Aquaculture nutrition, tập 16, số 4, 2010

A reference REF and three experimental diets, one with an increased protein to energy ratio PRO, another with an increased level of the amino acid histidine HIS and a third with suppleme

1 1,2 1 1 1 1

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, the Chinese Academy of Sciences,

responses and histological changes were investigated in

juve-nile gibel carp (Carassius auratus gibelio) Triplicate groups of

gibel carp (3.53 ± 0.02 g) were fed seven semipurified diets

(Diet 1 to 7) containing 3.20, 5.37, 7.08, 9.55, 12.70, 17.90 and

weight gain fed Diet 6 was 112.6% of that of control group

(Diet 1) after 3 months, but there was no significant difference

of weight gain between fish fed Diet 7 and the control group

Alanine aminotransferase (ALT) of fish hepatopancreas fed

Diet 7 was significantly higher than the control group

(P < 0.05), but no significant difference was observed in ALT

4, 5, 6 and 7) No significant histological lesions were identified

accumulated in hepatopancreas was logarithmically related to

mus-cles and ovaries of gibel carp fed Diet 3 to Diet 7 The present

residue of muscles and ovaries above the safety limitation of

European Union

KEY WORDS: aflatoxin B1, alanine aminotransferase, Carassius

auratus gibelio, residue, weight gain

Received 18 December 2008, accepted 13 March 2009

Correspondence: Dr Dong Han, State Key Laboratory of Freshwater and

Biotechnology, Institute of Hydrobiology, the Chinese Academy of

Sciences, Wuhan, Hubei, 430072, China E-mail: hand21cn@ihb.ac.cn

Aflatoxin, a polycyclic aromatic hydrocarbon, is produced

mainly by Aspergillus flavus and Aspergillus parasiticus,

which occurs naturally in several important plant feedstuffs

for poor growth, hepatotoxic, nephrotoxic, carcinogenic,mutagenic, teratogenic and cytotoxic properties (Halver1969; Jantrarotai & Lovell 1990; Cha´vez-Sa´nchez et al 1994;Sahoo et al 1996; Santacroce et al 2008) In studies of car-cinogenic effect, the liver of fish is one of the organs most

immuno-modulator in endotherms (Ottinger & Kaattari 1998; Sahoo

& Mukherjee 2001)

and is classified as group I carcinogen by International

contaminated feed by fish brings contamination of ucts, which may result in secondary contamination for

(Madhusudhanan et al 2006) Globally, there are strict

animal feed and food items (Gowda et al 2007) The

regulations, upper limit of most feed ingredients and

Administration (FDA) 1989) In many countries includingChina, there are no official safe levels for aflatoxins in fish

such as salmonoids (Hendricks et al 1977), channel catfish(Jantrarotai & Lovell 1990) and Nile tilapia (Tuan et al

good care of dietary ingredients Therefore, it is veryimportant and necessary to make clear the effect of low

.

2010 16; 335–342

. doi: 10.1111/j.1365-2095.2009.00669.x

Aquaculture Nutrition

Gibel carp, an omnivorous species, is an important

aqua-culture species in China and the annual production is more

than 3 million tonnes Increased use of plant ingredients in

aquafeed for gibel carp has intensified the potential for

aflatoxicosis The purpose of the present study is to

in gibel carp

MO, USA) According to the method of Sahoo & Mukherjee

was then added into the mixture After adding oil, the

chloroform was allowed to evaporate

Seven experimental diets (Diet 1-7) were formulated to

contain 3.20, 5.37, 7.08, 9.55, 12.70, 17.90 and 28.60 lg

experi-mental diets are shown in Tables 1 & 2 White fish meal and

fish oil were used as the main protein and lipid source Diets

Gibel carp was obtained from the hatchery of the Institute ofHydrobiology, the Chinese Academy of Sciences Before theexperiment, the juveniles were acclimated to the experimentalcondition for 2 weeks During the acclimation, fish were fedtwice daily with the control diet

The experiment was carried out in a flow-through system

volume: 140 L) Water flowing rate into each tank was

12D : 12L with the light period from 0800 to 2000

Before the experiment, fish were deprived of feed for 1 day

and randomly transferred into each tank During the ment, the fish were fed to apparent satiation twice a day (0900,1500), and daily feed intake was recorded The faeces wereremoved by siphoning twice a day just before each feeding

experi-Fish in each tank were batch-weighed every month after 1-day

on growth performance The trial lasted for 3 months

At the end of the experiment, 10 fish per tank were thetized with MS-222, and the length and weight of fish weremeasured Blood was withdrawn from dorsal vessels using the

serum The hepatopancreas of four fish were collected on ice,weighed and stored in liquid nitrogen for enzyme determina-tion The muscles, hepatopancreas, and ovaries of six fish were

three fish per tank were sampled for histological studies

Table 1 Formulation and chemical composition of the basal diet (in

dry weight)

Ingredient

Content (g kg)1)

FeSO 4 , 1250; C 6 H 10 CaO 6 Æ5H 2 O, 1750; ZnSO 4 Æ7H 2 O, 176.5; MnSO 4 Æ

4H 2 O, 81; CuSO 4 Æ5H 2 O, 15.5; CoSO 4 Æ6H 2 O, 0.5; KI, 1.5; starch, 225.

2 Vitamin premix (mg kg)1diet): thiamin, 20; riboflavin, 20;

pyri-doxine, 20; cyanocobalamine, 2; folic acid, 5; calcium patotheniate,

50; inositol, 100; niacin, 100; biotin, 5; starch, 3226; Vitamin A, 110;

Vitamin D 3 , 20; Vitamin E, 100; Vitamin K 3 , 10; Choline chloride,

1100.

Table 2 Supplemented and determined aflatoxin B 1 levels of the experimental diets

Experimental diets

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

Diet 7 Supplemented

AFB 1 (lg kg)1)

Determined AFB 1 (lg kg)1)

3.20 5.37 7.08 9.55 12.70 17.90 28.60

.

Serum analysis Serum superoxide dismutase (SOD) activity

was determined using the method of McCord & Fridovich

(1969) One unit of SOD activity is described as the amount

of sample required to cause 50% inhibition of the rate of

activity was measured using the method of Aebi (1984) in

which the initial rate of hydrogen peroxide decomposition is

determined One unit of catalase activity was defined as the

amount of enzyme that catalysed the decomposition of

rinsed with 0.65% physiological saline, and then

homoge-nized in five volumes v/w of ice-cold 0.65% physiological

saline using a hand held glass homogenizer on ice The

assays were conducted within 24 h after extraction

The enzyme activities of aspartate aminotransferase (AST)

and alanine aminotransferase (ALT) were measured at an

absorbance of 505 nm according to the method of Hørder &

Rej (1981) The protein content of the supernatant was

determined using Bradford assay with bovine serum albumin

as a standard, and the specific activity of the enzymes was

defined as activity unit per mg protein

The hepatopancreas samples were fixed in Bouins fluid for

12 h and dehydrated in a graded ethanol series The

hepa-topancreas slices were then embedded in paraffin, thin

sec-tioned to 7 lm, stained with hematoxylin and eosin (H & E)

and observed under light microscopy

Crude protein, lipid and energy content were analysed for

the experimental diets Dry matter content was determined

was analysed by the Kjeldahl method Crude lipid was

determined by ether extraction using a Soxtec system

(Soxtec System HT, 1043 Extraction Unit, Tecator,

Swe-den) and energy by bomb calorimeter (Phillipson

micro-bomb calorimeter; Gentry Instruments Inc., Aiken, SC,

USA)

hepatopancreas and ovaries of fish were carried out by the

enzyme-link immunosorbent assay (ELISA) method using acommercial assay kit (Brins-LivePro Biotechnology Corpo-ration, Beijing, China) For this determination, the absor-

spectrophotometer (PowerWave XS; BioTek Instruments

Duncans multiple range test was used to detect the cance of differences of means between groups followed by

proba-bility of P < 0.05 were considered statistically significant

During the experiment, the average weight gain (WG) of fish

concentra-tions (Table 3) After 1 month, fish WG increased with the

2 months, no significant difference was observed in WG

At the end of the experiment, WG of the fish fed Diet 7 wassignificant lower than that fed Diet 6 (P < 0.05) Table 3showed condition factor (CF) in the fish fed Diet 6 was sig-nificantly higher than CF in the other groups (P < 0.05).There was no significant effect on hepatosomatic index (HSI)and feed conversion ratio (FCR) between all groups(P > 0.05) There was no mortality observed during theexperiment

The activities of catalase (CAT), superoxide dismutase(SOD), aspartate aminotransferase (AST) and alanineaminotransferase (ALT) of gibel carp fed with different

(Diet 4, 5, 6 and 7) were significantly higher than the controldiet (P < 0.05) Hepatopancreas ALT activities of fish fedDiet 7 were significantly higher than the control (P < 0.05),while the activities of serum CAT and hepatopancreas ASTshowed no significant difference between different groups(P > 0.05)

.

After 3 months, no significant histological lesions were

identified in hepatopancreatic tissues of gibel carp between

and nuclei were uniform in size and shape and prominent

cytoplasm was present in most hepatocytes of the control fish

1

increased significantly and was logarithmically related to

ovary of fish was not detected in the control group and Diet

(Fig 3)

The phenomenon of hormesis for toxic or harmful stances, a dose–response characterized by a low dose stimu-lation and a high dose inhibition, has been widely discussed(Calabrese & Baldwin 1998, 2003; Calabrese 2005, 2008;

sub-Murado & Va´zquez 2007; Belz et al 2008) In the presentstudy, weight gain of fish fed Diet 6 was 112.6% of that ofthe control group after 3 months, but there was no significantdifference of weight gain between fish fed Diet 7 and thecontrol group This growth hormesis response was similar tothe results in chickens (Diaz et al 2008) However, decreasedgrowth was reported in many species fed different dietary

Han et al 2008) The reasons for the difference were ably that: (1) many reports studied the toxic effects of high

many research species were sensitive to even very low dietary

could be usually elicited for the low response of 10–20%

(Johnson & Bruunsgaard 1998) Fish fed Diet 7 showed the

during the experiment, which suggested that the hormesisresponse could change as experimental time went on Thecondition factor (CF) expressed the condition of fish, such asthe degree of well being, plumpness or fatness, and deter-mined from observed weights and fork length In this study,

Table 4 The activities of catalase (CAT), superoxide dismutase

(SOD), aspartate aminotransferase (AST) and alanine

aminotrans-ferase (ALT) of gibel carp fed with different concentrations of AFB 1

for 3 months (mean ± SE)*

AST (IU g)1pro)

ALT (IU g)1pro)

Average weight gain (%)1

* Different superscript letters within each row represent significant differences (P < 0.05).

1 Weight gain = 100 · (Final weight )initial weight)/(initial weight).

2 HSI (Hepatosomatic index) = 100 · (liver weight)/(fish weight).

3 CF (Condition factor) = 100 · final weight (g)/[fork length (cm)] 3

4 FCR (Feed conversion ratio) = total dry feed fed (g)/total wet weight gain (g).

.

there was a significant increase of CF in fish fed Diet 3 and

above and fish fed Diet 6 showed the highest CF It was

suggested that fish fed Diet 3 and above grew shorter in

shape than the control group

In this study, FCR was not significantly affected by the

finding in tilapia was reported that FCR of the control was

significantly lower than that of the group fed with 100 lg

dietary soybean meal was substituted by the contaminated palm kernel meal

fish (Tuan et al 2002) AST and ALT were used as the

Figure 1 Hepatopancreas of gibel carp

fed with (a) the control diet, (b) Diet 2,

(c) Diet 3, (d) Diet 4, (e) Diet 5, (f ) Diet

6 and (g) Diet 7 for 3 months H&E,

Bar = 50 lm.

.

biochemical indicators for hepatic damage (Cheng et al.

2001; Allameh et al 2005) In this study, the increase in ALT

damaged the hepatopancreas of gibel carp This result agreed

Kositcharoenkul 2006) Similarly, Han et al (2008) also

described a marked increase by 9.6% or 13.8% in serum

The increase in activation of SOD conferred protection

against oxidative damage (Mattson et al 1995) Many

peroxidation or chromosomal damage through the release ofoxygen free radicals (Amstad et al 1984; Shen et al 1994),which suggested that increasing oxidative damage might

supported the results found in this study that fish consumed

except increasing serum SOD activities On the other hand,Rastogi et al (2001) reported that SOD activity in liver ofrats was decreased when intraperitoneally administered at a

However, in this study, no significant changes were observed

in HSI and hepatopancreatic histology of gibel carp among

not cause the hepatopancreatic histological changes of gibel

fed for 6 months had been found to cause liver tumours inrainbow trout (Halver 1969) Our results indicated that gibelcarp is much less sensitive than rainbow trout to the histo-

the bile, liver, kidney and pyloric caeca of rainbow troutfollowing oral and intravenous administration over a period

of 8 days The finding was in close agreement with this study

that in muscles and ovaries On the other hand, Divakaran &

faeces, whole shrimp or tail muscle of Penaeus vannamei

were rapidly depleted and there was a very low potential for

Figure 2 The relationship between AFB1 in hepatopancreas (AH,

lg kg)1) and in diets (AD, lg kg)1).

Figure 3 AFB1 residue in muscle and ovary of gibel carp fed with

different dietary AFB1 for 3 months, and AFB1 residue in tissues of

fish was not detected in the control group and Diet 2.

.

hazard of secondary contamination from eating fish flesh for

human has been raised (Madhusudhanan et al 2006) In

adopted is 2 ppb posed on risk assessments (Food and

residue in muscles and ovaries of fish fed with more than

effectively observed from the growth of fish, especially at low

diet showed impaired physiological responses and more

limi-tation of European Union

The authors are grateful Guanghan Nie for his technical

help This study was supported by National Key Technology

R&D Program (2007BAD37B02, 2001BA505B06) and partly

by Natural Science Foundation of China (30700626) and

Key Project of Hubei Provincial Science and Technology

Department (2006AA203A01)

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Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting- Rotting-.

1,2 1 1

Research, Cleveland, Queensland, Australia

Several dietary strategies to ameliorate poorer growth

observed to occur at temperatures above the upper thermal

optima were examined with juvenile barramundi (Lates

calcarifer) A reference (REF) and three experimental diets,

one with an increased protein to energy ratio (PRO), another

with an increased level of the amino acid histidine (HIS) and

a third with supplementation of dietary nucleotides (NUC),

period Growth was affected by both temperature and diet

than those fed the NUC diet at the same temperature The

addition of the amino acid histidine to the diet did not

improve growth rates at either temperature At water

Other key factors including feed intake, feed conversion rate,

nutrient and energy retention and plasma enzymology were

also all affected by temperature and diet This study shows

that the use of a diet with an increased protein to energy ratio

provides significant benefits in terms of reducing the impact

of growth retardation at higher temperatures

KEY WORDS: Asian seabass, heat, stress, temperature

Received 26 September 2008, accepted 4 March 2009

Correspondence: Brett Glencross, CSIRO Marine and Atmospheric

Research, PO Box 120, Cleveland, QLD 4163, Australia E-mail: Brett.

Glencross@csiro.au

Barramundi production in Australia occurs in water

Williams 2001; Glencross 2006) However, optimal growth

of the fish begins to decline as the fish start to experience heatstress (Katersky & Carter 2005) Dietary strategies to improveperformance for this species have been examined at the lowertemperature spectrum, but little work has been performed atupper physiological ranges (Williams et al 2006)

Temperature stress manifests several key physiologicalresponses in fish, growth retardation and death being some ofthe extreme outcomes However, other key processes thatoccur include the development of cataracts, a reduction inprotein digestibility, an increase in endogenous proteinturnover and a decline in endogenous protein synthesis(Carter et al 2006; M Bermudes, personal communication).The increased rate of protein turnover during heat stresssuggests that an increase in the dietary protein to energy ratiomight also provide a means of improving the fishÕs response

to this higher level of protein turnover (Barnes et al 2006).However, studies with Atlantic salmon found no significantbenefit from changes to the dietary protein to energy ratio,although it was acknowledged that the range of dietaryprotein to energy ratios was not broad

With the higher turnover rate of protein, there is likely to

be an increased demand for amino acids in general andpotentially some specific ones more than others (Carter et al.2006) The observation of cataracts among barramundiunder heat stress draws analogies with other pathologicalcauses of cataracts, including a dietary deficiency of theamino acid histidine (Breck et al 2005)

Another dietary supplement that has also been used tosupport fish during periods of stress is nucleotides (Li &Gatlin 2006) Nucleotides have been regarded as conditional

or semi-essential nutrients for fish Their inclusion in fish dietshas been shown to enhance immunity and disease resistance

of fish by influencing the immune system Nucleotide

.

2010 16; 343–350

. doi: 10.1111/j.1365-2095.2009.00670.x

Aquaculture Nutrition

supplementation has also been shown to affect the growth and

differentiation of cells from the gastrointestinal tract of fish

(Uauy et al 1990), an organ that is critically affected by

increased levels of protein turnover during heat stress

This study examines a series of diets and the potential

benefits they provide to barramundi, being grown at

tem-peratures higher than their optimal range, compared with a

standard reference diet and their optimal growth

tempera-ture Each of the dietary treatments considered were included

based on their potential to address key effects of heat stress

previously identified A diet with an increased protein to

energy ratio (PRO) was included to see if this could

com-pensate for the increased rate of protein turnover and decline

in protein digestibility A diet fortified with histidine (HIS)

was included because of the reported benefits of this dietary

amino acid in reducing incidence of cataracts (Breck et al

2005) A third diet included an allocation of nucleotides

(NUC), which have been implicated in improving the ability

of fish to deal with stress (Li & Gatlin 2006)

Each of the four experimental diets was extruded using an

APV 19:25 laboratory-scale twin-screw feed extruder through

a 2.4 mm diameter die Following extrusion, the pellets were

dry-ing, and while the pellets were still warm, they were

vacuum-infused with the formulated oil allocation The diet complete

formulations and source of all of the ingredients used is

presented in Table 1 Composition of each of the

experi-mental diets is also presented in Table 1

This study used hatchery-reared barramundi (Lates calcarifer)

that were pregrown to an acclimation weight of around 15 g in

indoor 1000-L heated sea-water tanks For acclimation the

fish were split into two groups of four 150-L tanks of around

100 fish each One group of four tanks was maintained at

fish were grown The other group of four tanks had their water

stress (Mora & Maya 2006) Following the acclimation

period, 15 fish were each randomly allocated to each of the

12 tanks within their respective temperature blocks with an

overall initial weight of 17.9 ± 0.25 (mean ± SD, n = 360),

Table 1 Nutrient composition of the experimental diets

DM unless otherwise detailed

Estimated digestible protein 8

(MJ kg)1DM) 9

20.2 21.1 20.9 21.1 DP–DE (g MJ)1) 10 26.9 20.9 21.1 20.9

Sourced from Manildra, Auburn, New South Wales, Australia.

5 Sourced from Sigma, St Louis, Missouri, USA.

6 Sourced from Ridley Aquafeeds, Narangba, Queensland, lia, as Optimun TM

Austra-7 Vitamin and mineral premix includes (IU kg)1or g kg)1of mix): vitamin A, 2.5 mIU; vitamin D3, 0.25 mIU; vitamin E, 16.7 g;

pre-vitamin K3, 1.7 g; pre-vitamin B1, 2.5 g; pre-vitamin B2, 4.2 g; pre-vitamin B3,

25 g; vitamin B5, 8.3 g; vitamin B6, 2.0 g; vitamin B9, 0.8 g; vitamin B12, 0.005 g; biotin, 0.17 g; vitamin C, 75 g; choline, 166.7 g; ino- sitol, 58.3 g; ethoxyquin, 20.8 g; copper, 2.5 g; ferrous iron, 10.0 g;

magnesium, 16.6 g; manganese, 15.0 g; zinc, 25.0 g.

8 Based on protein digestibility of wheat gluten at 100%, wheat flour at 100% and fishmeal at 90%.

9 Based on energy digestibility of fish oil at 95%, wheat gluten at 95%, wheat flour at 50% and fishmeal at 95%.

10 DP–DE: digestible protein to digestible energy ratio Digestible protein and energy values are derived from McMeniman 1998.

PRO, high protein to energy ratio diet; NUC, nucleotide fortified diet; HIS, histidine fortified diet; REF, reference experimental diet;

DM, dry matter.

.

with initial block weights of 17.9 ± 0.24 for the 30C

water, controlled by a digital programmable logic

controller-managed solenoid and water mixing box prior to the water

being distributed to each tank] and continuous aerated

sea-water Over the course of the study, the temperatures for each

Pty Ltd, Lower Hutt, New Zealand) and individually weighed

to 0.1 g accuracy The fish were then allowed to regain

con-sciousness and equilibrium before being placed within their

designated tank At the end of the study, the fish were again

sedated and weighed as detailed previously

The fish were fed to apparent satiety once daily between 09:00

and 11:00 over a 28-day (4-week) period Apparent satiety was

determined by a loss in feeding activity, this was reached after

three feeding sessions over a 1-h period Any uneaten feed was

removed from each tank 1 h later and the uneaten portion was

dried and weighed to allow the determination of daily feed

intake based on correction factors for leaching losses

sus-tained over an equivalent period (Helland et al 1996)

At the end of the experiment, after the final weighing, three fish

from each tank were pooled and processed for sample analysis

The three fish were minced together after being dried of

residual surface moisture Samples of the mince were then

taken for dry matter analysis and another was frozen prior to

being freeze-dried Freeze-dried whole fish samples were

analysed for dry matter, nitrogen, ash, fat, amino acids and

energy Blood samples were collected from an additional three

fish from each tank using a 1-mL syringe and an 18G needle via

the caudal vein Blood from each of the three fish was pooled

tube, centrifuged at 1000g for 1 min to settle the erythrocytes

prior to it being frozen and sent for plasma analysis

All chemical analyses were performed by NATA (National

Association of Testing Authorities) accredited analytical

service providers (Chemistry Centre, East Perth, WA,Australia) Diet and fish samples were analysed for drymatter, ash, nitrogen, total lipids, amino acids and grossenergy content Dry matter was calculated by gravimetric

levels were calculated from the determination of total

composition of samples was determined by an acid hydrolysisprior to separation via high-performance liquid chromato-graphy The acid hydrolysis destroyed tryptophan making itunable to be determined using this method Crude fat content

of the diets was determined gravimetrically followingextraction of the lipids according to the method of Folch

gravimetri-cally following loss of mass after combustion of a sample in a

deter-mined by adiabatic bomb calorimetry

Samples of plasma were sent to the West Australian AnimalHealth Laboratories (South Perth, WA) for plasma enzymeassessment The assays were run on an Olympus AU400automated chemistry analyser (Olympus Optical Co Ltd.,Ngano, Japan) Each of the assays used was a standard kitdeveloped for the auto-analyser The tests performed includedalanine aminotransferance (ALT; Olympus Kit Cat No.OSR6107), direct bilirubin (Olympus Kit Cat No OSR6111),total bilirubin (Olympus Kit Cat No OSR6112), creatinekinase (CK; Olympus Kit Cat No OSR6179), gamma-glut-amyltransferase (GGT; Olympus Kit Cat No OSR6219),glutamate dehydrongenase (GLDH; Randox Kit Cat No.GL441), lactate dehydrogenase (LDH; Olympus Kit Cat

No OSR6128) and total protein (Olympus Kit Cat No.OSR6132)

Nitrogen, energy, lysine and histidine retention were mined based on the mass gain of each parameter over thecourse of the growth study, against the respective dietary in-take of each respective parameter All values were calculatedaccording to the following formula (Maynard & Loosli 1969):

Nc

 100;where Nt is the nutrient/energy content of the fish in a spe-cific replicate at time t and Ni is the mean initial nutrient/energy content of the fish from the beginning of the study(n = 3 replicates of three representative fish); Nc is theamount of nutrient/energy consumed by the fish from the

.

time of initial assessment to time t Determination of energy

and amino acid retentions was achieved the same way, but

with the substitution of the relevant energy or amino acid

criteria where the corresponding nitrogen criteria are

indi-cated in the equation

All values are means unless otherwise specified All data

were analysed for homogeneity of variances using CochranÕs

test Effects of diet, temperature and interactions were

of differences were further tested using a FisherÕs least

sig-nificant difference test based on critical ranges being set at

Significant effects of both diet and temperature were

observed on the final weight and weight gain of the fish

(Table 2) No interaction effect between diet and temperature

was observed for the final weight and weight gain Fish at

the only exception being the PRO diet, with which the

dif-ference in growth between the two temperatures was not

NUC diets grew significantly better than those fed the

tempera-tures, the fish fed the PRO diet grew significantly better than

those fed the NUC, HIS and REF diets

No significant effects of either diet and temperature, orinteraction terms were observed on feed intake by the fish(Table 2) Post-hoc analysis did identify that fish fed the

However, significant effects of diet, temperature and theinteraction terms were observed on the feed conversion rate

fed the PRO and NUC diets had significantly better FCRthan those fed the HIS or REF diet, but there were no dif-

temperatures, the fish fed the PRO and NUC diets had nificantly lower FCR than those fed the HIS and REF diets,while the FCR of the HIS diet was also lower than that of theREF diet

sig-There were no significant effects of diet or interaction term

on energy retention efficiency, but there was a significanteffect of temperature (Table 2) Energy retention was signif-

tem-perature, both the PRO and NUC diets had better energyretention than both the HIS and the REF diets Within the

energy retention efficiency There were no significant effects

of diet or interaction term on protein retention efficiency, butthere was a significant effect of temperature (Table 2) Within

better protein retention than the HIS diet, but not the REF

differences in protein retention efficiency

The retention of specific amino acids like lysine and tidine was significantly affected by the diet and interactionterms, but was not affected by temperature (Table 2) At

Table 2 Growth performance and feed utilization by fish fed the experimental diets

Temperature

Units

Pooled SEM

P-value

Initial weight g per fish 17.9 18.1 17.9 17.9 17.8 17.9 17.6 18.0 0.05 – – –

Final weight g per fish 61.7 a 61.5 a 56.6 ab 53.1 b 57.6 a 48.6 c 49.9 bc 47.7 c 1.32 0.016 0.001 0.347

Gain g per fish 43.7 a 43.5 a 38.6 ab 35.2 b 39.8 ab 30.7 c 32.4 bc 29.7 c 1.31 0.016 0.001 0.349

FCR Feed per gain 0.65 a 0.65 a 0.72 b 0.77 bc 0.72 b 0.75 b 0.82 c 0.98 d 0.02 0.000 0.000 0.004

Intake g per fish 28.3 a 28.4 a 27.7 a 27.0 ab 28.7 a 22.9 b 26.7 ab 29.2 a 0.64 0.393 0.431 0.195

Different superscripts indicate significant differences (P < 0.05) among treatments P-value (significance values) for temperature, diet and

interaction effects determined using two-way ANOVA ( MANOVA ) with FisherÕs least significant difference post-hoc tests.

PRO, high protein to energy ratio diet; NUC, nucleotide fortified diet; HIS, histidine fortified diet; REF, reference experimental diet.

.

in fish fed the PRO diet and lowest in fish fed the HIS or

between diets in terms of lysine and histidine retention

lowest retention efficiency was that of histidine from the HIS

diet and the highest was that of lysine from the PRO diet

Lipid retention was affected by the diet and interaction

terms, but there was no significant effect of temperature

(Table 2)

A series of significant effects on the plasma chemistry were

attributable to temperature, but no diet or interaction term

effects were noted (Table 3) Some discrete differences were

however observed at each temperature for specific

parame-ters

HIS diets had significantly lower CK activity than those fed

diet had significantly higher CK than those fed the PRO, HIS

and REF diets

had significantly lower ALT activity than those fed the NUC,

NUC diet had significantly higher CK than those fed the

PRO, HIS and REF diets

HIS and REF diets had significantly higher GLDH activities

tempera-tures, the fish fed the NUC diet had significantly higher

GLDH than those fed the PRO, HIS and REF diets

LDH activity also varied among treatments within

had significantly higher LDH than those fed the PRO, HIS

and REF diets However, there were no significant

of treatment, diet or interaction term Although total proteinlevels in the plasma showed no significant variability among

significantly lower plasma protein levels than those fed any ofthe other diets

This study examined the potential of a range of dietarystrategies to minimize the impact of elevated water temper-atures on performance parameters of barramundi Clear

interaction terms were identified These observations provide

a clear indication that dietary strategies can be used to reducethe effects of heat stress on growth In addition, clear benefits

of certain dietary strategies on growth even at optimal peratures were also identified

tem-Elevated temperature had a clear effect on all growthparameters, but not so on feed intake This observation isconsistent with those reported by Katersky & Carter (2005)

in feed intake In a similar study with Atlantic salmon, nobenefit was observed from increasing the dietary protein toenergy ratio (Barnes et al 2006) However, in contrast to thepresent study the range of dietary protein to energy ratioexamined in the study by Barnes et al (2006) was not as greatand this may have not allowed any significant effects to havebeen observed Consistent with the maintenance of feedintake levels, but with poorer growth the feed conversion of

poorer, similar to the observations of Katersky & Carter

Table 3 Plasma protein and enzyme activities from barramundi fed each of the dietary treatments

Temperature

Units

Pooled SEM

P-value

Creatinine kinase U L)1 9886 a 10 337 ab 9252 a 16 360 b 29 073 c 46 767 d 30 777 c 29 542 c 3299 0.535 0.000 0.391 Alanine aminotransferase U L)1 16 a 35 b 25 ab 33 b 56 c 68 d 48 c 48 c 5 0.667 0.016 0.831 Glutamate dehydrongenase U L)1 14 a 14 a 20 b 24 b 29 bc 46 d 34 c 33 c 3 0.344 0.000 0.127 Lactate dehydrogenase U L)1 3428 a 4130 a 4766 a 5060 a 11 033 b 16 950 c 10 594 b 10 927 b 1088 0.343 0.000 0.259 Total plasma protein g L)1 42 a 43 ab 44 ab 41 a 42 ab 33 c 45 b 40 a 1 0.238 0.315 0.257 Different superscripts indicate significant differences (P < 0.05) among treatments P-value (significance values) for temperature, diet and interaction effects determined using two-way ANOVA ( MANOVA ) with FisherÕs least significant difference post-hoc tests.

PRO, high protein to energy ratio diet; NUC, nucleotide fortified diet; HIS, histidine fortified diet; REF, reference experimental diet.

.

(2005) Retention of energy and protein was also significantly

impaired at the elevated temperatures (consistent with the

reten-tion of lysine and histidine were not significantly impaired by

elevated water temperatures, although certainly several of the

diets had lower lysine and histidine retention efficiencies at

apparent benefit from the inclusion of nucleotides on any

of the growth parameters could be ascertained at this

temperature

Several key dietary effects were observed in these studies that

have important implications for improving the performance

of barramundi diets An increase in the protein to energy

ratio significantly improved growth of fish relative to the

REF diet, but similar gains were also observed from the

NUC diet These improvements in performance associated

with a higher protein to energy ratio are consistent with

idealized diet specifications determined from modelling

studies with this species (Glencross 2008) Improvements in

growth with the addition of nucleotides to the diet also

provided significant benefit, equal to that achieved with the

increase in protein to energy ratio These observations

sup-port that at optimal growing conditions, where nutrient

demand is highest, that nucleotides may indeed be regarded

as conditionally or semi-essential nutrients (Li & Gatlin

2006) Importantly, it was noted that the addition of

the retention efficiencies of energy, protein, lysine and

histi-dine relative to the REF diet Further work identifying the

specific nature of this improvement in protein metabolism

with the inclusion of nucleotides is certainly warranted

A series of dietary effects on energy and nutrient retention

efficiencies were also observed High protein to energy ratios

were commensurate with improved energy and protein

retention and high lysine retention efficiency at both

nucleotides in the diet (NUC) also improved most retention

parameters compared with those observed in the REF diet

The addition of crystalline histidine to the diet (HIS) resulted

in significantly reduced histidine retention efficiencies at both

temperatures, but did not affect energy, protein or lysine

retention Comparison with the other treatments suggests

that this lower retention efficiency was as a result of a poorer

ability of the fish to use either elevated histidine levels or the

crystalline amino acids In other studies examining theretention of crystalline amino acids they have generally beenfound to be well utilized when included at low levels,equivalent to that of protein-bound amino acids (Williams

Key interaction terms were identified for the retention ciencies of both lysine and histidine The retention of lysine in

difference was not present In contrast to that observed withthe other three diets, an increase in the retention efficiency of

is unusual in that the retention characteristics of these aminoacids are counter to that of the total dietary protein content

some amino acids may be used more efficiently at highertemperatures when present at a lower ratio to energy content,while the total protein retention clearly shows no benefits,which suggests that other amino acids must be used sub-stantially less efficiently

No other interaction effects were observed for any of thegrowth parameter, supporting that growth was discretelyinfluenced by diet or temperature

The use of blood plasma enzymology provides an additionalinsight into the effects of each diet and temperature on thebiochemical processes occurring within the animal Each ofthe enzymes examined in this study are key marker enzymesfor particular cell types or metabolic pathways and as suchprovide key information on the effects of each of the treat-ments on certain tissues and metabolic processes

Elevated CK activities at the higher water temperatures areindicative of broad-scale muscle cell damage (Kopp et al

the PRO and HIS diets compared with the REF diet supportthat these diets reduce the level of muscle cell damage At

present and the NUC diet had an even further elevated level

of CK activity compared with the REF diet These vations support that at elevated temperatures the inclusion ofnucleotides may exacerbate muscle cell damage

obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser- obser-.

A similar elevation in ALT activity at the elevated water

temperatures indicates an increase in liver cell damage (Begum

2004; Refstie et al 2006; Saoudi et al 2008) At optimal water

temperatures, a reduction in ALT activity was observed

to energy ratio also reduced the level of ALT activity

sup-porting that the PRO diet limited the incidence of liver damage

at optimal water temperatures, but not at elevated water

temperatures The NUC diet while not exacerbating ALT

to the REF diet treatment at the same temperature

The increase in GLDH activities at elevated water

tem-peratures are consistent with the increased level of

endoge-nous protein turnover as this enzyme is involved in the

breakdown of amino acids by the reduction of glutamate to

a-ketoglutarate releasing ammonia and allowing access of

amino acids to the tricarboxylic acid cycle (Begum 2004) The

enzyme is also regarded as a marker enzyme of non-muscle

tissue (e.g liver and kidney) and therefore elevated levels

support that the tissue damage occurring is non-muscle

damage That the PRO and NUC diets had further reduced

accentu-ated protein retention and this is consistent with the observed

protein and amino acid retention data

LDH activities are generally indicative of tissue

degener-ation (Rao & Venkateswara 2006) The elevated levels of

damage and are also supportive of an increased level of

inclusion of dietary nucleotides exacerbated the effect of heat

on LDH activities, possibly indicating further tissue damage

from fish fed this diet

Plasma total protein levels were highly consistent at both

total protein of fish fed the NUC diet was observed This

reduction in plasma total protein levels is consistent with

(Refstie et al 2006)

No interaction terms among diet or temperature on any of

the plasma enzymology or plasma total protein levels were

observed

This study demonstrates that there is some potential to

improve the response of barramundi to thermal stress using

dietary strategies Increasing the protein to energy ratio

provides the best option of those examined to minimize

growth retardation owing to thermal stress Other tives such as dietary HIS and NUC fortification, whileimproving growth performance relative to the REF diet atoptimal temperatures, did not provide any benefit underconditions of heat stress

alterna-The authors acknowledge the support of Peter McCaffertyand Ken Dods at the Chemistry Centre of WA for per-forming some of the analytical work associated with thisproject They also thank Malcolm McGrath from the Wes-tern Australian Animal Health Laboratories for undertakingthe plasma chemistry work Dr Brian Jones provided edito-rial input The financial support of the Australian Centre forInternational Agricultural Research is also acknowledged

Barnes, J.C., Hauler, R.C & Carter, C.G (2006) The effect of tein : energy ratio on the growth and protein synthesis of Atlantic salmon (Salmo salar) at 19 C In: Proceedings of the International Society for Fish Nutrition and Feeding, p 56, Biarritz, France, 28 May–1 June INRA, St Pee Sur Nivelle, France.

pro-Begum, G (2004) Carbofuran insecticide induced biochemical alterations in liver and muscle tissues of the fish Clarius batrachus (linn) and recovery response Aquat Toxicol., 66, 83–92 Boonyaratpalin, M & Williams, K.C (2001) Asian seabass In: Nutrient Requirements and Feeding of Finfish for Aquaculture (Webster, C & Lim, C eds), pp 80–90 CABI Publishing, Wal- lingford, UK.

Breck, O., Bjerkas, E., Campbell, P., Rhodes, J.D., Sanderson, J & Waagbo, R (2005) Histidine nutrition and genotype affect cataract development in Atlantic salmon, Salmo salar L J Fish Dis., 28, 357–371.

Carter, C.G., Bridle, A.R., Ketersky, R.S., Barnes, J.C & Hauler, R.C (2006) Influence of nutrition and thermal stress on protein synthesis and degradation in fish In: Proceedings of the Interna- tional Society for Fish Nutrition and Feeding, p 57, Biarritz, France, 28 May–1 June INRA, St Pee Sur Nivelle, France Folch, J., Lees, 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.

Glencross, B.D (2006) Nutritional management of barramundi, Lates calcarifer – a review Aquacult Nutr., 12, 291–309 Glencross, B.D (2008) A factorial growth and feed utilisation model for barramundi, Lates calcarifer based on Australian production conditions Aquacult Nutr., 14, 360–373.

Helland, S., Grisdale-Helland, B & Nerland, S (1996) A simple method for the measurement of daily feed intake of groups of fish

.

Li, P & Gatlin, D.M (2006) Nucleotide nutrition in fish: current

knowledge and future applications Aquaculture, 251, 141–152.

Maynard, L.A & Loosli, J.K (1969) Animal Nutrition, 6th edn.

McGraw-Hill Book Co., New York, NY.

McMeniman, N (1998) Digestibility of feed ingredients in

barra-mundi In: Fishmeal Replacement in Aquaculture Feeds for

Barra-mundi Final Report of Project 93/120-03 (Williams, K.C ed.),

pp 16–37 Fisheries Research and Development Corporation,

Canberra, Australia.

Mora, C & Maya, M.F (2006) Effect of the rate of temperature

increase of the dynamic method on the heat tolerance of fishes.

J Thermal Biol., 31, 337–341.

Rao, J & Venkateswara, A (2006) Biochemical alterations in

euryhaline fish, Oreochromis mossambicus exposed to sub-lethal

concentrations of an organophosphorus insecticide,

monocroto-phos Chemosphere, 65, 1814–1820.

Refstie, S., Glencross, B., Landsverk, T., Sørensen, M., Lilleeng, E.,

Hawkins, W & Krogdahl, A (2006) Digestive function and

intestinal integrity in Atlantic salmon (Salmo salar) fed kernel meals and protein concentrates made from yellow or narrow- leafed lupins Aquaculture, 261, 1382–1395.

Saoudi, M., Abdelmouleh, A., Kammoun, W., Ellouze, F., Jamoussi,

K & El Feki, A (2008) Toxicity assessment of the puffer fish Lagocephalus lagocephalus from the Tunisian coast C R Biol.,

331, 611–616.

Uauy, R., Stringel, G., Thomas, R & Quan, R (1990) Effect of dietary nucleotides on growth and maturation of the developing gut in the rat J Paediatr Gastroenterol Nutr., 10, 497–503.

Williams, K.C., Barlow, C.G & Rodgers, L (2001) Efficacy of crystalline and protein-bound amino acids for amino acid enrich- ment of diets for barramundi/Asian seabass (Lates calcarifer Bloch) Aquacult Res., 32, 415–429.

Williams, K.C., Barlow, C.G., Rodgers, L & Agcopra, C (2006) Dietary composition manipulation to enhance the performance of juvenile barramundi (Lates calcarifer Bloch) reared in cool water.

Aquacult Res., 37, 914–927.

.

1 1 1 2,3 1

Australia

In this study, we undertook a series of experiments to

assess different faecal-collection methods and also the

digestibilities of a range of feed ingredients when fed

to Tra catfish (Pangasinodon hypothalamus) Apparent

digestibility coefficients for dry matter, energy and nitrogen

for a reference diet were also determined from faeces

collected by way of settlement at 2-h intervals from 2

to 24 h No significant effect of sample collection time

on the digestibility values was observed Stripping was

attempted, but was not considered a suitable method for

collecting digesta from Tra catfish Dissection collection

was also evaluated Values determined from dissection

collection were lower than those from using settlement

In a second experiment, a suite of test ingredients was

combined with a reference diet component in a 30 : 70

ratio to determine the ingredients for dry matter, energy

and nitrogen digestibilities Ingredients included were

trash-fish, defatted rice bran, wet full-fat rice bran, dried full-fat

rice bran, broken rice, cassava, soybean and fish meals

Collection of faeces by settlement was used for determining

the digestibility of each of the ingredients The results

showed that Tra catfish can efficiently digest protein and

energy from a wide range of feed ingredients and nutrient

sources

KEY WORDS: catfish, digestibility, faecal collection, methods,

Pangasinodon hypothalamus, Pangasius, rice bran

Received 12 December 2008, accepted 25 February 2009

Correspondence: Dr Brett Glencross, Brett PO Box 20, North Beach 6920.

E-mail: Brett.Glencross@csiro.au.

The culture of Pangasius catfish in the Mekong Delta region isincreasing in terms of both production tonnage and culture

815 000 tonnes, which accounted for approximately 80% ofthe total freshwater aquaculture production of the MekongDelta region (MOFI 2008) Of this production, the majority is

of Tra catfish, Pangasius hypothalamus The average feed costtypically comprises more than 80% of the total variable pro-duction costs in this industry, varying from 73.6% amongfarm-made feed category to 92.5% among manufacturedpellet feed farm category (Phuong et al 2007) However, there

is little data on the nutritional value of most common feedingredients used in diets for Tra catfish The evaluation of thedigestible protein and energy value of feed ingredients is crit-ical to the cost-effective formulation of modern aquaculturediets and is also an important part of the process in estab-lishing their nutritional value (Glencross et al 2007) How-ever, it is well known that the faecal-collection method caninfluence the digestibility assessment of a diet (Weatherup &McCracken 1998; Vandenberg & de la Noue 2001; Glencross

feasibility for collection of faecal samples from Pangasinodon

faecal-collec-tion methods to assess the digestible protein and energy value

of a range of feed ingredients widely used in feed formulationsfor Tra catfish in the Mekong Delta region of Vietnam

Experimental fish were hatchery reproduced at CanthoUniversity Fish were on-grown to test size (100 g) in

.

2010 16; 351–358

. doi: 10.1111/j.1365-2095.2009.00671.x

Aquaculture Nutrition

concrete tanks by feeding a commercial pelleted feed

(UniPresident, Dong Nai City, Vietnam) containing 32%

protein One week prior to start the experiment, fish were

transferred into experimental tank system for acclimation

and continued to fed the same diet prior to the introduction

of the experimental reference diet

The apparent digestibility coefficients (ADC) were estimated

for a reference (Table 1) and a series of test diets (Cho et al

1982) The reference diet was formulated to ensure that fish

obtained all essential nutrition for the normal growthaccording to currently understood Pangasius catfish dietaryrequirements (Hien & Yen 2005) The proximate composi-

por-tion of the reference diet was combined with each testingredient in a 70 : 30 ratio A total of eight test ingredientswere evaluated; these included: defatted rice bran (DRB), wetfull-fat rice bran (WFRB), dried full-fat rice bran (DFRB),fishmeal, broken rice, cassava meal, defatted soybean mealand trash-fish (Table 2) The diets were prepared by thor-oughly mixing the dry ingredients with oils and then addingdistilled water until a stiff dough was formed This was thenpassed through a screw-press with a 2.5-mm die before being

Trash-fish were included into the diet on a wet basis [allowing for anestimated 30% inclusion on a dry matter (DM) basis] beforethe diets were dried

For the settlement methodology studies, a series of ibility tanks of 180 L were used Each tank had a cylindro-conical base sloped at 360 fitted with a 65-mm diameter,250-mm long collection chamber that tapered into a 12-mmdiameter, 150-mm length of silicone tubing Continuouslyflowing, preheated (28 C) freshwater was filtered through

digest-a cotton filter, then digest-a didigest-atomdigest-aceous edigest-arth filter beforepassing on to the experimental tanks at a flow rate of

diffusers (Allan et al 1999) For dissection and strippingmethod assessment, fish from the same batch were main-tained at the same conditions, but in 600-L square concretetanks

Table 1 Formulations and composition of the reference diet (all

1 Vitamin and mineral premix includes (IU kg)1or g kg)1of

pre-mix): vitamin A, 400 000 IU; vitamin D3, 80 000 IU; vitamin E, 12 g;

vitamin K, 3, 2.4 g; vitamin B1, 1.6 g; vitamin B2, 3.0 g; vitamin B6,

1.0 g; niacin 1.0, vitamin B9, 0.8; vitamin B12, 0.004 g; folic acid,

0.032, biotin, 0.17 g; vitamin C, 60 g; choline, 4.8 g; inositol, 1.5 g;

ethoxyquin, 20.8 g; Copper, 10 g; ferrous iron, 20.0 g; magnesium,

16.6 g; manganese, 2.0 g; zinc, 11 g.

DM, dry matter.

Table 2 Composition (g kg)1 DM, unless otherwise detailed) of key feed ingredients for Tra catfish

Fishmeal Soybean TF WFRB DFRB DRB Cassava BR Ingredient specifications

TF, marine trash-fish; WFRB, wet full-fat rice bran; DFRB, dried full-fat rice bran; DRB, defatted

rice bran; BR, broken rice.

Fishmeal: Kiengiang Fish meal Company, Kiengiang province, Vietnam; Soybean: RAJA Fat and

feeds Private limited, India; Trash-fish: Local fishermen, Kiengiang province, Vietnam; WFRB,

DFRB, BR: Broken rice, cassava meal: Gentraco Feed, Cantho province, Vietnam; DRB: Cai Lan Oils

& Fats Industries Company Ltd, Can Tho Branch, Cantho City, Vietnam.

1 Carbohydrate based on dry matter (DM) ) (protein + ash + lipid).

.

This experiment was conducted in three settlement tanks.

Mass of faeces collected, ADC of DM, energy and nitrogen

for the reference diet were calculated and compared with

faeces collected by every 2 h after feeding (2, 4, 6, 8, 10, 12,

14, 16, 18, 20, 22, 24 h) Fish were stocked 7 days prior to the

start of the faecal-collection period to allow for

were stocked into each tank During this period, fish were fed

the reference diet (Table 1) Fish were fed to excess using by

food was removed, and the walls of the tank and the

settle-ment chamber were thoroughly cleaned to remove any faeces,

uneaten food or bacterial slime The silicone tubing into

prior to the removal of faeces to reduce bacterial

prolifera-tion, which can affect the composition of faeces (Spyridakis

The ADC of DM, energy and nitrogen for the reference diet

and two test ingredients (fish meal and soybean meal) were

calculated and compared using either settlement, stripping or

dissection faecal-collection technique Fish were stocked

7 days prior to the start of the faecal-collection period to

allow for acclimation to the experimental conditions and

each of the diets Twenty fish (91 g) were allocated to each

of nine settlement tanks Eighteen large 600-L square

con-crete tanks were stocked with 50 fish of the same sample size

as used in the nine settlement tanks

During this acclimation period, fish were fed each diet to

after the 12th hour of fish feeding, faecal collection was

commenced For the settlement technique, faecal samples

from different days but within each tank were pooled for

analysis For dissection techniques, all 50 fish within each

tank were killed, the distal intestine dissected and faeces were

expelled with gentle pressure into sample container For

stripping, all 50 fish were netted from each tank, the fish were

anaesthetized using MS-222, and then gentle pressure applied

to the abdomen near the distal intestine of the fish to expel

faeces onto the fingertips The faeces were placed into a

for analysis All fish were stripped only once During

pH between 7.7 and 8.3, and water temperature ranged from

This study consisted of nine treatments each allocated tothree randomly selected, replicate tanks A sample of thereference diet was combined with each test ingredient in a

70 : 30 ratio (Table 3) A total of eight test ingredients andone reference diet were evaluated The test ingredientsincluded; DRB, WFRB, DFRB, fishmeal, broken rice, cas-sava meal, soybean meal and trash-fish (Table 2) Faeceswere collected by settlement over a 12-h period The ADC ofeach of the test ingredients was calculated based on definingthe ADC of test diets and the ADC of reference diet based onthe proportion of reference diet and test ingredient in eachtest diet, and the proportion of nutrient/energy contributed

by each test ingredient in test diet (Sugiura et al 1998)

27 tanks) After 7 days acclimatization, faeces were collectedand pooled for each tank for 7 days Faeces were stored at)20 C before being dried for analysis Mean water tem-

Table 3 Formulations of the experimental diets (all values are in

vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita- vita-.

Diet and faecal samples were analysed for DM, chromium,

ash, phosphorus, nitrogen and gross energy content DM was

calculated by gravimetric analysis following oven drying at

bal-listic bomb calorimetry Protein levels were calculated from

the determination of total nitrogen by kjeldhal analysis,

acid-hydrolysis Soxhlet method Chromic oxide levels were

determined by spectrophotometric analysis following heating

in kjeldhal system with nitric acid and perchloric acid

Car-bohydrate was determined as the difference in DM content

minus protein, ash and fat All of these determinations were

conducted according to the methods specified by the AOAC

(2000)

Differences in the ratios of the parameters of DM, protein

or gross energy relative to chromium, in the feed and faeces

in each treatment were calculated to determine the apparent

parameters examined in each diet based on the following

formula

Diet 1 in each experiment is the referent diet The apparent

variable is based on the following formula (Eqn 1):

Crfaeces  Nutrientdiet

ð1Þ

chromium content of the diet and faeces respectively, and

parameter of concern (e.g protein or energy) in the diet and

faeces respectively With this formula, values range from 0 to

1 To achieve a percent apparent digestibility, the values

should be multiplied by 100

from the test ingredient included in the test diet at 30%

interest in the test diet ADC reference is the apparent

digestibility of the same nutrient from the reference diet,

which makes up 70% of the test diet (Eqn 2):

ð2Þ

nutrient from the test ingredient included in the test diet at

ingre-dient, test diet and basal diet respectively (Sugiura et al

1998) Where the inclusion level of the test ingredient varies,then the new ratio needs to be included in this equation inplace of the 70% and 30% values

The data were subjected to one-way analysis of variance(ANOVA; P < 0.05) Significant differences between treatmentmeans were compared using DuncanÕs multiple range test atthe 5% level of significance Data normality and homo-

USA) tests, version 13.0

Faecal quantities based on the amounts of faeces collected byway of settlement increased from the 2- to 14-h time pointafter feeding From the collections after 12- to 18-h timepoint, the faecal quantities collected were higher comparedwith the other time point, e.g 2- to 14-h time point (Fig 1)

After 18-h time point, the faecal quantities collected ished The ADC for DM calculated using faeces collected foreach separate 2-h interval showed no significant differencesacross time

dimin-0.00

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Time post feeding (h)

ADC Amount (g/tank)

Figure 1 Quantities of faeces collected and dry matter digestibility over varying settlement periods postfeeding No significant differ- ences in dry matter digestibility (apparent digestibility coefficient, ADC) were observed over time Each data point represents n = 3.

.

Stripping was identified as not a suitable method for

col-lecting digesta from Tra catfish There was difficulty in

obtaining sufficient sample because of the structure of the fish

intestinal tract and abdominal muscle layer However,

suffi-cient samples and faeces/digesta was able to be collected

using either the settlement or dissection methods Significant

differences between the settlement and dissection-collection

methods were noted on the digestibility traits of the diets

DM, protein and energy digestibilities of the diets calculated

from samples collected using dissection were consistently

lower than those obtained using settlement collection

However, the digestibility coefficients of the test ingredients

(fish meal and soybean meal) were unaffected by

faecal-collection method (Table 4)

study had a range of compositions (Table 2) Among

the protein source ingredients, the fish meal and trash-fish

had significantly different levels of protein (677 and

than wet rice bran and dry rice bran protein levels (134

which was less than that of the broken rice and cassava

on faecal samples collected using settlement techniques,

DM digestibilities of the ingredients varied substantially(Table 5) For protein feed ingredients, the total levels ofdigestible DM was the lowest for SBM (81.9%) and thehighest for both FM and trash-fish (87.7%), which wasfound to be a significant difference (P < 0.05) among thoseingredients DM digestibilities were significantly higher forbroken rice and cassava meal (83.2–90.7%) than rice bran,which ranged between 57% and 82% In rice bran group,WRB had a significantly (P < 0.05) higher DM digestibility(82%) in all Digestibility coefficients for energy were sig-nificantly higher for fish meal and trash-fish than for SBM.Protein digestibility was high for all protein ingredients

among those ingredients However, protein digestibility ofthe Cassava meal was the poorest (35.8%) of all theingredients evaluated Protein digestibility was similar forthe remaining ingredients (65.1–70.4%) and were not sig-nificantly different (P > 0.05)

This study examined some of the methodological ations for undertaking digestibilities studies with Pangasiuscatfish The practicalities and results from different faecal-collection methods, the time of postfeeding for optimal col-lection and the nutritive value of some key feed ingredientswere all examined

consider-Of the three methods for faecal/digesta collection attempted,stripping was considered an ineffective method of samplingdigesta from catfish This was because of difficulty inobtaining sufficient sample due to the structure of the fishintestinal tract and abdomen muscle layer Attempts toobtain digesta by stripping were also abandoned by Allan

quantities from silver perch (Bidyanus bidyanus) Despitethese problems, it is acknowledged that stripping and dis-section are the preferred methods for collecting faecal/digestasamples for fish species, especially where the faeces are

Table 4 Comparison of faecal-collection methods on diet and

ingredient digestibility parameters

Collection

methods

Dry matter (%)

Protein (%)

Energy (%) Reference diet digestibility

Values are means ± SD (n = 3) Different superscripts within rows

indicate significant differences between two collection methods at

P < 0.05.

.

loosely bound or contained substantial levels of

carbohy-drates (McMeniman & Sands 1996; Glencross et al 2005)

The ADCs calculated using digesta obtained by dissection

were considerably lower than those calculated using faeces

collected using the settlement method This was consistent

with other reports comparing active and passive collection

methods (Vandenberg & de la Noue 2001; Glencross et al

2005) Settlement collection was used as the preferred method

primarily due to the ability to collect a larger faecal sample

size without interfering or sacrificing fish and in the minimum

time However, collecting faeces after they have been voided

can result in leaching of DM and nutrients and potentially

lead to overestimation of digestibility values Collection

facilities that ensure rapid settlement of faeces (Cho &

Kaushik 1990; Satoh et al 1992; Hajen et al 1993) or

con-tinuous filtration from the water column (Choubert et al

1982; Spyridakis et al 1989) have been used as an attempt to

reduce this problem

Significant effects of faecal-collection method were also

noted on the digestibility assessment of the test ingredients

(fish meal and soybean meal) in this study This is consistent

with the findings of Glencross et al (2005) who also showed

that faecal-collection method affected ADCs of both the diets

and ingredients fed to rainbow trout

The similarity between the ADCs calculated from faeces

collected at 2-h intervals, and those calculated using faeces

collected over a 24-h period cumulatively after feeding,

supports that leaching from faeces in the collecting chamber

was not a significant problem over a 24-h period and that

there was no advantage in collecting samples more frequently

than a daily sample Similarly, Satoh et al (1992) found

minimal differences in lipid or protein digestibility when

faeces from rainbow trout were collected using settlement 3,

6, 9, 12 or 15 h after feeding Similar results were also

obtained when faeces from silver perch were collected using

settlement periods of 2–18 h after tanks were cleaned (Allan

The composition of the feedstuffs evaluated in this study isconsistent with the values reported in earlier publications(NRC 1993; Usmani et al 2003) for similar raw materials

The rice brans, cassava and broken rice are clearly, based ontheir low protein levels, primarily intended as potential energysources, whereas the trash-fish, fish meal and soybean mealconstitute the main protein sources The use of digestibilityinformation in least-cost diet formulation assumes that theADCs for separate ingredients are additive This was con-firmed for rainbow trout by separately measuring digestibility

of component ingredients of a reference diet and then paring the sum of these individual components on a propor-tional basis with direct measurement of the complete diet(Cho et al 1982) Results show that catfish have the tendency

com-to digest DM and protein in feedstuffs of animal origin moreefficiently than DM in feedstuffs of plant origin This is sug-gestive of a limited ability to digest non-starch polysaccha-rides by Pangasius catfish The DM ADC for fish meal was87.7% while that for soybean meal was only 81.9% Sullivan

& Reigh (1995) also observed that the DM digestibility ofMenhaden fish meal in hybrid striped bass (Morone soxetil-

However, the present study also found high digestibility ofprotein from soybean meal as well as fishmeal and trash-fishsources Similar observations have also been reported forchannel catfish, Ictalurus punctatus (Wilson & Poe 1985)

Lorico-Querijero & Chiu (1989) reported high digestibility ofboth plant and animal protein sources for tilapia, Oreochrimisniloticus, whereas in other studies on Mystus nemurus (Khan1994) and Cyprius carpio (Degani et al 1997), digestibilityvalues for fishmeal were found to be higher than that ofsoybean meal Allan et al (1999) reported that silver perch(B bidyanus) has higher DM ADCs for fishmeal higher thanthat of soybean, but protein ADC values were high and nosignificant differences were observed among those ingredients

Protein digestibility observed for rice bran in the presentstudy was similar to values reported for other species Clarias

Table 5 Apparent digestibility (%) specifications of test ingredients as determined using settlement faecal- collection methods

Values are means (n = 3) Different superscripts within rows indicate significant differences

between means among ingredients at P < 0.05.

TF, marine trash-fish; WFRB, wet full-fat rice bran; DFRB, dried full-fat rice bran; DRB, defatted

rice bran; BR, broken rice.

.

Channel catfish (Wilson & Poe 1985) The relatively low

values for protein digestibility are suggested to be artefacts

from the high levels of indigestible fibre present in the rice

bran acting as bulking agents with these ingredients and are

consistent with the low DM and energy digestibilities also

seen for these ingredients The low protein digestibility of the

cassava is unusual and suggests the influence of either

anti-nutritional factors such as protease inhibitors (Francis et al

2001) or protein damage from cooking (Glencross et al

2004) The high DM and energy digestibilities of broken rice,

cassava and wet rice bran that were noted in the present study

support that these ingredients are potentially useful energy

sources for use in Pangasisus diets In contrast, the

digest-ibility of DM and energy from DFRB and DRB were low and

indicative that these ingredients are less suitable as energy

sources

In conclusion, the collection of faeces by settlement was the

preferred method for determining digestibility in Tra

(Pangasius) catfish The results show that Tra catfish can

digest protein and energy from a wide range of feed

ingre-dients and nutrient sources These data can be used not only

to better define nutrient and energy requirements but also to

better formulate nutritionally efficient, cost-effective feeds for

this species However, further work is required to identify

and evaluate the nutritional value of additional protein

sources other than trash-fish and soybean and fish meals

Allan, G.L., Rowland, S.J., Parkinson, S., Stone, D.A.J &

Jantr-arotai, W (1999) Nutrient digestibility for juvenile silver perch

Bidyanus bidyanus: development of methods Aquaculture, 170,

131–145.

AOAC (2000) Official Methods of Analysis of the Association of

Official Analytical Chemists AOAC, Washington, DC.

Cho, C.Y & Kaushik, S.J (1990) Nutritional energetics in fish:

energy and protein utilisation in rainbow trout (Salmo gairdnerii).

World Rev Nutr Diet., 61, 132–172.

Cho, C.Y., Slinger, S.J & Bayley, H.S (1982) Bioenergetics of

sal-monid fishes: energy intake, expenditure and productivity Comp.

Biochem Physiol., 73B, 25–41.

Choubert, G., De la Noue, J & Luquet, P (1982) Digestibility in

fish: improved device for the automatic collection of feces

Aqua-culture, 29, 185–189.

Degani, G., Viola, S & Yehuda, Y (1997) Apparent digestibility

coefficient of protein sources for carp, Cyprinus carpio L Aquacult.

Res., 28, 23–28.

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

factors present in plant-derived alternate fish feed ingredients and

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

Glencross, B.D., Hawkins, W.E & Curnow, J.G (2004) Nutritional assessment of Australian canola meals I Evaluation of canola oil extraction method, enzyme supplementation and meal processing

on the digestible value of canola meals fed to the red seabream (Pagrus auratus, Paulin) Aquacult Res., 35, 15–24.

Glencross, B.D., Hawkins, W.E., Evans, D., McCafferty, P., Dods, K., 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 for aquaculture feeds Aquacult Nutr., 13, 17–34.

Hajen, W.E., Beames, R.M., Higgs, D.A & Dosanjh, B.S (1993) Digestibility of various feedstuffs by post-juvenile chinook salmon Oncorhynchus tshawytscha.in sea water: I Validation of technique Aquaculture, 112, 321–332.

Hien, T.T.T & Yen, D.T (2005) Study on Nutrition Requirement for 3 Species Cast Fish Pangasinodon bocourti, P conchophilusand and

P hypophthalmus Ministry of Education and Academic Project, 60

pp (in Vietnamese) CanTho University, CanTho City, Vietnam Khan, M.S (1994) Apparent digestibility coefficients for common feed ingredients in formulated diets for tropical catfish, Mystus nemurus (Cuvier & Valenciennes) Aquac Fish Manage., 25, 167– 174.

Lorico-Querijero, B.V & Chiu, Y.N (1989) Protein digestibility study in Oreochromis niloticus using chromic oxide indicator Asian Fish Sci., 2, 177–191.

McMeniman, N.P & Sands, N (1996) Estimation of in vivo digestibility of diets to barramundi Lates calcarifer In: Second World Fisheries Congress Proceedings, Vol 1 (Hancock, D.A & Beumer, J.P eds), pp 100–101 CSIRO Publishing, Melbourne, Vic., Australia.

MOFI (Ministry of Fisheries) (2008) Annual Report by the Ministry

of Fisheries (MOFI) MOFI, Hanoi, Vietnam (In Vietnamese) NRC (National Research Council) (1993) Nutrient Requirements of Fish National Academy Press, Washington, DC, 114 pp Phuong, N.T., Sinh, L.X., Thinh, N.Q., Chau, H.H., Anh, C.T & Hau, N.M (2007) Economics of aquaculture feeding practices: Viet Nam In: Economics of Aquaculture Feeding Practices in Selected Asian Countries (Hasan, M.R Ed.), pp 183–205 Tech- nical Paper No.505, FAO, Rome.

Satoh, S., Cho, C.Y & Watanabe, T (1992) Effect of faecal retrieval timing on digestibility of nutrients in rainbow trout diet with the Guelph and TUF feces collection systems Nippon Suisan Gakkai Shi, 58, 1123–1127.

Spyridakis, R., Metailler, J., Gabaudan, J & Riaza, A (1989) Studies on nutrient digestibility in European seabass Dicentrarchus labrax: I Methodological aspects concerning digesta collection Aquaculture, 77, 61–70.

Sugiura, S.H., Dong, F.M., Rathbone, C.K & Hardy, R.W (1998) Apparent protein digestibility and mineral availabilities in vari- ous feed ingredients for salmonid feeds Aquaculture, 159, 177– 202.

Sullivan, J.A & Reigh, R.C (1995) Apparent digestibility of selected feedstuffs in diets for hybrid striped bass (Morone saxatilis female

x Morone chrysops male) Aquaculture, 138, 313–322.

Usmani, N., Jafri, A.K & Khan, M.A (2003) Nutrient digestibility studies in Heteropneustes fossilis (Bloch), Clarias batrachus (Linnaeus) and C gariepinus (Burchell) Aquacult Res., 34, 1247– 1253.

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Vandenberg, G.W & de la Noue, J (2001) Apparent digestibility

comparison in rainbow trout (Oncorhynchus mykiss) assessed using

three methods of faeces collection and three digestibility markers.

Aquacult Nutr., 7, 237–245.

Weatherup, R.N & McCracken, K.J (1998) Comparison of

the estimation of digestibility of two diets for rainbow

trout, Oncorhynchus mykiss (Walbaum), using two markers and two methods of faeces collection Aquacult Res., 29, 527–

1 2 1 1 3

1

Laborato´rio de Enzimologia (LABENZ), Departamento de Bioquı´mica; Laborato´rio de Imunopatologia Keizo Asami (LIKA),

The aim of this study was to examine proteinases and

pep-tidases from the hepatopancreas of two sub-adult stages of

individuals (P < 0.05) The highest activity among

amino-acyl-b-naphthylamide substrates was found using alanine-,

arginine-, leucine- and lysine-b-naphthylamide There was a

positive correlation between the recommended concentration

of essential amino acids in penaeid shrimp feed and

amino-peptidase activity in both sub-adult stages Proteolytic

activity of F subtilis was strongly inhibited by specific trypsin

inhibitors The optimal temperature for trypsin,

chymo-trypsin and leucine aminopeptidase activity was between 45

inhibited by phenylmethylsulfonyl fluoride in both sub-adult

stages The use of tosyl-lysine-chloromethyl-ketone and

benzamidine caused strong inhibition of the proteolytic

bands Trypsin and chymotrypsin activity was the main

KEY WORDS

KEY WORDS: aminopeptidases, chymotrypsin, digestive

enzymes, Farfantepenaeus subtilis, southern brown shrimp,

trypsin

Received 6 November 2008, accepted 20 March 2009

Correspondence: Ranilson S Bezerra, Laborato´rio de Enzimologia –

LABENZ, Departamento de Bioquı´mica, Universidade Federal de

Per-nambuco, Cidade Universita´ria, Recife – PE 50670-420, Brazil E-mail:

ransoube@uol.com.br

The southern brown shrimp, Farfantepenaeus subtilis, is native

to the Atlantic coast of Central and South America, fromCuba down to Rio de Janeiro, and was one of the first species

to be farmed in Brazil, along with Farfantepenaeus brasiliensis,

The southern brown shrimp exhibits benthic omnivorousopportunistic feeding habits under semi-intensive conditions,although polychaetes and calanoid copepods seem to befavoured during all growth stages (Nunes & Parsons 2000).Despite its farming potential and attractive market fea-tures, the culture of F subtilis in semi-intensive conditions inBrazil has failed mainly due to low yields Studies carried out

by Brazilian farmers report a food conversion ratio rangingfrom 2.88 to 3.44 and a poor growth performance, thusgenerating low productivity The growth rate slows after theshrimp reach 6 g of body weight This suggests that the poorresults may be related to nutritional problems and ontoge-netic changes in the digestive enzyme metabolism (Maia &Nunes 2003)

Comprehension of digestion physiology and nutrientdigestibility remains a problem for the culture of F subtilis.Knowledge concerning the digestive system of this speciescan provide information applicable to food utilization Thus,the identification and characterization of digestive enzymesduring shrimp growth is an important step towards under-standing the digestive mechanisms and formulating feeds thatpromote better growth responses, as feed can be designedaccording to the digestive capacity (Lo´pez-Lo´pez et al 2005)

A number of studies have indicated properties of tive enzymes in shrimp and other crustaceans, such asproteases, carbohydrases, lipases and the digestibility offeed ingredients (Lemos et al 2000, 2004; Co´rdova-Murueta

diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges-.

2010 16; 359–369

. doi: 10.1111/j.1365-2095.2009.00673.x

Aquaculture Nutrition

et al 2003; Muhlia-Almaza´n et al 2003; Gaxiola et al.

2005; Lo´pez-Lo´pez et al 2005) However, synthesis

regula-tion and enzyme activity are species-specific (Ferna´ndez

Gimenez et al 2002) and it is therefore not possible to

extrapolate characteristics from one species to another This

peptidases as well as certain properties of these digestive

proteases from the hepatopancreas of the southern brown

shrimp, F subtilis, in two sub-adult stages These findings

provide basic information on protein digestion and will be

useful in further nutritional research

commercial fishery on the coast of Barra de Sirinhae´m

(836¢S; 351¢W), 100 km from the city of Recife in the

state of Pernambuco, Brazil All reagents used in the

enzyme assays were of analytical grade, purchased from

Sigma (St Louis, MO, USA) and Merck (Darmstadt,

Germany)

The specimens were transported alive to the Enzymology

Laboratory of the Universidade Federal de Pernambuco

Sixty-litre plastic bags were used for temporary storage at a

density of two specimens per bag in 12 L of salt water The

water was saturated with oxygen and a ratio of one-third

water to two-thirds oxygen was maintained in each bag

(approxi-mately 13 g of wet weight) The hepatopancreas from 20

0.60 g wet weight) specimens were dissected immediately

after killing by decapitation For each sub-adult stage,

four sets of five hepatopancreases were homogenized

Elvehjen tissue homogenizer (Bodine Electric Company,

Homogenates were centrifuged at 10 000 g for 25 min at

use The total soluble protein was determined following the

procedure described by Bradford (1976), using bovine serum

albumin as the standard protein

Non-specific proteolytic activity was assayed using azocasein

as substrate in a microcentrifuge tube Triplicate samples of

tri-chloroacetic acid (120 lL) was added to stop the reactionand the mixture was centrifuged at 8000 g for 5 min The

and absorbance was measured in a microtitre plate reader(Bio-Rad 680, Japan) at 450 nm against a similarly prepared

Previous experiments demonstrated that, for the first 60 min,the reaction carried out under these conditions follows firstorder kinetics Protease activity was expressed as units per

mg of protein One unit (U) of enzyme activity was defined asthe amount of enzyme required to hydrolyze azocasein and

Trypsin, chymotrypsin and leucine aminopeptidase activitywas determined in a 96-well microtitre plate, using ben-

succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAPNA) andleucine-p-nitroanilide (Leu-p-Nan) as specific substratesrespectively (Bezerra et al 2005) Triplicate samples of

BAPNA, SAPNA or Leu-p-Nan (30 lL) dissolved in

recorded at 405 nm using a microplate reader Activity was

activity was defined as the amount of enzyme required to

Aminopeptidase activity was also evaluated using acyl-b-naphthylamide as substrate The substrates used wereAla, Arg, Leu, Phe, Val, Ser, Gly, Ile, Tyr, His, Lys and Glu

equilibration, the enzyme (50 lL) was added After 120 min,the reaction was stopped by adding fresh Garnet reagent

mea-sured at 525 nm and the amount of b-naphthylamine wasdetermined using a standard b-naphthylamine curve Activity

.

of enzyme activity was defined as the amount of enzyme

(Oliveira et al 1999)

The following inhibitors prepared in DMSO at a final

fluoride (PMSF – serine proteases inhibitor),

tosyl-lysine-chloromethyl-ketone (TLCK) and benzamidine (both

tryp-sin inhibitors); Tosyl-phenylalanine chloromethyl ketone

(TPCK – chymotrypsin inhibitor); and bestatin

(aminopep-tidase inhibitor) (Bezerra et al 2005) Triplicate samples of

enzyme extract (25 lL) and inhibitors (25 lL) were placed in

Tris–HCl, pH 8.0, and the respective substrate (BAPNA –

PMSF, TLCK and benzamidine; SAPNA – PMSF and

TPCK; Leu-p-Nan – bestatin) and the proteolytic activity

were determined as described above The enzyme and

sub-strate blank were similarly assayed without enzyme and

substrate solution respectively The 100% values were

established using DMSO without inhibitors

The effects of pH and temperature on proteolytic activity of

the F subtilis enzyme extract were evaluated as described

Ther-mal stability was evaluated by assaying enzyme activity at

Proteases from F subtilis were studied by sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE),

using a 4% (w/v) stacking gel and 12.5% (w/v) separating gel

(Laemmli 1970) Enzyme preparations and molecular weight

markers (15 lL) (ovoalbumin – 46 kDa, glyceraldehyde

3-phosphate dehydrogenase – 36 kDa, carbonic anhydrase –

29 kDa, trypsinogen – 24 kDa and a-lactalbumin – 14.2 kDa)

were applied to each track of a vertical electrophoresis device

(Bio-Rad) The gels were stained for protein overnight in

acid acetic and methanol (65 : 10 : 25) and the background

of the gel was de-stained in the same solution without dye

Electrophoresis was performed at a constant current of 15 mA

Zymograms were also carried out, following the proceduredescribed by Garcia-Carren˜o et al (1993) After electro-

to remove the SDS The Triton X-100 was removed by

buffer, pH 8.0 The SDS-free, Triton X-100-free gels were

casein by the active fractions Finally, the gels were stainedand de-stained as described previously Thermal stability wasalso determined using the caseinolytic zymogram The samemethodology described above was employed, except thatsamples were pre-incubated at temperatures ranging from 25

were pre-incubated with serine protease and trypsin tors as described above (inhibition assays) Samples incu-bated with proteinase inhibitors were compared with control(without inhibitors) Decrease of the intensity or evanishment

inhibi-of the bands indicated inhibition inhibi-of proteolytic activity,identifying the type of enzyme

Data (mean ± standard deviation) processing was carried

Differences between mean values were analysed using theStudentÕs t-test Differences were considered significant when

pres-ence of trypsin-, chymotrypsin- and leucine like enzymes Trypsin-like (BAPNA) and chymotrypsin-like(SAPNA) activity was significantly higher (P < 0.05) in

aminopep-tidases was more evident with b-naphthylamide substrates(Table 1) Higher activity was found for basic (Arg-, Lys-)and non-polar (Ala-, Leu-) substrates Lower activityoccurred with aromatic (Tyr-), uncharged polar (Ser-) andnon-polar (Gly-) substrates While the activity was measured

.

for all aminoacyl-b-naphthylamide substrates used, some of

which (Glu-, Ile-, Phe-, His- and Val-b-naphthylamide)

showed negligible activity and were not reported There were

as substrates A positive correlation between the

recom-mended concentration of essential amino acids for penaeid

feed (Guillaume 1997) and aminopeptidase activity was

de-tected in both sub-adult stages, using

aminoacyl-b-naph-thylamide as substrates (Fig 1)

Digestive proteases were partially inhibited by PMSF

(using BAPNA and SAPNA as substrates) in both sub-adult

stages (Table 2) TLCK and benzamidine exhibited a strong

specimens TPCK affected chymotrypsin activity in both

sub-adult stages Leucine aminopeptidase activity was inhibited

The highest trypsin-like activity was obtained in a pH

aminopeptidase was 8.0 in both sub-adult stages (Fig 2)

Figure 3 displays the influence of temperature on lytic activity The optimal temperature for the trypsin-like

experi-mental conditions Trypsin-like activity dropped sharply

observed for the chymotrypsin-like enzymes Optimal

Residual trypsin-like activity was reduced to 15% after a

Table 1 Proteolytic activity in the hepatopancreas of

Farfantepena-eus subtilis SAS 6 and SAS 13 using specific and non-specific substrates

BAPNA, benzoyl- DL -arginine-p-nitroanilide – trypsin-specific

substrate; SAPNA,

succynil-alanine-alanine-proline-phenylalanine-p-nitroanilide; Leu-p-Nan, leucine-p-nitroanilide – leucine

amino-peptidase-specific substrate SAS 6 , approximately 6 g of wet

weight; SAS 13 , approximately 13 g of wet weight.

Values are shown as mean ± standard deviation (SD) of triplicates

of four crude extracts obtained from five hepatopancreases each.

Different italic superscript letters denote statistical differences

(P < 0.05).

–0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12

Arg

Lys Leu

Phe Val

Arg

Lys Leu

Phe Val

Figure 1 Correlation between aminopeptidase activity of enaeus subtilis SAS 6 (a) and SAS 13 (b) using aminoacyl-b-naph- thylamide as substrates and recommended concentration of essential amino acids for Penaeid shrimp feed Values are shown as mean ±

Farfantep-SD of triplicates of four crude extracts obtained from five pancreases each Concentration of amino acids expressed in % of crude protein: arginine (5.8), leucine (5.4), lysine (5.3), phenyllanine (4.0), valine (4.0) *Based on Guillaume (1997).

hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato- hepato-.

both SAS6 and SAS13 (Fig 4a) A similar profile was

obtained for the thermal stability of leucine aminopeptidase

Chy-motrypsin-like enzymes demonstrated higher heat resistance

than trypsin- and leucine aminopeptidase-like enzymes

(Fig 4b) No significant loss of activity appeared up to

value

com-mon pattern was observed concerning the number of bands

in both sub-adult stages Six bands ranging from 15.3 to

42.2 kDa were detected Three bands were found to have

molecular weights higher than 45 kDa and one band had a

molecular weight lower than 14.2 kDa

Proteolytic activity in both sub-adult stages was detected

Table 2 Effect of specific inhibitors on proteases of the

hepatopan-creas of Farfantepenaeus subtilis SAS 6 and SAS 13

PMSF, phenylmethylsulphonyl fluoride; TLCK, tosyl lysine

chlo-romethyl ketone; TPCK, tosyl phenylalanine chlochlo-romethylketone;

SAS 6 , approximately 6 g of wet weight; SAS 13 , approximately 13 g

of wet weight.

Values are shown as mean ± SD of triplicates of four crude extracts

obtained from five hepatopancreases each Different italic

super-script letters denote statistical differences (P < 0.05) Maximal

specific proteolytic activity (100%) was 7.03 mU mg)1for SAS 6 and

6.09 mU mg)1 for SAS 13 using BAPNA as substrate 100% was

4.40 mU mg)1for SAS 6 and 4.94 mU mg)1for SAS 13 using SAPNA

as substrate 100% was 0.29 mU mg)1for SAS 6 and 0.23 mU mg)1

for F subtilis adults using Leu-p-Nan as substrate.

1 PMSF inhibition using BAPNA as substrate.

2 PMSF inhibition using SAPNA as substrate.

020406080100120

pH

020406080100120

pH

020406080100120

pH (a)

Figure 2 Effects of pH on proteolytic activity of Farfantepenaeus

subtilis SAS 6 (d) and SAS 13 (s) using BAPNA (a), SAPNA (b) and

Leu-p-Nan (c) as substrates Values are shown as mean ± SD of

triplicates of four crude extracts obtained from five hepatopancreases

each The crude extract was incubated with the above substrates in

different assays at the indicated pH for 15 min and reactions were

measured at 405 nm Values are expressed as percentage of the

highest (100%) and were 7.20 mU mg)1for SAS 6 and 5.15 mU mg)1

for SAS 13 using BAPNA as substrate; 8.51 mU mg)1for SAS 6 and

4.96 mU mg)1 for SAS 13 using SAPNA; and 0.29 mU mg)1 for

SAS 6 and 0.25 mU mg)1for SAS 13 Leu-p-Nan as substrate

respec-tively SAS 6 : approximately 6 g of wet weight SAS 13 : approximately

13 g of wet weight.

.

(Fig 6b), whereas an extra band was detected in SAS6(white

dashed arrow, Fig 6a, lane 1) This proteolytic band is also

the number and intensity of bands were similar for both

Figure 7 displays the enzyme inhibition zymogram Allproteolytic bands were either totally or partially inhibited by

that most bands must be serine proteases No reaction wasrecorded in two bands using TLCK (lane 2) and in threebands using benzamidine (lane 3), suggesting a strong pres-ence of trypsin-like activity in the F subtilis enzyme extract

By comparing these results to those recorded in Fig 6, it is

(white arrow, Fig 7a, lane C) was inhibited by PMSF,TLCK and benzamidine The proteolytic bands that were notinhibited by trypsin inhibitors exhibited greater intensity(white dashed arrows, Fig 7a,b, lanes 2 and 3)

Studies on the characterization of digestive enzymes inshrimp are important to understanding their digestive phys-iology in comparative studies and represent basic informa-tion for further use of enzyme extracts as tools in the searchfor the proper nutrient sources in aquaculture The fact thatproteinases and peptidases are present in the hepatopan-creas of F subtilis is a relevant physiological advantage

Following hydrolysis of proteins by proteinases, peptidasessuch as aminopeptidases break long-chain peptides downinto smaller peptides and free amino acids, thereby enhancingthe absorption of these nutrients According to Zambonino-Infante & Cahu (2007), the activity of peptidases facilitatesthe assimilation of amino acids in the larvae of marine fishes

A better understanding of the digestive physiology of thisspecies is essential to the formulation of an adequate shrimpfeed In fact, knowledge on proteinase activity such as trypsin(which acts on basic amino acid residues) and chymotrypsin(which acts on aromatic amino acid residues) is important

Far-15 min and reactions were measured at 405 nm Values are expressed

as percentage of the highest (100%) and were 7.41 mU mg)1 for SAS 6 and 5.49 mU mg)1 for SAS 13 using BAPNA as substrate;

46.09 mU mg)1 for SAS 6 and 28.02 mU mg)1 for SAS 13 using SAPNA; and 0.46 mU mg)1for SAS 6 and 0.42 mU mg)1for SAS 13

Leu-p-Nan as substrate respectively SAS 6 : approximately 6 g of wet weight SAS 13 : approximately 13 g of wet weight.

.

information for diet formulation Species with low trypsin or

chymotrypsin activity may exhibit a deficiency in digesting

protein from diets with a high concentration of basic or

aromatic amino acid

The structure and morphology of the digestive system ofdecapod crustaceans are generally similar However, differ-ences are often observed at the biochemical level, two ofwhich are related to the cell pH of the midgut gland andenzyme expression (Saborowski et al 2008) According tothese authors, while crustaceans with a lower pH producecystein- and aspartic-proteinases, those with a higher pH(such as penaeid shrimp) predominantly express serine-proteinases

Trypsin- and chymotrypsin-like enzymes were identified inboth sub-adult stages of F subtilis The higher trypsin- and

related to the faster metabolism of younger organisms Thecrustacean digestive system generally exhibits a high con-centration of serine proteases, mainly trypsin and chymo-trypsin (Ferna´ndez et al 1997) Trypsin also plays animportant role in digestion through the activation of zymo-gens of both itself and other endopeptidases (Natalia et al.2004)

In this study, aminopeptidases were also observed in bothsub-adult stages As little information is available onaminopeptidases in shrimp, Leu-p-Nan and aminoacyl-b-naphthylamide substrates were used to provide a greaterunderstanding of these enzymes Aminopeptidases in the

strongly hydrolyzing alanine-, arginine-, leucine-, and b-naphthylamide substrates Greater hydrolysis of substratescontaining arginine, leucine and lysine were observed andthese amino acids are the same required at great concentra-tions in penaeid diets (5.8%, 5.4% and 5.3% of crude protein,respectively, according to Guillaume, 1997) These aminoacids correlated with the aminopeptidase activity using someb-naphthylamide substrates (Arg-, Leu-, Lys-, Phe- and Val-).The results of this study corroborate the requirementsreported in literature, as lysine and arginine are described asthe most limiting essential amino acids in commercial shrimpfeeds (Fox et al 1995) The correlation between recom-

lysine-020406080100

120

Temperature (°C)

020406080100

120

Temperature (°C)

020

.

mended concentrations of some dietary components, such as

lysine and arginine, and high aminopeptidase activity may be

related to the efficient digestion and incorporation of thesekey nutrients (Lemos & Nunes 2008) This interesting corre-lation has also been observed for F paulensis (Buarque et al

Figure 5 Electrophoresis of extract from Farfantepenaeus Subtilis

SAS 6 and SAS 13 Lanes: MW – molecular weight markers –

Ovoal-bumin (46 kDa), glyceraldehyde 3-phosphate dehydrogenase

(36 kDa), carbonic anhidrase (29 kDa), trypsinogen (24 kDa) and

a-lactalbumin (14.2 kDa); 1 – crude extract of F subtilis SAS 6 ;

2 – crude extract of F subtilis SAS 13 SAS 6 : approximately 6 g of wet

weight SAS 13 : approximately 13 g of wet weight.

Figure 7 Zymogram of proteolytic inhibition of hepatopancreas from Farfantepenaeus subtilis SAS 6 (a) and SAS 13 (b) using specific inhibi- tors Lanes: C – control (without inhibitors) Inhibition: 1 – PMSF;

2 – TLCK; 3 – benzamidine White dashed arrows (a,b) – bands not inhibited by trypsin inhibitors White arrow (a) – thermostable band (observed in Fig 5) from SAS 6 Black arrow (a) – extra proteolytic band inhibited by PMSF, TLCK and benzamidine in crude extract from SAS 6 SAS 6 : approximately 6 g of wet weight SAS 13 : approxi- mately 13 g of wet weight.

65 C; 6 – 75 C White dashed arrow – extra band observed in SAS 6

when compared to the protease pattern observed for SAS 13 White arrow – thermostable band in F subtilis SAS 6 SAS 6 : approximately

6 g of wet weight SAS 13 : approximately 13 g of wet weight.

.

et al.2002), as observed with the proteases in hepatopancreas

tissue of F subtilis in this study The strong inhibition by

TLCK and benzamidine (trypsin inhibitors) on the

proteo-lytic activity in both sub-adult stages indicates classical

trypsin activity (traditional mammalian trypsin) However,

TPCK was not efficient in inhibiting chymotrypsin activity in

the crude extract of both sub-adult stages This inhibitor has

also demonstrated a low effect on chymotrypsins in other

crustaceans (Garcia-Carren˜o et al 1994; Lemos et al 1999;

Ferna´ndez Gimenez et al 2002)

Classical protease inhibitors, such as PMSF, TLCK,

TPCK and benzamidine, are generally synthesized based on

the mammalian enzyme mechanism However, some studies

have found a low inhibitory effect by these molecules on

crustacean proteases (Garcia-Carren˜o et al 1994; Lemos et

evidence of the low compatibility of these enzymes with

mammalian proteases According to Dall & Moriarty (1983),

the digestive system of crustaceans is so different from that of

mammals that differences in the activity of their enzymes are

to be expected This may be important information for

aquaculture, mainly because the quality of shrimp feeds is

commonly evaluated using digestibility assays employing

mammalian enzymes (i.e pepsin test) In fact, these results

reinforce the advantage of the use of methodologies such

as pH-STAT, which evaluates digestibility by employing

enzyme extracts from the target shrimp species

Crustacean proteases generally exhibit the greatest activity

in the pH range from 5.5 to 9.0; trypsin activity is greatest

between pH 7.0 and 9.0 (Jiang et al 1991; Maeda-Martı´nez

to 10.0 (Saborowski et al 2004; Von Elert et al 2004) The

optimal pH for trypsin enzymes in F subtilis falls within this

interval Chymotrypsin-like enzymes also exhibited maximal

proteolytic activity in the alkaline range According to Dall &

Moriarty (1983), pH in gastric fluid during feeding may

fluctuate around neutrality and all digestive enzymes may be

active enough in this range for adequate digestion to occur

Trypsin-like enzymes in the hepatopancreas of both

val-ues correspond to those recorded for other crustaceans (from

retained about 15% of their activity after incubation for

stability was low at the same temperature, suggesting that

most of this enzyme must have been denatured

There is little information concerning the heat treatmentand temperature resistance of chymotrypsins from crusta-ceans Chymotrypsin activity from the gastric fluid of thecrab Cancer pagurus was extinguished after incubation at

chy-motrypsin of C pagurus seems to be less thermostable thanthe same enzyme in F subtilis (about 40% of initial value

A number of authors have studied aminopeptidases in fish(Refstie et al 2006) This demonstrates the importance ofunderstanding the role of aminopeptidases in the proteindigestion of aquatic organisms Galgani et al (1984) reportthe presence of leucine aminopeptidase in crude extract from

on aminopeptidases in shrimp with regard to their chemical characterization Further studies are required tocompare the physicochemical effects on aminopeptidases indifferent shrimp species Regarding the properties of leucine

respectively, and indicate a similar temperature denaturation

In the electrophoresis (SDS-PAGE) of the extracts from

pattern was observed in both samples (Fig 5) Two bandswere well visualized in the molecular weight range from 24 to

29 kDa, which is equivalent to the trypsin enzyme molecularweight reported in the literature for other aquatic animals(Kolodziejska & Sikorski 1996)

The thermal stability of proteolytic enzymes from F

demon-strated a similar band profile in both sub-adult stagesstudied However, one slight band with proteolytic activity at

that this enzyme remains active even under adverse ature conditions Moreover, an extra band was also observed

presence of one more protease in the hepatopancreas of

inhibited by PMSF, TLCK and benzamidine (white arrow,Fig 7), suggesting that it is a trypsin enzyme All bands

though the quantitative determination of proteolytic activitywas low at the same temperature In fact, the zymogramtechnique is more sensitive than the quantitative assays usingsoluble substrates (Lemos et al 2000)

Proteolytic bands inhibited by both PMSF (serine proteaseinhibitor) and TLCK or benzamidine (trypsin inhibitors)correspond to trypsin, which is a key enzyme in proteolytic

.

digestion Inhibition by PMSF alone indicates the presence

of chymotrypsin, another proteolytic enzyme present in the

hepatopancreas of penaeids (Lemos et al 2002)

This study demonstrated a large diversity of proteases in the

hepatopancreas of F subtilis, with the presence of trypsin,

chymotrypsin and aminopeptidases The presence of a high

content of proteinases and peptidases renders protein

diges-tion more efficient The most striking difference between

sub-adult stages was the greater chymotrypsin activity in the

possible to associate the protease metabolism with the

pre-viously observed slowdown in the growth rate in cultured

specimens (after reaching 6 g of body weight) Moreover, a

considerable large diversity of aminopeptidases was found in

both sub-adult stages The highest aminopeptidase activities

were observed using alanine-, arginine-, lysine- and

leucine-b-naphthylamide as substrates The proteolytic enzymes

stud-ied revealed optimal pH within the expected range for

decapod crustaceans, as described in the literature While the

trypsins and leucine aminopetidases found in this study

not thermostable at this temperature These findings provide

additional relevant information and could help to elucidate

the relationship between diet and the digestive potential of

F subtilis This study may be used as a comparative reference

for further feeding and nutrition studies on this species under

farming conditions

The authors would like to express their thanks to Mr

Otaviano Tavares da Costa, Rafael Padilha, Albe´rico

Espı´rito Santo and Joa˜o Virgı´nio for their technical

assis-tance This study was supported by the Financiadora de

Especial de Aqu¨icultura e Pesca – (SEAP/PR), Conselho

(CNPq), Fundac¸a˜o de Apoio a` Cieˆncia e Tecnologia do

Estado de Pernambuco (FACEPE) and Petro´leo do Brasil

S/A (PETROBRAS)

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.

1,2 1 1 1 1

APTA/SAA-SP – Ageˆncia Paulista de Tecnologia dos Agronego´cios/Po´lo Regional Noroeste Paulista, Votuporanga, Sa˜o Paulo, Brazil

To determine the digestible lysine requirement for pacu

juveniles, a dose–response feeding trial was carried out The

fish (8.66 ± 1.13 g) were fed six diets containing the

diet The gradual increase of dietary digestible lysine levels

of the parameters evaluated (P > 0.05) The increase of

improved weight gain (WG), specific growth rate (SGR),

protein productive value (PPV), protein efficiency rate

(PER), and apparent feed conversion rate (FCR), but was

lysine showed lower body lipid contents than fish in the other

treatments The digestible lysine requirement as determined

by the broken-line model, based on average WG values, was

were estimated based on the ideal protein concept and the

value determined for lysine

KEY WORDS: body composition, digestibility, ideal protein,

Received 9 October 2008, accepted 25 February 2009

Correspondence: APTA/SAA - SP - Ageˆncia Paulista de Tecnologia dos

Agronego´cios/Po´lo Regional Noroeste Paulista, Votuporanga, Sa˜o Paulo,

Brazil E-mail: abimorad@apta.sp.gov.br

Pacu (Piaractus mesopotamicus) is a tropical climate

migra-tory fish, native to the Basin that comprises the rivers Parana´,

Paraguay, and Uruguay in South America (Saint-Paul 1986)

Together with tambaqui (black-finned pacu) (Colossoma

macropomum) and its hybrid tambacu (C macropomum

as the most cultivated species in Brazil, after tilapia and carprespectively (Instituto Brasileiro do Meio Ambiente e dosRecursos Naturais Renova´veis (IBAMA) 2005) Suchimportance stems from its rapid growth, omnivorous feedingbehaviour, and excellent meat taste Several researches onpacu nutrition have been conducted with regard to digest-ibility coefficients of energy and protein from several ingre-dients (Abimorad & Carneiro 2004), protein requirement(Merola 1988; Carneiro et al 1994; Fernandes et al 2000),non-protein energy levels and sources (Pezzato et al 1992;

Abimorad et al 2007), and vitamin requirements (Martins1995; Belo et al 2005) However, few publications werefound on its amino acid requirements (Mun˜oz-Ramı´rez &

Carneiro 2002)

The traditional methodology used to determine the aminoacid requirements for fish is based on dose–response feedingexperiments for each amino acid, which is costly and time-demanding (Small & Soares 1998) In 1964, with the idealprotein concept proposed by Mitchell for swine and poultryand, later discussed by Fuller et al (1979), the all essentialamino acids (EAA) requirements could be expressed as anideal rate of a given amino acid in relationship to the EAAtotal in the animal tissue Consequently, the amino acidprofile of the skeletal muscle protein is the most used inresearches to represent amino acid requirements, since thattissue is substantially formed during growth (Fuller et al

1989; Small & Soares 1998; de la Higuera et al 1999; Portz &

Cyrino 2003; Abimorad et al 2008)

However, the muscle EAA profile only provides relativeEAA values, and does not quantify the values to be used inthe formulation of diets A simpler alternative would be todetermine the nutritional requirement of an essential aminoacid, generally the most limiting one, to estimate the otheramino acids requirements by of the ideal relationshipbetween the EAA of muscle (Twibell et al 2003; Wang et al

2005) Researches have reached the conclusion that lysine is

generally the first limiting amino acid in ingredients used for

feed manufacturing, and is also the EAA found at the highest

amounts in the carcasses of several fish species (Small &

Soares 2000), including pacu (Mun˜oz-Ramı´rez & Carneiro

2002), and therefore it is the reference amino acid used in this

type of study

Few studies have been conducted to determine digestible

amino acid requirements in fish (Wang et al 2005; Furuya

in diets is becoming increasingly important to formulate

more efficient feeds Therefore, the objective of this

investi-gation was to determine the adequate dietary digestible lysine

requirement for growth and protein utilization for pacu by

the dose–response method, and estimate the other EAA

requirements using the ideal protein concept

This study was carried out at Aquatic Organisms Nutrition

Laboratory from Aquaculture Center at Sa˜o Paulo State

University (CAUNESP – Jaboticabal, SP, Brazil), over a

90-day period

A total of 108 pacu juveniles (8.66 ± 1.13 g) were

dis-tributed into 18 cubic fibre cement tanks (100 L), in a

com-pletely randomized design with six treatments (6.8, 9.1, 11.4,

replicates, and six fish per experimental unit The tanks were

supplied with water from an artesian well, renovated at a rate

of approximately 10 times a day Each tank was supplied

with aeration system as well as a system that allowed water to

be drained directly from the bottom, to remove faeces and

food residues; in addition, these were siphoned once a week

Mean water physicochemical parameter values, measured

At the beginning of the study, 20 juveniles from the same

population used in the experiment were sacrificed

of initial body composition At the end of the study, six fish

from each tank, after a 24-h fast, were weighed, sacrificed,

and frozen to determine the body composition

A basal diet (Table 1) was formulated to contain

digestible energy; maintaining the same concentration ofnon-protein digestible energy (Abimorad et al 2007) and

The diets were either supplemented or not, with six levels of

other EAA were maintained at the same proportion based on

as in the muscle amino acid profile in relationship to theprotein level of the basal diet (Tacon 1987)

After finely ground, the ingredients in each diet were mixedmanually for 10 min, adding distilled water (40%, v/w) little

by little The diets were processed in a meat grinder (CAF22), forming 4–5 mm diameter granules, and dried in a

experimental period the fish were fed daily, twice a day (08:00and 18:00 h), until apparent satiety

A digestibility assay was carried out to determine thedigestible protein, energy, amino acids values in each exper-imental diet To accomplish that, 120 pacu juveniles(29.14 ± 4.97 g) were distributed into six feeding tanks

collect faeces After the fish were transferred from the feedingtanks into the collection tanks, the faeces were collectedrepeatedly at 30-min intervals and were stored in a refriger-ator Such procedure was repeated three times at 1-dayintervals, until the amount required for the analyses wasachieved

ADC values were calculated by the following formula:

dupli-in Parr bomb calorimeter at UNESPÕs Animal NutritionLaboratory, Jaboticabal, Brazil Dietary and faecal totalamino acids were measured by acid hydrolysis and ionicchange chromatographic (HPLC) at ITALÕs Chemistry

diets and faeces were determined by nitric-perchloric tion, according to Furukawa & Tsukahara (1966)

diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges- diges-.

The performance parameters were submitted to analysis of

were compared by DuncanÕs test (P < 0.05), using the

Cary, NC, USA) The broken-line models were applied for

the weight gain and apparent feed conversion to estimate the

most adequate level of digestible lysine (Portz et al 2000),

No mortality or visible external pathological signs were

observed in the fish during the experiment After the

deter-mination of the ADC values, digestible protein, energy

(Table 1) and amino acids values were calculated (Table 2)

The performance results of pacu juveniles-fed different

experimental diets are presented in Table 3 In general, the

growth performance of this study is in line with other studies

with pacu, in similar experimental conditions (Carneiro

There was no effect of the dietary treatments on feed intake(P > 0.05) The increasing in dietary digestible lysine level

weight gain (WG), specific growth rate (SGR), and proteinproductive value (PPV) The increase in digestible lysine level

WG, SGR, PPV, PER and FCR, without statistical ence from fish fed-diets containing a higher dietary digestible

showed smaller body lipid and higher body moisture contentrelative to the other treatments There was no effect of dietarytreatments on the body ash and protein content (Table 4)

The broken-line model was used to determine digestiblelysine requirement, based on the mean values for weight gainand apparent feed conversion rate The model estimated the

with greater weight gain, reaching a plateau at 51.15 g

Table 1 Experimental diets Diet

Digestible lysine levels (g kg)1)

* (Ingredient kg)1diet): Vitamins A = 600.000 IU; D 3 = 24.000 IU; E = 600 IU; K 3 = 120 mg;

Thi-amine = 180 mg; Riboflavin = 180 mg; Pyridoxine = 180 mg; B 12 = 480 mcg; C = 1.800 mg; Folic

Acid = 60 mg; Pantothenic acid = 480 mg; B.H.T = 1.47 g; Biotin = 6.0 mg; Inositol = 120 mg;

Nicotinamide = 840 mg; Choline = 4.8 g; Cobalt = 1.2 mg; Copper = 60 mg; Iron = 600 mg;

Iodine = 6.0 mg; Manganese = 180 mg; Selenium = 1.2 mg; Zinc = 600 mg; Vehicle q.s = 120 g.

.

(Fig 1), and 17.5 g kg)1 to respond with the best feed

con-version rate, reaching a plateau at 1.68 (Fig 2)

Table 5 presents essential amino acid values in muscle of

EAA ratio in muscle tissue (Arai1981), and estimation of other digestible essential amino

acids requirements by the ideal protein concept and the ADC

of individual amino acids

Most dose–response studies to determine amino acidrequirements for fish use purified or semi-purified diets,which can be harmful for growth because they reduce intake(Berge et al 2002), especially when they are deficient inessential amino acids, particularly lysine (Dabrowski et al

Table 2 Dietary crude and digestible

essential (EAA) and non-essential

(NEAA) amino acid values

Composition (g kg)1dry matter)

Digestible lysine levels (g kg)1)

EAA Arginine 12.0 (11.0) 11.9 (11.0) 11.9 (10.6) 11.9 (10.9) 11.9 (11.1) 11.8 (10.9) Histidine 5.3 (4.7) 5.3 (4.8) 5.3 (4.4) 5.2 (4.7) 5.2 (4.8) 5.2 (4.7) Isoleucine 8.1 (7.1) 8.1 (7.2) 8.1 (6.7) 8.1 (7.1) 8.0 (7.3) 8.0 (7.1) Leucine 26.1 (24.3) 26.1 (24.5) 26.0 (23.3) 26.0 (24.3) 25.9 (24.7) 25.8 (24.4) Lysine 8.2 (6.8) 10.4 (9.1) 13.1 (11.4) 14.5 (13.2) 17.2 (16.1) 21.0 (19.6) Methionine 4.4 (4.0) 4.4 (4.0) 4.4 (3.7) 4.4 (4.0) 4.4 (4.0) 4.3 (4.0) Phenylalanine 11.8 (108) 11.8 (10.9) 11.8 (10.4) 11.8 (10.8) 11.7 (11.0) 11.7 (10.8) Threonine 9.5 (8.4) 9.5 (8.5) 9.5 (8.1) 9.4 (8.4) 9.4 (8.6) 9.4 (8.3)

Valine 9.4 (8.2) 9.4 (8.4) 9.4 (7.8) 9.4 (8.3) 9.3 (8.5) 9.3 (8.2) NEAA

Aspartic acid 18.3 (16.1) 18.2 (16.3) 18.2 (15.6) 18.1 (16.1) 18.1 (16.5) 18.1 (16.0) Glutamic acid 45.7 (42.4) 45.6 (42.8) 45.5 (41.0) 45.4 (42.4) 45.3 (44.5) 45.1 (42.4) Alanine 16.5 (148) 16.5 (15.0) 16.4 (14.0) 16.4 (14.9) 16.4 (15.2) 16.3 (14.9) Cystine 2.2 (2.0) 2.2 (2.0) 2.2 (1.8) 2.2 (2.1) 2.2 (2.1) 2.2 (2.0) Glycine 10.5 (9.1) 10.5 (9.2) 10.5 (8.7) 10.5 (9.0) 10.4 (9.4) 10.4 (9.1) Proline 17.2 (16.0) 17.2 (16.2) 17.1 (15.5) 17.1 (16.3) 17.1 (16.3) 17.0 (16.1) Serine 11.9 (10.7) 11.9 (10.8) 11.8 (10.3) 11.8 (10.7) 11.8 (10.9) 11.7 (10.7) Tyrosine 8.3 (7.6) 8.2 (7.6) 8.2 (7.3) 8.2 (7.6) 8.2 (7.7) 8.1 (7.5) Crude AA (Digestible AA).

ND, not determined.

Table 3 Performance of pacu juveniles-fed diets containing different digestible lysine levels

Digestible lysine level (g kg)1)

(% day)1) 2

1.6 ± 0.3 b 1.8 ± 0.2 ab 1.9 ± 0.1 ab 1.9 ± 0.3 ab 2.2 ± 0.2 a 2.2 ± 0.1 a 0.0133 Feed intake (g/fish) 81.0 ± 6.4 79.2 ± 4.0 77.6 ± 2.3 80.7 ± 3.1 79.8 ± 3.8 71.7 ± 1.3 0.0951 Apparent feed

conversion rate 3

3.1 ± 0.6 a 2.7 ± 0.5 ab 2.4 ± 0.7 ab 2.5 ± 0.9 ab 1.8 ± 0.5 b 1.7 ± 0.3 b 0.0355 Protein efficiency rate 4 1.3 ± 0.3 c 1.5 ± 0.1 bc 1.6 ± 0.1 bc 1.8 ± 0.5 b 2.3 ± 0.2 a 2.5 ± 0.2 a 0.0006 Protein productive

value (%) 5

20.5 ± 3.9 c 23.3 ± 2.4 c 26.5 ± 3.6 c 28.6 ± 7.2 bc 36.2 ± 5.4 ab 40.5 ± 2.1 a 0.0008

Values are means of three replicates ± standard deviation Means followed by different letters on the row are statistically different (Duncan

P < 0.05).

Homogeneity of variance for all parameters were >0.05 by Brown & ForsytheÕs test.

1 Weight gain (WG) = (final weight ) initial weight).

2 Specific growth rate (SGR) = (log e weight final ) log e weight initial ) · 100/days.

3 Apparent feed conversion rate (FCR) = feed intake/weight gain.

4 Protein efficiency rate (PER) = weight gain/protein intake.

5 Protein productive value (PPV) = (Body protein final · weight final ) ) (Body protein initial · weight initial ) · 100/protein intake.

.

2007) In the present study, fish fed practical diets mented with synthetic amino acids and no effect on feed in-take was observed However, growth results indicated thatlysine is indispensable for pacu juveniles, which were able to

The digestible lysine requirement was determined by thebroke-line model for weight gain and apparent feed conver-sion rate response, because it provided better model fitting tothe data Equation fitting was verified by the least sum ofsquared deviations, F test significance, and coefficient ofdetermination (Shearer 2000; Murillo-Gurrea et al 2001)

considered more adequate than the one determined for FCR

response in relationship to the level determined for WG can

be explained by the fact that a diet with higher concentration

of a given nutrient, such as lysine, can provide better feedconversion rate, since no intake difference was observed(Maynard & Loosli 1974) On the other hand, to use more

synthetic lysine and the low increment on WG Therefore, itwas considered that the most adequate dietary digestible

Studies on amino acid requirements for fish are in generalconducted using nutrients in their crude form It is thereforedifficult to make comparisons with the digestible lysinevalue determined in our study Evaluation of optimal aminoacids concentration in percent of the dietary protein is

an additional way to make requirement data more parable (Santiago & Lovell 1988; Wilson 2003; Liebert &

com-Benkendorff 2007) Wang et al (2005) evaluated the ible lysine requirement of grass carp fry and estimated a

determined the digestible lysine requirement of Nile tilapia

shows that the crude lysine requirement for pacu juvenileswas 0.05% higher than for tilapia, but when the requirement

is expressed as digestible values, pacu proved more efficient inutilizing dietary lysine

Lysine level in relation to dietary protein estimated for

the values found by Jackson & Capper (1982) and Santiago

Table 4 Whole body composition of pacu juveniles-fed diets

con-taining different digestible lysine levels

Digestible

lysine

level

(g kg)1)

Composition (g 100 g)1body weight)

Values are means of three replicates ± standard deviation.

Means followed by different letters in the column are different by

DuncanÕs test (P < 0.05).

Homogeneity of variance for all parameters were >0.05 by Brown

& ForsytheÕs test.

Figure 1 Weight gain of pacu juveniles-fed diets containing different

digestible lysine levels.

X = 17.5

Y = 1.678 + 1.308 (17.5 –X)

P = 0.0023

Figure 2 Apparent food conversion rate of pacu juveniles-fed diets

containing different digestible lysine levels.

.