande

et al. (1982 in Maldonado et al. 1995), it is clear that major
amounts of bean tannins are located in the seed coat with
lower or negligible amounts in the cotyledons. Tannin content should be determined using another analytical method
for BM and the respective diets. Results for ANFs (Table 5)

Table 5 Proximate levels of anti-nutritional factors (ANFs) in the
experimental ingredients and diets
ANFs

Ingredients
Bean meal (BM)
Groundnut oilcake (GOC)
Sunflower oilcake, crude
(SFOC)
Sunflower oilcake, soaked
(SFOCS)
Sunflower oilcake, dehulled
(SFOCD)
Diets
D1: (SFOC)
D2: (SFOCS)
D3: (SFOC + BM)
D4: (SFOCD + BM)
D5: (SFOCS + GOC)
D6: (SFOCD + GOC + BM)

Trypsin
inhibitors
(IU g)1)

Phytate
(g kg)1)

Tannin
(g kg)1)

4305
4605
5547

27.68
37.15
36.00

nd
3.40
9.34

4999

40.63

7.77

4741

39.22

4.22

1838
2830
3749
5337
5104
5587

26.69
24.77
24.07
38.37
34.64
34.32

nd
nd
nd
nd
nd
nd

nd, not determined.

..............................................................................................

Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

in the ingredients suggested that those anti-nutrients were

elevated in diets 4 to 6 which contained a great number of
plant ingredients.
It has been reported that 5–6 g of phytic acid per kilogram can impair the growth of rainbow trout (Spinelli et al.
1983 in Richter et al. 2003), whereas 2% inclusion of
condensed tannin were shown to be tolerated without any
adverse eﬀect on growth (Becker & Makkar 1999 in Richter
et al. 2003). Even if ANF contents are higher in diets 4 to 6,
they did not have any apparent impact on the husbandry
performance of clarias juveniles, suggesting that these
ANFs were not the main issues inﬂuencing responses in the
present study.
Robinson et al. (1985) in Hendricks (2002), on the contrary, observed no eﬀect of trypsin inhibitor levels as high as
3.6 Trypsin Inhibitor Units (TIU) in an experiment with
channel catﬁsh. Contrary to the results of Garg et al. (2003)
on Indian carp Cirrhinus mrigala, ANF contents of our
experimental diets had no inﬂuence on palatability, the feed
intake of diets 4 and 6 being similar to that of diets 1 to 3.
Moreover, for juveniles fed the supplemented diets and the
non-supplemented diets containing FM, survival was 100%
and no deformity was reported, contrary to what had been
observed in Atlantic salmon (Salmo salar) and common carp
(Cyprinus carpio) when phytic acid level was increased in feed
(Ogino & Takeda 1976; Baeverfjord et al. 1998; Roy et al.
2002; Sugiura et al. 2004 in Helland et al. 2006). More
investigations are needed to determine the sensitivity of
African catﬁsh to these ANFs. The results of this study
would also suggest that BM would be a good substitute in
Clarias feeds, not only because of its lysine contribution, but
also thanks to its low content of ANFs.
In conclusion, plant ingredients can eﬃciently substitute

ﬁshmeal in African catﬁsh diets. Dehulling and cooking
processes improved digestibility of sunﬂower oilcake (SFOC)
and reduced some of its ANF contents, such as tannin and
trypsin inhibitors. The results of this study also suggest that
ﬁshmeal can be totally replaced by plant feedstuﬀs in Clarias
diets, assuming that a proper balance of the diﬀerent plant
ingredients is ensured, without AA supplementation.

The authors thank the General Commissariat for the
International Relationships (CGRI) and the Directorate of
International Relationships (DRI) of the French Speaking
Community Government and Ministry of Walloon of
Belgium, respectively, for ﬁnancial support to L. Nyinawamwiza and to the FUNDP-UNR project ÔFilie`re ClariasÕ.

We would like to acknowledge Mr Yves Beckers (Gembloux University) for gross energy determination in his
laboratory.

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Ó 2009 The Authors
Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 237–247

Aquaculture nutrition, tập 16, số 3, 2010

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