Availability of essential amino acids, nutrient utilisation and growth in juvenile black tiger shrimp, Penaeus monodon, following fishmeal replacement by plant protein

Aquaculture 322-323 (2011) 109–116

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Availability of essential amino acids, nutrient utilisation and growth in juvenile blacktiger shrimp, Penaeus monodon, following fishmeal replacement by plant protein

Lenaïg Richard a,b, Anne Surget a, Vincent Rigolet b,c, Sadasivam J. Kaushik a, Inge Geurden a,⁎a INRA, UR 1067 NuMeA (Nutrition, Metabolism and Aquaculture), F-64310 Saint-Pée-sur-Nivelle, Franceb UNIMA, 11 bis, rue Balzac, 75008 Paris, Francec AQUALMA, BP 93 Immeuble SCIM, 4 rue Galliéni, Mahajanga 401, Madagascar

Abbreviations: ADC, apparent digestibility coefficienCGM, corn gluten meal; FM, fishmeal; RSM, rapeseed mwheat gluten; WW, whole wheat.⁎ Corresponding author at: INRA,UR1067NuMeA(Nutrit

F-64310 Saint-Pée-sur-Nivelle, France. Tel.: +33 55951596E-mail address: [email protected] (I. Geurden)

0044-8486/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.aquaculture.2011.09.032

a b s t r a c t

Article history:Received 4 August 2011Received in revised form 22 September 2011Accepted 25 September 2011Available online 1 October 2011

Keywords:FishmealPlant proteinAmino acidShrimpDigestibility

Two trials with juvenile black tiger shrimp (Penaeus monodon) were undertaken to study the effects of repla-cing fishmeal by different levels of plant proteins on growth performances and nutrient utilisation of shrimpin semi-intensive conditions (Expt. 1) and on the availability of dietary nitrogen (N) and amino acids (Expt.2). Five isoproteic diets (on crude protein basis) were formulated to contain 34, 24, 16, 8, or 0% fishmeal, withfishmeal being replaced by a mixture of plant protein (corn gluten meal, wheat gluten, and rapeseed meal). InExpt. 1, the shrimp (initial body weight, IBW 1.5±0.1 g) were reared in earthen ponds for 144 days and fedone of the experimental diets. Apparent digestibility of nutrients and AA were assessed in Expt. 2, using 150 Ltanks and shrimp of 12.8±0.4 g IBW. After 144 days in grow-out ponds, shrimp fed the diet with 24% of fish-meal had similar growth as those fed the control diet containing 34% fishmeal (0% replacement). When 50%or more of the fishmeal were replaced, weight gain as well as N and energy gains significantly decreased. Di-gestibility of dry matter, protein and energy was also significantly lower in all fishmeal-replaced diets. In par-ticular, leucine digestibility decreased by 26% at 100% replacement, which was significantly correlated to anincreased incorporation of corn gluten meal. Our data confirm the need to improve our knowledge on AAavailability and raw material quality in order to improve fishmeal replacement in P. monodon diets.

t; EAA, essential amino acids;eal; SBM, soybean meal; WG,

ion,Aquaculture andGenomics),1; fax: +33 559545152..

rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Over the past 10 years, farmed shrimp production has expandedfrom 1.2 million to 4.7 million tonnes, increasing the demand for for-mulated shrimp feed (Tacon and Metian, 2008). To meet the high di-etary protein requirement of shrimp, commercial shrimp feeds areoften rich (25–50% of the diet) in fishmeal (FM), the preferred proteinsource due to its well-balanced essential amino acid (EAA) profile.As a consequence of the reduction of forage fisheries, production ofFM is levelling off (Naylor et al., 2009). Due to their wide availability,plant proteins from oil seed meals such as soybean meal, sunflowermeal, or pulses such as lupin or pea have been investigated as poten-tial fishmeal replacers in feed for both whiteleg shrimp (Litopenaeusvannamei) and black tiger shrimp (Penaeus monodon), reachinghigh levels of FM substitutions (Dayal et al., 2011; Forster et al.,2002; Paripatananont et al., 2001; Smith et al., 2007a; Sudaryonoet al., 1999) or even total FM substitution in L. vannamei (Amaya

et al., 2007a; Sookying and Davis, 2011). However, most of the re-search has been conducted under controlled laboratory conditionsrather than under field conditions (Amaya et al., 2007b; Sookyingand Davis, 2011), which makes it difficult to transfer to shrimp farm-ing industry.

In order to reach an adequate dietary EAA profile, a mixture of sev-eral plant proteins rather than a sole plant protein source is oftenused, as proposed for fish (Fournier et al., 2004; Gomes et al., 1995;Kaushik et al., 2004; Regost et al., 1999). Our previous work with P.monodon (Richard et al., 2010a) further indicated that both the pro-tein level and EAA profile should be considered together, as proteinaccretion in P. monodon was not reduced by feeding a high level(50% of diet) of an ‘imbalanced’ protein compared to an adequatelevel (30% of diet) of ‘balanced’ protein, suggesting that EAA require-ments should be expressed as a proportion of the diet or as a given in-take level (per unit body weight) rather than as a percentage ofdietary protein. It also implies that poor quality protein sources canbe included at higher than required protein levels in order to fulfilEAA requirements (Richard et al., 2010a), leading however to in-creased nitrogen excretion (Richard et al., 2010b). Besides, informa-tion on the availability of the EAA from the feed is also needed inorder to avoid an EAA deficiency and thus a suboptimal protein utili-sation by the shrimp. Earlier studies in shrimp reported big differ-ences in nutrient digestibility of plant proteins (Akiyama et al.,

Table 2Analysed amino acid composition of the experimental diets as g/100 g dry feed.

Diets

1 2 3 4 5 Requirements1

EAAArg 4.0 3.3 3.4 3.0 3.1 1.9His 1.6 1.4 1.5 1.3 1.4 0.8Ile 1.8 1.7 1.7 1.6 1.8 1.0Leu 2.9 3.6 3.5 4.3 4.8 1.7Lys 3.6 3.0 2.9 2.7 2.6 1.7*–2.1Met 1.0 0.9 0.8 0.8 0.8 0.8*–0.9Phe 1.7 1.8 1.9 2.0 2.3 1.4Thr 1.7 1.5 1.6 1.5 1.5 1.4Val 1.9 1.9 1.9 1.7 1.9 1.4

110 L. Richard et al. / Aquaculture 322-323 (2011) 109–116

1989; Brunson et al., 1997), partly attributed to the presence of anti-nutritional factors such as protease inhibitor, phytic acid or tannins(Cruz-Suarez et al., 2001; Francis et al., 2001; KumaraguruVasagam et al., 2007). The importance of considering nitrogen andEAA requirements in terms of available nutrients is substantiated bytwo recent survey studies in L. vannamei, showing large variations be-tween EAA availability of fishmeal and plant proteins (Lemos et al.,2009; Yang et al., 2009).

The main objective of this study was to evaluate the effect ofreplacing fishmeal by a mixture of plant proteins on the long termperformances of P. monodon reared under semi-intensive commercialpond conditions (pond study, Expt. 1). In addition, we examinedwhether changes in the protein source modified the availability ofthe dietary nitrogen and AA (digestibility study, Expt. 2).

NEAAAla 2.2 2.4 2.3 2.6 2.7Asp 3.4 3.1 3.2 2.9 2.9 Met+CysCys (theoretical)2 0.5 0.6 0.7 0.7 0.8 1.1*–1.3Glu 6.0 6.7 7.0 8.0 9.3Gly 2.2 1.9 1.9 1.6 1.6Pro 1.9 2.2 2.3 2.8 3.2Ser 1.8 1.8 1.9 2.1 2.2Tyr 1.3 1.3 1.4 1.6 1.8

1 Estimated EAA requirements (in g per 100 g DM) for P. monodon from the works ofMillamena et al. (1996a, b, 1997, 1998, 1999), except for values marked with *, estimatedin Richard et al. (2010a) bymeans of a factorial approach (estimatedprotein requirementswere 30% diet DM).

2 Calculated from the theoretical composition of the ingredients.

2. Material and methods

2.1. Diets

A practical diet (diet 1) was formulated to contain 34% fishmeal(FM) as the main protein source (59% of the dietary protein,Table 1). In four other diets, FM was gradually reduced to 24% (diet2), 16% (diet 3), 8% (diet 4) and 0% of the diet (diet 5), being replacedby a mixture of plant protein sources (corn gluten meal, rapeseedmeal, sorghum and wheat gluten) (Table 1). All five diets containedsoybean meal (SBM, from 19 to 25%) and krill and shrimp meal(each kept constant at 1%). The analysed EAA content of the five ex-perimental diets was above currently known requirements for P.monodon (Table 2). In order to meet the dietary lysine requirements,crystalline lysine was added in diets 4 and 5 (Table 1). All feeds wereindustrially manufactured by Nutrima (La Réunion, France), pelletedto be 2 mm and stored in sealed bags prior to shipping to the farmin Madagascar where they were stored in closed containers.

Table 1Composition and proximate analysis of the five diets fed to juvenile black tiger shrimp,P. monodon.

Diets

Ingredients (g/kg feed) 1 2 3 4 5

Fishmeal 340 240 160 80 0Squid meal 10 10 10 10 10Krill meal 10 10 10 10 10Soybean meal (48%) 215 191 250 200 234Whole wheat 350 200 92 160 60Wheat gluten (80%) 0 10 10 30 50Corn gluten meal (60%) 0 115 85 214 225Rapeseed meal 0 0 150 100 150Sorghum 0 150 150 100 150Fish oil 3 8 16 20 26Premix1 73 66 66 72 80Lysine 0 0 0 4 5

Proximate compositionDry matter (DM, %) 90.0 90.4 90.0 90.2 90.1Crude protein(N×6.25,% DM)

42.1 42.9 44.2 44.5 44.2

Crude lipid (% DM) 5.6 6.5 6.6 6.2 7.0Gross energy (kJ/g DM) 18.8 19.4 19.7 20.1 20.2Ash (% DM) 11.8 10.0 9.9 8.7 8.4Inert marker (%DM) 1.0 1.3 1.4 1.5 1.5

Leaching (%)2 6.3(2.1)a

3.6(1.5)b

5.3(0.8)ab

5.2(1.2)ab

6.2(0.4)a

1 Premix (micro-nutrient and additive mixture) contained (g/kg feed): cholesterol(1 or 8), de-oiled soy lecithin (10), mono Ca phosphate (10 to 24), Ca Propionate (2or 5), mineral and vitamin blends (20) and inert tracer (20).

2 Values are means and (SD) of five or six measurements. The P-value for the diet ef-fect was 0.0179. Values sharing a common letter are not significantly different.

2.2. Experimental designs

Two studies were performed at the facility of Aqualma (Unima,Madagascar): i) a 144-day grow-out feeding trial in 0.235 ha earthenponds and ii) a 35-day digestibility trial in 150 L indoor tanks.

2.2.1. Pond trial (Experiment 1)Juvenile black tiger shrimp (P. monodon) having an initial body

weight of 1.5±0.1 g were transferred into fifteen rectangular earthenponds (mean depth, 1.20 m; surface, 0.235 ha) and reared for144 days under semi-intensive conditions at a mean stocking densityof 7.1 shrimp/m2 (16,886 shrimps per pond on average). The pondswere characterised by a loam to sandy-loam soil. Two weeks priorto the start of the trial, ponds were prepared by draining the waterand the soil was tilled at a depth of 10 cm to improve oxidation andmineralisation of organic matter of the pond. Lime was applied onthe wet zone in the bottom of the ponds at a level of 1.5 kg/m². Oneweek later, the ponds were filled with water (30–32 ppt) pumpedfrom a common canal until an approximate level of 170 cm. Waterwas filtered at the entrance of the pond with a 300 μm nylon filter.Ponds were then fertilised with triple super phosphate (Ca(H2PO4)2.H20; 1 kg/ha) and urea (10 kg/ha) to stimulate natural pro-ductivity (planktonic bloom) in the pond.

Temperature and dissolved oxygen (DO) were measured twicedaily (4:00 am and 4:00 pm), whereas salinity, pH, water heightand turbidity (secchi disk) were measured every morning. Totalammonia-nitrogen, nitrite and nitrate were measured once a weekon water sample collected close to the exit side of the pond. Mechan-ical aeration was provided during the day when the levels of DOdropped below 3 mg/L.

Each diet was allocated to three replicate ponds. Feed was distribut-ed uniformly throughout the pond area three times a day (10:00, 14:00and 18:00). The feeding rate was based on feeding table of the farm andassumed a constant 100% survival. Average temperature, oxygen, salin-ity, pH and turbidity were 25.7±1.5 °C, 7.2±1.0 mg/L, 34.2±2.3 mg/L,8.7±0.3 and 38.9±18.7 cm, respectively.

During the trial, mean body weight was estimated every week bytaking two samples of 200 shrimp from each pond (one at the

111L. Richard et al. / Aquaculture 322-323 (2011) 109–116

entrance side, the other at the exit side of the pond). Final mean bodyweight was estimated after harvesting the whole pond (on average,14,597 shrimp per pond, equivalent to a final density of 6.2shrimp/m²), from a shrimp batch of 7 kg. At the beginning and atthe end of the trial, samples of 500 g food deprived shrimp (14 and12 h, respectively) were sacrificed in cold water and stored at −20 °C for later comparative carcass analyses (dry matter, nitrogen,lipid, ash and energy). The composition of the initial whole shrimpsamples for dry matter (g/100 g wet weight), protein (g/100 g wetweight), fat (g/100 g wet weight), energy (kJ/g wet weight) and ash(g/100 g wet weight) was: 23.2±1.2, 16.6±1.1, 1.4±0.2 4.5±0.3and 3.8±0.0, respectively. After 144 days of feeding, performance pa-rameters were calculated as:

– Survival (%): nf/ni×100– Weight gain (WG, kg): BIOMf−BIOMi+BIOMd

– Final mean body weight (Wf, g): (BIOMf×1000)/nf

– Final yield (kg/ha): BIOMf/0.235– Total dry feed supplied (TFS, kg): FEED×(100−DML)×DMF

– Dry matter supplied (% biomass/day): 100×(TFS/Δt)/BIOMm

– Dry matter supplied (g/shrimp/day): (1000×TFS)/(nm×Δt)– Pond-based feed efficiency (FE): WG/TFS– Pond-based corrected FE: WG/(TFS×ADMD)– Pond-based protein efficiency ratio (PER): WG/(TFS×% CP)– Pond-based corrected PER: WG/(TFS×% CP×APD)– Pond-based N retention (%): 100×[((Nf×Wf)−(Ni×Wi))]/

[((TFS×1000)×(% CP/6.25))/nm]– Pond-based corrected N retention (%):

100� Nf �Wfð Þ– Ni �Wið Þð Þ½ �= TFS� 1000� APD� %CP=6:25ð Þð Þ=nm½ �

– Pond-based energy retention (%): 100×[((Ef×Wf)−(Ei×Wi))]/[(TFS×1000×Efeed)/nm]

– Pond-based corrected energy retention (%): 100×[((Ef×Wf)−(Ei×Wi))]/[(TFS×1000×Efeed×AED)/nm]

Where ni, nf, nm are the initial, final and average number ofshrimp; BIOMi, BIOMf and BIOMd are the initial, final and dead shrimpbiomasses (kg); BIOMm is the mean biomass over the experimentalperiod ((BIOMi+BIOMf)/2); Wi and Wf are the initial and finalmean body weight (g); Δt the number of experimental days; FEEDis the total amount of feed distributed (kg); DML is the% of dry matterloss of feed after the leaching test; DMF is the dry matter content ofthe feed (g/100 g feed); Efeed and CP are the energy (kJ/g dry feed)and crude protein (g/100 g dry feed) content of the feed, respectively;ADMD, APD and AED, apparent digestible coefficient (ADC,%) for drymatter, protein and energy, respectively; Ei, Ni and Ef, Nf are the ener-gy and nitrogen content of shrimp (kJ/g fresh tissue and g/100 g freshtissue) at the beginning and the end of the trial, respectively.

2.2.2. Digestibility trial (Experiment 2)A five-week digestibility trial was carried out at the experimental

facility of Aqualma (Madagascar). An inert marker (SiO4, Sipernat®)was included at 2% in each of the five feeds (diets 1–5) to determineapparent digestibility coefficients. Seven shrimp (12.8±0.4 g) werestocked in 150 L covered plastic tanks (80×30.5 cm; diameter×-height). Each feed was randomly assigned to the tanks (nine replicatetanks per feed, at the exception of eight replicates for the controldiet). After 1 week of adaptation, shrimp were fed in excess fourtimes a day (7:00, 13:00, 19:00, 01:00) one of the experimentaldiets at a rate of 4%. Feed was distributed in three circular trays(20 cm diameter) and left for 1 h, after which all uneaten feed andfaeces were siphoned from the tanks. Three hours after the feed dis-tribution (10:00, 16:00, 22:00, 04:00), faeces were collected by si-phoning and immediately filtered through a 100 μm mesh nylonfilter and rinsed with distilled water before being stored at −20 °C

in aluminium cups for later analyses. Thirty minutes prior to the nextfeed distribution, the same procedure was repeated for each tank. Sili-cate (acid insoluble-ash) was used as the inert marker (Deering et al.,1996) and the content in the diets and faeceswas determined accordingto themethod of Atkinson (Atkinson et al., 1984). Apparent digestibilitycoefficients (ADC) of dry matter (ADMD), protein (APD), energy (AED)and amino acids were calculated as:

ADMD : 100× 1– Idiet=Ifaecesð Þð Þ

ADCnutrient : 100× 1– Xfaeces=Xdietð Þ× Idiet=Ifaecesð Þð Þ½ �

With Idiet and faeces: concentration of inert marker in the feed and

faeces samples g=100 g DMð ÞXdiet and faeces: studied nutrient content

of feed and faeces g=100 gDMð Þ:

2.3. Feed and faeces analyses

Water stability of feed was assessed using the methodology ofCruz-Suarez et al. (2001). Briefly, 5 g of feed (n=6) were allowedto stand in a 150 L tank filled with seawater (35 ppt) for 1 h. After-wards, feed was collected and dried for 48 h at 90 °C. The dry matterloss percentage (DML) was calculated as:

DML %ð Þ ¼ 100 × DMbefore–DMafterð Þ=DMbeforewith DMbefore and DMafter

the dry matter content of the feed before and after immersion in

water g=100 g feedð Þ:Feed intakes in both experiments were

corrected for the dry matter loss:

Feed samples were analysed for dry matter (105 °C for 24 h), ash(550 °C for 12 h), lipid (Soxtherm, Gerhardt, Germany), gross energy(bomb calorimeter IKA, Heitersheim, Germany), protein (N×6.25,Kjeldahl Nitrogen analyser 2000, Fison Instruments, Milano, Italy)and amino acids. Freeze-dried faeces samples were analysed for thesame components except lipid. Amino acid contents of the feedswere analysed using the AccQ.Tag method (Waters). Briefly, 100 mgof feed was hydrolysed with 25 mL of 6N HCl and 12.5 mL of 2-mercaptoethanol (23 h, 110 °C). After dilution (1/25 and 1/20, re-spectively), 10 μL of a standard 17 AA solution (Sigma) and hydro-lysed sample were derivatised by adding 70 μL of AccQ.Tag buffer(Waters) and 20 μL of AccQ.Fluor reagent (6-aminoquinolyl-N-Hydroxysuccinimidyl carbonate). 5 μL of sample was injected andanalysed by HPLC (column Symmetry C18 5 μm 3.9×150 mm) usingthree mobile phases (AccQ.Tag buffer, acetonitrile 100% and water,respectively) for an elution time of 45 min (flow rate of 1 mL per mi-nute; control temperature at 37 °C). The excitation and emissionwavelengths in the fluorescence detector were 250 and 395 nm,respectively.

2.4. Statistical analysis

Data from Expt. 1 and 2 were analysed by a one-way analysis ofvariance (ANOVA) using dietary treatment as factor (n=5 diets), fol-lowed by a comparison of means using Duncan's multiple range testin case of a significant effect (Pb0.05). The principal component anal-ysis (PCA) used the EAA as variables and the EAA contents of the rawmaterials (EAA, % ingredient protein) and of the faeces from shrimpfed each experimental diet (EAA, % faecal protein) as individuals.Eigen-values were analysed to extract the major principal compo-nents. Apparent digestibility coefficients of the individual EAA wereanalysed against changes in the relative dietary protein contributionof each major protein source, using linear regressions. ANOVA andPCA were performed using STATISTICA 8.0 software (StatSoft. Inc.,

112 L. Richard et al. / Aquaculture 322-323 (2011) 109–116

Tulsa, OK, USA). Linear regressions were performed using GraphPadPrism 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Growth and feed utilisation

The highest leaching coefficients (6.2–6.3%) were observed for thecontrol diet and diet 5, being different from that of diet 2 (3.6%)(Table 1, Pb0.05). Survival (85.2 to 87.3%) was not affected by thelevel of fishmeal in the diet (Table 3, PN0.05). Shrimp from the con-trol group (diet 1) had the highest biomass gain (310.9 kg), finalyield (1321 kg/ha) and final individual body weight (21.0 g). Whenreplacing 50% of the FM (diet 3), performances were significantly re-duced by 20% (Table 3, Pb0.05). The lowest growth was observed inshrimp fed diet 5 devoid of fishmeal (218 kg of biomass gain perpond; 15.1 g of final BW; 955 kg/ha of final yield) (Table 3, Pb0.05).The body composition of the shrimp was not affected by the replace-ment of dietary FM (Table 4, PN0.05). The pond-based nutrient reten-tion parameters (FE, PER, nitrogen and energy retentions) were allsignificantly higher for shrimp fed the diets 1 and 2 (0.6, 1.3–1.4,26.6–29.2 and 17.4–19.2%, respectively). When fed a FM-free diet(diet 5), nutrient retentions significantly decreased by 23–24%(Table 4, Pb0.05). However, there were no more significant differ-ences between the treatments when FE, PER and retentions were cor-rected for digestibility of dry matter, protein and energy, respectively(Table 4, PN0.05).

3.2. Digestibility trial (Expt. 2)

The apparent digestibility coefficients (ADC) of the control diet(diet 1) were 78.3%, 91.6% and 86.9% for dry matter, protein and en-ergy respectively (Table 5). Availability of individual amino acidsfrom the control diet (diet 1) ranged between 91.9 and 96.9%(Table 5). The ADCs were significantly affected by the dietary reduc-tion of fishmeal (Table 5, Pb0.05). When 25% of the fishmeal wasreplaced by plant protein (diet 2), digestibility decreased by 9% com-pared to the control diet. Among the AA, availability of leucine wasthe most affected (−14.5%), followed by proline (−11.3%), phenylal-anine (−10.6%) and alanine (−10.9%). When 100% of FM wasreplaced (diet 5), ADC of dry matter, protein and energy decreasedby 20, 16.8 and 17.1%, respectively. The maximal loss of AA availabil-ity (−26%) was observed for leucine (Table 5).

Table 3Effect of fishmeal replacement on growth performance of P. monodon reared in earthen po

Diets

1 2

Mean SD Mean SD

Performances and feed allocation(per pond)

Survival (%) 87.3 6.1 86.6 3.1Initial biomass (kg/pond) 24.1 1.0 24.8 2.3Final biomass (kg/pond) 310.5 11.0 a 282.5 2.3Biomass gain (kg/pond) 310.9 9.9 a 281.2 2.7Final yield (kg/ha) 1321 47 a 1202 10Total feed supplied (TFS, kg DM/pond) 518.1 13.4 a 500.2 14.6

Performances and feed allocation(per shrimp)

Initial mean body weight (g/shr) 1.4 0.1 1.5 0.1Final mean body weight (g/shr) 21.0 1.3 a 19.8 0.5Dry matter (DM) supplied (% biomass per day) 2.2 0.1 2.3 0.1DM supplied (g/shrimp/day) 0.23 0.01 a 0.23 0.01

1 Values are means of three replicate ponds per treatment.2 P-values are given by the one way ANOVA. Mean values with different superscripted le

EAA compositions of the protein sources and the faecal proteinwere analysed using a multivariate analysis (principal componentanalysis, PCA) presented in Fig. 1. The space is described by a numberof dimensions defined by the variables (in our case, 9 dimensions forthe 9 studied EAA). The first two principal components (PC1 and PC2)accounted for 87% of the total variability. Therefore, the data (EAAcomposition of ingredients and faeces) were analysed in a factorialplane characterised by PC1 and PC2 (Fig. 1). The first axis (PC1, hori-zontal) discriminates ingredients and faeces based mostly on threo-nine, arginine, lysine, valine and methionine with a respectivecontribution of 16.1, 15.7, 14.5, 13.9% and 12.4% (Fig. 1). The secondaxis (PC2, vertical) is mainly built on differences in leucine content(42.4% of contribution, Fig. 1). The three other EAA did not signifi-cantly contribute to the extracted PCs. On the first axis, fishmeal(FM), rapeseed meal (RM) and soybean meal (SBM) are grouped to-gether, being rich in most of the above cited EAA, while wheat ingre-dients (whole wheat and wheat gluten), also grouped together, werenot highly discriminated (close to the 0, 0 axes intercept). Corn glutenmeal (CGM) in particular was clearly separated from the other pro-tein sources, reflecting its low lysine and high leucine content. Faeceswere highly discriminated along PC2, with faeces of the control group(0% replacement, F1) being in opposition with those of the 75 and100% replacement groups (F4 and F5) (Fig. 1), which indicate alower leucine content in the faecal protein from the control groupthan from the two other groups fed increased amounts of CGM richin leucine (close following PC2). To further investigate the relativecontribution of ingredients to the digestibility of the whole diets, lin-ear regression analyses were done using each ingredient's relativecontribution to dietary protein content and the apparent EAA digest-ibility coefficients of the diets (Table 6). The results indicate that bothfishmeal (FM) and whole wheat (WW) utilisation are positively cor-related to EAA availability (Table 6, Pb0.05), FM contributing howevermore than WW to EAA availability (0.84bR²b0.95 and 0.75bR²b0.83,respectively). All other ingredients (CGM, RM, and WG) are negativelycorrelated with EAA availability (Table 6, Pb0.05). Among them, in-creased incorporation of CGM contributed the most to the loss of avail-ability for all EAA (0.80bR²b0.89, Table 6).

4. Discussion

Most studies on fishmeal replacement in feed for penaeid shrimphave been conducted under controlled indoor conditions and overrelatively short periods (Amaya et al., 2007b). To our knowledge,this is the first study on a long-term effect of fishmeal replacement

nds during 144 days (Expt. 1)1.

3 4 5

Mean SD Mean SD Mean SD P-values2

85.8 3.4 85.2 3.7 87.0 14.3 0.996125.0 1.1 24.5 2.4 24.4 2.3 0.9785

ab 249.9 22.4 bc 235.9 35.2 bc 224.4 42.5 c 0.0167ab 247.2 18.4 bc 233.3 32.7 c 218.0 23.5 c 0.0014ab 1063 95 bc 1004 150 bc 955 181 c 0.0166ab 482.2 6.3 b 473.0 20.8 bc 446.1 16.4 c 0.0017

1.5 0.1 1.4 0.1 1.4 0.1 0.8544a 17.2 1.1 b 16.2 1.6 bc 15.1 0.3 c 0.0003

2.5 0.2 2.6 0.3 2.5 0.3 0.1456a 0.21 0.01 ab 0.21 0.00 bc 0.20 0.01 c 0.0053

tters were significantly different between groups (Pb0.05).

Table 4Final whole body composition and nutrient retention of shrimp P. monodon reared for 144 days in earthen ponds (Expt. 1)1.

Diets

1 2 3 4 5

Final body composition (g/100 g wet weight) Mean SD Mean SD Mean SD Mean SD Mean SD P-values 2

Dry matter 27.8 0.8 27.7 0.4 27.0 1.4 27.9 1.9 27.7 1.9 0.9465Protein 20.7 0.8 20.6 0.2 20.3 0.8 21.3 1.3 20.7 1.4 0.8193Fat 2.7 0.3 2.9 0.2 2.8 0.1 2.8 0.6 2.7 0.3 0.8859Ash 3.2 0.1 3.4 0.2 3.2 0.3 3.4 0.2 3.7 0.1 0.1666Gross energy (kJ/g wet shrimp) 5.9 0.2 5.9 0.1 5.8 0.3 5.9 0.4 5.9 0.5 0.9934

Nutrient retention parametersPond-based FE 0.60 0.01 a 0.56 0.02 ab 0.51 0.03 bc 0.49 0.05 c 0.49 0.03 c 0.0070Pond-based FE corrected for digestibility 0.77 0.02 0.82 0.03 0.84 0.06 0.80 0.08 0.84 0.06 0.4825Pond-based PER 1.43 0.03 a 1.31 0.04 a 1.16 0.08 b 1.11 0.11 b 1.10 0.08 b 0.0011Pond-based PER corrected for digestibility 1.56 0.03 1.59 0.05 1.43 0.10 1.41 0.14 1.47 0.10 0.1560Pond-based N gain (mg/shrimp/day) 4.48 0.19 a 4.14 0.13 a 3.50 0.34 b 3.47 0.57 b 3.10 0.16 b 0.0022Pond-based N retention (% intake) 29.2 0.7 a 26.6 1.0 ab 23.1 2.6 b 23.2 3.5 b 22.4 0.6 bc 0.0102Pond-based N retention (% digestible N intake) 31.9 0.8 32.2 1.2 28.4 3.2 29.6 4.5 30.0 0.8 0.3879Pond-based energy gain (kJ/shr/day) 0.82 0.02 a 0.77 0.03 a 0.65 0.07 b 0.62 0.11 b 0.58 0.04 b 0.0034Pond-based E retention (% GE intake) 19.2 0.4 a 17.4 0.5 ab 15.5 1.9 b 14.8 2.4 b 14.5 0.6 bc 0.0110Pond-based E retention (% DE intake) 22.1 0.5 22.1 0.6 20.9 2.5 20.1 3.3 20.8 0.9 0.6646

1 Values are means of three replicate ponds per treatment.2 P-values are given by the one way ANOVA. Mean values with different superscripted letters were significantly different between groups (Pb0.05).

113L. Richard et al. / Aquaculture 322-323 (2011) 109–116

by plant protein in the black tiger shrimp P. monodon using practicaldiets under commercial rearing conditions. After 144 days of earthenpond rearing, average survival (87%) and FE (0.60) of P. monodon fedthe control diet (34% fishmeal) were comparable to values reportedfor L. vannamei reared under intensive pond conditions over long pe-riods (Casillas-Hernandez et al., 2007; Sookying and Davis, 2011)using diets containing little or no FM. Also the final pond yield forthe control diet (1.3 tonnes/ha), when corrected for differences in ini-tial stocking density (7.1 in our study relative vs. 15 or 35 shrimp/m²in the latter studies), was similar to that reported in both latter stud-ies with L. vannamei (approximately 1 to 1.6 tonnes/ha). In contrast,the values of nitrogen retention (29–32%) as found here were slightlyabove those reported for P. monodon fed a commercial diet either

Table 5Effect of fishmeal replacement by plant protein mixture on the apparent digestible coefficie

Diets

1 2 3

Mean SD Mean SD Mean

DM 78.3 1.3 a 68.9 3.8 b 61.3Protein 91.6 0.5 a 82.7 1.7 b 81.4Energy 86.9 0.9 a 78.8 1.9 b 74.0

EAAArg 96.6 0.3 a 93.1 0.7 b 92.1His 94.7 0.4 a 89.0 1.1 b 87.5Ile 95.4 0.2 a 87.5 1.3 b 86.1Leu 95.1 0.3 a 80.6 1.7 b 78.1Lys 96.9 0.2 a 94.9 0.7 b 93.9Met 95.7 0.2 a 89.6 1.1 b 88.2Phe 94.3 0.5 a 83.7 1.4 b 81.5Thr 94.0 0.4 a 86.5 1.1 b 84.1Trp – – –

Val 94.6 0.2 a 86.9 1.1 b 85.0

NEAAAla 94.7 0.4 a 83.8 0.9 b 80.9Asp 92.6 0.5 a 86.1 1.2 b 84.1Glu 95.5 0.3 a 85.8 0.9 b 84.4Gly 91.9 0.5 a 87.2 1.2 b 84.8Pro 93.3 0.6 a 82.0 1.4 b 79.8Ser 92.7 0.6 a 83.3 1.3 b 80.8Tyr 95.0 0.5 a 85.1 1.4 b 82.9

Mean values with different superscripted letters were significantly different between group

under intensive outdoor conditions (22–24%) (Briggs and Funge-Smith, 1994; Jackson et al., 2003) or under controlled indoor condi-tions (24.7%) (Richard et al., 2010a). The lower stocking density ofshrimp in semi-intensive ponds generally enables shrimp to feedmore on natural biota (microbial community and phytoplankton)known to recycle dietary and faecal N wastes, which improves appar-ent N retentions (Burford and Williams, 2001; Burford et al., 2002;Jackson et al., 2003; Teichert-Coddington et al., 2000). The highwater renewal and absence of natural biota in the 150 L tanks usedby Richard et al. (2010a) most likely contributed to reduced N reten-tion (24.7%) in our previous study. Also, low quality of feed or ingre-dients, overfeeding as well as poor water stability of the pellets canlead to reduced N retention (Burford and Williams, 2001).

nts (ADC, %) for dry matter, protein, energy and amino acids in P. monodon (Expt. 2).

4 5

SD Mean SD Mean SD

4.5 c 61.8 4.5 c 58.3 3.9 c2.0 b 78.5 2.4 c 74.8 2.4 d2.4 c 73.5 2.8 c 69.8 2.1 d

1.1 b 90.7 0.5 c 89.6 0.5 c2.2 bc 85.3 0.7 cd 84.6 1.5 d2.8 b 81.6 0.4 c 80.0 2.4 c3.4 b 72.4 1.4 c 69.3 2.7 c1.0 b 94.5 0.2 b 93.9 0.4 b1.5 b 85.2 0.6 c 82.1 0.7 d3.4 b 77.6 1.4 c 75.3 2.6 c2.4 b 81.2 1.3 c 78.0 1.5 d

– –

2.9 b 80.5 0.3 c 79.0 2.1 c

2.4 b 75.7 0.6 c 72.4 2.5 d2.2 b 81.1 1.4 c 78.4 1.5 d2.3 b 80.7 1.1 c 79.6 1.8 c2.4 bc 84.0 1.3 c 82.2 1.2 c3.2 bc 77.1 1.6 cd 75.4 2.3 d2.6 bc 78.0 1.2 c 74.9 1.6 d2.9 b 79.7 1.2 c 77.1 1.7 c

s (Pb0.05).

FM

SBM

WW

WG

CGM

RM

111

22

23 3

3

4

4

4

5

5

5

Principal component 1: 64.58%

Prin

cipa

l com

pone

nt 2

: 22

.16%

FM

SBM

WW

WG

CGM

RM

111

22

23 3

3

4

4

4

5

5

5

(Leu)

(Arg, Lys , Met, Thr , Val)

Fig. 1. Principal component analysis of the essential amino content (% protein) of the raw materials and of the shrimp faeces (% protein). CGM, corn gluten meal: FM, fishmeal; RM,rapeseed meal; SBM, soybean meal; WG, wheat gluten; WW, whole wheat; 1–5, faeces of shrimp fed the diets 1 to 5. The vectors represent the relative contribution of the EAAfound to be discriminating (variables) to the two extracted principal components.

114 L. Richard et al. / Aquaculture 322-323 (2011) 109–116

The mixture of plant protein ingredients in this study successfullyreplaced up to 25% of the fishmeal (24% dietary fishmeal) without anyadverse effect on shrimp performances. However, the significant reduc-tion in growth with FM replacement level of 50% or higher (diets con-taining 16, 8 or 0% of FM) is not in accordance with previous findingsof successful growth of P. monodon fed 14% FM (Smith et al., 2007a),6% FM (Sudaryono et al., 1999) or even 5% FM (Biswas et al., 2007), inassociation with 30% lupin seed meal, 40% lupin kernel meal and 62%soybean meal+L-lysine, respectively. In these studies, the utilisationof more than 10% of other marine protein feedstuffs (i.e., squid or

Table 6Parameter estimates obtained from the linear regressions between the ADC value of each essdietary protein content (Expt. 2).

Parameters Raw materials Arg His Ile

Slope FM 0.12 0.17 0.26WW 0.64 0.95 1.38WG −0.58 −0.83 −1.32CGM −0.17 −0.24 −0.38RSM −0.28 −0.41 −0.60

Intercept FM 89.2 83.4 78.9WW 88.9 82.8 78.4WG 95.2 92.2 92.5CGM 95.9 93.3 94.0RSM 94.7 91.5 90.9

R² FM 0.913 0.843 0.892WW 0.817 0.789 0.749WG 0.666 0.593 0.675CGM 0.848 0.802 0.874RSM 0.590 0.547 0.524

P-value FM *** *** ***WW *** *** ***WG ** ** **CGM *** *** ***RSM ** ** **

*Pb0.05.**Pb0.01.***Pb0.001.

shrimp meal) most likely facilitated higher FM replacement levels(Smith et al., 2000). Overall, higher levels of fishmeal substitutionby plant proteins have been reported in white shrimp, L. vannamei.Using rapeseed (canola) meal, soybean meal and wheat flour (Suarezet al., 2009) or fermented grains and wheat gluten (Molina-PovedaandMorales, 2004), 6% FM successfully maintained L. vannamei growthperformances. After 81 days of rearing in a green water semi-closedrecirculating tank system,Amaya et al. (2007a) even observed nodiffer-ence in growth between L. vannamei fed a 9% FM diet and a diet whereFM was replaced by solvent extracted soybean meal, corn gluten meal

ential amino acid and the relative contribution of each major protein source to the total

Leu Lys Met Phe Thr Val

0.42 0.05 0.22 0.31 0.26 0.262.37 0.32 1.19 1.76 1.43 1.42−2.16 −0.20 −1.15 −1.58 −1.31 −1.32−0.61 −0.06 −0.32 −0.45 −0.37 −0.38−1.00 −0.14 −0.52 −0.75 −0.64 −0.6267.2 93.5 81.9 73.7 77.4 77.765.8 93.1 81.6 72.6 76.8 77.389.5 95.8 93.7 90.1 91.0 91.591.9 96.1 94.8 91.9 92.5 93.287.1 95.9 92.4 88.5 89.9 90.20.898 0.596 0.954 0.876 0.927 0.9040.825 0.835 0.792 0.814 0.815 0.7610.681 0.341 0.745 0.650 0.677 0.6570.863 0.457 0.894 0.831 0.847 0.8710.548 0.568 0.576 0.551 0.607 0.555*** ** *** *** *** ****** *** *** *** *** ***** * *** ** ** ***** ** *** *** *** ***** ** ** ** ** **

115L. Richard et al. / Aquaculture 322-323 (2011) 109–116

and sorghum and 1% squid meal. Similarly, Sookying and Davis (2011)showed nobenefit on growth performance of L. vannamei for a diet con-taining about 10% FM compared with diets containing no FM and highlevels of soybean meal in combination with poultry by-product meal,distiller's dried grains or pea meal. Whether such differences in re-sponse between L. vannamei and P. monodon to low dietary levels ofFM are due to inter-species differences or due to the availability of nu-trients from the different feeds is worthy of investigation.

Among those factors susceptible to decrease growth of shrimp fol-lowing the substitution of fishmeal by plant protein sources, the pres-ence of anti-nutritional factors, a low digestibility or a low palatabilityof proteins leading to reduced feed intake have been identified(Sudaryono et al., 1999) as in teleost fish (Espe et al., 2007). In thepond trial, the amount of feed distributed was adjusted on a weeklybasis to the pond's biomass. As such, the mean daily feed supplyrecalculated over the experimental period did not vary among treat-ments, being 2.2, 2.3, 2.5, 2.6 and 2.5% of the shrimp average biomassfor diets 1 to 5, respectively. Moreover, whereas it is not possible toaccurately monitor actual feed consumptions in ponds, the carefulmeasurement of intakes during the small scale trial (Expt. 2) didnot reveal any particular palatability problem or feed wastage withthe plant-protein diets. Further leaching tests showed that thewater stability of the feed pellets was not negatively affected by theplant protein sources. Also, the FE (0.49–0.60) and PER (1.10–1.43)values from the pond study were in line with previous results on P.monodon (Bautista-Teruel et al., 2003; Paripatananont et al., 2001;Sudaryono et al., 1999), whereas energy retentions were slightlylower than values reported for L. vannamei (Suarez et al., 2009).

Since the quality of a dietary protein is determined not only by theanalysed concentration in nitrogen and EAA but also by their avail-ability, estimation of apparent digestibility coefficients has been un-dertaken in L. vannamei (Cruz-Suarez et al., 2009; Lemos et al.,2009; Yang et al., 2009). As such, several studies stated that differ-ences in nutrient availability between protein sources may affectthe success of fishmeal replacement strategies (Cruz-Suarez et al.,2009; Smith et al., 2007b; Sudaryono et al., 1999; Yang et al., 2009).However, quantitative data on nitrogen and EAA availability from al-ternative shrimp feed or from individual ingredients for shrimp re-mains limited. In this study, apparent digestibility of nutrients wasstrongly reduced at all fishmeal replacement levels. Dry matter(DM), protein and energy digestibility were 78.3%, 91.6% and 86.9%,respectively when fed the control diet (34% FM) and decreased by9% after replacing 25% of the dietary fishmeal (i.e. from 34 to 24%FM). At total fishmeal replacement, digestibility of DM was only58.3% and that of protein 74.8%, the latter value being similar tothat reported in juvenile L. vannamei (75.5%) fed a 30% corn glutenmeal based diet (Lemos et al., 2009) but lower than those reportedby Bautista-Teruel et al. (2003) in P. monodon fed a diet containing33% feed pea meal and 17% marine protein diets (77.3 and 87.5%, re-spectively for DM and protein). Although the current feed formula-tions (varying levels of various ingredients) were not specificallydesigned to identify the individual contribution of each proteinsource to the observed changes in digestibility (Brunson et al.,1997), some indirect assumptions can be made. First, the similarityin digestibility values between diets 2 and 3 and also between diets4 and 5 suggests that rapeseed meal, which compensated for theextra fishmeal replacement between these diets, did not negativelyaffect nutrient uptakes. The same is true for soybean meal, whichmoreover was added at a comparable level as in the control diet.Good results with rapeseed (Buchanan et al., 1997), soybean meal(Alvarez et al., 2007) or both (Suarez et al., 2009) were already ob-served in shrimp. Second, the classification of ingredients and faecesaccording to their AA profile (principal component analysis) clearlypointed towards a low digestibility of corn gluten meal (CGM),whose AA profile highly reflected that of the faecal protein. The lowdigestibility of CGM is further substantiated by the negative

correlations between the EAA availability coefficients and the contri-bution of CGM to total dietary protein supply (from 0% in the controldiet to 18% in diet 2 and 37% in diet 5). This was especially marked forleucine, the availability of which decreased by 14.5% after replacingonly 25% of the FM (diet 2). These results are surprising as corn glutenmeal is usually found to be a highly digestible protein source in tele-osts such as tilapia (Wu et al., 1995), rainbow trout (Gaylord et al.,2010), Atlantic cod (Tibbetts et al., 2006) or seabream (Pereira andOliva-Teles, 2003). In turbot, however, inclusion of CGM has beenshown to reduce the overall protein digestibility as well as that of sev-eral EAA in particular of leucine, decreasing from 94% to 68% whenCGM totally replaced FM (Regost et al., 1999). Recent reports of di-gestibility data in L. vannamei also showed the low apparent digest-ibility of i) protein from CGM (59%) relative to that of the other testingredients (mostly above 80%) (Lemos et al., 2009) as well as of ii)dietary amino acids when provided by CGM compared to mostother protein sources (Yang et al., 2009). For example, digestibilitycoefficients in the latter study dropped from 95 to 71% for Met, 72to 34% for Cys, 76 to 55% for Tyr, 83 to 69% for Pro and 81 to 65% forLeu when feeding corn gluten meal vs. fishmeal to L. vannamei(Yang et al., 2009). Furthermore, the low growth performance seenin L. vannamei fed a diet containing 15% CGM (Forster et al., 2002)possibly also stems from a reduced availability of protein and EAAfrom CGM. Nevertheless, it can not be excluded that the low digest-ibility of protein and AA when using high levels of CGM in shrimp,as in the present study and in previous reports for L. vannamei(Lemos et al., 2009; Yang et al., 2009), stems from a low quality ofthe raw material as suggested earlier in turbot fed high levels ofCGM (Regost et al., 1999). These observations deserve particular at-tention in further studies using CGM in shrimp feed.

Once corrected for faecal losses, the digestible EAA concentrationswere consistent with estimates for P. monodon using semi-purifieddiets under laboratory conditions (Chen et al., 1992; Millamenaet al., 1996b, 1997, 1998, 1999; Richard et al., 2010a). Only the avail-able methionine content in diets 3 to 5 was lower than our estimateof methionine requirement (0.8% diet DM) in P. monodon (Richardet al., 2010a). We recently demonstrated that up to 50% of the methi-onine requirement of P. monodon for growth can be spared by dietarycystine, while keeping the total sulphur AA at 1.1% of diet DM (Rich-ard et al., 2011). This finding is of interest in view of the increased useof plant protein sources in shrimp feed, most of these being charac-terised by a higher cysteine level and lower methionine over cysteineratio than fishmeal. However, cyst(e)ine availability has been shownto vary depending on the ingredient, as shown in L. vannamei, being81% for extruded soybean meal and only 34% for CGM (Yang et al.,2009). Since we did not measure cyst(e)ine availibility in the presentstudy and since diets were not supplemented with methionine (ana-lysed ‘crude’ Met level was above the Met requirement of 0.8 g/100 gdiet DM), a deficiency in total sulphur AA cannot be ruled out for thelow FM diets. These data highlight the need to determine the avail-ability of EAA together with that of cyst(e)ine to improve the use ofplant proteins as a substitute to fishmeal in diets of P. monodon.

Acknowledgements

The authors acknowledge Frédéric Terrier and Peyo Aguirre fortheir help during diet manufacturing and to Marie Jo Borthaire andVincent Michel for assistance with the laboratory analyses. Specialthanks are due to Christian Ramamonjisoa (Aqualma facility) for histechnical assistance. L.R., S.K. and I.G designed the study. L.R. did thedata analysis. L.R., S.K. and I.G contributed to the drafting of thepaper. A.S. did the AA analyses. L.R. and V.R. contributed to the orga-nisation of the experiment in Madagascar. There are no contractualagreements for the presented data which might cause conflicts of in-terest. The authors acknowledge UNIMA and institutional funds from

116 L. Richard et al. / Aquaculture 322-323 (2011) 109–116

INRA for funding this study and ANRT (France) for the scholarship toL.R. (CIFRE PhD Research Grant).

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