Aquaculture 322-323 (2011) 109–116

Contents lists available at SciVerse ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Availability of essential amino acids, nutrient utilisation and growth in juvenile black
tiger 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, France
b
UNIMA, 11 bis, rue Balzac, 75008 Paris, France
c
AQUALMA, BP 93 Immeuble SCIM, 4 rue Galliéni, Mahajanga 401, Madagascar

a r t i c l e i n f o a b s t r a c t

Article history: Two trials with juvenile black tiger shrimp (Penaeus monodon) were undertaken to study the effects of repla-
Received 4 August 2011 cing fishmeal by different levels of plant proteins on growth performances and nutrient utilisation of shrimp
Received in revised form 22 September 2011 in semi-intensive conditions (Expt. 1) and on the availability of dietary nitrogen (N) and amino acids (Expt.
Accepted 25 September 2011
2). Five isoproteic diets (on crude protein basis) were formulated to contain 34, 24, 16, 8, or 0% fishmeal, with
Available online 1 October 2011
fishmeal being replaced by a mixture of plant protein (corn gluten meal, wheat gluten, and rapeseed meal). In
Keywords:
Expt. 1, the shrimp (initial body weight, IBW 1.5 ± 0.1 g) were reared in earthen ponds for 144 days and fed
Fishmeal one of the experimental diets. Apparent digestibility of nutrients and AA were assessed in Expt. 2, using 150 L
Plant protein tanks and shrimp of 12.8 ± 0.4 g IBW. After 144 days in grow-out ponds, shrimp fed the diet with 24% of fish-
Amino acid meal had similar growth as those fed the control diet containing 34% fishmeal (0% replacement). When 50%
Shrimp or more of the fishmeal were replaced, weight gain as well as N and energy gains significantly decreased. Di-
Digestibility 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 an
increased incorporation of corn gluten meal. Our data confirm the need to improve our knowledge on AA
availability and raw material quality in order to improve fishmeal replacement in P. monodon diets.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Over the past 10 years, farmed shrimp production has expanded
from 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 are
often rich (25–50% of the diet) in fishmeal (FM), the preferred protein
source due to its well-balanced essential amino acid (EAA) profile.
As a consequence of the reduction of forage fisheries, production of
FM is levelling off (Naylor et al., 2009). Due to their wide availability,
plant proteins from oil seed meals such as soybean meal, sunflower
meal, or pulses such as lupin or pea have been investigated as poten-
tial fishmeal replacers in feed for both whiteleg shrimp (Litopenaeus
vannamei) and black tiger shrimp (Penaeus monodon), reaching
high levels of FM substitutions (Dayal et al., 2011; Forster et al.,
2002; Paripatananont et al., 2001; Smith et al., 2007a; Sudaryono
et al., 1999) or even total FM substitution in L. vannamei (Amaya
Abbreviations: ADC, apparent digestibility coefficient; EAA, essential amino acids;
CGM, corn gluten meal; FM, fishmeal; RSM, rapeseed meal; SBM, soybean meal; WG,
wheat gluten; WW, whole wheat.
⁎ Corresponding author at: INRA, UR 1067 NuMeA (Nutrition, Aquaculture and Genomics),
F-64310 Saint-Pée-sur-Nivelle, France. Tel.: +33 559515961; fax: +33 559545152.
E-mail address: geurden@st-pee.inra.fr (I. Geurden).
et al., 2007a; Sookying and Davis, 2011). However, most of the re-
search has been conducted under controlled laboratory conditions
rather than under field conditions (Amaya et al., 2007b; Sookying
and 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 often
used, 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 protein
accretion in P. monodon was not reduced by feeding a high level
(50% of diet) of an ‘imbalanced’ protein compared to an adequate
level (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 of
dietary protein. It also implies that poor quality protein sources can
be included at higher than required protein levels in order to fulfil
EAA 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 in
order 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.,

0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2011.09.032
110 L. Richard et al. / Aquaculture 322-323 (2011) 109–116

1989; Brunson et al., 1997), partly attributed to the presence of anti- Table 2
nutritional factors such as protease inhibitor, phytic acid or tannins Analysed amino acid composition of the experimental diets as g/100 g dry feed.

(Cruz-Suarez et al., 2001; Francis et al., 2001; Kumaraguru Diets

Vasagam et al., 2007). The importance of considering nitrogen and
1 2 3 4 5 Requirements1
EAA requirements in terms of available nutrients is substantiated by
EAA
two recent survey studies in L. vannamei, showing large variations be-
Arg 4.0 3.3 3.4 3.0 3.1 1.9
tween EAA availability of fishmeal and plant proteins (Lemos et al., His 1.6 1.4 1.5 1.3 1.4 0.8
2009; Yang et al., 2009). Ile 1.8 1.7 1.7 1.6 1.8 1.0
The main objective of this study was to evaluate the effect of Leu 2.9 3.6 3.5 4.3 4.8 1.7
replacing fishmeal by a mixture of plant proteins on the long term Lys 3.6 3.0 2.9 2.7 2.6 1.7*–2.1
Met 1.0 0.9 0.8 0.8 0.8 0.8*–0.9
performances of P. monodon reared under semi-intensive commercial Phe 1.7 1.8 1.9 2.0 2.3 1.4
pond conditions (pond study, Expt. 1). In addition, we examined Thr 1.7 1.5 1.6 1.5 1.5 1.4
whether changes in the protein source modified the availability of Val 1.9 1.9 1.9 1.7 1.9 1.4
the dietary nitrogen and AA (digestibility study, Expt. 2).
NEAA
Ala 2.2 2.4 2.3 2.6 2.7
Asp 3.4 3.1 3.2 2.9 2.9 Met+Cys
2. Material and methods Cys (theoretical)2 0.5 0.6 0.7 0.7 0.8 1.1*–1.3
Glu 6.0 6.7 7.0 8.0 9.3
2.1. Diets Gly 2.2 1.9 1.9 1.6 1.6
Pro 1.9 2.2 2.3 2.8 3.2
Ser 1.8 1.8 1.9 2.1 2.2
A practical diet (diet 1) was formulated to contain 34% fishmeal Tyr 1.3 1.3 1.4 1.6 1.8
(FM) as the main protein source (59% of the dietary protein, 1
Estimated EAA requirements (in g per 100 g DM) for P. monodon from the works of
Table 1). In four other diets, FM was gradually reduced to 24% (diet Millamena et al. (1996a, b, 1997, 1998, 1999), except for values marked with *, estimated
2), 16% (diet 3), 8% (diet 4) and 0% of the diet (diet 5), being replaced in Richard et al. (2010a) by means of a factorial approach (estimated protein requirements
by a mixture of plant protein sources (corn gluten meal, rapeseed were 30% diet DM).
2
meal, sorghum and wheat gluten) (Table 1). All five diets contained Calculated from the theoretical composition of the ingredients.

soybean 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. 2.2. Experimental designs
monodon (Table 2). In order to meet the dietary lysine requirements,
crystalline lysine was added in diets 4 and 5 (Table 1). All feeds were Two studies were performed at the facility of Aqualma (Unima,
industrially manufactured by Nutrima (La Réunion, France), pelleted Madagascar): i) a 144-day grow-out feeding trial in 0.235 ha earthen
to be 2 mm and stored in sealed bags prior to shipping to the farm ponds and ii) a 35-day digestibility trial in 150 L indoor tanks.
in Madagascar where they were stored in closed containers.
2.2.1. Pond trial (Experiment 1)
Juvenile black tiger shrimp (P. monodon) having an initial body
Table 1 weight of 1.5 ± 0.1 g were transferred into fifteen rectangular earthen
Composition and proximate analysis of the five diets fed to juvenile black tiger shrimp, ponds (mean depth, 1.20 m; surface, 0.235 ha) and reared for
P. monodon.
144 days under semi-intensive conditions at a mean stocking density
Diets of 7.1 shrimp/m 2 (16,886 shrimps per pond on average). The ponds
Ingredients (g/kg feed) 1 2 3 4 5 were characterised by a loam to sandy-loam soil. Two weeks prior
to the start of the trial, ponds were prepared by draining the water
Fishmeal 340 240 160 80 0
Squid meal 10 10 10 10 10
and the soil was tilled at a depth of 10 cm to improve oxidation and
Krill meal 10 10 10 10 10 mineralisation of organic matter of the pond. Lime was applied on
Soybean meal (48%) 215 191 250 200 234 the wet zone in the bottom of the ponds at a level of 1.5 kg/m². One
Whole wheat 350 200 92 160 60 week later, the ponds were filled with water (30–32 ppt) pumped
Wheat gluten (80%) 0 10 10 30 50
from a common canal until an approximate level of 170 cm. Water
Corn gluten meal (60%) 0 115 85 214 225
Rapeseed meal 0 0 150 100 150 was filtered at the entrance of the pond with a 300 μm nylon filter.
Sorghum 0 150 150 100 150 Ponds were then fertilised with triple super phosphate (Ca
Fish oil 3 8 16 20 26 (H2PO4)2.H20; 1 kg/ha) and urea (10 kg/ha) to stimulate natural pro-
Premix1 73 66 66 72 80 ductivity (planktonic bloom) in the pond.
Lysine 0 0 0 4 5
Temperature and dissolved oxygen (DO) were measured twice
daily (4:00 am and 4:00 pm), whereas salinity, pH, water height
Proximate composition
and turbidity (secchi disk) were measured every morning. Total
Dry matter (DM, %) 90.0 90.4 90.0 90.2 90.1
Crude protein 42.1 42.9 44.2 44.5 44.2 ammonia-nitrogen, nitrite and nitrate were measured once a week
(N × 6.25,% DM) on water sample collected close to the exit side of the pond. Mechan-
Crude lipid (% DM) 5.6 6.5 6.6 6.2 7.0 ical aeration was provided during the day when the levels of DO
Gross energy (kJ/g DM) 18.8 19.4 19.7 20.1 20.2 dropped below 3 mg/L.
Ash (% DM) 11.8 10.0 9.9 8.7 8.4
Inert marker (%DM) 1.0 1.3 1.4 1.5 1.5
Each diet was allocated to three replicate ponds. Feed was distribut-
ed uniformly throughout the pond area three times a day (10:00, 14:00
Leaching (%)2 6.3 3.6 5.3 5.2 6.2
and 18:00). The feeding rate was based on feeding table of the farm and
(2.1)a (1.5)b (0.8)ab (1.2)ab (0.4)a
assumed a constant 100% survival. Average temperature, oxygen, salin-
1
Premix (micro-nutrient and additive mixture) contained (g/kg feed): cholesterol
ity, pH and turbidity were 25.7 ± 1.5 °C, 7.2± 1.0 mg/L, 34.2 ± 2.3 mg/L,
(1 or 8), de-oiled soy lecithin (10), mono Ca phosphate (10 to 24), Ca Propionate (2
or 5), mineral and vitamin blends (20) and inert tracer (20). 8.7 ± 0.3 and 38.9 ± 18.7 cm, respectively.
2
Values are means and (SD) of five or six measurements. The P-value for the diet ef- During the trial, mean body weight was estimated every week by
fect was 0.0179. Values sharing a common letter are not significantly different. taking two samples of 200 shrimp from each pond (one at the
 L. Richard et al. / Aquaculture 322-323 (2011) 109–116
entrance side, the other at the exit side of the pond). Final mean body
weight was estimated after harvesting the whole pond (on average,
14,597 shrimp per pond, equivalent to a final density of 6.2
shrimp/m²), from a shrimp batch of 7 kg. At the beginning and at
the end of the trial, samples of 500 g food deprived shrimp (14 and
12 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 shrimp
samples for dry matter (g/100 g wet weight), protein (g/100 g wet
weight), 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 ± 0.3
and 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
½ððNf
Wf Þ–ðNi
Wi ÞÞ=½ðTFS
1000
APD
ð%CP=6:25ÞÞ=nm 
– 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 of
shrimp; BIOMi, BIOMf and BIOMd are the initial, final and dead shrimp
biomasses (kg); BIOMm is the mean biomass over the experimental
period ((BIOMi + BIOMf)/2); Wi and Wf are the initial and final
mean body weight (g); Δt the number of experimental days; FEED
is the total amount of feed distributed (kg); DML is the% of dry matter
loss of feed after the leaching test; DMF is the dry matter content of
the 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 dry
matter, 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 fresh
tissue) 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 determine
apparent digestibility coefficients. Seven shrimp (12.8 ± 0.4 g) were
stocked in 150 L covered plastic tanks (80 × 30.5 cm; diameter × -
height). Each feed was randomly assigned to the tanks (nine replicate
tanks per feed, at the exception of eight replicates for the control
diet). After 1 week of adaptation, shrimp were fed in excess four
times a day (7:00, 13:00, 19:00, 01:00) one of the experimental
diets 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 and
faeces 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 nylon
filter and rinsed with distilled water before being stored at −20 °C
111
in aluminium cups for later analyses. Thirty minutes prior to the next
feed 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 faeces was determined according
to the method of Atkinson (Atkinson et al., 1984). Apparent digestibility
coefficients (ADC) of dry matter (ADMD), protein (APD), energy (AED)
and amino acids were calculated as:
ADMD : 100×ð1–ðIdiet =Ifaeces ÞÞ
ADCnutrient : 100×½1–ððXfaeces =Xdiet Þ×ðIdiet =Ifaeces ÞÞ
With Idiet and faeces : concentration of inert marker in the feed and
faeces samplesðg=100 g DMÞXdiet and faeces : studied nutrient content
of feed and faeces ðg=100 gDMÞ:
2.3. Feed and faeces analyses
Water stability of feed was assessed using the methodology of
Cruz-Suarez et al. (2001). Briefly, 5 g of feed (n = 6) were allowed
to 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 matter
loss percentage (DML) was calculated as:
DMLð%Þ ¼ 100 ×ðDMbefore –DMafter Þ=DMbeforewith DMbefore and DMafter
the dry matter content of the feed before and after immersion in
water ðg=100 g feedÞ: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 the
same components except lipid. Amino acid contents of the feeds
were analysed using the AccQ.Tag method (Waters). Briefly, 100 mg
of 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 and
analysed by HPLC (column Symmetry C18 5 μm 3.9 × 150 mm) using
three 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 emission
wavelengths 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 of
variance (ANOVA) using dietary treatment as factor (n = 5 diets), fol-
lowed by a comparison of means using Duncan's multiple range test
in case of a significant effect (P b 0.05). The principal component anal-
ysis (PCA) used the EAA as variables and the EAA contents of the raw
materials (EAA, % ingredient protein) and of the faeces from shrimp
fed 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 were
analysed against changes in the relative dietary protein contribution
of each major protein source, using linear regressions. ANOVA and
PCA 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 GraphPad
Prism 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 the
control diet and diet 5, being different from that of diet 2 (3.6%)
(Table 1, P b 0.05). Survival (85.2 to 87.3%) was not affected by the
level of fishmeal in the diet (Table 3, P N 0.05). Shrimp from the con-
trol group (diet 1) had the highest biomass gain (310.9 kg), final
yield (1321 kg/ha) and final individual body weight (21.0 g). When
replacing 50% of the FM (diet 3), performances were significantly re-
duced by 20% (Table 3, P b 0.05). The lowest growth was observed in
shrimp fed diet 5 devoid of fishmeal (218 kg of biomass gain per
pond; 15.1 g of final BW; 955 kg/ha of final yield) (Table 3, P b 0.05).
The body composition of the shrimp was not affected by the replace-
ment of dietary FM (Table 4, P N 0.05). The pond-based nutrient reten-
tion parameters (FE, PER, nitrogen and energy retentions) were all
significantly 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, P b 0.05). 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, P N 0.05).
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 acids
from 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, P b 0.05). When 25% of the fishmeal was
replaced by plant protein (diet 2), digestibility decreased by 9% com-
pared to the control diet. Among the AA, availability of leucine was
the most affected (− 14.5%), followed by proline (−11.3%), phenylal-
anine (− 10.6%) and alanine (− 10.9%). When 100% of FM was
replaced (diet 5), ADC of dry matter, protein and energy decreased
by 20, 16.8 and 17.1%, respectively. The maximal loss of AA availabil-
ity (−26%) was observed for leucine (Table 5).
EAA compositions of the protein sources and the faecal protein
were analysed using a multivariate analysis (principal component
analysis, PCA) presented in Fig. 1. The space is described by a number
of dimensions defined by the variables (in our case, 9 dimensions for
the 9 studied EAA). The first two principal components (PC1 and PC2)
accounted for 87% of the total variability. Therefore, the data (EAA
composition of ingredients and faeces) were analysed in a factorial
plane 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 respective
contribution of 16.1, 15.7, 14.5, 13.9% and 12.4% (Fig. 1). The second
axis (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, were
not highly discriminated (close to the 0, 0 axes intercept). Corn gluten
meal (CGM) in particular was clearly separated from the other pro-
tein sources, reflecting its low lysine and high leucine content. Faeces
were highly discriminated along PC2, with faeces of the control group
(0% replacement, F1) being in opposition with those of the 75 and
100% replacement groups (F4 and F5) (Fig. 1), which indicate a
lower leucine content in the faecal protein from the control group
than from the two other groups fed increased amounts of CGM rich
in leucine (close following PC2). To further investigate the relative
contribution of ingredients to the digestibility of the whole diets, lin-
ear regression analyses were done using each ingredient's relative
contribution to dietary protein content and the apparent EAA digest-
ibility coefficients of the diets (Table 6). The results indicate that both
fishmeal (FM) and whole wheat (WW) utilisation are positively cor-
related to EAA availability (Table 6, P b 0.05), FM contributing however
more than WW to EAA availability (0.84b R²b 0.95 and 0.75 b R²b 0.83,
respectively). All other ingredients (CGM, RM, and WG) are negatively
correlated with EAA availability (Table 6, P b 0.05). Among them, in-
creased incorporation of CGM contributed the most to the loss of avail-
ability for all EAA (0.80 b R² b 0.89, Table 6).
4. Discussion
Most studies on fishmeal replacement in feed for penaeid shrimp
have been conducted under controlled indoor conditions and over
relatively short periods (Amaya et al., 2007b). To our knowledge,
this is the first study on a long-term effect of fishmeal replacement

Table 3
Effect of fishmeal replacement on growth performance of P. monodon reared in earthen ponds during 144 days (Expt. 1)1.

Diets

1 2 3 4 5

Mean SD Mean SD Mean SD Mean SD Mean SD P-values2

Performances and feed allocation

(per pond)
Survival (%) 87.3 6.1 86.6 3.1 85.8 3.4 85.2 3.7 87.0 14.3 0.9961
Initial biomass (kg/pond) 24.1 1.0 24.8 2.3 25.0 1.1 24.5 2.4 24.4 2.3 0.9785
a ab bc bc c
Final biomass (kg/pond) 310.5 11.0 282.5 2.3 249.9 22.4 235.9 35.2 224.4 42.5 0.0167
a ab bc c c
Biomass gain (kg/pond) 310.9 9.9 281.2 2.7 247.2 18.4 233.3 32.7 218.0 23.5 0.0014
a ab bc bc c
Final yield (kg/ha) 1321 47 1202 10 1063 95 1004 150 955 181 0.0166
a ab b bc c
Total feed supplied (TFS, kg DM/pond) 518.1 13.4 500.2 14.6 482.2 6.3 473.0 20.8 446.1 16.4 0.0017

Performances and feed allocation

(per shrimp)
Initial mean body weight (g/shr) 1.4 0.1 1.5 0.1 1.5 0.1 1.4 0.1 1.4 0.1 0.8544
a a b bc c
Final mean body weight (g/shr) 21.0 1.3 19.8 0.5 17.2 1.1 16.2 1.6 15.1 0.3 0.0003
Dry matter (DM) supplied (% biomass per day) 2.2 0.1 2.3 0.1 2.5 0.2 2.6 0.3 2.5 0.3 0.1456
a a ab bc c
DM supplied (g/shrimp/day) 0.23 0.01 0.23 0.01 0.21 0.01 0.21 0.00 0.20 0.01 0.0053
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 (P b 0.05).
 L. Richard et al. / Aquaculture 322-323 (2011) 109–116 113

Table 4
Final 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
2
Final body composition (g/100 g wet weight) Mean SD Mean SD Mean SD Mean SD Mean SD P-values

Dry matter 27.8 0.8 27.7 0.4 27.0 1.4 27.9 1.9 27.7 1.9 0.9465
Protein 20.7 0.8 20.6 0.2 20.3 0.8 21.3 1.3 20.7 1.4 0.8193
Fat 2.7 0.3 2.9 0.2 2.8 0.1 2.8 0.6 2.7 0.3 0.8859
Ash 3.2 0.1 3.4 0.2 3.2 0.3 3.4 0.2 3.7 0.1 0.1666
Gross 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 parameters

a ab bc c c
Pond-based FE 0.60 0.01 0.56 0.02 0.51 0.03 0.49 0.05 0.49 0.03 0.0070
Pond-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 0.4825
a a b b b
Pond-based PER 1.43 0.03 1.31 0.04 1.16 0.08 1.11 0.11 1.10 0.08 0.0011
Pond-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 0.1560
a a b b b
Pond-based N gain (mg/shrimp/day) 4.48 0.19 4.14 0.13 3.50 0.34 3.47 0.57 3.10 0.16 0.0022
a ab b b bc
Pond-based N retention (% intake) 29.2 0.7 26.6 1.0 23.1 2.6 23.2 3.5 22.4 0.6 0.0102
Pond-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 0.3879
a a b b b
Pond-based energy gain (kJ/shr/day) 0.82 0.02 0.77 0.03 0.65 0.07 0.62 0.11 0.58 0.04 0.0034
a ab b b bc
Pond-based E retention (% GE intake) 19.2 0.4 17.4 0.5 15.5 1.9 14.8 2.4 14.5 0.6 0.0110
Pond-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 (P b 0.05).

by plant protein in the black tiger shrimp P. monodon using practical
diets under commercial rearing conditions. After 144 days of earthen
pond rearing, average survival (87%) and FE (0.60) of P. monodon fed
the control diet (34% fishmeal) were comparable to values reported
for 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 for
the 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/m²
in 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 slightly
above those reported for P. monodon fed a commercial diet either
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 of
shrimp in semi-intensive ponds generally enables shrimp to feed
more 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 high
water renewal and absence of natural biota in the 150 L tanks used
by 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 can
lead to reduced N retention (Burford and Williams, 2001).

Table 5
Effect of fishmeal replacement by plant protein mixture on the apparent digestible coefficients (ADC, %) for dry matter, protein, energy and amino acids in P. monodon (Expt. 2).

Diets

1 2 3 4 5

Mean SD Mean SD Mean SD Mean SD Mean SD

DM 78.3 1.3 a 68.9 3.8 b 61.3 4.5 c 61.8 4.5 c 58.3 3.9 c
Protein 91.6 0.5 a 82.7 1.7 b 81.4 2.0 b 78.5 2.4 c 74.8 2.4 d
Energy 86.9 0.9 a 78.8 1.9 b 74.0 2.4 c 73.5 2.8 c 69.8 2.1 d

EAA
Arg 96.6 0.3 a 93.1 0.7 b 92.1 1.1 b 90.7 0.5 c 89.6 0.5 c
His 94.7 0.4 a 89.0 1.1 b 87.5 2.2 bc 85.3 0.7 cd 84.6 1.5 d
Ile 95.4 0.2 a 87.5 1.3 b 86.1 2.8 b 81.6 0.4 c 80.0 2.4 c
Leu 95.1 0.3 a 80.6 1.7 b 78.1 3.4 b 72.4 1.4 c 69.3 2.7 c
Lys 96.9 0.2 a 94.9 0.7 b 93.9 1.0 b 94.5 0.2 b 93.9 0.4 b
Met 95.7 0.2 a 89.6 1.1 b 88.2 1.5 b 85.2 0.6 c 82.1 0.7 d
Phe 94.3 0.5 a 83.7 1.4 b 81.5 3.4 b 77.6 1.4 c 75.3 2.6 c
Thr 94.0 0.4 a 86.5 1.1 b 84.1 2.4 b 81.2 1.3 c 78.0 1.5 d
Trp – – – – –
Val 94.6 0.2 a 86.9 1.1 b 85.0 2.9 b 80.5 0.3 c 79.0 2.1 c

NEAA
Ala 94.7 0.4 a 83.8 0.9 b 80.9 2.4 b 75.7 0.6 c 72.4 2.5 d
Asp 92.6 0.5 a 86.1 1.2 b 84.1 2.2 b 81.1 1.4 c 78.4 1.5 d
Glu 95.5 0.3 a 85.8 0.9 b 84.4 2.3 b 80.7 1.1 c 79.6 1.8 c
Gly 91.9 0.5 a 87.2 1.2 b 84.8 2.4 bc 84.0 1.3 c 82.2 1.2 c
Pro 93.3 0.6 a 82.0 1.4 b 79.8 3.2 bc 77.1 1.6 cd 75.4 2.3 d
Ser 92.7 0.6 a 83.3 1.3 b 80.8 2.6 bc 78.0 1.2 c 74.9 1.6 d
Tyr 95.0 0.5 a 85.1 1.4 b 82.9 2.9 b 79.7 1.2 c 77.1 1.7 c

Mean values with different superscripted letters were significantly different between groups (P b 0.05).
114 L. Richard et al. / Aquaculture 322-323 (2011) 109–116

11
1

FM
Principal component 2: 22.16%

WW 3
2
3 3
2
WG
RM SBM 2
44
4 55
5

(Leu) CGM

Principal component 1: 64.58% (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 EAA
found to be discriminating (variables) to the two extracted principal components.

The mixture of plant protein ingredients in this study successfully
replaced up to 25% of the fishmeal (24% dietary fishmeal) without any
adverse 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 findings
of 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), in
association with 30% lupin seed meal, 40% lupin kernel meal and 62%
soybean meal+ L-lysine, respectively. In these studies, the utilisation
of more than 10% of other marine protein feedstuffs (i.e., squid or
shrimp meal) most likely facilitated higher FM replacement levels
(Smith et al., 2000). Overall, higher levels of fishmeal substitution
by plant proteins have been reported in white shrimp, L. vannamei.
Using rapeseed (canola) meal, soybean meal and wheat flour (Suarez
et al., 2009) or fermented grains and wheat gluten (Molina-Poveda
and Morales, 2004), 6% FM successfully maintained L. vannamei growth
performances. After 81 days of rearing in a green water semi-closed
recirculating tank system, Amaya et al. (2007a) even observed no differ-
ence in growth between L. vannamei fed a 9% FM diet and a diet where
FM was replaced by solvent extracted soybean meal, corn gluten meal

Table 6
Parameter estimates obtained from the linear regressions between the ADC value of each essential amino acid and the relative contribution of each major protein source to the total
dietary protein content (Expt. 2).

Parameters Raw materials Arg His Ile Leu Lys Met Phe Thr Val

Slope FM 0.12 0.17 0.26 0.42 0.05 0.22 0.31 0.26 0.26
WW 0.64 0.95 1.38 2.37 0.32 1.19 1.76 1.43 1.42
WG − 0.58 − 0.83 − 1.32 − 2.16 − 0.20 − 1.15 − 1.58 − 1.31 − 1.32
CGM − 0.17 − 0.24 − 0.38 − 0.61 − 0.06 − 0.32 − 0.45 − 0.37 − 0.38
RSM − 0.28 − 0.41 − 0.60 − 1.00 − 0.14 − 0.52 − 0.75 − 0.64 − 0.62
Intercept FM 89.2 83.4 78.9 67.2 93.5 81.9 73.7 77.4 77.7
WW 88.9 82.8 78.4 65.8 93.1 81.6 72.6 76.8 77.3
WG 95.2 92.2 92.5 89.5 95.8 93.7 90.1 91.0 91.5
CGM 95.9 93.3 94.0 91.9 96.1 94.8 91.9 92.5 93.2
RSM 94.7 91.5 90.9 87.1 95.9 92.4 88.5 89.9 90.2
R² FM 0.913 0.843 0.892 0.898 0.596 0.954 0.876 0.927 0.904
WW 0.817 0.789 0.749 0.825 0.835 0.792 0.814 0.815 0.761
WG 0.666 0.593 0.675 0.681 0.341 0.745 0.650 0.677 0.657
CGM 0.848 0.802 0.874 0.863 0.457 0.894 0.831 0.847 0.871
RSM 0.590 0.547 0.524 0.548 0.568 0.576 0.551 0.607 0.555
P-value FM *** *** *** *** ** *** *** *** ***
WW *** *** *** *** *** *** *** *** ***
WG ** ** ** ** * *** ** ** **
CGM *** *** *** *** ** *** *** *** ***
RSM ** ** ** ** ** ** ** ** **

*P b 0.05.
**P b 0.01.
***P b 0.001.
 L. Richard et al. / Aquaculture 322-323 (2011) 109–116
and sorghum and 1% squid meal. Similarly, Sookying and Davis (2011)
showed no benefit on growth performance of L. vannamei for a diet con-
taining about 10% FM compared with diets containing no FM and high
levels 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 of
FM 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 palatability
of proteins leading to reduced feed intake have been identified
(Sudaryono et al., 1999) as in teleost fish (Espe et al., 2007). In the
pond trial, the amount of feed distributed was adjusted on a weekly
basis to the pond's biomass. As such, the mean daily feed supply
recalculated 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 biomass
for diets 1 to 5, respectively. Moreover, whereas it is not possible to
accurately monitor actual feed consumptions in ponds, the careful
measurement of intakes during the small scale trial (Expt. 2) did
not reveal any particular palatability problem or feed wastage with
the plant-protein diets. Further leaching tests showed that the
water stability of the feed pellets was not negatively affected by the
plant 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 slightly
lower than values reported for L. vannamei (Suarez et al., 2009).
Since the quality of a dietary protein is determined not only by the
analysed 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 affect
the 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 was
strongly 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 by
9% after replacing 25% of the dietary fishmeal (i.e. from 34 to 24%
FM). At total fishmeal replacement, digestibility of DM was only
58.3% and that of protein 74.8%, the latter value being similar to
that reported in juvenile L. vannamei (75.5%) fed a 30% corn gluten
meal based diet (Lemos et al., 2009) but lower than those reported
by Bautista-Teruel et al. (2003) in P. monodon fed a diet containing
33% 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 specifically
designed to identify the individual contribution of each protein
source to the observed changes in digestibility (Brunson et al.,
1997), some indirect assumptions can be made. First, the similarity
in digestibility values between diets 2 and 3 and also between diets
4 and 5 suggests that rapeseed meal, which compensated for the
extra fishmeal replacement between these diets, did not negatively
affect nutrient uptakes. The same is true for soybean meal, which
moreover 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 faeces
according to their AA profile (principal component analysis) clearly
pointed towards a low digestibility of corn gluten meal (CGM),
whose AA profile highly reflected that of the faecal protein. The low
digestibility of CGM is further substantiated by the negative
115
correlations between the EAA availability coefficients and the contri-
bution of CGM to total dietary protein supply (from 0% in the control
diet to 18% in diet 2 and 37% in diet 5). This was especially marked for
leucine, the availability of which decreased by 14.5% after replacing
only 25% of the FM (diet 2). These results are surprising as corn gluten
meal 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 and
Oliva-Teles, 2003). In turbot, however, inclusion of CGM has been
shown to reduce the overall protein digestibility as well as that of sev-
eral EAA in particular of leucine, decreasing from 94% to 68% when
CGM 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 test
ingredients (mostly above 80%) (Lemos et al., 2009) as well as of ii)
dietary amino acids when provided by CGM compared to most
other protein sources (Yang et al., 2009). For example, digestibility
coefficients in the latter study dropped from 95 to 71% for Met, 72
to 34% for Cys, 76 to 55% for Tyr, 83 to 69% for Pro and 81 to 65% for
Leu when feeding corn gluten meal vs. fishmeal to L. vannamei
(Yang et al., 2009). Furthermore, the low growth performance seen
in L. vannamei fed a diet containing 15% CGM (Forster et al., 2002)
possibly also stems from a reduced availability of protein and EAA
from 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 of
the raw material as suggested earlier in turbot fed high levels of
CGM (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 concentrations
were consistent with estimates for P. monodon using semi-purified
diets under laboratory conditions (Chen et al., 1992; Millamena
et al., 1996b, 1997, 1998, 1999; Richard et al., 2010a). Only the avail-
able methionine content in diets 3 to 5 was lower than our estimate
of methionine requirement (0.8% diet DM) in P. monodon (Richard
et al., 2010a). We recently demonstrated that up to 50% of the methi-
onine requirement of P. monodon for growth can be spared by dietary
cystine, 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 use
of plant protein sources in shrimp feed, most of these being charac-
terised by a higher cysteine level and lower methionine over cysteine
ratio than fishmeal. However, cyst(e)ine availability has been shown
to vary depending on the ingredient, as shown in L. vannamei, being
81% for extruded soybean meal and only 34% for CGM (Yang et al.,
2009). Since we did not measure cyst(e)ine availibility in the present
study and since diets were not supplemented with methionine (ana-
lysed ‘crude’ Met level was above the Met requirement of 0.8 g/100 g
diet DM), a deficiency in total sulphur AA cannot be ruled out for the
low 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 of
plant proteins as a substitute to fishmeal in diets of P. monodon.
Acknowledgements
The authors acknowledge Frédéric Terrier and Peyo Aguirre for
their help during diet manufacturing and to Marie Jo Borthaire and
Vincent Michel for assistance with the laboratory analyses. Special
thanks are due to Christian Ramamonjisoa (Aqualma facility) for his
technical assistance. L.R., S.K. and I.G designed the study. L.R. did the
data analysis. L.R., S.K. and I.G contributed to the drafting of the
paper. A.S. did the AA analyses. L.R. and V.R. contributed to the orga-
nisation of the experiment in Madagascar. There are no contractual
agreements 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 to
L.R. (CIFRE PhD Research Grant).
References
Akiyama, D.M., Coelho, S.R., Lawrence, A.L., Robinson, E.H., 1989. Apparent digestibility
of feedstuffs by the marine shrimp Penaeus vannamei Boone. Nippon Suisan Gakkai
55, 91–98.
Alvarez, J.S., Hernandez-Llamas, A., Galindo, J., Fraga, I., Garcia, T., Villarreal, H., 2007.
Substitution of fishmeal with soybean meal in practical diets for juvenile white
shrimp Litopenaeus schmitti (Perez-Farfante and Kensley, 1997). Aquaculture Re-
search 38, 689–695.
Amaya, E.A., Davis, D.A., Rouse, D.B., 2007a. Alternative diets for Pacific white shrimp
(Litopenaeus vannamei). Aquaculture 262, 419–425.
Amaya, E.A., Davis, D.A., Rouse, D.B., 2007b. Replacement of fish meal in practical diets
for the Pacific white shrimp (Litopenaeus vannamei) reared under pond conditions.
Aquaculture 262, 393–401.
Atkinson, J.L., Hilton, J.W., Slinger, S.J., 1984. Evaluation of acid-insoluble ash as an in-
dicator of feed digestibility in rainbow trout Salmo gairdneri. Canadian Journal of
Fisheries and Aquatic Sciences 41, 1384–1386.
Bautista-Teruel, M.N., Eusebio, P.S., Welsh, T.P., 2003. Utilization of feed pea, Pisum sati-
vum, meal as a protein source in practical diets for juvenile tiger shrimp, Penaeus
monodon. Aquaculture 225, 121–131.
Biswas, P., Pal, A.K., Sahu, N.P., Reddy, A.K., Prusty, A.K., Misra, S., 2007. Lysine and/or
phytase supplementation in the diet of Penaeus monodon (Fabricius) juveniles: ef-
fect on growth, body composition and lipid profile. Aquaculture 265, 253–260.
Briggs, M.R.P., Funge-Smith, S.J., 1994. A nutrient budget of some intensive marine
shrimp ponds in Thaïland. Aquaculture Research 25, 789–811.
Brunson, J.F., Romaire, R.P., Reigh, R.C., 1997. Apparent digestibility of selected ingredi-
ents in diets for white shrimp Penaeus setiferus L. Aquaculture Nutrition 3, 9–16.
Buchanan, J., Sarac, H.Z., Poppi, D., Cowan, R.T., 1997. Effects of enzyme addition to ca-
nola meal in prawn diets. Aquaculture 151, 29–35.
Burford, M.A., Williams, K.C., 2001. The fate of nitrogenous waste from shrimp feeding.
Aquaculture 198, 79–93.
Burford, M.A., Preston, N.P., Glibert, P.M., Dennison, W.C., 2002. Tracing the fate of 15N-
enriched feed in an intensive shrimp system. Aquaculture 206, 199–216.
Casillas-Hernandez, R., Nolasco-Soria, H., Garcia-Galano, T., Carrillo-Farnes, O., Paez-Osuna,
F., 2007. Water quality, chemical fluxes and production in semi-intensive Pacific white
shrimp (Litopenaeus vannamei) culture ponds utilizing two different feeding strategies.
Aquacultural Engineering 36, 105–114.
Chen, H.Y., Leu, Y.T., Roelants, I., 1992. Quantification of arginine requirements of juve-
nile marine shrimp, Penaeus monodon using microencapsulated arginine. Marine
Biology 114, 229–233.
Cruz-Suarez, L.E., Ricque-Marie, D., Tapia-Salazar, M., McCallum, I.M., Hickling, D.,
2001. Assessment of differently processed feed pea (Pisum sativum) meals and ca-
nola meal (Brassica sp.) in diets for blue shrimp (Litopenaeus stylirostris). Aquacul-
ture 196, 87–104.
Cruz-Suarez, L.E., Tapia-Salazar, M., Villarreal-Cavazos, D., Beltran-Rocha, J., Nieto-
Lopez, M., Lemme, A., Ricque-Marie, D., 2009. Apparent dry matter, energy, protein
and amino acid digestibility of four soybean ingredients in white shrimp Litope-
naeus vannamei juveniles. Aquaculture 292, 87–94.
Dayal, J.S., Rajaram, V., Ambasankar, K., Ali, S.A., 2011. Sunflower oil cake as a replace-
ment for fishmeal in feeds of tiger shrimp, Penaeus monodon reared in tanks and in
net cages. Indian Journal of Geology-Marine Science 40 (3), 460–470.
Deering, M.J., Hewitt, D.R., Sarac, H.Z., 1996. A comparison of inert markers used to es-
timate protein digestibility in the leader prawn Penaeus monodon. Journal of the
World Aquaculture Society 27 (1), 103–106.
Espe, M., Lemme, A., Petri, A., El-Mowafi, A., 2007. Assessment of lysine requirement for
maximal protein accretion in Atlantic salmon using plant protein diets. Aquacul-
ture 263, 168–178.
Forster, I., Dominy, W., Tacon, A.G., 2002. The use of concentrates and other soy prod-
ucts in shrimp feeds. In Avances en Nutricion Acuicola VI. Memorias del VI Simpo-
sium Internacional de Nutricion Acuicola. 3–6 Septiembre 2002, Cancún, Quintana
Roo, México. Cruz-Suarez, L.E., Ricque-Marie, D., Gaxiola-Cortes, M.G., Simoes, N.
(ed.), 527–540.
Fournier, V., Huelvan, C., Desbruyeres, E., 2004. Incorporation of a mixture of plant
feedstuffs as substitute for fish meal in diets of juvenile turbot (Psetta maxima).
Aquaculture 236, 451–465.
Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-
derived alternate fish feed ingredients and their effects in fish. Aquaculture 199,
197–227.
Gaylord, T.G., Barrows, F.T., Rawles, S.D., 2010. Apparent amino acid availability from
feedstuffs in extruded diets for rainbow trout Oncorhynchus mykiss. Aquaculture
Nutrition 16 (4), 400–406.
Gomes, E.F., Rema, P., Kaushik, S.J., 1995. Replacement of fish meal by plant proteins in
the diet of rainbow trout (Oncorhynchus mykiss): digestibility and growth perfor-
mance. Aquaculture 130, 177–186.
Jackson, C., Preston, N.P., Thompson, P.J., Burford, M.A., 2003. Nitrogen budget and ef-
fluent nitrogen components at an intensive shrimp farm. Aquaculture 218,
397–411.
Kaushik, S.J., Coves, D., Dutto, G., Blanc, D., 2004. Almost total replacement of fish meal
by plant protein sources in the diet of a marine teleost, the European seabass,
Dicentrarchus labrax. Aquaculture 230, 391–404.
Kumaraguru Vasagam, K.P., Balasubramanian, T., Venkatesan, R., 2007. Apparent di-
gestibility of differently processed grain legumes, cow pea and mung bean in
black tiger shrimp, Penaeus monodon Fabricius and associated histological anoma-
lies in hepatopancreas and midgut. Animal Feed Science and Technology 132,
250–266.
Lemos, D., Lawrence, A.L., Siccardi, A.J., 2009. Prediction of apparent protein digestibil-
ity of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with
species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei.
Aquaculture 295, 89–98.
Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996a. Methionine requirement
of juvenile tiger shrimp Penaeus monodon Fabricius. Aquaculture 143, 403–410.
Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996b. Valine requirement of
postlarval tiger shrimp, Penaeus monodon Fabricius. Aquaculture Nutrition 2,
129–132.
Millamena, O.M., Bautista, M.N., Reyes, O.S., Kanazawa, A., 1997. Threonine require-
ment of juvenile marine shrimp Penaeus monodon. Aquaculture 151, 9–14.
Millamena, O.M., Bautista-Teruel, M.N., Reyes, O.S., Kanazawa, A., 1998. Requirements
of juvenile marine shrimp, Penaeus monodon (Fabricius) for lysine and arginine.
Aquaculture 164, 95–104.
Millamena, O.M., Teruel, M.B., Kanazawa, A., Teshima, S., 1999. Quantitative dietary re-
quirements of postlarval tiger shrimp, Penaeus monodon, for histidine, isoleucine,
leucine, phenylalanine and tryptophan. Aquaculture 179, 169–179.
Molina-Poveda, C., Morales, M.E., 2004. Use of a mixture of barley-based fermented
grains and wheat gluten as an alternative protein source in practical diets for Lito-
penaeus vannamei (Boone). Aquaculture Research 35, 1158–1165.
Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin, D.M.,
Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite re-
sources. Proceedings of the National Academy of Sciences 106, 15103–15110.
Paripatananont, T., Boonyaratpalin, M., Pengseng, P., Chotipuntu, P., 2001. Substitution
of soy protein concentrate for fishmeal in diets of tiger shrimp Penaeus monodon.
Aquaculture Research 32, 369–374.
Pereira, T.G., Oliva-Teles, A., 2003. Evaluation of corn gluten meal as a protein source in
diets for gilthead sea bream (Sparus aurata L.) juveniles. Aquaculture Research 34,
1111–1117.
Regost, C., Arzel, J., Kaushik, S.J., 1999. Partial or total replacement of fish meal by corn
gluten meal in diet for turbot (Psetta maxima). Aquaculture 180, 99–117.
Richard, L., Blanc, P.-P., Rigolet, V., Kaushik, S.J., Geurden, I., 2010a. Maintenance and
growth requirements for nitrogen, lysine and methionine and their utilisation effi-
ciencies in juvenile black tiger shrimp, Penaeus monodon, using a factorial ap-
proach. The British Journal of Nutrition 103, 984–995.
Richard, L., Vachot, C., Brèque, J., Blanc, P.-P., Rigolet, V., Kaushik, S.J., Geurden, I., 2010b.
The effect of protein and methionine intake on glutamate dehydrogenase and ala-
nine aminotransferase activities in juvenile black tiger shrimp Penaeus monodon.
Journal of Experimental Marine Biology and Ecology 391, 153–160.
Richard, L., Vachot, C., Surget, A., Rigolet, V., Kaushik, S.J., Geurden, I., 2011. The effect of
choline and cystine on the utilisation of methionine for protein accretion, remethy-
lation qnd trans-sulfuration in juvenile shrimp Penaeus monodon. The British Jour-
nal of Nutrition 106, 825–835.
Smith, D.M., Tabrett, S.J., Irvin, S.J., Barclay, M., 2000. Fishmeal replacement research for
shrimp feed in Australia. In In: Crus-Suarez, L.E., Ricque-Marie, D., Tapia-Salazar,
Olvera-Novoa, M.A., Civera-Cerecedo, R. (Eds.), Avances en Nutricion Acuicola V.
Memorias del V Simposium Internacional de Nutricion Acuicola, pp. 277–286.
19–22 Noviembre, 2000. Merida, Yucatan, Mexico.
Smith, D.M., Tabrett, S.J., Glencross, B.D., 2007a. Growth response of the black tiger
shrimp, Penaeus monodon fed diets containing different lupin cultivars. Aquacul-
ture 269, 436–446.
Smith, D.M., Tabrett, S.J., Glencross, B.D., Irvin, S.J., Barclay, M.C., 2007b. Digestibility of
lupin kernel meals in feeds for the black tiger shrimp, Penaeus monodon. Aquacul-
ture 264, 353–362.
Sookying, D., Davis, D.A., 2011. Pond production of pacific white shrimp (Litopenaeus
vannamei) fed high levels of soybean meal in various combinations. Aquaculture
319 (1–2), 141–149.
Suarez, J.A., Gaxiola, G., Mendoza, R., Cadavid, S., Garcia, G., Alanis, G., Suarez, A., Faillace, J.,
Cuzon, G., 2009. Substitution of fish meal with plant protein sources and energy bud-
get for white shrimp Litopenaeus vannamei (Boone, 1931). Aquaculture 289, 118–123.
Sudaryono, A., Tsvetnenko, E., Evans, L.H., 1999. Evaluation of potential of lupin meal as
an alternative to fish meal in juvenile Penaeus monodon diets. Aquaculture Nutri-
tion 5, 277–285.
Tacon, A.G.J., Metian, M., 2008. Global overview on the use of fish meal and fish oil in
industrially compounded aquafeeds: trends and future prospects. Aquaculture
285, 146–158.
Teichert-Coddington, D.R., Martinez, D., Ramírez, E., 2000. Partial nutrient budgets for
semi-intensive shrimp farms in Honduras. Aquaculture 190, 139–154.
Tibbetts, S.M., Milley, J.E., Lall, S.P., 2006. Apparent protein and energy digestibility of
common and alternative feed ingredients by Atlantic cod, Gadus morhua (Linnaeus,
1758). Aquaculture 261 (4), 1314–1327.
Wu, Y.V., Rosati, R.R., Sessa, D.J., Brown, P.B., 1995. Evaluation of corn gluten geal as a
protein source in tilapia diets. Journal of Agricultural and Food Chemistry 43,
1585–1588.
Yang, Q., Zhou, X., Zhou, Q., Tan, B., Chi, S., Dong, X., 2009. Apparent digestibility of selected
feed ingredients for white shrimp Litopenaeus vannamei, Boone. Aquaculture Re-
search 41, 78–86.