Nutrition of Litopenaeus Vannamei Reared

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Nutrition of Litopenaeus vannamei

reared in tanks or in ponds
gérard Cuzon
Aquaculture
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Aquaculture 235 (2004) 513 – 551
www.elsevier.com/locate/aqua-online
Nutrition of Litopenaeus vannamei reared in tanks

or in ponds
Gérard Cuzon a,*, Addison Lawrence b, Gabriela Gaxiola c,
Carlos Rosas c, J. Guillaume d
a
Ifremer BP7004 Taravao, Tahiti, French Polynesia
b
Texas A&M University, Port Aransas, TX, USA
c
Facultad de Ciencias, UNAM, Mexico
d
Inra/Ifremer BP 70, 29080 Plouzané, France
Received 12 December 2002; received in revised form 1 December 2003; accepted 17 December 2003
Abstract
The Pacific white shrimp Litopenaeus vannamei (Boone) is the most important shrimp
species cultivated in the Americas. More than 90% of the shrimp cultivated in 1998 on the
American continent (132 000 tons) was L. vannamei. L. vannamei is the species cultivated
especially in Ecuador and Mexico. Its growth potential is quite good and it is a hardy
species in spite of virus diseases, such as IHHN or, to a certain extent, other pathogens
such as WSSv (depending on pond management). Regarding feed, it represents the species
with the best opportunity at reduced protein level; plant protein and carbohydrates available
in their habitat to be channeled into tissue. A review of some requirements of juveniles is
provided while emphasis is put on cost reduction, pond utilization and, generally speaking,
on sustainable production. From an experimental point of view, nutritional requirements for
protein, copper, zinc, calcium, phosphorous and some vitamins are examined based on data
accumulated by Texas A&M, Ifremer/COP/Tahiti, Ifremer/Brest, UNAM/Mexico, and several
other research centers. Pond production results are extended to pens or ponds as well as to
some feeding aspects in large semi-intensive ponds or super-intensive conditions. Experi-
ments conducted with a ‘‘bacterial flock’’ (moulinettes) evidenced the beneficial role of
bacteria.
If temperature is around 27 jC, it is feasible to target for a double crop a year. A well-
balanced feed, in super-intensive culture is achieved without use of additives (probiotics,
immunostimulants). L. vannamei is the species in which most research has been concentrated
to understand better shrimp nutrition and physiology, and in which the relationship between
* Corresponding author. Tel.: +689-54-60-80; fax: +689-54-60-99.

E-mail address: gcuzon@ifremer.fr (G. Cuzon).
0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2003.12.022
514 G. Cuzon et al. / Aquaculture 235 (2004) 513–551
nutrition and genetics can currently be studied thanks to the existence of highly inbred
strains.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Larvae; Juveniles; Breeders; Shrimp; Peneid; Nutrition
1. Introduction
Nutrition and feeding of L. vannamei (synonym: P. vannamei) have received a great deal
of attention (Cuzon et al., 1985; Pedrazzoli et al., 1998) over the last 20 years. Scientists,
farmers, feed manufacturers, as well as suppliers of feed ingredients, all contributed to the
better feeding of juveniles raised under semi-intensive or intensive conditions. Also
included are pathologists who have been trying to boost the resistance of shrimp though
substances or nutrients to minimize mortalities due to pathogens (Vibrio, viruses, etc.). Is
there a need for another review on the topic? Since the Kagoshima workshop in 1995, there
have been new considerations regarding nutrition and feeding which are worth looking into.
Under tropical environments many studies (Aquacop, 1989) have been performed leading
to a super intensive method with 20 tons/ha/year and 70% survival; however, sustainability
is still a matter of discussion and will be addressed herein. All the work done in Texas
presented here provides an interesting approach linking tank and pond results. Reduction of
protein content for environmentally friendly feeds is a solid ground, and digestibility
(ADC), as well as protein energy (P/E), experiments would contribute greatly. The review
on some requirements such as copper, vitamin C under ascorbate phosphate form,
cholesterol, essential fatty acids is needed for a more accurate formulation for each stage
of development. In the early stages of shrimp cultivation, there were trends in nutrition
leading to the inclusion of an ingredient as a carrier of a major nutrient; for example, sesame
meal for arginine, or alfalfa concentrate for carotene, bringing about some complex
formulations. Today these essential elements are better known and, hence, feed can be
more accurately formulated. Although fish meal, for example, is still widely used, little by
little, nutritionists should reach a way to replace it at least partially with plant proteins. This
paper provides a wide array of information on nutrition and feeding of L. vannamei, starting
from its initial stage in 1975 when cooperation between Ralston Purina and CNEXO/COP
Tahiti started until 2001, with the introduction of genetics to nutrition, relating wild and
domestic shrimp from a nutrient utilization point of view. Some data will be considered
with a critical eye but what is expected through this paper is to apprehend a whole period of
research development on one species that has become one of the most ‘‘productive’’
species.
2. Protein
A reasonable amount of work has been done on the digestibility of nutrients in L.

vannamei: ADC of protein sources and amino acids was first documented by Akiyama et
G. Cuzon et al. / Aquaculture 235 (2004) 513–551 515
al. (1991), Smith et al. (1985) showed a difference in the digestive capacity among 4-g, 12-
g, and 20-g shrimp with an improvement in protein ADC in 4-g animals.
Cousin et al. (1993) showed that small or larger sized L. vannamei responded similarly
to several fish meals (fresh, moderately fresh, or stale). If differences in ADC potential
exist between larvae and postlarvae, these differences tend to disappear between postlarvae
and juveniles. Proteins will act according to their dietary level in a range from 10% to 50%
(Table 1) and depending on the nature of protein. Native protein seems better hydrolyzed
than processed ones (Zwilling, 1981). Is there a relation between digestive capacity and
growth performance? At first glance it seems the case: at constant feed intake, more
digestive nutrient absorbed more growth.
Although pepsin does not exist due to the lack of stomach, endo- and exo-peptidases
are present; trypsin (Galgani et al., 1985), chymotrypsin (Le Chevalier and van Worm-
houdt, 1998), and an inhibitor of trypsin that controls digestive functions (Garcia-Carreno
et al., 1997) have been identified.
A measure of the degree of hydrolysis (DH) with the pH stat method (Esquerra et al.,
1987) helped address the potential of digestibility of a feedstuff particularly fish meal. The
in vitro DH results should be positively correlated with in vivo measurements of apparent
protein digestibility (APD) in order to validate the method. The low digestibility of some
fish meals has been attributed to excessive heat treatment, which reduces the nutritional
value of dietary protein by causing cross-link reactions or amino acid racemization (Lemos
et al., 2000). What effect on growth? By and large, L. vannamei juveniles raised in
different conditions (tanks, raceways, moulinettes, earthen ponds) were fed MR25 in the
early 1970s, regular grower feeds up to 40% protein distributed in earthen ponds from
1972 –1982 in Aquacop facilities, experiments in tanks containing as much as 60% protein
such as the Nippai diet and observations expressed the fact that it was difficult to exceed
the overall capacity of the digestive tract to digest protein since levels up to 60% of the diet
were apparently digested. However, considering the omnivorous behavior turning to
herbivorous trend (digestive enzymes profile evolve accordingly), experimental results
provided a possibility for dietary protein limitation in spite of the fact that it is very
difficult to address protein intake.
2.1. Relative protein requirement
For juveniles, Colvin and Brand (1977) reported less than 30% to be the protein
requirement while Kureshy and Davis (2002) found a maximum protein requirement at
32% for juveniles and sub-adults of L. vannamei. The first report of protein requirement
for postlarvae, raised in tanks during one month (Colvin and Brand, 1977), indicated 30 –
35%. Velasco et al. (2001) examined the effect of dietary protein, energy level, and water
quality on postlarvae, in an attempt to verify growth in a range of 10– 33% of crude
protein and 3– 7% lipid level (Fig. 1). Survival was about 80% for all treatments; growth
Table 1
L. vannamei (0.58 g) fed 25%CP diet with different levels of soybean meal (SBM)
% SBM in diet 15 30 45 53
% weight gain 250 300 350 310
Fig. 1. L. vannamei postlarvae relative growth rate (RGR) according to lipid (3, 7, 11), protein levels (10, 15, 25,
35) and DE.
did not differ except for the 10% protein level, which was significantly lower. In two
experiments, in a re-circulating system, they found an optimal dietary crude protein level
at 21.4% (exp 1) and 24.5% (exp 2). But they did not estimate protein and energy/day per
gram of animal with diets averaging about 16 MJ kg 1 DE. The absolute protein
requirement for maintenance and maximal growth has also been reported for juveniles
and sub-adults of L. vannamei.
Maintenance protein requirement was of 1.8– 3.8 mg of dietary protein per gram of
body weight per day (mgDP kg BW day 1) for juveniles and 1.5– 2.1 mg DP g 1 BW
day. for sub-adults. Maximal growth was observed with 46 mg DP g 1 BW day for
juveniles and 24 mg DP g 1 BW day for subadults, both fed 32% of dietary protein
(Kureshy and Davis, 2002).
Sub-adults are prone to discriminate according to the quality of the diet during a 60-day
trial at 27 jC (Aquacop, 1972; unpublished results). The quality of the diet in terms of
balance between protein (35% CP) and digestible energy (DE) (14 kJ g 1) with a P/E ratio
of 22 and some fiber content kept survival above 80%, FCR 2.5 (Fig. 2). Further
Fig. 2. Growth rate of L vannamei fed on dry diets (Aquacop, 1972).

Fig. 3. IGR (%/day) of juveniles L. vannamei fed semi-purified diets containing # P/E ratios from menhaden fish
oil (5 – 11%).
assessment of constraints such as protein and energy led to optimal growth or FCR
(Cousin, 1995) confirmation of the growth potential of wild L. vannamei. The protein/
energy ratio has been extensively covered by several authors (for review, see Cousin,
1995) and a review published by Cuzon and Guillaume (1997) in which the optimal P/E
required by L. vannamei and L. stylirostris was determined for optimal growth or FCR.
The final objective is to produce an absolute requirement in terms of milligram of
protein per 100 g of shrimp biomass. Aranyakananda and Lawrence (1993) obtained
maximal growth with 8 mg protein kJ 1. Lawrence et al. (unpublished results, Fig. 3)
studied dietary requirement of juveniles and optimal protein/energy ratio, using three
levels of crude protein (25%, 35%, and 45%) and dietary energy ranged from 14 to 15
kJ g 1 DE. Juvenile L. vannamei utilized feeds more efficiently when ingestion rate (mg
feed/g shrimp/2 h), feeding frequency (15/day), and daily ration size increased (Fig. 4).
A maximal dietary protein requirement of 15% seems questionable at an energy level
around 15 kJ g 1 DE, to sustain optimal growth without a considerable increase in feed
Fig. 4. IGR of juveniles L. vannamei fed semi-purified diets supplemented with freeze-dried Artemia and five
lipid levels (2 – 14%).
intake, and a protein/energy ratio calculated at 70 mg kJ 1 DE. An interesting approach

was developed based on a series of experiments with constant protein, constant energy
or constant P/E ratio, which led to a three-dimension contour lines representation where
an area of optimal growth and optimal FCR was identified. For 7– 8 g juveniles, values
in terms of DP and DE were calculated at 40% protein and 17 kJ g 1, expressed in
terms of 23 mg protein kJ 1 DE. A daily intake of 1.2 g protein (DP) and 140 kJ (DE)
should be considered adequate for a biomass of 100 g shrimp. The optimal protein/
energy ratio was of 24 mg kJ 1 DE. In this latter study, protein and energy retention
seemed low (20%) as compared to findings in rainbow trout (40 – 60%), but shrimp like
any crustacean export cast at each molt, which can represent an energetic expenditure of
1.4 kJ or 26% of the overall intermolt energy gain (Read and Caulton, 1980). Protein
requirement is probably not as highly correlated with protein accretion as it is in
vertebrates due to chitin synthesis, chitin is a polysaccharide containing nitrogen
equivalent to 43% crude protein.
In general, however, values on protein requirements for L. vannamei support the idea of
a possible replacement of marine by plant protein sources. Fish meal incorporation can get
sustainable optimum growth rate and performances in tanks that were found different
according to diets composition (Fig. 5). Soybean meal at 45% incorporation of a diet with
25% CP showed an optimum in terms of weight gain (Lawrence et al., 1995) (Table 1).
Davis et al. (2002) tested L. vannamei juveniles fed different processed pea meals at 5%,
10%, and 20% of inclusion level, and found that when it was extruded or micronized, their
protein and energy content were highly digestible without apparent adverse effects on
growth or survival. Cruz-Suarez et al. (2001) found an optimal P/E ratio (20 mg kJ 1 DE)
for juveniles fed diets containing 25– 35% protein with pea meal as dominant plant protein
source.
A large amount of experimental work has been performed on the requirements of L.
vannamei. A wide range of practical formulations developed by feed manufacturers have
been identified, emphasizing in the beginning, on water stability of pellets, based on
farmers’ recriminations.
Total crude protein level in shrimp feeds faced some drastic drop and trials led to
enhanced ingestion rates. The potential for growth is there, and the ability of L.
vannamei to digest large amounts of plant proteins is worth taken into consideration
for future feeds.
Fig. 5. Incidence of nature of protein on juveniles growth.

3. Amino acids (AA)
Although protein in fish meal has been considered to be a highly suitable dietary
ingredient for growth in finfish, the number of test proteins used in crustaceans
nutritional studies is almost as numerous as the number of investigators. Several
reference proteins have been proposed, such as a proportional composition of AA
found in the whole body of short-necked clam (Venerupis decussata) would be
satisfactory or isolated from crab (Cancer irroratus), and this ingredient used as a
source of protein to determine protein requirement of different species, given non-
protein energy sources and other nutrients were the same. Use of such «ideal protein»
in diets of similar ingredients composition by investigators may modify the hierarchy
of requirement among species. For example, determination of protein requirement of
Marsupenaeus japonicus has changed when substituting crab protein for casein. It
remains that many authors took shrimp tail muscle protein as a reference. The actual
contribution of a protein towards satisfying the AA requirement can be shown with a
beneficial effect of AA supplementation. Such positive effect has been achieved with
casein + methionine. Hew (1983) improved casein with lysine and arginine addition.
Colvin and Brand (1977) found a lysine requirement (0.5% of protein) with a growth
plateau and concluded that lysine was not limiting. Adding 1% arginine, phenylala-
nine, leucine, or isoleucine led to growth promotion. Lim (1993) reported a positive
effect of crystalline AA supplementation in a diet for L. vannamei. On the other hand,
many attempts to improve AA balance proved unsuccessful: with a purified diet,
crystalline arginine was 90 times less efficient than protein bound arginine to meet the
requirement. Then, some authors tested plastein or microcapsules of arginine added to
a casein based diet. Chen et al. (1992) improved biological value of casein with
microcapsules of arginine and estimated the need at 2.5% diet. Fox et al. (1995b)
determined the lysine requirement for juveniles L. vannamei with AA covalently
bound to wheat gluten.
Casein supplemented with encapsulated amino acids can serve as a reference protein.
Failure to improve the feeding value of protein is sometimes due to leaching or lack of
palatability of the diet. But the deficiency in arginine is noticeable because apart from its
contribution to protein synthesis, this amino acid functions as a phosphagen for muscle
contraction.
Satisfaction of the requirement for AA should be based on their biological availability.
Availability is affected or reduced by three main factors: (i) incomplete digestion, (ii)
presence of inhibitors, (iii) damage to protein. It is more important than using the total
amount of each in different feedstuffs.
Hence, there has been an emphasis not only on the amino acid profile of the diet but on
the use animal protein, such as fish meal, which contributes balanced essential amino
acids. On the contrary, plant proteins for example may provide lower digestibility or
contain a limiting amino acid.
Protein represent about 16% of the mass of a shrimp. After a meal, protein synthesis
increases and proteolysis decreases. There is a protein gain and the muscle mass is
preserved. As in fish, only 40% of dietary protein is retained and 60% is lost or
oxidized.
3.1. Essential amino acids requirement
– Qualitatively: Using a diet with acetate 14C administered to shrimp, 10 EAA were
identified, the same as in fish.
– Quantitatively: until recently there was a lack of quantitative data on EAA requirement
for peneid species, except for P. monodon.
In contrast to similar studies on trout or carp, whole protein and crystalline EAA from
test diets with a basal level, of all EAA except the one to be tested, resulted is poor growth
due to poor feed intake and subsequent leaching of AAs.
The profile of muscle amino acids (Table 2) has been used as a reference for diet
formulation even though in most diets the preponderance of fish meal rendered a fair
amount of essential AA to sustain optimal growth.
3.1.1. Imbalance diets

Zein, deficient in lysine and tryptophan, and casein, deficient in arginine, have been
used as test proteins when supplemented with crystalline AAs. But digestibility of these
proteins was well below 90%, generating difficulties in interpretation.
3.1.2. Oxidation method

After several weeks of acclimation on a defined diet, 14C lysine was introduced in the
pellet and output of radioactive 14CO2 was measured. At low lysine intake, a low rate of
14
CO2 expiration was recorded. Expiration of CO2 increased as dietary lysine increased
used for protein synthesis, up to the requirement level or above. Excess lysine intake
above the requirement for protein deposition increased the expiration of 14CO2. One
criticism of the method is related to pool size and specific radioactivity, which are lower at
higher lysine intake.
3.1.3. Indirect oxidation method

Aside from lysine, another amino acid (14C phenylalanine) was injected. Both
phenylalanine and tryptophan have also been used (Amouroux et al., 1997). At low
Table 2
Indispensable amino acids (AA) composition of shrimp tail muscle in % protein (Lim, 1993) and diet25 in % as fed
Muscle Diet25
Lysine 6.4 1.6
Arginine 7.5 1.9
Histidine 1.9 0.5
Leucine 6.5 1.6
Isoleucine 3.6 0.9
Valine 3.8 1
Threonine 3.4 0.6
Methionine 2.6 0.7
(Met + cystine) 3.3 0.8
Phenylalanine 3.6 0.9
Tryptophan 1.1 0.3
lysine intake, growth and protein synthesis are limited, so the excess amino acid is
oxidized and a high level of 14CO2 was measured. At breakpoint, there is enough lysine
for more protein synthesis, and requirement is satisfied. Trials revealed difficulties in
applying the protocol to shrimp. It was necessary to increase metabolism by increasing
temperature (24 –27 jC), trying to get animals to eat two meals with hot AA, increasing
specific radioactivity in pellets (0.08 – 1.5 ACie) and controlling ‘‘rapid’’ feed intake.
Amino acid oxidation after a meal will increase if intake exceeds requirement or an
imbalance in amino acid pool occurs probably because the incoming dietary amino acids
are oxidized at a higher rate in shrimp than in fish.
Amouroux et al. (1997) were unable to use an indirect oxidation method for AA
determination mainly due to the difficulties encountered in controlling feed intake (reduced
leaching, keeping enough specific activity, and/or recovering at least 80% radioactivity).
Muscle turnover was found to be low (KS = 9.7) in L. vannamei (Mente and Houlihan,
2002) and, just as in fish, a reduced amino acid loss through oxidation has been confirmed.
Oxidation of amino acids, protein loss (exuvia), synthesis of N-compound from AA and
protein turnover contribute to maintenance. But protein turnover is not likely to be a big
factor in AA losses (Cowey, 1994, personal communication).
3.2. Values obtained for essential amino acid (Table 2)
Discrepancies among values found by different authors have received attention and are
related to several aspects: (i) Imprecision in the feeding curve and feed intake. (ii)
Different growth rates (Table 3) have been obtained even in the same species of shrimp or
different stages of development have been used; for example, early postlarvae or juveniles.
Low rates of growth can produce high requirement levels; and it is only with high rates of
growth that more satisfactory values can be obtained. In case of slow growth, maintenance
requirement for growth is somewhat larger and may affect the results. (iii) Diets containing
large amount of crystalline AA yield poorer growths than whole protein.
3.3. Whole body amino acid composition or basis for essential amino acid pattern
A high correlation between the dietary AA profile and that of the whole body has been
assumed. For example, the essential amino acids profile of whole shrimp can help
calculate an essential amino acid index. However, shrimp meal has not proven to be an
ideal protein source for crustaceans as fish meal has been for finfish. The strange
Table 3
Growth: weight gain/% weight gain considering 2 mg copper from basal diet
mg/kg supplement GM %
0 2.88 5
8 3.10 5.6
16 2.35 5.8
32 3.42 6
64 3.50 6.2
126 3.43 6
unsuitability of shrimp meal is perhaps due to the fact that shrimp meals are principally
derived from the hepatopancreas and exoskeleton (except the one from Houma, LA).
Therefore, shrimp meals are composed of a proportionately large amount of N-containing
chitin, which contributes to the calculated «crude protein» content of this ingredient, and
content in true protein is low.
Mimicking the shrimp muscle amino acid profile (larvae, juveniles, adults) has been
tried by several authors. Amino acid composition of the tail protein of L. vannamei (Lim,
1993) revealed that shrimp tail muscle is particularly rich in arginine and this is the reason
why so many diets are limiting in this AA. This has been revealed many times with
experiments conducted in clear water. The amino acid composition of the tail muscle
protein is a good indicator to formulate a balanced feed in terms of amino acid
composition matching with shrimp amino acid requirement. Very often when examining
essential amino acids composition of experimental diets arginine was found to be the first
limiting factor; lysine came next; its requirement was examined using intact sources or free
amino acids (Fox et al., 1995a).
In fish, Cowey (1992) reported that using conventional practical diets, after 4 weeks,
the quantity of amino acids in the body indicated an increase in amino acids and values for
AA requirements could be deduced. However this approach is not acceptable for shrimp
because only 40% of dietary protein is retained and 60% is lost or oxidized. Hence, it is
difficult to take into account that loss when using accretion of amino acids in the body as
an index for AA requirements.
Deficiency of amino acid does not lead to deficiency signs or disease, except for one
case of caudal erosion due to tryptophan deficiency, but rather to poor growth even when
considering amino acids, such as methionine or lysine, which have been identified to exert
precise actions in fish.
3.4. Absorption of amino acids
Digestive gland (HP) is the principal site of nutrient absorption and authors report 53%
total digestibility for 15 AA in the foregut and 48% in the midgut, while no evidence for
assimilation of AA could be found in the hindgut. Part of it is chitinized and secreted as a
peritrophic membrane; hence, absorption is probably nil. Among the crystalline amino
acids tested, metionine was absorbed first and without synchronization with another amino
acid. The poor utilization, 11% of crystalline amino acids, was explained by the absence of
simultaneity in the inputs. Such statement can be moderated with the actual consideration
that in case of multiple meals in a day, as occurs in shrimp, deficiency would be
overlapped between one intake and the next.
Leucine, metionine, and valine are known to exert competitive or reversible inhibition
in fish (Cowey and Forster, 1971). In P. marginatus, studies on glycine transport by the
isolated midgut revealed an energy-dependence at lower AA concentrations. Alanine was
a non-competitive inhibitor of mucosal glycine whereas proline was fully competitive.
Aliphatic, neutral amino acids, and histidine were more potent inhibitors of glycine influx
than aromatic neutral and anionic amino acids. Lysine transport across the mucosal border
of perfused gut from freshwater shrimp revealed an apparent adaptation of the high-
affinity carrier process for very low lysine concentrations.
3.5. Metabolism of amino acids
Pattern of N excretion: the role of gills and liver in excretion is probably the same as in
fish. Ammonia – N excretion varied according to dietary protein level. For example, adult
L. stylirostris excreted 0.6 mg N g dry w 1 h 1 after acclimated 40 days to a diet
containing 50% protein (Gauquelin et al., 2003). Calculated O/N values (< 20– 25) clearly
indicated a use of protein as energetic substrate. It was observed that excretion rate
increased when shrimp were fed ad libitum when compared to animals fed two meals a
day. The size of the ration and feed intake was related to ammonia –N excretion level. The
reductive amination of ketoglutarate in the muscle of P. monodon is catalyzed by L-GDH
and from a-ketoglutarate and NH4 +.
Transamination, mainly between glutamic acid and pyruvic acid, with the formation of
alanine, has been shown in different species of crustaceans (Rosas et al., 2002).
Chitin formation: Horst (1989) evidenced in L. vannamei shrimp the association between
chitin and protein synthesis. Analysis of cuticle revealed 73% (by weight) amino acids and
27% N-acetylglucosamine. Major amino acids in the cuticle were: Asp and Asn, Glu and
Gln, Thr, Ala, His, and Leu. A hypothesis is favored that crustacean chitin is secreted as a
glycoprotein complex and concurrent protein synthesis is required for chitin deposition to
continue. Basic amino acids are considered essential in particular lysine because of the
special need for lysine. Lysine is needed at high levels by fast growing animals. Free amino
acid concentration (2– 20 Amol/ml) increase in hemolymph before exuviation.
3.6. Metabolism of certain amino acids in shrimp
Arginine metabolism takes place in the HP. The question arises about the possible
antagonism of Lys-Arg, such as occurs in mammals, where an increased amount of Lys in
the diet triggers a falling off in growth rate and appetite. Moreover, additional Lys
enhances the activity of arginase, so Arg in the diet is unavailable to the animal. An
absence of Lys-Arg antagonism has been found in channel catfish. Tryptophan presents a
catabolism complicated with several steps from Trp to CO2. For many AA, the first
breakdown step is by deamination or transamination. Trp is the first reaction carried out by
trp pyrolase (oxidation). There is no evidence that nicotinic acid is formed from trp as in
mammals. There is no report on AA deficiency signs in shrimp. Methionine (Met) can be
converted to cysteine, thus providing all the needed cysteine to shrimp, but cysteine will
spare methionine. If marginally deficient in Met, then there is a recycling of homocystine
to meet this deficiency and, in case of excess, the pathway will turn to cysteine and taurine.
Regulated mechanisms exist around homocystine, and S-adenosyl met (SAM) can donate
S groups of carbonyl groups to make polyamines.
3.7. Non-hormonal control of protein and amino acid metabolism
Allosteric enzymes can modulate their activity with positive (ADP, AMP) or negative
(GTP) modulators. If ADP levels are increased in cells, GDH is stimulated and there is a
deamination of glutamate to keto-glutarate which enters the TCA cycle. It is an effective
mechanism for increasing activity. Coarse control functions in mammals, enzymes
activities can change 10 times according to the level of protein in the diet. In shrimp or in
fish this coarse control does not operate. Finally, a fine control (control by Km) is
plausible: there is an effect of substrate concentration on enzymes activities. Requirement
of amino acids can be determined by direct oxidation and, in fish, an increase of substrate
degradation measured by the expired 14CO2 has been shown, its value corresponding to
the AA requirement of the species. Unfortunately, this method was difficult to implement
in shrimp (Amouroux et al., 1997).
3.8. Conclusion
Aquatic animals have a higher protein requirement than terrestrial animals. The aquatic
food web with scarcity of carbohydrates (CHO) and abundance of lipids and protein is
responsible for the common trend of aquatic organisms to use protein as an energy source
(Guillaume, 1999). The protein sparing-effect of lipid and CHO has been insufficiently
evaluated and recently CHO was found useful to spare protein with a greater ability in wild
shrimp than in farmed ones (Arena, 2002). Almost all studies on protein requirements have
focused on the level of production with best weight gain but it is clear that protein for
optimal FCR, optimal protein retention and optimal body composition, must be consid-
ered. Results from studies in tanks are not fully representative of a requirement in ponds
and especially in intensive systems, where a bacterial flock can bring sustainable amount
of organic carbon (7 mg l 1).
Finally, the use of plant protein as a replacement for fish meal, with cereals with low
lysine or legumes with low metionine will necessitate knowledge on amino acid
supplementation.
4. Carbohydrates
The capacity to digest common ingredients is important in L. vannamei juveniles. Many

authors (Cousin et al., 1993; Davis and Arnold, 1993; Cruz et al., 1996) have studied
digestion of carbohydrates. Juveniles and sub-adults fed at high starch levels, around 40%,
gave an apparent digestive coefficient that reflects an increase in digestibility (Fig. 6).
Starch has been shown to be digested as well, either raw or pre-cooked, and this digestive
property represents an interesting aspect in relation to the amount of plant sources that can
be used in a regular grower diet.
Digestive enzymes in L. vannamei are classified in three groups, i.e., proteases (endo,
such as trypsin and chymotrypsin; exo, such as amino peptidases; Zwilling et al., 1981;
astacin, a protease of low molecular weight), carbohydrases (a-amylases and laminar-
inases), and lipases.
Amylase, one of the most studied digestive enzymes in L. vannamei (Le Moullac et al.,
1994; van Wormhoudt et al., 1995; Van Wormhoudt and Bellon-Humbert, 1996),
represents 1% of the crude hepatopancreas (HP) extract. Starch splitting enzyme can
hydrolyse 1,4V linkages on both sides of the 1,6V branching points producing oligosac-
charides that are hydrolysed to glucose by maltase (Ceccaldi, 1997). In shrimp, it appears
likely that starch digestion is accomplished by the initial soaking of food in the anterior
Fig. 6. ADC starch (80 – 90) versus dietary starch levels. ADC protein versus dietary starch levels.
chamber and subsequently through the grinding action of the gastric mill that precedes the
action of amylase in the hepatopancreas tubules.
With other nutrients such as glucose, the latter can decrease activity of hexokinase
enzymes that phosphorylate glucose in hepatic cells.
Enzyme activity is dependent on internal factors such as ontogenesis, feeding activity,
intermolt period and diet composition. Ontogenesis of enzymes is evidenced by a
progressive synthesis during development; chymotrypsin has been found with two isoforms
in the adult stage and one isoform in the larval stage, whereas trypsin and amylase remain the
same. Induction of digestive enzymes is dependent on the stage of larval development. Rate
of ingestion, which is high during night time, corresponds to maximal proteolytic activity
(Hernandez Cortes et al., 1999) and a rhythm of enzyme activities was found in M. japonicus
revealing a peak at dawn for this burrowing species. The molting cycle affects enzyme
activities, at stages A –B, they are lower than at stages C or D1 when feeding activity is
intense and reserves are built-up in preparation for molting. Starch levels had no effect on
amylase activity except if measured at low levels (Van Wormhoudt et al., 1980). During
starvation, shrimp amylase production is completely depressed.
By and large, a-amylase may be a poor indicator of diet according to Forster and
Gabbott (1971) even though a-amylase expression varied with the level of dietary protein
(casein) presenting two isoforms at 25% versus one isoform at 40% protein (Le Moullac et
al., 1994).
Digestion of starch as the main ingredient is complex. The range of starch digestion is
related to dietary level between 10% and 50%, and source (potato starch with large
granules is less digested than cornstarch with smaller granules and wheat starch).
Concerning texture, either native or gelatinised, wheat starch was found highly digestible
(90%) under amylase and glycosidase actions. Shrimp amylases are highly efficient in
digesting wheat starch (Cousin et al., 1993). According to Le Moullac et al. (1994) starch
levels between 1% and 20% in microparticulate diets have no influence on amylase
specific activity in L. vannamei larvae.
If no dietary requirement for carbohydrate can be expressed sensu stricto; among
carbohydrates, simple sugars have been shown to produce deleterious effects on growth
of juveniles. Complex carbohydrates promote growth and feed manufacturers widely use
this source: (i) it helps maintain the texture of the pelleted feed and (ii) it serves as a relative
low cost energy source. Among several varieties of starches, wheat starch was found to be
well digested by L. vannamei juveniles. L. vannamei, through the domestication process, has
lost 95% of alleles for the a-amylase gene in comparison with the wild population (as
evidenced by the Remote Frequence Length Polymorphism method) raising questions on the
level of amylase polymorphism (Arena, 2001). In case of a highly inbred strain of L.
vannamei, as in Tahiti (Aquacop, 2001, unpublished results), for more than 23 generations,
the concern is that this could cause a possible reduction in growth performance due to this
loss of heterozygosity. Through domestication, could shrimp have selected alleles that adapt
to a heavy load of glucose from a high starch diet, for example? Research along this topic is
being performed to identify a possible decrease in digestibility of starch or selection of a
genotype that would still allow shrimp to digest starch and utilize glucose at metabolic levels
(Arena, 2001, personal communication).
Dietary starch leads to glucose that enters different metabolic pathways by phosphor-
ylation; this has still to be documented and some measurement of enzymatic activities at
the intermediary metabolic level must be done. Shrimp are diabetic-like animals,
hyperglycemic with hemolymph levels of glucose as high as 1.5 g/l in post-prandial
status indicate a need for energy source, a special requirement linked to the build-up of the
new exoskeleton after each molt, or a difficulty to handle a large load of hemolymph
glucose. Another alternative could be glucose synthesis from glycogenic amino acids,
although shrimp seem to use energy derived from amino acids more efficiently rather than
energy coming from glucose.
Domestication of L. vannamei seems to limit the genomic capability of shrimp for the
utilization of nutrients from the diet. It is necessary to know the expression of the
phenotype through the genome environment interaction to respond and adjust to this
limitation; for example, more dietary protein may be needed than more carbohydrate.
5. Lipids
ADC of lipids (Cousin, 1995; Merican and Shim, 1997) provide an indication of action
of lipases and esterases during the digestive process; esterases release short chain fatty
acids (Galois, 1983). The utilization of some nutrients may vary by influencing enzymes
of intermediary metabolism, for example, an excess dietary fatty acids can reduce citrate
lyase activity; and excess dietary, lipids tend to reduce lipogenic enzyme activity.
Triacylglycerols (TG) are fed to shrimp in the form of fish oil (either pure or contained
in fish meal). An emulsification process in the digestive gland (hepatopancreas) leads to
the formation of micelles, these, in turn, would liberate fatty acids through lipase action in
M- and R-cells. Fatty acids are resynthesized as di- or tri-acylglycerols leading to neutral
lipid or phospholipids, the latter class of lipids being prominent during ovarian develop-
ment. As in vertebrates, TG containing a fatty acid in position 2 of the glycerol moiety
would be liberated more rapidly than fatty acids in position 1 or 3. Hence, fatty acid
utilization will vary according to lipid source and ratio between lipid classes.
The effects of feeding various sources of dietary lipid on weight gain, feed conversion,
survival, and fatty acid composition of juveniles of L. vannamei have been studied by Lim
et al. (1997). Shrimp fed a diet containing menhaden fish oil had the best growth and
survival, followed by linseed oil (source of linolenic acid, LNA). Both n-6 and n-3 HUFAs
are essential for juvenile L. vannamei, however, n-3 fatty acids promot faster growth than
n-6 fatty acids. More recently, Gonzalez-Felix et al. (2003a) examined the nutritional value
of linoleic (LOA) and linolenic acid. Juvenile L. vannamei’s dietary requirements were not
demonstrated however the results showed a greater nutritional value of n-3-HUFAs
(arachidonic acid, EPA, DHA) which produced significantly higher final weight and
instantaneous growth rate (IGR). In brief, PUFAs (LOA + LNA) were inferior to HUFAs
(AA + EPA + DHA) and such relation is linked to a limited ability to elongate and
desaturate the n-3 linolenic fatty acid chain. This was reflected in tissue fatty acid
composition (Gonzalez-Felix et al., 2003b) with a profile of shrimp tissues reflecting the
composition of the dietary lipids.
5.1. Phospholipids (PL)
Coutteau et al. (2000) showed that a 1.5% supplement of purified soybean phospha-
tidylcholine (PC) significantly improved growth and reduced sensitivity to osmotic stress
compared to that from marine fish roe.
Gong et al. (2000b) found no dietary requirement of PC, but if combined at 0.4% and
0.5% with phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in a 25:22
proportion (0.8% and 1.7%), a significant growth promotion was observed on shrimp.
The recommended level of phospholipid supplementation in diets for L. vannamei
juveniles ranged from 3% to 5%, which can be supplied as 3 –5% lecithin type I (98% of
phospholipids) or 4 –7% lecithin type II (71% phospholipids).
5.2. Phospholipids and cholesterol
Gong et al. (2000a) observed that without dietary phospholipid supplementation,

shrimp required 0.4% cholesterol from the diet at maximal IGR but the dietary cholesterol
requirement increased as dietary phospholipid level increased. D’Abramo (1997) sug-
gested that the role of lecithin is to disperse and transport cholesterol to the tissues. This is
in agreement with the interaction of cholesterol and de-oiled soy lecithin requirement as
observed by Gong et al. (2000a). Dietary cholesterol and PL interact to improve growth
and contribute to the retention of total lipid and triacylglycerols in the hepatopancreas and
of cholesterol in the muscle of L. vannamei juveniles. The dietary cholesterol requirement
of L. vannamei juveniles is affected by dietary PL. Optimal growth of L. vannamei was
achieved with 0.4%, 0.1%, 0.1%, and 0.05% supplemental cholesterol at dietary PL levels
of 0%, 2%, 3%, and 5%, respectively. The dietary PL requirements based on growth were
5% without cholesterol supplementation, 3 –5% with cholesterol supplementation of up to
0.2% of basal diet, and 3% with 0.4% cholesterol included in the diet.
5.3. Requirement for cholesterol
Among sterols, cholesterol is essential, and although phytosterols have been shown to
be used by shrimp to synthesize cholesterol, there is a need for dietary cholesterol in
crustaceans (Kanazawa et al., 1971; Teshima and Kanazawa, 1971) at a level varying from
0.5% to 1.5% of the diet depending on rearing conditions. Duerr (1995, personal
communication) added another dimension to the concept of requirement, revealing a
difference in requirement not only due to environmental conditions (Fig. 7) but also to
growth rate related to somatic tissue build up. This has also been observed in trout while
determining the vitamin C requirement (Woodward, 1992, personal communication). A
greater need for cholesterol in shrimp growing faster in outdoor conditions than indoor
conditions (green water versus clear water) has been observed.
The relation between cholesterol and the molting process in crustaceans is described as
the lipocytic index, a ratio between cholesterol and phosphatides (Renaud, 1949), which at
the level of the hypodermis would trigger water absorption in preparation for exoskeleton
retrieval.
From an anatomical point of view, shrimp possess an HP, corresponding to 4% total
body weight; there is no real adipose tissue and lipid accumulation, if any, occurs in HP (5
mg lipid/100 mg fresh tissue) or ovary during reproduction period. Lipogenic enzymes
could be inhibited in the presence of excess of fatty acids, similar to what is found in fish,
except for AMP citrate lysase. And, at least during the juvenile phase (1– 20 g), shrimp
will meet its energy needs with enough fatty acids to extract long chain polyunsaturated
fatty acids (20:5n-3 and 22:6n-3) and cholesterol. A dietary intake of 24 mg triacylgly-
cerols per day for a 10-g shrimp in pond conditions is common.
5.4. Lipid transport
The physiological status of the animal, for example, whether females are ablated or not,
is to be considered. In eyestalk ablated females (for maturation purpose), fatty acids will be
converted to phospholipids that combine with lipoproteins to form HDL. HDLs are
thought to have a dual function: (i) to transport phospholipids for ovary development
(Harrison, 1990) and (ii) to play a role in immune defense mechanisms. Teshima and
Kanazawa (1971) observed a transfer of lipid from the HP to the ovary in ablated females:
according to lipid class, phospholipids were mainly affected. In intact animals, fatty acids
will form neutral lipids that will to be used in energy metabolism (Galois, 1983), each
Fig. 7. Cholesterol requirement of L. vannamei reared indoors and outdoors (Duerr, 1995, unpublished results).
mole of palmityl-CoA would generate through h-oxidation a large amount of energy in the
form of 129 mol of ATP.
Interestingly, lipids are not needed during the juvenile stages at a level above 5 –8% and
attempts to explain this situation are discussed elsewhere (Cuzon et al., in press(b)). For
example, a 65% protein, 4% CHO diet, would leave room for only 8% TG to provide
essential fatty acids and phospholipids to meet growth requirements of juveniles.
L. vannamei were tested for astaxanthin requirement in a practical diet (Aquacop, 1989,
unpublished result) in indoor and outdoor tanks and no difference was observed from a
growth point of view during a two month trial. Doses between 0 and 100 mg astaxanthin/
kg diet did not affect either growth rate or pigmentation. There was an accumulation in the
hepatopancreas and hypodermis but the protective role was more pronounced than any
other function. Similarly, during nauplius stages, a better survival of pigmented L.
vannamei nauplii could be related to the enrichment of astaxanthin in the breeders diet.
Today, carotenoids are considered for their putative role in the immune response.
6. Vitamins
In this section, due to problems linked to vitamin contribution of natural biota in ponds,
the over fortification by feed manufacturers and the leaching in seawater, we will deal only
with ascorbic acid which in the early stages of shrimp nutrition was provided in high doses
(up to 10 000 mg/kg diet) for optimal growth until the advent of the stable form (stay-C),
supplementation and the drastic decreased (50 –100 mg/kg diet). In pond conditions, trials
with or without vitamin supplementation showed little difference. In terms of immune
response, megadoses can provide some protection, at least in what is called a functional
diet providing some protection against viral diseases or possibly against syndromes due to
vibriosis.
Vitamin C is needed by shrimp at levels below those used in early studies of nutritional
requirements (Guary et al., 1974). Studies conducted recently with stable forms of vitamin
C are more reliable ( Moreau et al., 1998). Survival is dramatically improved by vitamin C
above 30 mg/kg diet (He and Lawrence, 1993a) (Fig. 8).
For postlarvae of L. vannamei, culture tests with vitamin C-supplemented micro-bound
diets (Kontara et al., 1997) confirmed the hypothesis that dietary ascorbic acid (2000 mg/
kg) improved the overall physiological condition of animals as measured by salinity stress
and optimal resistance to Vibrio harveyi infection. However, at PL36 (2 mg diet wt),
Lavens et al. (1999) did not find differences among treatments in stress resistance to
salinity shock of postlarvae fed 130 mg ascorbic acid as ascorbyl-2 monophosphate.
In juveniles (0.24 g), the effect of ascorbyl-2-polyphosphate (AAPP) on survival,
growth, and tissue ascorbic acid was examined, AAPP requirement for normal growth and
survival was found to be between 31 and 62 mg/kg diet. There were no differences in
growth and survival of shrimp fed diets containing either 15 or 25 AAPP in the diet.
Measurement of ascorbic acidic (AsA) activity in feeds indicated that 90% of it remained
after feed processing and storage. AsA activity in tissue was proportional to AAPP level in
the diet. Tissue AsA activity suggested that there may have been a difference in bio-
availability between the two AAPP levels tested.
Fig. 8. Effect of AAPP on survival of L. vannamei juveniles.
In 0.1 ha ponds, experiments with juveniles indicated a 2-g growth rate per week.
Production was the same with or without vitamin C added to the feed (13 mg/kg from feed
ingredients versus about 240 mg vitamin C kg feed). Growth of 2 g per week and 82%
survival were obtained with a production in 8 weeks of almost 4.0 tons/ha/crop.
Today, considerations have turned to vitamin E requirement, coinciding with the
immune response and deactivation of peroxides that tend to destroy cell membranes
(Scott et al., 1982).Vitamin E was tested to find an optimal incorporation related to growth
(He and Lawrence, 1993b) and authors found a similar value to fish, i.e., 99 mg/kg of diet,
and this value could change if dietary lipid is increased. Among liposoluble vitamins (A,
D, E, and K), feeding or vitamin E free diets resulted is the lowest survival.
7. Unknown growth factor
The improvement in growth rate with squid extract fed to juveniles, raised in indoor
tanks with clear water, was not a consequence of molt frequency (same intermolt
length in control and treated) but rather the weight increase at molt was significantly
higher for shrimp fed squid extract (Cruz et al., 1987). This evidence was obtained
because it is possible to raise L. vannamei individually, in contrast to L. stylirostris
that did not respond in such environmental conditions (Aquacop and Cruz, 1989,
personal communication). Lipid fractions as well as protein fractions of squid extracts
were investigated. Lipid fractions contained greater than 80% phospholipids. Squid oil
has been used as a regular source of triacylglycerols for feeding breeders in captivity
(6– 12% of the diet). Sterols did present some positive effects on growth and success
at molt.
A significant effect of squid was clearly evident in the protein fraction (Guillaume,
1999): at 16% there was a supply of amino acids adding to the squid effect but at 1.5%
factor, the effect cannot be due to nutrients in surplus (Fig. 9). The observation justifies the
regular use for squid meal in shrimp feeds. Various types of shrimp feeds and in particular
Fig. 9. Squid concentration (0 – 16%) and stocking density (17 and 45 shrimp/m2) on weight gain in grams.
during maturation include squid; squid factor as well as high levels of phospholipids could
be involved in ovary development. The effect of protein level and squid percentage on
pond production of L. vannamei has been shown with the following ratios of protein/
squid: 35:3, 40:5, 45:10, and 45:15. Again, there was a growth of 2 g per week with no
differences between 35% and 45% dietary protein or between 3% and 15% squid meal in
the diet. Growth promotion was recorded with krill meal in replacement for 2% fish meal.
Krill may act as an attractant, improve palatability or may contain an unidentified growth
factor which improves FCR (Lopez et al., 1998).
8. Minerals and trace elements
Davis et al. (1992) reviewed this topic for all macrominerals and some trace elements
for L. vannamei, hence, a summary will be made only on zinc, copper, and phosphorus.
Phosphorus is an element shrimp cannot find in reasonable amount in seawater. In contrast
to calcium, phosphorus has to be added to the diet, but according to the pH of the anterior
part of the stomach differences are expected in relation to the nature of the phosphate salt
(Velasco et al., 2001). Various types and levels of dietary phosphorus were tested to
compare growth of PL and accumulation of assimilated dietary phosphorus in water.
Survival and growth were significantly different among treatments. But growth was
significantly lower in re-circulating tanks (Fig. 10). Dissolved reactive phosphorus (DRP
accumulation) was significantly higher for the diet with NaH2PO4 than with CaHPO4.
DRP accumulation was significantly different with Na2HPO4 (disodium phosphate). DRP
significantly increased with increased levels of NaH2PO4.
For juvenile L. vannamei, the presence of phytic phosphorus has been reported to
depress growth rate. Feed intake and FCR were markedly increased when sodium phytate
was added.
However, sodium phytate even at high feeding levels did not decrease calcium
deposition, contrary to what is known in vertebrates (Civera and Guillaume, 1989).
Dietary essentiality of copper has been demonstrated. Copper is a trace element that
might be deficient in a grower diet. Such a deficiency could be unfavorable to juveniles that
Fig. 10. Sources of phosphates NaH2PO4, Na2HPO4 or CaHPO4 at 0.8% and growth (RGR) on juveniles raised in
static or re-circulating water.
need to synthesize hemocyanin in their hemolymph. Hemocyanin includes a heme contain-

ing copper. This element is involved in hematopoiesis as well as collagen and elastin
synthesis. From a general point of view, it is known that high copper levels are toxic, normal
seawater contains 0.5 mg copper/l and levels in coastal estuaries are 0.6 mg copper/l. EDTA
is used in shrimp hatcheries to reduce copper in seawater. In experiments with L. vannamei
(Davis et al., 1993), the basal diet contained 2 mg copper/kg of diet (Table 3).
Test diets were supplemented with 0.2– 128 mg copper/kg diet by replacing cellulose
with cupric sulfate heptahydrate. Ten days prior to the start of the experiment, 20-day-old
postlarvae, with an initial wet weight of 2.6 mg, were fed the basal diet lacking copper
supplementation but containing 2 mg Cu/kg of diet. Growth rate in these conditions
increased with increasing supplemental copper levels up to 30 mg (total 32 mg copper/kg
diet). Percent change in weight for 32 mg copper/kg diet was 6.2%. No additional on
growth was evidenced for copper levels from 32 to 128 mg copper/kg.
It has been accepted that about 60 mg of copper/kg diet in the form of copper sulfate
should be maintained in pelleted feeds, provided daily, during the growing phase.
Fig. 11. ADC in % for zinc and phosphate with and without phytate in the diet at 1.5%.
Table 4
Hepatopancreatic zinc levels (Ag/g d.wt) considering 18 mg/kg supplemental Zn from base diet
Mg/kg 0% phytate 1.5% phytate
18 120 80
33 137
48 141
78 143 105
218 120
Zinc is a cofactor of several enzyme systems, such as hydrogenase enzymes, alcohol,

glutamate, lactate, glyceraldehyde-3-P, malic, ribonuclease, carboxypeptidase, aldolase,
DNA polymerase, carbonic anhydrase and alkaline phosphatase (Davis et al., 1992).
Bioavailability of zinc in fish meals is relatively low and inversely related to the tri-
calcium phosphate content of fish meal (Fig. 11). The bioavailability of zinc is lower in
white fish meal than in brown fish meal. Phytate has been shown to decrease
bioavailability of zinc in a number of terrestrial animals. For L. vannamei, Davis et
al. (1992) have shown that, in the absence of phytate, a supplemental level of 15 –18
mg zinc/kg basal diet (33 mg total zinc/kg diet) satisfies the zinc requirement. However,
in the presence of 1.5% phytate, 200 mg zinc/kg diet supplemental level to the 18 mg
zinc/kg basal diet (total 218 mg zinc/kg diet) satisfied the zinc requirement (Table 4).
Sodium phytate may enhance growth of Japanese shrimp (P. japonicus) if it is added at
low levels in the presence of calcium (calcium absorbed from the sea water is not
sufficient) to avoid tissue mineralization. In addition, furthermore, the capacity of
phytoacid to hydrolyze rather than to complex zinc has been suggested (Civera and
Guillaume, 1989).
Hepatopancreatic zinc is a sensitive indicator of the dietary zinc requirement rather than
the difference in carapace mineralization, growth or survival, which are not good
indicators of dietary zinc requirements. Phytates reduce hepatopancreatic zinc levels and
zinc bioavailability and phosphate availability, although regarding the latter there may be
species differences (Table 4).
9. Nutrition and rearing systems for L. vannamei juveniles
Extensive system and ‘‘moulinette’’ meet the same needs in terms of a preponderance
for input from what is called natural productivity; nutrition is a challenge and absence of
pathogenicity is significant (Table 5).
On the contrary, the semi-intensive system developed in Ecuador, Mexico, Colombia,
and other countries, provided a solution where feed impact is preponderant; natural
productivity, in terms of contribution to tissue build-up, has not always been very well
assessed (20 – 40%). Furthermore, the absence of equilibrium in the environment and
sludge accumulation in ponds makes this system less sustainable than expected.
Therefore, the heavy contribution of the quality of feed, and its stability in particular,
is a major pre-requisite that increases cost and leads to consider alternative rearing
systems.
Table 5
Comparative conditions of nutrition in three different rearing systems
Extensive Semi-intensive Intensive (moulinette)
3 4
Bacteria/ml 10 10 108 (bacterial flock)
Productivity +++ + ++ + +
Production 0.3 tons/ha 3 – 4 tons/ha >25 tons/ha
Protein (fish meal) < 10% 25 – 35% 20 – 25%
Protein unicellular ++
Vitamin + ++
Protection vs. pathogen +++
Effluents discharge 0 +++ 0
Water exchange % >40% 40 0
Gut filling temporary temporary constant
Type of feed pellet/collect MR20,25
9.1. Extensive
Extensive rearing at 1 shrimp/m2 can represent a condition for breeders production in

captivity. This condition is particularly appreciated because: (i) animals can grow rapidly
to maturation size, (ii) at this density the probabilities of having animals contaminated by a
pathogenic bacterium such as V. penaeicida or V. pulchritudo are lower. Even in earthen
ponds, extensive rearing is a solution for having breeders ready as quickly as possible in
case of a selective program to keep variability or to select families for their growth
potential. At Ifremer/Cop/Tahiti, for example, L. vannamei have been reproduced for 24
generations in captivity based on this technique and the level of variability is now at a low
level without impairing the reproductive capacity of this strain.
9.2. Moulinette (concept of external rumen)
L. vannamei remain the species with the world record in terms of production under
super-intensive conditions, which emphasizes the capability of this species to adapt to a
wide range of rearing conditions.
This system, developed by Aquacop et al. (1987), Aquacop (1989) and Ralston
Purina (1975), received this name because the water movement inside the tank and
convection cells created a permanent re-suspension of organic particles, called
‘‘bacterial flock’’. This flock cleared the medium and the ‘‘moulinette’’ system
designed under the perspective of raising shrimp at high density in a system in
which clearance was achieved in the tank itself. Shrimp were raised in a medium that
could be considered an external rumen rich in several bacterial ecotypes, contributing
to the nitrification process.
At that time, there was no consideration for pathogens. Today, rearing systems, in
particular the ‘‘moulinette system’’ and its derivatives, are strongly considered as a way of
coping with pathogens, and, hence, improve overall survival compared to semi-intensive
cultures. Considerations on bacterial ecology have led to the concept of volume occupied
by non-pathogenic bacteria, leaving limited space for pathogens and their proliferation
(Table 6).
Table 6
Parameters controlled at production level with semi-commercial feeds in Tahiti
Feed for vanna 40 – 42%CP Feed for vanna 38 – 35%CP
1990 – 1995 1996 – 1997
Rearing cycle in months 5 5
Final survival in % 33 – 42 28 – 25
Mortality %week 3–5 3–5
Growth g/week 1.2 1.2
Final weight in g 18 18
FCR 1.9 – 3.0 1.6 – 2.5
Mud accumulation m3/cycle 12 – 13 12
Water renewal % 5 – 20 [15] 5 – 20 [15]
Induction mortality at transfer 2 – 3% 2 – 3%
Final weight at harvest 20 – 21 g 18 – 20 g
Production m ton/year 10 – 15 10 – 12
Stocking density/m2 100 – 120 Id
300 – 400
Production (NH3 – NH+4) – N 168 g/J/ha basically lessened
Production (NH3 – NH+4) – N 25 kg/ha/cycle
Regarding sustainability of such a system, the question arose on whether starting

‘‘moulinettes’’ might not be as reliable as thought initially. It is true that difficulties might
come up, especially in small volumes, but as soon as animals were stocked, feed provided
and aeration set, the bacterial flock settled quite rapidly with 12 ecotypes identified in the
water and six ecotypes in the digestive tract of shrimp (Ecotron, 1988, personal
communication). There is equilibrium between bacteria in the water and bacteria in the
gut. In the gut they can increase up to 108/ml as compared to 103/ml in lagoon water.
Bacteria can colonize feces that are ingested by shrimp (coprophagy). The medium in
equilibrium is convenient, acceptable for juveniles up to maturation. These rearing systems
are still in use to produce breeders in captivity with animals performing quite well during
the reproductive process (Aquacop, 1991).
9.3. Clear water, high water renewal, and ‘‘moulinettes’’
Conditions encountered most often in large shrimp pond farms of productive countries
and called semi-intensive system (Table 6) involve high water renewal. Compared to other
systems, such as tambaks, ‘‘moulinettes’’ provided a good opportunity to further
improvement rearing systems considering environment regulation aspects and the spread
of viral disease, as well as different epizootic spreads in several productive areas of
tropical countries (Table 5).
Among these three systems, it is clear that advantages are not concentrated in one
system and considerations are needed according to the site and the level of regulation;
nutritional considerations being more linked with health of animals. A better adjustment of
daily of feed ration in relation to the energy required for growth (Bureau et al., 2000) is
recommended to reduce feed wastage.
From a nutritional point of view, there are difficulties to relate tank and pond results
mainly due to the interference of natural productivity. Its contribution is quite difficult to
address even though the problem has been studied with the stable isotope technique, but
variability in bloom evolution is still too high and unpredictable.
Rearing conditions still remain such that resistance to viral infections and proliferation
of pathogens are common. This has led to a search for effective substances to boost the
immune response and to manage ponds under different conditions (biosecurity rules),
requiring less water renewal and providing room for more friendly bacterial populations.
10. Nutrition and feeding in tanks or in ponds
Daily feed ration is changed according to biomass, but feeding frequency can shift feed
intake level and, of course, attractants will favor phagostimulation, such as Artemia
nauplii. Ingestion rate with a semi-purified diet increased from 2 to 2.5 mg feed/2 shrimp/2
h with 0 and 2% freeze dried Artemia, respectively. Artemia meal is affordable today at
low percentage, even in commercial shrimp feeds to maintain palatability of the diet and
enhance feed consumption, which would, reduce residence time in the water, avoiding
pond bottom pollution and preserving feed quality.
Two important points have been shown concerning feeding: (i) satiation feeding
produces the best weight increase over 30 days (Fig. 12) and, in practice, the more one
moves away from ad libitum feeding the less weight gain, (ii) two meals are better than
one, with two meals ad libitum, growth rate increased and FCR remained stable (Cam et
al., 1995). From a practical point of view, the more you let shrimp ingest the feed the better
the growth rate. Lawrence et al. (1995) showed that 15 times a day feeding of a 15% CP
diet coincided, in terms of shrimp growth, with two to three times a day feeding of a 30%
Fig. 12. Average weight gains in g/month according to four feeding levels and ad libitum (Cam et al., 1995).
CP diet (in terms of feed intake, the amount of protein ingested was about the same).
However, data on FCR were not analyzed. Hence, the following conclusions can be
drawn: (i) with frequent feeding, the concept of equal of inputs becomes less valid, (ii)
with frequent feeding in experimental tanks, shrimp go back to situations encountered in
the wild where they exhibit a nibbling activity. Such a behavior for feeding activity brings
continuously minute amounts of food to the gut, leading to a regular diffusion of nutrients
through the absorptive part of the hepatopancreas and midgut to the hemolymph. Also,
proteolytic activity may follow the rate of ingestion, which is maximum for compounded
feed between 20:00 and 22:00 h (Hernandez Cortes et al., 1999).
Results from Cam et al. (1995), on ration levels for P. vannamei, revealed experimen-
tally the difficulty encountered while working with shrimp in contrast to working with
fish. This difficulty lies in the correct assessment of feed intake, and the adaptation to feed
texture (collet versus pellet) on the other to allow feed to be swallowed as straightforward
as possible (Cuzon and Williams, 2001).
11. Abiotic factors and nutrition
In the last 20 years, several worker have assessed the environmental variables that are
optimal for L. vannamei physiology and consequently for its growth. White shrimp L.
vannamei have been cultivated in different environments reflecting the estuarine and/or
marine environments where farms are located. Since the beginning of shrimp culture, it
has been hypothesized that nutritional requirements can change according to variations in
abiotic factors, such as salinity, temperature, dissolved oxygen, etc., since all these
parameters alone and/or together modify the energetic requirements of shrimp. Unfortu-
nately, there are not many studies relating nutrition with environmental factors and its
effect on growth rate of shrimp. The bulk of information describes the effects of salinity,
temperature, dissolved oxygen, or salinity– temperature combinations on growth, survival,
or physiological conditions. Rosas et al. (1998, 2001b) showed a relationship between
salinity and diet manipulation.
At different salinities, carbohydrates, in particular starch, interacted with growth
performance. Salinity affects the distribution of many marine and estuarine organisms.
The life cycle of many peneid shrimp, including L. vannamei occurs between marine and
estuarine environments. Reactions to salinity change with age of shrimp due to its
physiological condition. Resistance to a saline shock by L. vannamei postlarvae changes
with age: a 2 h LC50 value of 20% for PL4 to 2% for PL20 (Aquacop et al., 1991). In a
more recent study, using similar methodology, L. vannamei postlarvae exposed to low
salinity, the 2 h-LC50 decreased from 17% for both PL1 and PL2 to 14%, 10%, 8%, 5%,
and 3% for PL3, PL4, PL5, PL6 and PL7, respectively. Thus salinity appeared as a stressor
which could be used as a rapid, inexpensive and simple test for L. vannamei postlarvae. L.
vannamei 8-day old postlarvae are more sensitive to salinity changes than 22-day old
postlarvae (Ogle et al., 1992), since younger shrimp exhibited a lower survival (20%) than
older shrimp (84%) under 16%. Bouaricha et al. (1994) observed that osmotic regulation is
related to gill structure development and Na+ – K+ ATPase activity in shrimp; maximal
Na+ – K+ ATPase activity was observed in P. japonicus after PL5. Although there are
differences between optimal growth and salinity, the fastest growth rate for L. vannamei
has been obtained in salty water. At low salinity, shrimp use protein as a source of amino
acids to maintain osmotic pressure and for growth. Dietary protein levels affect growth rate
of L. vannamei exposed at 40% and 15% salinities. The fastest growth rate was observed
in shrimp fed 50% dietary protein and maintained in low salinity as compared to shrimp
fed 30% dietary protein and maintained at low or high salinity (Rosas et al., 2001a).
Higher growth rate coincided with higher hemolymph protein concentration, indicating
that protein metabolism is enhanced when shrimp are fed high protein levels. High
hemolymph protein concentration indicates that hemolymph, through hemocyanin, is able
to store protein after salinity acclimation (Marangos et al., 1989). After a salinity change,
the loss of FAAs from muscle results in their release into the blood and the additional
osmotic load as the blood level would increase water inflow from an external medium.
Transfer of FAAs to the gastric fluids in the digestive gland provides a means of
minimizing this additional load. The digestive gland is thought to be the site for
hemocyanin synthesis, and products of this synthesis might cause an increase in
hemolymph protein content when the FAAs are transferred from the blood. The increase
in digestive gland weight (HSI = 4) reported in M. japonicus after a salinity change can be
used to confirm this mechanism. Whatever mechanism is used to excrete the osmotically
active nitrogenous compounds in L. vannamei, it is clear that adaptation to lower salinity
involves a loss of organic molecules from the body. Although production of these
substances by the tissue appears to be intracellular, ultimately they have to be supplied
by the food.
At low salinity, the high growth rate observed in L. vannamei fed diets with high
protein levels resulted from the dietary amino acids as osmotic effectors, which reduced
the loss of amino acids from muscle, and promoted growth. In such circumstances, muscle
weight loss reported in other shrimp species after a salinity change (Marangos et al., 1989)
might be compensated through an increase in dietary protein level. Since hemocyanin is
the most abundant protein in hemolymph and can be used by shrimp as a storage protein,
hemolymph elevation observed in L. vannamei juveniles was due to hemocyanin.
Depending on salinity, this hemocyanin could be used as a source of osmotic effectors
or as an energy source. Crustacean hemocyanin could be useful as an adaptive molecule to
environmental changes, since crustaceans can synthesize one or more hemocyanin types
allowing adaptation to their own particular ecological, behavioral, and physiological
milieu. As with hemoglobin in mammals, a nutritional role can be included in the
crustacean hemocyanin. An increase in protein content of hemolymph related with
hemocyanin has been reported in L. vannamei maintained in low salinity and fed high
protein diets (Rosas et al., 2001b). In shrimp little is known about the time needed to
accomplish the physiological adjustments to salinity changes: 4 days were enough to
obtain full ionic regulation in juvenile M. plebejus Hess, P. esculentus Haswell, F.
merguiensis de Man, and M. bennettae (Dall and Smith, 1981). L. setiferus postlarvae ,
PL10 to PL14, can tolerate diluted media (5x)and were able to adapt in only 2 h to
changes in salinity (Rosas et al., 1998).
In L. vannamei juveniles, acclimated for 4 days at different salinities (Rosas et al.,
2001c), the apparent heat increment increased as salinity decreased, with the largest values
recorded in shrimp acclimated to 5%. Heat increment increased as dietary protein
increased (Rosas et al., 1996; Taboada et al., 1998) and salinity decreased (Rosas et al.,
2001b); amino acid deamination and anabolism were the main contributors to heat
increment variation (Beamish and Trippel, 1990). After a salinity drop, heat increment
increased as a consequence of dietary protein degradation, amino acids were used to
maintain osmotic pressure (Claybrook, 1983). L. vannamei juveniles are well-adapted to
tolerate salinity changes when exposed to rapid or progressive changes. Though at 15%,
shrimp require a greater quantity of energy to maintain homeostasis, they are more
efficient due to an increment in respiratory metabolism together with an increment of food
consumption, revealing an increase in energy expenditure linked to heat increment.
The isosmotic point has been associated with optimal conditions for several peneid
shrimp regarding growth. However, Brito et al. (2001) showed that there was no relation
between the isosmotic point and growth rate because the optimal salinity for growth differs
among species. At the same time, isosmotic points are the same regardless of the species.
The osmotic gradient produced with a salinity less than the isosmotic point improves
assimilation mechanisms with more energy ingested and lower physical activity (save
energy for growth). Optimal salinities for growth of L. vannamei juveniles are 5% and
15% under laboratory conditions (Samocha et al., 1998; Rosas et al., 2001d), even if
shrimp are infected with IHHN virus producing a runt deformity syndrome (Bray et al.,
1994).
Temperature affects shrimp physiological status; it has a major effect on metabolism
(Prosser, 1973) because it controls enzymatic activity, food consumption and, growth rate.
Optimal temperature for L. vannamei juveniles changes with age (Wyban et al., 1995).
Optimal temperature for shrimp below 5 g may be greater than 30 jC, whereas for larger
shrimp optimal temperature is about 27 jC. L. vannamei postlarvae are sensitive to
temperature changes between 23 and 27 jC. Feeding rate, and feeding behavior observed
in farms were tightly related to temperature (Villarreal et al., 1994). This relation is in line
with variations in energy demand controlled by temperature. Furthermore, oxygen
consumption increased between 0.36 mg O2/h/g at 20 jC to 1 mg O2/h/g at 32 jC with
temperature in L. vannamei juveniles (Villarreal et al., 1994), revealing an increased
energy demand with increasing temperature.
Energy increment enhanced feeding rate affects protein retention. L. vannamei
ammonia excretion increases according to temperature in a range of 24 jC (9– 8.7 Ag
N – NH3/g/h), 28 jC (22 –13 Ag N –NH3/g/h), and 32 jC (23 – 17 Ag N – NH3/g/h) at 10%,
25%, and 40%, indicating that protein metabolism is affected by temperature in this
salinity range (Jiang et al., 2000). For crustaceans, proteins appear to be a major source of
metabolic energy and nitrogen excretion results from the catabolism of dietary and
endogenous amino acids (Claybrook, 1983). Changes in ammonia excretion rate may
reflect the response of shrimp’s metabolic rate to temperature and its ability to adapt
environmental changes. Using the Q10 coefficient indicated that L. vannamei nitrogen
excretion was lower between 24 and 28 jC than between 28 and 32 jC, and low
temperature could affect shrimp protein metabolism (Jiang et al., 2000).
Variation in dissolved oxygen are a common occurrence in aquaculture. Low oxygen
tension can reduce growth and feed digestibility (Seidman and Lawrence, 1986; Rosas et
al., 1998) and cause mortality in farmed shrimp. In L. vannamei, lethal level of O2 is about
1 mg O2/l. The effect of dissolved oxygen (DO) on an organism has been classified as a
limiting factor and it does not act directly on growth or feed digestibility, but instead on
metabolism. DO decreased the respiratory metabolism, as well as feeding and growth rates
in shrimp exposed to 2 mgO2/l (Rosas et al., 1998). Low DO does not damage any
physiological or biochemical response of shrimp, but limits the aerobic metabolism
through oxygen reduction at the last electron receptor of the respiratory chain. L.
vannamei juveniles exposed to sublethal DO levels recovered if adequate DO levels
were provided (Charmantier et al., 1994). Sublethal DO levels between 1.5 and 3 mg/
l caused a decrease in the hyper- and hypo-osmoregulatory capacity of shrimp, which was
time-dependent and could be recovered after 24 h at oxygen saturation. DO level in
intensive shrimp culture ponds depends on water exchange, amount of food, phyto-
plankton bloom, and aeration. DO limits food consumption and growth. An equation to
predict the DO level was developed by Hopkins et al. (1991) based on the amount of feed
applied (kg/ha per day) per unit aeration (hp/day/ha) in L. vannamei culture: F/
A = 29 (4.3 DO), where F is the 5-day average feed rate (kg/ha per day), A is the
aeration rate (hp per day/ha), and DO is the dawn dissolved oxygen (mg/l). To determine
the aeration rate required for a given daily feed amount, a minimum acceptable dissolved
oxygen must be targeted. If, for example, 3.0 mg/l is taken as an acceptable minimum
(Seidman and Lawrence, 1986), 3.0 can be substituted in the above equation and solve
for F/A = 16 kg/hp. It means that for every horsepower of aeration equipment in the pond,
a maximum of 16 kg of feed can be applied each day with reasonable expectation that the
dawn DO concentration will be at least 3.0 mg/l.
All abiotic factors exert an impact on the metabolic rate of shrimp and the nutritive
requirements should be covered consequently; for example, at low temperature a more
protein dense diet will maintain animals and reduce mortality, oxygen will control feed
intake, and nutrients will be used for to the growth efficiently according to a range of
abiotic factor variations corresponding to the specific organism.
Aside from abiotic factors, internal factors and biotic factors should be taken into
account for their respective influence on metabolic rate, growth, and reproduction.
12. Special feeds
Special feeds represent a category of feeds produced in a limited amount and with less
cost consideration than for a grower feed because of the paramount importance of efficacy
of such feeds during a short period of time.
12.1. Larval feed and feeding
Few reports on nutrient requirements of L. vannamei larvae are available (Smith et al.,
1993; Pedrazolli et al., 1998).
Both of these reports were produced without using live food, however larval growth fed
microparticles was significantly lower than those fed live feed. It is for this reason that
research on larval nutrition has focused on the study of replacing formulated feeds for live
feed and the digestive physiology at different shrimp stages. Substitution of microparticles
for live feed has been shown with larvae fed with spray dried algae and Artemia that
exhibited 84% survival with 66% replacement of algae (Biedenbach et al., 1990), whereas
Galgani and Aquacop (1988) obtained a 42% survival of postlarvae when larvae were fed
with microparticles; however, growth was lower than that observed with algae.
Different feeding schedules have been reported for L. vannamei larvae, aside from
MBD, microparticles, or microcapsules.
Aquacop (1977) developed a feeding schedule for L. vannamei larvae leading to a
production of post-larvae P1 in 12 days of culture at 28 jC. Kuban et al. (1985) tested
four feeding schedules of live feed using phytoflagellates, diatoms or phytoflagellates and
Artemia or diatoms and Artemia for L. vannamei larvae, and concluded that the best
combination was diatoms and Artemia nauplii, the latter was added during ZII substage.
Larvae fed diatoms with or without Artemia metamorphosed faster than larvae fed
phytoflagellate. However, Durruty Lagunes (2001) and Brito et al. (2001) demonstrated a
reduction in the use of algae and Artemia nauplii with good survival and growth rates if
an alternative schedule included diets or rotifers in the live feed sequence. Live feed
sequence has been optimized to provide reliable results in the laboratory. But, algae vary
in composition whether taken in log phase or in stationary phase. Lipid composition can
differ significantly and, most of the time, hatcheries are unable to control this quality
factor. Little consideration has been given to bacteria’s contribution of bacteria (Castille
and Lawrence, 1979); some limited trials with friendly bacteria growing on saccharose
and glucose, as dissolved components in seawater, as well as some amino acids, have
been made, revealing that all those aspects contribute to the beneficial use of balanced
inert particles.
This explains a predilection for the use of microparticulated, microbound diets or
microcapsules instead of algae (serving as a reference). Their composition has been
considerably improved in recent years, though phospholipid requirements were not always
covered, even though maturation of the gut seems relevant during this phase of
development. Microparticles were used mainly with mass-production of postlarvae in
commercial hatcheries and the perspective looks promising to fully replace live feed
sequence by microcapsules. Producers are prone to go along in this direction with an
eventual help of probiotics.
12.2. Microcapsules
Most of the work on nutrient requirements have coming from Kanazawa’s team using
the M. japonicus model and different microparticles to replace algae and Artemia.
Person-Le Ruyet (1987) proposed as future research priorities the optimization of water
stable microfeeds to provide total replacement of live feed in hatcheries, incorporation of
attractants and gut enzyme stimulants, as well as modification of hatchery systems to
optimize use of inert particles. Jones et al. (1987) obtained 90% survival for PL5 – 7’s from
larvae fed a combination of Frippak microcapsules, algae, and Artemia at 3– 5/ml, whereas
80% survival was obtained in those fed microcapsules and algae, without Artemia.
Pedroza-Islas et al. (in press) used an artificial diet for shrimp larvae of L. vannamei that
was microencapsulated with a polysaccharide blend (66% gum arabic, 17% mesquite gum
and 17% maltodextrin 10 DE). The microencapsulated diet (MD) was fed to mysis alone,
and as co-feeds with the microalgae Chaetoceros cerastosporum and Tetraselmis chuii
(Mixed), and compared with a live diet (control) of C. cerastosporum, T. chuii and Artemia
nauplii. No significant differences ( P>0.05) were detected in the growth rates, develop-
ment and quality indexes of the larvae fed the three experimental diets. All the diets
supported survival percentages of over 90%. Shrimp larvae fed the microencapsulated and
mixed diets had a higher specific trypsin activity and soluble protein content than those fed
the live diet. Amylase activity decreased in the mixed and microencapsulated diet
apparently by the carbohydrate composition of the diet. The results showed that it is
possible to substitute a live diet by a microencapsulated one with a wall composition made
up by a polysaccharide blend in Litopenaeus vannamei mysis.
Using purified microparticulated diets, Durruty Lagunes (2001) reported a 30% dietary
protein as optimal for protozoea whereas mysis required 50% CP. However, Le Moullac et
al. (1994) determined that a diet containing 60% casein and 10% starch produced the
highest larval growth in protozoea of this species.
Lipid-wall microcapsules embedded in gelled food particles (mixture of alginate and
gelatin named CXMs) were tested in larvae of L. vannamei for delivery of nutrients
(Villamar and Langdon, 1994) with a potential usefulness in research on the nutrition of
aquatic suspension feeders. In practice, it should now be possible to formulate and deliver
a complete artificial diet, with all the potentially essential dietary constituents. The
difficulty of total substitution of artificial for live feed, either microparticles or micro-
capsules, has been reported on the lack of digestive capabilities of larvae.
Le Moullac et al. (1994) studied the activity of digestive enzymes (trypsin, chymo-
trypsin, and amylase) in relation to types food in L. vannamei larval stages. Micro-
particles containing casein caused a decrease in soluble protein content and enzyme
activities. Specific trypsin activity but not chymotrypsin was correlated to casein levels
( P < 0.01) in microparticles (10% and 60% casein). Regarding protein sources, squid
meal stimulated significantly chymotrypsin activity ( P < 0.02) whereas trypsin activity
decreased ( P < 0.05) with fish protein soluble concentrate (CPSP80). Durruty Lagunes
(2001) reported a peak in amylase activity (glucosidase activity in larvae of L. vannamei
was also reported) in protozoeanII fed 40% protein. At mysisIII, the peak activity of all
three enzymes was observed in larvae fed 30% and 50% protein. The peak coincided
with the best growth and survival. Ravallec-Ple et al. (2001) studied the influence of
incorporation of hydrolysates made with purified shrimp chymotrypsin. Peptides acted
positively upon growth, survival, and digestive enzyme activities of L. vannamei larvae.
For example, cod hydrolysate added to microparticulated diets stimulated significantly
growth and digestive enzyme activity. The presence and action of peptides of low
molecular weight related to growth factors and secretagogue agents increased larvae
growth and survival.
12.3. Postlarvae (PLs)
During the 1980s, PLs raised in hatcheries tended to be disliked by local farmers in
Ecuador. They preferred wild PLs that revealed to be more hardy animals, possessed more
genetic variability, and according to experts more genes are inherited and activated (from
an ontogenetic point of view), which has been favorable in terms of gene activation. PLs
obtained from a hatchery are considered of less potential. In hatcheries, a lack of algae at
zoeal stages or a distribution of microparticles without enough peptides could reduce gene
expression, coding for example for digestive enzymes. Such peptides are said to possess a
so-called secretagogue effect (Gallardo et al., 2001).
Diets related to growth and digestive enzyme activities in L. vannamei postlarvae (Brito
et al., 2001) were fed to PLs ranging from PL1 to PL10. Postlarvae received algae for a
given time, together with a dry diet acting partially to replace Artemia nauplii. In fact, it
was an association of three food classes (algae, dry diet, and Artemia) that provide the
highest growth performance in PLs. An adaptation, from a biochemical point of view, was
developed and the change was evidenced through the determination of digestive enzyme
activities. For example, with trypsin, an induction of enzyme activity occurred when PLs
were fed a diet rich in animal protein, Artemia with or without dry diet, but trypsin-like
activity was 10 times higher in PLs fed Artemia than in PLs fed artificial diets. On the
contrary, diets including algae did not enhance enzyme activity. Similar results were found
in P. indicus (Jones et al., 1987). It was emphasized (Brito et al., 2001) that trypsin activity
induction does not run in parallel with growth rate; PLs fed animal protein did not grow
more than PLs fed a mixture of the three sources (algae, Artemia, microparticles). Aside
from these ‘‘traditional sources’’, the role of bacteria can be mentioned in systems such as
‘‘moulinettes’’, where a bacterial flock (bacteria, microalgae such as diatoms, zooplankton
such as rotifers, copepods, mysidacea, organic matter in micro-beadlets) provide nutrients;
soluble substances, such as hexoses, could be taken up (Castille and Lawrence, 1979) with
a beneficial effect on PLs development.
12.4. Spawners
There are few reports on the biochemical composition and energetic content of
shrimp eggs. Galois (1983) analyzed palaemonid eggs with an average fresh weight of
50 Ag which contained 10 Ag lipid with 6 Ag PL and 4 Ag TG. Among the fatty acids,
16:0, 18:1 and 18:2n-6 accounted for 1/3 each. During vitellogenesis, there was, a
transfer of TG from the HP to ovaries, mainly from 0.6 to 176 mg %body fresh weight.
At spawning, this would represent 0.7 J/egg and between 200 000 and 450 000 eggs are
spawned according to the size of the female. The initial consideration of such basic data
is helpful inorder to understand the needs of spawners in order to produce quality eggs
and viable larvae.
A review of shrimp breeder nutrition by Waldroup (1978) led to the following
applications.
From an energetic point of view, shrimp breeder, weighing 55 g (BW) at approximately
32 weeks of age, has a basal net energy requirement (NEm) calculated according to an
allometric equation NEm = 0.457 BW (kg)0.6. Thus, in a shrimp weighing 55 g, NEm =
0.46 0.11 = 0.1 kcal/shrimp/day or 0.4 kJ/shrimp/day. Since this is approximately 82%
of the NEm value, then, 0.4/0.82 = 0.5 kJ = NEm. Adding 50% of this value for activity,
the total NE for activity in non-spawning shrimp is 0.5 + 24 = 24.5 kJ/shrimp/day. The
energy content of an egg is around 0.12 J. Thus the total NE requirement of a L.
vannamei shrimp at 100% production, is 24.5 + 0.12 = 24.6 kJ/day at 28 jC or if one
considers that energy of a spawn is 0.12 200 000 = 24 kJ, then 0.62 + 24 = 24.6 kJ/day.
However, these shrimp are still growing, body gain is approximately 2 g over a 4-week
period = 0.07 g gain/day. Gain = 18% protein*4 kcal + 4% fat*9 kcal = 0.08 or 0.3 kJ. Total
ME required = 24.62 + 0.3 = 25 kJ/shrimp/day for the period of 32 –36 weeks of age. In
terms of feed intake, it led to a quantity equal to 2.3% biomass/day.
A review of the role of nutrition and feeding during the reproductive period (Harrison,
1990) provided a reasonable amount of data to be able to formulate a balanced dry feed for
L. vannamei breeders even if a certain amount of fresh food was still needed for the week
preceding spawning. Recently, Artemia biomass has been favored (Naessens et al., 1997)
and in many hatcheries handling broodstock includes the use of blood worms, mussels, or
squid. Squid and the so-called ‘‘second squid factor’’ linked to L. vannamei maturation
(Mendoza, 1989) was found to increase ovary development. Consequently, maturation of
L. vannamei is considered from two different approaches according to location and
availability or lack of natural marine food. Vitamin C or vitamin E in relation to the
biochemical composition on spawning eggs (Racotta et al., 2000). Fortification with
vitamins is advised with a particular emphasis on vitamins C (100 –400 ppm) and E (300
ppm), An adequate diet must include quality protein sources at high levels (above 50%)
and a substantial amount of marine lipid (13%) and phospholipid (3 –5%), cholesterol
(0.5 – 1%), and carotenoids (50 ppm) in order to cope with the rapid transfer from the
digestive gland to ovaries during vitellogenesis. Taking into account all these observations,
several articles (Palacios, 2000; Chamberlain and Lawrence, 1981) referring to the
influence of phospholipids and highly unsaturated fatty acids (Cahu et al., 1989; 1994;
Lytle et al., 1988) have been published. It is definitely possible to sustain spawning,
quality of eggs, and viable postlarvae with an adequately formulated diet (Aquacop, 1977;
Cuzon et al., in press(a)).
13. Conclusion
How does L. vannamei differ from other peneid shrimps? Is there a real difference
between L. vannamei and other peneid species?
This is a primitive species in terms of ecological classification together with L.
stylirostris (Kurata, 1975, personal communication) and its gut physiology is similar to
other peneid species. Range of digestive enzymes is the same. Habitat is similar but
feeding habits can change. For example, in ponds, stomach content of juveniles revealed a
large proportion of a benthonic diatoms (Navicula sp.). Hence, from a point of view of
requirement, L. vannamei could be classified as a species with lower relative protein
requirements than L. stylirostris when reared is tanks. However, when placed in intensive
systems with bacterial flock, L. vannamei can graze on bacteria attached to organic
particles as does L. stylirostris. L. vannamei can be fed numerous meals a day, juveniles
grow well at 27 jC and protein deposition is optimal: if low protein (25%) and low
digestible energy (13 kJ/g) diets are provided, then, there is a maximal nitrogen retention
efficiency (70%) as described by Cousin (1995). Protein degradation remains low as
protein turnover is low (Mente and Houlihan, 2002). Recently, a strong protein sparing
effect with starch was evidenced (Arena, 2001). This effect is clearly shown in wild
specimens; this ability to derive energy from starch seems to be lessened if highly inbred
strains are tested. Whether this depression in growth is related to a severe loss of
polymorphism (95% loss of allelic frequency on the a-amylase gene) is too early to
conclude, but further studies are in progress. L. vannamei has been used as a model for
many nutrition works and the extension of its culture, the range of ingredients selected, the
rearing systems selected, prove the potential of this species to convert plant protein
sources. At present, the level of knowledge is satisfactory for juvenile growth, but little is
known of the beneficial effects of nutrition on immune responses, and in the face of the
spreading of epizootics, such as WSSv, there is an urgent need for balanced diets not only
for growth but also for shrimp health protection. This review, with its emphasis on
nutrition is only a part of the issues necessary for improved shrimp culture, which involves
genetics and a better understanding of the defense mechanisms; besides, last but not least,
the subtle management of ponds, representing a complex ecosystem, is another important
approach to be taken into account.
Abbreviations
CXMs lipid-walled microcapsules embedded in gelled food particles
HP for hepatopancreas
TG for triacylglycerides
DP dietary protein
CP for crude protein
PLs for postlarvae
PL for phospholipids
FCR for food conversion ratio
CPSP80 for soluble fish protein concentrate 80% crude protein
MBD for micro binding diets
DH for degree of hydrolysis
ADC apparent digestive coefficient
UNAM Universidad Nacional Autónoma de México
CNEXO Centre national pour l’exploitation des Oceans
OI’s for Oceanic Institute
AHI apparent heat increment or SDA specific dynamic action or HiE
IGR instantaneous growth rate
RGR relative growth rate
ppt for part per thousand (x)
EAA for essential amino acids with an another denomination as indispensable AA
amino acids are denominated as Lys for lysine, Arg for arginine, His for histidine,
Leu for leucine, Ile for isoleucine, Thr for threonine, Phe for phenylalanine, Trp
for tryptophan, Met for methionine and tVal for valine
Acknowledgements
The authors are very much indebted to Texas A&M University for contributions and
release of valuable information, colleagues of Ifremer/Brest for valuable suggestions in the
preparation of the manuscript, UNAM and Ifremer (Aquacop) for common scientific data
shared for this review.
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Nutrition of Litopenaeus Vannamei Reared

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Nutrition of Litopenaeus vannamei reared in tanks

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Keywords: Larvae; Juveniles; Breeders; Shrimp; Peneid; Nutrition

A reasonable amount of work has been done on the digestibility of nutrients in L.

2.1. Relative protein requirement

Fig. 2. Growth rate of L vannamei fed on dry diets (Aquacop, 1972).

intake, and a protein/energy ratio calculated at 70 mg kJ 1 DE. An interesting approach

Fig. 5. Incidence of nature of protein on juveniles growth.

3. Amino acids (AA)

3.1. Essential amino acids requirement

3.1.1. Imbalance diets

3.1.2. Oxidation method

3.1.3. Indirect oxidation method

3.2. Values obtained for essential amino acid (Table 2)

3.4. Absorption of amino acids

3.5. Metabolism of amino acids

3.6. Metabolism of certain amino acids in shrimp

3.7. Non-hormonal control of protein and amino acid metabolism

The capacity to digest common ingredients is important in L. vannamei juveniles. Many

5.1. Phospholipids (PL)

5.2. Phospholipids and cholesterol

Gong et al. (2000a) observed that without dietary phospholipid supplementation,

5.3. Requirement for cholesterol

5.4. Lipid transport

Fig. 8. Effect of AAPP on survival of L. vannamei juveniles.

7. Unknown growth factor

8. Minerals and trace elements

need to synthesize hemocyanin in their hemolymph. Hemocyanin includes a heme contain-

Zinc is a cofactor of several enzyme systems, such as hydrogenase enzymes, alcohol,

9. Nutrition and rearing systems for L. vannamei juveniles

Extensive rearing at 1 shrimp/m2 can represent a condition for breeders production in

9.2. Moulinette (concept of external rumen)

Regarding sustainability of such a system, the question arose on whether starting

9.3. Clear water, high water renewal, and ‘‘moulinettes’’

10. Nutrition and feeding in tanks or in ponds

11. Abiotic factors and nutrition

12. Special feeds

12.1. Larval feed and feeding

12.3. Postlarvae (PLs)

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