Aquaculture nutrition, tập 16, số 3, 2010

at 3 and 28 g L1salinity to deter-mine apparent crude protein digestibility ACPD, energy digestibility AED and amino acid availability AAAA from soybean meal SBM, soy protein isolate SPI

Trang 1

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

FL, USA

Two experiments were conducted with Florida pompano,

Trachinotus carolinusL at 3 and 28 g L)1salinity to

deter-mine 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)1salinity, 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

plant-based 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: marty.riche@ars.usda.gov

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

estimated 227 000 kg total annual catch; however, because ofits highly prized taste and texture it maintains a high marketdemand Florida pompano tolerate a wide range of salinities,stress, readily consume pelleted rations, successfully breed incaptivity, and are an excellent candidate for aquaculture(McMaster et al 2004) There is increased interest in rearingeuryhaline species such as Florida pompano in freshwater orlow-salinity conditions However, nutritionally balanceddiets do not exist for Florida pompano presenting anobstacle to development of large-scale commercial produc-tion 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 increasesustainability and profitability (Glencross et al 2007) Inaddition to palatability and anti-nutritional concerns, use ofingredients as alternatives to fishmeal is limited by unknownavailability of nutrients Apparent digestibility coefficients offeed ingredients exist for only a few fish species, but notFlorida pompano To develop low-cost, low-polluting dietsthat achieve maximum efficiency, nutrient requirements andnutrient availability from dietary ingredients must be deter-mined to implement least-cost formulation of economicaland balanced diets

There is also evidence that salinity aﬀects nutrient ibility (Lall & Bishop 1976; MacLeod 1977; Dabrowski et al.1986; Krogdahl et al 2004) Digestibility in Golden-lineseabream, Sparus sarba (Forsskal) was higher in low salinityrelative to isosmotic or full-strength seawater (Woo & Kelly1995) Similarly, protein digestibility in milkﬁsh, Chanoschanos (Forsskal) was elevated in freshwater relative tosaltwater (Ferraris et al 1986) Zeitoun et al (1973) alsosuggested that protein requirements of rainbow trout,Oncorhynchus mykiss(Walbaum) were higher with increasingsalinity

digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest- digest-.

2010 16; 223–230

Aquaculture Nutrition

Trang 2

We hypothesized protein digestibility and amino acid (AA)

availability would be diﬀerent in low-salinity adapted Florida

pompano than saltwater adapted Florida pompano

There-fore, the objective was to determine apparent digestibility of

crude protein (CP), energy, and AA availability from

soy-bean meal (SBM), soy protein isolate (SPI) and corn gluten

meal (CGM) at both 3 and 28 g L)1salinity representing the

known range of salinity supporting normal growth of Florida

pompano

Florida pompano broodstock were spawned at the USDA,

Agricultural Research Services Center for Reproduction and

Larviculture, Fort Pierce, Florida, USA Postlarval juveniles

were reared at 28C and 30 g L)1salinity 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 28C and 7 g L)1salinity

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)1salinity

Two 8750 L recirculating systems with sand, bead, cartridge

and carbon ﬁltration, and ultraviolet light sterilization were

used Both systems were maintained at 28C 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

Mid-land, Decatur, IL, USA) and CGM (Rangen, Inc.) were

substituted at 300 g kg)1for 300 g kg)1of the reference diet

utilizing a modiﬁed diet replacement method All diets

incorporated yttrium oxide (Y2O3) at 5 g kg)1of the diet as

an inert marker Feed ingredients were ground via

ham-mermill (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 60C 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

commercial diet and allowed a 4-day acclimation to the newenvironment At initiation of the experiment, ﬁsh wereswitched to their assigned experimental diet and fed 4.7%

body weight per day divided between a morning and noon feeding Faecal samples were collected on day 5 andday 7 of being fed the experimental diets Faecal sampleswere collected 3–4 h following the morning feeding on day ofcollection

after-Prior to faecal collection, ﬁsh were anaesthetized with

75 mg L)1 tricaine methanesulphonate (MS-222; WesternChemical, Inc., Ferndale, WA, USA) Upon induction ofstage IV anaesthesia, the area around the anus was dried with

a towel and faecal samples collected by gentle expression ofthe lower gastrointestinal tract (Austreng 1978) Immatureﬁsh were used and care was taken not to contaminate sam-ples with urine Following collection, ﬁsh were resuscitatedand placed back into the experimental unit Faeces collected

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)1dry diet)

Reference diet

Test diets

Menhaden meal (low temperature) 1 338.5 237.0 Soybean meal (solvent extracted) 2 221.0 154.7

Porcine blood meal (spray dried) 2 30.0 21.0

1 Special Select, Omega Protein, Inc., Houston, TX, USA.

2 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)1premix):

USB, Cleveland, OH, USA.

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

.

2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230

No claim to original US government works

Trang 3

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 ingredients

contri-bution 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

adiabatic bomb calorimetry (Parr 1266; Parr InstrumentsCo., Moline, IL, USA) Ash was determined followingincineration at 600C for 2 h (AOAC 2002) Crude lipidwas determined gravimetrically following chloro-form : methanol extraction (Bligh & Dyer 1959) in aSoxhlet apparatus Crude ﬁbre was determined by a com-mercial 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 110C for 24 h Aseparate aliquot was analysed for cysteine (Cys) and methi-onine (Met) following performic acid oxidation to cysteicacid and methionine sulphone Amino acids were separatedusing a C-18 reverse phase HPLC column and quantiﬁedwith a photodiode array detector following postcolumnderivatization with ninhydrin

Table 2 Analysed composition (g kg)1)

of test ingredients and experimental

diets fed to Florida pompano

Test ingredient

Reference diet

CGM diet

SBM diet

SPI diet

CGM 1 SBM 2 SPI 3

5-28-242 5-04-612 – Proximate components

Dehulled, solvent-extracted soybean meal; Rangen, Inc., Buhl, ID, USA.

3 Soy protein isolate, Pro-Fam , Archer Daniels Midland, Decatur, IL, USA.

4 Nitrogen-free extract (100 ) moisture ) crude protein ) crude lipid ) ash ) fibre).

5

Aspartic acid + asparagine.

6 Glutamic acid + glutamine.

.

Trang 4

Diﬀerences in apparent nutrient availability were analysed

using the model statement for a Latin square design:

Yijk¼ l þ Iiþ columnjþ rowkþ eijk;

where I represents the main eﬀect of test ingredient, column

represents variation due to tank, and row represents

varia-tion due to week Analysis was performed using the general

linear model procedure ofSASwith 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

pair-wise contrasts between the three ingredients were evaluated

Signiﬁcance was reported at P < 0.05 unless otherwise

sta-ted Where analysis indicated row or column eﬀects in

3 g L)1salinity (alanine) or 28 g L)1(glutamic acid +

glu-tamine) no further analysis was conducted as row and

col-umn both represent restrictions on randomization making

the F-test questionable Regression analysis was performed

with test ingredient protein and energy as independent

vari-ables and apparent energy digestibility (AED) as the

depen-dent 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)1for 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)1as CaCO3 at 3 g L)1

salinity and from 6.62 to 7.95 mg L)1and 86 to 139 mg L)1

as CaCO3at 28 g L)1salinity 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

signiﬁ-cantly 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

statistically diﬀerent, the overall pattern suggests that AAAAappears higher from soy products than CGM at low salinity

in agreement with ACPD The availability of Metapproached 100% for all ingredients Overall mean AAAAwas similar to ACPD for all test ingredients, they were 81.2%

and 81.9% (CGM), 93.6% and 92.2% (SBM), 93.8% and93.1% (SPI) for AAAA and ACPD respectively

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)1salinity

Test ingredient

3 g L)1 28 g L)1 3 g L)1 28 g L)1Reference diet 72.8 (0.5) 74.7 (1.1) 71.3 (1.2) 72.3 (0.7) Corn gluten meal 81.9 (4.2) b 83.4 (2.9) a 77.4 (4.2) b 77.4 (3.4) a

Soybean meal 92.2 (2.0)a 87.1 (3.6)a 70.5 (6.5)b 62.2 (4.0)bSoy protein isolate 93.1 (1.9)a 85.0 (3.5)a 93.4 (2.5)a 78.1 (4.1)aMean 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)1salinity

Amino acids

Reference

Indispensable Arginine 83.3 (1.0) 73.5 (23.3) 102.0 (7.4) 95.3 (1.4) Histidine 80.3 (0.6) 76.8 (6.1) 103.3 (9.0) 92.5 (4.5) Isoleucine 80.4 (1.2) 68.1 (10.4) 91.8 (11.7) 96.4 (1.7) Leucine 86.1 (0.3) 88.1 (4.3) 92.1 (1.1) 94.7 (1.0) Lysine 81.9 (1.1) 76.2 (17.9) 100.0 (4.7) 94.1 (2.6) Methionine 1 83.0 (1.8) 100.0 (2.8) 110.1 (6.0) 105.7 (8.1) Phenylalanine 84.0 (0.5) 83.2 (6.7) b 97.0 (3.5) a 95.1 (2.7) a

Threonine 75.3 (1.0) 81.0 (9.4) 92.3 (6.0) 89.9 (6.4) Valine 82.2 (2.0) 81.6 (7.9) 85.6 (8.5) 98.6 (3.1) Dispensable

Alanine 81.5 (1.3) 91.5 (5.0) 89.7 (10.9) 96.4 (2.0) Asx 2 73.9 (0.3) 79.3 (7.1) 87.7 (3.8) 90.9 (4.1) Cysteine 1 84.5 (0.5) 67.8 (10.9) 91.5 (3.3) 82.8 (5.2) Glx3 80.0 (0.6) 86.5 (3.7)b 94.0 (3.1)a 93.8 (3.1)aGlycine 71.8 (1.4) 72.8 (9.8) 71.7 (12.3) 88.4 (2.7) Proline 73.5 (0.7) 84.2 (3.5) 88.9 (2.5) 93.1 (3.4) Serine 79.3 (1.3) 84.9 (7.3) 94.5 (4.4) 93.6 (3.5) Tyrosine 83.1 (1.0) 84.8 (9.2) 99.4 (7.1) 92.9 (3.4) Overall

2 Aspartic acid + asparagine.

3 Glutamic acid + glutamine.

.

Trang 5

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

diﬀer-ences were detected at 28 g L)1salinity 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 &

Oliva-Teles 1999; Sa´ et al 2006) The reason is unclear;however, the values in this study are similar to previouslyreported values (75.8% and 73.3% for ACPD and AEDrespectively) for juvenile Florida pompano fed the same dietformulation (Riche, new characters, 2009)

Poor digestibility is one reason attributed to low feedeﬃciency (FE) in Florida pompano (Tatum 1973; McMaster1988; Lazo et al 1998; Weirich et al 2006) However, SBMdigestibility and AAAA at 3 g L)1 salinity were similar tothat 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)1salinity was the same as reported for gilthead seabream,Sparus aurataL (Lupatsch et al 1997) Although ACPD forSBM was similar to that reported for haddock, Melano-grammus aegleﬁnus L (92.2%) and Cobia, RachycentroncanadumL (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 energyvalues from SBM were also reported in European seabass,Dicentrarchus labraxL (da Silva & Oliva-Teles 1998) and reddrum, Sciaenops ocellatus L (Gaylord & Gatlin 1996)

De Silva & Perera (1984) suggested that lower proteindigestibility occurs in diets with higher protein However, inthis study no diﬀerence in protein digestibility between soyproducts was detected at either salinity despite 130 g kg)1higher protein in the SPI diet Conversely, in this study AEDwas directly proportional to dietary protein (r2= 1.00) andinversely 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 nega-tively correlated to dietary carbohydrate and positively cor-related to dietary protein and lipid (Sullivan & Reigh 1995).Carbohydrate digestibility in Florida pompano is about 50%(Williams et al 1985) underscoring its limited availabilityand impact on energy digestibility

Florida pompano have short digestive tracts Intestinaltransit time for a fish meal/SBM diet was reported as 3 h inseawater at 29–31C (Williams et al 1985) This was laterconfirmed using the same dietary formulation serving as thereference diet in this study (Riche, new characters, 2009) Theshort transit may result in limited enzymatic contact timeattenuating digestion and absorption of nutrients, possiblycausing the poor FE reported for Florida pompano.Faecal stripping was initiated 3 h postprandially Consis-tent results with previous trials (Riche, new characters, 2009)coupled with the small SEM of coefficients in the referencediet suggests that this was appropriate for the reference diet.However, the high SEM of coefficients associated with the

Table 5 Mean (SEM; n = 4) apparent amino acid availability

(AAAA) coeﬃcients (%) for soybean meal (SBM), soy protein

iso-late (SPI) and corn gluten meal (CGM) in Florida pompano

Trachinotus carolinus adapted to 28 g L)1salinity

Different superscripts across a row indicate significant differences

between ingredients tested (P < 0.05).

1 Not statistically evaluated due to insufficient material for suitable

Trang 6

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

(Glen-cross 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

digest-ibility of CGM was similar in haddock, but ACPD in

had-dock 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 wholebody AA proﬁle exhibited only 60% of the weight gain ofpompano fed a menhaden meal based diet with the same AAproﬁle (Riche; unpublished data) Results from this studysuggest that poor weight gain previously observed was due inpart to lower AA availability from CGM

Apparent availability of Met was high for all test dients, as it was in cobia (Zhou et al 2004) The Met avail-ability from test ingredients evaluated in low salinity was100–110%, suggesting enhanced availability from the otherprotein sources used in the test diets However, cautionshould be exercised in interpreting the Met values as insuf-ﬁcient material in some cases limited the number of samplesfor estimating means

ingre-Signiﬁcantly, lower apparent Lys availability was observedfrom CGM than SBM at the higher salinity (P < 0.05) andappeared 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, Seriolaquinqueradiata (Temminck & Schlegel) (Masumoto et al

1996), but the opposite of cobia (Zhou et al 2004) andAtlantic salmon (Anderson et al 1992) Lower Lys avail-ability from CGM relative to the soy products may be anartefact of lower Lys in CGM Analysis of test ingredientsindicated Lys was 53.6, 30.6 and 10.0 g kg)1dry matter forSPI, SBM and CGM respectively At low dietary Lys,endogenous sources account for more of the recovered Lysmasking true availability and depressing apparent availabil-ity The 10% increase in true Lys availability over apparentLys availability from CGM in red sea bream (Yamamoto

et al 1998) and yellowtail (Masumoto et al 1996) supportthis hypothesis

The low CGM coefficients and high variability for Arg(SEM of 23.3%) and Lys (SEM of 17.9%) in the low salinitytreatment are attributable to high recovery of these AA inone faecal sample resulting in AAAA for that replicate of6.5% and 29.1% for Arg and Lys respectively Removal ofthat sample from consideration would have resulted incoefficients of 95.8% and 91.9% for Arg and Lys, respec-tively, which are similar to the other ingredients Althoughresiduals of the coefficients tested as outliers (Snedecor &

Cochran 1967), the coefficients were not removed fromanalysis because row and column effects could not be ruledout Moreover, it is possible the coefficients could representtrue variability in AAAA for a marine species held at lowsalinity

Although the experimental design precludes statisticalanalysis of test ingredients between the two salinities, the .

Trang 7

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

fur-ther 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

sug-gests 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

pom-pano 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

collec-tion, 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

plant-based proteins This work was funded in part by the Link

Foundation Additional funding was provided by the

USDA/Agricultural Research Service Project No

6225-63000-007-00D 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

Agri-culture are oﬀered on a non-discriminatory basis without

regard to race, colour, national origin, religion, sex, maritalstatus, or handicap

Allan, G.L., Parkinson, S., Booth, M.A., Stone, D.A.J., Rowland, S.J., Frances, J & Warner-Smith, R (2000) Replacement of ﬁsh meal in diets for Australian silver perch, Bidyanus bidyanus: I Digestibility of alternative ingredients Aquaculture, 186, 293–310 Anderson, J.S., Lall, S.P., Anderson, D.M & Chandrasoma, J (1992) Apparent and true availability of amino acids from common feed ingredients for Atlantic salmon (Salmo salar) reared in sea water Aquaculture, 108, 111–124.

AOAC (Association of Official Analytical Chemists) (2002) Official Methods of Analysis of the Association of Analytical Chemists, 17th edn Association of Official Analytical Chemists, Washington, DC, USA.

Austreng, E (1978) Digestibility determination in ﬁsh using chromic oxide marking and analysis of contents from diﬀerent segments of the gastrointestinal tract Aquaculture, 13, 265–272.

Bligh, E.G & Dyer, W.J (1959) A rapid method of total lipid extraction and purification Can J Biochem Phys., 37, 911–917 Cheng, Z.J & Hardy, R.W (2003) Effects of extrusion processing of feed ingredients on apparent digestibility coefficients of nutrients for rainbow trout (Oncorhynchus mykiss) Aquacult Nutr., 9, 77– 83.

Dabrowski, K., Leray, C., Nonnotte, G & Colin, D.A (1986) tein digestion and ion concentrations in rainbow trout (Salmo gairdneri Rich.) digestive tract in sea- and fresh water Comp Biochem Physiol., 83A, 27–39.

Pro-De Silva, S.S & Perera, M.K (1984) Digestibility in Sarotherodon niloticus fry: eﬀect of dietary protein level and salinity with further observations on variability in daily digestibility Aquaculture, 38, 293–306.

Ferraris, R.P., Catacutan, M.R., Mabelin, R.L & Jazul, A.P (1986) Digestibility in milkfish, Chanos chanos (Forsskal): effects of protein source, fish size and salinity Aquaculture, 59, 93–105 Forster, I (1999) A note on the method of calculating digestibility coefficients of nutrients provided by single ingredients to feeds of aquatic animals Aquacult Nutr., 5, 143–145.

Gaylord, T.G & Gatlin, D.M., III (1996) Determination of ibility coeﬃcients of various feedstuﬀs for red drum (Sciaenops ocellatus) Aquaculture, 139, 303–314.

digest-Glencross, B.D., Carter, C.G., Duijster, N., Evans, D.R., Dods, K., McCafferty, P., Hawkins, W.E., Maas, R & Sipsas, S (2004) A comparison of the digestibility of a range of lupin and soybean protein products when fed to either Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss) Aquaculture, 237, 333–346 Glencross, B., Evans, D., Dods, K., McCafferty, P., Hawkins, W., Maas, R & Sipsas, S (2005) Evaluation of the digestible value of lupin and soybean protein concentrates and isolates when fed to rainbow trout, Oncorhynchus mykiss, using either stripping or settlement faecal collection methods Aquaculture, 245, 211–220 Glencross, B.D., Booth, M & Allan, G.L (2007) A feed is only as good as its ingredients – a review of ingredient evaluation strategies for aquaculture feeds Aquacult Nutr., 13, 17–34.

Hajen, W.E., Higgs, D.A., Beames, R.M & Dosanjh, B.S (1993) Digestibility of various feedstuﬀs by post-juvenile Chinook salmon (Oncorhynchus tshawytscha) in sea water Measurement of digestibility Aquaculture, 112, 333–348.

Krogdahl, A˚., Sundby, A & Olli, J.J (2004) Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) digest and

.

Trang 8

metabolize nutrients diﬀerently Eﬀects of water salinity and

die-tary starch level Aquaculture, 229, 335–360.

Lall, S.P & Bishop, F.J (1976) Studies on the nutrient requirements

of rainbow trout, Salmo gairdneri, grown in sea water and fresh

water In: Advances in Aquaculture: FAO Technical Conference on

Aquaculture, Kyoto, Japan 26 May–2 June 1976 (Pillay, T.V.R &

Dill, W.A eds), pp 580–584 Fishing News Books, Farnham,

England.

Lazo, J.P., Davis, D.A & Arnold, C.R (1998) The eﬀects of dietary

protein level on growth, feed eﬃciency and survival of juvenile

Florida pompano (Trachinotus carolinus) Aquaculture, 169, 225–

232.

Lupatsch, I., Kissil, G.W., Sklan, D & Pfeffer, E (1997) Apparent

digestibility coeﬃcients of feed ingredients and their predictability

in compound diets for gilthead seabream, Sparus aurata L.

Aquacult Nutr., 3, 81–89.

MacLeod, M.G (1977) Eﬀects of salinity on food intake, absorption

and conversion in the rainbow trout Salmo gairdneri Mar Biol.,

43, 93–102.

Masumoto, T., Ruchimat, T., Ito, Y., Hosokawa, H & Shimeno, S.

(1996) Amino acid availability values for several protein sources

for yellowtail (Seriola quinqueradiata) Aquaculture, 146, 109–119.

Maynard, L.A & Loosli, J.K (1969) Animal Nutrition, 5th edn.

McGraw-Hill Book Company, New York, NY, USA, 613 pp.

McMaster, M.F (1988) Pompano aquaculture: past success and

present opportunities Aquacult Mag., 14, 28–34.

McMaster, M.F., Kloty, T.C & Coburn, J.F (2004) Pompano

mariculture – 2004 Aquacult Mag., 30, 25–29.

Peres, H & Oliva-Teles, A (1999) Eﬀect of dietary lipid level on

growth performance and feed utilization by European sea bass

juveniles (Dicentrarchus labrax) Aquaculture, 179, 325–334.

Riche, M (2009) Evaluation of digestible energy and protein for

growth and nitrogen retention in juvenile Florida pompano,

Trachinotus carolinus J World Aquacult Soc., 40, 45–57.

Sa´, R., Pousa˜o-Ferreira, P & Oliva-Teles, A (2006) Eﬀect of dietary

protein and lipid levels on growth and feed utilization of white sea

bream (Diplodus sargus) juveniles Aquacult Nutr., 12, 310–321.

Santinha, P.J.M., Medale, F., Corraze, G & Gomes, E.F.S (1999)

Eﬀects of the dietary protein: lipid ratio on growth and nutrient

utilization in gilthead seabream (Sparus aurata L.) Aquacult.

Nutr., 5, 147–156.

da Silva, J.G & Oliva-Teles, A (1998) Apparent digestibility

coef-ﬁcients of feedstuﬀs in seabass (Dicentrarchus labrax) juveniles.

Aqua Living Resour., 11, 187–191.

Snedecor, G.W & Cochran, W.G (1967) Statistical Methods, 6th

edn The Iowa State University Press, Ames, IA, USA, 593 pp.

Sullivan, J.A & Reigh, R.C (1995) Apparent digestibility of selected

feedstuﬀs in diets for hybrid striped bass (Morone saxatilis

$ · Morone chrysops #) Aquaculture, 138, 313–322.

Tatum, W.M (1973) Comparative growth of pompano notus carolinus) in suspended cages receiving diets of a ﬂoating trout chow with those receiving a mixture of 50% trout chow and 50% sinking ration In: Proceedings of the 4th Annual Meeting of the World Mariculture Society (Avault, J.W &

(Trachi-Boudreaux, E eds), pp 125–141 Louisiana State University, Baton Rouge, LA, USA.

Tibbetts, S.M., Milley, J.E & Lall, S.P (2006) Apparent protein and energy digestibility of common and alternative feed ingredients by Atlantic cod, Gadus morhua (Linnaeus, 1758) Aquaculture, 261, 1314–1327.

Tibbetts, S.M., Lall, S.P & Milley, J.E (2004) Apparent digestibility

of common feed ingredients by juvenile haddock, Melanogrammus aegleﬁnus L Aquac Res., 35, 643–651.

Watanabe, W.O (1995) Aquaculture of the Florida pompano and other jacks (Family Carangidae) in the Western Atlantic, Gulf of Mexico, and Caribbean basin: status and potential In: Culture of High-value Marine Fishes (Main, K.L & Rosenfeld, C eds),

pp 185–205 Oceanic Institute, Honolulu, HI, USA.

Weirich, C.R & Riche, M (2006) Acute tolerance of juvenile Florida pompano, Trachinotus carolinus L to ammonia and nitrite at various salinities Aquac Res., 37, 855–861.

Weirich, C.R., Groat, D.R., Reigh, R.C., Chesney, E.J & Malone, R.F (2006) Eﬀect of feeding strategies on production characteristics and body compoasition of Florida pompano reared

in marine recirculating systems N Am J Aquacult., 68, 330–

Wu, X., Liu, Y., Tian, L., Mai, K & Yang, H (2006) Apparent digestibility coeﬃcients of selected feed ingredients for yellowﬁn seabream, Sparus latus J World Aquacult Soc., 37, 237–245.

Yamamoto, T., Ikeda, K., Unuma, T & Akiyama, T (1997) Apparent availabilities of amino acids and minerals from several protein sources for ﬁngerling rainbow trout Fish Sci., 63, 995–

1001.

Yamamoto, T., Akimoto, A., Kishi, S., Unuma, T & Akiyama, T.

(1998) Apparent and true availabilities of amino acids from several protein sources for ﬁngerling rainbow trout, common carp, and red sea bream Fish Sci., 64, 448–458.

Zeitoun, I.H., Halver, J.E., Ullrey, D.E & Tack, P.I (1973) ence of salinity on protein requirements of rainbow trout (Salmo gairdneri) ﬁngerlings J Fish Res Board Can., 30, 1867–1873.

Inﬂu-Zhou, Q., Tan, B., Mai, K & Liu, Y (2004) Apparent digestibility of selected feed ingredients for juvenile cobia Rachycentron canadum.

Aquaculture, 241, 441–451.

.

Trang 9

1,2 1,3 1 1 1 1 1

1

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

A 9-week feeding experiment was conducted to determine the

dietary biotin requirement of Japanese seabass, Lateolabrax

japonicusC 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 28.0C, 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

experi-ment, ﬁsh fed Diet 1 developed severe biotin deﬁciency

syn-dromes 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

effi-ciency ratio and protein effieffi-ciency 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

concentra-tion (0–6.1 lg g)1) signiﬁcantly increased with the

supple-mentation 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)1biotin for maximal growth

(Lateolabrax japonicus)

Received 9 April 2008, accepted 7 January 2009

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

Biotin is a water-soluble vitamin generally included in min B complex and functions in several speciﬁc carboxyla-tion and decarboxylation reactions It is part of thecoenzymes of several carboxylation enzymes ﬁxing CO2, such

vita-as propionyl CoA in the formation of propionic acid, CoA carboxylase and pyruvate carboxylase Carboxylaseﬁxation of CO2 to form methylmalonyl CoA is involved inthe carboxylation and decarboxylation of tricarboxylic acids.Biotin is also involved in the syntheses of fatty acids, lipidsand citrulline

acetyl-As biotin is one of the most important and expensivevitamins added to aquafeeds, it is necessary to quantify theminimum requirement of biotin to manufacture cost-eﬀectivecommercial feeds However, several factors have been proved

to influence the need for dietary biotin in animals Forexample, high dietary lipid has been shown to obscure theeffects of biotin in rats, chicks and brook trout (Jacobs et al.1970; Marson & Donaldson 1972; Poston & McCarteney1974) The quantitative requirement of biotin for optimalgrowth has only been investigated in few species of fish Forexample, 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)1for hybridtilapia (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 cally important food ﬁsh with fast growth and high marketvalue, and now has been widely cultured in China However, .

economi-2010 16; 231–236

Trang 10

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

suit-able 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

nutri-tional 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

knowl-edge, no information is available on its dietary biotin

requirement Hence, the present investigation was

under-taken to determine the optimum dietary biotin requirement

on the basis of weight gain (WG) of juvenile Japanese

sea-bass

Six isonitrogenous and isoenergetic diets were formulated

with graded levels of biotin (Table 1) Vitamin-free casein

(Sigma, Chemical, St Louis, MO, USA) and gelatin (Sigma,Chemical) were used as protein source, dextrin (ShanghaiChemical Co., Shanghai, China) as carbohydrate source, andmenhaden ﬁsh oil (Food grade) and soybean oil (Food grade)

as lipid sources Amino acid (Shanghai Chemical Co.) ture was added to simulate the whole body amino acid pat-tern of Japanese seabass ﬁngerling (Mai et al 2006) Biotin(Sigma) was added to the test diets at the expense of cellulose

mix-to provide concentrations of 0, 0.01, 0.05, 0.25, 1.25 and6.25 mg kg)1 diet The biotin concentrations in the dietsdetermined 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 through220-lm mesh and thoroughly mixed with biotin, then withmenhaden fish oil and soybean oil Finally, cold water wasadded to produce stiff dough, which was subsequently pel-leted with an experimental diet mill [F-26 (II), South ChinaUniversity of Technology, China] and dried for about 12 h in

a ventilated oven at 45C After drying, the diets werebroken up and sieved into proper pellet size The sizes ofpellets were 1.5· 3.0 and 2.5 · 4.0 mm All the diets weresealed in bags and stored at)15 C until used

Experimental ﬁsh were obtained from a commercial farm inYantai, 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 tothe experimental diet and the rearing conditions At the start

of the experiment, the ﬁsh were fasted for 24 h and weighedafter being anesthetized with eugenol (1 : 10 000) (ShanghaiReagent Corp., Shanghai, China) Juvenile Japanese seabasswith 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 30fish and provided with continual aeration The fish were fed

by hand twice daily at 08:00 and 17:00 hours respectively Toprevent the waste of dietary pellets, ﬁsh were slowly hand-fedlittle by little to apparent satiation on the basis of visualobservation of ﬁsh feeding behaviour The feeding trial lastedfor 9 weeks, from weeks 1 to 4, 1.5-mm pellets were fed;

thereafter, 2.5-mm pellets were fed until the end of theexperiment During the experimental period, feed consump-tion was recorded daily The number and weight of dead ﬁshwere recorded and a natural photoperiod was maintained

Water ﬂow rate through each tank was 2 L min)1 Water

Table 1 Formulation and proximate composition of the basal diet

(g kg)1dry matter)

Ingredients

Content (g kg)1)

1 Amino acid mixture (g kg)1diet): 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)1permix): NaF, 0.2 g; KI, 0.08 g; CoCl 2 Æ6H 2 O

(1%), 5.0 g; CuSO 4 Æ5H 2 O, 1.0 g; FeSO 4 ÆH 2 O, 8.0 g; ZnSO 4 ÆH 2 O, 3.0 g;

MnSO 4 ÆH 2 O, 1.5 g; MgSO 4 Æ7H 2 O, 120.0 g; Ca (H 2 PO 4 ) 2 ÆH 2 O, 750.0 g;

NaCl, 10.0 g; Zoelite, 101.22 g.

3 Vitamin premix (mg kg)1diet): B 1 , 25 mg; B 2 , 45 mg; B 6 , 20 mg;

B 12 , 0.1 mg; pantothenic acid, 60 mg; niacin acid, 200 mg; folic

Trang 11

temperature ranged from 25.0 to 28.0C, 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)1during 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

termi-nation 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 NFwere initial and ﬁnal number of ﬁsh; WI

and WFwere initial and ﬁnal weight of ﬁsh in g; FTwas total

FI on dry basis in g; PTwas protein intake on dry basis in g;

Twas 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 usingSPSS 10.0 for windows Differences between the means weretested by Tukey’s multiple range test The level of significancewas chosen at P < 0.05 After comparing the sum of squaresabout regression (SSR) between broken-line regressionmodel and second-order polynomial regression model, thebroken-line model (Robbins et al., 1979), which gave theleast value of SSR and the highest estimation coefficient (R2),was used to estimate the optimal requirements of dietarybiotin 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 syndromescharacterized 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 showany deficiency syndrome

It can be seen from Table 2 that with the increase of dietarybiotin level, FI of fish was significantly increased (Diets 1–4)and then levelled off when the dietary biotin level reached0.247 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 thefish feeding on Diet 4 (0.247 mg biotin kg)1diet) The sur-vival rate of fish fed the biotin-deficient diet (73.3%) was thelowest 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)1BW day)1) of Japanese seabass fed experimental diets with graded biotin levels for 9 weeks 1

Diets

FI (g kg)1

BW day)1) Diet 1 (0) 2.26 ± 0.03 4.46 ± 0.07 d 73.3 ± 6.67 b 97.2 ± 2.99 d 0.41 ± 0.04 b 1.10 ± 0.04 c 21.42 ± 1.41 b

Diet 2 (0.010) 2.26 ± 0.03 7.36 ± 0.17 c 91.1 ± 2.22 ab 225.5 ± 7.59 c 0.70 ± 0.04 ab 1.50 ± 0.02 b 25.46 ± 0.94 a

Diet 3 (0.049) 2.26 ± 0.03 14.59 ± 0.22 a 88.9 ± 2.22 ab 545.4 ± 9.73 a 0.85 ± 0.05 a 1.97 ± 0.03 a 28.22 ± 1.11 ab

Diet 4 (0.247) 2.26 ± 0.03 13.58 ± 0.32 ab 95.6 ± 2.22 a 500.7 ± 14.01 ab 0.79 ± 0.05 a 2.00 ± 0.04 a 31.70 ± 2.33 a

Diet 5 (1.238) 2.26 ± 0.03 13.66 ± 0.44ab 86.7 ± 3.85ab 504.4 ± 19.27ab 0.76 ± 0.10a 1.93 ± 0.02a 30.83 ± 1.77aDiet 6 (6.222) 2.26 ± 0.03 12.83 ± 0.13b 88.9 ± 5.88ab 467.6 ± 5.68b 0.75 ± 0.09a 2.01 ± 0.02a 30.89 ± 2.81a

ANOVA

1 Values are means ± S.E.M of three replicate groups (n = 3).

2 Mean values with different superscript letter in the same column differ significantly (P < 0.05).

.

Trang 12

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

die-tary 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)1diet (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

(141.9–146.9 g kg)1), crude lipid (49.9–56.3 g kg)1), ture (761.3–776.8 g kg)1) and ash content (45.1–45.9 g kg)1)

mois-Liver biotin concentration (0.0–6.1 lg g)1), however, icantly increased with increasing dietary biotin level(P < 0.05), and no tissue saturation was observed within therange of dietary biotin levels

signif-This study demonstrates that biotin is one of the essentialvitamins for Japanese seabass Deficiency syndromes such ashigh mortality, poor growth, anorexia, atrophy and darkskin colour were observed in Japanese seabass fed on biotin-deficient diet (Diet 1) for 6 weeks (plus the acclimation per-iod of 2 weeks) Similarly, Halver (1989) found that Pacificsalmonids fed biotin-deficient diets showed skin disorders,atrophy, convulsions and loss of appetite

It was also found that dietary biotin-deficiency resulted indeficiency signs in Asian catfish and Indian catfish, whichwere characterized by convulsions, heavy mortality, listless-ness, anorexia, poor FI and feed conversion, dark skin col-our, 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 ofnutrients are accepted in principle as a method for deter-mining dietary nutrient requirements Nutrient require-ments in ﬁsh can be estimated by either broken-lineregression analysis (Robbins et al 1979) or polynomialregression analysis (Zeitoun et al 1976) Comparisonsbetween broken-line regression model and second-orderpolynomial regression model have been made before theanalysis of optimal dietary biotin requirement on the basis

of WG The sum of squares about regression and thecoeﬃcient 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)

1 Values are means ± SEM of three replicate groups (n = 3).

2 Mean values with different superscript letter in the same column differ significantly (P < 0.05).

.

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

Trang 13

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

Japa-nese 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)1for 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

biotin-free 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

con-tent 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) Theyreported the body biotin concentration of Atlantic salmon,hybrid tilapia and the liver biotin level of Asian catfishincreased significantly with the increasing of dietary biotinlevel without tissue saturation In this study, higher survivalrate and no biotin deficiency syndrome were observed in thetreatments when liver biotin concentrations were more than0.5 lg g)1(Diet 2)

In rats (Jacobs et al 1970), chicks (Marson & Donaldson1972) and trout (Poston & McCarteney 1974), the dietarylipid content has been shown to obscure eﬀects of biotindeﬁciency However, in this study, the basal diet contained

432 g kg)1 crude protein and 125 g kg)1 crude lipid inmeeting 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 dietwith 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 andXiao, 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 alltheir help during the experiment

Ai, Q., Mai, K., Li, H., Zhang, L., Duan, Q., Tan, B., X, W., Ma, H., Zhang, W & Liufu, Z (2004a) Eﬀects of dietary protein to energy ratios on growth and body composition of juvenile Japanese seabass, Lateolabrax japonicas Aquaculture, 230, 507–516.

Ai, Q., Mai, K., Zhang, C., Xu, W., Duan, Q., Tan, B & Liufu, Z (2004b) Eﬀects of dietary vitamin C on growth and immune response of Japanese seabass, Lateolabrax japonicus Aquaculture,

242, 489–500.

AOAC (1995) Oﬃcial Methods of Analysis of AOAC International Vol I Agriculture Chemical; Contaminants, Drug, 16th edn AOAC International, Arlington, VA.

Baker, D.H (1986) Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrient.

J Nutr., 116, 2339–2349.

Castledine, A.J., Cho, C.Y., Slinger, S.J., Hicks, B & Bayley, H.S (1978) Inﬂuence of dietary biotin level on growth, metabolism and pathology of rainbow trout J Nutr., 108, 698–711.

Gao, C., Liu, Q., Liang, Y & Han, A (1998) The optimum content

of protein and fat in the artiﬁcial diet fed to cultured juvenile seabass (Lateolabrax japonicus) Mar Fish Res., 19, 81–85 (In Chinese with English abstract).

Gu¨nther, K.D & Meyer-Burgdorff, K.H (1990) Studies on biotin supply to mirror carp (Cyprinus Carpio L) Aquaculture, 84, 49– 60.

.

Trang 14

Halver, J.E., ed (1989) The vitamins In: Fish Nutrition, 2nd edn,

pp 57–60 Academic Press Inc., San Diego, CA, USA.

Hong, H., Lin, L., Chen, X., Hu, J & Zhou, L (1999) Studies on the

optimal content and protein sparing eﬀect of lipid in artiﬁcial

foodstuﬀ for Lateolabrax japonicus J Jimei Univ., 4, 41–44 (In

Chinese with English abstract).

Hu, J., Chen, X & Hong, H (1995) Evaluation of soybean cake as a

substitute for partial ﬁsh meal in artiﬁcial diets for Lateolabrax

japonicus J Oceanogr Taiwan Strait, 14, 418–421 (In Chinese

with English abstract).

Jacobs, R., Kilburn, E & Majerus, P.W (1970) Acetyl CoA

car-boxylase The eﬀect of biotin deﬁciency on enzyme in rat liver and

adipose tissue J Biol Chem., 245, 6462–6467.

Kashiwada, K., Kanazawa, A & Techima, S (1971) Studies on the

production of B vitamins intestinal bacteria VI: production of folic

acid by intestinal bacteria of carp Memoits Fac Fish Kagoshima

Univ., 20, 185–189.

Lahely, S., Ndaw, S., Arella, F & Hasselmann, C (1999)

Determi-nation of biotin in foods by high-performance liquid

chromato-graphy with post-column derivatization and ﬂuorimetric detection.

Food Chem., 65, 253–258.

Lin, L.M., Hu, J.C & Hong, H.X (1994) The most suitable content

of protein in the artiﬁcial feed to culture Lateolabrax japonicus.

J Xiamen Fish Coll., 16, 6–10 (In Chinese with English abstract).

Lovell, R.T & Buston, J.C (1984) Biotin supplementation of

prac-tical diets for channel catﬁsh J Nutr., 114, 1092–1096.

Maeland, A., Waagbo, R., Sandnes, K & Hjeltnes, B (1998) Biotin

in practical ﬁsh-meal based diet for Atlantic salmon Salmo salar L.

fry Aquacult Nutr., 4, 241–247.

Mai, K., Zhang, L., Ai, Q., Duan, Q., Zhang, C., Li, H., Wan, J &

Liufu, Z (2006) Dietary lysine requirement of juvenile Japanese

seabass, Lateolabrax japonicus Aquaculture, 258, 535–542.

Marson, J.V & Donaldson, W.E (1972) Fatty acid synthesising

systems in chick liver Inﬂuences of biotin deﬁciency and dietary

fat J Nutr., 102, 667–672.

Mohamed, J.S (2001) Dietary biotin requirement determined for

Indian catﬁsh, Heteropneustes fossilis (Bloch), ﬁngerlings

Aqua-cult Res., 32, 709–716.

Mohamed, J.S., Ravisankar, B & Ibrahim, A (2000) Quantifying the dietary biotin requirement of the catﬁsh Clarias batrachus.

Aquacult Int., 8, 9–18.

Ogino, C., Watanabe, T., Kakino, J., Iwanaga, N & Mizuno, M.

(1970) B vitamin requirements of carp: III Requirement of biotin.

Bull Jpn Soc Sci Fish., 36, 734–740 (In Japanese with English abstract).

Pan, Y., Wang, F & Liu, H (2000) Optimal proportion of ﬁsh meal and soybean cake in formulated diets of juvenile sea perch Lateolabrax japonicus J Dalian Fish Coll., 15, 157–163 (In Chinese with English abstract).

Poston, H.A (1976) Optimum level of dietary biotin for growth, feed utilization and swimming stamina of ﬁngerling lake trout (Salv- elinus namaycush) J Fish Res Board Can., 33, 1803–1806.

Poston, H.A & McCarteney, T.H (1974) Eﬀect of dietary biotin and lipid on growth, stamina, lipid metabolism and biotin-containing enzymes in brook trout J Nutr., 104, 315–322.

Robbins, K.R., Norton, H.W & Baker, D.H (1979) Estimation

of nutrient requirements from growth data J Nutr., 109, 1710–

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.

.

Trang 15

1,2 3 1 4 1

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 SciencesAgronomiques de Gembloux, Gembloux, Belgium; 4 Unite´ de Biochimie de la Nutrition, Faculte´ dInge´nierie Biologique,Agronomique et Environnementale, Louvain-la-Neuve, Belgium

The study was undertaken to evaluate the growth

perfor-mance 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

deter-mine 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)1FM) 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

maxi-mum 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

ADC values for neutral detergent fibre (NDF), dry matter,energy, protein and amino acids (AA) There were no sig-nificant differences in protein ADCs (88–90%) with increasedlevels of dietary vegetable ingredients Both soaking anddehulling of sunflower before incorporation helped in thereduction of NDF, antitrypsin and tannins Digestibility ofall AA was generally high, greater than 90% for bothindispensable and non-indispensable AA Based on the dataobtained, it was possible to totally replace menhaden fishmeal with a mixture of vegetable proteins (72% of totaldietary protein) when diets contained a relatively low per-centage of animal protein (28% based on blood meal andchicken viscera meal) without negative effects

coeﬃcient, Clarias gariepinus, feed utilization, growth formances, sunﬂower oilcake

per-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: patrick.kestemont@fundp.ac.be

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

2010 16; 237–247

Trang 16

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

accom-plished 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;

Four-nier 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

palat-ability 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

diges-tion (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

biologi-cal availability of macromolecules such as proteins and

car-bohydrates (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

inhib-itors, 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

absorp-tive 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 byimproving digestibility and to partly reduce the presence ofANFs include a wide range of processing techniques such ascooking, dehulling, germination, roasting, extrusion, soakingand recently extrusion cooking (Akpapunam & Sefa-Dedeh1997; 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 formulationshould be based on nutrient bioavailability, reliable data onthe digestibility of diﬀerent ingredients for each species mightwell be considered as a necessary prerequisite However,potential interactions among ingredients should also beconsidered

Fish meal (FM), the conventional dietary protein source incatﬁsh feed (40–60% of the total protein) (Van Weerd 1995)

is totally imported in Rwanda, soybean is scarce while ﬂower oil cake is available and less expensive (Nyina-wamwiza et al 2007) Moreover, it has been demonstratedthat dietary incorporation of soybean meal, groundnut cakeand winged bean improved the growth performance, feedintake and feed eﬃciency (FE) of Clarias gariepinus (Balogun

sun-& Ologhobo 1989; Degani et al 1989; Hoﬀman et al 1997;

Fagbenro 1998, 1999) Our knowledge on anti-nutrienteﬀects in African catﬁsh is very poor

Based on the foregoing, several objectives were identiﬁed inthis 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 evaluatethe resulting influence on the growth response, protein utili-zation and FE of C gariepinus fingerlings Among the testedingredients, sunflower oilcake was especially investigated byapplying different processing methods such as soaking ordehulling, and by combining it with other by-products Asnutrients are not available to an animal before they areabsorbed in the digestive tract, in a second stage, apparentdigestibility coefficients (ADCs) for dry matter, protein,energy, fibre and AA in experimental diets was studied

Experiment 1: growth and feed utilization In the firstexperiment, fish were obtained by artificial reproductionfrom broodstock cultured in earthen ponds at the RwasaveFish Culture Station of the National University of Rwanda(Butare District) At 3–4 g body weight, fish were acclima-tized to the experimental conditions for 3 weeks in plastictanks and received a mixture of the six experimental diets in .

Trang 17

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

dis-tributed 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 ± 1.5C, dissolved oxygen and pH ranged

from 3.1 to 6.0 mg L)1and 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

digest-ibility 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,

Uni-versite´ 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

con-stantly 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 faecesfrom the system The faecal samples collected from each tankwere frozen daily At the end of the digestibility trial, thepooled faeces from each tank were freeze-dried prior toanalysis for chromic oxide, protein, AA, ﬁbre and energy

Six diets were formulated containing graded levels of FM Aﬁrst diet with sunﬂower oilcake from hulled and unsoakedseeds (SFOC) containing only 25% of FM was formulated asreference In the second diet, SFOC was soaked in water for

24 h before incorporation in the diets (SFOCS) in order todiminish ANFs and to improve the feed intake (Amrish 2002)

In the third diet, SFOC level was reduced and it was mixed withbean 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) islow, whereas its content in methionine is high On the contrary,the lysine content of Phaseolus seeds is relatively high, theamount 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 requirementfor lysine estimated at 4.8% of protein for Clarias hybrids(Unprasert 1994 in Wilson 2002) Webster & Lim (2002) foundlysine to be the main limiting AA in Channel catfish Ictaluruspunctatus and perhaps in other warmwater fish as well(Robinson et al 1980 in Wilson 2002) Groundnut (Arachishypogea) oilcake (GOC) was used as a substitute for fish-meal because of its high-crude protein content (480 g kg)1).Because of the potentially higher digestibility of dehulledsunflower meal/oilcake (SFOCD), and the food intake pref-erences (Gill et al 2006), for the fourth diet, fishmeal wasreduced to 150 g kg)1 Finally, fishmeal was reduced to 0%

by using a mixture of local ingredients such as BM, GOCand 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 wereselected from local markets in Rwanda, partly based on theirpotential as cheap and readily available protein sources.All diets were analysed for proximate composition usingstandard methods given in AOAC (1980) and results arepresented in Tables 1 and 2

All collected ingredients were cooked in a pressure cookerfor 1–2 h at 100C with addition of a few volumes of water,followed by sun drying Before mixing, ingredients wereground, mixed thoroughly with water, made into spaghetti(2 mm diameter), and converted into pellets after sun drying. .

Trang 18

For the digestibility experiment, 15 g kg)1of 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 105C

for 24 h Total lipids of ﬁsh carcass were extracted with

chloroform/methanol/water (10 : 10 : 9, vol/vol/vol)

accord-ing 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 550C

Gross energy of the diets and faeces was determined using

an adiabatic bomb calorimeter 1241, Parr Instrument pany, Moline-Illinois-USA) Neutral detergent ﬁbre (NDF)and acid detergent ﬁbre (ADF) in diets and faeces weremeasured by the method of Goering & van Soest (1970)

Com-Chromic oxide was estimated spectophotometricallyfollowing the method of Furukawa & Tsukahara (1966)

Total AA contents of diets (Table 2) and faecal samplesfrom each tank were measured by ion-exchange chromatog-raphy, Biochrom 20 Plus-Amino Acid Analyser, BiochromLtd, Cambridge, UK (Moore et al 1958) For sulphur AA,samples were ﬁrst oxidized by a performic acid-phenol tooxidize methionine and cystine to methionine sulphone andcysteic acid, respectively (Lewis 1966) These oxidized sam-ples, as well as unoxidized samples, were hydrolysed in 6 NHCl, for 24 h at 110C Norleucine was used as an internal

Table 1 Composition of the six mental diets

experi-25% Fish meal

15% Fish meal 0% Fish meal Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6

SFOC SFOCS

SFOC + BM

SFOCD + BM

SFOCS +

BM + GOC

SFOCD + GOC + BM Ingredients (g kg)1diet)

Crude protein (g kg)1dry matter)3 367 351 384 378 350 381

Gross energy

(kJ g)1dry matter) 3

NDF, neutral detergent fibre; ADF, acid detergent fibre.

1 Mineral mixture INRA Belgium, MLNP 763, (composition per kilogram: dibasic calcium

phos-phate: 500 g; calcium carbonate: 215 g; sodium chloride: 40 g; potassium chloride: 90 g;

mag-nesium 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.

.

Trang 19

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

sec-ondary (imino acid, proline and hydroxy-proline)

Trypto-phan could not be analysed because of its destruction during

acid hydrolysis

Among the multiple ANFs that can be found in the

veg-etable 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-DL-arginine-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

experi-mental 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 perkilogram of sample analysed, catechin being the standardtannin used

Phytic acid contents were determined according to themethod of March et al (1995) The method of proportionconsists ﬁrstly in isolating phytates, after their extraction insulphuric acid, in the form of iron (III) phytate Secondly,NaOH and water were added to this solid iron (III) salt inorder to precipitate hydrated iron (III) oxide and liberate thephytate The absorbance was measured at 400 nm against areagent blank Phytic acid contents were expressed as grams

of phytic acid per kilogram of sample analysed

Fish performance was determined using the following mulae:

for-Weight gainð%Þ ¼ 100 ðWf WiÞ=Wi

where Wiand Wfis the initial and ﬁnal body mass (g)

Specific growth rateðSGR; % per dayÞ

¼ 100 ½lnðWfÞ lnðWiÞ=Dtwhere Wiand Wfis the initial and ﬁnal mean body mass (g)andDt is the duration of experiment

Feed efficiency (FE)¼ ðFB IBÞ=TFIwhere FB is the ﬁnal biomass per tank (g), IB is the initialbiomass per tank (g) and TFI is the total food intake (g)

Table 2 Proximate amino acids

compo-sition of the experimental diets (g per

16 g N)

Amino acid (% dry matter)

25% Fish meal 15% Fish meal 0% Fish meal Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6

SFOC SFOCS

SFOC + BM

SFOCD + BM

SFOCS +

BM + GOC

SFOCD + GOC + BM

Trang 20

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 matterð%Þ ¼ 100 ½1 ðDiÞ=ðFiÞ

The ADCs of proteins, AA, ﬁbre and energy were

calcu-lated as follows (Cho & Slinger 1979):

ADC¼ 100 ½1 ðF =DÞ 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

dif-ference) 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

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

and those fed other diets In contrast, no significant ferences were obtained between fish fed diet 6 containingdehulled SFOC (0% FM) and fish fed diets containing 15

dif-or 25% FM On the contrary, the best overall growthresponse was obtained in fish fed diet 3 (25% fishmeal whenSFOC was reduced to 25%) Similar results were observedfor FE and PER with a significant reduction observed fordiet 5 No significant differences were observed between fishfed diets 1 and 2 containing unsoaked and soaked SFOC,respectively

Apparent digestibility coefficients for dry matter, protein,energy, fibre and AA in diets consumed by C gariepinusfingerlings are shown in Table 4 ADCs of dry matter andgross energy were significantly affected by experimentaldiets (P < 0.05), generally high for the diets containingdehulled SFOC and especially lowered by increased inclu-sion of hulled SFOC meal in the diet Diet 3 gave inter-mediate 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 insolubleﬁbres (NDF) in diets 4 and 6, whereas it was lowest in diet

5 In comparing diets 1 and 2, soaking process increasedADC values of NDF, dry matter, gross energy, proteinand AA ADCs There were no significant differences inADCs (88–90%) of protein with increased level of vege-table ingredients in diets (>0.05) Digestibilities of all AAwere generally high, over 90% for indispensable and non-indispensable AA little affected by experimental diet

Indeed, digestibilities of three IAA (isoleucine, methionineand threonine) were higher in diets 1 and 2, but lower indiet 5

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

Parameters

Initial body weight (g) 7.54 ± 0.08 7.51 ± 0.02 7.56 ± 0.04 7.44 ± 0.09 7.43 ± 0.03 7.45 ± 0.04

Final body weight (g) 42.8 ± 13.1 ab 42.1 ± 9.7 ab 53.3 ± 11.8 a 38.7 ± 2.3 abc 26.0 ± 4.4 c 35.6 ± 5.1 bc

Protein efficiency ratio 3.11 ± 0.47a 3.22 ± 0.37a 3.16 ± 0.39a 2.97 ± 0.08ab 2.21 ± 0.37c 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).

.

Trang 21

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)1diet 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 plantby-product content While the 2 first diets contained onlysunflower oilcake, a part of that oilcake was substituted by

BM in the third diet It was apparent that Clarias ﬁngerlingsmight be sensitive to a large (higher than 25.7%) inclusion ofhulled sunﬂower oil cake for several reasons Firstly, because

of the high fibre content in SFOC, and secondly because thecomplementary nature of SFOC and BM leads to a betteressential AA balance Differences between diets 5 and 6 canonly be explained by the dehulling of sunflower Fishmealcan thus be totally replaced by a combination of groundnutoilcake, BM and sunflower oilcake, providing that sunfloweroilcake is dehulled before its incorporation into the diet.Diets 1 and 2 provided similar results; the soaking of sun-flower oilcake did not affect these results, whereas dehullingimproved its nutritive value

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

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

Parameters

SFOCS + BM + GOC

SFOCD + GOC + BM

Digestibility of dry matter (%) 60.5 ± 1.3 c 64.2 ± 1.1 bc 67.1 ± 1.4 ab 72.1 ± 1.9 a 65.0 ± 4.4 bc 70.9 ± 2.6 ab

Digestibility of crude protein (%) 87.7 ± 3.0 88.8 ± 0.6 87.6 ± 0.8 88.5 ± 1.8 88.0 ± 0.8 89.5 ± 0.5 Digestibility of gross energy (%) 72.4 ± 1.9 bc 74.4 ± 0.6 bc 77.0 ± 2.5 ab 81.3 ± 0.7 a 71.0 ± 3.9 c 78.6 ± 2.9 a

Values are given as mean ± standard deviation.

1 Indispensable amino acid (IAA).

.

Trang 22

matter digestibility coeﬃcients ranged from a minimum of

60.5% (D1) to a maximum of 72.1% (D4) All diets

con-taining 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

dif-ferent anti-nutrients during the pre-treatment process,

espe-cially 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

con-trary, 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

feed-stuﬀs 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% forcottonseed cake and similar to the 75.8 and 79% (except forD1 and D5) for groundnut cake, sunflower cake and soybeancake, respectively, reported for C gariepinus (Fagbenro1998) Bjo¨rck et al (1984 in Cheng & Hardy 2003) suggestedthat the increased soluble fibre portion would improve ADCs

of fibre and thus increase digestible energy, becausenon-ruminant animals (such as pigs) could utilize the fibre tomeet 30–50% of their energy needs via fermentation tovolatile fatty acids Results of the present study suggest thatthis is not true in African catfish

Anti-nutritional factors are present in sunﬂower oilcakes andgroundnut oilcake in similar proportions, whereas BM con-tained less phytic acid and displayed less antitryptic activity

Both soaking and dehulling of sunﬂower before tion helped in the reduction of trypsin inhibitors and tanninsbut not phytate It was not possible to assay tannins in BMbecause of pigment interference According to Deshpande

incorpora-et al.(1982 in Maldonado et al 1995), it is clear that majoramounts of bean tannins are located in the seed coat withlower or negligible amounts in the cotyledons Tannin con-tent should be determined using another analytical methodfor 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 Trypsin inhibitors (IU g)1)

Phytate (g kg)1)

Tannin (g kg)1) Ingredients

Trang 23

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

kilo-gram 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

con-trary, 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

Nyina-wamwiza and to the FUNDP-UNR project Filie`re Clarias

We would like to acknowledge Mr Yves Beckers loux University) for gross energy determination in hislaboratory

(Gemb-Abdel-El-Samei, M.H & Lasztity, R (1984) Comparative study on the amino acids composition in three local Phaseolus vulgaris seeds varieties Z Lebensm Unters Fosch., 178, 24–26.

Aganda, A.A & Mosase, K.W (2001) Tannin content, nutritive value and dry matter digestibility of Lonchocarpus capassa, Zizyphus mucronata, Sclerocarya birrea, Kirkia acuminata and Rhus lancea seeds Anim Feed Sci Technol., 91, 107–113 Akpapunam, M.A & Sefa-Dedeh, S (1997) Some physicochemical properties and anti-nutritional factors of raw, cooked and germinated Jack bean (Canavalia ensiformis) Food Chem., 59, 121–125.

Alonso, R., Orue, E & Marzo, F (1998) Eﬀects of extrusion and conventional processing methods on protein and antinutritional factor contents in pea seeds Food Chem., 63, 505–512.

Alonso, R., Aguire, A & Marzo, F (2000) Eﬀects of extrusion and traditional processing methods on antinutrients and in vitro digestibility of protein and starch in faba and kidney beans Food Chem., 68, 159–165.

Amrish, K.T (2002) Inﬂuence of water soaking of mustard cake

on glucosinolate hydrolysis Anim Feed Sci Technol., 99, 215– 219.

AOAC (1980) Oﬃcial methods of analysis In: Association of Oﬃcial Analytical Chemists (Horwitz, W ed.), 13th edn, p 1018 Wash- ington, D.C.

Balogun, M.A & Ologhobo, D.A (1989) Growth performance and nutrient utilization of ﬁngerling Clarias gariepinus (Burchell) fed raw and cooked soy bean diets Aquaculture, 76, 119–126 Bergmeyer, H.U (1965) Methods of Enzymatic Analysis Academic Press, New York, p 555.

Brown, P.B., Strange, R.J & Robbins, K.R (1985) Protein ibility coefficients for rearing channel catfish fed high protein feedstuffs Prog Fish Cult., 47, 94–97.

digest-Burel, C., Boujard, T., Tulli, F & Kaushik, S.J (2000) Digestibility

of extrudes peas, extruded lupin, and rapeseed meal in rainbow trout (Oncorhynchus mykiss) and turbo (Psetta maxima) Aqua- culture, 188, 285–298.

Cheng, Z.J & Hardy, R.W (2003) Eﬀect of extrusion processing

of feed ingredients on apparent digestibility coeﬃcients on nutrients for rainbow trout (Oncorhynchus mykiss) Aquac Nutr.,

9, 77–83.

Cho, C.Y & Slinger, S.J (1979) Apparent digestibility measurement

in feedstuffs for rainbow trout in: Proceeding of the World posium on Finfish Nutrition and Fishfeed Technology (Harver, J & Tiews, K eds), Vol 2, pp 239–247 Heenemann, Berlin Chong, A.S.C., Hashim, R & Ali, A.B (2002) Assesment of dry matter and protein digestibilities of selected raw ingredients by discus fish (Symphysodon aequifasciata) using in vivo and in vitro methods Aquac Nutr., 8, 229–238.

Sym-Choubert, G., de la Noue, J & Luquet, P (1982) Digestibility in ﬁsh: improved device for the automatic collection of faeces Aquacul- ture, 29, 185–189.

Dagnelie, P (1975) The´orie et Me´thodes Statistiques, Vol II Presses Agronomiques de Gembloux, Belgique, pp 463.

Degani, G., Ben-Zvi, Y & Levanon, D (1989) The eﬀect of diﬀerent protein levels and temperatures on feed utilisation, growth and

.

Trang 24

body composition of Clarias gariepinus (Burchell 1822)

Aquacul-ture, 76, 293–301.

Desphande, S.S & Cheryan, M (1985) Evaluation of vanillin assays

for tannin analysis of dry beans J Food Sci., 50, 905–910.

Egounlety, M & Aworth, O.C (2003) Eﬀect of soaking, dehulling

and fermentation with Rhizopus oligosporus on the

oligo-saccharides, trypsin inhibitor, phytic acid and tannins of soybean

(Glycine max Merr.), cowpea (Vigna unguiculata L Walp) and

groundbean (Macrotyloma geocarpa Harms) J Food Eng., 56,

249–254.

Fagbenro, O.A (1996) Apparent digestibility of crude protein

and gross energy in some plant and animal-based feedstuﬀs by

Clarias isheriensis (Siluriformes: Clariidae) J Appl Ichthyol., 12,

67–68.

Fagbenro, O.A (1998) Apparent digestibility of various oilseed

cakes/meals in African catﬁsh diets Aquac Int., 6, 317–322.

Fagbenro, O.A (1999) Comparative evaluation of heat-processed

winged bean (Psophocarpus tetragonolobus) meals as partial

replacement for ﬁsh meal in diets for the African catﬁsh (Clarias

gariepinus) Aquaculture, 170, 297–305.

Folch, J., Lee, M & Sloane-Stanley, G.H (1957) A simple method

for the isolation and puriﬁcation of total lipids from animal

tissues J Biol Chem., 226, 497–509.

Fournier, V., Huelvan, C & Desbruyeres, E (2004) Incorporation of

a mixture of plant feedstuﬀs as substitute for ﬁsh meal in diets of

juvenile turbot (Psetta maxima) Aquaculture, 236, 451–465.

Francis, G., Makkar, H.P.S & Becker, K (2001) Antinutritional

factors present in plant-derived alternate ﬁsh feed ingredients and

their eﬀects in ﬁsh Aquaculture, 199, 197–227.

Furukawa, A & Tsukahara, H (1966) On the acid digestion of

chromic oxide as an index substance in the study of digestibility of

ﬁsh feed Bull Japan Soc Sci Fish., 32, 502–506.

Garg, S.K., Alok, K & Bhatnagar, A (2003) Oilcakes as protein

sources in supplementary diets for the growth of Crrhinus mrigla

(Ham) ﬁngerlings: laboratory and ﬁeld studies Bioresour

Tech-nol., 86, 283–291.

Gill, N., Higg, D.A., Skura, B.J., Rowshandeli, M., Dosanjh, B.,

Mann, J & Gannam, A.L (2006) Nutritive value of partially

dehulled and extruded sunﬂower meal for post-smolt Atlantic

salmon (Salmo salar L.) in sea water Aquac Res., 37, 1348–1359.

Goering, H.K & van Soest, P.J (1970) Forage Fiber Analyses

(Apparatus, Reagents, Procedure and Some Applications)

Agri-culture Handbook No 379ARS USDA, Washington, D.C.

Gomes, E.F., Rema, P & Kaushik, S.J (1995) Replacement of ﬁsh

meal by plant proteins in the diet of rainbow trout (Oncorhynchus

mykiss): digestibility and growth performance Aquaculture, 130,

177–186.

Helland, S., Denstadli, V., Witten, P.E., Hjelde, K., Storebakken, T.,

Skrede, A., Asgard, T & Baeverfjord, G (2006) Hyper dense

vertebrae and mineral content in Atlantic salmon (Salmo salar L.)

fed diets with graded levels of phytic acid Aquaculture, 261, 603–

614.

Hendricks, J.D (2002) Adventitious toxins In: Fish Nutrition

(Halver, J.E & Hardy, R.W eds), 3rd edn, pp 143–159 Academic

Press, London.

Hoﬀman, L.C., Prinsloo, J.F & Rukan, G (1997) Partial

replace-ment of ﬁsh meal with either soybean meal, brewers yeast or

tomato meal in the diets of African sharptooth catﬁsh Clarias

gariepinus Water S.A., 23, 181–186.

Huisman, J.P., Van der Poel, A.F.B & Liener, I.E (eds) 1989)

Recent Advances of Research in Antinutritional Factors in Legume

Seeds Pudoc, Wageningen, p 389.

Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau,

B & Laroche, M (1995) Partial or total replacement of ﬁsh meal

by soybean protein on growth, protein utilisation, potential estrogenic or antigenic eﬀects cholesterolemia and ﬂesh quality

in raimbow trout, Oncorhynchus mykiss Aquaculture, 133, 257–

274.

Kaushik, S.J., Cove`s, D., Dutto, G & Blanc, D (2004) Almost total replacement of ﬁsh meal by plant protein sources in the diet of a marine teleost, the European seabass, Dicentrarchus labrax.

Kenan, K & Yasar, O¨ (2005) Apparent digestibility of selected feed ingredients for Nile tilapia (Oreochromis niloticus) Aquaculture,

250, 308–316.

Koplik, R., Mestek, O., Kominkova, J., Borkova, M & Suchanek,

M (2004) Eﬀect of cooking on phosphorus and trace elements species in peas Food Chem., 85, 31–39.

Krogdahl, A (1989) Alternative protein sources from plants contain antinutrients aﬀecting digestion in salmonids In:

Proceedings of the Third International Symposium on Feeding and Nutrition in Fish (Takeda, M & Watanabe, T eds), pp 253–

261 Toba, Japan.

Lewis, O (1966) Short ion-exchange column method for the mation of cystine and methionine Nature, 209, 1239–1241.

esti-Lienner, I.E (1980) Toxic Constituents of Plant Foodstuﬀs, 2nd edn.

Academic Press, New York, p 502.

Maldonado, H.G., Castellanos, J & Gonzalez de Mejia, E (1995) Relationship between theoretical and experimentally detected tannin content of common beans (Phaseolus vulgaris L.) Food.

Chem., 55, 333–335.

Mambrini, M & Kaushik, S.J (1995) Indispensable amino acid requirement of ﬁsh: correspondence between quantitative data and amino acid proﬁles of tissue proteins J Appl Ichthyol., 11, 240–

247.

March, J.G., Villacampa, A.I & Grases, F (1995) spectrophotometric determination of phytic acid with phytase from Aspergillus ﬁcuum Anal Chim Acta, 300, 269–272.

Enzymatic-Masumoto, T., Ruchimat, T., Ito, Y., Hosokawa, H & Shimeno,

S (1996) Amino acid availability values for several protein sources for yellowtail (Seriola quinqueradiata) Aquaculture, 146, 109–119.

Moore, S., Spackman, D & Stein, W (1958) Chromatography of amino acid on polystyrene sulfonated resins Anal Chem., 30, 1185–1200.

Nibedita, M & Sukumar, B (2003) Extrusion cooking technology employed to reduce the anti-nutritional factor tannin in sesame (Sesamum indicum) meal J Food Eng., 56, 201–202.

Nyina-wamwiza, L., Wathelet, B & Kestemont, P (2007) Potential of local agricultural by-products for the rearing

of African catﬁsh, Clarias gariepinus in Rwanda: eﬀects on growth, feed utilization and body composition Aquac Res., 38, 206–214.

Oellermann, L.K & Hecht, T (2000) Comparison of the fillet yield, protein content and amino acid profile of Clarias gariepinus and the Clarias gariepinus · Heterobranchus longifilis hybrid Aquac.

Res., 31, 553–556.

Regost, C., Arzel, J & Kaushik, S.J (1999) Partial or total replacement of ﬁsh meal by corn gluten meal for turbot (Psetta maxima) Aquaculture, 180, 99–117.

Richter, N., Siddhuraju, P & Becker, K (2003) Evaluation of nutritional quality of moringa (Moringa oleifera Lam) leaves as an alternative protein source for Nile tilapia (Oreochromis niloticus L.) Aquaculture, 217, 599–611.

.

Trang 25

Robaina, L., Izquierdo, M.S., Moyano, F.J., Socorro, J., Vergara,

J.M., Montero, D & Fernandez Palacios, H (1995) Soybean and

lupin seed meals as protein sources in diets for gilthead seabream

(Sparus aurata): nutritional and histological implication

Aqua-culture, 130, 219–233.

Sajjadi, M & Carter, C.G (2004) Eﬀect of phytic acid and phytase

on feed intake, growth digestibility and trypsin activity in Atlantic

salmon (Salmo salar, L.) Aquac Nutr., 10, 135–142.

Sauvant, D., Perez, J.M & Tran, G (2002) Tables de composition

et de valeur nutritive des matie`res premie`res destine´es aux animaux

de´levage INRA, Paris, p 301.

Sen, M & Bhattacharyya, D.K (2000) Nutritional quality of ﬂower seed protein fraction extracted with isopropanol Plant Foods Hum Nutr., 55, 265–278.

sun-Van Weerd, J.H (1995) Nutrition and growth in Clarias species.

A review Aquat Living Resour., 8, 395–401.

Webster, C.D & Lim, C (2002) Nutrient Requirements and Feeding

of Finﬁsh for Aquaculture CABI Publishing, p 418.

Wilson, R.P (2002) Amino acids and proteins in: Fish Nutrition (Halver, J.E & Hardy, R.W eds), 3rd edn, pp 143–159 Academic Press, London.

.

Trang 26

1,2 1,3 1,4 1,5 1,6 1

1

Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN, USA; 2 Present address, Indiana

Soybean Alliance, Indianapolis, IN, USA;3 Present address, Rock Springs Farm, Delphi, IN, USA;4 Present address, Eli

Lilly and Company, Lafayette, IN, USA;5 Present address, Fernandina Beach, FL, USA;6 Present address, University of

Hawaii, Hawaii Institute of Marine Biology, KaneÕohe, HI, USA

Rapid methods of estimating dietary essential amino acid

(EAA) requirements might facilitate increases in aquaculture

production, particularly for new or emerging industries We

conducted a 12-week feeding study to test the hypothesis that

whole body EAA concentrations and the quantiﬁed

methi-onine requirement could be used to predict the remaining

dietary EAA requirements for juvenile all-female yellow

perch Six puriﬁed diets were developed and fed to triplicate

groups of ﬁsh for 12 weeks The diets contained the EAA

profile of fishmeal (FM), the profile as predicted by whole

body analysis, the quantiﬁed methionine requirement and

resulting A/E ratios (PRED), PRED + 20% of all EAA

(PRED20), PRED + 40% of all EAA (PRED40),

PRED + 20% threonine, isoleucine and tryptophan

(PRED320), and PRED + 40% threonine, isoleucine and

typtophan (PRED340) Mean weight gain and feed

con-sumption were signiﬁcantly higher in ﬁsh fed PRED20

(35.7 ± 3.2 and 55.0 ± 5.3 g, respectively) than in ﬁsh fed

FSM (25.1 ± 0.4 g wt gain, 41.0 ± 1.9 g cons), PRED

(23.4 ± 2.3 g wt gain, 40.1 ± 4.2 g cons) and PRED340

(22.9 ± 3.3 g wt gain, 35.0 ± 3.8 g cons) There was no

significant difference in feed efficiency among treatments We

recommend an EAA proﬁle similar to PRED20 for feeding

all-female juvenile yellow perch

perch

Received 23 July 2008, accepted 29 January 2009

Correspondence: P.B Brown, Purdue University, 715 West State Street,

West Lafayette, Indiana 47907-2061, USA E-mail:pb@purdue.edu

Dietary essential amino acid (EAA) requirements have beenquantified for a small percentage of fish raised in aquaculture(NRC 1993) The lack of quantified requirements poseschallenges for emerging aquaculture industries Feed accep-tance, weight gain, feed conversion ratios and other moresubtle biological responses may not be optimized when fishare fed inappropriate concentrations or ratios of EAA

Additionally, feeding excess concentrations of EAA canresult in increased ammonia excretion and degrade waterquality (Cai et al 1996; Yang et al 2002) The most commonmethod for quantifying dietary EAA requirements is theclassic dose–response approach using experimental dietscontaining graded concentrations of a speciﬁc EAA (Wilson2002); however, quantifying all 10 EAA requirements by thatmethod is time consuming and costly A more rapid method

of estimating optimal dietary EAA concentrations in dietswould be beneﬁcial, particularly for emerging aquacultureindustries

Phillips & Brockway (1956) were apparently the first topropose analysis of EAA concentrations in fish tissues as amethod of determining protein quality in feedstuffs Rumsey

& Ketola (1975) extended that reasoning and used wholebody and egg EAA concentrations to predict the ability ofvarious dietary protein sources to meet the dietary EAArequirements of trout and salmon Composite samples ofthree species of trout (rainbow, Oncorhynchus mykiss,brown, Salmo trutta and brook trout, Salvelinus fontinalis)were used along with published egg EAA concentrations forrainbow trout (Suyama & Ogino 1958) and Atlantic sal-mon, Salmo salar (Cowey et al 1962) Fish gained signiﬁ-cantly more weight when the EAA concentrations matched

2010 16; 248–253 .doi: 10.1111/j.1365-2095.2009.00659.x

.

2009 The Authors Journal compilation 2009 Blackwell Publishing LtdAquaculture Nutrition

Trang 27

the EAA concentrations in trout eggs However, the basal

diet used in that study contained 80% soybean meal, a

dietary concentration higher than that tolerated by trout

(Kaushik et al 1995) Ogata et al (1983) used the A/E ratio

(individual EAA concentration divided by the sum of all

EAA concentrations plus cystine and tyrosine) proposed by

Arai (1981) and reported improved weight gain in cherry,

Oncorhynchus masou and amago salmon, Oncorhynchus

rhodurus, fed dietary EAA concentrations that

corre-sponded to whole body EAA A/E ratios compared to ﬁsh

fed a diet matching ﬁsh meal EAA ratio Cowey & Tacon

(1983) and Wilson & Poe (1985) presented graphical

rela-tionships indicating positive correlations between whole

body EAA and quantiﬁed dietary EAA requirements in

common carp, Cyprinus carpio and channel catﬁsh, Icalurus

punctatus, respectively Correlation coeﬃcients of whole

body EAA concentrations to dietary requirements (0.96)

were higher than for egg concentrations (0.68) in channel

catﬁsh Higher correlation coeﬃcients between whole body

and dietary requirements were also reported for common

carp and coho salmon (Oncorhynchus kisutch) when

com-pared to egg EAA concentrations (see Wilson 2002)

Mambrini & Kaushik (1995) reviewed the available data

and concluded that whole body EAA concentrations were

the best choice for predicting dietary EAA needs While

whole body concentrations of EAA provide the relative

ratio of EAA to each other, they do not indicate a

quan-titative dietary concentration The ratios should be used in

conjunction with a quantiﬁed requirement of at least one

EAA to establish dietary concentrations

Ngamsnae et al (1999) used the quantiﬁed arginine (arg)

requirement to predict EAA requirements for silver perch

(Bidyanus bidyanus) They then quantiﬁed the phenylalanine

(phe) requirement and reported good agreement between the

predicted phe requirement and the requirement quantiﬁed in

the separate growth study Twibell et al (2003) reported

whole body concentrations of EAA for hybrid striped bass

Morone chrysops· Morone saxatillis and, using the

quanti-ﬁed dietary requirement for lysine, developed and tested

puriﬁed diets containing varying concentrations of EAA

Fish fed the experimental diet containing 120% of the

pre-dicted EAA concentrations plus additional threonine (thr),

isoleucine (ile) and tryptophan (trp) gained more weight than

fish fed EAA profiles matching fish meal or 120% of the

predicted values Thr, ile and trp are turned over at a faster

rate than lys in some terrestrial vertebrates (Baker 1991;

Adeola 1998); thus, the quantiﬁed EAA used to predict

the other dietary EAA concentrations is an important

All-female yellow perch juveniles were obtained from acommercial producer (Coolwater Farms, LLC, Cambridge,

WI, USA) and transported to the Purdue University culture Research Facility All ﬁsh were quarantined for twomonths prior to experimentation Fish were maintained at

Aqua-22C during quarantine and fed a salmonid diet (Nelson andSons, Inc., Murray, UT, USA) to apparent satiation twiceper day Transport, quarantine and experimental proce-dures followed Purdue Animal Care and Use Committeerequirements

A closed recirculating system was used for the feeding trial.The system contained individual aquaria Each aquariumheld 76 L of water The system was equipped with foursubmerged filters and one biofilter for solids removal andoxidation of nitrogenous wastes, respectively Water waspumped from the biofilter through a sand filter for secondarysolids removal Flow rate to individual tanks was

1 L min)1 The diurnal light/dark cycle was 16 h light/8 hdark Water temperature was maintained at 22 ± 1C.Groups of 12 ﬁsh were randomly stocked into 18 aquaria.Fish were allowed to acclimatize to the new system and theirrespective cohorts for 1 week, during which they were fed acommercial salmonid diet (Nelson and Sons, Inc., Murray,

UT, USA) Experimental diets were assigned to triplicategroups of ﬁsh and replicate groups were fed their respectivediets for an additional week All ﬁsh were fed twice per day toapparent satiation during acclimatization to the new systemand diets Following the acclimatization period, the number

of ﬁsh was reduced to 10 Average individual ﬁsh weightranged from 25.8 to 26.8 g Fish were fed their respectiveexperimental diets to apparent satiation twice daily for the12-week experimental period Feed consumption was deter-mined daily by weighing feed containers designated for eachreplicate, feeding and then reweighing the container.Water quality was monitored daily and was similar topreviously reported studies with yellow perch (Twibell &Brown 1997) Dissolved oxygen concentrations were

‡7.2 mg L)1throughout the feeding trial Ammonia-N andnitrite-N did not exceed 0.63 and 0.076 mg L)1, respectively. .

Trang 28

At the end of the study, all ﬁsh were fasted for 24 h.

Surviving ﬁsh within each replicate group were counted and

weighed in a tared vessel

During the quarantine period, a composite sample of six

perch from the initial group was fasted for 24 h, sampled and

prepared for EAA analysis All ﬁsh were pooled, ground and

dried at 100C Duplicate analysis of EAA was conducted

on dried samples by a commercial laboratory (New Jersey

Feed Labs, Trenton, NJ, USA) using standard methods

(AOAC 2003) Results of that analysis were used to establish

A/E ratios (Table 1)

Vitamins, casein, gelatin, dextrin, carboxymethylcellulose

(CMC), crystallineL-amino acids and cellulose were obtained

from US Biochemical (Cleveland, OH, USA) Menhaden oil,

ascorbic acid and reagent grade minerals were obtained from

Omega Protein (Reedville, VA, USA), Roche Inc (Nutley,

NJ, USA) and Sigma Chemical (St Louis, MO, USA),

respectively Choline chloride and soybean lecithin were

obtained from Bio-Serv (Frenchtown, NJ, USA)

Six puriﬁed diets (Brown et al 1996) were formulated to

supply 330 g protein kg diet)1(Table 2) Diet one (FM) was

formulated to mimic the EAA A/E proﬁle of herring ﬁsh

meal (NRC 1993) Diet two (PRED) contained the EAA

proﬁle as predicted by whole body analysis of EAA, the

quantiﬁed requirement for met (Twibell et al 2000) and the

calculated A/E ratios for the remaining EAA Diets 3 and 4

contained the predicted values, described above, plus an

additional 20% (PRED20), or 40% (PRED40) of all EAA

Diets 5 and 6 contained the predicted values from above plus

an additional 20 (PRED320) or 40% (PRED340) of thr, ile

and trp Availability of all amino acids was assumed to be

92% The non-essential amino acid (NEAA) premix wasadjusted to maintain isonitrogenous diets Diets contained

120 g lipid (menhaden oil) kg diet)1and 150 g carbohydrate(dextrin) kg diet)1 Nutritionally complete vitamin andmineral premixes were added to all diets (Twibell et al 2000)

Diets were mixed, neutralized, pelleted, dried and storedusing previously reported methods (Twibell et al 2000)

Mean weight gain, feed consumption, feed efficiency andsurvival were analyzed as a completely randomized designusing individual tanks as an experimental unit Analyses wereconducted using one-way ANOVA (Rao 1998) with the Sta-tistical Analysis System, version 8.2 (SAS Institute Inc.,Cary, NC, USA) Student–Newman–Keuls test was used toseparate significant differences in mean values Significantdifference was accepted at P < 0.05

The mean lysine concentration in whole perch was higherthan any other EAA (Table 1), followed by leucine andarginine Concentrations of tryptophan and methionine werethe lowest EAA concentrations

Mean weight gain and feed consumption were significantlydifferent between treatments (Table 3) Fish fed PRED20exhibited significantly higher weight gain than fish fed FM,PRED or PRED340 There were no significant differences inweight gain between fish fed PRED20, PRED40 andPRED320 There were no significant differences in weightgain between fish fed PRED40, PRED320, FM, PRED andPRED340 Mean consumption of PRED20 was significantlyhigher than consumption of PRED340 There were no sig-nificant differences in consumption of experimental diets byperch fed PRED20, PRED40, PRED320, FM and PRED

There were no signiﬁcant diﬀerences in consumption byperch fed PRED40, PRED320, FM, PRED and PRED340

There were no significant differences in feed efficiency amongtreatments with all values ranging from 0.58 to 0.65 Survival

of fish did not differ among treatments and only four fishdied during the course of the 12-week study

The results of this study indicate that whole body EAAconcentrations and resulting A/E ratios in conjunction withthe quantiﬁed requirement for met can be used to predict adietary EAA proﬁle appropriate for juvenile yellow perch

Table 1 Yellow perch whole body amino acid concentrations

(g kg)1), A/E ratio of each amino acid to methionine and the

cor-responding predicted dietary concentration (g kg)1)

Amino acid

Carcass concentration

Ratio

to Met

Dietary level

Trang 29

However, this is the second report indicating that some

die-tary EAA concentrations may be underestimated by this

method Twibell et al (2003) reported that the arg

require-ment was underestimated by using whole body EAA

con-centrations and the quantiﬁed lys requirement for hybrid

striped bass and found that additional thr, ile and trp werebeneﬁcial With yellow perch, an additional 20% of all EAAwas beneﬁcial and there is no clear indication which onesmay have been underestimated Supplemental thr, ile and trp

at 20 and 40% above predicted concentrations did not

Table 3 Mean (±SEM) weight gain (g),

consumption (g), feed efﬁciency (g gain/

g consumed) and survival (%) of

all-female yellow perch fed puriﬁed diets

Table 2 Composition of diets (g kg )

fed to juvenile all-female yellow perch.

Diets contained the essential amino acid

(EAA) proﬁle of herring ﬁsh meal

(FSM), the predicted EAA

concentra-tions based on whole body EAA A/E

ratios and a quantiﬁed methionine

requirement (PRED), PRED plus an

1 EAA premix 1 contained (g 100 g)1 premix): 15.6 L -lysine-HCI, 13.6 L -arginine – HCI, 11.9 L -leucine, 7.3 L -isoleucine, 10.1 L -valine, 5.3 L -methionine, 2.3 L -cyst(e)ine, 6.2 L -phenylalanine, 4.5 L -tyrosine, 1.9 L -trypotophan, 3.9 L -histidine, 7.2 L -threonine, 10.2 cellulose.

2 EAA premix 2 contained (g 100 g)1premix): 15.9 L -lysine-HCI, 13.2 L -arginine-HCI, 9.9 L -leucine, 6.2 L -isoleucine, 6.4 L -valine, 3.6 L -methionine, 1.2 L -cyct(e)ine, 6.4 L -phenylalanine, 4.5 L -tyrosine, 1.6 L -tryptophan, 4.4 L -histidine, 6.8 L -threonine, 19.9 cellulose.

3 EAA premix 3 contained (g 100 g)1premix): 10.1 L -lysine-HCI, 8.0 L -arginine-HCI, 6.5 L -leucine, 4.0 L -isoleucine, 4.3 L -valine, 2.4 L -methionine, 0.7 L -cyst(e)ine, 4.1 L -phenylalanine, 3.0 L -tyrosine, 1.0 L -tryptophan, 2.8 L -histidine, 4.3 L -threonine, 48.8 cellulose.

4 EAA premix 4 contained (g 100 g)1premix): 12.3 L -lysine-HCI, 9.5 L -arginine-HCI, 9.1 L -leucine, 4.2 L -isoleucine, 5.3 L -valine, 2.9 L -methionine, 0.8 L -cyst(e)ine, 5.1 L- phenylalanine, 3.4 L- tyrosine, 1.2 L- tryptophan, 3.4 L- histidine, 5.3 L- threonine, 37.5 cellulose.

5 NEAA premix contained (g 100 g)1 premix): 16.7 L- glycine, 16.7 L- alanine, 16.7 L- serine, 16.7 L- aspartic acid, 16.7 L- glutamic acid, 16.7 L- proline.

6 Mineral and vitamin premixes were the same as reported by Twibell et al (2000).

7 Contained 350 g kg)1ascorbic acid equivalents (Roche Inc., Nutley, NJ, USA).

8 Soybean lecithin contained 700 g kg)1 phosphatidylcholine and 120 g kg)1 dylcholine (Bio-Serv, Frenchtown, NJ, USA).

lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati- lysophosphati-.

Trang 30

improve responses The predicted arg requirement (1.8%) is

higher than the quantiﬁed requirement of 1.4% from growth

studies (Twibell & Brown 1997) Thus, while several authors

have argued that EAA concentrations of whole ﬁsh are

similar (Gatlin 1987; Ng & Hung 1994; Ramseyer & Garling

1994; Kaushik 1998; Portz & Cyrino 2003; Gurure et al

2007), taking the next step and predicting dietary EAA

requirements apparently does not account for some key

factor(s) Assumed availability values may be inﬂuencing the

challenges in predicting dietary needs Endogenous turnover

of EAA may also be impacting extrapolation from whole

body EAA concentrations to dietary EAA concentrations

(Baker 1991)

To date, the quantiﬁed arg requirement has been used to

predict the phe requirement of silver perch (Ngamsnae et al

1999), the lys requirement has been used to predict the

remaining nine dietary EAA requirements for hybrid striped

bass (Twibell et al 2003), and the met requirement has been

used to predict the remaining nine EAA requirements for

yellow perch There is no clear indication which initial

quantiﬁed dietary EAA requirement is best for predicting the

remaining dietary EAA concentrations However, this

method assumes similar endogenous turnover of the

quan-tiﬁed EAA relative to the remaining predicted EAA

Knowledge of EAA turnover in ﬁshes would be a valuable

piece of information for extending this approach

Weight gain of yellow perch fed PRED20 was signiﬁcantly

higher than ﬁsh fed FSM This is the second report (Twibell

et al.2003) in which predicted dietary EAA concentrations

from whole body EAA concentrations resulted in higher

weight gain than ﬁsh fed a ﬁsh meal control pattern of EAA

The speciﬁc reasons for this improvement are not clear, but

indicate that the ﬁsh meal pattern is not optimal for either

hybrid striped bass or yellow perch Modiﬁcation of the

predicted concentrations that resulted in higher weight gain

was diﬀerent for the two species Supplemental thr, ile and

trp were necessary for hybrid striped bass, which may reﬂect

a similar situation identiﬁed in poultry (Baker 1991) with

respect to endogenous turnover relative to lys In yellow

perch, supplemental EAA at 20% over predicted values was

necessary, yet the predicted dietary EAA concentrations were

in excess of quantiﬁed dietary EAA requirements for arg, and

supplemental thr, ile and trp were not beneﬁcial Comparison

of values from Table 1, expressed as a percentage of dietary

crude protein, to known dietary EAA requirements for ﬁsh

(Wilson 2002) indicate that all are above or within the range

reported for ﬁshes, with the exception of phe A focussed

eﬀort on aromatic EAA requirements in perch might be

beneﬁcial to further reﬁne this approach

Perch fed PRED20 consumed significantly more feed thanthose fed PRED340 Numerically, they also consumed morethan the other treatments, and over the course of a grow-outcycle a significant difference may be manifested The reasonfor this difference is unknown, but may be associated with adifference in flavour between the diets Amino acids can act

as feed attractants resulting in increased consumption(Harada 1989) Mixtures of various amino acids and otherﬂavour additives may elicit higher feed intake than individualamino acids (Takaoka et al 1995; Papatryphon & Soares2000) In previous work with yellow perch, ﬂavour additivesthat contained mixtures of various compounds elicited higherconsumption than a control (Gould et al 2003) Anotherpossible reason for the higher consumption of PRED20 isthat optimal dietary EAA balance impacts consumption de

la Higuera et al (1999) reported increased consumption inEuropean eel (Anguilla anguilla) when an EAA-deficient dietwas supplemented with the deficient EAA However, it is notpossible to distinguish the effects of EAA deficiency fromflavour of their experimental diet with and without supple-mental amino acids

Using the quantiﬁed dietary requirement for yellow perchand calculating A/E ratios from whole bodies, an optimalEAA pattern was developed Supplementation of all EAA by20% over predicted values was necessary to achieve maxi-mum response, but additional thr, ile and trp were appar-ently not necessary These data will be useful for formulatingdiets for this emerging aquaculture species

This research was partially supported by the AgriculturalResearch Program, Purdue University (IND 059054)

Adeola, O (1998) Bioavailability of tryptophan in soybean meal and trypotophan retention in the carcasses of four-week-old ducks.

Cai, T., Wermerskirchen, J & Adelman, I.R (1996) Ammonia excretion rate indicates dietary protein adequacy for ﬁsh Prog.

Fish-Cult., 58, 124–127.

.

Trang 31

Cowey, C.B & Tacon, A.G.J (1983) Fish nutrition – relevance to

invertebrates in: Proceedings of the Second International

Confer-ence on Aquaculture Nutrition: Biochemical and Physiological

Approaches to Shellﬁsh Nutrition (Pruder, G.D., Landgon, C &

Conklin, D eds), Special publication number 2, pp 13–30

Loui-siana State University, Baton Rouge, LA, USA.

Cowey, C.B., Daisley, K.W & Parry, G (1962) Study of amino

acids, free or as components of protein, and some B vitamins in the

tissues of the Atlantic salmon, Salmo salar, during spawning

migration Comp Biochem Physiol., 7, 29–38.

Gatlin, D.M III (1987) Whole-body amino acid composition and

comparative aspects of amino acid nutrition of the goldﬁsh, golden

shiner and fathead minnow Aquaculture, 60, 223–229.

Gould, N.L., Glover, M.M., Davidson, L.D & Brown, P.B (2003)

Dietary ﬂavor additives inﬂuence consumption of feeds by yellow

perch Perca ﬂavescens J World Aquacult Soc., 34, 412–417.

Gurure, R., Atkinson, J & Moccia, R.D (2007) Amino acid

com-position of Arctic charr, Salvelinus alpinus (L.) and the prediction

of dietary requirements for essential amino acids Aquacult Nutr.,

13, 266–272.

Harada, K (1989) Feeding attraction activities of L -dipeptides for

abalone, oriental weatherﬁsh, and yellowtail Bull Jap Soc Sci.

Fish., 55, 1629–1634.

de la Higuera, M., Akharbach, H., Hidalgo, M.C., Peragon, J.,

Lupianez, J.A & Garcia-Gallego, M (1999) Liver and white

muscle protein turnover rates in the European eel (Anguilla

anguilla): eﬀects of dietary protein quality Aquaculture, 179, 203–

216.

Kaushik, S.J (1998) Whole body amino acid composition of

Euro-pean seabass (Dicentrarchus labrax), gilthead seabream (Sparus

aurata) and turbot (Psetta maxima) with an estimation of their

IAA requirement proﬁles Aquat Liv Res., 11, 355–358.

Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau,

B & Laroche, M (1995) Partial or total replacement of ﬁsh meal

by soybean protein on growth, protein utilization, potential

estrogenic or antigenic eﬀects, cholesterolemia and ﬂesh quality in

rainbow trout, Onchorhynchus mykiss Aquaculture, 133, 257–274.

Mambrini, M & Kaushik, S.J (1995) Indispensible amino acid

requirements of ﬁsh: correspondence between quantitative data

and amino acid proﬁles of tissue proteins J Appl Ichthyol., 11,

240–247.

Ng, W.K & Hung, S.S.O (1994) Amino acid composition of whole

body, egg and selected tissues of white sturgeon (Acipenser

trans-montanus) Aquaculture, 126, 329–339.

Ngamsnae, P., de Silva, S.S & Gunasekera, R.M (1999) Arginine

and phenylalanine requirement of juvenile silver perch Bidyanus

bidyanus and validation of the use of body amino acid composition

for estimating individual amino acid requirements Aquacult.

Papatryphon, E & Soares, J.H Jr (2000) Identiﬁcation of feeding stimulants for striped bass Morone saxatillis Aquaculture, 185, 339–352.

Phillips, A.M & Brockway, D.R (1956) The nutrition of trout II Protein and carbohydrate Prog Fish-Cult., 18, 159–164 Portz, L & Cyrino, J.E.P (2003) Comparison of the amino acid contents of roe, whole body and muscle tissue and their A/E ratios for largemouth bass Micropterus salmoides (Lacepede, 1802) Aquacult Res., 34, 585–592.

Ramseyer, L.J & Garling, D.L Jr (1994) Amino acid composition of the ovaries, muscle, and whole body of yellow perch Prog Fish- Cult., 56, 175–179.

Rao, P.V (1998) Statistical Research Methods in the Life Sciences Brooks/Cole Publishing Company, Paciﬁc Grove, CA, USA Rumsey, G.L & Ketola, H.G (1975) Amino acid supplementation

of casein in diets of Atlantic salmon (Salmo salar) fry and of soybean meal for rainbow trout (Salmo gairdneri) ﬁngerlings.

J Fish Res Board Can., 32, 422–426.

Suyama, M & Ogino, C (1958) Changes in chemical compositions during development of rainbow trout eggs Bull Jap Soc Sci Fish., 23, 785–788.

Takaoka, O., Takii, K., Nakamura, M., Kumai, H & Takeda, M (1995) Identiﬁcation of feeding stimulants for tiger puﬀer Fisheries Science, 61, 833–836.

Twibell, R.G & Brown, P.B (1997) Dietary arginine requirement of juvenile yellow perch J Nutr., 127, 1838–1841.

Twibell, R.G., Wilson, K.A & Brown, P.B (2000) Dietary sulfur amino acid requirement of juvenile yellow perch fed the maximum cystine replacement value for methionine J Nutr., 130, 612–616 Twibell, R.G., Grifﬁn, M.E., Martin, B., Price, J & Brown, P.B (2003) Predicting dietary essential amino acid requirements for hybrid striped bass Aquacult Nutr., 9, 373–381.

Wilson, R.P (2002) Amino acids and protein In: Fish Nutrition (Halver, J.E & Hardy, R.W eds), 3rd edn, pp 144–181 Academic Press Inc., New York, NY, USA.

Wilson, R.P & Poe, W.E (1985) Relationship of whole body and egg essential amino acid patterns to amino acid requirement patterns in channel catﬁsh Ictalurus punctatus Comp Biochem Physiol., 80B, 385–388.

Yang, S., Liou, C & Liu, F (2002) Eﬀects of dietary protein level on growth performance, carcass composition and ammonia excretion

in juvenile silver perch Bidyanus bidyanus Aquaculture, 213, 363– 372.

.

Trang 32

1 1 1,2 1 1,2

1

Institute of Animal Nutrition, Sichuan Agricultural University, Sichuan, Yaan, China, 2 Engineering Research Center for

Animal Desease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Yaan, China

This experiment was conducted to evaluate the eﬀects of

die-tary pyridoxine on disease resistance, immune responses and

intestinal microﬂora of ﬁsh A total of 1050 Jian carp

(11.71 ± 0.05 g) were randomly distributed into seven

groups, feeding diets containing graded levels of pyridoxine

(0.2, 1.7, 3.2, 5.0, 6.3, 8.6 and 12.4 mg kg)1diet) After 80 days

of feeding, a challenge trial was conducted by injection of

Aeromonas hydrophilafor 17 days Results indicated that with

increasing dietary pyridoxine concentration up to 5.0 mg kg)1

diet, survival rate after challenge with A hydrophila and

phagocytic activity of leukocyte were improved (P < 0.05),

and plateaued thereafter (P > 0.05) Red blood cell and white

blood cell counts were lowest when fed the diet containing

1.7 mg pyridoxine kg)1diet Haemagglutination titre,

lyso-zyme activity, acid phosphatase activity, total iron-binding

capacity, antibody titre and immunoglobulin M content

fol-lowed the similar pattern to that observed with survival rate

Aeromonas hydrophila, Escherichia coli and Lactobacillus

counts in intestine were not aﬀected by dietary pyridoxine

concentration (P > 0.05) These results suggested that

pyri-doxine could enhance immune response of ﬁsh

microﬂora, Jian carp, pyridoxine

Received 11 August 2008, accepted 29 January 2009

Correspondence: Xiao-Qiu Zhou, Institute of Animal Nutrition, Sichuan

Agricultural University, Yaan 625014, China E-mail: zhouxq@sicau.

edu.cn

Pyridoxine has been demonstrated to be an essential dietary

nutrient for normal growth of ﬁsh as it participates in the

metabolism of proteins and amino acids in the form ofprosthetic group of enzymes (Ogino 1965; Shiau & Wu 2003)

Previous article shows that pyridoxine can improve growthperformance and enhance amylase, Na+, K+-ATPase,Gamma-glutamyl transpeptidase (c-GT) and alkaline phos-phatase activities in intestine of juvenile Jian carp (He et al

2008, in press) However, the relationship between ine and immune responses of carp remains unknown

pyridox-Nutritional status is an important factor inﬂuencingimmune defence of animals (Chen et al 2005) Vitamins Cand E can enhance disease resistance and immune responses

of ﬁsh (Hardie et al 1991; Anbarasu & Chandran 2001;

Wang et al 2006a) However, limited information is able on effect of dietary pyridoxine concentration related todisease resistance and immune responses in fish ThoughHardy et al (1979) found a protective effect of dietary vita-min B6 on chinook salmon challenged with a virulent strain

avail-of Vibrio anguillarum, resistance to furunculosis avail-of Atlanticsalmon following immunization with Aeromonas salmonicidawas not influenced by dietary pyridoxine concentration(Albrektsen et al 1995) Granulocytes and monocytes/mac-rophages are key cells involved in part of non-specificimmune responses in fish (Dalmo et al 1997), while non-specific immune responses system contains various defencehumeral components such as agglutinin, lysozyme and metalion binding proteins (Dalmo et al 1997; Sakai 1999) Pyri-doxine plays an important role in modulating the non-specific immune responses of abalone, Haliotis discus hannaiIno (Chen et al 2005) However, immune responses ofAtlantic salmon following immunization with Vibrio sal-monicida were not influenced by dietary pyridoxine concen-tration (Albrektsen et al 1995) It should be noted that thediet of Atlantic salmon was a practical diet (containing 70%

ﬁsh meal) which might contain high pyridoxine tion and achieve requirement for optimal immune responses,

concentra-2010 16; 254–261 .doi: 10.1111/j.1365-2095.2009.00660.x

.

Trang 33

while the diet of abalone was a puriﬁed diet (Albrektsen et al.

1995; Chen et al 2005)

Eﬀect of nutrient on intestinal microﬂora is also valuable to

investigate as intestine is a barrier for microbial invasion

(Dalmo et al 1997), and intestinal microﬂora is sensitive

to dietary changes (Ringø & Olsen 1999)

Mannanoligo-saccharide and methionine could promote the growth and

reproduction of beneﬁcial bacteria and depress harmful

intestinal bacteria in juvenile Jian carp (Zhou & Li 2004; Tang

et al.2008, in press) However, there are few reports on the

effect of dietary pyridoxine on intestinal microflora in fish

The present study was part of a larger study which

involved the determination of the pyridoxine requirement

(He et al 2008, in press) and explores the relationship

between dietary pyridoxine concentration and disease

resis-tance, immune responses and intestinal microﬂora of juvenile

Jian carp

The basal diet was formulated to contain approximately

308.1 g crude protein kg)1 diet and 48.6 g crude lipid kg)1

diet (Table 1) Casein, gelatin, ﬁsh meal and soy protein

concentrate were used as dietary protein sources; ﬁsh oil and

soybean oil, corn starch and a-starch were used as dietary

lipid and carbohydrate sources, respectively The basal diet

contains 0.6 mg pyridoxine kg)1diet which was calculated

according to the analysed pyridoxine concentration of

ingredients Pyridoxine hydrochloride (PN, Sigma, St Louis,

MO, USA) was added to the test diets to provide graded

concentration The analysed pyridoxine concentrations of the

seven diets were 0.2 (unsupplemented), 1.7, 3.2, 5.0, 6.3, 8.6

and 12.4 mg kg)1diet After being prepared completely, the

diets were air-dried at room temperature and stored at

)20 C until used

Juvenile Jian carp (Cyprinus carpio var Jian) obtained from

the Yaan ﬁsheries were used in this experiment and

accli-matized to laboratory conditions and fed the pyridoxine-free

basal diet (Table 1) for 4 weeks At the end of the

acclima-tization period, a total of 1050 Jian carp (11.71 ± 0.05 g)

were randomly distributed into each of 21 glass aquaria

(90 cm W· 30 cm L · 40 cm H) Each aquarium was

ran-domly assigned to one of three replicates of the seven dietary

treatments The aquarium was connected to a closed water

and oxygen auto-supplementing system Water exchangerates in each aquarium were maintained at 1.2 L min)1andthe water was drained through bioﬁlters to remove solidsubstances and reduce ammonia concentration The watertemperature and pH were 24 ± 1C and 7.0 ± 0.3,respectively The experimental units were under natural lightand dark cycle

After 80 days of feeding, 10 ﬁsh with similar body weightobtained from each aquarium were moved to the respectivelylabelled new tank and acclimatized to the experimentalcondition for 5 days Fish were infected by intraperitoneal

Table 1 Composition and nutrients content of experimental diets

Ingredients

Diets (g kg)1, dry diet)

2 Per kilogram of pyridoxine-free vitamin premix (g kg)1): retinyl acetate (500 000 IU g)1), 0.800 g; cholecalciferol (500 000 IU g)1), 0.480 g; DL -a-tocopherol acetate (50%), 20.00 g; menadione (50%), 0.200 g; thiamin nitrate (98%), 0.063 g; riboflavine (80%), 0.875 g; cyanocobalamin (10%), 0.010 g; ascorbyl acetate (92%), 7.247 g; calcium- D -pantothenate (98%), 3.337 g; niacin (98%), 2.857 g; D -biotin (20%), 0.500 g; meso-inositol (98%), 44.898 g; folic acid (96%), 0.521 g All ingredients were diluted with corn starch to 1 kg.

3 Per kilogram of pyridoxine hydrochloride premix (g kg)1): each treatment group containing pyridoxine hydrochloride 0, 0.0579, 0.1406, 0.2233, 0.3059, 0.3886 and 0.5954 g, respectively Each pyridoxine hydrochloride mixture was diluted with corn starch to

1 kg.

.

Trang 34

injection with A hydrophila (A hydrophila strain, which was

obtained from Huazhong Agricultural University in China),

by the modiﬁed method of Yang et al (2008) The dose is

concentration of 1010cfu 0.5 mL ﬁsh)1, which was enough to

activating the immune system and consequently enable the

investigation of eﬄuent on reactivity against a threatening

disease according to our preliminary study data (Yang et al

2008) The challenge trial was conducted for 17 days at the

time antibody content was highest according to data (J Jiang

& X.-Q Zhou, unpublished data) in our laboratory and

experiment conditions were the same to feeding trial except

that water temperature was maintained at 25 ± 1C

At the end of the feeding trial, 15 ﬁsh were collected from

each aquarium Head kidney and spleen were removed and

weighed Blood was collected from caudal vein by syringe

with heparin as anticoagulant from ﬁve ﬁsh of each aquarium

for determining red blood cell (RBC) count, white blood cell

(WBC) count and phagocytic activity (PA) of leukocytes Six

hours after last feeding, nine ﬁsh collected from each

aquarium were anaesthetized with benzocaine (50 mg L)1),

then the ventral belly surface of ﬁsh was opened and intestine

were sampled into sterile containers by sterile knife for

enu-meration of intestinal microﬂora

The number of dead ﬁsh was recorded during challenge

trial to calculate survival rate At the end of the challenge

trial, ﬁve ﬁsh per aquarium were anaesthetized with

benzocaine (50 mg L)1) after 12 h of the last feeding Blood

was collected from caudal vein of carps, stored at 4C

overnight and centrifuged at 3000 g for 10 min, then stored

at)20 C until it was analysed for immune parameters

Red blood cell and WBC count were measured ing to the method of Yang et al (2008) PA of leukocyteswas determined by a modiﬁed method described by Kim &

accord-Austin (2006) Serum agglutination activity was determinedusing haemagglutination assay which was modiﬁed fromBarracco et al (1999) and Sritunyalucksana et al (1999)

Serial two-fold dilutions of serum following immunizationfrom all groups were diluted using phosphate buﬀeredsaline (PBS, pH 7.2) in U-shaped bottom microtitrewells to which an equal volume of freshly prepared 2%

erythrocyte suspension (rabbit in PBS) was added andincubated for 2 h at 25C Titres were recorded as thereciprocal of the highest dilution showing agglutination

Lysozyme activity (LA) of the serum following zation was measured by the method of Ellis (1990) Acidphosphatase activity (ACP) of the serum following immu-nization was spectrophotometrically measured according toPipe (1990) Total iron-binding capacity (TIBC) wasdetermined by the method of Soldin et al (2004) Anti-body titre (Ab titre) was determined using a microagglu-tination test by the method of Vivas et al (2004)

immuni-Immunoglobulin M (IgM) level was determined by themethod of Takemura (1993) Intestinal microﬂora wasenumerated using the pour plate method of Spanggaard

et al (2002) Pyridoxine concentration in ingredients anddiets were analysed by microbiological assay procedure(AOAC 2000)

Data on total number of dead ﬁsh in each aquarium duringthe challenge trial, head kidney weight and spleen weightwere used to calculate:

Survival rate¼ 100 1 Total no of dead fish in each aquarium during the challenge trial

No of fish in each aquarium

Head kidney index (HKI, &Þ ¼The head kidney weight 1000

Wet body weight

Spleen index (SI, &Þ ¼The spleen weight 1000

Wet body weight

Phagocytic activity of leukocyte¼No of leukocyte phagocytized E:coli commune 100

Total no of leukocyte .

Trang 35

All data were subjected to one-way analysis of variance

sig-niﬁcant diﬀerences among treatment groups, and probability

value P < 0.05 indicates a signiﬁcant diﬀerence

The mortality rose to 79.3% on the 60th day in the plemented treatment group (0.2 mg pyridoxine kg)1diet) andthere was no survival ﬁsh at the end of 80 days feeding trial.The remaining treatments readily accepted the test diets andtheir growth response ranged from 454 to 527% increase inbody weight at the end of the feeding trial (He et al 2008,

unsup-in press)

Survival rate of juvenile Jian carp challenged with

A hydrophila is presented in Fig 1 Results indicated thatvaccinated survival rate in low pyridoxine concentrationtreatment group (1.7 and 3.2 mg kg)1diet) were signiﬁcantlylower than that in treatment groups fed the diet containing

‡6.3 mg kg)1diet (P < 0.05)

Head kidney index and SI of juvenile Jian carp fed thediets containing varying levels of pyridoxine are presented inTable 2 Results indicated that the HKI in treatment groupfed the diet containing 1.7 and 2.3 mg kg)1diet were higherthan that in treatment groups fed the diet containing

‡5.0 mg kg)1diet (P < 0.05) With increasing dietary doxine levels up to 5.0 mg kg)1 diet, SI was improved(P < 0.05), and no diﬀerences were found with furtherincrease of pyridoxine levels (P > 0.05)

pyri-Red blood cell counts, WBC counts and PA of leukocyteare presented in Table 3 Results indicated that RBC in the

a

bc c

0 20

Figure 1 Survival rate (%) of juvenile Jian carp (Cyprinus carpio var.

Jian) challenged with Aeromonas hydrophila and fed diets containing

graded levels of pyridoxine (PN, mg kg)1) Values are means ± SD

of three groups of ﬁsh (n=3) Values with diﬀerent superscipt letters

are signiﬁcantly diﬀerent (P < 0.05).

Table 2 Head kidney index (HKI, &)

and spleen index (SI, &) of juvenile Jian

carp (Cyprinus carpio var Jian) fed diets

containing graded levels of pyridoxine

Table 3 Red blood cell (RBC, 1012L)1),

white blood cell (WBC, 1010L)1) and

phagocytic activity (PA) of leukocyte

(%) of juvenile Jian carp (Cyprinus

car-pio var Jian) fed diets containing graded

levels of pyridoxine (PN, mg kg)1)

RBC 1.9 ± 0.0c 2.3 ± 0.0a 2.3 ± 0.0a 2.2 ± 0.0ab 2.2 ± 0.0ab 2.2 ± 0.1bWBC 1.7 ± 0.0d 3.1 ± 0.1c 3.9 ± 0.2b 4.2 ± 0.0a 4.1 ± 0.1a 4.1 ± 0.1a

PA 13.3 ± 3.3c 28.3 ± 2.9b 33.3 ± 1.7a 35.0 ± 1.7a 34.4 ± 1.0a 35.6 ± 1.0aValues are means ± SD of three groups of fish (n = 3) Values within the same row having different superscript alphabets are significantly different (P < 0.05).

Table 4 Non-speciﬁc immunity

param-eters of juvenile Jian carp (Cyprinus

carpio var Jian) challenged with

Aero-monas hydrophila and fed diets

contain-ing graded levels of pyridoxine (PN,

Trang 36

lowest pyridoxine concentration treatment group

(1.7 mg kg)1 diet) was lower than that in other treatment

groups (P < 0.05), and high pyridoxine concentration

treatment group (12.4 mg kg)1diet) was lower than 3.2 and

5.0 mg kg)1diet groups (P < 0.05) With increasing dietary

pyridoxine levels up to 6.3 mg kg)1diet, WBC was improved

(P < 0.05), and no diﬀerences were found with further

increase of pyridoxine levels (P > 0.05) PA of leukocyte was

increased with increasing pyridoxine levels up to 5.0 mg kg)1

diet (P < 0.05), and no diﬀerences were found with further

increase of pyridoxine levels (P > 0.05)

Haemagglutination titre (HA), LA, ACP and TIBC of

juvenile Jian carp challenged with A hydrophila are presented

in Table 4 Results indicated that with increasing dietary

pyridoxine levels up to 5.0 mg kg)1diet, HA, LA, ACP and

TIBC were improved (P < 0.05), and no diﬀerences were

found with further increase of pyridoxine levels (P > 0.05)

Antibody titre and IgM content of juvenile Jian carp

challenged with A hydrophila are presented in Table 5

Results indicated that with increasing dietary pyridoxine

levels up to 5.0 mg kg)1diet, Ab titre and IgM content got to

maximum, and no diﬀerences were found with further

increase of pyridoxine levels (P > 0.05)

Aeromonas hydrophila, E coli and Lactobacillus counts in

whole intestine of juvenile Jian carp are presented in Table 6

Results showed that no diﬀerences were found for

A hydrophila, E coli and Lactobacillus counts in whole

intestine among all treatment groups (P > 0.05)

Survival rate, especially after challenge may reﬂect disease

resistance of ﬁsh (Erdal et al 1991) Our study showed that

with increasing dietary pyridoxine concentration up to6.3 mg kg)1 diet, survival rate was improved Hardy et al

(1979) also found protective eﬀect of dietary vitamin B6

on chinook salmon challenged with a virulent strain of

V anguillarum However, resistance to furunculosis ofatlantic salmon following being challenged with A salmoni-cidawas not inﬂuenced by dietary pyridoxine concentration(Albrektsen et al 1995)

Head kidney and spleen are important haemopoietic tissuesfor ﬁsh (Agrawal & Mahajan 1983) The present results indi-cate that supplement of adequate pyridoxine concentrationcan promote growth of spleen HKI, in this study, declinedwith increasing dietary pyridoxine levels in treatment groupsfed the£5.0 mg kg)1diet, which may be explained by the factthat common carp was oedema when the intake was inade-quate pyridoxine (Ogino 1965) Why spleen did not follow asimilar pattern to that of head kidney is unknown

The innate immune system is composed of cellular andhumoral components (Budinõ et al 2006) There are two mainissues when it comes to the cellular immunity: one is thenumber of immune cells involved in the immune response, andthe other is the activity of these cells (Wang et al 2006b) Thenumber of erythrocytes in fish may affect the oxygen exchangeand the capacity for oxygen transport, causing stress undercertain conditions and making fish more susceptible to diseases(Benfey & Biron 2000) In the present study, RBC of juvenileJian carp decreased with the inadequate dietary pyridoxineconcentration There were similar reports on Channa punctatus(Agrawal & Mahajan 1983), Indian catfish (Mohamed 2001)and grouper (Huang et al 2005) However, Chen et al (2005)found that total haemocyte count of abalone was not signifi-cantly affected by the dietary treatments It should be notedthat fish blood contains haemoglobin but abalone contains

Table 5 Speciﬁc immunity parameters

of juvenile Jian carp (Cyprinus carpio var Jian) challenged with Aeromonas hydrophila and fed diets containing graded levels of pyridoxine (PN, mg kg)1)

Ab titre 14.4 ± 3.6b 28.8 ± 7.2b 44.8 ± 17.5a 38.4 ± 14.3a 44.8 ± 17.5a 38.4 ± 14.3a

IgM 86.0 ± 4.4c 156.2 ± 5.8b 179.8 ± 8.7a 182.2 ± 12.3a 177.1 ± 8.8a 180.1 ± 9.9a

Ab titre, antibody titre; IgM, Immunoglobulin M (mg L)1serum).

Values are means ± SD of three groups of fish (n = 3) Values within the same row having

different superscript alphabets are significantly different (P < 0.05).

Table 6 Aeromonas hydrophila, chia coli and Lactobacillus counts (log 10 cfu g)1) in intestine of juvenile Jian carp (Cyprinus carpio var Jian) fed diets containing graded levels of pyridoxine (PN, mg kg)1)

A hydrophila 8.0 ± 0.1a 8.0 ± 0.6a 8.2 ± 0.3a 8.5 ± 0.1a 8.4 ± 0.0a 8.1 ± 0.3a

E coli 7.5 ± 0.1 a 7.2 ± 0.2 a 7.4 ± 0.2 a 7.3 ± 0.1 a 7.3 ± 0.2 a 7.2 ± 0.2 a

Lactobacillus 7.3 ± 0.4 a 7.1 ± 0.0 a 7.2 ± 0.1 a 7.4 ± 0.3 a 7.5 ± 0.1 a 7.3 ± 0.2 a

Values are means ± SD of three groups of fish (n = 3) Values within the same row having

different superscript alphabets are significantly different (P < 0.05).

.

Trang 37

haemocyanin Furthermore, the present results showed that

RBC in high pyridoxine concentration treatment group

(12.4 mg kg)1diet) was lower than that in normal treatment

groups There was diﬀerent result in channel catﬁsh fed diets

which were containing 20 mg kg)1 or higher levels of

pyridoxine appear normochromic anaemia, but RBC not

changed (Andrews & Murai 1979) The reason for why RBC in

high pyridoxine concentration treatment group declined is

unknown In the present study, WBC was signiﬁcantly aﬀected

by dietary pyridoxine concentration, which was in consistent

with the report that slight leukopenia (8.3% decrease)

accompanied with granulocytosis and thrombopenia had been

observed in Channa punctatus fed with a pyridoxine-deﬁcient

diet for 8 months (Agrawal et al 1983) Phagocytosis is one of

the ﬁrst defence mechanisms of immune system against an

invading pathogen (Dunier et al 1995) In the present study,

with the levels of pyridoxine from 1.7 up to 5.0 mg kg)1diet

had signiﬁcantly increased rate of phagocytosis, which was

supported by Chen et al (2005) in abalone

The ﬁsh defence system is basically similar to that

described in mammals (Albrektsen et al 1995; Sakai 1999)

Teleosts also have various humoral defence components such

as agglutinin, complements, lysozyme and metal ion binding

proteins (Dalmo et al 1997; Sakai 1999) Agglutinins are

proteins or glycoproteins that can be directed against various

saccharide moieties on cell surfaces (Fisher & Dinuzzo 1991;

Anderson 1996) So it acts as opsonins and causes

aggrega-tion by binding to proteins/glycoproteins and /or

carbohy-drate moieties that are free in solution or are constituents of

microbes (Ingram 1980; Alexander & Ingram 1992) In our

study, HA was signiﬁcantly aﬀected by dietary pyridoxine

concentration, and similar results were also reported in

abalone (Chen et al 2005) As HA may have strong

rela-tionship with phagocytic ratio due to agglutinins role as

opsonins in the process of phagocytosis (Olafsen et al 1992;

Orda´s et al 2000), the increased HA was conﬁrmed by the

increased PA of leukocyte

Lysosomal ACP is widely believed to be a valuable

indi-cator of macrophage activation and activated macrophages

often display increased lysosomal enzyme activity (Secombes

1986; Chung & Secombes 1987; Dalmo et al 1997) Our

study showed that ACP activities of serum that followed

immunization were repressed when Jian carp were fed the

diet containing inadequate pyridoxine There are similar

reports that ACP activity in serum of abalone had some

positive correlations with dietary pyridoxine concentration,

although no signiﬁcant diﬀerence was observed (Chen et al

2005) Lysozyme can cleave the glycosidic bonds in the

peptidoglycan layer of certain Gram-positive bacteria and

has antimicrobial activity against several Gram-negativebacteria (Dalmo et al 1997) In the present study, LA wassignificantly affected by dietary pyridoxine concentration.But LA in serum of abalone and head kidney of Atlanticsalmon was not affected by dietary pyridoxine concentration(Albrektsen et al 1995; Chen et al 2005) The difference mayderive from different species of fish and tissue

Iron-binding proteins such as apotransferrins, min and metallothionein which can inhibit the growth ofbacteria have been identified in fish (Dalmo et al 1997).Total iron-binding capacity (TIBC) can represent iron-binding proteins concentration in serum (Soldin et al 2004).The results of the present study indicated that TIBC of theserum followed immunization in treatment groups contain-ing 1.7 mg pyridoxine kg)1 diet was the lowest among alltreatment groups There is no report about the relationshipbetween dietary pyridoxine concentration and iron-bindingproteins concentration in serum of fish

ceruloplas-As advanced vertebrates, fish produce specific responsesafter being stimulated by antigen IgM is the main immu-noglobulin present in teleosts (Watts et al 2001) Our studyshowed that Ab titre and IgM content of the serum thatfollowed immunization were significantly affected by dietarypyridoxine concentration But specific antibody response ofAtlantic salmon immunization with V salmonicida was notinfluenced by dietary pyridoxine concentration (Albrektsen

et al.1995) The diﬀerence may be derived from the fact thatthe diet of Atlantic salmon was a practical diet and contained

a large amount of ﬁsh meal (700 g kg)1) which containedhigh pyridoxine concentration and reached the requirement

of optimal antibody production

The mechanisms that immune responses were aﬀected bydietary pyridoxine concentration may be related to pyri-doxine participating in the metabolism of proteins, aminoacids and one carbon unit, and participating antioxidantreaction Pyridoxine participates in metabolism of proteinsand amino acids as a prosthetic group of enzymes (Ogino1965; Shiau & Wu 2003), which may promote synthesis ofimmune defence components which are mainly proteins.Pyridoxine also aﬀected the activity of serine hydroxymeth-yltransferase to participate in production of one carbon unitwhich involves in synthesising DNA and RNA (Trakatellis &Dimitriadou 1992, 1997; Perry et al 2007) Pyridoxine has adirect antioxidant activity of preventing superoxide radicalformation, glycated haemoglobin formation and erythrocytelipid peroxidation during glucose autoxidation (Jain & Lim2001)

With the rapidly developing ﬁsh farming industry, ing bacterial disease is very important Aeromonas hydrophila .

Trang 38

avoid-may cause infections in ﬁsh and is generally associated with

small surface lesions, sloughing of scales, local haemorrhage

and septicaemia (Balca´zar et al 2008) Escherichia coli are

the other important harmful bacteria Lactobacillus is

dominant bacteria in the intestine of carp, which could

depress the growth of baneful bacteria by decreasing pH in

intestine and through the shielding competition (Guo 2002)

Moreover, Lactobacillus can produce bacteriocin-like

sub-stances controlling overgrowth of potentially pathogenic

bacteria (Boris & Barbes 2000) Aeromonas hydrophila, E coli

and Lactobacillus counts in intestine of juvenile Jian carp

were not aﬀected by dietary pyridoxine concentration in our

condition

In summary, disease resistance, non-speciﬁc immune and

speciﬁc immune functions of Jain carp were signiﬁcantly

enhanced by dietary pyridoxine, but the underlying

molecu-lar mechanism requires more study With immunity

enhancement, pyridoxine is necessary for ﬁsh to maintain

normal immune responses and disease resistance

The work was supported by Program for Chang Jiang

Scholars and Innovative Research Team in University

(PCSIRTO555) The authors would like to thank the

per-sonnel of these teams for their kind assistance

Agrawal, N.K & Mahajan, C.L (1983) Haematological and

hae-matopoietic studies in pyridoxine deﬁcient ﬁsh, Channa punctatus

Bloch J Fish Biol., 22, 91–103.

Albrektsen, S., Glette, J., Waagbø, R & Sandnes, K (1995)

Inﬂu-ence of dietary vitamin B6 on tissue vitamin B6 contents and

immunity in Atlantic salmon, Salmo salar L Aqua Res., 26, 331–

339.

Alexander, J.B & Ingram, G.A (1992) Noncellular nonspeciﬁc

defence mechanisms of ﬁsh Annu Rev Fish Dis., 2, 249–279.

Anbarasu, K & Chandran, M.R (2001) Eﬀect of ascorbic acid on

the immune response of the catﬁsh, Mystus gulio (Hamilton), to

diﬀerent bacterins of Aeromonas hydrophila Fish Shellﬁsh Immun.,

11, 347–355.

Anderson, R.S (1996) Interactions of Perkinsus marinus with humoral

factors and hemocytes of Crassostrea virginica J Shellﬁsh Res., 15,

127–134.

Andrews, J.W & Murai, T (1979) Pyridoxine requirements of

channel catﬁsh J Nutr., 109, 533–537.

AOAC (Association of Oﬃcial Analytical Chemists) (2000) Oﬃcial

Methods of Analysis of the AOAC International, 17th edn AOAC

International, Gaithersburg, MD, USA.

Balca´zar, J.L., Vendrell, D., Blas, I.D., Zarzuela, I.R., Muzquiz, J.L.

& Girones, O (2008) Characterization of probiotic properties of

lactic acid bacteria isolated from intestinal microbiota of ﬁsh.

Barracco, M.A., Medeiros, I.D & Moreira, F.M (1999) Some haemato-immunological parameters in the mussel Perna perna.

Fish Shellﬁsh Immun., 9, 387–404.

Benfey, T.J & Biron, M (2000) Acute stress response in triploid rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis) Aquaculture, 184, 167–176.

Boris, S & Barbes, C (2000) Role played by lactobacilli in trolling the population of vaginal pathogens Microbes Infect., 2, 543–546.

con-Budin˜o, B., Cal, R.M., Carla Piazzon, M & Lamas, J (2006) The activity of several components of the innate immune system in diploid and triploid turbot Comp Biochem Phys A., 145, 108–

Dunier, M., Vergnet, C., Siwicki, A.K & Verlhac, V (1995) Effect of lindane exposure on rainbow trout (Oncorhynchus mykiss) immunity IV Prevention of nonspecific and specific immunosuppression by dietary vitamin C (ascorbate-2-polyphosphate) Ecotox.

Erdal, J.L., Evensen, O., Kaurstad, O.K., Lillehaug, A., Solbakken,

R & Thorud, K (1991) Relationship between diet and immune response in Atlantic salmon (Salmo salar L.) after feeding various levels of ascorbic and omega-3 fatty acids Aquaculture, 98, 363–

Hardy, R.W., Halver, J.E & Brannon, E.L (1979) Eﬀect of dietary protein level on the pyridoxine requirement and disease resistance

of chinook salmon in: Proceedings of the World Sympositim on Finﬁsh Nutrition and Fishfeed Technology (Halver, J.E & Tiews, K.

ed.) pp 1, 253–260, Berlin, June 20–23 June, Heenemann Verlagsgesellschaft, Berlin.

He, W., Zhou, X.Q., Feng, L., Jiang, J & Liu, Y (2008) Dietary pyridoxine requirement of juvenile Jian carp (Cyprinus carpio var.

Jian) Aqua Nutr., doi: 10.1111/j.1365-2095.2008.00604.x.

Huang, J.W., Tian, L.X., Du, Z.Y., Yang, H.J & Liu, Y.J (2005) Pyridoxine deﬁciency of grouper, Epinephelus coioides: physiological and biochemical alteration Fish Physiol Biochem., 31, 333–

.

Trang 39

glucose-treated human erythrocytes Free Radic Biol Med., 30,

232–237.

Kim, D.H & Austin, B (2006) Innate immune responses in rainbow

trout (Oncorhynchus mykiss, Walbaum) induced by probiotics.

Fish Shellﬁsh Immun., 21, 513–524.

Mohamed, J.S (2001) Dietary pyridoxine requirement of the Indian

catﬁsh, Heteropneustes fossilis Aquaculture, 194, 327–335.

Ogino, C.B (1965) Vitamin requirements of carp, Cyprinus carpio: I.

Deﬁciency symptoms and requirements of vitamin B6 Nippon

Suisan Gakk., 31, 546–551.

Olafsen, J.A., Fletcher, T.C & Grant, P.T (1992) Agglutinin activity

in Paciﬁc oyster (Crassostrea gigas) hemolymph following in vivo

Vibrio anguillarum challenge Dev Comp Immun., 16, 123–138.

Orda´s, M.C., Orda´s, A., Beloso, C & Figueras, A (2000) Immune

parameters in carpet shell clams naturally infected with Perkinsus

atlanticus Fish Shellﬁsh Immun., 10, 597–609.

Perry, C., Yu, S., Chen, J., Matharu, K.S & Stover, P.J (2007)

Eﬀect of vitamin B6 availability on serine

hydroxymethyltrans-ferase in MCF-7 cells Arch Biochem Biophys., 462, 21–27.

Pipe, R.K (1990) Hydrolytic enzymes associated with the granular

haemocytes of the marine mussel Mytilus edulis Histochem J., 22,

595–603.

Ringø, E & Olsen, R.E (1999) The eﬀect of diet on aerobic bacterial

ﬂora associated with intestine of Arctic charr (Salrelinus alphinus

L.) J Appl Microbiol., 86, 22–28.

Sakai, M (1999) Current research status of ﬁsh immunostimulants.

Secombes, C.J (1986) Macrophage activation during experimental

allergic orchitis in rainbow trout (Salmo gairdneri) Dev Com.

Immun., 10, 539–546.

Shiau, S.Y & Wu, M.H (2003) Dietary vitamin B6 requirement of

grass shrimp, Penaeus monodon Aquaculture, 225, 397–404.

Soldin, O.P., Bierbower, L.H., Choi, J.J., Thompson-Hoffman, S &

Soldin, S.J (2004) Serum iron, ferritin, transferrin, total

iron binding capacity, hs-CRP, LDL cholesterol and

magne-sium in children; new reference intervals using the Dade

Dimen-sion Clinical Chemistry System Clin Chim Acta, 342, 211–217.

Spanggaard, B., Huber, I., Nielsen, J., Nielsen, T., Appel, K.F &

Gram, L (2002) The microﬂora of rainbow trout intestine: a

comparison of traditional and molecular identiﬁcation

Aquacul-ture, 182, 1–15.

Sritunyalucksana, K., Sithisarn, P., Withayarnkul, B & Flegel,

T.W (1999) Activation of prophenoloxidase, agglutinin and

antibacterial activity in haemolymph of the black tiger prawn,

Penaeus monodon, by immunostimulants Fish Shellﬁsh Immun.,

9, 21–30.

Takemura, A (1993) Changes in an immunoglobulin M (IgM)-like protein during larval stages in tilapia Oreochromis mossambicus Aquaculture, 115, 233–241.

Tang, L., Wang, G.H., Jiang, J., Feng, L., Liu, Y., Li, S.H., Kuang, S.Y & Zhou, X.Q (2008) Eﬀect of methionine on intestinal enzymes activities, microﬂora and humoral immune of juvenile Jian carp (Cyprinus carpio var Jian) Aqua Nutr., doi: 10.1111/j.1365- 2095.2008.00613.x.

Trakatellis, A & Dimitriadou, A (1992) Eﬀect of pyridoxine ciency on immunological phenomena Postgrad Med J., 68, S70– S77.

deﬁ-Trakatellis, A & Dimitriadou, A (1997) Pyridoxine deﬁciency: new approaches in immunosuppression and chemotherapy Postgrad Med J., 73, 17–22.

Vivas, J., Riano, J., Carracedo, B., Razquin, B.E., Lo´pez-Fierro, P.

& Naharro, G (2004) The auxotrophic aroA mutant of monas hydrophila as a live attenuated vaccine against A salmonicida infections in rainbow trout (Oncorhynchus mykiss) Fish Shellﬁsh Immun., 16, 193–206.

Aero-Wang, Z.L., Mai, K.S., Liufu, Z.G., Ma, H.M., Xu, W., Ai, Q.H., Zhang, W.B., Tan, B.P & Wang, X.J (2006a) Eﬀect of high dietary intakes of vitamin E and n-3 HUFA on immune responses and resistance to Edwardsiella tarda challenge in Japanese ﬂounder (Paralichthys olivaceus, Temminck and Schlegel) Aqua Res., 37, 681–692.

Wang, W.B., Wang, Y P., Hu, W., Li, A.H., Cai, T.Z., Zhu, Z.Y & Wang, J.G (2006b) Effects of the ‘‘all-fish’’ growth hormone transgene expression on non-specific immune functions of common carp, Cyprinus carpio L Aquaculture, 259, 81–87.

Watts, M., Munday, B.L & Burke, C.M (2001) cDNA sequences and organization of IgM heavy chain genes in two holostean ﬁsh Dev Comp Immunol., 19, 153–164.

Yang, Q.H, Zhou, X.Q, Jiang, J & Liu, Y (2008) Eﬀect of dietary vitamin A deﬁciency on growth performance, feed utilization and immune responses of juvenile Jian carp Aquac Res., 39, 902–906.

Zhou, X.Q & Li, Y.N (2004) The eﬀect of Bio-Mos on intestinal microﬂora and immune function of juvenile Jian carp (Cyprinus carpio var Jian) in: Nutritional Biotechnology in the Feed and Food Industries, Proceedings of Alltechs 20th Annual Symposium (Suppl 1: Abstracts of posters presented), p 109 Lexington, Ky, 24–26 May.

.

Trang 40

1 2 2,3 1 4

2

1

Universidade do Algarve, Faro, Portugal; 2 IFAPA – CIFPA-ƠAgua del PinoÕ, Huelva, Spain; 3 Centro Oceanogra´ﬁco de

Canarias, IEO, Santa Cruz de Tenerife, Spain;4 Departamento de Biologia Animal, Facultad de Biologı´a, Universidad de La

Laguna, La Laguna, Spain

(Correction added on 1 June 2009, after ﬁrst online publication: The header ƠLETTER TO THE EDITORÕ was removed from

the ﬁrst page of this article.)

The eﬀects of feeding three natural frozen diets, grass

shrimp (Palaemonetes sp.), crayﬁsh (Procambarus clarkii)

and ﬁsh (Sardina pilchardus) and two semi-humid artiﬁcial

diets (based on ﬁsh or shrimp powder) to the cuttleﬁsh,

Sepia ofﬁcinalis, were analysed Growth rate and feeding

rate [FR; % body weight (BW) day)1] and food

conver-sions (FC, %) were determined Cuttleﬁsh fed shrimp grew

larger (3.8% BW day)1) and had the highest FC, followed

by those fed crayﬁsh, and sardine The highest FR was

obtained for cuttleﬁsh fed crayﬁsh (10.5% BW day)1)

Although both artiﬁcial diets were accepted, none

pro-duced growth Digestive gland-to-body weight ratio (DG/

BW ratio) was calculated for animals fed each diet A

positive correlation (r = 0.94) between cuttleﬁsh ingestion

FR and DG weight was obtained Mortality occurred

mainly during the last week, and some cannibalism

occurred among cuttleﬁsh fed artiﬁcial diets Finally, lipid

composition of diets, DG and mantle of each group were

analysed Sardine diet was characterized by high levels of

triacylglycerol (TG), whereas the main diﬀerence between

shrimp and crayﬁsh was the higher n-3/n-6 ratio found in

shrimp Changes in the lipid composition of DG were

related to diet, but did not correlate with growth data A

strong loss of TG in the DG of artiﬁcial diets groups was

notable No diﬀerences in mantle lipid composition among

the natural diets were found, but artiﬁcial diet groups

showed higher contents of neutral lipids in their mantle

respect to natural diets According to results obtained,

crayﬁsh (P clarkii) could be used as an alternative prey for

rearing S ofﬁcinalis compared with shrimp Artiﬁcial diets

showed the worst eﬀects in growth and mortality as well asthe stronger inﬂuence on DG and mantle lipid composition

of cuttleﬁsh

gland, growth, lipid composition, mantle

Received 19 August 2008, accepted 22 January 2009 Correspondence: Eduardo Almansa, Centro Oceanogra´ﬁco de Canarias, IEO, Avd 3 de Mayo n73, 38005, Santa Cruz de Tenerife, Canary Islands, Spain E-mail: eduardo.almansa@ca.ieo.es

Sepia ofﬁcinalis is one of the most easily cultured pods This species has been maintained and/or cultured inaquaria world over since the late 1960s (Richard 1971;

cephalo-Pascual 1978; Boletzky & Hanlon 1983; Forsythe et al 1994;

Lee et al 1998; Domingues et al 2001a,b, 2002, 2003a); it ishighly adaptable to life in captivity and suitable for aqua-culture because it has (1) large eggs, (2) high hatchling sur-vival, (3) sedentary behaviour, (4) tolerance to crowding,handling and shipping, (5) acceptance of non-living foodsand (6) most importantly, it is easy to reproduce in captivity(Forsythe et al 1994)

Appropriate and inexpensive diets are basic requirementsfor the success in commercial aquaculture (Chen & Long1991) Cephalopods can be maintained or grown with severalnatural live or dead preys (Boletzky & Hanlon 1983; Toll &

Strain 1988; DeRusha et al 1989; DiMarco et al 1993;

Domingues et al 2002, 2005) Saltwater crustaceans, mainlyshrimp, have been the diets that promote better growth

2010 16; 262–275 .doi: 10.1111/j.1365-2095.2009.00661.x

.

Định dạng
Số trang	111
Dung lượng	10,14 MB