N° d’ordre : 4256

THÈSE

PRÉSENTÉE A

L’UNIVERSITÉ BORDEAUX 1

ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE

Par Lenaïg RICHARD

POUR OBTENIR LE GRADE DE

DOCTEUR

SPÉCIALITÉ : Sciences des Aliments et Nutrition

Conséquences métaboliques du remplacement de la farine de poisson par

des protéines végétales chez la crevette géante tigrée (Penaeus monodon)

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Metabolic consequences of fishmeal replacement by plant proteins in the

black tiger shrimp (Penaeus monodon)

Soutenue le : 3 Mai 2011 Devant la commission d’examen formée de : M. HIGUERET, Paul (Professeur Université Bordeaux 1) Président du jury Mme ESPE Marit (Chercheur, NIFES, Norvège) Rapporteur M. BUREAU, Dominique (Chercheur, Université de Guelph, Canada) Rapporteur Mme MAMBRINI, Muriel (Directrice de recherche, INRA) Examinatrice Mme GEURDEN, Inge (Chargé de recherche, INRA) Directrice scientifique Mr KAUSHIK, Sadasivam (Directeur de recherche, INRA) Directeur de thèse Mr RIGOLET, Vincent (UNIMA, Directeur d’exploitation, AQUALMA) Invité

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A mon grand-père

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REMERCIEMENTS

Cette thèse a été financée par l’entreprise UNIMA France, grâce à une bourse CIFRE délivrée par l’Agence Nationale de la Recherche et de la Technologie (ANRT) pour une durée de 3 ans. Mes premiers remerciements vont à mes encadrants, le Dr Kaushik et le Dr Geurden, qui ont été des piliers essentiels à la réussite de ce projet. Je vous remercie sincèrement pour ces trois années, pour nos discussions, pour votre confiance, soutien et implication dans ce travail, ainsi que pour votre grande patience. Je tiens aussi à remercier particulièrement Christiane Vachot, pour son aide si précieuse dans le travail de laboratoire à St-Pée et Madagascar, et plus particulièrement pour les analyses enzymatiques. Aussi, un grand merci à Jeanine Brèque pour son aide sur l’étude des acides aminés. Tous les essais présentés dans cette thèse ont été réalisés sur les fermes d’AQUALMA (Besalampy et Mahajamba), implantées à Madagascar. Je voudrais remercier tous mes collègues malgaches qui ont rendus possible l’accomplissement de ce projet, et notamment le personnel de la ferme de Besalampy. Plus particulièrement, un grand merci à Vincent Rigolet, Christian, Philippe, Léon Paul, Abel, Cartier, Panchu et Pierre-Philippe pour leur aide dans la mise en place et le suivi des essais. Côté basque, je remercie tout le personnel de la station INRA de St-Pée pour ces trois ans de convivialité, de bonne humeur, et d’échanges, et notamment Marie-Jo, Laurence, Geneviève, Stéphanie, Peyo, Maïté, et Panxoa. Je tiens aussi à remercier très sincèrement tous les stagiaires/techniciens/thésards/post-docs croisés au laboratoire pour les bons moments passés ensemble, et particulièrement Karine, Mélanie, et Claudia. Enfin et le plus important à mes yeux, je tiens à remercier ma famille et mes amis pour leur inconditionnelle confiance, compréhension, et pour leur présence à mes côtés durant ces trois années. Un grand merci !

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TABLE OF CONTENT

List of publications…………………………………………………………………..……..9 List of abbreviations……………………………………………………………………....11 List of tables……………………………………………………………………………....13 List of figures…………………………………………………………………….…….…15 Chapter 1…………………………………………………………………...17 1.1. Shrimp biology and production……………………………………………....19

1.2. Utilisation of dietary protein and amino acids in shrimp……………………..22 1.3. Protein and amino acid requirements of shrimp……………………………...30 1.4. Protein sources in shrimp feed: fish meal and alternatives to fish meal……...40 1.5. Objectives…………………………………………………………………….46

Chapter 2…………………………………………………….……….….....47 2.1. Présentation de l’article…………………………………….…….…….......…49 2.2. Abstract……………………………………………………..…….………..…52 2.3. Introduction………………………………………………..………….……....53 2.4. Material and methods……………………………………….………………...55 2.5. Results……………………………………………………….….………….....63 2.6. Discussion………………………………………………….……....……...…..67 2.7. Acknowledgements………………………………………….….…....…...…..75 Chapter 3…………………………………………………………………...77 3.1. Présentation de l’article…………………………………….…….……...……79 3.2. Abstract……………………………………………………….…….……...…82 3.3. Introduction………………………………………………….…….…...……..83 3.4. Material and methods…………………………………………….…………...84 3.5. Results………………………………………………………….……………..93 3.6. Discussion……………………………………………………….…...…….....99 3.7. Acknowledgements…………………………………………….……………104 Chapter 4………………………………………………………....……….105 4.1. Présentation de l’article…………………………………………….………..107 4.2. Abstract…………………………………….………….………….…..……..110 4.3. Introduction………………………………….………….……………...……111 4.4. Material and methods………………………….…………….…………...….114 4.5. Results………………………………………….……………………………117 4.6. Discussion…………………………………..………………………...……...122 4.7. Conclusions……………………………….…….……………...………..…...128 4.8. Acknowledgements…………………….……….…………………………...128 Chapter 5……………………………………………………………….…129 5.1. Présentation de l’article…………………………………….………..………131 5.2. Abstract…………………………………………………….…………..……134 5.3. Introduction…………………………………………………..………..…….135

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5.4. Material and methods…………………………………………………..……136 5.5. Results…………………………………………………………....………….143 5.6. Discussion…………………………………………………….......…………148 5.7. Conclusions………………………………………………………………….152 5.8. Acknowledgements………………………………………………………….153

Chapter 6…………………………………………………....………...…..155 6.1. Protein requirements…………………………………………………...……157 6.2. Amino acid requirements…………………………………………………....165 Chapter 7……………………………………………………….……..…..175 7.1. Conclusions………………………………………………….…………...….177 7.2. Perspectives………………………………………………….……………....177 References…………………………………………….…………...………179

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LIST OF PUBLICATIONS

Peer-reviewed papers

Richard, L, Blanc, P-P, Rigolet, V, Kaushik, SJ, Geurden, I. 2010. Maintenance and

growth requirements for nitrogen, lysine and methionine and their utilisation efficiencies in

juvenile black tiger shrimp, Penaeus monodon, using a factorial approach. Brit J Nutr 103,

984-995

Richard, L, Vachot, C, Brèque, J, Blanc, P-P, Rigolet, V, Kaushik, SJ, Geurden, I. 2010.

The effect of protein and methionine intake on glutamate dehydrogenase and alanine

aminotransferase activities in juvenile black tiger shrimp Penaeus monodon. J Exp Mar Biol

Ecol 391, 153-160

Richard, L, Vachot, C, Surget, A, Rigolet, V, Kaushik, SJ, Geurden, I. 2011. The effect of

choline and cystine on the utilisation of methionine for protein accretion, remethylation and

transsulfuration in juvenile shrimp Penaeus monodon (accepted in Brit J Nutr).

Richard, L, Surget, A, Rigolet, V, Kaushik, SJ, Geurden, I. 2011. Fishmeal replacement

by plant protein affects essential amino acid availability in juvenile black tiger shrimp,

Penaeus monodon (to be submitted to Aquaculture)

Proceedings oral presentations

Richard, L, Blanc, P-P, Rigolet, V, Kaushik, SJ, Geurden, I. Estimation of maintenance and

growth requirements for protein, lysine, and methionine in juvenile Black Tiger Prawn,

Penaeus monodon using a factorial approach. World Aquaculture, 25-29 September 2009,

Veracruz, Mexico.

Richard, L, Vachot, C, Surget, A, Rigolet, V, Kaushik, SJ, Geurden, I. Regulation of total

sulfur amino acids pathways by methionine, cysteine and choline in juvenile tiger prawn

Penaeus monodon. 14th Symposium ISFNF, 31st May-4th June 2010, Qingdao, China.

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Richard, L, Vachot, C, Brèque, J, Blanc, P-P, Rigolet, V, Kaushik, S, Geurden, I. Effect of

protein and methionine intake on glutamate dehydrogenase (GDH) and alanine

aminotransferase (ALAT) activities in juvenile black tiger shrimp Penaeus monodon. 3rd

EAAP international symposium, 6-10th September 2010, Parma, Italy.

Proceedings poster presentations

Richard, L, Blanc, P-P, Borthaire, MJ, Rigolet, V, Kaushik, SJ, Geurden, I. Estimation des

besoins d’entretien et de croissance en protéine, lysine, et méthionine chez des juvéniles de

crevette tigrée Penaeus monodon utilisant une approche factorielle. 2èmes Journées de la

recherche filière piscicole, 1-2 juillet 2009, Paris

Richard, L, Vachot, C, Brèque, J, Blanc, P-P, Rigolet, V, Kaushik, SJ, Geurden, I. Effets de

l’ingestion de protéine et méthionine sur l’activité de la glutamate déshydrogenase et de

l’alanine aminotransferase chez les juvéniles de crevette tigrée, Penaeus monodon. Journée

scientifique de l’Ecole Doctorale SVS, 28 avril 2010, Arcachon.

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LIST OF ABBREVIATIONS AA, amino acids ADC, apparent digesible coefficient Ala, alanine ALAT, Alanine aminotransferase ANFs, antinutritional factors ANOVA, analysis of variance Arg, arginine Asp, asparagine BHMT, betaine-homocysteine methyltransferase BLM, broken line model BUN, branchial and urinary losses BW, body weight CBS, cystathionine β-synthase CC, choline chloride CGM, corn gluten meal Cit, citrulline CP, crude protein (PB, protéine brute) Cys, cysteine Cyss, cystine DGC, daily growth coefficient DE, digestible energy Diff, differenciation DM, dry matter (MS, matière sèche) DP, digestible protein (PD, protéine digestible) DR, dose response EAA, essential amino acids (AAE, acides aminés essentiels) Ez, enzyme FAA, free amino acids FCR, feed conversion ratio FE, feed efficiency FI, feed intake FM, fishmeal GDH, Glutamate dehydrogenase GE, gross energy Glu, glutamate Gln, glutamine Gly, glycine Hcy, homocysteine He, hemolymph His, histidine HP, high protein diet HR, holocrine release IBW, initial body weight (PMI, poids moyen initial) Ile, isoleucine kg, kilogram kJ, kilojoule L, liter Leu, leucine LP, low protein diet Lys, lysine Met, methionine mg, milligram MP, medium protein diet MS, methionine synthase N, nitrogen NEAA, non essential amino acids (AANE, acides aminés non essentiels) NH3, ammonia

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NO2, nitrite NO3, nitrate NP, non protein diet O2, oxygen Orn, ornithine PC, phosphatidylcholine PER, protein efficiency ratio Phe, phenylalanine PL, phospholipid ppt, pert per thousand Pro, proline Prot, protein Quad-1, model quadratic one slope RSM, rapeseed meal Ser, serine SGR, specific growth rate Shr, shrimp SKM, saturation kinetic model SOR, sorghum Tau, taurine TFAA, Total Free Amino Acids Thr, threonine Trp, tryptophan TSAA (or SAA), total sulphur amino acids Tyr, tyrosine Val, valine WBP, whole body protein WG, wheat gluten WG, weight gain (%) WW, whole wheat

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LIST OF TABLES

CHAPTER 1

Table 1.1. Comparison of the main shrimp farming systems (adapted from Tacon, 2002) Table 1.2. Classification of the main digestive enzymes in crustacean Table 1.3. Some data on free amino acid concentrations (µM/L plasma and µM/g tissue) in different tissues of

shrimp compared to that in rat Table 1.4.Summary data on protein requirement estimates of different species of Penaeid shrimp (from (Shiau,

1998, Velasco et al., 2000) Table 1.5. Summary data on essential amino acid (EAA) requirement estimates for Penaeid shrimp Table 1.6. Requirements for several fish species (NRC, 1993) Table 1.7. Protein-bound amino acid concentrations in different tissues of shrimp (g/100 dry tissue) Table 1.8. Plant proteins commonly used in animal feeds (Francis et al., 2001, Venero et al., 2008)

CHAPTER 2

Table 2.1. Formulation and analysed composition of the ten experimental semi-purified diets fed to P.monodon juveniles for 6 weeks

Table 2.2. Formulation of the AA blend added to the semi-purified casein-based diets (g/kg diet) Table 2.3. Analysed AA composition of the ten experimental diets (g/16g N) Table 2.4. Survival, feed intake, growth and nutrient utilisation in juvenile P.monodon fed the semi-purified

diets for 6 weeks Table 2.5. Parameters estimated by fitting the four regression models through the experimental data using N

gain (g/kg BW per d) as the response parameter and the different intake levels of nitrogen, lysine or methionine (g/kg BW per d) as input parameter in P.monodon.

Table 2.6. Estimated requirements for N equilibrium (maintenance, M) and maximal N gain (N growth, G) for N, protein, lysine and methionine using the four regression models for juvenile P.monodon

CHAPTER 3

Table 3.1. Composition and proximate analysis of the five diets fed to juvenile black tiger shrimp P.monodon Table 3.2. Analysed amino acid composition of the experimental diets as g/100g dry feed and as g/16g N (in

brackets) Table 3.3. Effect of replacing fishmeal by plant protein mixture on water quality of the earthen ponds (Expt. 1) Table 3.4. Effect of fishmeal replacement on growth performance of P.monodon reared in ponds during 144

days (Expt. 1) Table 3.5. Final whole body composition and nutrient retention of shrimp reared for 144 days in earthen ponds

(Expt. 1) Table 3.6. Effect of fishmeal replacement by plant protein mixture on the apparent digestible coefficients (ADC,

%) for dry matter, protein, energy and amino acids in P.monodon (Expt. 2) Table 3.7. Parameter estimates obtained from the linear regressions between the ADC value of each essential

amino acid and the relative contribution of each protein source to the dietary protein content (Expt. 2) Table 3.8. Parameter estimates obtained with a broken line regression using the weight gain (%) of juvenile

P.monodon reared in cages for 49 days as a response criteria (Expt. 3) CHAPTER 4

Table 4.1. Formulation and analysed composition of the experimental six semi-purified diets fed to P.monodon juveniles for 6 weeks

Table 4.2. Analysed AA composition of the experimental diets as g/kg feed and as g/16 g N (within brackets)

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Table 4.3. Weight gain, nitrogen and methionine intakes, nitrogen gain, and hepatosomatic index (HSI) in juvenile P.monodon fed one of the six semi-purified diets for 6 weeks

Table 4.4. ALAT activities in three organs of juvenile P.monodon fed low, medium or high protein diets with adequate or deficient levels of methionine during six weeks

Table 4.5. GDH activity in three organs of juvenile P.monodon fed low, medium or high protein diets with adequate or deficient levels of methionine

CHAPTER 5

Table 5.1. Formulation of the experimental semi-purified diets fed to juvenile P. monodon for 5 weeks Table 5.2. Analysed chemical composition of the experimental semi-purified diets fed to juvenile P.monodon

for 5 weeks Table 5.3. Effect of Met and choline on growth and metabolic parameters of juvenile P.monodon fed the

experimental diets during 35 days Table 5.4. Effect of Met and cystine on growth and metabolic parameters of juvenile P.monodon fed the

experimental diets during 35 days

CHAPTER 6

Table 6.1. Comparisons of CP requirements and performances between fish, terrestrial vertebrates and shrimp Table 6.2. Comparison of enzyme activities in several species and tissues Table 6.3. EAA requirement estimates in P. monodon obtained in chapters 2 and 3 Table 6.4. Comparison of activity levels of BHMT and CBS in digestive gland and muscle (mean ± standard

error) Table 6.5. Comparison of dietary composition of practical diets before and after correction for digestibility

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LIST OF FIGURES

CHAPTER 1

Fig. 1.1. Life cycle of penaeid shrimp (adapted from Bailey-Brock and Moss, 1992; Whitaker, 1999) Fig. 1.2. Evolution of the production of shrimp and prawn (× 1000 tons) (FAO, 2011b) Fig. 1.3. Evolution of the total capture production of shrimp and prawn (× 1000 tons) (FAO, 2011b) Fig. 1.4. Morphology and digestive physiology of penaeid shrimp (After Dall et al., 1990 and Ceccaldi, 1997). Fig. 1.5. Cell differentiation and function in a digestive gland tubule and the digestive process in shrimp. Fig. 1.6. Ammoniogenesis from amino acids catabolism in crustacean. Fig. 1.7. Comparison of the whole body EAA composition of fish and shrimp (Kaushik, personal data). Fig. 1.8. Differences in response criteria to determine AA requirements (Kaushik, 1986). Fig. 1.9. Linear and curvilinear regressions at different nutrient intakes. Fig. 1.10. Criteria of selection for a suitable alternative protein source Fig. 1.11. Variation in amino acid profile of some plant proteins based on the AA profile of fishmeal (base 100)

(data adapted from NRC, 1993; Sujak et al., 2006). Fig. 1.12. Methionine metabolism pathways in mammals.

CHAPTER 2

Fig. 2.1. Effect of dietary levels of protein and lysine (a) and protein and methionine (b) on daily individual nitrogen gain (mg/d) of juvenile P.monodon fed the semi-purified diets for 6 weeks.

Fig. 2.2. Linear broken line regressions (BLM) of nitrogen gain vs. nitrogen intake (a), lysine intake (b) and methionine intake (c) in juvenile P.monodon.

Fig. 2.3. Non-linear regressions obtained with the logistic model of nitrogen gain vs. nitrogen intake (a), lysine intake (b) and methionine intake (c) and their respective marginal efficiencies in juvenile P.monodon.

CHAPTER 3

Fig. 3.1. Principal component analysis of the essential amino content (% protein) of the raw materials and of the shrimp faeces (% protein).

Fig. 3.2. Effect of dietary replacement of fishmeal by plant protein (0% or 50%) and of feeding level (% normal feeding rate) on weight gain (a), survival (b), feed efficiency (c), and protein efficiency ratio (d) of P.monodon reared in cages for 49 days (Expt. 3).

CHAPTER 4

Fig. 4.1. Postprandial changes (from 0 to 6 hours after feeding) in total free amino acid levels in the hemolymph of juvenile P.monodon fed the three semi-purified diets CP10, CP30 and CP50. Values represent means with standard deviations (n=2 replicate analyses on samples pooled per treatment)

Fig. 4.2. Correlation between the quantity of nitrogen distributed (mg/g shrimp) and ammonia accumulation (mg NH3-N/g shrimp) during nine hours.

Fig. 4.3. Changes in total activity (mean ± SEM) of GDH (blue shading) and ALAT (purple dashed shading) in shrimp muscle between low/intermediate and high/intermediate protein levels.

CHAPTER 5

Fig. 5.1. Effect of dietary levels of choline, CC (A) and of cystine, Cyss (B) on daily individual N gain (mg/shrimp/day) of juvenile Penaeus monodon fed the semi-purified diets adequate (CTL) limiting (30 or 50%) in SAA (Met+Cys) during five weeks.

CHAPTER 6

Fig. 6.1. Relation between growth rate and N requirement estimates for P. monodon from studies in tanks (chapter 2) or cages (chapter 3) and for L. vannamei (recalculated from Kureshy and Davis, 2002).

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Fig. 6.2. Comparison of efficiency of digestible protein utilisation between juvenile and subadult L. vannamei and juvenile P. monodon.

Fig. 6.3. Focus on the effect of changing N intake from medium to low on N gain, marginal efficiency of N utilisation (%) and changes in GDH activity.

Fig. 6.4. Logistic regression of the Lysine intake vs. N gain and simultaneous marginal efficiency of N utilisation (chapter 2) in P. monodon (a) and rat (b) (Gahl et al., 1994).

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Chapter 1

General Introduction

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1.1. Shrimp biology and production

1.1.1. Taxonomy and biology

The black tiger shrimp (Penaeus monodon, Fabricius 1798) belongs to the largest Phylum in

the animal kingdom, the Arthropoda, characterized by the presence of paired appendages and

a protective cuticle or exoskeleton that covers the whole animal. The subphylum Crustacea is

made up of over 40.000 species, most of them aquatic. Within the class Malacostraca, the

family Penaeidae (Rafinesque, 1815) together with crayfish, lobster and crab, belong to the

order Decapoda and the suborder Dendrobranchiata (Brusca and Brusca, 1990). As other

dendrobranchiate shrimp, P. monodon is characterised by i) gills composed of a main axis

(carrying blood vessels) and two principal side filaments, ii) an external fertilisation of the

eggs, iii) the hatching of embryos as nauplii and iv) a relatively large adult size (over 30 cm

long) (Brusca and Brusca, 1990). Like most arthropods, Penaeids have an open circulatory

system composed of hemocytes and hemolymph which surrounds all cells. The copper-based

protein hemocyanin is the main constituent of hemolymph and has an important role as

oxygen carrier (Dall et al., 1990).

P. monodon is naturally found in tropical waters, on the coasts of South-East Asia, East

Africa and Australia (FAO, 2011a). During its life cycle, penaeid shrimp go through five

main life stages known as egg, larva (nauplius, protozoeal, mysis), post-larva, juvenile and

adult, which take place in different environments (from coastal estuaries to marine waters)

(Fig.1.1). Being euryhaline, P. monodon adapts easily to salinity fluctuations (Chien, 1992).

This is especially true for the juvenile stages of P. monodon, exhibiting normal

osmoregulation at salinities ranging between 3 and 50 parts per thousand (ppt) (Cheng and

Liao, 1986). Under farming conditions, salinity for optimal growth of P. monodon ranges

between 10 and 30 ppt (Liao and Murai, 1986).

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Fig. 1.1. Life cycle of penaeid shrimp (adapted from Bailey-Brock and Moss, 1992; Whitaker, 1999)

1.1.2. Pattern of growth of crustaceans

In crustaceans, tissue growth is continuous while the ‘external’ growth is discontinuous due to

series of moults (i.e., ecdysis) during which the animal casts off the old exoskeleton (i.e.,

exuvia) (Hartnoll, 1983). Simple linear growth models or models based on daily growth

coefficients (DGC) appear however appropriate to predict growth of P. monodon, at least

during the first 100-150 days of rearing (Bureau et al., 2000).

The moulting cycle is under hormonal control regulated by both stimulating and inhibiting

hormones, located in the Y-organ (in the thorax) and X-organ (or sinus gland, in the eyestalk),

respectively (Chang, 1995). Each moulting cycle comprises four main stages (pre-moult,

moult, post-moult and intermoult) distinguishable by criteria such as exoskeleton hardness or

changes in the organisation of the setae from uropods (i.e., A-B stages for post-moult, C stage

for intermoult, D stage for pre-moult and E stage for moult) (Drach, 1939, Promwikorn et al.,

2004). Several physiological changes are associated with the moulting cycle in shrimp

(Regnault, 1987, Chang, 1995, Promwikorn et al., 2004) with significant changes in the

nutritional status during the moult cycle as crustaceans build up nutrient reserves before the

moult, stop eating during the moult and start feeding again in the post-moult stage (Al-

Mohanna and Nott, 1989). For these reasons, animals in intermoult (recognised

physiologically stable) are preferentially used for nutritional studies (Cousin, 1995).

Offshore

Estuaries

Coastal

Eggs

Nauplius (5 stages)

Protozoea (3 stages)

Mysid (3 stages) Post larvae

(2 stages) Juvenile

Sub adult

Adult

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1.1.3. Shrimp production

For the past thirty years, aquaculture has rapidly increased, in particular shrimp production

with a mean annual growth rate of 18% (FAO, 2010). In 2008, shrimp farming accounted for

about 50% (Fig. 1.2 and 1.3) of the total shrimp production (FAO, 2010), with marine shrimp

such as whiteleg shrimp (Litopenaeus vannamei) and black tiger shrimp (Penaeus monodon)

representing together 3 millions tonnes or 93% of the total aquaculture production in 2008

(Fig.1.2) (FAO, 2011b).

0

500

1000

1500

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2500

3000

3500

4000

1980

1982

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1986

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1990

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1996

1998

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2002

2004

2006

2008

Tota

l aqu

acul

ture

pro

duct

ion

(x 1

000

tons

)

Others

Black tiger shrimp (P.monodon)

Whiteleg shrimp (L.vannamei)

Fig. 1.2. Evolution of the production of shrimp and prawn (× 1000 tons) (FAO, 2011b)

0

500

1000

1500

2000

2500

3000

3500

1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2008

Tota

l cap

ture

(x 1

000

tons

)

Fig. 1.3. Evolution of the total capture production of shrimp and prawn (× 1000 tons) (FAO, 2011b)

While some countries such as Madagascar managed to develop a recognisable production of

shrimp (~ 8000 tonnes in 2006) (Tacon and Metian, 2008), the Asia-Pacific region (China,

Thailand, Vietnam, Indonesia and India) remains the main producer (88%) of shrimp which

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globally accounts for around 3.2 million of tonnes (FAO, 2011b). Based on stocking density

and some other criteria, shrimp farming is categorised as extensive, semi-intensive and

intensive (Table 1.1). Although the extensive system contributed to around 50% of the world

production in 1999 (Tacon, 2002), the proportion of farms using complete feeds is expected to

increase up to 95% by 2020 (Tacon and Metian, 2008). Among the several challenges facing

shrimp farming, the development of disease-free species as well as the improvement of

domestication technology and the need for replacement of fishmeal in the feed must be

highlighted (FAO, 2011a). Regarding the latter, a better understanding of the nutritional

requirements under practical farming (including the role of natural productivity such as

microorganisms and algae) must also be emphasised to efficiently replace marine protein in

shrimp diet and maximise nutrient utilisation (Tacon, 2002).

Table 1.1. Comparison of the main shrimp farming systems (adapted from Tacon, 2002) Farming systems

Extensive System Semi-intensive system

Intensive System

Ponds size (ha) Earthen ponds:

Up to > 100 Earthen ponds:

< 1-20 Earthen ponds, raceways, tanks: 0.1-2 ha

Water exchange (%/day) Low (0-5) 5-20 High (25-100) Stocking densities (shrimp/m²) Low (< 5) 5-25 > 25 Artificial aeration None Partial or continuous Partial or continuous Fertilisation of ponds Little or none Yes Yes Complete feed Little or none Yes Yes Yield (kg shrimp/ha/year) < 1000 1000-5000 > 5000

1.2. Utilisation of dietary protein and amino acids in shrimp

1.2.1. Digestive anatomy and physiology

The anatomy and function of the digestive tract of shrimp have been described in detail by

(Dall and Moriarty, 1983) and by Ceccaldi (1997). In summary, the digestive tract in shrimp is

divided into three main compartments which are the foregut (mouth, oesophagus, stomach),

the midgut (intestine and digestive gland, also called hepatopancreas) and the hindgut (rectum

and anus) (Fig. 1.4). Nutrient digestion (enzymatic degradation) and absorption occur in the

midgut and especially in the digestive gland (Dall and Moriarty, 1983). Absorption is unlikely

to occur in the foregut or hindgut, both being covered with a cuticle layer which prevents

direct contact between cells and lumen. The overall uptake of nutrients seems fast, as labelled

food is observed in tissue one hour after feeding and completely absorbed after 4-6 hours (Dall

et al., 1990). A further specificity of shrimp is that the intestinal epithelium secretes mucus

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which first coats the chyme leaving the stomach and then serves as a building block of the

peritrophic membrane (i.e., chitin pellicula) of the faeces (Guillaume and Ceccaldi, 1999). Yet

another original aspect of crustacean digestive tract is that most reserves of the animal are

accumulated here and are utilised at each moulting cycle to build up new tissues after ecdysis.

Fig. 1.4. Morphology and digestive physiology of penaeid shrimp (After Dall et al., 1990 and Ceccaldi, 1997). Ac, anterior caecum; Af, antennal flagellum; Am, appendix masculine; Anf, antenullar flagellum; C, carapace; Cp, cardiac pocket; Dg, digestive gland; E, eyes; Ex, exopod of pereopod; In, intestine; Lo, lateral ossicules; M, mouth; Mt, medium tooth; O, oesophagus; P, pereopods; Pc, pyloric chamber; Pl, pleopod; R, rostrum; Rs, rostral spine; T, telson; U, uropods; Vo, ventral ossicules.

The process of digestion comprises several steps. Feed particles are first brought to the mouth

thanks to specific appendages (e.g., maxilla, maxillipeds, etc), before being swallowed and

sent through oesophagus where regular peristaltic contractions lead them to the cardiac pocket

of the stomach. Once in the stomach, the feed particles are ground by calcified teeth and

ossicles (constitutive of the gastric mill). Afterwards, the feed particles combined with

enzymes secreted by the digestive gland enter the pyloric chamber where the particles are

filtered, the coarser ones being sent back to the proximal part and the finer ones (few microns)

passing to the digestive gland (Guillaume and Ceccaldi, 1999). The digestive gland, which

accounts for 2 to 6% of the body mass (Ceccaldi, 1997), is composed of 2-3 lobes further

divided into ducts and tubules associated with connective tissue (Ceccaldi, 1997). Four types

of epithelial cells are distinguished in the tubule: E (Embryonalzellen), R (Restzellen),

F(Fibrenzellen) and B (Blasenzellen). It is commonly accepted that E-cells differentiate into

two lines, R-cells (reserve) and F-cells (secretion) (Fig. 1.5). The F-cells can be further

differentiated into B-cells (mature F-cells with a large vacuole). However, some controversy

exists regarding the latter type of cells, some authors suggesting they originate directly from E-

cells (Vogt, 1993). Although E cells are non-specialised, R cells are used for absorption and

storage of nutrients such as lipid droplets, glycogen and minerals, while F cells secrete

Cephalothorax Abdomen

C R

Rs

E

Anf

Af

P

Ex

P Am Pl

U

T

Ac Cp

O

M Pc Dg

Mt

Lo Vo

In

24

digestive enzymes (Dall and Moriarty, 1983, Ceccaldi, 1997, Guillaume and Ceccaldi, 1999).

The function of B-cells is subject to question, being used either for absorption and secretion

(Dall and Moriarty, 1983) or for degradation as implied by more recent work (Vogt, 1993).

Fig. 1.5. Cell differentiation and function in a digestive gland tubule and the digestive process in shrimp. Abs, absorption; D, degenerescence; Diff, differentiation; Ez, enzyme; He, hemolymph; HR, holocrine release; 1, other road for B-cell differentiation proposed by Vogt (1993).

Table 1.2. Classification of the main digestive enzymes in crustacean Types Substrates Names Organs

Zinc Astacin 1,2 Digestive gland (but stored in stomach)

Endoproteases Serine

Trypsin-like 2, 3, 4

Chymotrypsin-like 2, 3, 4, 5 Digestive gland

Exoproteases Carboxypeptidases A and B 2, 5

Aminopeptidases (leucine) 5

Dipeptidases 2 Digestive gland

1 Bond and Beynon (1995); 2 Guillaume and Ceccaldi (1999); 3 Sriket et al.(2011); 4 Van Wormhoudt (1974); 5 Vega-Villasante et al.(1995).

The enzymes involved in protein hydrolysis in crustaceans are presented in Table 1.2. As

pepsin is absent from proteolytic secretion in crustaceans (Galgani et al., 1983, Vega-

Villasante et al., 1995), protein digestion is mostly done by serine endoproteases and

exoproteases (Omondi, 2005). Trypsin-like enzyme is usually found and can represent up to

one third of the soluble protein of digestive gland (Guillaume and Ceccaldi, 1999).

Chymotrypsin-like activity is also found in shrimp digestive gland (van Wormhoudt, 1974,

Lumen

Apex

Abs

Abs

Chyme, particle

Digestion

Abs

He He

STOMACH Midgut

Enzymes

F Ez

B

F

Pinocytosis

HR

1

Diff. Diff.

R

E E

R

R

Midgut

D

25

Sriket et al., 2011). In general, enzyme activities depend on several factors such as moult

cycle (Fernández et al., 1997), growth stages (Fang and Lee, 1992; Jones et al., 1997) or

dietary composition (van Wormhoudt et al., 1980, Le Moullac et al., 1996, Guzman et al.,

2001). For instance, a decrease of the amylase to total protease ratio was observed in P.

monodon until PL stage, remaining low at juvenile and adult stages, during which

chymotrypsin activity is higher than that of trypsin (Fang and Lee, 1992). Also, Van

Wormhoudt et al. (1980) observed the highest protease activity when the shrimp Palaemon

serratus was fed a diet containing 45% protein, compared to other dietary protein levels.

1.2.2. Nitrogen uptake and utilisation in shrimp

Under natural conditions, penaeid shrimp usually feed on small invertebrates, molluscs or

crustaceans rich in protein. As a consequence, protein substrate is recognised as the major

dietary nutrient and energy substrate in shrimp (Marte, 1980, Dall et al., 1990, Rosas et al.,

1995, Rosas et al., 2002). Stable isotope methods (e.g., using 15N isotopes) have been

successfully used to measure the fate of the ingested N (Preston et al., 1996). In P. monodon,

nitrogen (N) retention is usually quite low, ranging from 20 to 35 % depending on the rearing

conditions (Burford et al., 2002, Jackson et al., 2003, Thakur and Lin, 2003). A small fraction

of N is lost as indigestible faecal N, which also contains non-protein nitrogen from the chitin

pellicula of faeces (cf. paragraph 1.2.1). Once digested, amino acids are released into the

hemolymph (mechanisms not yet clearly explored) to the target organs for protein synthesis.

The free amino acid (FAA) pool is in a dynamic status reflecting dietary supply, endogenous

protein degradation, uptake for synthesis and protein accretion in shrimp (Mente et al., 2002)

as in other animals. Despite much work and interest on protein nutrition in shrimp, available

data on FAA are however limited and summarised in Table 1.3. The FAA pool in shrimp,

mostly constituted of non-essential AA (NEAA), is found in higher concentrations than in

vertebrates (Claybrook, 1983). Whereas the FAA concentrations in shrimp muscle are around

230 µmole/g wet weight (Table 1.3), FAA levels have been reported to be ~20 µmole/g wet

quadriceps muscle in rat (Lunn et al., 1976) and around 30-40 µmole and 30-60 µmole/g wet

white muscle in rainbow trout (Oncorhynchus mykiss) and gilthead seabream (Sparus aurata),

respectively (Yamamoto et al., 2000, Gomez-Requeni et al., 2004).

26

Table 1.3. Some data on free amino acid concentrations (µM/L plasma and µM/g tissue) in different tissues of shrimp compared to that in rat

Rat a P. monodon b M. nipponense c P. japonicus d

EAA Pl M L He G DG M M M DG

ARG 55 0.2 - 48.9 1.5 9.1 34.7 29.8 55.8 9.4

HIS 80 2.0 0.9 14 0.1 1.4 0.2 5.1 1.3 1.3

ILE 119 0.08 0.3 19.4 0.1 3.2 0.2 4.1 0.7 2.6

LEU 203 0.1 0.4 36.6 0.2 8.7 0.4 8.4 1.2 6.7

LYS 658 2.0 1.8 23.7 0.1 9.5 0.3 6.1 1.9 8.6

MET 95 0.3 0.5 1.2 0 0.7 0 4.8 0.3 1

PHE 70 0.08 0.2 11.2 0 4 0.1 4.8 0.3 ** 3.6

THR 750 3.4 3.8 13.8 0.2 4.4 0.3 20.4 - 3

TRP - - - - - - - 2.1 **

VAL 270 0.3 0.5 36.9 0.2 4.1 0.4 7.6 1.6 3.7

NEAA

ALA 463 3.6 2.6 106.9 1.3 7.2 6.8 22.5 9.4 8.7

ASP 61 0.9 4.4 42.1 1.4 2.6 2.9 5.2 0.2 2.2

CIT - - - 1.8 0 0 0.1 -

CYS 32 0.2 0.1 0.5 0 1.4 0 -

GLU 26 2.1 3.9 2.7 4.5 2.9 14.6

GLN 245

3.2 6.6

228.5 1.2 4.1 4.2 - 12.3 2.7

GLY 154 2.1 1.6 89 0.8 6.1 180.8 54.8 129.6 6.9

ORN - - - 5.6 0.1 0 0.1 -

PRO 301 1.1 0.3 97.3 0.6 3.2 1.1 39.5

SER 265 1.5 1.4 46.6 0.3 3.8 0.7 8.5 1.3 3.8

TYR 128 0.2 0.2 6.6 0.1 3.7 0.2 - 1.6 4.1

ASN - - - 59.5 0.3 2.1 0.9 - 1.1 2.1

TAU - - - 276.7 11.2 17.8 2.5 - 12.5 53.1 Total FAA

3949 21.3 25.6 1193 21.8 101 239.6 228.2 233.7 138.1 a Lunn et al. (1976) (mean values over 24 hours); b Chen and Chen (2000) (after 24 hours exposure to a 0.002 mM ammonia solution); c Wang et al. (2004) (reared 14 days at 14 ppt salinity); d Marangos et al. (1989) (** is the Met+Trp concentrations). DG, digestive gland; G, gills; He, hemolymph; L, liver; M, muscle; Pl, plasma.

27

The FAA pool was found to represent between 38 and 53% of the whole protein-bound AA

pool in L. vannamei (Mente et al., 2002), while it was about 20% of the protein-bound AA

pool in muscle of Macrobrachium nipponense (Wang et al., 2004). It is generally the highest

in muscle (80-385 µmole/g fresh weight), followed by digestive gland (2-fold lower), gills

and hemolymph (2-6µmole/mL) (Claybrook, 1983). Among the FAA, glycine, alanine,

proline and arginine represent around 80% of the muscle FAA pool of P. monodon (Fang et

al., 1992), a proportion which seems to be conserved in many crustacean species (Claybrook,

1983, Fang et al., 1992). The FAA profile of the muscle mostly reflects the FAA profile of the

whole body, given the relative proportion of the muscle to the whole body mass (Claybrook,

1983). While the nutritional status (Dall et al., 1990) as well as the dietary protein source

(Mente et al., 2002) is known to influence FAA concentration in hemolymph or whole body

mass, the time course of changes occurring in the FAA pool of shrimp are little investigated.

Another point of interest with regard to the FAA pool is its possible involvement in

osmoregulation. The wide capacity for osmoregulation in euryhaline Penaeid shrimp is also

associated with changes in FAA such as glycine, alanine, proline, glutamic acid, aspartic acid

and taurine, considered to be major osmotic effectors (Dall, 1975; Dall et al., 1990; Chen and

Chen, 2000). In P. monodon, salinity (15, 30, or 45 ppt) modifies the FAA concentration but

not the relative FAA profile, being the lowest in shrimp acclimatised to 15 ppt (Fang et al.,

1992). Claybrook (1983) suggested that either excretion, incorporation into protein, or

catabolism (oxidation or non protein N compounds synthesis) was responsible for the overall

loss of FAA in hyposmotic conditions. Similar observations can also be made based on data

of Wang et al. (2004) with M. nipponense grown at different salinities ranging from 0 to 20

ppt, with the effects more pronounced in salinities above 14 ppt.

1.2.3. Ammoniogenesis and nitrogen excretion

Part of the nitrogen is also lost through branchial and urinary excretions (“BUN”). Like

teleosts, penaeid shrimp are ammoniotelic, with more than 70% of excreted nitrogen being in

the form of ammonia, followed by amino acids (< 10%), urea (1-5%) and uric acid (Regnault,

1987, Liou et al., 2005). The major part of ammonia is excreted through branchial epithelium

by passive diffusion and active ionic exchange (Regnault, 1987; Greenaway, 1991).

Nitrogen excretion in decapod crustaceans has been reviewed by Claybrook (1983) and

Regnault (1987) and is being summarized here in Fig. 1.6. Ammonia originates from the

catabolism of nitrogenous compounds such as AA (dietary and endogenous) and nucleic acids

28

(Regnault, 1987; Greenaway, 1991; Kaushik, 1998a). Like in other species, shrimp can derive

energy from the degradation of AA which are used either to produce pyruvate and acetyl CoA

or enters directly the Krebs cycle (Claybrook, 1983). AA catabolism occurs through two main

pathways known as direct deamination and transdeamination, among which only few AA

(serine and proline) can undergo the former (Regnault, 1987). Transdeamination, commonly

observed in crustaceans (Regnault, 1987), is catalysed by the aminotransferases such as

alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) and glutamate

dehydrogenase (GDH). Aminotransferases have been detected in several penaeid species

(Claybrook, 1983, Chien et al., 2003, Pan et al., 2003) with GDH being found in crustaceans

(Claybrook, 1983) at various concentrations depending on species (Batrel and Regnault,

1985; King et al., 1985) and tissues (Chaplin et al., 1970; Li et al., 2009). Aminotransferases

catalyse the transfer of the amino group to a ketoacid (resulting principally into L-glutamate

formation) (Regnault, 1987), while GDH catalyses the reversible conversion of glutamate into

ketoacid and ammonia (Greenaway, 1991). Although it was first believed that only the

reductive function of GDH (ammonia elimination) occurred in shrimp (Regnault, 1987), later

studies also demonstrated the oxidative function of GDH leading to ammoniogenesis (Batrel

and Regnault, 1985; King et al., 1985; Regnault and Batrel, 1987; Greenaway, 1991). Prior to

excretion, some enzymes are thought to play a role in ammonia detoxification (Greenaway,

1991). In crabs, high activity of both glutamine synthetase and glutaminase have been

detected (in muscle and gills, respectively), suggesting detoxification through glutamine

formation, similarly to mammals (King et al., 1985). However, GDH is considered the key

regulator of nitrogen catabolism (Bidigare and King, 1981, King et al., 1985) as its activity

accounts for most of the ammonia excretion (Batrel and Regnault, 1985).

In shrimp, like in most teleosts, environmental factors such as temperature (Regnault, 1987),

salinity (Chen and Lai, 1993) and ambient ammonia (Regnault, 1987, Chen and Nan, 1993),

but also the moult cycle (Regnault, 1979) affect ammonia excretion. Also, dietary factors,

especially dietary protein levels (Koshio et al., 1993a) are known to affect the quantity of

excreted ammonia. Despite some studies (Batrel and Regnault, 1985, Regnault, 1987, Rosas

et al., 2001a), the respective effect of such factors on the activity of enzymes involved in

ammonia formation and/or removal is less well characterised and deserves further

investigation.

29

Fig. 1.6. Ammoniogenesis from amino acids catabolism in crustacean. 1, aminotransferase enzymes (ALAT, ASAT); 2, glutamate dehydrogenase; 3, glutamine synthetase; 4, glutaminase

In teleost, nitrogen excretion can be reduced by optimising the digestible protein (DP) to

digestible energy (DE) ratio, through a decrease in DP and concomitant increase in non-

protein DE (i.e., fat or carbohydrates) (Cho and Bureau, 2001). In shrimp, fat does not appear

to be an ideal energy source due to the limited capacity for lipid utilisation and storage,

resulting in a low “protein-sparing action” of dietary fat in shrimp (Cousin, 1995, Hu et al.,

2008). In contrast, dietary starch has been reported to improve protein utilisation in P.

monodon (Shiau and Peng, 1992) or L. vannamei (Cruz-Suarez et al., 1994). However, the

protein sparing capacity through changes in DP/DE ratio appears to be species-dependent

(Rosas et al., 2001b). Precise quantitative data on the possible effects of dietary DP/DE ratio

on nitrogen excretion in shrimp is however missing. In fish, the lack of regulation of AA

catabolism in response to changes in dietary protein intake has been proposed to explain their

high protein requirements (Walton and Cowey, 1982a). Our knowledge on the capacity of

FAA hemolymph

Dietary AA Endogenous AA

glutamate

α -ketoglutarate

NH4+ + NADH

H2O + NAD+

NH4+ + glutamate

glutamine

2 3

NH4+

Mitochondria

α-ketoglutarate

α-ketoacid

glutamate

1

Intracellular FAA pool

NH3 Direct deamination

α-ketoacids

Gills

Water

glutamine

NH4+ + glutamate

NH3

4

Cytosol

30

shrimp to adjust dietary AA catabolism to changes in dietary protein levels or sources is also

limited.

1.3. Protein and amino acid requirements of shrimp

Dietary protein or essential amino acid (EAA) requirements represent the quantity of protein

or AA to be ingested to support the physiological needs of the animal (e.g., for maintenance or

maximal growth), integrating their bioavailability and efficiency of utilisation (Reeds, 2001).

1.3.1. Quantitative data on protein and amino acid requirements

Dietary protein requirements reported for the main cultured penaeid species range between 36

and to 50% diet (Table 1.4). The variation may be attributed to differences in feeding habit

between the species (Kanazawa, 1989), growth stages (Cuzon and Guillaume, 1999),

environmental factors such as salinity (Shiau and Chou, 1991; Shiau et al., 1991), feeding

levels (Kureshy and Davis, 2002, Venero et al., 2007), type of dietary protein used (Koshio et

al., 1993a, Guillaume, 1997) or the differences in methods of determination of the

requirements very much similar to what has been reported with finfish (Wilson, 2002).

As with other animal species, dietary proteins should supply penaeid shrimp with specific

amino acids (AA) in the right amounts and relative quantities. Very early, the qualitative

needs for the EAA were determined using radioactive 14C labelled individual AA in P.

serratus (Cowey and Forster, 1971) and P. monodon (Coloso and Cruz, 1980). These EAA

are Arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine

(Met), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) and valine (Val) (Kanazawa,

1989), the same 10 EAA as found in mammals and teleosts (Cuzon and Guillaume, 1999).

Two AA (cystine and tyrosine) are considered semi-essential as they can be synthesised from

methionine and phenylalanine, respectively (Cuzon and Guillaume, 1999). The use of semi-

purified experimental diets for EAA studies in shrimp has been facilitated by coating or

microencapsulating the crystalline AA prior to adding them to the diet (Chen et al., 1992;

Millamena et al., 1996a, 1996b, 1997, 1998, 1999, Alam et al., 2005), which improved the

growth of the shrimp probably by retarding the absorption of the crystalline AA (Deshimaru,

1976). Most of the EAA research for P. monodon has been performed in the 1990’s by

Millamena and co-workers using semi-purified diets (Table 1.5). By applying the dose-

response method, they found that requirements for Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp

31

and Val were similar to values obtained for finfish (Table 1.6), probably due to similarities in

the whole body AA profile between shrimp and fish (Fig. 1.7)

Table 1.4. Summary data on protein requirement estimates of different species of Penaeid shrimp (from (Shiau, 1998, Velasco et al., 2000)

Penaeus spp. Requirement % References

P. aztecus 40 Venkataramiah et al., 1975

51 Zein-Eldin and Corliss, 1976

P. californiensis 35 Colvin and Brand, 1977

P. indicus 43 Colvin, 1976

P. japonicus 50 Deshimaru and Kuroki, 1975a

52–57 Deshimaru and Yone, 1978a

45–55 Teshima and Kanazawa, 1984

P. merguiensis 34–42 Sedgwick, 1979

P. monodon 45–50 Lee, 1971

40 Alava and Lim, 1983

40–50 Bautista, 1986

40–44 Shiau et al., 1991a

36–40 Shiau and Chou, 1991

P. setiferus 28–32 Andrews et al., 1972

30 Lee and Lawrence, 1985

P. stylirostris 35 Colvin and Brand, 1977

L. vannamei 30 Colvin and Brand, 1977

> 36 Smith et al., 1985

20.2-24.5% Velasco et al., 2000

Table 1.5. Summary data on essential amino acid requirement estimates for Penaeid shrimp Requirements

Essential amino acids Species Growth stages % diet (g/16g N) protein (CP, % diet) Methods References

ARG P. monodon juvenile 2.5 5.47 45 DR Chen et al. (1992) ARG 1.85 5.3 35 DR Millamena et al. (1998) HIS 0.8 2.2 35-40 DR Millamena et al. (1999)

ILE 1.01 2.7 35-40 DR Millamena et al. (1999)

LEU 1.7 4.3 35-40 DR Millamena et al. (1999)

LYS 2.08 5.2 40 DR Millamena et al. (1998)

MET P. monodon PL20 0.89 2.4 37 DR Millamena et al. (1996a)

MET+CYS 1.3 3.5 37 DR Millamena et al. (1996a)

PHE 1.4 3.7 35-40 DR Millamena et al. (1999)

THR 1.4 3.5 40 DR Millamena et al. (1997)

TRP 0.2 0.5 35-40 DR Millamena et al. (1999)

VAL 1.35 3.4 40 DR Millamena et al. (1996b)

ARG M. japonicus juvenile 2.66 5.32 50 DR Alam et al. (2004)

ARG 1.4-1.8 2.9-3.6 50 Teshima et al. (2002)

HIS 0.5-0.7 1.1-1.4 50 Teshima et al. (2002) ILE 1.1-1.4 2.3-2.9 50 Teshima et al. (2002) LEU 1.7-2.1 3.4-4.3 50 Teshima et al. (2002) LYS M. japonicus juvenile 1.7-2.0 3.2-4.0 50 WBP Teshima et al. (2002) PHE 1.3-1.6 2.6-3.2 50 Teshima et al. (2002) MET 0.6-0.8 1.3-1.6 50 Teshima et al. (2002) THR 1.1-1.4 2.3-2.9 50 Teshima et al. (2002) TRP 0.3-0.4 0.6-0.8 50 Teshima et al. (2002) VAL 1.2-1.5 2.4-3.1 50 Teshima et al. (2002) 4.5 35 Fox et al. (1995)

LYS L.vannamei juvenile 5.2 DR Fox et al. (1995)

4.7 45 Fox et al. (1995)

THR L. vannamei juveniles 1.4 3.8 36 DR Huai et al. (2009)

DR, dose-response; WBP, whole body protein

Table 1.6. Requirements for several fish species (NRC, 1993)

ARG HIS ILE LEU LYS MET +CYS

PHE +TYR THR TRP VAL

Rainbow Trout %DM 1.5 0.7 0.9 1.4 1.8 1.0 1.8 0.8 0.2 1.2

% CP 3.9 1.8 2.4 3.7 4.7 2.6 4.7 2.1 0.5 3.2

% DP 4.4 2.1 2.6 4.1 5.3 2.9 5.3 2.4 0.6 3.5

Pacific Salmon %DM 2.0 0.6 0.8 1.3 1.7 1.4 1.7 0.8 0.2 1.1

% CP 5.4 1.6 2.0 3.5 4.5 3.6 4.6 2.0 0.4 2.9

% DP 6.0 1.8 2.2 3.9 5.0 4.0 5.1 2.2 0.5 3.2

Common Carp %DM 1.3 0.6 0.8 1.0 1.7 0.9 2.0 1.2 0.2 1.1

% CP 3.7 1.8 2.2 2.9 5.0 2.7 5.7 3.4 0.7 3.1

% DP 4.3 2.1 2.5 3.3 5.7 3.1 6.5 3.9 0.8 3.6

Tilapia %DM 1.2 0.5 0.9 1.0 1.4 0.9 1.6 1.0 0.3 0.8

% CP 3.7 1.5 2.7 3.0 4.5 2.8 4.8 3.3 0.9 2.4

% DP 4.2 1.7 3.1 3.4 5.1 3.2 5.5 3.8 1.0 2.8 CP, crude protein; DM, dry matter; DP, digestible protein

0

2

4

6

8

10

12

Arg His Ile Leu Lys Thr Trp Val SAA Phe+Tyr

Fish

Shrimp

g/16gN

Fig. 1.7. Comparison of the whole body EAA composition of fish and shrimp (Kaushik, personal data).

1.3.2. Maintenance and growth requirements

The maintenance requirement for protein represents the point of N balance (Moughan,

2003), which is reached when N intake equals obligatory N losses. In aquatic animals,

the main components of obligatory N losses are the branchial and urinary excretion of

AA, the synthesis of non-protein N compounds (e.g., carnitine, glutathione) and the AA

breakdown for e.g. glucose or energy production (Rollin, 1999). In fish, maintenance

requirement is usually estimated by measuring the amount of endogenous N excretion

(fecal, urinary and branchial losses) in starved animals or, more indirectly, by measuring

N retention (N consumed/N retained in the body) at different N intake levels (Wilson,

34

2002). In crustaceans, only few studies have investigated the level of maintenance using

direct (Koshio et al., 1993b) or indirect measurement (Kureshy and Davis, 2002).

In growing animals, the maintenance fraction fluctuates between 10% and 20-30% of the

total N requirement, being higher at low than high protein deposition (Rollin, 1999,

Carter and Hauler, 2011). There are also inter-species differences in the relative

proportion between N required for maintenance and growth (Fournier et al., 2002). The

N requirement for growth (above maintenance) is a function of the productive status of

the animal. It is composed of the AA requirement for protein accretion, the use of AA

for synthesis of non-protein compounds needed during growth, the preferential and the

inevitable AA catabolism, the latter being defined as the degradation of AA when both

protein and energy levels are adequate (Moughan, 2003). In fish as in mammals, several

questions persist regarding the ‘inevitable’ relative to the ‘preferential’ AA catabolism

and the possible dietary regulation by the amount and/or type of AA ingested (Rollin,

1999). In shrimp, to our knowledge, no information is available regarding the relative

contribution of these different allocations to growth requirement or on the marginal

efficiency of N or AA utilisation for growth.

1.3.3. Methodological issues related to estimation of AA requirements in

shrimp

1.3.3.1. Dose-response approach

Most of the requirement studies in shrimp are based on the dose response approach, by

feeding diets containing graded levels of the studied AA while measuring a biological

response such as nitrogen (or AA) accretion and body growth or direct or indirect AA

oxidation (Fig. 1.8).

Fig. 1.8. Differences in response criteria to determine AA requirements (Kaushik, 1986).

Type III: Oxidation of tracer AA

Type II : Plasma FAA

Type I : Growth, N gain

AA intake

Response

Requirement

35

Despite the extensive utilisation of the dose-response approach, several limitations have

been pointed out in all animals including fish (Rollin, 1999, Rollin et al., 2003), the

most important being the variation in the EAA profile between diets. To prevent this

problem, Gous and Morris (1985) proposed the ‘diet dilution’ technique, in which diets

are prepared by mixing a summit diet (with 180% of the required level of all AA but the

one studied) with different proportions of a protein-free dilution diet (Gous and Morris,

1985).

For analysing response data, several linear or non-linear regression models can be used

(Robbins et al., 2006). In studies with shrimp, both broken line (Millamena et al.,

1996a, 1998; Kureshy and Davis, 2002; Huai et al., 2009) and quadratic regressions

(Millamena et al., 1997, 1999) have been used for the evaluation of AA and protein

requirements. Linear models, such as broken-line regression, rely on the assumption

that the animal responds to graded intake levels with a constant efficiency until a

maximal growth level is reached after which efficiency becomes instantaneously zero.

Such an assumption of a constant marginal efficiency has been criticised from a

physiological point of view (Mercer et al., 1978, 1982), suggesting that curvilinear

regressions would be more adequate, like for enzyme kinetics or AA degradation (Fuller

and Garthwaite, 1993; Gahl et al., 1994). Several such non-linear kinetic models have

been proposed, such as the four-parameter saturation kinetic model (Mercer et al.,

1978), exponential model (Fuller and Garthwaite, 1993) or logistic model (Gahl et al.,

1991, 1994, 1996). Using the saturation kinetic or logistic model, distinction can be

made between intake for maximal efficiency of utilisation and that at which the animal

is the most sensitive to nutrient changes (Mercer, 1982). In parallel, the logistic model

integrates the law of diminishing returns, defined as the continuous decrease in response

as intakes approach the requirement for maximal response (Gahl et al., 1991). However,

none of these regressions appears completely satisfactory to describe a biological

response over nutrient intakes varying from zero to excess. This was illustrated in pigs

(Fig. 1.9), for which using N intake and N gain, three intake zones could be

distinguished and fitted with different models (Fuller and Garthwaite, 1993). These are

i) zone 1 (from 0 to the maintenance intake) where the efficiency of the limiting AA is

considered constant and close to 1. When using N gain as the response criterion, this

might not be true if a shift occurs in the first limiting AA between maintenance and

growth (Fuller and Garthwaite, 1993); ii) zone 2 (above maintenance) where the animal

response is mostly linear and depends on protein quality; iii) zone 3 (at high intakes)

36

where efficiency decreases progressively following the law of diminishing returns (grey

circle).

In addition, requirement estimates are influenced by the choice of response criterion or

parameter (body mass gain, N gain or AA gain) and by the number of experimental data

available throughout the range of intakes (Gahl et al., 1994; Shearer, 2000). Also, a non-

linear regression leads to an asymptotical approach of the maximum response with the

choice for maximum criteria (90, 95% or more of the asymptote) influencing the

estimation of the requirement (Rodehutscord et al., 1995a). In fish, the saturation kinetic

model (Mambrini and Kaushik, 1995a; Fournier et al., 2002; Bodin et al., 2009) or

exponential (Rodehutscord et al., 1995a, 1995b, 1997) have been used to evaluate N or

AA requirements. Similarly to previous work on pigs (Heger et al., 2002, 2003, 2008),

constant efficiency of AA utilisation was found in fish between maintenance and almost

maximum growth using AA gain as response (Hauler and Carter, 2001a; Rollin et al.,

2003; Abboudi et al., 2006, 2007; Bodin et al., 2008; Carter and Hauler, 2011). It

follows from the above that the choice of the regression model must be related to the

question addressed in an experiment, regardless of the species considered.

Fig. 1.9. Linear and curvilinear regressions at different nutrient intakes. M, maintenance requirement; XR, requirement for maximal growth; 1, 2, 3, the different intake zones.

1.3.3.2. Factorial approach

The factorial approach was designed to integrate the physiological process of

maintenance and growth into the estimation of requirement (Shearer, 1995). This

1 3 2

asymptote

Maximal

XR M

asymptote

0

0

0

37

approach has been used in fish (Shearer, 1995; Lupatsch et al., 2001; Bodin et al., 2008)

and crustaceans (Teshima et al., 2001, 2006; Tzafrir-Prag et al., 2010).

In mammals, an alternative was proposed by Fuller et al. (1989) to determine

maintenance and growth requirements simultaneously for both protein and AA. The

method originated from the difference observed between the AA pattern required for

maintenance and for protein accretion. Fuller et al. (1989) considered that the total

requirement was thus affected by a change in the relative contribution of one of these

components. In this approach, the authors also assumed that the relationship between N

(or AA) intake and N retention was linear. The maintenance requirement was then

calculated as the intake at which N retention was equal to zero (zero N balance), while

requirement for growth (for 1g protein accretion) was estimated from the reciprocal of

the regression coefficient (1/a). This method has been successfully applied in fish

(Gatlin et al., 1986; Mambrini and Kaushik, 1995a; Fournier et al., 2002; Hauler and

Carter, 2001a, 2007, 2011).

1.3.3.3. Ideal protein profile

Another alternative to the dose-response approach for determining individual EAA

requirements relies on the ideal protein concept. The ideal protein is defined as an AA

profile where all the EAA are equally limiting (for both maintenance and growth) so

that EAA are required in constant proportion of protein (Boisen et al., 2000). Very

early, it was shown that the whole body protein-bound AA composition is stable and

does not change much between different species of fish (Mambrini and Kaushik, 1995b)

and that there is a good correlation between the whole body EAA composition and that

of the EAA requirement pattern. It is generally assumed that the whole body EAA

profile can be considered as the ideal protein for fish (Mohanty and Kaushik, 1991,

Kaushik, 1998b; Furuya et al., 2004; Tibaldi and Kaushik, 2005) and shrimp

(Peñaflorida, 1989; Alam et al., 2002; Teshima et al., 2002). A comparison of data on

whole body protein-bound AA profiles of different shrimp (Table 1.7) shows much

homogeneity and there is also much similarity between the AA profiles of fish and

shrimp (Fig. 1.7). The application of the ideal profile concept however necessitates

quantitative data on the requirement for at least one EAA (usually lysine), all the others

being calculated relative to the ideal EAA profile. In pigs, the ideal protein was

determined by linear regression between AA intake and N retention, assuming that N

retention was reduced by the first limiting AA (Wang and Fuller, 1989). Moreover, this

38

approach considers that the efficiency of utilisation of the first limiting EAA decreases

with increasing dietary protein content, due to an overall increase in AA catabolism

(Cowey and Cho, 1993). This implies that a perfect balance between dietary AA must

be supplied in the dietary protein source (cf. paragraph 1.3.3.5.) in order to achieve an

optimal protein growth.

Table 1.7. Protein-bound amino acid concentrations in different tissues of shrimp (g/100 dry tissue)

EAA M. nipponense a

(muscle) M. japonicus b (whole body)

P. monodon c (whole body)

ARG 4.0 3.5 3.6 3.7 3.2 3.8 2.7 3.4 3.2

HIS 1.4 1.1 1.2 1.5 1.1 1.5 1.0 1.3 1.0

ILE 2.6 1.2 1.4 1.2 1.3 1.4 1.2 1.2 1.8

LEU 4.9 2.4 2.6 2.3 2.4 2.6 2.1 2.3 2.9

LYS 5.2 2.7 3.1 2.8 2.7 3.0 2.4 2.8 3.0

MET 0.5 0.8 0.9 0.7 0.8 0.8 0.7 0.7 1.0

PHE 2.6 2.2 2.1 2.0 2.0 2.2 1.8 1.9 1.7

THR 2.0 1.4 1.5 1.4 1.4 1.4 1.2 1.3 1.6

TRP 2.4 0.5 0.6 0.6 0.4 0.5 0.4 0.3 0.4

VAL 2.7 1.5 1.4 1.3 1.4 1.4 1.3 1.3 2.0

NEAA ALA 3.6 2.2 2.6 2.2 2.1 2.1 1.8 2.4 2.4

ASP 7.6 3.3 3.6 3.4 3.4 3.6 3.0 3.4 4.0

GLU 12.1 6.4 7.0 6.3 6.4 6.8 5.8 6.5 6.4

GLY 2.9 4.0 4.7 3.6 3.6 4.6 3.8 3.8 3.2

PRO 0.3 2.5 2.8 2.3 2.6 2.3 1.9 2.2 1.6

SER 2.7 1.3 1.4 1.2 1.3 1.3 1.2 1.3 1.6

TYR - 2.2 2.0 2.3 2.0 2.2 2.0 1.9 1.6 a Wang et al. (2004) (reared at 14 ppt salinity); b Alam et al. (2002); c Millamena et al. (1998)

1.3.3.4. Variation of dietary nutrient density

Since shrimp culture takes place in natural ponds, the optimal daily supply of protein is

a major decision tool for farm management (Venero et al., 2007). As inappropriate

protein quality or quantity supplies can lead to environmental loads in terms of N and

increase production costs, estimation of requirement was approached using different

dietary protein contents at suboptimal feeding levels (Kureshy and Davis, 2002; Venero

et al., 2007). Both studies showed that feed allowance could be reduced by using more

dense diets (high levels of protein), as suggested for fish (Cho and Bureau, 2001).

1.3.3.5. Mode of expression of data on requirements

Protein or AA requirements for shrimp are expressed in relative (% diet, % protein)

(Millamena et al., 1996a, 1996b, 1997, 1998, 1999) or absolute terms (g/kg body weight

39

per day, g/shrimp per day) (Teshima et al., 2001; Kureshy and Davis, 2002; Teshima et

al., 2006). Expressing requirement as a percentage of the diet assumes that the dietary

protein content has no effect on the efficiency of utilisation of the first limiting AA, or

inevitable AA loss. A series of studies with rainbow trout (Encarnacao et al., 2004,

2006) have suggested that non-protein energy sources affect efficiency of lysine

utilisation, but not the lysine requirement for growth. However, in a more recent work,

Carter and Hauler (2011) did not find any difference in lysine utilisation in Atlantic

salmon as affected by dietary DP/DE ratios.

In fish, the expression of AA requirements related to dietary protein is based on the

assumption that the efficiency of utilisation of the first limiting AA decreases with

increasing protein levels, or that the inevitable AA loss is related to the overall AA

catabolism (Cowey and Cho, 1993). However, recent results in fish indicate similar

efficiency of lysine utilisation when fed either a low or a high protein diet (Bureau and

Encarnacao, 2006; Bodin et al., 2009).

To reduce inter-studies variation, Hauler and Carter (2001b) recommended expressing

AA requirement as g AA per g gain. Also, the authors suggested using preferentially

AA or protein gain to correct for the change in whole body composition. Regarding

shrimp, the latter concern might not be as important as in fish since the lipid content of

the shrimp carcass stays more or less constant over time due to limited storage capacity

(cf. paragraph 1.2.3).

1.3.3.6. Bioavailability of amino acids

Meeting the animal’s requirement for EAA through dietary protein sources requires

precise knowledge on the biological / nutritional value of the protein source. In shrimp,

the digestibility of proteins or the availability of AA has been estimated both in vitro

(Lemos et al., 2004, 2009) and in vivo. The in vitro technique, which uses enzyme

extracts of the shrimp digestive gland (Ezquerra et al., 1997) allows a rapid screening of

protein digestibility of the ingredients and has been found to reflect in vivo digestibility

values (Lemos et al., 2009). In the in vivo approach, digestibility is measured either

gravimetrically or indirectly by incorporation of an indigestible, physiologically inert

marker (e.g., chromic oxide, ytterbium, acid-insoluble ash) into the feed, the indirect

method being recommended for shrimp (Smith and Tabrett, 2004) as in the case of

finfish. Besides being inert and indigestible, the marker must be non toxic and

40

transported through the gut at the same rate as the food digesta (Smith and Tabrett,

2004). Chromic oxide is the most commonly used marker in digestibility studies with

shrimp (Smith and Tabrett, 2004; Cruz-Suarez et al., 2001, 2007, 2009). Although

apparent digestibility values in shrimp have been found to be little influenced by the type

of marker (Deering et al., 1996; Smith and Tabrett, 2004), the use of acid-insoluble ash

(AIA) has some advantages such as low cost, easy to handle and analyse, favouring its

utilisation (Goddard and McLean, 2001). Information on protein and AA availabilities in

shrimp is extremely limited (Lemos et al., 2009; Yang et al., 2009), despite the

demonstration of the considerable variation in nutrient digestibility between protein

sources (Akiyama et al., 1989; Brunson et al., 1997; Yang et al., 2009).

1.4. Protein sources in shrimp feed: fish meal and alternatives to

fish meal

1.4.1. Fishmeal replacement and utilisation of plant protein sources

Traditionally, formulated feed for shrimp contain high levels of fishmeal (FM) ranging

from 25 to 50% of the diet (Amaya et al., 2007). From a recent global survey on the use

of FM in feeds for farmed finfish and shrimp (Tacon and Metian, 2008), it appears that

in 2010, between 820 to 860 thousand tons of FM is used for marine shrimp farming,

representing about 27% of the global FM production. With increasing intensification

and the increased reliance on formulated feeds for shrimp farming which is expected to

increase by 72% in 2020, there is a prospective increase in demand for FM (Tacon and

Metian, 2008). There is hence an urgent need to look for reliable alternative protein

sources for shrimp production. A viable alternative protein source must fulfil several

requirements (dashed zone, Fig. 1.10), being i) nutritionally suitable for the animal

(digestible, adequate nutrient profile, low anti-nutritional factors, etc), ii) economically

advantageous (competitive and steady price, ready availability, easy to handle, store

and use), iii) environmental friendly (minimal pollution, limiting ecosystem stress), iv)

and acceptable by the consumer (origin of product, human health) (Naylor et al., 2009).

41

Fig. 1.10. Criteria of selection for a suitable alternative protein source

Of the different protein sources, protein-rich ingredients which are of interest for

inclusion in feeds for finfish or shrimp are mostly obtained from animal products of

marine (fishmeal, shrimp meal, krill meal, etc) or terrestrial (meat and bone meal,

hydrolysed feather meal, poultry by-product meal, blood meal) origin. Although poultry

by-products appear to hold much promise in L. vannamei (Davis and Arnold, 2000), due

to societal pressure as well as legal restrictions in the use of terrestrial animal proteins

(Naylor et al., 2009), plant proteins constitute an ideal alternative for the replacement of

FM.

Table 1.8. Plant proteins commonly used in animal feeds and the associated ANFs (Francis et al., 2001; Venero et al., 2008a) Protein sources Plant ingredients Anti-nutritional factors

Oilseeds

Soybean meal Cottonseed meal Rapeseed/canola meal Peanut meal Sunflower meal Linseed meal Sesame meal

PI, LEC, PA, SAP, POE, AV, ALL PA, POE, GOS, AV, CA PI, GLUC, PA, TAN PI, SAP, AI PA, PI

Leguminous seeds Lupins Field/feed pea meal Cowpea

PI, SAP, POE, ALK PI, LEC, TAN, CY, PA, SAP, AV

Leguminous leaf meals

Leucaena leucocephala Alfalfa

MIM PI, SAP, POE, AV

By-products of the brewery industry

Distillers’ dried grains Distillers’ dried grains with solubles

Protein isolates or concentrates

Corn gluten meal Wheat gluten Soy, canola, potato and pea protein concentrates

AI, arginase inhibitor; ALL, allergens; ALK, alkaloids; ANFs, antinutritional factors; AV, antivitamins; CA, cyclopropenoic acid; CY, cyanogens; LEC, lectins; GLUC, glucosinolates; GOS, gossypol; MIM, mimosine; PA, phytic acid; PI, protease inhibitor; POE, phytoestrogens; SAP, saponins; TAN, tannins.

Nutrition Economics

Consumer Environment

42

As with other animals, diverse plant protein sources are already being used in the feeds

for shrimp. Soybean meal, lupin and pea have been largely investigated as potential

fishmeal replacers in L. vannamei and P. monodon, reaching successful growth with

only 17.5% (Paripatananont et al., 2001), 14% (Smith et al., 2007a) or 6% fishmeal

(Sudaryono et al., 1999). Even total fishmeal replacement was achieved using soybean

meal for L. vannamei reared in ponds (Amaya et al., 2007). As highlighted by the latter

authors, most of the research has been however conducted under controlled conditions,

which makes it difficult to transfer to the shrimp industry. Further replacement studies

should be, thus, conducted under conditions mimicking those of commercial farms.

1.4.2. Limitations to plant protein incorporation

A major issue with the use of plant protein sources is that linked with the presence of

different antinutritional factors (Table 1.8) known to affect negatively animal growth by

hampering i) protein digestion (protease inhibitor, tannins, lectins) or ii) mineral

utilisation (phytic acid, gossypol, glucosinolates) (Lim, 1996), iii) by acting as

antivitamins or iv) affecting palatability (alkaloids, saponins). However, physical

treatments (dehulling, autoclaving, soaking), by reducing or eliminating antinutritional

factors (Cruz-Suarez et al., 2001; Kumaraguru Vasagam et al., 2007; Kaushik and

Hemre, 2008), or enzyme addition (Buchanan et al., 1997) can improve the nutritional

value of plant proteins. Further studies on the effect of antinutritional factors on protein

digestibility are however needed. Secondly, palatability may be low with high levels of

plant protein, decreasing feed intake (Lim et al., 1997; Molina-Poveda and Morales,

2004) as seen in fish (Espe et al., 2007). Heat treatment or micronisation may improve

palatability through the removal of inhibiting substances (Venero et al., 2008a). Thirdly,

the EAA profile of plant proteins is often less suitable than that of marine protein

(Peñaflorida, 1989; Venero et al., 2008a). While some amino acids such as cystine and

leucine appear in larger proportions in plant proteins than in fishmeal (Fig. 1.11), some

others are clearly limiting, among which methionine, lysine, threonine and arginine can

be distinguished (Venero et al., 2008a). Several studies have indicated that

supplementation of diet with crystalline lysine improved performances of shrimp fed

plant proteins deficient in lysine (Forster et al., 2002; Biswas et al., 2007). Also, in

order to improve the EAA profile of the diet, combinations of several proteins can be

used, as suggested for fish (Regost et al., 1999; Fournier et al., 2004; Kaushik et al.,

2004). In fish, the incorporation of a mixture of EAA balanced plant proteins allowed to

reduce fishmeal incorporation to 10% (Fournier et al., 2004) or even 5% (Kaushik et al.,

43

2004; Espe et al., 2006). Although similar conclusions were reached for L. vannamei

(Molina-Poveda and Morales, 2004), further investigation on the utilisation of plant

proteins by shrimp is required in order to replace more dietary fishmeal and especially

in P. monodon diets.

0

50

100

150

200

250

Arg His Ile Leu Lys Met Cys Phe Tyr Thr Trp Val

Sorghum Rapeseed meal Soybean meal Lupin meal Corn gluten meal

Fig. 1.11. Variation in AA profile (in % of dry ing redient) of some plant proteins based on the AA profile of fishmeal (100 baseline) (data adapted from NRC, 1993; Sujak et al., 2006).

1.4.3. Limiting amino acids: specific role of sulphur amino acids (SAA)

As mentioned above, the use of plant protein sources involves possible deficiencies in

one or more EAA. While lysine is an EAA involved mainly with protein synthesis and

protein growth in a direct manner, methionine (Met) has a number of physiological

roles. Besides its role as an EAA in protein synthesis, methionine serves as a precursor

of several sulphur-containing compounds and as a methyl group donor (Finkelstein et

al., 1988).

The former function enables the synthesis of the amino acid cysteine (Cys), considered

as semi-essential. The capacity for biosynthesis of cysteine reported in crustaceans

(Claybrook, 1983) is believed to follow the same pathways (transmethylation and

transsulfuration) as in vertebrates for which SAA has been well described by

Finkelstein and Martin (1984) (Fig 1.12). In summary, methionine is converted to S-

adenosylmethionine (SAM) which is further methylated into homocysteine (Hcy) by

transmethylation reactions. Hcy is at the branch point of the Met metabolism because it

can either by transsulfurated irreversibly into cysteine (through cystathionine gamma-

Fishmeal Basis

44

lyase and cystathionine beta-synthase, CBS) or remethylated into methionine by the

betaine-homocysteine methyltransferase (BHMT) or the folate-vitamin B12-dependent

Met synthase (MS). In vertebrates, cysteine can be either oxidised or used for protein,

glutathione or taurine synthesis formed mostly through cysteine sulfinic acid and

regulated by the cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase

(CSD) enzymes (Jacobsen and Smith, 1968). In invertebrates such as shrimp, taurine is

an important osmoregulator (Claybrook, 1983) but conflicting literature exists regarding

its capacity of biosynthesis (Smith et al., 1987, Shiau and Chou, 1994). The Met-sparing

effect of cyst(e)ine has been clearly demonstrated in fish (Walton and Cowey, 1982b;

Kim et al., 1992) but has never been investigated for shrimp species despite the fact that

increasing dietary inclusion levels of plant proteins will modify the Met/Cys ratios of

shrimp diets (cf. paragraph 1.4.2).

The second major metabolic function of Met is to provide methyl groups through its

conversion into SAM, the major methylating agent in biological processes such as DNA

methylation (Tesseraud et al., 2009) or synthesis of phospholipids (PL) such as

phosphatidylcholine (PC) (Michael et al., 2006). Besides its role as a constituent of PC,

choline is also a precursor of the neurotransmitter acetylcholine and betaine (Simon,

1999). Betaine can be used as a methyl source for remethylation of Hcy through a

specific pathway, the BHMT pathway (Fig. 1.12). In shrimp, a dietary essentiality of

choline has been demonstrated only in some species (Shiau, 1998) such as P. japonicus

(Kanazawa et al., 1976) and P. monodon (Shiau and Lo, 2001). While interaction

between dietary Met and choline has been observed in fish (Kasper et al., 2000; Wu and

Davis, 2005) as well as in shrimp (Michael et al., 2006), the possible sparing of choline

by betaine has been demonstrated only in fish (Kasper et al., 2002; Wu and Davis,

2005). Since betaine is often used in shrimp diets to improve palatability (Penaflorida

and Virtanen, 1996; Saoud and Davis, 2005), a better understanding of the interaction

between dietary supplies of Met, choline and betaine on the growth response, together

with a characterisation of enzymes involved in methionine metabolism, may help to

improve formulations of shrimp feed.

45

Fig. 1.12. Methionine metabolism pathways in mammals. BHMT: Betaine Homocysteine Methyltransferase; CBS: Cystathionine beta synthase; CγL: Cystathionine gamma lyase; CDO: Cysteine dioxygenase; CSD: Cysteine sulfinate decarboxylase; Cys: cysteine; Cyss: cystine; Hcy: Homocysteine; Met: Methionine; MS: Methionine Synthase. The light blue circles represent the transmethylation enzymes; the blue green, the remethylating enzymes; the purple, the transsulfurating enzymes; the dark blue, the cysteine degrading enzymes.

MS

DMG

BHMT

Betaine aldehyde

BETAINE

CHOLINE

N5,N10-methyltetrahydrofolate

N5- methyltetrahydrofolate

Tetrahydrofolate

S-adenosylméthionine (SAM)

S-adenosylhomocystéine (SAH)

HOMOCYSTEINE

METHIONINE (Met)

Cystine (Cyss) Glutathion

Oxidation

Cysteine sulfinic acid

CDO

Cysteic acid

TAURINE

Sulfite

β-sulfinly pyruvic acid

CYSTEINE (Cys)

Cystathionine

CBS sérine

Cγγγγ L

Cysteamine

Hypotaurine

CSD

Pyruvate sulfate

46

1.5. Objectives

Due to economic and sustainability considerations, formulated feed for shrimp rely more

and more on the use of alternatives to fish meal, such as plant protein sources which are

often limiting in lysine and methionine. For shrimp, information is rather scarce on the

availability of AA from alternative proteins and on the regulation of AA metabolism by

dietary factors. A better understanding is needed to optimise feed formulations and feed

management in shrimp farms and thus the sustainability of shrimp production.

Therefore, the aims of this thesis are i) to study the potential reduction of fishmeal as

protein source in diets for shrimp P.monodon and ii) to improve our understanding of

AA utilisation when supplied at imbalanced levels through inappropriate protein and/or

AA dietary concentrations.

Beginning with a literature overview of protein and AA nutrition of shrimp (Chapter 1),

the requirements as well as utilisation efficiency for protein, lysine and methionine were

assessed in P. monodon using semi-purified diets (Chapter 2). The next chapter

compares the long-term growth performances of P. monodon under semi-intensive

commercial conditions fed practical diets in which fishmeal is gradually replaced by a

mixture of plant proteins, with specific attention to possible changes in protein and AA

availability (Chapter 3). In a next step, the ability of P. monodon to adapt its AA

catabolism to dietary changes in protein quantity or quality (EAA profile) was

investigated (Chapter 4). To predict the possible effects of a low methionine/high

cystine supply brought by plant protein, sulphur AA metabolism was studied in P.

monodon fed semi-purified diets in view of a possible methionine-sparing effect of

cystine and choline (Chapter 5). Finally, the results are discussed in Chapter 6 before

proposing some perspectives for further research on shrimp nutrition (Chapter 7).

47

Chapter 2

Determination of requirements and efficiency of utilisation of essential amino acids and nitrogen

in juvenile Penaeus monodon

**** Détermination des besoins en acides aminés essentiels

et azote, et de leur efficacité d’utilisation chez les juvéniles P. monodon

48

49

2.1. Présentation de l’article

Pour améliorer les niveaux de remplacement de la farine de poisson par des sources

protéiques alternatives, et donc afin d’améliorer les formulations alimentaires

appliquées aux crevettes d’élevage, il est nécessaire de mieux définir les besoins

nutritionnels de l’animal. Dans cette étude, l’objectif principal était donc d’estimer les

besoins en protéine, lysine et methionine chez des juvéniles de crevette Penaeus

monodon. Pour ce faire, nous avons utilisé une approche factorielle en appliquant la

technique de la « suppression d’AAE » (EAA deletion technique), permettant d’estimer

simultanément le besoin en protéine et en AAE. De plus, grâce à ce design

expérimental, nous pouvons étudier l’interaction éventuelle entre l’apport protéique et

l’apport en AAE chez la crevette.

Les juvéniles (poids moyen initial, PMI, de 2.4g) ont été nourris pendant six semaines

avec un des dix aliments semi-purifiés (NP, LP, LPL, LPM, MP, MPL, MPM, HP,

HPL, HPM). L’azote alimentaire est fourni par de la caséine et un mélange d’acides

aminés cristallins (le ratio caséine/AA cristallins est égal à 55/45). Les aliments ont été

formulés pour apporter quatre niveaux différents de protéine brute (5, 14, 34, et 54% de

l’aliment sec), ainsi que deux niveaux en lysine et méthionine (adéquat ou 30%

déficitaire, exprimé en pourcentage de protéine brute, PB). Dans tous les aliments, le

ratio cystine/méthionine est maintenu constant (0.3). Les besoins ont été estimés en

étudiant la relation entre l’ingéré de protéine/lysine/méthionine (g/kg poids vif (PV) par

jour) et l’accrétion azotée (gain N, en g/kg PV/jour), à l’aide de modèles de régression

linéaire (Broken-line) et non linéaire (quadratic, logistic, Mercer).

Cette étude est la première à fournir les besoins d’entretien pour les protéines brutes

(PB), la lysine et la méthionine, chez l’espèce P. monodon. Basés sur la régression

logistique, nos résultats indiquent que le besoin est de 4.5 g de PB/kg PV/jour,

représentant 19% du besoin pour la croissance maximale (23.9 g/kg PV/jour). Pour la

lysine et la méthionine, les besoins d’entretien sont de 0.2 et 0.1 g /kg PV/jour,

respectivement, ce qui représente 14-17% du besoin de croissance maximale (1.4 et 0.7

g/kg PV/jour, respectivement). Exprimés en terme relatif (% PB), les besoins en lysine

et méthionine pour la croissance maximale sont de 5.8% et 2.9%. Ces valeurs sont

semblables à celles obtenues dans des études antérieures sur des post-larves de P.

monodon. L’efficacité d’utilisation marginale (au dessus de l’entretien) pour l’accrétion

azotée atteint un maximum de 38%, 0.77, et 1.62 pour l’azote, la lysine et la

méthionine, respectivement. La loi des rendements décroissants est observée à partir

d’un ingéré représentant 40% de celui nécessaire pour la croissance maximale. Enfin,

50

nos résultats indiquent une interaction significative entre la teneur alimentaire en

protéine brute et méthionine sur l’accrétion azotée. Ainsi, les besoins en acides aminés,

ne peuvent être exprimés en pourcentage de protéine brute, qu’à des ingérés protéiques

inférieurs à celui nécessaire pour la croissance maximale ; ceci suggérant d’exprimer les

besoins en termes absolus (g/kg poids vif par jour).

51

Maintenance and growth requirements for nitrogen, lysine

and methionine and their utilisation efficiencies in juvenile

black tiger shrimp, Penaeus monodon

using a factorial approach

Lenaïg Richard

Pierre-Philippe Blanc

Vincent Rigolet

Sadasivam J. Kaushik

Inge Geurden

Published in: British Journal of Nutrition (2010), 103: 984-995

52

2.2. Abstract

We used a factorial approach to distinguish maintenance from growth requirements for

protein, lysine and methionine in the black tiger shrimp, Penaeus monodon. Juvenile P.

monodon (initial weight 2.4 g) were fed during six weeks one of ten semi-purified diets

based on casein and purified amino acids (AA) as nitrogen (N) source. The diets

contained four levels of crude protein (CP, from 5 to 54% diet dry matter) with two

levels (%CP) of lysine or methionine (normal or 30% deficient). Requirements were

determined using linear and non-linear regression models. We could thus obtain the first

ever data on maintenance (N equilibrium) requirements for CP and AA in P. monodon.

CP requirements for maintenance (4.5 g/kg body weight (BW) per d) represented

approximately 19% of the CP requirement for maximal N gain (23.9 g/kg BW per d).

The marginal efficiency of utilisation reached a maximum of 38% for N, 0.77 for lysine

and 1.62 for methionine using N gain as response. Lysine requirements were 0.20 g/kg

BW per d for N maintenance and 1.40 g/kg BW per d for maximal N gain. Methionine

requirements were 0.11 g/kg BW per d for N maintenance and 0.70 g/kg BW per d for

maximal N gain. The lysine (5.8%) and methionine (2.9%) requirements for maximal N

gain, expressed as % of protein requirement, agree with literature data using a dose-

response technique with smaller P. monodon. The observed interaction between dietary

CP and methionine for N gain demonstrates that requirements for essential AA

(expressed as %CP) cannot be evaluated separately from CP requirements.

Keywords: crustaceans; protein requirement; indispensable amino acids; logistic

model; marginal utilisation efficiency

53

2.3. Introduction

The black tiger shrimp (Penaeus monodon) is the second most cultured crustacean

species worldwide (FAO, 2007). Due to the importance of protein for shrimp growth, its

high cost in formulated feeds and the environmental implications of N losses, it is

essential to gain a better understanding of N requirements and N utilisation in P.

monodon. Available data on crude protein requirements (CP, % diet dry matter, DM) of

P. monodon show a large degree of variability, i.e. from 36-40 % (Shiau and Chou,

1991) up to 50 % (Shiau, 1998). Several factors, e.g. differences in protein source,

dietary energy level, life stage, rearing conditions and, in particular, differences in feed

intake (FI), can explain some of this variation (Rollin et al., 2003). The confounding

effect of FI on dietary protein requirement estimates has also been illustrated with the

pacific white shrimp, Litopenaeus vannamei (Kureshy and Davis, 2002; Venero et al.,

2007). The latter authors (Venero et al., 2007) demonstrated that maximum weight gain

could be obtained by a wide range of dietary CP levels (30-40 % DM diet) at different

feed allowances (50, 75 and 100% of typical daily intakes), underlining the importance

of expressing protein requirements on absolute basis rather than as a percentage of the

diet. The use of the factorial approach, which allows the distinction between

maintenance and growth for estimating protein requirements, has been initially

developed for terrestrial animals (Fuller et al., 1989; Wang and Fuller, 1989) and has

also been applied for teleost fish (Gatlin et al., 1986; Fournier et al., 2002; Abboudi et

al., 2006). For shrimp, data on protein requirements using the factorial approach have

been documented for two marine species: L. vannamei (Kureshy and Davis, 2002) and

the Kuruma prawn Marsupenaeus japonicus (Teshima et al., 2001), reported as having

more carnivorous feeding habits than L. vannamei, and for the freshwater prawn

Macrobrachium rosenbergii, with herbivorous-omnivorous feeding habits (Teshima et

al., 2006). For P. monodon, so far, no studies applied the factorial approach in order to

distinguish maintenance from growth protein requirements.

In studies on amino acid (AA) requirements, the dietary AA profile is mostly based on

either shrimp whole body or tail muscle composition as the reference (Millamena et al.,

1998; Alam et al., 2002). With the increasing use of plant-based proteins in shrimp

feed, as an alternative to marine protein sources (fish, shrimp or squid meal), lysine

and methionine will be the first two limiting essential amino acids (EAA) (Gatlin et al.,

2007). Data on the requirements of P. monodon for lysine and methionine are limited

to the studies of Millamena et al. (Millamena et al., 1996, 1998), who estimated

54

requirements of post-larval P. monodon based on growth response using diets with

fixed CP level (37 or 40%) and different levels of lysine and methionine. The dose

response method has been criticised since the EAA balance differs in each of the test

diets, in contrast to the ‘diet dilution’ technique which is based on a ‘summit’ diet with

high-protein level being diluted with a protein-free or low-protein diet of similar EAA

profile (Gous, 2007). Furthermore, a combination of both methods (Gous and Morris,

1985; Wang and Fuller, 1989), using diets in which the protein level is ‘diluted’ by

non-protein nutrients while creating an EAA deficiency at each of the tested protein

levels, has been used in studies with fish (Fournier et al., 2002; Abboudi et al., 2006;

Abboudi et al., 2007). This provides simultaneous estimations of protein and EAA

requirements in a single study, and enables to analyze the relationship between the

level of EAA and that of total CP. In studies on fish, as in other animals, some

controversy exists on potential interactions between levels of EAA and CP on for

instance the utilisation efficiency of the first limiting EAA, possibly leading to wrong

estimations of requirements (Heger et al., 2003; Abboudi et al., 2007). Another point,

not examined in shrimp, concerns the validity of applying the ideal protein pattern in

high protein diets, i.e. the question whether the different EAA are always required as a

constant proportion of crude protein or not. This point has received a lot of attention in

broilers (Morris et al., 1999; Sterling et al., 2003) and is of practical relevance for diets

in which poor quality protein sources are included at higher than normal levels to

provide a minimal level of EAA in the diet (% diet), resulting in imbalanced dietary

AA profiles (% CP).

The aim of this study was to determine, within a single feeding trial, the

requirements for protein and for two EAA, lysine, and methionine, for maintenance (N

equilibrium) and maximal body protein deposition of juvenile P. monodon and to study

the possible relationship between the limiting EAA and dietary CP supply. Different

mathematical models were used to determine the requirements, giving consideration to

the basic assumptions behind and to obtain original data on efficiency of N and AA

utilisation in shrimp.

55

2.4. Materials and methods

2.4.1. Experimental diets

Ten semi-purified diets were formulated with four different levels of crude protein

(CP) and two levels of lysine or methionine (adequate or 30% deficient). The nitrogen

source was casein and a blend of crystalline AA in a 55:45 ratio (casein: AA blend,

Tables 2.1 and 2.2). Diets NP (no-protein), LP (low protein), MP (medium protein) and

HP (high protein) were formulated to supply 0, 1.6, 4.8 and 8.1 % N, respectively. Fish

protein soluble concentrate (20 g/kg diet) and an attractant mix (glucosamine, taurine,

betaine, glycine, and alanine) (15 g/kg diet) were included in all diets in order to

improve palatability, which provided an additional N source in all diets, including in

the ‘protein-free’ NP diet (0.8% N, Table 2.1). Gelatinised starch levels compensated

for varying CP levels. The four diets NP, LP, MP and HP had a similar AA

(indispensable and non-indispensable) profile (% CP, Table 2.3), which was based on

the AA composition of shrimp whole body (“ideal AA profile”, compiled from

literature). The six other diets, LPL, LPM, MPL, MPM, HPL, HPM, were formulated

to be 30 % deficient in either lysine (diets L) or methionine (diets M) (Table 2.3). The

AA deficiencies (relative to the AA profile of the adequate diets) were obtained by

replacing the tested AA from the AA blend by non essential AA (NEAA) in order to

keep diets isonitrogenous at each level of total N supply. The ratio of

cystine:methionine was kept constant at approximately 0.3 in all diets so that the levels

of cystine were proportionally lower in the methionine-deficient diets (Table 2.2). Each

AA blend (Table 2.2) was coated with agar (Mambrini and Kaushik, 1995a) dissolved

in warm water (30°C; pH=5) before being mixed with the other ingredients. The

experimental diets were manufactured by Institut National de la Recherche

Agronomique (INRA) at the experimental facility of Donzacq (France). Ingredients

such as casein, cholesterol, lecithin, sodium alginate, cellulose, fish protein soluble

concentrate, gelatinised starch and attractants were first mixed together and

homogenised before adding the coated AA blend and the fish oil. After thorough

mixing, feed was pelleted (meat grinder, 3 mm), dried at 40°C and stored in sealed

bags prior shipping to the experimental site in Madagascar, where it was stored at 4°C.

56

Table 2.1. Formulation and analysed composition of the ten experimental semi-purified diets fed to P. monodon juveniles for 6 weeks Diets 1

Ingredients (g/kg diet) NP LP LPM LPL MP MPM MPL HP HPM HPL Casein 2 0 62 62 62 186 186 186 310 310 310 Amino acid mix 3 0 45 45 45 135 135 135 225 225 225 Cholesterol 2 20 20 20 20 20 20 20 20 20 20 Soybean lecithin 4 20 20 20 20 20 20 20 20 20 20 Fish oil 5 60 60 60 60 60 60 60 60 60 60 Sodium alginate 4 50 50 50 50 50 50 50 50 50 50 Mineral mix 6 50 50 50 50 50 50 50 50 50 50 Vitamin mix 7 50 50 50 50 50 50 50 50 50 50 Agar 2 15 15 15 15 15 15 15 15 15 15 Cellulose 20 20 20 20 20 20 20 20 20 20 Fish protein soluble concentrate 5 20 20 20 20 20 20 20 20 20 20 Gelatinised corn starch 8 680 573 573 573 359 358 359 144 144 144 Attractant mix 9 15 15 15 15 15 15 15 15 15 15 Analysed chemical composition Dry matter (DM. % diet) 89.6 89.3 90.6 90.4 90.6 89.8 89.5 89.0 89.2 89.1 N (% DM) 0.82 2.31 2.71 2.31 5.45 5.44 5.28 8.52 8.65 8.62 Crude protein (N x 6.25, % DM) 5.1 14.4 16.9 14.4 34.1 34.0 33.0 53.2 54.1 53.9 Lysine (% DM) 0.18 0.91 1.04 0.66 2.35 2.39 1.62 3.63 3.68 2.56 Methionine (% DM) 0.10 0.44 0.38 0.47 0.96 0.69 0.99 1.54 1.12 1.60 Crude lipid (% DM) 6.8 6.8 7.0 7.4 7.8 6.9 7.7 7.5 7.5 7.7 Ash (% DM) 5.7 5.7 5.8 5.7 6.0 6.0 6.0 5.9 6.0 6.1 Gross energy (kJ/g DM) 18.6 19.0 19.3 19.2 20.2 20.1 19.6 21.3 21.0 21.0 1 NP, non-protein; LP, low protein; LPM, methionine-deficient low-protein diets; LPL, lysine-deficient low-protein diets; MP, medium protein; MPM, methionine-deficient medium protein diets; MPL, lysine-deficient medium-protein diets; HP, high protein; HPM, methionine-deficient high-protein diets; HPL, lysine-deficient high-protein diets. 2 Acros France; 95% stabilized cholesterol; glycine 98%; D-glucosamine 98% HCL; Agar powder; pure casein (CAS 9000-71-9) 3 Eurolysine and Acros (see Table 2.2. for details) 4 Louis François (St Maur, France) 5 Sopropêche (Lorient, France) 6 supplied the following (to provide g/kg mixture): magnesium oxyde, 124; calcium carbonate, 215; potassium chloride, 90; sodium chloride, 40; potassium iodide, 40 mg; copper sulfate, 3; cobalt sulfate, 20 mg; ferric sulfate, 20; manganese sulfate, 3; zinc sulfate, 4; dibasic calcium phosphate, 500; sodium fluoride, 1. 7 supplied the following (to provide g/kg mixture): retinyl acetate (A), 0.172; thiamin (B1), 0.1; riboflavin (B2) (80%), 0.5; nicotinic acid (B3), 1; calcium-panthotenate (B5) (98%), 2; pyridoxine (B6), 0.3; Inositol (B7), 30; biotin (B8) (2%), 1; folic acid (B9), 0.1; vitamin B12 (1g/kg), 1; ascorbic acid (C) (35%), 14.29; cholecalciferol (D3), 0.006; tocopheryl acetate (E), 3.7 ; menadione (K3) (50%), 2; choline chloride (60%), 167. 8 Roquette (Lestrem, France) 9 contained glucosamine, taurine, betaine, glycine and alanine as 5:3:3:2:2

Table 2.2. Formulation of the AA blend added to the semi-purified casein-based diets (g/kg diet) Diets 1

Amino acids LP LPM LPL MP MPM MPL HP HPM HPL Arg 2 7.3 7.3 7.3 21.8 21.8 21.8 36.3 36.3 36.3

His 3 0.4 0.4 0.4 1.1 1.1 1.1 1.8 1.8 1.8

Ile 4 1.0 1.0 1.0 3.1 3.1 3.1 5.1 5.1 5.1

Leu 4 2.1 2.1 2.1 6.4 6.4 6.4 10.7 10.7 10.7

Lys 3 3.4 3.4 0.2 10.3 10.3 0.6 17.2 17.2 1.0

DL-Met 3 0.9 0.0 0.9 2.8 0.0 2.8 4.7 0.0 4.7

Phe 4 1.2 1.2 1.2 3.6 3.6 3.6 6.0 6.0 6.0

Tyr 4 0.4 0.5 0.7 1.3 1.6 2.0 2.1 2.7 3.3

Trp 4 0.5 0.5 0.5 1.5 1.5 1.5 2.5 2.5 2.5

Val 4 0.7 0.7 0.7 2.0 2.0 2.0 3.4 3.4 3.4

Asp 4 5.8 6.1 6.4 17.3 18.2 19.1 28.8 30.3 31.9

Thr 4 1.5 1.5 1.5 4.4 4.4 4.4 7.3 7.3 7.3

Ser 4 0.3 0.4 0.5 1.0 1.3 1.6 1.6 2.1 2.7

Glu 4 2.8 3.2 3.8 8.4 9.7 11.3 14.0 16.2 18.8

Pro 4 5.7 5.8 6.0 17.0 17.5 18.0 28.3 29.1 30.0

Gly 2 6.3 6.5 6.7 18.8 19.4 20.1 31.3 32.3 33.5

Ala 4 3.9 4.1 4.3 11.8 12.3 12.8 19.6 20.5 21.4

Cys 4 0.9 0.4 0.9 2.6 1.3 2.8 4.3 2.1 4.7

Total (g/kg diet) 45.0 45.1 45.0 135.0 135.3 135.0 225.0 225.5 225.0 1 LP, low protein; LPM, methionine-deficient low-protein diets; LPL, lysine-deficient low-protein diets; MP, medium protein; MPM, methionine-deficient medium protein diets; MPL, lysine-deficient medium-protein diets; HP, high protein; HPM, methionine-deficient high-protein diets; HPL, lysine-deficient high-protein diets. 2 Acros, L-Arg 98%, Gly 98% 3 Eurolysine 4 Jerafrance

Table 2.3. Analysed AA composition of the ten experimental diets (g/16g N) Diets g/16g N NP LP LPM LPL MP MPM MPL HP HPM HPL Arg 3,7 8,0 8,1 8,0 8,4 8,0 8,3 7,9 7,7 7,8

His 0,9 1,6 1,6 1,6 1,8 1,9 1,8 1,8 1,8 1,8

Ile 2,2 3,5 3,6 3,3 3,7 3,8 3,8 3,7 3,8 3,7

Leu 4,4 7,2 7,3 7,4 7,7 7,8 7,7 7,9 7,9 7,9

Lys 3,5 6,3 6,1 4,6 6,9 7,0 4,9 6,8 6,8 4,8

Met 2,0 3,0 2,2 3,3 2,8 2,0 3,0 2,9 2,1 3,0

Phe 2,6 3,9 3,8 4,2 4,2 4,3 4,3 4,2 4,1 4,6

Tyr 1,5 2,7 2,7 2,7 3,2 3,3 3,3 3,4 3,4 3,4

Val 2,6 4,2 4,3 4,1 4,5 4,7 4,6 4,4 4,4 4,4

Asp 5,7 9,8 9,8 10,0 10,0 9,9 10,2 9,5 9,4 9,7

Thr 2,4 3,8 3,9 3,7 3,9 3,9 3,9 3,8 3,8 3,7

Ser 2,4 3,4 3,4 3,3 3,5 3,7 3,6 3,5 3,7 3,5

Glu 8,3 14,6 14,8 14,9 14,7 15,0 15,0 15,6 16,0 16,0

Pro 4,6 10,6 10,7 10,5 11 11,1 11,3 10,9 10,9 10,9

Gly 9,4 8,7 8,5 8,7 6,6 6,0 6,1 7,0 6,9 7,0

Ala 7,4 7,2 7,1 7,4 6,0 5,7 6,0 5,7 5,6 5,9

Cys 0,7 0,9 0,7 1,1 0,9 0,6 1,0 0,8 0,6 0,9

58

2.4.2. Experimental animals

The feeding trial was undertaken at the hatchery facilities of Aqualma, Madagascar.

Forty three 150 L fibreglass tanks (80×30.5; diameter × height) were used and stocked

each with 15 juvenile (85 days old post-larvae) Penaeus monodon (initial mean weight:

2.36 g ± 0.10), reared in earthen ponds for 66 days prior to the experiment. At the start

of the study, juveniles were individually weighed and allocated to the respective tanks.

During the ten days of adaptation, all groups were fed diet MP at an initial ration level

of 2% biomass per day. During the growth trial, which lasted six weeks, each of the ten

experimental diets was distributed to four replicate groups allocated randomly. A

commercial practical diet (CP: 46%; crude fat: 7%; Nutrima, La Réunion) was included

as a control with three replicates to follow overall performance.

Sea water was filtered through a 50µ sand filtration system prior to distribution into the

tank. The minimum water exchange was 40% of the tank volume and was done every

morning before the first feeding. Temperature, pH and oxygen concentration of the

water were measured twice daily (morning at 04:00 and afternoon at 17:00 hours).

Salinity was recorded daily at 16:00 hours. Analyses for nitrate-N, nitrite-N, and

ammonia-N in the water in all the 43 tanks were made every week using commercial

kits (Nitraver®5 Nitrate, Nitriver®3 Nitrite, and salicylate kit; Hach, Loveland, CO,

USA). Average pH, oxygen concentration, temperature and salinity of the water were

8.0 ± 0.1, 5.7 ± 0.5 mg/L, 30.1 ± 1.6 °C and 30.8 ± 1.8 ppt, respectively. Total ammonia

nitrogen, nitrate (N-NO3) and nitrite (N-NO2) were 0.17 ± 0.26, 0.88 ± 0.47, and 0.15 ±

0.22 mg/L respectively.

Special care was taken to assess actual feed consumption and to adapt feed distributions

to the demands of each group. Feed distributions were done four times daily (08:00;

13:00; 18:00; 23:00 hours). At each distribution, feed was deposited into two small

circular trays (20 cm diameter) placed inside the tank. Two hours after each feed

distribution, the trays were checked and left-overs were counted, collected in a box and

stored at -20°C until the next collecting time. A code was established to determine the

amount of feed to be distributed at the next meal:

0: all the feed was consumed, the next distribution increased by 30% more than the previous one

1: less than 3 pellets remained in the tray; the next distribution was of equal amount

2: more than 3 pellets remained in the tray; the next distribution was decreased by 30%

59

All feed leftovers were collected from each tray and pooled per tank on a weekly basis.

Dry matter content of each uneaten feed sample was determined by drying to a constant

weight at 90°C for 48 hours. We could thus estimate actual FI for each tank on a weekly

basis. Nitrogen, lysine and methionine intakes were calculated from the total FI

measured.

Biomass of each tank was measured every two weeks during the whole trial. Mortality

was checked before and after each feeding period; dead animals were removed and

weighed. Exuvia were removed from the tanks as soon as they were observed in order

to avoid the animals to feed on them.

Performance calculations were as follows:

Survival rate (%): 100 × final number/initial number

Total dry matter feed intake (TFI, g DM): dry feed distributed– dry recovered feed

Daily feed intake (DFI, g DM/kg BW per d): TFI / (average BW × average nb × 42 d)

Specific growth rate (SGR) (% / d): 100 × (ln(BWF)-ln(BWI)) / 42 d

Feed efficiency (FE): (BF-BI + BD) / TFI

N intake (g/kg BW per d): DFI × %Nfeed

N gain (g/kg BW per d): ((N Shrimp F × BWF)-(N Shrimp I × BWI)) / (average BW × 42 d)

N retention: N gain / N intake × 100

Where BWI, BWF: initial and final body weight (g) and average BW (kg): ((BWI + BWF)/(1000 ×

2)); BF, BI, and BD: initial, final and dead biomasses (g); nb, number of shrimp; Nfeed, N content of

the feed (g/100 g DM); NShrimp I and NShrimp F: initial and final N content of shrimp (g/100g

fresh matter).

2.4.3. Proximate analysis of diets and shrimp whole body

At the beginning of the study, 15 shrimp (12-h feed deprived) were selected for whole

body composition analyses. At the end of the trial, a pool of 8 shrimp (12-h feed

deprived) was analysed per treatment (2 shrimp per tank). All samples were kept at -

20°C prior to analyses. Whole shrimp were ground and analysed for dry matter before

being freeze-dried. Gross energy content of feed samples was analysed using an

adiabatic bomb calorimeter (IKA). Feed samples as well as freeze-dried whole body

samples were analysed for dry matter (105 °C for 24h), ash (550 °C for 12h), lipid

(Soxtherm, Gerhardt, Germany), and protein (N × 6.25, Kjeldahl Nitrogen analyser

2000, Fison Instruments, Milan, Italy). Based on comparative carcass analyses, gain and

retention values were computed. The AA composition of the diets was analysed

(AgroBio Laboratory, Rennes, France) after hydrolysis (6N HCl, 110°C, 23 hours).

After evaporation, samples were analysed in an automatic AA analyser (Biochrom-30,

60

Biochrom Ltd, Cambridge, UK) using a sodium high resolution protein hydrolysate

column (resin ultra pac8). The amino acids were derivatised with ninhydrin and

quantified at 570 nm and 440 nm for proline.

2.4.4. Data analysis

All data were analysed by a one-way analysis of variance (ANOVA) using dietary

treatment as factor (n=11 diets) or by a two-way ANOVA using the level of protein and

of EAA (lysine or methionine) as independent factors (n=6 diets), followed by the

comparison of means using the Duncan’s multiple range test in case of a significant

effect (P<0.05). ANOVA were performed using STATISTICA 5.0 software (StatSoft.

Inc., Tulsa, OK, USA).

Four regression models were used to estimate the maintenance (X-value for zero gain)

and growth requirements. The first models use a broken line (BLM) and a quadratic

regression (Quad-1) (Robbins et al., 2006):

BLM model : Y = L + U.(X-R) if X > R, (X-R) = 0

Quadratic with one slope: Y = L + U. (X-R). (X-R) if X > R, (X-R) = 0

Where Y, dependent variable as N gain (g/kg BW per d); X, independent variable as N,

lysine or methionine intake (g/kg BW per d); U, slope of the first segment (nutrient

utilisation efficiency); R, breakpoint X value (maximal growth requirement value); L,

plateau value.

Maintenance requirements (Y=0) were calculated as: X = (UR – L) / U with the BLM

model and as X = R+(-L/U)1/2 with the quadratic model.

The third model used was the four parameters saturation nutrient kinetic model (SK-

4) (Mercer, 1982):

Y = [(B × K0.5 n) + (Rmax × Xn)] / (K0.5

n + Xn)

Where Y, dependent variable as N gain (g/kg BW per d); X, independent variable as N,

lysine or methionine intake (g/kg BW per d); B, intercept on y-axis for X = 0; K0.5,

concentration for ½(R+B); Rmax, maximum Y response; n, apparent kinetic order.

Maintenance requirement (Y=0) was calculated as: X = K0.5 × (-B/Rmax) (1/n). Total

requirement was estimated at 95% of the maximum gain (Rmax) (Rodehutscord et al.,

1995) following the above equation. Therefore, requirement (g/kg BW per d) was

calculated as:

61

X = [(K0.5n × (Rlim-B))/(Rmax- Rlim)] (1/n) with Rlim = Rmax× 0.95

The fourth model was logistic model (Gahl et al., 1991) and described as:

Y = [Rmax +((b × (1+c)-Rmax) × e(-kX))] / (1+(c × e(-kX)))

Where Y, dependent variable as N gain (g/kg BW per d); X, independent variable as N,

lysine or methionine intake (g/kg BW per d); b, intercept on y-axis for X = 0; Rmax,

maximum Y response; c, shaping parameter that locates the inflection point; k, scaling

parameter.

Maintenance requirement (Y=0) was calculated as X = (-1/k) × Ln (-Rmax/(b+bc- Rmax)).

Overall requirement (g/kg BW per d), estimated at 95% Rmax (Finke et al., 1989) was

calculated as:

X = [Ln (Rmax – Rmax 0.95) – Ln (Rmax 0.95 × c –b –bc + Rmax)]/ (-k)

Marginal efficiency of nutrient utilisation was calculated as the first derivative dY/dX,

according to the following equation:

dY/dX = [-k × (b+bc-Rmax).e(-kX) + kc.Rmax. e

(-kX)) / (1+c × e(-kX))²

Protein requirements were determined using data from NP, LP, MP, and HP treatments.

Lysine requirements were determined using data from treatments NP, LP, LPL, MP,

MPL, HP, HPL (excluding the methionine deficient diets) and methionine requirements

using those from treatments NP, LP, LPM, MP, MPM, HP, HPM (excluding the lysine

deficient diets). Four replicate tanks for each treatment (n=4) were included in the

analysis, except for HP (n=3) where one tank had to be excluded because of

cannibalism. Graphical presentations and parameter estimates were made using

GraphPad Prism 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

Table 2.4. Survival, feed intake, growth and nutrient utilisation in juvenile P. monodon fed the semi-purified diets for 6 weeks

Survival (%) Final BW (g) SGR (%/d) FI (g/kg BW per d) FE N retention (%)

Dietary treatments 1 Mean 2 SD 2 Mean SD Mean SD Mean SD Mean SD Mean SD

NP 80.0 14.4 2.3 e 0.1 -0.01 f 0.13 45.3 c 2.6 -0.001 e 0.031 -18.7 c 10.7

LP 83.3 8.6 3.2 d 0.2 0.67 e 0.19 64.4 ab 8.3 0.099 d 0.014 13.5 b 2.3

LPM 90.0 12.8 3.6 cd 0.3 1.00 d 0.27 71.5 ab 5.1 0.134 d 0.041 13.1 b 4.1

LPL 90.0 15.9 3.5 d 0.3 0.90 de 0.15 64.8 ab 6.2 0.134 d 0.019 13.7 b 2.0

MP 81.7 17.5 5.2 ab 0.1 1.82 ab 0.02 66.5 ab 1.8 0.273 bc 0.011 16.2 b 0.6

MPM 78.3 18.4 4.1 c 0.2 1.36 c 0.15 62.5 ab 9.0 0.219 c 0.011 13.9 b 1.1

MPL 78.3 10.0 4.5 bc 0.3 1.58 bc 0.24 61.4 b 7.8 0.255 bc 0.012 16.8 b 0.8

HP 80.0 20.0 4.9 b 0.2 1.65 bc 0.27 67.1 ab 1.6 0.235 c 0.055 10.9 b 1.3

HPM 80.0 14.4 5.3 ab 0.6 1.90 ab 0.30 66.7 ab 4.3 0.270 bc 0.022 11.5 b 0.8

HPL 66.7 9.4 5.7 a 0.7 2.08 a 0.22 73.0 a 10.7 0.290 b 0.062 10.6 b 2.1

Commercial 86.7 6.7 5.2 ab 0.3 2.01 a 0.14 34.4 d 1.5 0.529 a 0.045 24.7 a 2.3

Effect of diet (one-way ANOVA) 3 0.594 <0.000 <0.000 <0.000 <0.000 <0.000

Effect of lysine and protein (two-way ANOVA) 4

Protein 0.210 <0.000 <0.000 0.236 <0.000 <0.000

Lysine 0.574 0.375 0.111 0.890 0.114 0.858

Protein x lysine 0.402 0.003 0.010 0.352 0.131 0.903

Effect of methionine and protein (2-way ANOVA) 4

Protein 0.625 <0.000 <0.000 0.513 <0.000 0.010

Methionine 0.866 0.597 0.705 0.725 0.664 0.428

Protein x methionine 0.808 0.000 0.004 0.194 0.009 0.403 1 NP, non-protein; LP, low protein; LPM, methionine-deficient low-protein diets; LPL, lysine-deficient low-protein diets; MP, medium protein; MPM, methionine-deficient medium protein diets; MPL, lysine-deficient medium-protein diets; HP, high protein; HPM, methionine-deficient high-protein diets; HPL, lysine-deficient high-protein diets. 2 n=4 per diet, except for HP and commercial diets for which n=3. a-f Mean values with different superscript letters were significantly different between groups (P < 0.05) 3 P values given by the 1-way ANOVA (n=11 diets). 4 P values given by the 2-way ANOVA (n=6 diets per analysis). NP and commercial diet were excluded from the test, as well the met-deficient diets from the ANOVA on the effect of lys and the lys-deficient diets from the ANOVA on the effect of met.

2.5. Results

Table 2.4 shows the performances of the shrimp analysed by a one-way (effect

treatment, n=11) and two-way ANOVA (effect dietary protein and EAA level, n=6

diets). Results from the one-way ANOVA showed no significant effect of the dietary

treatment on the survival of the shrimp (Table 2.4). Feed intakes (g/kg BW per d) in

shrimp fed diets LP, MP and HP were higher than when fed diet NP or the practical diet

(Table 2.4). The lowest final BW was found for shrimp fed the NP and LP diets (2.3 to

3.6 g) and the highest for those fed diets MP, the three HP diets and the practical diet

(4.9 to 5.7 g), which were not significantly different. The deficient-HP diets and the

non-deficient MP diet led to similar growth rates, which were not different from growth

rates obtained with the practical diet (Table 2.4). Among the experimental diets, diets

LP and NP provided a significantly lower FE than diets MP or HP. The two-way

ANOVA of the data in Table 2.4 showed a significant effect of the protein level on final

BW, SGR and FE (HP = MP > LP), but not on survival or FI. No significant effect of

the EAA deficiency could be detected on any of the parameters (Table 2.4).

Interestingly, for both EAA, the 2-way ANOVA indicated a significant interaction

between the levels of protein and EAA for growth performances (final BW and SGR),

which was more pronounced (lower P-value) for methionine than for lysine (Table 2.4).

The interaction between the level of methionine and protein regarding growth, and also

FE, can be explained by the fact that methionine deficiency reduced growth and FE, at

the medium protein level (MPM vs. MP), but not at the higher or lower protein levels

(Table 2.4).

The analysis of whole body composition of the shrimp (data not shown) revealed a low

lipid content (0.3-0.6 g/100 g shrimp) and an ash content of 3.4-4.0 g/100 g shrimp,

without visible effect of the dietary treatment. There was a positive linear relationship

(R2=0.96) between the concentration of dietary and whole body protein, which was

17.4, 19.1, 20.2 and 21.9 g/100 g shrimp for diets NP , LP, MP and HP, respectively. As

intended, N intakes (data not shown) differed largely between the dietary treatments,

being significantly higher with diet HP (5.7 g/kg BW per d) than with diets MP or LP

(3.6 and 1.5 g/kg BW per d, respectively), and the lowest value being with the NP

treatment (0.4 g/kg BW per d). Daily N gain (per unit BW) was significantly affected by

N intake: HPM-fed animals had the highest N gain (0.67 g/kg BW per d), whereas NP-

fed animals had a daily N loss of 0.07 g/kg BW per d. Daily N gains of shrimp fed diets

HP and MP were not significantly different from the N gain obtained with the

64

commercial diet (0.59-62 g/kg BW per d). Lysine or methionine deficiency did not

affect the level of N intake or N gain (P > 0.05). However, a significant interaction was

found between dietary protein and methionine level on the daily N gain (P = 0.0026),

related to the fact that the deficiency in methionine (Fig. 2.1b) affected N growth only at

the medium protein level. Although the interaction between lysine and protein levels

was not significant (P = 0.055), N gains presented in Fig. 2.1a show a similar tendency

with the effect of a lysine deficiency being more pronounced at the medium than at

lower or higher dietary protein levels.

Fig. 2.1. Effect of dietary levels of protein and lysine (a) and protein and methionine (b) on daily individual nitrogen gain (mg/d) of juvenile P. monodon fed the semi-purified diets for 6 weeks. LP, low protein; LPL, lysine-deficient LP; MP, medium protein; MPL, lysine-deficient MP; HP, high protein; HPL, lysine-deficient HP; LPM, methionine-deficient LP; MPM, methionine-deficient MP; HPM, methionine-deficient HP. Values are means (n=4 per treatment, except for HP where n=3), with standard deviations represented by vertical bars. a,b,c Mean values with unlike letters are significantly different (P < 0·05; one-way ANOVA). P-values of the two-way ANOVA (protein × lysine) are as follows: protein, P < 0·0001; lysine, P = 0·936; protein × lysine, P = 0·055. P-values of the two-way ANOVA (protein × methionine) are as follows: protein, P < 0·0001; methionine, P = 0·503; protein × methionine, P = 0·002.

65

Table 2.5. Parameters estimated by fitting the four regression models through the experimental data using N gain (g/kg BW per d) as the response parameter and the different intake levels of nitrogen, lysine or methionine (g/kg BW per d) as input parameter in P. monodon. (Mean values with their standard errors)

BLM, broken line model; Quad-1, quadratic model with one slope; SK-4, four parameters saturation kinetic model; df, degrees of freedom

Estimated requirement Models Parameter estimates Nitrogen Lysine Methionine

Mean SE Mean SE Mean SE

L 0.60 0.02 0.62 0.01 0.63 0.02

U 0.24 0.03 0.65 0.04 1.35 0.10

R 3.14 0.25 1.14 0.05 0.56 0.03

df 12 24 24

R² 0.98 0.96 0.95

BLM

Sy,x 0.04 0.05 0.06

L 0.64 0.02 0.63 0.02 0.66 0.03

U -0.03 0.00 -0.26 0.04 -1.02 0.17

R 5.03 0.39 1.75 0.14 0.90 0.07

df 12 24 24

R² 0.98 0.96 0.95

Qua

d-1

Sy,x 0.04 0.06 0.06

B -0.07 0.02 -0.06 0.03 -0.07 0.03

K0,5 1.74 0.12 0.62 0.05 0.31 0.02

n 3.11 0.75 2.48 0.50 2.90 0.59

Rmax 0.65 0.04 0.68 0.04 0.68 0.04

df 11 23 23

R² 0.99 0.95 0.96

SK

-4

Sy,x 0.04 0.06 0.06

Rmax 0.63 0.03 0.64 0.02 0.65 0.03

b -0.12 0.03 -0.09 0.04 -0.10 0.04

c 8.61 7.57 6.85 4.92 7.65 5.71

k 1.41 0.42 3.69 0.87 7.57 1.89

df 11 23 23

R² 0.99 0.96 0.96

Logi

stic

Sy,x 0.04 0.06 0.06

66

Table 2.6. Estimated requirements for N equilibrium (maintenance, M) and maximal N gain (N growth, G) for N, protein, lysine and methionine using the four regression models for juvenile P. monodon

Requirement estimates BLM 1 Quad-1 SK-4 Logistic

N (g/kg BW per d) M 0.65 0.63 0.86 0.73

G 3.15 5.03 4.65 3.82

Protein (g/kg BW per d) 2 M 4.06 3,94 5.36 4.53

G 19.66 31.46 29.05 23.89

M/G (%) 20.64 12.52 18.47 18.98

Lysine (g/kg BW per d) M 0.18 0.18 0.24 0.20

G 1.14 1.75 2.11 1.40

M/G (%) 15.92 10.34 11.33 14.46

Methionine (g/kg BW per d) M 0.10 0.10 0.14 0.11

G 0.56 0.90 0.90 0.70

M/G (%) 17.45 10.80 16.00 16.39

Lys/Met ratio M 1.86 1.86 1.66 1.77

G 2.04 1.94 2.35 2.01

Lys (%CP) 3 M 4.49 4.60 4.45 4.46

G 5.82 5.56 7.25 5.85

Met (%CP) 3 M 2.41 2.47 2.67 2.51

G 2.86 2.86 3.09 2.91 1 BLM, broken line model; Quad-1, quadratic model with one slope; SK-4, four parameters saturation kinetic model. 2 N requirement × 6.25 3 Calculated ratio = 100 × (estimated essential amino acid requirement/estimated protein requirement)

2.5.1. Protein and EAA requirement estimates

The four models used to estimate the requirements for N, lysine and methionine all gave

a satisfactory regression coefficient (0.95 < R² < 0.99, Table 2.5). The SK-4 model gave

the highest and the broken line method (BLM) the lowest N requirement estimates

(Tables 2.5 and 2.6). Based on the contrasting assumptions inherent to the model

regarding nutrient utilisation efficiency (constant or diminishing returns) (Gahl et al.,

1991), we decided to further comment the results obtained only with the BLM (Fig. 2.2)

and the logistic (Fig. 2.3) model.

Nitrogen requirements for maintenance (N equilibrium) and maximal N gain were

estimated to be 0.6-0.7 g N/kg BW per d and 3.1-3.8 g N/kg BW per d, respectively,

corresponding to CP levels of 4.1-4.5 g and 19.7 to 23.9 g/kg BW per d (Table 2.6, Fig.

2.2a and 2.3a). Regarding the requirements for lysine, using N gain as response, both

models led to similar estimates, being 0.18-0.20 g/kg BW per d for maintenance and

1.14-1.40 g/kg BW per d for maximal N gain (Table 2.6, Fig. 2.2b and 2.3b).

Methionine requirement for maintenance or N balance was found to be 0.10-0.11 g/kg

BW per d, whereas that found for maximum N gain ranged from 0.56 to 0.70 g/kg BW

per d (Table 2.6, Fig. 2.2c and 2.3c). Based on the above data, we could estimate the

need as percentage of protein requirement, which was 5.8% for lysine 2.9% for

methionine (Table 2.6). Maintenance requirements for protein represented 19-21% of

67

the protein requirement for maximum growth, whereas the maintenance/growth ratio

was 14.5-15.9% for lysine and between 16.4 and 17.4% for methionine (Table 2.6).

2.5.2. Marginal efficiencies of N and EAA utilisation

The instantaneous marginal efficiency of N utilisation, calculated from the parameters

obtained with the logistic model, showed a maximum efficiency of 38%, occurring at an

N intake level of 39% of that needed for maximum N gain (Fig. 2.3a). The BLM

estimate of constant N utilisation efficiency between maintenance and maximal growth

was 24% (Table 2.5). Lysine marginal efficiency, determined after plotting N gain

against lysine intakes, peaked at 0.77 at a lysine intake level corresponding to 35.7% of

the predicted requirement for maximum N gain (Fig. 2.3b). Methionine marginal

efficiency reached a maximum of 1.62 corresponding to 37.1% of the requirement

estimated for maximum N gain (Fig. 2.3c).

2.6. Discussion

The survival and whole body mass increases of the shrimp fed the semi-purified MP

and HP diet are comparable to those reported in other feeding trials with P. monodon of

similar size ranges (Glencross et al., 1999; Glencross and Smith, 1999). Only few

studies with shrimp species applied a factorial approach to determine the part of N

losses to be attributed to maintenance or to estimate N utilisation between maintenance

and maximal growth response (Kureshy and Davis, 2002; Teshima et al., 2001, 2006).

As for EAA requirements, there is to our knowledge, no such data available for

crustaceans. Some of the methodological dissimilarities between the present and latter

studies with shrimp susceptible to affect requirement estimates (Fuller and Garthwaite,

1993; Bodin et al., 2009), concern the feed supply, response criterion and mathematical

model. In the present study, the different diets were fed on an ad libitum basis with

careful monitoring of intakes instead of supplying a single diet at fixed ratios as in the

study with L. vannamei (Kureshy and Davis, 2002). In line with Teshima et al. (2001,

2006), protein accretion (daily N gain per unit BW) was used as response rather than

total weight (Kureshy and Davis, 2002), considered less pertinent as response criterion

than N accretion (Gahl et al., 1991; Rodehutscord et al., 1997). Regarding the model,

we decided to focus on a linear (broken line) and non-linear (logistic) regression

because of the contrasting assumptions regarding the marginal utilisation efficiency, the

former assuming constant efficiency and the latter being recommended in vertebrates

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for the determination of diminishing returns when approaching maximal intake (Gahl et

al., 1994).

Fig. 2.2. Linear broken line regressions (BLM) of nitrogen gain vs. nitrogen intake (a), lysine intake (b) and methionine intake (c) in juvenile P. monodon. The parameters of the regression equations and the requirement estimates are summarised in Tables 2.5 and 2.6.

Fig. 2.3. Non-linear regressions obtained with the logistic model of nitrogen gain vs. nitrogen intake (a), lysine intake (b) and methionine intake (c) and their respective marginal efficiencies in juvenile P. monodon. The six essential amino acid-deficient diets were excluded from the model (a). The three methionine and lysine-deficient diets were excluded from model (b) and (c), respectively. The parameters of the regression equations and the requirement estimates are summarised in Tables 2.5 and 2.6. Marginal instantaneous utilisation efficiency is defined as the incremental responses in nitrogen gain per incremental unit of nitrogen intake (a), lysine intake (b) and methionine intake (c).

70

2.6.1. Protein requirements for maintenance and growth

The maintenance requirement for protein, defined as the amount of protein ingested by

the shrimp to maintain its N equilibrium (N synthesis equals N breakdown), was

estimated from both models at the X-intercept level. The protein requirements for

maintenance for P. monodon estimated in the present study (2.4 g IBW) ranged between

4.1 and 4.5 g CP/kg BW per d, showing only minor variations according to the model

used. These estimates are superior to data from juvenile L. vannamei (1.8-3.8 g CP/kg

BW per d) using zero BW gain as response parameter (Kureshy and Davis, 2002). The

low protein maintenance requirement of 0.2 g CP/kg BW per d reported for M.

rosenbergii (IBW: 0.15 g) (Teshima et al., 2006) and of 1.1 g CP/kg BW per d for M.

japonicus (IBW: 1.69 g) (Teshima et al., 2001) reflects in fact the daily obligatory N

losses at zero feed intake, which was assumed as reflecting maintenance requirements.

If calculated in the same manner, daily N losses in our study were 0.16 g/kg BW per d,

equivalent to 0.98 g CP/kg BW per d, a value close to that reported for M. japonicus

(Teshima et al., 2001).

Protein requirement for maximum N gain in P.monodon juveniles ranged between 19.7-

23.9 g CP/kg BW per d, in line with the protein requirement for growth (20.5-23.5 g

CP/kg BW per d) reported for sub-adult L. vannamei (Kureshy and Davis, 2002). The

protein requirements of 7.1 g CP/kg BW per d found for M. rosenbergii (Teshima et al.,

2006) and of 10 g CP/kg BW per d for M. japonicus (Teshima et al., 2001) are lower

than in the present study, possibly due to the relatively poor growth in their studies as

compared to ours. It is also worth noting that in our study growth of the shrimp fed the

MP and HP semi-purified diets was similar to that obtained with a commercial practical

diet.

The ratio between maintenance and growth requirement, which reflects the proportion

of a nutrient used by an animal to maintain N balance as compared to that needed for

maximal N gain, was about 20%. This is much higher than the maintenance/growth

ratio reported for L. vannamei juveniles (3.9 to 8.8%) and subadults (6.4 to 10.2%)

(Kureshy and Davis, 2002), suggesting a relatively higher protein requirement for

maintenance of N balance in juvenile P. monodon. However the present

maintenance/gain ratio is within the same range as found in juvenile teleost fish, i.e.

12.3% for Atlantic salmon (Salmo salar) fry (Abboudi et al., 2006), 15.1% in Channel

catfish (Ictalurus punctatus) fingerlings (Gatlin et al., 1986), 16.7% in juvenile Nile

tilapia (Oreochromis niloticus) (Kaushik et al., 1995), or 21% for juvenile two-banded

seabream (Diplodus vulgaris) (Ozorio et al., 2009).

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2.6.2. Efficiency of protein utilisation for maximal N gain

The study of marginal N utilisation efficiency, which reflects the efficiency of N

utilisation between maintenance and maximum growth, depends on the biological

assumption of constant efficiency or not, and thus the mathematical model. We found

that marginal efficiency of N utilisation in P. monodon reached a maximum of 38% at

39% of the maximal growth requirement, after which N utilisation efficiency decreased

to 5.6% at maximal N gain intake levels. Diminishing returns are important to consider

from an economical perspective for choosing the requirement, with estimates for

optimal utilisation efficiency occurring at lower N intakes than for optimal growth

(Gahl et al., 1994). The average value of the marginal (instantaneous) efficiencies

obtained with the logistic model was 24.9%, which agrees well with the constant

efficiency of 24% obtained with broken line model (U estimate). The comparison with

other data from shrimp is difficult since no data on the regression coefficients were

given (simple linear regression) (Teshima et al., 2001, 2006) or since weight gain rather

than N gain/kg BW was used as response criterion without precision of efficiency

values (Kureshy and Davis, 2002). However, the present N utilisation efficiency (24%)

is inferior to values similarly obtained by broken line regression (Bodin et al., 2009) or

simple linear regression (Fournier et al., 2002) in other species, such as teleost fish

showing N utilisation efficiencies as high as 37.9% for gilthead seabream (Sparus

aurata) (Fournier et al., 2002) or 39.6% for European seabass (Dicentrarchus labrax)

(Fournier et al., 2002) and 34-44% for rainbow trout (Oncorhynchus mykiss) (Fournier

et al., 2002; Bodin et al., 2009).

A more common parameter in nutritional studies on shrimp is total N retention, i.e. the

ratio of total N gain to cumulated N intakes, without identification of the part of N

losses due to maintenance. N retentions varied between 11 (HP diets) and 17% (MPL).

Low N retentions, comprised between 10 and 15% of N intakes, were also reported for

adult L. stylirostris under laboratory conditions (Gauquelin et al., 2007). The N retention

obtained with the commercial treatment (24.7%) agrees with N retentions in intensive

shrimp farms in which approximately 20% of total dietary N input was recovered in the

harvested P. monodon (Briggs and Funge-Smith, 1994; Jackson et al., 2003). Higher N

retentions of up to 31% for P. monodon (Thakur and Lin, 2003) or up to 46 % of N

input for post-larval L. vannamei (Perez-Velasquez et al., 2008) have been attributed to

the natural productivity (development of bacteria and phytoplankton populations) taking

place in a static system (without water renewal) which constitutes a source of N intake

and, hence, might overestimate N retention. In our study, the water renewal (>40 % of

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each tank per day) is believed to have maintained the recycling of N wastes through

natural productivity close to zero. Although special care was taken for monitoring

intakes, efficiencies of N utilisation with the experimental diets might be slightly

underestimated due to some unseen feed losses or to leaching related to the slow

feeding behaviour of the shrimp, as underlined before by others (Teshima et al., 2001).

Also the loss of exuvia during the growth of the animal, not taken into consideration in

the present work, accounts for a part of N losses which leads to underestimation of

actual N retentions.

2.6.3. Lysine and methionine requirements for N maintenance and maximal

N gain

Data obtained here on lysine and methionine requirements for maintenance are the first

ever estimates of EAA requirements for N balance in crustacean shrimp. The proportion

of ingested AA spent to cover the N maintenance requirement as compared to the

requirement for maximal N gain was 14.5-15.9% for lysine, and 16.4-17.4% for

methionine. The similarity in maintenance contributions to total requirement for both

EAA differs from the lower maintenance contribution for lysine than methionine

reported in rainbow trout (4% for lysine vs. 10% for methionine) (Rodehutscord et al.,

1997) or in growing pigs (6% for lysine vs. 12% for sulphur AA) (Heger et al., 2002). In

this respect, it would be of interest to evaluate the contributions at zero AA gain given

the possible underestimation of requirement based on N gain, related to shifts in the

mobilisation of the type of body protein at intake levels near maintenance (Gahl et al.,

1994; Edwards et al., 1999).

Expressing the current lysine and methionine requirements for maximal N gain (1.1-1.4

and 0.6-0.7 g/kg BW per d, respectively) as a proportion of protein requirement enables

comparisons with the two other studies available in literature on lysine and methionine

requirements for growth of the same species. Expressed this way, the lysine and

methionine requirements for maximal N gain (5.8 % and 2.9 % of the protein

requirement) are very close to the lysine requirement of 5.2 % and the methionine

requirement of 2.4 % reported for post-larval P. monodon (approx. 20 mg IBW) using

the dose-response technique (Millamena et al., 1996, 1998). The above requirements,

however, exceed those found for L. vannamei, being 4.5-5.2 % for lysine (Fox et al.,

1995) and only 1.26 % for methionine (Fox et al., 2006). Expressing maintenance

requirements the same way (g/16g N), the proportion of protein requirement for

maintenance covered by both EAA was only slightly less than that seen for maximal

73

growth, being 4.5 % for lysine (using both models) and 2.4-2.5% for methionine. Lower

contribution of EAA to maintenance than to growth (g/16g N) (Mercer, 1982; Fuller et

al., 1989; Chung and Baker, 1992; Fournier et al., 2002) can be partly explained by the

sparing of EAA due to the preferential oxidation of NEAA at N equilibrium, as

suggested very early in the rats displaying lower requirements for EAA than for NEAA

near maintenance (Dreyer, 1976) and in chickens for which dispensable AA were

suggested to maintain and replete protein reserves (Shapiro and Fisher, 1962). In

salmon fry, NEAA enabled a reduction in N losses, suggesting their implications in

protein metabolism under maintenance conditions (Abboudi et al., 2009).

The ideal protein concept has been applied in EAA requirement studies for both

terrestrial and aquatic species (Mambrini and Kaushik, 1995b; Kaushik, 1998b; Boisen

et al., 2000; Furuya et al., 2004), including crustaceans (Millamena et al., 1998; Alam et

al., 2002). In the current study, the AA profile of the balanced diets (LP, MP and HP)

was based on a range of published AA profiles of P. monodon whole body. The created

deficiency in lysine was probably not sufficient to impact growth performances of the

shrimps, although a tendency for lower N gain was observed at the medium protein

level. In contrast, methionine deficiency affected significantly N gains, however, only at

the medium protein level (MP), but not with the low (LP) or high-protein (HP) diets.

This interaction between methionine and dietary protein indicates that requirements for

EAA, when expressed as % CP, should be evaluated together with requirements for

protein, as in the present and some other mentioned studies (Wang and Fuller, 1989;

Fournier et al., 2002; Bodin et al., 2009; Thu et al., 2009).

Interestingly, growth of the shrimp was not depressed by supplying excess N.

Moreover, at intake levels exceeding N requirements, imbalances in the dietary EAA

profile did not negatively affect feed intake or growth, as shown by the similarity in

performances between shrimp fed the methionine-deficient (HPM) or the balanced high

protein (HP) diet. This observation hence suggests that the ideal protein concept of ‘a

perfect and constant ratio among individual EAA and dietary N’ (Boisen et al., 2000)

should be applied only up to the N intake level providing maximal N gain, in line with

broiler studies showing that lysine requirements are to be expressed as % CP at intake

levels below but not above protein requirement (Urdaneta-Rincon et al., 2005). Also in

kittens, increasing dietary CP while keeping methionine levels (% diet) constant did not

reduce growth or feed intake (Strieker et al., 2007), in contrast to earlier findings,

referred to as AA imbalances, in rat and other animals (Harper et al., 1970). These

74

aspects are important to consider when poor quality proteins are included at higher than

normal levels to provide a minimal level of EAA in the diet.

2.6.4. Efficiency of lysine and methionine utilisation for N gain

For vertebrates, a large debate continues to exist as to whether marginal EAA utilisation

for growth (N or AA gain) is constant (Edwards and Baker, 1999; Edwards et al., 1999;

Heger et al., 2002, 2003; Rollin et al., 2003, 2006; Abboudi et al., 2006; Hauler et al.,

2007) or not (Heger and Frydrych, 1985; Rodehutscord et al., 1995, 1997; Gahl et al.,

1991, 1994; Fatufe and Rodehutscord, 2005; Peres and Oliva-Teles, 2008; Bodin et al.,

2009). Because of this uncertainty and the fact that there is currently no such

information for crustaceans, we compared efficiency values for both EAA obtained with

the broken line and logistic regression, which fitted the data equally well (R2 > 0.95).

Based on the logistic model, the instantaneous efficiency (N gain per g AA intake)

reached a maximum of 0.77 for lysine and of 1.62 for methionine, whereas diminishing

returns in N gain began, respectively, at an intake level of 35.7% and 37.1% of that

required for maximal N gain. This observation is consistent with data from growing rats

in which diminishing return responses in N gain started for each of the ten EAA at less

than 40% of maximum gain (Gahl et al., 1991). In the same line, highest efficiencies

were observed in rats fed diets providing 30 to 60% of the requirement of the limiting

EAA (Heger and Frydrych, 1985). Assuming a linear relation between AA intake and N

gain and thus a constant efficiency value, the marginal efficiency (N gain per g AA

intake) of AA utilisation in the present study was 0.65 for lysine and 1.35 for

methionine (U slopes BLM). Regarding the efficiency of methionine utilisation, studies

in terrestrial animals suggest that the growth response to changes in methionine intake

depends on the presence of other dietary substrates (cystine, choline, betaine), which

may spare the use of dietary methionine for metabolic processes (such as

transmethylation to S-adenosylmethionine and transulphuration to cysteine) other than

for lean body growth (Heger et al., 2008). In this respect, in the presence of excess

cystine, methionine retention in growing pigs was found to be a linear function of

methionine intake (ranging from 45 to 90% of the requirement) (Chung and Baker,

1992a). When both cystine and methionine are limiting, e.g. as in the present study by

keeping cystine/methionine (0.3/1) ratios constant, the increased demand for non-

protein synthesis may result in decreased methionine utilisation (Heger et al., 2008). For

P. monodon or other shrimp species, the relative contribution of cystine to the total

sulphur AA requirement and the effect of cystine on methionine utilisation still remain

to be elucidated.

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2.7. Acknowledgements

The authors acknowledge the team of the Aqualma facility, with a special thanks to

Christian Ramamonjisoa, Abel Randrianandrazana and Andry Rakotojaona for their

technical assistance. From the INRA team, special thanks are due to Christiane Vachot,

Fred Terrier and Peyo Aguirre for their help during diet manufacturing and to Marie Jo

Borthaire for assistance with the laboratory analyses. P.-P. B and V.R. contributed to

the organisation of the experiments in Madagascar. S.J.K. and I.G. designed the study.

L.R. did the data analysis. L.R., S.J.K. and I.G. contributed to the drafting of the paper.

There are no contractual agreements for the presented data which might cause conflicts

of interest. The authors acknowledge UNIMA and institutional funds from INRA for

funding the present study and Association Nationale de la Recherche et Technologie

(ANRT, France) for the scholarship to L.R. (CIFRE PhD Research Grant).

76

77

CHAPTER 3

Effect of dietary fishmeal replacement by plant protein on requirement and availability of essential amino acids in juvenile Penaeus

monodon

***** Effets du remplacement de la farine de poisson par des protéines végétales sur les besoins et la disponibilité des

acides aminés essentiels, chez les juvéniles Penaeus monodon

78

79

3.1. Présentation de l’article

Nous avons précédemment déterminé les besoins en protéine, lysine et méthionine des

juvéniles P. monodon, élevés en conditions contrôlées (bacs de 150 L) et nourris avec

des aliments semi-purifiés (chapitre 2). Suivant ces résultats, cette deuxième étude a été

réalisée pour pouvoir comparer les résultats obtenus en milieu contrôlé et en milieu

d’élevage, afin de faciliter le transfert de connaissances. Les objectifs de cette étude

étaient i) de déterminer le niveau optimal de remplacement de la farine de poisson par

un mélange de protéines végétales en condition d’élevage (bassins en terre), ii) tout en

caractérisant l’effet de chaque aliment sur la disponibilité (digestibilité) des nutriments,

et iii) sur les besoins en acides aminés essentiels et protéine.

Cinq aliments isoprotéiques ont été formulés pour remplacer 0, 25, 50, 75, ou 100% de

la farine de poisson par un mélange de protéines végétales (gluten de maïs, gluten de

blé, colza, sorgho). Dans le premier essai, l’effet du remplacement de la farine de

poisson pendant un cycle complet d’élevage a été étudié sur des juvéniles P. monodon

(PMI de 1.5 ± 0.1g) stockés pendant 144 jours dans des bassins en terre (0.2350 ha), et

nourris avec un des cinq aliments. Dans un deuxième essai, la digestibilité apparente des

aliments a été mesurée sur des crevettes (PMI de 12.8 ± 0.4g) stockés dans des bacs de

150 L. Enfin, dans le troisième essai, les besoins en acides aminés essentiels disponibles

et protéines digestibles ont été évalués sur des juvéniles P. monodon (PMI de 4.5 ±

0.2g) stockés pendant 49 jours dans des cages de 3 m², immergées dans un bassin

d’élevage. Les animaux ont été rationnés avec l’aliment contrôle (100 % de la farine de

poisson) ou l’aliment substitué à 50%, appliquant 15, 45, 75 ou 100% du taux de

nourrissage normalement utilisé.

Les résultats indiquent qu’après 144 jours de grossissement, seules les crevettes

nourries avec l’aliment contenant 24% de farine de poissons ont des performances

semblables à celles nourries avec l’aliment contrôle (34% de farine de poisson, 0%

remplacement). Quand 50% ou plus de la farine de poisson est remplacé, on observe

une diminution significative (> 20%) du gain de poids, de l’accrétion protéique et

énergétique. La digestibilité apparente est significativement réduite dans les aliments

substitués pour la matière sèche (jusqu’à -20%), protéine (jusqu’à -17%), et des acides

aminés (P< 0.05), et en particulier celle de la leucine qui diminue de 26% dans l’aliment

totalement remplacé. Cette perte de digestibilité est significativement corrélée à

l’incorporation du gluten de maïs. Enfin, en utilisant un modèle de régression broken-

line, le type de protéine (farine de poisson ou végétale) n’affecte pas les besoins

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nutritionnels. Ces derniers, en terme digestible, sont estimés à 4.47 % matière sèche

(MS) pour l’azote, et à 2.62, 1.05, 1.18, 2.01, 2.35, 0.63, 1.13, 1.09, and 1.27 % MS,

respectivement pour l’arginine, l’histidine, l’isoleucine, la leucine, la lysine, la

méthionine, la phénylalanine, la thréonine et la valine. Ces résultats sont semblables à

ceux obtenus avec des post-larves P. monodon, à l’exception de la méthionine, dont le

besoin semble plus faible dans cette étude, probablement due à la teneur en cystine des

aliments.

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Fishmeal replacement by plant protein affects essential amino

acid availability in juvenile black tiger shrimp,

Penaeus monodon

Lenaïg Richard

Anne Surget

Vincent Rigolet

Sadasivam J. Kaushik

Inge Geurden

To be submitted

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3.2. Abstract

Three trials with juvenile black tiger shrimp (Penaeus monodon) were undertaken to

study the effects of replacing fishmeal by different levels of plant proteins on the

performances of the shrimp in semi-intensive conditions (Expt. 1), on the availability of

dietary nitrogen and essential amino acids (EAA) for the shrimp (Expt. 2) and on their

requirements for growth (Expt. 3). Five diets were formulated to replace 0, 25, 50, 75,

and 100% of the fishmeal by a mixture of plant protein (corn gluten meal, wheat gluten,

rapeseed meal and sorghum). In Expt. 1, the shrimp (initial body weight, IBW 1.5 ±

0.1g) were reared in earthen ponds for 144 days and fed one the experimental diets.

Apparent digestibility of nutrients and EAA were assessed in Expt. 2 using 150 L tanks

and shrimp of 12.8±0.4g IBW. In Expt.3, shrimp (IBW, 4.5 ± 0.2g) were reared in 3 m²

cages for 49 days and fed either the 0 or 50% fishmeal replaced diet at 15, 45, 75 or

100% of the usual feeding rate to determine the nutrient requirements. After 144 days in

grow-out ponds, shrimp fed the diet with 24% of fishmeal had similar growth as those

fed the control diet containing 34% fishmeal (0% replacement). When 50% or more of

the fishmeal was replaced, weight gain as well as N and energy gains significantly

decreased. Digestibility of dry matter, protein and energy was also significantly lower in

all fishmeal-replaced diets. In particular, leucine digestibility decreased by 26% at

100% replacement, which was significantly correlated to an increased incorporation of

corn gluten meal. Using a broken line regression, the source of protein (fishmeal vs.

plant proteins) did not affect nutrient requirement estimates. Digestible nitrogen

requirement was estimated as 4.47 g/100g dry matter (DM). Requirements for arginine

(Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),

phenylalanine (Phe), threonine (Thr) and valine (Val) were 2.62, 1.05, 1.18, 2.01, 2.35,

0.63, 1.13, 1.09, and 1.27 % DM, respectively.

Key-words: fishmeal, plant protein, amino acid, shrimp, digestibility

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3.3. Introduction

Over the past ten years, farmed shrimp production has expanded from 1.2 million to 4.7

million tonnes, increasing the demand for formulated shrimp feed (Tacon and Metian,

2008). To meet the high dietary protein requirement of shrimp, commercial shrimp

feeds are often rich (25-50% of the diet) in fishmeal (FM), the preferred protein source

due to its well-balanced essential amino acid (EAA) profile. As a consequence of the

reduction of forage fisheries, production of fishmeal is levelling off, decreasing its

availability and increasing its price (Naylor et al., 2009). Due to their wide availability

(Naylor et al., 2009), plant proteins such as soybean meal, lupin, and pea have been

investigated as potential fishmeal replacers in feed for shrimp such as whiteleg shrimp

(Litopenaeus vannamei) and black tiger shrimp (Penaeus monodon), reaching

successful substitution up to 40 or 75% (Sudaryono et al., 1999; Paripatananont et al.,

2001; Forster et al., 2002; Smith et al., 2007a). However, most of the research has been

conducted under controlled rather than practical conditions (Amaya et al., 2007), which

makes it difficult to transfer to shrimp industry.

In order to reach an adequate dietary EAA profile, a mixture of several plant proteins

rather than a sole plant protein source is often used, as proposed for fish (Gomes et al.,

1989; Regost et al., 1999; Fournier et al., 2004; Kaushik et al. 2004). Our previous work

with P. monodon (Richard et al., 2010a) also indicated that both the protein level and

EAA profile should be considered together, as protein accretion in P. monodon was not

reduced by feeding a high level (50%) of ‘imbalanced’ protein compared to an adequate

(30%) level of ‘balanced’ protein, suggesting that EAA requirements should be

expressed as a proportion of the diet or as a given intake level (per unit body weight)

rather than as a percentage of dietary protein. It also implies that a poor quality protein

sources can be included at higher than required protein levels in order to fulfil EAA

requirements. In order to avoid an EAA deficiency and thus a suboptimal protein

utilisation by the shrimp, information on the availability of the EAA from the feed is

also needed. Earlier studies in shrimp reported important differences in nutrient

digestibility of plant proteins (Akiyama et al., 1989; Brunson et al., 1997), partly

attributed to the presence of anti-nutritional factors such as protease inhibitor, phytic

acid, or tannins (Cruz-Suarez et al., 2001; Francis, 2001; Kumaraguru Vasagam et al.,

2007). The importance of considering nitrogen and EAA requirements in terms of

available nutrients is substantiated by two recent survey studies in L. vannamei,

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showing large variations between EAA availability of fishmeal and plant proteins (Yang

et al., 2009; Lemos et al., 2009).

The main objective of this study was to evaluate the effect of replacing fishmeal by a

mixture of plant proteins on the long term performances of P. monodon reared in semi-

intensive commercial conditions (pond study, Expt. 1). In addition, we examined

whether the protein source modified i) the availability of dietary nitrogen and AA

(digestibility study, Expt. 2) and ii) the growth requirements of available nitrogen and

EAA (cage study, Expt. 3).

3.4. Material and methods

3.4.1. Diets

A commercial-like control diet (FM34) was formulated to contain 34% FM as the main

protein source (59% of the dietary protein, Table 3.1). In four other diets, FM was

gradually reduced to 24 (FM24), 16 (FM16), 8 (FM8), and 0% of the diet (FM0) being

replaced by a mixture of plant protein sources (corn gluten meal, rapeseed meal,

sorghum, and wheat gluten) (Table 3.1). All five diets contained soybean meal (SBM,

from 19 to 25%) and krill and shrimp meal (KM and SM, each kept constant at 1%).

The analysed essential amino acid content of the five experimental diets was above

known requirements for P. monodon (Table 3.2). In order to meet the dietary lysine

requirements, crystalline lysine was added in diets FM8 and FM0 (Table 3.1). All feeds

were industrially manufactured by Nutrima (La Réunion, France), pelleted to be 2 mm

and stored in sealed bags prior to shipping to the farm in Madagascar where they were

stored in closed containers.

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Table 3.1. Composition and proximate analysis of the five diets fed to juvenile black tiger shrimp P.monodon

Fishmeal replacement (%) 0 25 50 75 100 Diets FM34 FM24 FM16 FM8 FM0 Ingredients (g/kg feed) Fishmeal 340 240 160 80 0 Squid Meal 10 10 10 10 10 Krill meal 10 10 10 10 10 Soybean meal (48%) 215 191 250 200 234 Whole wheat 350 200 92 160 60 Wheat gluten (80%) 0 10 10 30 50 Corn gluten meal (60%) 0 115 85 214 225 Rapeseed meal 0 0 150 100 150 Sorghum 0 150 150 100 150 Fish oil 3 8 16 20 26 Basal mixture1 73 66 66 72 80 Lysine 0 0 0 4 5

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

42.1 42.9 44.2 44.5 44.2

Crude lipid (% DM) 5.6 6.5 6.6 6.2 7.0 Gross energy (kJ/g DM) 18.8 19.4 19.7 20.1 20.2 Ash (% DM) 11.8 10.0 9.9 8.7 8.4 Inert Marker (%DM) 1.0 1.3 1.4 1.5 1.5 Leaching (%)2 6.3 (2.1) a 3.6 (1.5) b 5.3 (0.8) ab 5.2 (1.2) ab 6.2 (0.4) a

1 Basal mixture contains cholesterol, de-oiled soy lecithin, mono Ca phosphate, Ca Propionate, mineral and vitamin mixtures, and inert tracer 2 Values are means and (SD) of five or six measurements. The P-value for the diet effect was 0.0179 Table 3.2. Analysed amino acid composition of the experimental diets as g/100g dry feed and as g/16g N (in brackets) Diets

FM34 FM24 FM16 FM8 FM0 Requirements 1 EAA Arg 4.0 (9.4) 3.3 (7.7) 3.4 (7.7) 3.0 (6.8) 3.1 (6.9) 1.9 (5.3) His 1.6 (3.9) 1.4 (3.3) 1.5 (3.3) 1.3 (2.9) 1.4 (3.3) 0.8 (2.2) Ile 1.8 (4.2) 1.7 (4.0) 1.7 (3.9) 1.6 (3.6) 1.8 (4.2) 1.0 (2.7) Leu 2.9 (7.0) 3.6 (8.4) 3.5 (8.0) 4.3 (9.7) 4.8 (10.8) 1.7 (4.3) Lys 3.6 (8.6) 3.0 (7.0) 2.9 (6.6) 2.7 (6.0) 2.6 (5.9) 2.1 (5.2 - 5.8 2) Met 1.0 (2.3) 0.9 (2.1) 0.8 (1.9) 0.8 (1.9) 0.8 (1.8) 0.9 (2.4 - 2.9 2) Phe 1.7 (4.0) 1.8 (4.3) 1.9 (4.2) 2.0 (4.6) 2.3 (5.1) 1.4 (3.7) Thr 1.7 (4.0) 1.5 (3.6) 1.6 (3.6) 1.5 (3.4) 1.5 (3.4) 1.4 (3.5) Val 1.9 (4.5) 1.9 (4.3) 1.9 (4.2) 1.7 (3.9) 1.9 (4.4) 1.4 (3.4) NEAA Ala 2.2 (5.3) 2.4 (5.6) 2.3 (5.2) 2.6 (5.8) 2.7 (6.1) Asp 3.4 (8.1) 3.1 (7.3) 3.2 (7.3) 2.9 (6.5) 2.9 (6.6) Met+Cys Cys (theoretical) 3 0.5 (1.3) 0.6 (1.4) 0.7 (1.5) 0.7 (1.6) 0.8 (1.7) 1.3 (3.5)

Glu 6.0 (14.3) 6.7 (15.6) 7.0 (15.8) 8.0 (18.0) 9.3 (20.9) Gly 2.2 (5.3) 1.9 (4.5) 1.9 (4.3) 1.6 (3.7) 1.6 (3.7) Pro 1.9 (4.5) 2.2 (5.2) 2.3 (5.2) 2.8 (6.4) 3.2 (7.3) Ser 1.8 (4.2) 1.8 (4.3) 1.9 (4.3) 2.1 (4.7) 2.2 (4.9) Tyr 1.3 (3.0) 1.3 (3.2) 1.4 (3.3) 1.6 (3.6) 1.8 (4.0)

1 Estimated EAA requirements for P. monodon, from the works of (Millamena et al., 1996a, b, 1997, 1998, 1999) 2 Richard et al. (2010a) 3 calculated from theoretical composition of ingredients

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3.4.2. Experimental designs

Three studies were performed at the facility of Aqualma (Unima, Madagascar): i) a 144-

day grow-out feeding trial in 0.235 ha earthen ponds, ii) a 35-day digestibility trial in

150 l indoor tanks, and iii) a 49-day trial using 3 m² outdoor cages kept inside earthen

ponds.

3.4.2.1. Pond trial (Experiment 1)

Juvenile shrimp P. monodon (initial body weight of 1.5 ± 0.1 g) were transferred into

fifteen rectangular earthen ponds (mean depth, 1.20m; surface, 0.235 ha) and reared for

144 days under semi-intensive conditions at a mean stocking density of 7.1 shrimp/m²

(16886 shrimps per pond on average). The ponds were characterised by a loam to

sandy-loam soil. Two weeks prior to the start of the trial, ponds were prepared by

draining the water and the soil was tilled at a depth of 10 cm to improve oxidation and

mineralisation of organic matter of the pond. Lime was applied on the wet zone in the

bottom of the ponds at a level of 1.5 kg/m². One week later, the ponds were filled with

water (30-32 ppt) pumped from a common canal until an approximate level of 170 cm.

Water was filtered at the entrance of the pond with a 300 µm nylon filter. Ponds were

then fertilised with triple super phosphate (Ca(H2PO4)2.H20; 1kg/ha) and urea (10kg/ha)

to stimulate natural productivity (planktonic bloom) in the pond.

Temperature and dissolved oxygen (DO) were measured twice daily (4:00 am and 4:00

pm), whereas salinity, pH, water height, and turbidity (secchi disk) were measured

every morning. Total ammonia-nitrogen, nitrite and nitrate were measured once a week

on water sample collected close to the exit side of the pond. Mechanical aeration was

provided during the day when the levels of DO dropped below 3 mg/L.

Each diet was allocated to three replicate ponds. Feed was distributed uniformly

throughout the pond area three times a day (10:00, 14:00, and 18:00). The feeding rate

was based on feeding table of the farm and assumed a constant 100% survival.

During the trial, mean body weight was estimated every week by taking two samples of

200 shrimp from each pond (one at the entrance side, the other at the exit side of the

pond). Final mean body weight was estimated after harvesting the whole pond (on

average, 14597 shrimp per pond, equivalent to a final density of 6.2 shrimp/m²), from a

shrimp batch of 7 kg. At the beginning and at the end of the trial, samples of 500g food

deprived shrimp (14 and 12 hours, respectively) were sacrificed in cold water and stored

at -20°C for later comparative carcass analyses (dry matter, nitrogen, and energy). After

144 days of feeding, performance parameters were calculated as:

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- Survival (%): nf / ni × 100

- Weight gain (WG, kg): BIOMf – BIOMi + BIOMd

- Final mean body weight (Wf, g): BIOMf / nf

- Final yield (kg/ha): BIOMf / 0.235

- Total dry feed supplied (TFS, kg): FEED × (1- DML) × DMF

- Dry matter supplied (% biomass / day): (TFS/∆t) / BIOMm × 100

- Dry matter supplied (g/shrimp/day): ((1000 × TFS) / (nm × ∆t))

- Feed efficiency (FE): WG / TFS

- Corrected FE: WG / (TFS × ADMD)

- Protein efficiency ratio (PER): WG/ (TFS × % CP)

- Corrected PER: WG / (TFG × %CP × APD)

- N retention (%): [((Nf × Wf) – (Ni × Wi))] / [(TFS × (CP/6.25)) / nm]

- Corrected N retention (%):

[((Nf × Wf) – (Ni × Wi))] / [((TFS × %CP × APD)/6.25) / nm]

- Energy retention (%): [((Ef × Wf) – (Ei × Wi))] / [(TFS × Efeed) / nm]

- Corrected Energy retention (%): [((Ef × Wf) – (Ei × Wi))] /[(TFS × Efeed × AED) / nm]

Where ni and nf are the initial and final number of shrimp; BIOMi, BIOMf, and BIOMd

are the initial, final, and dead shrimp biomasses (kg); BIOMm is the mean biomass over

the experimental period ((BIOMi+BIOMf)/2); Wi and Wf are the initial and final mean

body weight (g); ∆t the number of experimental days; FEED is the total amount of feed

distributed (kg); DML is the % of dry matter loss of feed after the leaching test; DMF is

the dry matter content of the feed (g/100g feed); Efeed and CP are the energy (kJ/g dry

feed) and crude protein (g/100g dry feed) content of the feed; ADMD, APD, and AED,

apparent digestible coefficient (ADC, %) for dry matter, protein, and energy,

respectively; nm is the mean number of shrimp during the experimental period; Ei, Ni

and Ef, Nf are the energy and nitrogen content of shrimp (kJ/g fresh tissue and g/100g

fresh tissue) at the beginning and the end of the trial, respectively.

3.4.2.2. Digestibility trial (Experiment 2)

A five-week digestibility trial was carried out at the experimental facility of Aqualma

(Madagascar). An inert marker (SiO4, Sipernat®) was included at 2% in each of the five

feeds (FM34, FM24, FM16, FM8, and FM0) to determine apparent digestibility

coefficients. Seven shrimp (12.8 ±0.4g) were stocked in 150 L covered plastic tanks (80

× 30.5 cm; diameter × height). Each feed was randomly assigned to the tanks (nine

replicate tanks per feed, at the exception of eight replicates for the control diet). After

one week of adaptation, shrimp were fed in excess four times a day (7:00, 13:00, 19:00,

88

01:00) one of the experimental diets at a rate of 4%. Feed was distributed in three

circular trays (20 cm diameter) and left for one hour, after which all uneaten feed and

faeces were siphoned from the tanks. Three hours after the feed distribution (10:00,

16:00, 22:00, 04:00), faeces were collected by siphoning and immediately filtered

through a 100 µm mesh nylon filter and rinsed with distilled water before being stored

at -20°C in aluminium cups for later analyses. Thirty minutes prior to the next feed

distribution, the same procedure was repeated for each tank. Marker (acid-insoluble ash)

content of diets and feces was determined according to the method of Atkinson

(Atkinson et al., 1984). Apparent digestibility coefficients (ADC) of dry matter

(ADMD), protein (APD), energy (AED), and amino acids were calculated as:

ADMD: 100 × (1- (Idiet /Ifaeces))

ADCnutrient: 100 × [1- ((Xfaeces/Xdiet) × (Idiet /Ifaeces))]

With Idiet and faeces: concentration of inert marker in the feed and faeces samples (g/100g DM)

Xdiet and faeces: studied nutrient content of feed and faeces (g/100g DM)

3.4.2.3. Out-door cage trial (Experiment 3)

Twenty-four juvenile Penaeus monodon (IBW: 4.5 ± 0.2g) were stocked in closed

submerged 3 m² cages (2 × 1.5 × 0.5 m; length × width × height; 1 mm polyethylene

cover) placed in an earthen pond, at 15 cm above the bottom. Adaptation lasted two

weeks during which the shrimp were fed the control diet (FM34 diet). During the 49-

day experiment, diet FM34 and FM16 were fed to four replicate cages at a feeding rate

of 15, 45, 75, or 100% of the basal level (7% biomass per day). The basal feeding level

was chosen based on the usual feeding practice during the warm water season. Every

week, dead shrimp were removed (to avoid consumption of exuvia) and shrimp biomass

weighing to adjust the feed ration for the following week. Averaged 49-day trial values

for temperature, oxygen, pH, salinity and turbidity (secchi disk) were 30.1±1.3°C,

5.4±0.9 mg/L, 8.5±0.2, 17.0±3.6 ppt, 33.6±22.6 cm, respectively.

Feed was distributed in a circular tray three times per day (08:00, 15:00, and 22:00), and

consumption checked after each feeding time, leading to a consumption score (0: 100%

consumed; 1: 50% consumed; 2: 0% consumed). The apparent feed intakes were

corrected for the consumption score and the digestible nutrient intake calculated based

on the ADC obtained from the experiment 2. Shrimp biomass was measured weekly,

and the performance parameters were calculated as:

- Survival (%): nf / ni × 100

- Weight gain (% IBW): (Wf – Wi)/ Wi × 100)

- Feed efficiency: (BIOMf – BIOMi + BIOMd) / (FEED × (1-DML%) × DMF × ADMD)

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- Protein efficiency: (BIOMf – BIOMi + BIOMd) / (FEED × (1-DML%) × DMF × CP

×APD)

Where ni and nf are the initial and final number of shrimps; Wi and Wf are the initial and final mean body

weight (g); BIOMi, BIOMf, and BIOMd are the initial, final, and dead shrimp biomasses (g); FEED is the

total amount of feed distributed (g); DML% is the dry matter loss of feed after the leaching test; DMF is

the dry matter content of the feed (g/100g feed)., ADMD and APD are the apparent digestibility

coefficient for dry matter and protein, respectively (%).

3.4.3. Feed and faeces analyses

Water stability of feed was assessed using the methodology of Cruz-Suarez et al.

(2001). Briefly, five grams of feed (n=6) were allowed to stand in a 150 L tank filled in

with seawater (35 ppt) for one hour. Afterwards, feed was collected and dried for 48

hours at 90°C. The dry matter loss percentage (DML) was calculated as:

DML (%) = 100 × (DMbefore– DMafter) / DMbefore with DMbefore and DMafter the dry matter

content of the feed before and after immersion in water (g/100g feed). Feed intakes in

all experiment were corrected for the dry matter loss.

Feed samples were analysed for dry matter (105°C for 24 h), ash (550°C for 12 h), lipid

(Soxtherm, Gerhardt, Germany), gross energy (bomb calorimeter IKA, Heitersheim,

Germany), protein (N × 6·25, Kjeldahl Nitrogen analyser 2000, Fison Instruments,

Milano, Italy), and amino acids. Freeze-dried faeces samples were analysed for the

same components except lipid. Amino acid contents of the feeds were analysed using

the AccQ.Tag method (Waters). Briefly, 100 mg of feed was hydrolysed with 25 mL of

6N HCl and 12.5 mL of 2-mercaptoethanol (23h, 110°C). After dilution (1/25 and 1/20,

respectively), 10 µL of a standard 17 AA solution (Sigma) and hydrolysed sample were

derivatised by adding 70 µL of AccQ.Tag buffer (Waters) and 20 µL of AccQ.Fluor

reagent (6-aminoquinolyl-N-Hydroxysuccinimidyl carbonate). 5 µL of sample was

injected and analysed by HPLC (column Symmetry C18 5 µm 3.9 × 150 mm) using

three mobile phases (AccQ.Tag buffer, acetonitrile 100% and water, respectively) for an

elution time of 45 minutes (flow rate of 1 mL per minute; control temperature at 37°C).

The excitation and emission wavelengths in the fluorescence detector were 250 and 395

nm, respectively.

3.4.4. Statistical analysis

Data from Expt. 1 and 2 were analysed by a one-way analysis of variance (ANOVA)

using dietary treatment as factor (n=5 diets), followed by a comparison of means using

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Duncan’s multiple range test in case of a significant effect (P < 0.05). In Expt. 3, a

broken-line regression was used to determine the requirement for digestible protein and

essential amino acids for maximal weight gain based on the following equation:

Y = L + U. (X-R) if X > R, (X-R) = 0

With Y = dependent variable as weight gain (%) X = independent variable as digestible nutrient (% DM) U = slope or efficiency of utilisation L = maximal Y response (plateau value) R = maximal requirement

The dietary nutrient content used in the regression was calculated as: AA% × ADCAA ×

FR% where AA% is the dietary AA content (% DM), ADCAA is the apparent digestibility

coefficient of the AA, and FR% is the level of feeding (15, 45, 75, or 100% of normal).

The principal component analysis (PCA) used essential amino acids as variables and

EAA content of raw materials (EAA, % ingredient protein) and of faeces from shrimp

fed each experimental diet (EAA, % faecal protein) as individuals. Eigen-values were

analysed to extract the major principal components. Apparent digestibility coefficients

of the individual EAA were analysed against changes in the relative dietary protein

contribution of each ingredient, using linear regressions.

ANOVA and PCA were performed using STATISTICA 8.0 software (StatSoft. Inc.,

Tulsa, OK, USA). Broken-line regression and linear regressions were performed using

GraphPad Prism 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

Table 3.3. Effect of replacing fishmeal by plant protein mixture on water quality of the earthen ponds (Expt. 1) 1 Water parameters

Diets Temperature (°C) Oxygen (mg/L) Salinity (ppt) pH Turbidity (cm) FM34 25.7±0.0 7.3±0.0 34.1±0.3 8.7±0.0 40.1±1.3 FM24 25.8±0.2 7.1±0.1 34.3±0.3 8.6±0.1 36.4±3.7 FM16 25.7±0.1 7.2±0.3 34.2±0.1 8.7±0.0 38.9±1.6 FM8 25.7±0.1 7.2±0.2 34.1±0.4 8.7±0.1 38.9±1.5 FM0 25.7±0.1 7.2±0.2 34.0±0.3 8.7±0.1 37.0±2.5

P-values 2 0.9346 0.4758 0.7941 0.7186 0.3260 1 Values are means ± SD of three replicate ponds per treatment 2 P-values are given by the one way ANOVA.

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Table 3.4. Effect of fishmeal replacement on growth performance of P.monodon reared in ponds during 144 days (Expt. 1) 1 Diets

FM34 FM24 FM16 FM8 FM0 Mean SD Mean SD Mean SD Mean SD Mean SD P-values 2

Performances and feed gift (per pond)

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

Initial mean body weight (g/shr) 1.4 0.1 1.5 0.1 1.5 0.1 1.4 0.1 1.4 0.1 0.8544 Final mean body weight (g/shr) 21.0 1.3 a 19.8 0.5 a 17.2 1.1 b 16.2 1.6 bc 15.1 0.3 c 0.0003 DM supplied (% biomass per day) 2.2 0.1 2.3 0.1 2.5 0.2 2.6 0.3 2.5 0.3 0.1456 DM supplied (g/shr/day) 0.23 0.01 a 0.23 0.01 a 0.21 0.01 ab 0.21 0.00 bc 0.20 0.01 c 0.0053

1 Values are means of three replicate ponds per treatment 2 P-values are given by the one way ANOVA. Mean values with different superscript letters were significantly different between groups (P < 0.05)

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Table 3.5. Final whole body composition and nutrient retention of shrimp reared for 144 days in earthen ponds (Expt. 1)1 Diets

FM34 FM24 FM16 FM8 FM0 Final body composition (g/100g wet weight) Mean SD Mean SD Mean SD Mean SD Mean SD P-values 2

Dry matter 27.8 0.8 27.7 0.4 27.0 1.4 27.9 1.9 27.7 1.9 0.9465 Crude ash 3.2 0.1 3.4 0.2 3.2 0.3 3.4 0.2 3.7 0.1 0.1666 Crude protein 20.7 0.8 20.6 0.2 20.3 0.8 21.3 1.3 20.7 1.4 0.8193 Crude lipid 2.7 0.3 2.9 0.2 2.8 0.1 2.8 0.6 2.7 0.3 0.8859 Gross energy (kJ/ g wet shrimp) 5.9 0.2 5.9 0.1 5.8 0.3 5.9 0.4 5.9 0.5 0.9934 Nutrient retention parameters FE 0.60 0.01 a 0.56 0.02 ab 0.51 0.03 bc 0.49 0.05 c 0.49 0.03 c 0.0070 FE corrected for digestibility 0.77 0.02 0.82 0.03 0.84 0.06 0.80 0.08 0.84 0.06 0.4825 PER 1.43 0.03 a 1.31 0.04 a 1.16 0.08 b 1.11 0.11 b 1.10 0.08 b 0.0011 PER corrected for digestibility 1.56 0.03 1.59 0.05 1.43 0.10 1.41 0.14 1.47 0.10 0.1560 N gain (mg/shrimp/day) 4.48 0.19 a 4.14 0.13 a 3.50 0.34 b 3.47 0.57 b 3.10 0.16 b 0.0022 N retention (% intake) 29.2 0.7 a 26.6 1.0 ab 23.1 2.6 b 23.2 3.5 b 22.4 0.6 bc 0.0102 N retention (% digestible N intake) 31.9 0.8 32.2 1.2 28.4 3.2 29.6 4.5 30.0 0.8 0.3879 Energy gain (kJ/shr/day) 0.82 0.02 a 0.77 0.03 a 0.65 0.07 b 0.62 0.11 b 0.58 0.04 b 0.0034 E retention (% GE intake) 19.2 0.4 a 17.4 0.5 ab 15.5 1.9 b 14.8 2.4 b 14.5 0.6 bc 0.0110 E retention (% DE intake) 22.1 0.5 22.1 0.6 20.9 2.5 20.1 3.3 20.8 0.9 0.6646

1 Values are means of three replicate ponds per treatment 2 P-values are given by the one way ANOVA. Mean values with different superscript letters were significantly different between groups (P < 0.05)

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3.5. Results

3.5.1. Performances

Dietary fishmeal replacement levels did not significantly affect the water quality

parameters of the outdoor ponds during the 144 days of feeding (Table 3.3, P > 0.05).

Average temperature, oxygen, salinity, pH and turbidity were between 25.7-25.8°C, 7.1-

7.3 mg/L, 34.0-34.3 ppt, 8.6-8.7, and 36.4-40.1 cm, respectively (Table 3.3). The

highest leaching coefficients (6.2-6.3%) were observed for the control and FM0 feeds,

being different from that of FM24 (3.6%) (Table 3.1, P < 0.05). Survival (85.2 to

87.3%) was not affected by the level of fishmeal in the diet (Table 3.4, P > 0.05).

Performances of shrimp were significantly affected by the dietary replacement of

fishmeal (Table 3.4, P < 0.05). Shrimp from the control group (FM34) had the highest

biomass gain (310.9 kg), final yield (1321 kg/ha), and final individual body weight

(21.0g). When replacing 50% of the fishmeal (FM16 diet), performances were

significantly reduced of 20% in (Table 3.4, P < 0.05). The lowest growth was observed

in shrimp fed FM0 diet (218 kg of biomass gain per pond; 15.1g of final BW; 955 kg/ha

of final yield) (Table 3.4, P < 0.05). However, body composition of the shrimp was not

affected by the replacement of dietary fishmeal (Table 3.5, P > 0.05). The nutrient

retention parameters (FE, PER, nitrogen and energy retentions) were all significantly

higher for shrimp fed the FM34 and FM24 diets (0.6, 1.3-1.4, 26.6-29.2, and 17.4-

19.2%, respectively).When fed a totally FM-free diet (FM0), nutrient retention

significantly decreased of 23-24% (Table 3.5, P < 0.05). However, there was no more

significant differences between the treatments when FE, PER, and retentions were

corrected for digestibility of dry matter, protein, and energy, respectively (Table 3.5, P

> 0.05).

3.5.2. Digestibility trial (Expt. 2)

The apparent digestibility coefficients (ADC) of the control diet (FM34) were 78.3%,

91.6%, and 86.9% for dry matter, protein and energy respectively (Table 3.6).

Digestibility of individual amino acids from the control diet FM34 ranged between 91.9

and 96.9% (Table 3.6). The ADCs were significantly affected by dietary FM

incorporation levels (Table 3.6, P < 0.05). When 25% of the fishmeal was replaced by

plant protein (FM24 diet), digestibility decreased by 9% compared to control diet.

Among the AA, availability of leucine was the most affected (-14.5%), followed by

proline (-11.3%), phenylalanine (-10.6%), and alanine (-10.9%). When 100% of FM

94

was replaced (FM0 diet), ADC of dry matter, protein and energy decreased by 20, 16.8,

and 17.1%, respectively. The maximal loss of AA availability (- 26%) was observed for

leucine (Table 3.6).

Table 3.6. Effect of fishmeal replacement by plant protein mixture on the apparent digestible coefficients (ADC, %) for dry matter, protein, energy and amino acids in P.monodon (Expt. 2)

Diets FM34 FM24 FM16 FM8 FM0 Mean SD Mean SD Mean SD Mean SD Mean SD DM 78.3 1.3 a 68.9 3.8 b 61.3 4.5 c 61.8 4.5 c 58.3 3.9 c Protein 91.6 0.5 a 82.7 1.7 b 81.4 2.0 b 78.5 2.4 c 74.8 2.4 d Energy 86.9 0.9 a 78.8 1.9 b 74.0 2.4 c 73.5 2.8 c 69.8 2.1 d EAA Arg 96.6 0.3 a 93.1 0.7 b 92.1 1.1 b 90.7 0.5 c 89.6 0.5 c His 94.7 0.4 a 89.0 1.1 b 87.5 2.2 bc 85.3 0.7 cd 84.6 1.5 d Ile 95.4 0.2 a 87.5 1.3 b 86.1 2.8 b 81.6 0.4 c 80.0 2.4 c Leu 95.1 0.3 a 80.6 1.7 b 78.1 3.4 b 72.4 1.4 c 69.3 2.7 c Lys 96.9 0.2 a 94.9 0.7 b 93.9 1.0 b 94.5 0.2 b 93.9 0.4 b Met 95.7 0.2 a 89.6 1.1 b 88.2 1.5 b 85.2 0.6 c 82.1 0.7 d Phe 94.3 0.5 a 83.7 1.4 b 81.5 3.4 b 77.6 1.4 c 75.3 2.6 c Thr 94.0 0.4 a 86.5 1.1 b 84.1 2.4 b 81.2 1.3 c 78.0 1.5 d Trp - - - - - Val 94.6 0.2 a 86.9 1.1 b 85.0 2.9 b 80.5 0.3 c 79.0 2.1 c NEAA Ala 94.7 0.4 a 83.8 0.9 b 80.9 2.4 b 75.7 0.6 c 72.4 2.5 d Asp 92.6 0.5 a 86.1 1.2 b 84.1 2.2 b 81.1 1.4 c 78.4 1.5 d Glu 95.5 0.3 a 85.8 0.9 b 84.4 2.3 b 80.7 1.1 c 79.6 1.8 c Gly 91.9 0.5 a 87.2 1.2 b 84.8 2.4 bc 84.0 1.3 c 82.2 1.2 c Pro 93.3 0.6 a 82.0 1.4 b 79.8 3.2 bc 77.1 1.6 cd 75.4 2.3 d Ser 92.7 0.6 a 83.3 1.3 b 80.8 2.6 bc 78.0 1.2 c 74.9 1.6 d Tyr 95.0 0.5 a 85.1 1.4 b 82.9 2.9 b 79.7 1.2 c 77.1 1.7 c

EAA compositions of the protein sources and the faecal protein were analysed using a

multivariate analysis (principal component analysis) presented in Fig. 3.1. The space is

described by a number of dimensions defined by the variables (in our case, 9

dimensions for the 9 studied essential amino acids, EAA). The first two components

(PC1 and PC2) described together 87% of the total variability. Therefore, the data (EAA

composition of ingredients and faeces) were analysed in a factorial plane characterised

by PC1 and PC2 (Fig.3.1). The first axis (PC1, horizontal) discriminates ingredients and

faeces based mostly on threonine, arginine, lysine and valine, and methionine with a

respective contribution of 16.1, 15.7, 14.5, 13.9%, and 12.4% (Fig. 3.1). The second

axis (PC2, vertical) is mainly built on differences in leucine content (42.4% of

contribution, Fig. 3.1). On the first axis, fishmeal (FM), rapeseed meal (RM) and

soybean meal (SBM) are grouped together, being rich in most of the above cited EAA,

while wheat ingredients (whole wheat and wheat gluten), also grouped together, were

not highly discriminated (close to the 0, 0 axes intercept). Sorghum (SOR) and corn

95

gluten meal (CGM) in particular was clearly separated from the other protein sources,

reflecting its low lysine and high leucine content. Faeces were highly discriminated

along PC2, with faeces of the control group (0% replacement, F1) being in opposition

with those of the 75 and 100% replacement groups (F4 and F5) (Fig. 1), which indicates

a lower leucine content in the faecal protein from the control group than from the two

other groups, fed increased amounts of CGM and SOR rich in leucine (close following

PC2). To further investigate the relative contribution of ingredients to the digestibility

of the whole diets, linear regression analyses were done using each ingredient’s relative

contribution to dietary protein content and the apparent EAA digestibility coefficients of

the diets (Table 3.7). The results indicate that both fishmeal (FM) and whole wheat

(WW) utilisation are positively correlated to EAA availability (Table 3.7, P < 0.05), FM

contributing however more than WW to EAA availability (0.84 < R² < 0.95 and 0.75 <

R² < 0.83, respectively). All other ingredients (CGM, RM, WG, and SOR) are

negatively correlated with EAA availability (Table 3.7, P < 0.05). Among them,

increased incorporation of CGM contributes the most to the loss of availability for all

EAA (0.80 < R² < 0.89), with the exception of lysine to which SOR is strongly

correlated (R² = 0.74) (Table 3.7).

FM

SBM

WW

WG

CGM

RM

SOR

F1F1

F1

F2

F2

F2

F3F3

F3

F4F4

F4

F5

F5

F5

Principal component 1: 64%

Prin

cipa

l com

pone

nt 2

: 23

%

FM

SBM

WW

WG

CGM

RM

SOR

F1F1

F1

F2

F2

F2

F3F3

F3

F4F4

F4

F5

F5

F5

Fig. 3.1. Principal component analysis of the essential amino content (% protein) of the raw materials and of the shrimp faeces (% protein). CGM, corn gluten meal: FM, fishmeal; RM, rapeseed meal; SBM, soybean meal; SOR, sorghum; WG, wheat gluten; WW, whole wheat; F1-F5, faeces of shrimp fed the diets with FM replaced from 0 to 100%. The vectors represent the relative contribution of the major discriminating EAA (variables) to the two principal component analysis.

ARG

LYS

THR

VAL

LEU

MET

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Table 3.7. Parameter estimates obtained from the linear regressions between the ADC value of each essential amino acid and the relative contribution of each protein source to the dietary protein content (Expt. 2) Parameters raw materials ARG HIS ILE LEU LYS MET PHE THR VAL Slope FM 0.12 0.17 0.26 0.42 0.05 0.22 0.31 0.26 0.26 WW 0.64 0.95 1.38 2.37 0.32 1.19 1.76 1.43 1.42 WG -0.58 -0.83 -1.32 -2.16 -0.20 -1.15 -1.58 -1.31 -1.32 CGM -0.17 -0.24 -0.38 -0.61 -0.06 -0.32 -0.45 -0.37 -0.38 RSM -0.28 -0.41 -0.60 -1.00 -0.14 -0.52 -0.75 -0.64 -0.62 SOR -1.25 -1.92 -2.71 -4.78 -0.68 -2.28 -3.53 -2.76 -2.75 Intercept FM 89.2 83.4 78.9 67.2 93.5 81.9 73.7 77.4 77.7 WW 88.9 82.8 78.4 65.8 93.1 81.6 72.6 76.8 77.3 WG 95.2 92.2 92.5 89.5 95.8 93.7 90.1 91.0 91.5 CGM 95.9 93.3 94.0 91.9 96.1 94.8 91.9 92.5 93.2 RSM 94.7 91.5 90.9 87.1 95.9 92.4 88.5 89.9 90.2 SOR 96.2 93.9 94.3 93.4 96.9 95.0 93.1 93.0 93.4 R² FM 0 .913 0 .843 0 .892 0 .898 0 .596 0 .954 0 .876 0 .927 0 .904 WW 0 .817 0 .789 0 .749 0 .825 0 .835 0 .792 0 .814 0 .815 0 .761 WG 0 .666 0 .593 0 .675 0 .681 0 .341 0 .745 0 .650 0 .677 0 .657 CGM 0 .848 0 .802 0 .874 0 .863 0 .457 0 .894 0 .831 0 .847 0 .871 RSM 0 .590 0 .547 0 .524 0 .548 0 .568 0 .576 0 .551 0 .607 0 .555 SOR 0 .614 0 .623 0 .565 0 .657 0 .741 0 .573 0 .645 0 .597 0 .562

P-value FM *** *** *** *** ** *** *** *** *** WW *** *** *** *** *** *** *** *** *** WG ** ** ** ** * *** ** ** ** CGM *** *** *** *** ** *** *** *** *** RSM ** ** ** ** ** ** ** ** ** SOR ** ** ** ** *** ** ** ** ** * P < 0.05 ** P < 0.01 *** P < 0.001

97

Table 3.8. Parameter estimates obtained with a broken line regression using the weight gain (%) of juvenile P.monodon reared in cages for 49 days as a response criteria (Expt. 3)

Best-fit values Standard error

Diets EAA L U R L U R R² ARG 203.20 79.96 2.74 3.96 6.89 0.15 0.980 HIS 203.20 199.40 1.10 3.96 17.18 0.06 0.980 ILE 203.20 183.50 1.19 3.96 15.80 0.07 0.980 LEU 203.20 109.30 2.00 3.96 9.42 0.11 0.980 LYS 203.20 87.29 2.51 3.96 7.52 0.14 0.980 MET 203.20 332.60 0.66 3.96 28.65 0.04 0.980 PHE 203.20 192.50 1.14 3.96 16.58 0.06 0.980 THR 203.20 193.40 1.13 3.96 16.66 0.06 0.980

FM34 VAL 203.20 169.40 1.29 3.96 14.59 0.07 0.980 ARG 139.70 61.02 2.30 3.08 6.52 0.16 0.972 HIS 139.70 151.20 0.93 3.08 16.15 0.07 0.972 ILE 139.70 129.70 1.08 3.08 13.85 0.08 0.972 LEU 139.70 69.37 2.02 3.08 7.41 0.14 0.972 LYS 139.70 70.08 2.00 3.08 7.49 0.14 0.972 MET 139.70 256.00 0.55 3.08 27.35 0.04 0.972 PHE 139.70 127.10 1.10 3.08 13.58 0.08 0.972 THR 139.70 145.50 0.96 3.08 15.54 0.07 0.972

FM16 VAL 139.70 121.20 1.16 3.08 12.94 0.08 0.972 FM34 NITROGEN 203.20 49.72 4.40 3.96 4.28 0.25 0.980 FM16 NITROGEN 139.70 33.30 4.20 3.08 3.56 0.30 0.972

Fig. 3.2. Effect of dietary replacement of fishmeal by plant protein (0% or 50%) and of feeding level (% normal feeding rate) on weight gain (a), survival (b), feed efficiency (c), and protein efficiency ratio (d) of P. monodon reared in cages for 49 days (Expt. 3). For each parameter, P-values indicate the results of the two-way ANOVA. Bars represent mean values ± standard deviation (n=4). Mean values with different superscript letters are significantly different (P < 0.05).

a) b)

c) d)

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3.5.3. Nutrient requirement estimations

Survival of shrimp was not affected by feeding levels (Fig. 3.2b, P > 0.05) and varied

between 91.7 and 97.9%. There was a significant interaction between diets and feeding

levels on weight gain (Fig. 3.2a, P < 0.05). The maximal weight gain was observed in

shrimp fed the FM34 diet at 75 and 100% (200.5-205.9%) and those fed FM16 diet at

100% (143.8%) (Fig. 3.2a). Feed efficiency and protein efficiency ratio were

significantly affected by feeding level for both diets. Shrimp fed at 15% had the highest

FE and PER values (1.0 and 1.8, respectively) while those from shrimp fed at 100% was

only 0.7 and 1.3, respectively for FE and PER (Fig. 3.2c and 3.2d). PER was also

significantly affected by the type of diet (P < 0.05) and was the highest in shrimp fed

the FM34 diet (1.7 vs. 1.5, respectively for FM34 and FM16 fed shrimp). Digestible

nitrogen and EAA requirement (g/100 g DM) were determined using the weight gain (%

initial BW) as the response criterion (Table 3.8; Fig. 3.3). The maximum response (L

value) was significantly higher for the shrimp fed the FM34 diet than those fed the

FM16 diet (Table 3.8, P < 0.05). The type of protein (fishmeal or plant protein) did not

significantly affect the estimate of digestible N or EAA requirement for maximal weight

gain (R value, P > 0.05, Table 3.8). Thus, a common R estimate was calculated from the

regression, giving a N requirement of 4.47 g/100g DM. In the same manner,

requirements for arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine

(Lys), methionine (Met), phenylalanine (Phe), threonine (Thr) and valine (Val) were

2.62, 1.05, 1.18, 2.01, 2.35, 0.63, 1.13, 1.09, and 1.27 % DM, respectively (Fig. 3.3).

99

Fig. 3.3. Estimation of the requirements for digestible EAA expressed as g/100g DM

3.6. Discussion

Most studies on fishmeal replacement in feed for penaeid shrimp have been conducted

under controlled indoor conditions and over relatively short periods (Amaya et al.,

2007). To our knowledge, this is the first study on a long-term effect of fishmeal

replacement by plant protein in the black tiger shrimp P. monodon using practical diets

under commercial rearing conditions. After 144 days of earthen pond rearing, average

survival (87%) and FE (0.60) of P. monodon fed the control diet (34% fishmeal) were

comparable to values reported for L. vannamei in an intensive 203-day pond trial

(Casillas-Hernandez et al., 2007). Also the final pond yield for the control diet (1.3

tonnes /ha), when corrected for differences in initial stocking density (7.1 in our study

relative to 15 shrimp per m² in the latter study), was similar to that found for L.

vannamei (1.6 tonnes /ha, Casillas-Hernandez et al., 2007). In contrast, the values of

nitrogen retention (28-32%) as found here were slightly above those reported for P.

monodon fed a commercial diet either under intensive outdoor conditions (22-24%)

(Briggs and Funge-Smith, 1994, Jackson et al., 2003) or under controlled indoor

100

conditions (24.7%) (Richard et al., 2010a). The lower stocking density of shrimp in

semi-intensive ponds generally enables shrimp to feed more on natural biota (microbial

community and phytoplankton) known to recycle dietary and faecal N wastes, which

improves apparent N retentions (Teichert-Coddington et al., 2000; Burford and

Williams, 2001; Burford et al., 2002; Jackson et al., 2003). The high water renewal and

absence of natural biota in the 150 L tanks used by Richard et al. (2010a) most likely

contributed to lower the N retention (24.7%) of the shrimp in the previous study. Also,

low quality of feed or ingredients, overfeeding as well as poor water stability of the

pellets can lead to reduced N retention (Burford and Williams, 2001).

The mixture of plant protein ingredients in this study successfully replaced up to 25% of

the fishmeal (24% dietary fishmeal) without any adverse effect on shrimp

performances. However, growth was significantly reduced at a fishmeal replacement

level of 50% or higher (diets containing 16, 8 or 0% of FM). These results are not in

accordance with previous findings of successful growth in P.monodon fed 14% FM

(Smith et al., 2007a), 6% FM (Sudaryono et al., 1999) or even 5% FM (Biswas et al.,

2007), in association with 30% lupin seed meal, 40% lupin kernel meal, and 62%

soybean meal+L-lysine, respectively. In the former studies, the utilisation of more than

10% of other marine protein (i.e., squid or shrimp meal) most likely facilitated higher

FM replacement levels (Smith et al., 2000). Overall, higher levels of fishmeal

substitution by plant proteins have been reported in white shrimp, L vannamei. Using

rapeseed (canola) meal, soybean meal and wheat flour (Suarez et al., 2009) or fermented

grains and wheat gluten (Molina-Poveda and Morales, 2004), 6% FM successfully

maintained L. vannamei growth performances. Under practical pond conditions, even

total FM replacement (using soybean meal, corn gluten meal and sorghum) was

achieved without affecting performances of L. vannamei (Amaya et al., 2007). Such

difference in response between L. vannamei and P. monodon to low dietary levels of

FM warrants a critical analysis of factors involved in feed utilization.

Several factors may be responsible for the decrease in growth of the shrimp following

the substitution of fishmeal by the plant protein sources, such as the presence of anti-

nutritional factors, a low digestibility or a low palatability of proteins leading to reduced

feed intake (Espe et al., 2007). In the pond trial, the amount of feed distributed was

adjusted on a weekly basis to the pond’s biomass. As such, the mean daily feed supply

recalculated over the experimental period did not vary among treatments, being 2.2, 2.3,

2.5, 2.6, and 2.5% of the shrimp average biomass for diets FM34, FM24, FM16, FM8,

101

and FM0, respectively. Moreover, whereas it is not possible to accurately monitor actual

feed consumptions in ponds, the careful measurement of intakes during the small scale

trials (Expts. 2 and 3) did not reveal any particular palatability problem or feed wastage

with the plant-protein diets. Further leaching tests showed that the water stability of the

feed pellets was not negatively affected by the plant protein sources. Also, the FE (0.49-

0.60) and PER (1.10-1.43) values from the pond study were in line with previous results

on P. monodon (Sudaryono et al., 1999, Bautista-Teruel et al., 2003, Paripatananont et

al., 2001), whereas energy retentions were slightly lower than values reported for L.

vannamei (Suarez et al., 2009).

The quality of a dietary protein is determined not only by the analysed concentration in

nitrogen and EAA but also by their availability, mostly measured through estimation of

apparent digestibility coefficients (Cruz-Suarez et al., 2009, Lemos et al., 2009, Yang et

al., 2009). As such, several studies stated that differences in nutrient availability

between protein sources may affect the success of fishmeal replacement strategies

(Cruz-Suarez et al., 2001; Cruz-Suarez et al., 2009; Smith et al., 2007b; Yang et al.,

2009; Sudaryono et al., 1999). However, quantitative data on nitrogen and EAA

availability from alternative shrimp feed or from individual ingredients for shrimp is

limited. In this study, apparent digestibility of nutrients was strongly reduced at all

fishmeal replacement levels. Dry matter (DM), protein and energy digestibility were

78.3%, 91.6%, and 86.9%, respectively when fed the control diet (34% FM) and

decreased by 9% after replacing 25% of the dietary fishmeal (24% FM). At total

fishmeal replacement, digestibility of DM was only 58.3% and that of protein 74.8%,

the latter value being similar to that reported in juvenile L. vannamei (75.5%) fed a 30%

corn gluten meal based diet (Lemos et al., 2009) but lower than those reported by

Bautista-Teruel et al. (2003) in P.monodon fed a diet containing 33% feed pea meal and

17% marine protein diets (77.3 and 87.5%, respectively for DM and protein). Although

the current feed formulations (varying levels of various ingredients) were not

specifically designed to identify the individual contribution of each protein source to the

observed changes in digestibility (Brunson et al., 1997), some indirect assumptions can

be made. First, the similarity in digestibility values between diets FM24 and FM16 and

also between FM8 and FM0 suggests that rapeseed meal, which compensated for the

extra fishmeal replacement between these diets, did not negatively affect nutrient

uptakes. The same is true for soybean meal, which moreover was added at a comparable

level in the control diet. Good results with rapeseed (Buchanan et al., 1997), soybean

meal (Alvarez et al., 2007) or both (Suarez et al., 2009) were already observed in

102

shrimp. Second, the classification of ingredients and faeces according to their AA

profile (principal component analysis) points towards a low digestibility of two protein

sources, i.e. corn gluten meal (CGM) and sorghum, whose AA profiles highly reflected

those of the faecal protein. The low digestibility of CGM is further substantiated by the

negative correlations between the EAA availability coefficients and the contribution of

CGM to total dietary protein supply. This was especially marked for leucine whose

digestibility decreased by 14.5% after replacing only 25% of the FM. These findings are

consistent with recent reports of digestibility data in L. vannamei showing the low

apparent digestibility of i) CGM protein (59%) relative to that of the other test

ingredients (mostly above 80%) (Lemos et al., 2009) and also of ii) dietary amino acids

when provided by CGM compared to most other protein sources (Yang et al., 2009).

For example, digestibility coefficients in the latter study dropped from 95 to 71% for

Met, 72 to 34% for Cys, 76 to 55% for Tyr, 83 to 69% for Pro, and 81 to 65 % for Leu

when feeding corn gluten meal vs. fishmeal to L. vannamei (Yang et al., 2009).

Furthermore, the low growth performance seen in L. vannamei fed a diet containing

15% CGM possibly also stems from a reduced availability of protein and EAA from

CGM (Forster et al., 2002). The low ADC of the diet using high level of corn gluten

meal in shrimp, also seen in turbot (Regost et al., 1999), contrasts with earlier results on

teleost fish (Wu et al., 1995; Pereira et al., 2003) and requires further investigation.

Once corrected for faecal N losses, the digestible protein content of our diets ranged

from 33.1% (diet FM0) to 38.6% DM (diet FM34), respectively, which should be

adequate for P. monodon according to our previous estimations using semi-purified

diets (Richard et al., 2010a). Applying the apparent digestibility coefficients to

parameters reflecting feed utilization (PER and N or energy retentions) in shrimp from

the pond trial showed that these stayed slightly superior at the high relative to low FM

levels, although without statistical difference. In contrast, in the cage study, PER values

(corrected for digestibility) remained significantly higher in shrimp fed the diet FM34

vs. diet FM16, being respectively 1.7 and 1.5, even if comparable to values found in the

pond study. It is also worth noting here that the relative difference in growth was similar

between shrimp fed diets FM34 and FM16 in the pond (reduced by 25%) and in the

cage study (reduced by 28%, at 100% of normal feeding level), confirming the

possibility of using experimental cages in commercial ponds to simulate diet-induced

differences in growth.

103

In the cage trial (Expt. 3), the shrimp were fed graded levels of diets FM34 and FM16 in

an attempt to evaluate the growth requirement for digestible protein and EAA under

practical conditions, using two types of protein sources. Interestingly, weight gain was

similar between shrimp fed FM34 at 45% and those fed FM16 at 75%, showing that

feed allowance can be reduced by using a balanced protein source, similarly to previous

findings in L.vannamei (Venero et al., 2007, 2008b). In line with the pond trial, the

plateau value (L value) obtained with the broken line regressions indicates a lower

growth for shrimp fed diet FM16 compared to those receiving diet FM34. The

replacement of 50% of the dietary fishmeal (FM16) did however not significantly

modify the estimated requirement levels (R values) of digestible nitrogen and digestible

EAA, consistent with findings in teleosts showing that the origin of the plant proteins

(e.g. wheat gluten vs. corn gluten) only marginally influences EAA requirement

estimates (Thu et al., 2007). Although the EAA requirements were consistent with

estimates for P. monodon using semi-purified diets under laboratory conditions (Chen et

al., 1992; Millamena et al., 1996b, 1997, 1998, 1999; Richard et al., 2010a), methionine

requirement (0.63% DM) was lower than the 0.89 % DM value previously reported by

Millamena et al, (1996a) and in our own earlier work (Richard et al., 2010a), in which

dietary cystine level was, respectively, 0.41% and 0.30% DM (at adequate Met level). It

is recognised in vertebrates that part of the methionine requirement for growth can be

spared by dietary cyst(e)ine, as reported in pig (Chung and Baker, 1992b) or even

teleost fish (Walton and Cowey, 1982b; Kim et al., 1992). For penaeid shrimp little is

known on the possible interacting effect of dietary cyst(e)ine on methionine

requirements. The current lower requirement may result from a higher dietary cysteine

content, especially in the diet rich in plant protein. Nevertheless, cyst(e)ine availability

in L. vannamei is reported to be highly variable between ingredients, being 81% for

extruded soybean meal and only 34% for CGM (Yang et al., 2009). Thus, further

evaluation of cystine availability is needed to improve the use of plant proteins in diets

of P. monodon together with specific studies on the methionine-sparing effect of cystine

(part of our own ongoing research).

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3.7. Acknowledgements

The authors acknowledge Frédéric Terrier and Peyo Aguirre for their help during diet

manufacturing and to Marie Jo Borthaire and Vincent Michel for assistance with the

laboratory analyses. Special thanks are due to Christian Ramamonjisoa (Aqualma

facility) for his technical assistance.

L.R., S.K. and I.G designed the study. L.R. did the data analysis. L.R., S.K. and I.G

contributed to the drafting of the paper. A.S. did the AA analyses. L.R. and V.R.

contributed to the organisation of the experiment in Madagascar. There are no

contractual agreements for the presented data which might cause conflicts of interest.

The authors acknowledge UNIMA and institutional funds from INRA for funding this

study and ANRT (France) for the scholarship to L.R. (CIFRE PhD Research Grant).

105

CHAPTER 4

Capacity of Penaeus monodon to regulate the amino acid catabolism in response to variation in

dietary protein and methionine supplies

**** Capacité de régulation du catabolisme des acides aminés chez Penaeus monodon, en réponse à une variation de l’apport alimentaire en protéine et

méthionine

106

107

4.1. Présentation de l’article

Dans les études précédentes, nous avons établi les besoins nutritionnels en protéine et

AAE pour des crevettes P. monodon juvéniles, élevées en conditions contrôlées

(chapitre 2) ou en bassin d’élevage (immergées dans des cages, chapitre 3). Les résultats

présentés au chapitre 2 ont indiqué que l’accrétion protéique est affectée simultanément

par les apports alimentaires en protéine (10, 30, 50%) et méthionine (adéquat ou 30%

carencé). Afin de mieux caractériser le métabolisme azoté de la crevette P. monodon,

nous avons donc évalué l’influence de telles variations alimentaires sur les enzymes

impliquées dans le catabolisme des acides aminés (transdéamination). Ainsi, l’activité

de l’alanine aminotransférase (ALAT, E.C.2.6.1.2) et glutamate déshydrogénase

(E.C.1.4.1.3.) a été mesurée quatre heures après le repas dans les branchies, la glande

digestive, et le muscle abdominal des crevettes juvéniles P. monodon.

Les crevettes (PMI de 2.4g) ont été nourries pendant six semaines avec un des six

aliments semi-purifiés contenant 14, 34, ou 54% de protéine brute (à base de caséine et

d’acides aminés cristallins), adéquat ou carencé (30%) en méthionine (le ratio

cystine/méthionine étant maintenu constant à 0.3).

Pour les deux enzymes, les résultats indiquent que l’activité est la plus élevée dans le

muscle, suivi des branchies et de la glande digestible, dans laquelle la GDH est

faiblement détectée. L’activité totale de l’ALAT est significativement inférieure (P <

0.05) chez les crevettes nourries avec 14% de protéine comparée à celle nourries avec

54% protéine, mais pas différente de celle des crevettes nourries avec 34% de protéine.

Comparée à l’activité observée chez les crevettes nourries avec 34% de protéine,

l’activité de la GDH dans le muscle diminue de 35% avec 14% de protéine et augmente

de 26% avec l’aliment à 54% de protéine (P < 0.05). Dans les branchies, nous

observons une interaction significative entre le niveau alimentaire de protéine et de

méthionine sur la GDH (P < 0.05). A 34% de protéine et seulement à ce niveau

protéique, l’activité de la GDH augmente quatre fois sa valeur basale (adéquat en

méthionine) quand l’aliment est carencé en méthionine.

En conclusion, nos résultats suggèrent pour la première fois une capacité de régulation

positive et négative des enzymes du catabolisme azoté dans le muscle de P. monodon,

en fonction du niveau protéique alimentaire. Tandis que le muscle semble être l’organe

majeur de la transdéamination, les branchies quant à elles semblent impliquées dans la

formation d’ammoniaque, quand un acide aminé essentiel est limitant (ici la

méthionine).

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109

The effect of protein and methionine intake on glutamate

dehydrogenase and alanine aminotransferase activities in

juvenile black tiger shrimp Penaeus monodon

Lenaïg Richard

Christiane Vachot

Jeanine Brèque

Pierre-Philippe Blanc

Vincent Rigolet

Sadasivam J. Kaushik

Inge Geurden

Published in: Journal of Experimental Marine Biology and Ecology (2010), 391:

153-160

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4.2. Abstract

This study evaluates the influence of both dietary protein and methionine on amino acid

trans- and deamination (alanine aminotransferase, ALAT and glutamate dehydrogenase,

GDH) in three tissues (muscle, digestive gland, gills) of the marine black tiger shrimp

(Penaeus monodon). Shrimp (2.4g) were fed one of the six semi-purified diets

containing 14, 34 or 54% crude protein (% dry matter) with two levels of methionine

(normal or 30% reduced) for 6 weeks. Both ALAT and GDH activities were the highest

in the muscle. ALAT activity in muscle significantly decreased when feeding the low

versus high protein diets. Compared to those fed the intermediate protein level, GDH

activity in muscle decreased (by 35%) when fed the low and increased (by 26%) when

fed the high protein diets (P < 0.05). A significant interaction between dietary protein

and methionine was observed on GDH activity in gills which, due to the relative

methionine deficiency, increased 4-fold at the intermediate protein level. In summary,

our results demonstrate for the first time the capacity of up- and down- regulation of

enzyme activity by dietary protein levels in the muscle of P. monodon, and the active

role played by branchial tissue in ammoniogenesis in response to a relative essential

amino acid (methionine) deficiency.

Keywords: crustaceans; ALAT; GDH; dietary protein level; essential amino acids;

amino acid catabolism

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4.3. Introduction

Despite its importance in aquaculture as the second most cultured marine shrimp

species worldwide (FAO, 2007), data on protein and amino acid (AA) metabolism are

limited in the black tiger shrimp, Penaeus monodon. In natural conditions, P. monodon

is known to have a predator-like feeding behaviour (Marte, 1980). This has been

highlighted by the high protein requirement (23.9 g protein/kg BW/d) found in our

earlier study of which 20% was used for maintenance (Richard et al., 2010a). In the

same study, methionine requirement for optimal N gain was found to be 2.9% of crude

protein (CP), which agrees well with previous estimations of 2.4% CP for P. monodon

postlarvae (Millamena et al., 1996). Furthermore, using a broken line regression,

marginal efficiency of N utilisation (between maintenance and optimal growth) for N

gain was found to be only 24%, whereas total N retentions (N gain/N intake) ranged

between 11 and 17% (Richard et al., 2010a). Other studies on crustacean shrimp species

similarly reported relatively low N retentions, comprised between 10 and 15% for

Litopenaeus stylirostris (Gauquelin et al., 2007) or around 20% for P. monodon reared

in intensive commercial conditions (Briggs and Funge-Smith, 1994; Jackson et al.,

2003). These results imply the loss of a significant portion of the ingested nitrogen into

the environment, which is not used for body protein accretion. In decapod crustaceans,

ammonia is the main form of metabolic N excretion (Regnault, 1987). The identified

pathways for AA degradation in crustaceans are generally considered to correspond to

those of vertebrates (Claybrook, 1983). The detection of both alanine aminotransferase

(ALAT, EC 2.6.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.3) in several

crustacean species (Claybrook, 1983; Mayzaud and Conover, 1988: Chien et al., 2003;

Li et al., 2009) support this idea. While ALAT enables the transamination of AA, GDH

plays a central role in the flux of ammonia in the free AA pool as it catalyses the

transformation of glutamate into ketoacid and ammonia and vice versa (Greenaway,

1991). It was initially believed that only the reductive function leading to glutamate

synthesis occurred in crustacea (Claybrook, 1983). However, later studies (Batrel and

Regnault, 1985; King et al., 1985; Regnault, 1987; Greenaway, 1991) have

demonstrated also the oxidative function of GDH, underlining the central role of GDH

in crustaceans as in other species not only in ammonia uptake but also in ammonia

production.

Most studies on the activities of ALAT (Galindo-Reyes et al., 2000; Chien et al, 2003;

Pan et al., 2003) or GDH (Regnault, 1987; Rosas et al., 2001a) in crustaceans have dealt

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with the effect of environmental factors (salinity, ambient ammonia) on free AA

metabolism (osmoregulation, ammonia detoxification). Only few have documented the

effect of dietary CP supply. Among these, Litopenaeus vannamei has been reported to

increase its branchial GDH activity (towards glutamate synthesis) when fed a 50%

compared with 30% protein diet (Rosas et al., 2001), whereas ALAT activity in the

freshwater prawn Macrobrachium rosenbergii remained unchanged when fed a 25 or

35% protein diet (Manush et al., 2005). The adaptive response of AA catabolic enzymes

when fed with low versus high protein diets is a question of interest, considering the

high protein requirement observed for P. monodon. It has been suggested that the high

protein requirement of teleosts possibly reflects their incapacity to adapt their AA

catabolism to reduced dietary protein levels (Rumsey et al., 1981; Walton and Cowey,

1982), as shown by the inability to downregulate GDH or ALAT in different fish

species (Cowey and Cho, 1993; Gouillou-Coustans et al., 2002; Gomez-Requeni et al.,

2003). This has also been suggested to explain the high protein requirement in terrestrial

carnivores such as the cat, Felis silvestris catus (Rogers et al., 1977; Tews et al., 1984).

In warm-blooded vertebrates, essential amino acid (EAA) requirements are generally

positively correlated with the dietary CP intake (Morris et al., 1999). As such, each of

the EAA should be provided as a constant proportion of dietary CP, in line with the

‘ideal protein’ concept (Boisen et al., 2000). Furthermore, when dietary CP is increased

without a concomitant increase in the limiting EAA the consequent AA imbalance

results in reduced protein deposition and growth. This has been attributed to

impairments in appetite and in the utilisation of the limiting EAA (Harper et al., 1970),

related to the overall high activity of enzymes involved in AA catabolism when

confronted with excess dietary protein, leading to the loss of the limiting EAA (Morris

et al., 1999). Such AA imbalance phenomenon has not been observed in our recent

feeding trial with P. monodon, at least not for feed intake, growth or N gain (Richard et

al., 2010a).

The objectives of this study were (1) to characterize the activity of GDH and ALAT in

three different tissues of P. monodon, (2) to examine whether the activity of both

enzymes is regulated by dietary protein and methionine intake with a specific attention

to the interaction between dietary protein level and methionine deficiency.

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Table 4.1. Formulation and analysed composition of the experimental six semi-purified diets fed to P. monodon juveniles for 6 weeks

Diets

CP10 CP10m CP30 CP30m CP50 CP50m

Protein levels Low Intermediate High

Methionine levels Normal 30% less Normal 30% less Normal 30% less

Casein (g/kg feed) 1 62 186 310

Amino acid mix (g/kg feed) 45.1 45.1 135.2 135.5 225 225.6

Arginine 1 7.3 7.3 21.8 21.8 36.3 36.3

Histidine 2 0.4 0.4 1.1 1.1 1.8 1.8

Isoleucine 3 1.0 1.0 3.1 3.1 5.1 5.1

Leucine 3 2.1 2.1 6.4 6.4 10.7 10.7

Lysine 2 3.4 3.4 10.3 10.3 17.2 17.2

DL-Methionine 2 0.9 0.0 2.8 0.0 4.7 0.0

Phenylalanine 3 1.2 1.2 3.6 3.6 6.0 6.0

Tyrosine 3 0.4 0.5 1.3 1.6 2.1 2.7

Tryptophan 3 0.5 0.5 1.5 1.5 2.5 2.5

Valine 3 0.7 0.7 2.0 2.0 3.4 3.4

Aspartic acid 3 5.8 6.1 17.3 18.2 28.8 30.3

Threonine 3 1.5 1.5 4.4 4.4 7.3 7.3

Serine 3 0.3 0.4 1.0 1.3 1.6 2.1

Glutamic acid 3 2.8 3.2 8.4 9.7 14 16.2

Proline 3 5.7 5.8 17.0 17.5 28.3 29.1

Glycine 1 6.3 6.5 18.8 19.4 31.3 32.3

Alanine 3 3.9 4.1 11.8 12.3 19.6 20.5

Cystine 3 0.9 0.4 2.6 1.3 4.3 2.1

Gelatinised corn starch (g/kg feed) 4 573 359 144

Basal mixture (g/kg feed) 5 320 320 320

Analysed chemical composition

Dry matter (DM, % diet) 89.3 90.6 90.6 89.8 89.0 89.2

Crude protein (N x 6.25, % DM) 14.4 16.9 34.1 34.0 53.2 54.1

Methionine content (% DM) 0.44 0.38 0.96 0.69 1.54 1.12

Crude lipid (% DM) 6.8 7.0 7.8 6.9 7.5 7.5

Ash (% DM) 5.7 5.8 6.0 6.0 5.9 6.0

Gross energy (kJ/g DM) 19.0 19.3 20.2 20.1 21.3 21.0 1 Acros France: pure casein (CAS 9000-71-9) ; Arginine 98% ; Glycine 98% 2 Eurolysine 3 Jerafrance 4 Roquette (Lestrem, France) 5 Basal mixture (g/kg feed): 95% stabilised Cholesterol (20), Soybean lecithin (20), Fish oil (60), Sodium alginate (50), Mineral mix (50), Vitamin mix (50), Agar powder (15), Cellulose (20), Fish protein soluble concentrate (20), Attractant mixture (15): D-glucosamine 98% HCL, taurine, betaine, glycine 98% and alanine as 5:3:3:2:2.

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Table 4.2. Analysed AA composition of the experimental diets as g/kg feed and as g/16 g N (within brackets)

Diets CP10 CP10m CP30 CP30m CP50 CP50m

Arginine 11.6 (8.0) 13.7 (8.1) 28.7 (8.4) 27.4 (8.0) 42.2 (7.9) 41.8 (7.7)

Histidine 2.3 (1.6) 2.8 (1.6) 6.0 (1.8) 6.3 (1.9) 9.3 (1.8) 9.6 (1.8)

Isoleucine 5.1 (3.5) 6.1 (3.6) 12.6 (3.7) 13.0 (3.8) 19.9 (3.7) 20.6 (3.8)

Leucine 10.4 (7.2) 12.3 (7.3) 26.2 (7.7) 26.6 (7.8) 41.9 (7.9) 42.4 (7.9)

Lysine 9.1 (6.3) 10.4 (6.1) 23.5 (6.9) 23.9 (7.0) 36.3 (6.8) 36.8 (6.8)

Methionine 4.4 (3.0) 3.8 (2.2) 9.6 (2.8) 6.9 (2.0) 15.4 (2.9) 11.2 (2.1)

Phenylalanine 5.6 (3.9) 6.4 (3.8) 14.4 (4.2) 14.8 (4.3) 22.5 (4.2) 21.9 (4.1)

Valine 6.1 (4.2) 7.3 (4.3) 15.3 (4.5) 15.9 (4.7) 23.4 (4.4) 24.0 (4.4)

Aspartic acid 14.2 (9.8) 16.6 (9.8) 34.2 (10.0) 33.7 (9.9) 50.8 (9.5) 50.7 (9.4)

Threonine 5.5 (3.8) 6.5 (3.9) 13.1 (3.9) 13.3 (3.9) 20.2 (3.8) 20.7 (3.8)

Serine 4.9 (3.4) 5.8 (3.4) 11.9 (3.5) 12.6 (3.7) 18.8 (3.5) 19.8 (3.7)

Glutamic acid 21.1 (14.6) 25.1 (14.8) 50.1 (14.7) 51.0 (15.0) 83.0 (15.6) 86.7 (16.0)

Proline 15.3 (10.6) 18.2 (10.7) 37.6 (11.0) 37.8 (11.1) 58.1 (10.9) 59.0 (10.9)

Glycine 12.5 (8.7) 14.4 (8.5) 22.4 (6.6) 20.5 (6.0) 37.2 (7.0) 37.3 (6.9)

Alanine 10.5 (7.2) 12.0 (7.1) 20.4 (6.0) 19.3 (5.7) 30.5 (5.7) 30.4 (5.6)

Cystine 1.4 (0.9) 1.3 (0.7) 3.0 (0.9) 2.1 (0.6) 4.4 (0.8) 3.2 (0.6)

4.4. Materials and methods

4.4.1. Experimental diets

We applied a 3 x 2 experimental design, in which six diets (CP10, CP10m, CP30,

CP30m, CP50, CP50m) were formulated to contain one of the three crude protein levels

10, 30 or 50%. Within each dietary protein level, methionine (Met) was supplied either

in adequate proportion (based on the whole shrimp body AA profile compiled from

literature) or 30% less of the adequate proportion in order to create a relative deficiency

(Table 4.1). The reduction in methionine was done concomitantly with that of cystine

(Cys) in order to keep a constant Cys:Met ratio of 0.3. Nitrogen was supplied through a

blend of casein and crystalline AA (55:45) in each diet. The crystalline AA were coated

with agar (1.5g/100g feed) prior to mixing with the other ingredients to prevent leaching

(Richard et al., 2010a). Table 4.2 presents the analysed AA profile of the six diets.

4.4.2.Experimental animals and rearing conditions

Juvenile P. monodon (2.4 g ± 0.1, Aqualma hatchery, Madagascar) were reared in

circular 150 l fibreglass covered tanks (15 shrimp per tank) for six weeks, following a

10-day adaptation period during which they were fed the CP30 diet at 2% biomass per

d. Four replicate groups were used for each diet. Average pH, oxygen concentration,

temperature and salinity of the water were, respectively, 8.0 ± 0.1, 5.7 ± 0.5 mg/L, 30.1

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± 1.6 °C and 30.8 ± 1.8 ppt. Water exchange was applied on a daily basis, at a minimum

of 40% for each tank.

The shrimp were fed ad libitum four times a day (08:00; 13:00; 18:00; 23:00) using two

circular trays in each tank. Feed was left within the tank for 2 hours, after which feed

left-over was collected and stored at -20°C. Weekly apparent dry matter intake was

estimated after drying the collected uneaten feed to a constant weight (48h, 90°C).

Nitrogen intake was calculated from the total feed intake measured. Every two weeks,

biomass of each tank was weighed. Molt and mortality were checked daily, removing

and weighing the dead animals and exuvia. Moulting stage was recorded according to

the classification of Drach (1939) and moulting shrimp were discarded from later

biochemical analyses. Diet and whole body composition (12-h feed deprived shrimp)

analyses were done as previously described (Richard et al., 2010a).

4.4.3. Hemolymph total amino acids

The shrimp were caught by a hand net and weighed individually. Hemolymph was

sampled from six shrimp at each hour (0, 2, 3, 4, 5, and 6 hours after the last feeding)

for treatments CP10, CP30, and CP50 and at 0, 4 and 6 hour after feeding for the

methionine deficient groups (CP10m, CP30m, CP50m treatments). Hemolymph was

collected by puncture between cephalothorax and first pair of pleopods with a 1 mL

syringe (needle 25G 5/8” 0.5 x 16 mm). To prevent clogging, the syringes were rinsed

with a sodium citrate solution at 12.5% (Rosas et al., 2001a). Once collected, the

hemolymph was immediately centrifuged during 5 minutes at 5000 g, and stored at -20

°C. The six hemolymph samples at each hour were pooled per treatment in order to get

sufficient volume for total free AA (TFAA) analyses. Samples were treated by ultra-

filtration to eliminate protein, with a centrifugation at 2000 g during 2 hours followed

by a filtration (filters Amicon-Microcon, YM 100000 dalton). Five hundred µL of water

and 300 µL of ninhydrine reagent (Sigma, 2% solution) were added to 100 µL of filtrate

and left during 10 min at 100 °C. 1.5 mL of ethanol was added progressively to stop the

reaction. After 30 min in the dark, absorbance was measured at 570 nm using a

spectrophotometer (Shimadzu, UV-160A). Results were expressed in µmol.mL-1 of

hemolymph. Each analysis was performed in duplicate.

4.4.4. GDH and ALAT activities

Digestive gland, gills and abdominal muscle were carefully dissected from the same

animals used for hemolymph sampling at four hours after feeding. All organs were

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immediately weighed and frozen in dry ice prior to stocking at -80 °C. Frozen tissues

were homogenized in 10 volumes of ice-cold buffer (30 mM HEPES, 0.25 mM

saccharose, 0.5 mM EDTA, 5 mM K2HPO4, 1 mM dithiotreitol (DTT, pH 7.4) with

Ultra-Turrax (16000 round per minute). Alanine aminotransferase (ALAT, EC 2.6.1.2.)

activity was measured on homogenates first centrifuged at 1000 g for 10 min at 4 °C to

eliminate fat matter and then at 10000 g for 20 min at 4 °C. Afterwards, ALAT activity

was determined in the supernatant using a commercial kit (Biomerieux, France, ref.

63312).

For analysis of glutamate dehydrogenase (GDH, EC 1.4.1.3.), a mitochondrial enzyme,

supernatants were treated by ultrasound (1 min, 1 s pulse, amplitude 50, Vibra Cell

72405, Bioblock Scientific, France) before centrifugation (1000 g for 10 min at 4 °C ),

followed by a second centrifugation (15000 g for 20 min at 4 °C). GDH activity was

then measured using the following conditions: 175 mM Tris, 100 mM Semi-carbazine,

1.1 mM NAD, 1 mM ADP, 5 mM L-leucine, 100 mM L-glutamate. The assay of GDH

was based on measurement of the oxidation of L-glutamate into α-ketoglutarate:

GDH L-glutamate + NAD + H2O α-ketoglutarate + NH4 + NADH ADP, leucine, semi-carbazine The reaction being reversible, addition of semi-carbazine allowed orienting it towards

NADH and ammonia synthesis. Addition of ADP and leucine stabilised the enzyme.

Enzyme activity was determined from the slope of NAD reduction recorded during 10

minutes at 340 nm (37 °C) on microplate (final volume = 280 µL) with a

spectrophotometer. Enzyme activity units (IU), defined as µmoles of substrate

converted to the product per minute at the assay temperature (37 °C), were expressed

per mg of protein and per g of tissue. Protein content was determined by the method of

Bradford (1976), using bovine serum albumin (BSA) as standard.

4.4.5. Ammonia (NH3-N) excretion

At the middle of the trial, all tanks were sampled for NH3-N measurement. One tank

without shrimp was used as a blank. The first sampling was done for all tanks just after

a water renewal and prior the first feeding (shrimp unfed for 9 hours). Nine hours after

feeding the usual rations a second water sample was taken from all tanks. All samples

were stored in plastic flasks at 4 °C, containing chloroform to prevent bacterial

development. Ammonia concentrations were measured by the indophenol blue method

(Koroleff, 1983).

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4.4.6. Statistical analysis

Results are presented with means and standard deviations (SD). All parameters were

analysed by a two-way ANOVA (STATISTICA 5.0 software, StatSoft Inc., Tulsa OK,

USA) with dietary protein level (low, intermediate, and high) and methionine level

(normal, deficient) as main factors. Six samples per dietary treatment were considered

in the statistical analysis of ALAT and GDH activities (n=6), except for the HP-

treatment (n=5). Regarding GDH activity in digestive gland, only three samples (n=3)

were analysed because of the extremely low activity (limit of detection) in all

treatments. For all analyses, differences were considered significant at P < 0.05. Post-

hoc comparisons were made using a Duncan’s multiple range test in case of a

significant effect. The correlation between nitrogen intake and ammonia excretion or

TFAA concentrations were estimated by linear regression analysis using GraphPad

Prism 4.00 for Windows (GraphPad Software, San Diego CA, USA).

Fig. 4.1. Postprandial changes (from 0 to 6 hours after feeding) in total free amino acid levels in the hemolymph of juvenile P. monodon fed the three semi-purified diets CP10, CP30 and CP50. Values represent means with standard deviations (n=2 replicate analyses on samples pooled per treatment)

4.5. Results

4.5.1.Growth and nitrogen utilisation

No significant differences in apparent feed intake were observed between the ten groups

(Table 4.3, P > 0.05). As intended, shrimp receiving the CP50 diet had the highest

nitrogen intake (g/kg BW per day) followed by those fed the CP30 and CP10 diets

(Table 4.3, P < 0.05). Methionine intake was in the following order: CP50, CP50m,

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CP30, CP30m, CP10 = CP10m. Dietary protein and methionine levels had an

interacting effect on both relative weight gain and absolute daily nitrogen gain (Table

4.3, P < 0.05) as a significant decrease in weight gain (-18%) and N gain (-33%) could

be observed but only at the intermediate protein level (30%) between shrimp fed the

methionine deficient (CP30m) compared to those fed the normal methionine diet

(CP30). The hepatosomatic index (HSI) was not significantly affected by either the

dietary protein level or methionine content of the diets (Table 4.3, P > 0.05).

4.5.2. Changes in hemolymph total free amino acids (TFAA)

Data on postprandial changes of the TFAA concentrations in hemolymph of shrimp fed

the CP10, CP30, and CP50 diets are presented in Fig. 4.1. The lowest TFAA

concentrations were found for the animals fed the low protein diets (1.11 to 1.51

µmol/mL) in contrast to those fed the high protein diet (2.06 to 3.93 µmol/mL). At 0, 2

and 3 hours postfeeding, there was a positive correlation between nitrogen intake (g/kg

BW/d) and the TFAA concentration in hemolymph (0.994 < R² < 0.998; P < 0.05). The

highest concentration was observed two hours after feeding (Fig. 4.1) for the shrimp fed

the CP50 and CP30 diets (3.93 and 2.52 µmol/mL, respectively) and were back to initial

level three hours after feeding regardless of the dietary treatment. Also a positive

correlation between methionine intake and TFAA level was observed four hours

postfeeding (data not shown, R² = 0.80; P < 0.05).

4.5.3. Ammonia excretion

Shrimp fed the high protein diet demonstrated the highest ammonia excretion followed

by those fed the intermediate and low protein diets (P < 0.05). Moreover, a positive

correlation was observed between the quantity of nitrogen distributed (mg/g shrimp)

and cumulative ammonia excretion (mg NH3-N/g shrimp) between 0 and 9 hours

postfeeding (Fig. 4.2, P < 0.05). The correlation indicated that approximately 29% of

the ingested nitrogen was excreted as ammonia. No effect of the dietary methionine

level was observed on ammonia-N excretion (P > 0.05).

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Fig. 4.2. Correlation between the quantity of nitrogen distributed (mg/g shrimp) and ammonia accumulation (mg NH3-N/g shrimp) during nine hours. Y= 0.29 (± 0 .11) x + 0.06 (± 0.14), R²= 0.261, P-value: 0.0126

4.5.4.GDH and ALAT enzyme activities in abdominal tail muscle, gills and

digestive gland

Specific activities of ALAT were the highest in muscle (191 mU/mg protein) and gills

(148 mU/mg protein), which represent around 3-fold the values measured in digestive

gland (68 mU/mg protein) (P < 0.05). Whereas ALAT activities in the digestive gland

or gills were not affected by the dietary protein level, significant differences in muscle

total ALAT activity were observed between shrimp fed the CP50 (9.7 U/g muscle) and

CP10 diets (7.3 U/g muscle) (Table 4.4, P < 0.05; Fig. 4.3), albeit the total activity

variation was only -17% and +9.4%, respectively, for shrimp fed the low vs.

intermediate and high vs. intermediate protein level diets (Fig. 4.3). No effect of the

relative methionine deficiency could be detected neither on total or specific ALAT

activities (Table 4.4).

Table 4.3. Weight gain, nitrogen and methionine intakes, nitrogen gain, and hepatosomatic index (HSI) in juvenile P. monodon fed one of the six semi-purified diets for 6 weeks

Weight gain (%) 1 HSI (%) 2 Apparent feed intake (g DM/kg BW/d) 3

Nitrogen intake (g DM/kg BW/d)

Methionine intake (g DM/kg BW/d)

Absolute N gain (mg/shr/d) 4

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

CP10 133.08 c 10.51 3.17 0.61 64.40 8.35 1.49 0.19 0.28 e 0.04 0.57 c 0.18

CP10m 152.76 bc 16.44 2.91 1.11 71.48 5.11 1.94 0.14 0.27 e 0.02 0.76 c 0.26

CP30 215.27 a 1.99 2.49 0.51 66.51 1.82 3.63 0.1 0.64 c 0.02 2.24 a 0.03

CP30m 177.09 b 11.52 2.78 0.39 62.50 9.01 3.4 0.49 0.43 d 0.06 1.49 b 0.15

CP50 200.94 ab 23.09 2.60 0.29 67.06 1.61 5.71 0.14 1.03 a 0.02 2.28 a 0.27

CP50m 223.63 a 27.89 2.43 0.33 66.66 4.29 5.77 0.37 0.75 b 0.05 2.60 a 0.51

Protein 5 < 0.000 0.116 0.513 <0.000 < 0.000 < 0.000

Methionine 0.848 0.810 0.725 0.455 < 0.000 0.503

Protein × methionine 0.004 0.511 0.194 0.086 < 0.000 0.003 1 Weight gain (% initial weight): (BWfinal / BW initial) × 100 2 Hepatosomatic index (HSI): (digestive gland weight / shrimp weight) × 100 3 Apparent feed intake: (g DM/kg BW/d): [(FG-FU)/ (aBW × aN) × 42 days)] 4 Absolute Nitrogen gain (mg/shr/d): [(N shr/final × BWfinal) – (N shr/initial × BW initial)] / 42 days × 1000 With BW initial: mean initial body weight (g); BWfinal: mean final body weight (g); FG: Feed gift (g DM); FU: Feed uneaten (g DM); aBW: Average mean body weight (kg); aN: Average shrimp number; N shr/initial and N shrimp/final: itrogen content of shrimp at the beginning and end of the experiment (g/100 g DM)

5 P-values given by the 2-way ANOVA. Data were analysed by a 1-way ANOVA in case of a significant interaction and annotated with letters. In each column, different letters depict significantly different groups (P < 0.05, 1-way ANOVA)

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Specific activities of GDH (Table 4.5) were significantly higher in muscle (10.2 mU/mg

protein) than in gills (7.3 mU/mg protein). GDH activity was close to zero in the

digestive gland, irrespective of the treatment (0.03 mU/mg protein, Table 4.5).

Compared to shrimp fed the CP30 diet (0.51 U/g muscle), total GDH activity in the

muscle was significantly lower when fed the diet CP10 (0.33 U/g muscle, -35%

variation) and significantly (P < 0.05; Table 4.5 and Fig. 4.3) higher when fed diet CP50

(0.65 U/g muscle, +26.4% variation). Muscle GDH was not sensitive to a dietary

reduction in methionine. In contrast, the 2-way ANOVA on GDH activity in the gills

indicated a significant interaction between protein and methionine (Table 4.5, P < 0.05).

There was a 4-fold increase in gill GDH activity due to the relative methionine

reduction in shrimp fed the diet with the intermediate protein level (0.28 and 1.15 U/g

gill for CP30 and CP30m fed shrimp, respectively). In contrast, the reduction in dietary

methionine level did not affect gill GDH activity at low or high dietary protein levels.

Table 4.4. ALAT activities in three organs of juvenile P. monodon fed low, medium or high protein diets with adequate or deficient levels of methionine during six weeks

MUSCLE GILLS DIGESTIVE GLAND

U/g tissue mU/mg protein U/g tissue mU/mg protein U/g tissue mU/mg protein

Mean 1 SD Mean SD Mean SD Mean SD Mean SD Mean SD

CP10 6.8 1.8 189.8 41.0 8.8 2.2 166.5 57.6 3.4 4.6 62.6 83.6

CP10m 7.9 1.3 188.8 42.4 7.4 1.5 124.2 33.3 3.5 4.3 67.3 79.7

CP30 8.9 2.3 202.5 56.6 8.6 2.7 139.6 31.2 4.4 4.8 80.5 80.8

CP30m 8.7 2.2 182.4 56.7 10.3 0.7 149.8 28.4 2.5 1.5 45.6 28.6

CP50 9.8 1.1 194.8 28.7 8.5 3.9 166.8 87.5 2.9 4.1 54.1 79.5

CP50m 9.5 2.4 187.0 63.5 8.6 4.0 143.5 74.3 3.9 3.2 93.5 95.9 Prot 2 0.023 0.988 0.466 0.882 0.999 0.940

Methionine 0.786 0.578 0.895 0.334 0.834 0.910

Prot × Met 0.602 0.895 0.390 0.510 0.652 0.526 1 Means and standard deviation (n=6 per treatment, except for the HP where n=5) 2 P-values given by the 2-way ANOVA (n=6 diets per analysis).

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Table 4.5. GDH activity in three organs of juvenile P. monodon fed low, medium or high protein diets with adequate or deficient levels of methionine

MUSCLE GILLS DIGESTIVE GLAND

U/g tissue mU/mg protein

U/g tissue mU/mg protein

U/g tissue mU/mg protein

Mean 1 SD Mean SD Mean SD Mean SD Mean SD Mean SD

CP10 0.34 0.17 8.5 4.3 0.3 b 0.2 5.3 b 3.3 ND 0.0 ND 0.0

CP10m 0.33 0.15 7.4 2.8 0.5 b 0.3 7.1 ab 4.9 ND 0.0 ND 0.0

CP30 0.48 0.14 9.7 2.5 0.3 b 0.3 4.3 b 5.4 ND 0.0 ND 0.0

CP30m 0.55 0.14 11.0 3.0 1.2 a 0.4 13.5 a 2.8 0.005 0.0 0.1 0.1

CP50 0.65 0.17 12.6 3.2 0.5 b 0.5 9.9 ab 9.4 ND 0.0 ND 0.0

CP50m 0.65 0.11 12.4 1.9 0.3 b 0.4 4.1 b 4.3 0.006 0.0 0.1 0.2 Protein 2 < 0.000 0.005 0.057 0.454 0.617 0.618

Methionine 0.729 0.983 0.028 0.341 0.183 0.183

Prot × Met 0.730 0.605 0.003 0.007 0.617 0.618 1 Means and standard deviation (n=6 per treatment, except for the HP where n=5); ND: not detected 2 P-values given by the 2-way ANOVA (n=6 diets per analysis). Data were analysed by a 1-way ANOVA in case of a significant interaction and annotated with letters. In each column, different letters depict significantly different groups (P < 0.05, 1-way ANOVA)

Fig. 4.3. Changes in total activity (mean ± SEM) of GDH (blue shading) and ALAT (purple dashed shading) in shrimp muscle between low/intermediate and high/intermediate protein levels. For each protein level, values represent the change in total activity, calculated as: (ACTX-ACT CP30)/( ACTCP30) × 100 with ACTX = total activity at X protein level (X= 10%, n=12; or 50%, n=12) and ACTCP30 = total activity at medium protein level (n=12). The observed changes were significant for GDH but not for ALAT total activity in muscle.

4.6. Discussion

4.6.1. Enzyme activities in abdominal tail muscle, gills and digestive gland

The existence and functionality of both ALAT and GDH have been demonstrated in a

large variety of crustaceans (Claybrook, 1983 for review; Regnault, 1987). In the

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present study, ALAT activity was detected in all three tissues of juvenile P. monodon.

ALAT specific activities as found here in the digestive gland were lower than in gills

and muscle, but were more than 10-fold higher than those reported in the digestive

gland of the freshwater crab Chasmagnathus granulatus (Dewes et al., 2006). In

mammals as well as in teleosts, ALAT activity is mostly hepatic, being around 10-fold

higher in teleosts (Kirchner et al., 2003; Martin et al., 2003; Peres and Oliva-Teles,

2007) than that in the digestive gland of P. monodon, as observed here.

GDH activity in P. monodon, which was detected in muscle and gills but not in

digestive gland, was the highest in muscle. This confirms previous findings in

crustaceans, where muscle has been recognised as the main site for ammoniogenesis

(Greenaway, 1991). This is illustrated in crabs by the high GDH and glutamine

synthetase activities in muscle (King et al., 1985). Also in the freshwater crab

Oziotelphusa s. senex, GDH oxidative activity is higher in muscle and gills than in

digestive gland (Ramamurthi et al., 1982). The low GDH activity observed in digestive

gland also agrees with the recent study of Li et al. (2009) in L. vannamei, showing a

GDH activity of only 0.2 mU/mg protein in digestive gland as compared with

approximately 10 mU/mg protein in muscle. As in the present study, the latter authors

measured the oxidative function of GDH activity. Thus, these results would indicate

that digestive gland is little or not involved in ammonia production as previously

suggested in decapod crab (King et al., 1985). However, this absence of detectable

GDH activity in digestive gland contrasts with data in vertebrates, including fish, where

the liver is recognised as the main site of AA metabolism and which displayed hepatic

GDH activities (oxidative function) of almost 100 mU/mg protein in rainbow trout

(Martin et al., 2003) and up to 200 mU/mg protein in turbot, Scophthalmus maximus

(Gouillou-Coustans et al., 2002; Peres and Oliva-Teles, 2005). P. monodon muscle

GDH activities found in the present study (between 7.4 and 12.6 mU/mg protein) are in

line with observations in other decapod species, being 26 mU/mg protein in Cancer

pagurus (Regnault, 1993) and 9.5 mU/mg protein in L. vanammei (Li et al., 2009), and

comparable to the value of 17 mU/mg protein observed in muscle of African lungfish,

Protopterus dolloi (Frick et al., 2008). However, these levels are still lower than those

observed in skeletal muscle of rats and chicks, of 62.5 and 40.7 mU/mg protein,

respectively (Zhou and Thompson, 1996).

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4.6.2. Up and down regulation of enzyme activities by crude protein (CP)

level

Dietary CP requirements have been suggested to reflect the ability of an animal to adapt

its AA catabolism to changes in protein intake. In this respect, the high CP requirement

in species with carnivorous feeding habits, such as the cat (Rogers et al., 1977) or the

rainbow trout (Cowey and Cho, 1993), has been partly attributed to a lack of

downregulation of enzymes involved in nitrogen removal and thus a limited capacity to

conserve nitrogen at restricted protein intakes. Similar absences of adaptation of both

GDH and ALAT activities to low protein diets have been reported in rainbow trout fed

27 compared to 55% CP (Kirchner et al., 2003) or in turbot fed diets containing 15.6

compared to 50% CP (Gouillou-Coustans et al., 2002). In P. monodon or other

crustacean species with high dietary protein needs, little is known on the adaptive

response of GDH or ALAT to a restricted dietary protein supply.

In our previous modelling study, using the same three protein levels (10, 30 and 50%),

we determined diet CP30 as being the closest to meeting the protein requirement of

juvenile P. monodon (Richard et al., 2010a). In the same study, maximum marginal

efficiency of N utilisation was the highest at an N intake level coinciding with that

obtained with diet CP10. Considering the latter pattern in N utilisation efficiency, as

well as the positive correlation between N intake and total free AA in the hemolymph of

the shrimp, activity of enzymes involved in AA catabolism was expected to be modified

when feeding diet CP10 compared to diet CP30. We indeed observed a significant

reduction (35%) of GDH activity in the abdominal tail muscle in shrimp fed the low

compared to intermediate protein diet, whereas the decrease (17%) in the activity of

ALAT was not significant. Also in the freshwater prawn M. rosenbergii, ALAT activity

was found to be unmodified when lowering the dietary protein level from 35 to 25%

(Manush et al., 2005), although this could be explained by the small variation in dietary

protein. In contrast, the adaptive downregulation of muscle GDH activity as observed

here indicates that P. monodon is able to reduce AA deamination when a lower

substrate is available. This differs from observations in teleosts, where hepatic GDH

activity does not adapt to restricted protein intakes (Cowey and Cho, 1993; Kirchner et

al., 2003). In teleost fish, however, contrary to the shrimp in our study (undetectable

GDH in digestive gland), highest GDH activity was found in liver rather than in muscle

or gills (Frick et al., 2008). In crustaceans, the muscle free AA pool reacts rapidly (1 to

4h following the meal) to changes in dietary AA supplies, especially when fed a diet

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rich in crystalline AA (Fox et al., 2009). Moreover, in P. monodon at a salinity of 30 ppt

(as in the present study and close to iso-osmotic point for this species), the free AA

content in muscle was reported to be 50 times higher than in hemolymph (Fang et al.,

1992). As such, it is not surprising that adaptive changes in GDH activity occurred in

the muscle, being the major tissue regarding protein deposition and having the largest

free AA pool (Claybrook, 1983; Houlihan et al., 1986; Ballantyne, 2001; Wang et al.,

2004). Batrel et Regnault (1985) demonstrated that in the shrimp Crangon crangon L.,

starvation markedly decreased GDH oxidative activity in whole body. The present

changes in muscle GDH activity suggest that dietary composition (more or less protein)

affects the oxidative activity of this enzyme, at the muscle level.

In the present study, increasing the dietary protein above the requirement level, i.e. from

30 (diet CP30) to 50% (diet CP50) resulted in a similar whole body protein accretion.

This demonstrates the capacity of the shrimp to cope with N intakes above the

requirement for growth, which implies that the shrimp successfully eliminated the

excess of ingested nitrogen. In our experiment, the increase (9.4%) of ALAT activity in

muscle of shrimp fed diet CP50 compared to CP30 was not significant (only significant

between the two extreme diets CP50 vs. CP10). In contrast, activities of GDH were

significantly increased (26%) in tail muscle of shrimp fed diet CP50 compared to diet

CP30. Therefore the higher ammonia excretion in shrimp fed the high protein diets

might be, at least partially, due to the increase in GDH activity in muscle, the major

body free AA pool. The observed change in muscle GDH activity (26% upregulation)

however seems small as compared to changes in GDH activity in liver of teleosts. For

example, at protein intakes above growth requirement, rainbow trout increased hepatic

GDH and ALAT activity by 200 and 160%, respectively, when fed a 61 vs. 46% CP diet

(Sanchez-Muros et al., 1998). However, when comparing enzyme activities in different

organs, it is important to consider the relative size of the tissue. In shrimp, as in teleosts,

digestive gland or liver accounts for approximately 2-6% and muscle for approximately

50% of the body mass (Ceccaldi, 1997; Ballantyne, 2001). As such, the higher

amplitude of activity in liver than in muscle seen in teleosts might be related to the

smaller body mass contribution of liver. Also, a lower relative change in enzyme

activity in muscle than in liver might be equally effective in dealing with changes in

dietary AA supply.

Our earlier study (Richard et al., 2010a) estimated nitrogen retention (gain/intake) to be

between 11 and 17% for juvenile P.monodon fed the high and intermediate protein

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diets. This low nitrogen retention values seem to be in line with the 10 to 15% N

retention obtained in L. stylirostris (Gauquelin et al., 2007). In the present study,

cumulated ammonia excretion represented however only 29% of the distributed quantity

of nitrogen. Regarding the high proportion of crystalline AA in the experimental diets,

part of the ingested AA may have been excreted as such. In fact, Regnault (1987)

indicated that AAs are the second most important nitrogenous waste in crustacean

(around 10% of the total excreted nitrogen). Other reasons for the low recovery of

supplied nitrogen in the form of ammonia-N may be related to leaching of N in the

water despite the coating of AA with agar, or to bacterial degradation. Moreover, in

shrimp, ammonia and fructose-6P can react to form glucosamine (GlcN) and H2O

through the action of the reversible enzyme glucosamine-6P-deaminase. Then,

glucosamine can be acetylated to form N-acetylglucosamine (GlcNAc) which enters

chitin composition. For fast growing juvenile shrimp, it could be hypothesised that part

of the formed NH3 is oriented towards glucosamine production to be detoxified and

stored. Although this was not investigated in the present study, Regnault (1996)

suggested that in blue crabs the NH3 utilisation by formation of GlcNAc was not

important enough to reduce the excreted ammonia load. This should be investigated by

complementary measurements on P. monodon to elucidate the ammonia fate regulation,

in relation to dietary protein levels.

4.6.3. Methionine imbalance and enzyme activities

To our knowledge, this is the first ever study in a crustacean to assess the effect of

changes in an essential AA, here dietary methionine level, on the response of enzymes

involved in trans- or deamination. In fish, both the type (protein-bound vs. crystalline)

and quantity of AA supplied affect liver GDH activity rather than ALAT (Peres and

Oliva-Teles, 2006); although in rainbow trout, arginine in excess did not clearly modify

GDH activity (Fournier et al., 2003).

In the present study, the 30% methionine reduction did not modify the activity of ALAT

at none of the three dietary protein levels and in none of the three organs studied. In

contrast, dietary methionine deficiency significantly modified GDH activity, however

only at the 30% protein level (diet CP30m) and only in the gills, where GDH activity

increased by 400% compared to the balanced diet CP30. This observation was

accompanied by a significantly lower growth or N gain in shrimp fed diet CP30m,

whereas the relative methionine deficiency, as illustrated by the significant interaction,

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had no effect on growth or N gain at the other protein levels. The decreased

performance due to the relative methionine deficiency at that particular (30%) protein

level close to requirement indicates that, as expected, the methionine supply was below

the requirement for maximal body protein synthesis. Because body protein accretion

requires each indispensable AA at a specific level, the low methionine supply in diet

CP30m prevented the utilisation of the other AA for protein synthesis. As a

consequence, those AA in relative excess have to be eliminated, reflected here by an

increase in branchial GDH activity. In aquatic crustaceans, gills are recognized as the

main site for ammonia excretion (Greenaway, 1991). The present 4-fold increase in

branchial GDH activity due to the relative methionine deficiency demonstrates that gills

might also play an active role in ammoniogenesis, in addition to their well-known role

in ammonia excretion as suggested before in fish (Ballantyne, 2001). It remains

however unclear why only gill and not muscle GDH activity responded to the

indispensable AA imbalance, especially when taking into account the adaptive response

of GDH activity in muscle to changes in the level of dietary protein supply, as observed

here. In our study, the 30% methionine reduction did not affect enzyme activities at the

high dietary protein level. When feeding this methionine-deficient high protein diet

(CP50m), all AA, except methionine, are provided in excess of the required levels. As

such, the specific effect on GDH activity due to the relative methionine deficiency

becomes fairly small as compared to the effect of the overall nitrogen excess, which

significantly increased AA deamination.

The observation that relative methionine deficiency reduced the growth of the shrimp at

the intermediate but not at the high protein intake level contrasts with reports in

vertebrates of impaired appetite and reduced growth when fed high protein diets with

imbalanced AA profiles (Harper et al., 1970). From a nutritional perspective, our results

indicate that a protein source which does not supply the ideal AA profile (poor quality

protein) can be incorporated in shrimp diets at a level exceeding the animal’s protein

requirement in order to compensate for a specific AA deficiency without affecting

performances. This is illustrated here by the identical performances between shrimp fed

diet CP30 and CP50m. However, from an environmental perspective, a higher amount

of protein than required for growth leads to a marked increase in ammonia excretion.

This is to be considered when formulating shrimp diets, since the higher ammonia

excretion has direct negative consequences on the environment.

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4.7. Conclusions

The low to undetectable activity levels of ALAT and GDH in P. monodon digestive

gland suggest a minor role of this tissue in AA trans- and de-amination compared to

muscle and gills. Dietary changes in the level of CP and methionine had a more

pronounced effect on GDH than on ALAT activity. Our data suggest for the first time

that the high protein requirement of P. monodon is not related to an enzymatic disorder

in the regulation of AA deamination since P. monodon appears able not only to up- but

also to down-regulate muscle GDH activity to changes in dietary protein supply.

Moreover, the increase in branchial GDH activity as a response to a dietary methionine

deficiency (seen only at a protein intake level close to requirement) highlights the active

role of gill tissue in ammonia formation.

4.8. Acknowledgements

The authors acknowledge Frédéric Terrier and Peyo Aguirre for their help during diet

manufacturing and to Marie Jo Borthaire for assistance with the laboratory analyses.

Special thanks are due to Christian Ramamonjisoa, Abel Randrianandrazana and Andry

Rakotojaona (Aqualma facility) for their technical assistance.

S.K. and I.G. designed the study. L.R. did the data analysis. L.R., S.K. and I.G.

contributed to the drafting of the paper. C.V. and L.R. did the enzymatic analyses. J.B.

did the hemolymph TFAA analyses. L.R., P.P.B. and V.R. contributed to the

organisation of the experiment in Madagascar. There are no contractual agreements for

the presented data which might cause conflicts of interest. The authors acknowledge

UNIMA and institutional funds from INRA for funding this study and ANRT (France)

for the scholarship to L.R. (CIFRE PhD Research Grant).

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CHAPTER 5

Effect of dietary methionine, cystine and choline on growth and regulation of methionine

metabolism of Penaeus monodon

**** Effets de l’apport alimentaire en méthionine, cystine,

et choline sur la croissance et la régulation du métabolisme de la méthionine chez Penaeus monodon

130

131

5.1. Présentation de l’article

Dans les chapitres précédents, nous avons déterminé les besoins nutritionnels en

méthionine en relation avec le taux de protéine alimentaire (chapitre 2), et démontré les

conséquences d’une carence en méthionine sur le catabolisme des AA (chapitre 4) des

juvéniles P. monodon. Cependant, ces résultats n’intègrent pas l’effet possible de la

teneur en cystine dans l’aliment, car les rapports cystine/méthionine étaient maintenus

constants à un niveau semblable à celui mesuré dans la farine de poisson (0.3). Du fait

du profil en AA différent des sources protéiques végétales (faible en méthionine, riche

en cystine), il apparaît indispensable de mieux caractériser le rôle de la cystine dans le

métabolisme des AA soufrés chez la crevette. De plus, les résultats obtenus lors du

remplacement de la farine de poisson en conditions d’élevage (chapitre 3), indiquent un

besoin en méthionine plus faible que celui obtenu précédemment, probablement du à

une concentration en cystine plus élevée dans l’aliment.

L’objectif de cette étude est donc d’évaluer la capacité d’épargne de la méthionine par

la cystine (et la choline) sur l’accrétion protéique et le métabolisme de la méthionine

(reméthylation et transsulfuration) chez les juvéniles P.monodon. Pour cela, les

crevettes (PMI de 3.3g) ont été élevées pendant 35 jours en conditions contrôlées (bacs

150 L). Trois aliments semi-purifiés (caséine + acides aminés cristallins) isoprotéiques

(30% de protéine brute) sont formulés pour apporter trois niveaux en acides aminés

soufrés (AAS : méthionine + cystine) : adéquat (CTL), 30% carencé (DEF30), ou 50%

carencé (DEF50). Dans chacun de ces aliments, le ratio cystine/méthionine est maintenu

constant à 0.4, et l’apport en choline est adéquat (3g/kg aliment). Trois aliments

similaires (CTL+CC, DEF30+CC, DEF50+CC) sont formulés pour contenir de la

choline en excès (7 g/kg aliment). Enfin, dans deux aliments supplémentaires carencés

en méthionine, la cystine est ajoutée en excès pour maintenir l’apport en AAS à un

niveau adéquat (1.1-1.2 g/100g MS) (aliments DEF30+cyss et DEF50+cyss avec un

rapport cystine/Met respectif de 0.9 et 1.6).

Les résultats indiquent que l’accrétion azotée est significativement affectée par la

carence en AAS (P < 0.05). En revanche, l’ajout de choline et cystine dans les aliments

carencés en méthionine permet de maintenir l’accrétion azotée au même niveau que

celui du groupe contrôle. Ainsi, nous démontrons que la méthionine utilisée pour

l’accrétion azotée peut être épargnée jusqu’à 50% par un excès de choline ou de cystine.

Pour la première fois, nous détectons l’activité de la BHMT et CBS dans la glande

digestive de la crevette P. monodon. Seule la BHMT semble répondre à une carence

alimentaire en AAS. En revanche, un apport excessif de cystine ou choline ne modifie

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pas le niveau d’activité enzymatique pour la reméthylation ou la transsulfuration de la

méthionine. Nos résultats suggèrent aussi une capacité de synthèse de la taurine à partir

de la cystine quand l’aliment est carencé en méthionine (30%). D’autres recherches

doivent être conduites afin de mieux caractériser la régulation métabolique de l’épargne

de la méthionine, et la possible biosynthèse de taurine chez P. monodon.

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The effect of choline and cystine on the utilisation of

methionine for protein accretion, remethylation and

transsulfuration in juvenile shrimp Penaeus monodon

Lenaïg Richard

Christiane Vachot

Anne Surget

Vincent Rigolet

Sadasivam J. Kaushik

Inge Geurden

Accepted in British Journal of Nutrition

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5.2. Abstract

This 35-day feeding experiment examined in juvenile shrimp Penaeus monodon (3.3g

initial body weight) the effects of methionine (Met), choline and cystine on protein

accretion and the activity of two key-enzymes of remethylation (betaine-homocysteine

methyltransferase, BHMT) and transsulfuration (cystathionine β-synthase, CBS). The

interaction between methionine and choline was tested using semi-purified diets

adequate, 30 or 50% limiting in total sulphur amino acid (SAA) content with a constant

cystine/Met ratio. The diets contained either basal or excess choline (3 g vs. 7 g/kg

feed). Cystine was added to two other 30 and 50% Met-limiting diets to adjust the SAA

supply to that of the control diet in order to evaluate the interaction between methionine

and cystine. As expected, nitrogen accretion was significantly lower with the SAA-

limiting diets but increased back to control levels by the extra choline or cystine,

demonstrating their sparing effect on Met utilisation for protein accretion. We show for

the first time, the activities of BHMT and CBS in shrimp digestive gland. Only BHMT

responded to the SAA-deficiencies whereas the extra choline and cystine did not

stimulate remethylation or downregulate transsulfuration. Our data also suggest the

capacity of P. monodon to synthesise taurine, being significantly affected by the cystine

level in the 30% SAA-limiting diets. Further research is warranted to better understand

the metabolic regulation of taurine synthesis in shrimp and of the observed Met-sparing

effects.

Keywords: crustacean; sulphur amino acid; methionine utilisation; methionine sparing;

taurine.

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5.3. Introduction

The marine black tiger shrimp Penaeus monodon is the world’s second most cultured

crustacean species (FAO, 2007). For crustacean shrimp, as for farmed finfish, plant

protein sources are increasingly included in the feeds in order to reduce the reliance on

wild-caught marine protein sources. However, the replacement of fish or shrimp meal

by plant protein sources changes the amino acid (AA) profile of the diet, with

methionine (Met) as one of the first-limiting essential AA (Davis and Arnold, 2000;

Nguyen and Davis, 2009). In P. monodon fed an optimal crude protein (CP) level, we

previously noted that a 30% Met-deficiency diminished protein accretion (Richard et

al., 2010a) and increased deamination (Richard et al., 2010b), suggesting a change in

AA catabolism in shrimp receiving an imbalanced dietary Met supply. In P. monodon,

or crustacean species in general, not much is known on the metabolic utilisation of Met

besides its need for protein synthesis. In contrast, the importance of Met as a methyl-

group donor for methylation reactions and as a precursor of other sulfur-containing

compounds, such as cysteine or taurine, is well recognised in vertebrates (Finkelstein et

al., 1986, 1988; Stipanuk, 2004; Baker, 2006; Espe et al., 2008). As such, homocysteine

(Hcy), at the branch point of the three major pathways of Met metabolism

(transmethylation, remethylation and transsulfuration), is often regarded as a regulatory

component of Met metabolism since Hcy can be either transsulfurated for cysteine

production (catalysed by cystathionine β-synthase, CBS) or remethylated into Met. In

rat liver, the remethylation of Hcy into Met occurs by two different pathways using

either the folate-vitamin B12 dependent enzyme methionine synthase (MS) or betaine-

homocysteine-methyltransferase (BHMT) (Stipanuk, 2004), which appear to contribute

equally to the regeneration of Met (Finkelstein and Martin, 1984).

We recently evaluated the Met requirement for maximal protein gain in P. monodon to

be 0.56 g per kg body weight per day, corresponding to a dietary level of 0.8% Met (%

DM) (Richard et al., 2010a). This value is close to the Met requirement value of 0.9%

found for post-larval P. monodon (Millamena et al., 1996), but lower than the 1.3-1.4%

Met requirement values reported for juvenile P. monodon (Liou and Yang, 1994) or

kuruma shrimp, Marsupenaeus japonicus (Teshima et al., 2002; Michael et al., 2006).

The total sulphur AA (SAA) requirements estimated at 1.1% (0.8 % Met) by ourselves

in an earlier study (Richard et al., 2010a) and 1.3% (0.9% Met) by Millamena and

coworkers (Millamena et al., 1996) included 0.3% and 0.4% of cystine, respectively.

Little attention has been paid to the interaction between dietary Met and cyst(e)ine when

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determining Met requirements in shrimp, despite the ample evidence of the Met-sparing

effect of cystine in vertebrates such as teleosts (Walton and Cowey, 1982b; Cowey et

al., 1992; Nguyen and Davis, 2009), birds (Garber and Baker, 1971; Sasse and Baker,

1974) or mammals (Teeter et al., 1978; Chung and Baker, 1992c) where 50% or more of

the requirement for Met can be covered by a dietary supply of cystine.

Dietary choline has been found to improve growth providing free methyl groups

(Emmert et al., 1998; Wu and Davis, 2005; Dilger et al., 2007) thus exerting a sparing

effect on Met utilisation (Simon, 1999; Wu and Davis, 2005; Michael et al., 2006). As

BHMT is directly involved in the remethylation of Met through betaine, this enzyme is

suggested to have a dual role: in the catabolism of choline (betaine) and / or

conservation of Met depending on the dietary level of Met (Simon, 1999).

For crustaceans, there is no information on the nutritional regulation of enzymes

involved in SAA metabolism. In this study, we examined the potential sparing effect of

dietary choline and cystine on the utilisation of Met for protein accretion in juvenile

shrimp P. monodon and their effect on the activity of two enzymes of Met metabolism

involved in remethylation (BHMT) and transsulfuration (CBS).

5.4. Material and methods

Within one experiment, we investigated in shrimp the Met-sparing effect i) of dietary

choline when Met was either adequate (CTL) or limiting (30 and 50%) in the diet (diets

CTL, CTL+CC, DEF30, DEF30+CC, DEF50, and DEF50+CC) and ii) of cystine added

to the 30 and 50% Met-limiting diets (diets DEF30, DEF30+ Cyss, DEF50, and

DEF50+ Cyss). The first series of diets was formulated to contain decreasing levels of

SAA with a constant cystine/Met ratio. The second series contained similar levels of

total SAA, by modifying the cystine/Met ratio.

5.4.1. Experimental diets

Eight semi-purified iso-nitrogenous diets were formulated to supply three dietary SAA

levels and two choline levels (Tables 5.1 and 5.2). The dietary crude protein (CP) level

was based on our previous study with juvenile P. monodon (Richard et al., 2010a) and

formulated to be 35% CP as fed (38% diet DM). Nitrogen was supplied by casein and a

crystalline AA blend at a ratio of 43:57 (Table 5.1). The diets were manufactured by

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Institut National de la Recherche Agronomique (INRA) at the experimental facility of

Donzacq (France). The crystalline AA blend was coated with 2% agar dissolved in

warm water (pH 7, 40°C). Glucosamine and fish protein soluble concentrate (CPSP 90)

were added to improve feed palatability. Casein, cholesterol, soybean lecithin, sodium

alginate, cellulose, CPSP 90, starch, glucosamine, minerals and vitamins were first

mixed together and homogenised before adding the coated AA, fish oil and choline

chloride. After thorough mixing, feed was pelleted (3 mm), dried at 40°C and shipped to

experimental facility in Madagascar, where it was stored at 4°C.

5.4.1.1. Study of the Met-sparing effect of choline

Three diets were formulated to be adequate (diets CTL) or 30 or 50% limiting in Met

(diets DEF30 and DEF50, respectively). In these diets (CTL, DEF30 and DEF50), the

ratio of Cystine:Met was kept constant at 0.4 so that total SAA levels decreased

according to the Met level (from 1.2 to 0.6 g/100g DM, Table 5.2). The adequate Met

level (0.8 g/100g DM) corresponds to the requirement value determined in our previous

study (Richard et al., 2010a). Three supplemental diets (CTL+CC, DEF30+CC,

DEF50+CC) were formulated to supply choline in excess (7000 mg/kg dry feed).

Choline chloride (CC), an efficient choline source in shrimp (Michael et al., 2006), was

used in the diets. The basal level of choline in the diet was 3000 mg/kg DM, as in our

previous study (Richard et al., 2010a).

5.4.1.2. Study of the Met-sparing effect of cystine

We used the same two diets as previously described (DEF30 and DEF50). In two other

diets, cystine (cyss) was added in excess (DEF30+Cyss and DEF50+Cyss) in order to

adjust the total SAA supply to that of the diet CTL (1.1-1.2 g/100g DM). In these diets,

the Cystine:Met ratios were 0.9 for diet DEF30+cyss and 1.6 for diet DEF50+cyss.

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Table 5.1. Formulation of the experimental semi-purified diets fed to juvenile P. monodon for 5 weeks Ingredients (g/kg diet) Diets

Basal mixture 1 621

Casein 2 160

AA mixture 212

Arginine HCl 3 31.7

Histidine HCl 3 3.4

Isoleucine 3 6.4

Leucine 3 12.5

Lysine 4 7.5

DL-methionine 4 From 0.1 to 4.4 5

Phenylalanine 3 6.9

Threonine 3 7.2

Tryptophan 3 2.4

Valine 3 5.8

Alanine 2 16.2

Aspartic acid 3 25.6

Cystine 3 From 1.2 to 7.2 5

Glutamic acid 3 24.5

Glycine 2 23.9

Proline 3 20.5

Serine 3 4.8

Tyrosine 3 5.2

Choline chloride 2 6 0-7

1 The basal mixture supplied (in g/kg diet as fed): Gelatinised starch, 310; Fish oil, 59; Soy lecithin, 20; Sodium alginate, 49; Cellulose, 20; Agar, 20; stabilised Cholesterol, 15; Fish protein concentrate CPSP 90, 20; D-Glucosamine 98% HCl, 8; Mineral mixture, 50 ; Vitamin mixture, 50. The mineral and vitamin mixtures were as presented in Richard et al. (2010a) and supplied 167 g/kg mixture of choline chloride (60%). 2 Acros (France); 95% pure casein (CAS 9000-71-9) 3 Jerafrance (France) 4 Eurolysine (France) 5 The crystalline AA mixture supplied methionine at 4.5, 2.3, and 0.1 g/kg feed and cystine at 2.9, 2.0, and 1.2 g/kg feed in the control, 30 and 50% Met-limiting diets, respectively. For the cystine-enriched diets, the AA mixture supplied 5.1 and 7.2 g/kg feed, respectively in the 30% and 50% Met-limiting diets. An increase in the amount of non-essential AA compensated for varying Met and cystine levels. 6 Choline chloride (99%) was supplemented at 7g/kg feed in choline-enriched diets. Gelatinised starch (in basal mixture) compensated for the choline addition.

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Table 5.2. Analysed chemical composition of the experimental semi-purified diets fed to juvenile P. monodon for 5 weeks

Diets 1

CTL CTL + CC

DEF30 DEF30 + CC

DEF50 DEF50 + CC

DEF30 + Cyss

DEF50 + Cyss

DM (% diet) 89.6 90.1 90.6 90.2 90.2 90.1 90.5 90.1

Crude protein (N x 6.25, % DM)

37.5 38.4 37.7 38.3 38 38.1 37.8 38.7

Crude fat (% DM) 8.5 8.9 10.3 9.7 9.9 9 9.1 8.7

Ash (% DM) 5.9 6.0 5.9 6.0 5.9 5.9 5.8 6.0

Gross Energy (kJ/g DM) 19.8 19.8 19.8 19.7 19.9 19.8 19.8 19.7

Choline (% DM) 0.34 0.75 0.29 0.74 0.34 0.71 0.28 0.21

Amino acids (g/kg DM)

Arg 46.4 45.6 43.5 43.9 44.1 46.6 43.3 48.2

His 7.1 5.7 6.0 5.9 6.3 7.0 6.1 7.0

Ile 14.5 14.5 12.9 13.5 12.7 14.7 13.3 14.5

Leu 26.1 25.9 23.0 23.7 23.8 26.5 23.6 26.6

Lys 22.9 22.7 20.2 21.1 21.0 22.4 20.5 22.0

Met 8.3 8.3 5.6 5.9 4.2 4.4 5.8 4.5

Phe 14.8 14.5 11.8 13.4 13.5 14.9 12.9 15.0

Thr 15.6 15.3 13.9 14.2 14.3 15.2 14.0 15.5

Val 16.3 15.7 16.6 14.8 13.4 15.3 15.1 15.5

Ala 23.3 22.7 21.5 22.0 22.5 23.6 20.3 23.4

Asp 37.0 35.6 34.7 34.8 35.5 36.9 32.7 35.5

Cystine 3.4 3.2 2.6 2.7 1.8 1.7 5.4 7.2

Glu 55.7 54.2 51.2 52.3 54.0 56.9 50.3 54.6

Gly 27.4 27.4 26.3 26.3 26.5 28.0 25.9 27.8

Pro 38.7 37.6 33.6 34.0 35.8 38.4 33.3 39.1

Ser 13.0 12.9 12.2 12.6 12.7 13.5 12.1 13.0

Tyr 14.2 14.0 12.4 12.9 12.6 13.8 13.6 16.0

Ratio Cystine:Met 0.4 0.4 0.5 0.5 0.4 0.4 0.9 1.6

Total SAA supply (% DM) 1.2 1.1 0.8 0.9 0.6 0.6 1.1 1.2 1 CTL: control diet, adequate in Met or SAA; DEF30: 30% Met- or SAA-limiting diet; DEF50: 50% Met- or SAA-limiting diet; DEF30+CC and DEF50+CC: with excess choline chloride (CC); DEF30+Cyss and DEF50+Cyss: with extra cystine in order to adjust the SAA level to that in control diet.

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5.4.2. Animal husbandry

Juvenile P.monodon (3.3 ± 0.1g, Aqualma hatchery, Madagascar) were reared in

circular 150 l fibreglass covered tanks (15 shrimp per tank) for five weeks, following a

7-day adaptation period during which they were fed the control diet (diet CTL) at 3.5%

biomass/day. Four replicate groups were used for each diet. Average temperature,

oxygen concentration, pH and salinity of the water were, respectively, 29.8 ± 1.2 °C, 6.1

± 0.4 mg/L, 8.0 ± 0.1 and 35.0 ± 0.5 g/L. Water was exchanged on a daily basis, at a

minimum of 40% for each tank.

The shrimp were fed ad libitum four times a day (08:00; 13:00; 18:00; 23:00) using

three circular trays in each tank. After 2 hours, feed was removed and the left-over

carefully collected and stored at -20°C. Weekly apparent dry matter intake was

calculated after drying the uneaten feed to a constant weight (48h, 90°C). Every week,

biomass of each tank was weighed. Mortality was checked daily and dead shrimp were

removed and weighed. Shrimp in ecdysis were not considered for final sampling. A

representative sample of 12-h feed-deprived shrimp was taken at the start (25 shrimp)

and the end of the experiment (5 shrimp from each of the four replicate tanks per dietary

treatment) for whole body composition analysis. All samples were kept at -20°C prior to

analyses. Feed and whole carcasses were analysed for dry matter (105°C, 24 h), ash

(550°C, 12 h), and protein (N × 6.25, Kjeldahl Nitrogen Analyser 2000, Fison

Instruments, Milan, Italy). Feed samples were analysed for choline content at the

laboratory IPL (Bordeaux, France). Daily growth coefficient (DGC, %) was calculated

as: (Wf1/3 - Wi

1/3) /∆t) × 100 with Wi and Wf representing mean initial and final body

weights (g), respectively and ∆t, the duration of the growth trial (35 days). Nitrogen

gain (mg/shrimp/day) was calculated as ((Nf × Wf) – (Ni × Wi))/∆t × 1000 with Ni and

Nf, the nitrogen content of shrimp at the beginning and the end of the experiment

(g/100g fresh matter).

5.4.3. Enzyme activities

5.4.3.1. Tissue sampling and preparation

At sampling days, the shrimp were allowed to eat for one hour before the excess feed

was removed from the tank. Three hours after feed removal, shrimp were sampled;

digestive gland were quickly dissected out, immediately weighed and frozen in dry ice

prior to stocking at -80°C. Frozen tissues were homogenised in 10 volumes of ice-cold

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phosphate buffer (0.04M, pH 7.4, EDTA 1mM, DTT 1 mM) with Ultra-Turrax (16000

rpm). The homogenates were centrifuged at 1000 × g (4°C for 10 min). The supernatant

fraction was then centrifuged at 45000 × g (4°C for 20 min). Samples were analysed for

betaine-homocysteine methyltransferase (BHMT, EC.2.1.1.5) and cystathionine β-

synthase (CBS, EC.4.2.1.22) activities. Protein content of the digestive gland was

determined using Bradford method with bovine serum albumin as standard. From

preliminary results, we determined the optimal protein concentration to be 1 mg protein

per tube for both BHMT and CBS activity determinations and the sample volume to be

added in each tube was adjusted to this protein concentration.

5.4.3.2. Enzyme activity determinations

Measurements of BHMT activity were based on Finkelstein and Mudd (1967) and

Lambert et al. (2002). The following compounds were incubated for 90 min at 37°C in a

volume of 467.5 µL: 37.5 µL of 466.6 mM potassium phosphate buffer (pH 7.4), 35 µL

of 100 mM DL-homocysteine (Sigma-Aldrich, France), 55 µL of 59 mM betaine 14CH3

(ARC, France) at 2500 Bq. The volume of each tissue extract was adjusted according to

its protein concentration and homogenisation buffer (0.04 mM potassium phosphate

buffer, pH 7.4; 1 mM EDTA; 1 mM DTT) was added to compensate volume if

necessary. After incubation, the reaction was stopped by adding 62.5 µL of cold water

and 280 µL of the mixture was pipetted into 10 mL polypropylene columns column (0.8

x 4 cm; Biorad, France) containing 2.5 mL of Dowex® ion exchange resin (1X4 (Cl-),

200-400 mesh, Sigma Aldrich, France). The non-converted betaine was eluted with 10

mL of water, and the labelled products with 10 mL of 1 M acetic acid (from preliminary

tests, acetic acid gave a 85% product recovery).

Measurements of CBS activity were based on Mudd et al. (1965) and Lambert et al.

(2002). The following compounds were incubated for 120 min at 37°C in a volume of

400 µL: 100 µL of mix reagent (buffer with 1.2 M Tris-HCl, pH 8.3 and 20 mM EDTA;

500 mM DL-homocysteine; 0.6 mM pyridoxal phosphate), 20 µL of 50 mM L-serine 3-14C (ARC, France) at 2500 Bq. The added volume of each tissue extract was adjusted

according to its protein concentration and homogenisation buffer (0.03 mM potassium

phosphate buffer, pH 6.9; 1 mM EDTA; 1 mM DTT) was added to compensate volume

if necessary. After incubation, the reaction was stopped by adding 400 µL of cold

trichloroacetic acid 10% and all samples were centrifuged for 5 min at 6500 rpm. 500 µL

of supernatant fraction was pipetted into 20 mL of water. The mixture was eluted to the

column containing Dowex® ion exchange resin (50WX4 (H+), 200-400 mesh, Sigma

Aldrich, France) by rinsing consecutively with 18 mL of water, 35 mL of 0.4 M HCl, 10

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mL of water and 4 mL of 2M NH4OH. Preliminary tests indicated a recovery of 54%

with HCl and NH4OH. Radioactivity of samples was measured in a Packard Tri-Carb

liquid scintillation counter after addition of 10 mL of Ultima-Gold™ reagent (Perkin

Elmer, France). All analyses were done in duplicate and a blank value obtained from

incubation of a heat-inactivated extract was subtracted for each sample. BHMT and

CBS activities are expressed as unit (U) per mg protein where one unit is defined as one

nmole of betaine and serine transformed in 90 and 120 min reaction, respectively.

5.4.4. Amino acid analyses

5.4.4.1. Feed protein bound amino acids (AA)

100 mg of feed was hydrolysed (23h, 110°C) with 25 mL of 6N HCl and 12.5 mL of 2-

mercaptoethanol. For the analysis of cystine (through cysteic acid measurement),

mercaptoethanol was replaced by a solution of phenol 1% and sodium azide 0.2%. After

dilution (1/20), 10 µL of the hydrolysed sample was derivatised by adding 70 µL of

AccQ.Tag buffer (Waters) and 20 µL of AccQ.Fluor reagent (6-aminoquinolyl-N-

Hydroxysuccinimidyl carbonate) in a 0.5 mL microtube. A diluted (1/25) standard

solution of 17 AA (Sigma) was derivatised in the same way. 5 µL of each sample was

then analysed by HPLC (column Symmetry C18 5 µm 3.9 × 150 mm) using three

mobile phases (AccQ.Tag buffer, acetonitrile 100% and water, respectively) with a total

elution time of 45 minutes. The AA separations were done using a flow rate of 1 mL per

minute and the control temperature was set at 37°C. Wavelengths for excitation and

emission in the fluorescence detector were 250 and 395 nm, respectively.

5.4.4.2. Hemolymph free amino acids (AA)

Hemolymph of six shrimp per treatment (from those sampled for enzyme activity

determination) was taken by puncture between the cephalothorax and first pair of

pleopods using a 1 mL syringe (needle 25G 5/8” 0.5 x 16 mm). Plasma was obtained

following immediate centrifugation (5 min, 5000 g) and stored at -20 °C. 100 µL of

each plasma sample was then centrifuged (1h30, 2000 g, 15°C) followed by a filtration

(filters Amicon-Microcon, YM 100 kd) to eliminate protein. Samples were then

derivatised following the AccQ.Tag method by Waters and injected in the HPLC

column as previously described. AA concentrations are expressed as µmol/L of

hemolymph.

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5.4.5. Statistical analysis

The effects of dietary methionine and choline on metabolism and performances were

analysed by a two-way ANOVA using methionine (adequate, 30% limiting, and 50%

limiting) and choline (adequate or excess) as independent factors (n=6 diets). The effect

of methionine and cystine dietary levels was analysed by a two-way ANOVA using

methionine level (30 and 50% limiting) and cystine levels (normal or excess) (n=4

diets). For the amino acid and enzyme analyses, outliers (detected by scatterplot with

box plots) were excluded from the analysis. Outlier and extreme points were described

as followed:

Outliers: value > UBV + oc * (UBV – LBV) and value < LBV – oc* (UBV-LBV)

Extremes: value > UBV + 2oc * (UBV – LBV) and value < LBV – 2oc* (UBV-LBV)

Where LBV is the lower value of the box plot (25th percentile); UBV is the upper value of the box plot

(75th percentile), oc is the outlier coefficient (constant: 1.5).

For body composition and performances, four replicates per treatment were considered

in the analysis, except for diet DEF30 (n=3). Because the dietary treatments did not

affect hepatic somatic index and hepatic protein concentration, the enzyme activities

were compared in term of units per mg protein. All analyses were performed using

STATISTICA 5.0 software (StatSoft, Inc., Tulsa, OK, USA). Data were analysed by

Duncan’s multiple range test in case of a significant effect (P < 0.05).

5.5. Results

5.5.1. Performances

The survival (91-98%) of the shrimp was not affected by the dietary treatments (Tables

5.3 and 5.4, P > 0.05). Growth parameters and feed efficiency were significantly higher

in shrimp fed the excess dietary choline compared to those fed the basal choline diets

(Table 5.3, P < 0.05). The 30% Met-limiting diet significantly reduced the daily growth

coefficient (DGC, %) and feed efficiency of shrimp compared to the CTL diet (Table

5.3). Surprisingly, growth parameters of shrimp fed the 50% Met-limiting diets were

intermediate between those of shrimp fed the CTL and 30% Met-limiting diet. We

observed a significant interaction between Met and choline for individual nitrogen gain

(Fig. 5.1, P < 0.05). This interaction should be attributed to the fact that the extra

choline improved nitrogen gain in shrimp fed the Met-limiting diets but not in shrimp

fed the Met-adequate CTL diet. The addition of cystine significantly improved the DGC

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and feed efficiency of shrimp, irrespective of the level of Met-limitation (Table 5.4, P <

0.05). N gains significantly increased by the extra dietary cystine at both Met levels (30

and 50% limiting) (Fig. 5.1, P < 0.05).

5.5.2. Methionine metabolism

Both BHMT and CBS were found to be active in the digestive gland of P. monodon

shrimp with high intra-treatment variability. BHMT activity was significantly affected

by the dietary Met or SAA level (Table 5.3). The DEF30-fed shrimp had a significantly

lower BHMT activity compared to those fed the CTL diet (17 vs. 33 U/mg protein,

respectively). No significant difference in BHMT activity was found between shrimp

fed the CTL and DEF50 diets (33 vs. 36 U/mg protein, respectively). Surprisingly,

dietary choline addition did not significantly affect BHMT activity, although there was

a tendency (P = 0.126) for lower BHMT activity in shrimp fed the excess choline level

at the adequate (CTL) and 30% Met-limiting levels (DEF30) (Table 5.3). CBS activity

was not affected by either the Met or choline levels in the diets (Table 5.3, P > 0.05).

The activity in digestive gland varied from 3 to 7 U/mg protein, respectively, for

DEF30+CC and DEF50 fed shrimp. No significant effect of extra dietary cystine could

be detected on BHMT or CBS activity (Table 5.4, P > 0.05).

Free methionine, homocysteine and serine concentrations in hemolymph were not

significantly affected by any of the dietary treatments, although free methionine slightly

decreased with the Met-limiting diets (11 and 10 µmole/L vs. 14 µmole/L in the CTL

diet) and free serine tended to decrease in hemolymph of shrimp fed the 30% Met-

limiting diet (Table 5.3, P = 0.072). Cysteine was not detected by the HPLC technique

which might reflect its very low concentration. Cystine, cysteic acid and taurine

concentrations were significantly affected by the dietary Met level, but not by dietary

choline level (Table 5.3). Concentrations of cystine and cysteic acid were significantly

higher in hemolymph of shrimp fed the CTL compared to those fed diet DEF30

(respectively 3.2 vs. 2.1 µmole/L for cystine; and 8.5 vs. 4.5 µmole/L for cysteic acid).

Hemolymph taurine concentration was significantly higher with the DEF50 compared

to the DEF30 diet (635.5 vs. 521.0 µmole/L). The addition of cystine to the DEF30 diet

increased hemolymph cystine and taurine concentrations in shrimp (2.2 vs. 3.7 and

523.4 vs. 732.3 µmole/L, respectively for cystine and taurine). However, no increase

was observed at the 50% Met-deficiency level (Table 5.4).

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Fig. 5.1. Effect of dietary levels of choline, CC (a) and of cystine, Cyss (b) on daily individual N gain (mg/shrimp/day) of juvenile P. monodon fed the semi-purified diets adequate (CTL) limiting (30 or 50%) in SAA (Met+Cys) during five weeks. Data represent mean values with SD (n=4 per treatment, except for DEF30 where n=3). Means sharing a common letter are not significantly different (P > 0.05, 1-way ANOVA).

Table 5.3. Effect of Met and choline on growth and metabolic parameters of juvenile P. monodon fed the experimental diets during 35 days Diets P-values

CTL CTL+CC DEF30 DEF30+CC DEF50 DEF50+CC Met CC Met × CC

Performances 1 Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Survival (%) 91.7 3.2 96.7 3.3 91.1 4.4 95 3.2 96.7 3.3 98.3 1.7 0.373 0.197 0.864

Final body weight (g) 5.7 0.1 5.9 0.1 5.1 0.1 5.6 0.3 5.4 0.1 5.8 0.2 0.092 0.020 0.704

Daily growth coefficient (%)2 0.82 0.05 0.93 0.01 0.62 0.04 0.79 0.05 0.73 0.04 0.84 0.06 0.009 0.003 0.786

Apparent feed intake (mg/shrimp/d) 3

276 8 258 3 245 18 257 20 253 16 303 18 0.248 0.249 0.093

Feed efficiency 0.24 0 0.29 0 0.2 0 0.25 0 0.23 0 0.23 0 0.006 0.003 0.062

Enzyme activites (U/mg protein) 4

BHMT 42 8 26 5 22 5 13 4 36 9 37 3 0.012 0.126 0.363

n= 6 n=7 n=6 n=6 n=8 n=7

CBS 5 1 5 1 5 2 3 1 7 3 4 1 0.626 0.419 0.867

n=5 n=4 n=6 n=5 n=6 n=4

Free hemolymph amino acids (µmole/L) 5

Methionine 13.6 1.1 11.9 2 10.9 (5) 1.5 7.8 1 9.7 2.4 10 .0 (5) 1.6 0.105 0.281 0.622

Homocysteine 3.5 0.8 3.8 0.5 3.6 0.6 4.1 1.2 4 0.8 3.7 0.8 0.957 0.833 0.897

Serine 46.8 8 49.7 11 36.8 4.8 29.1 2.3 49 4.7 48.4 (5) 11 0.072 0.769 0.772

Cysteine ND 6 ND ND ND ND ND ND ND ND ND ND

Cystine 3.1 (5) 0.2 3.2 0.3 2.2 0.2 2.0 (5) 0.5 2.5 0.2 2.7 0.3 0.001 0.961 0.787

Cysteic acid 9.8 (5) 1.3 7.3 1.4 5.1 0.7 3.8 0.7 4.4 1.4 4.1 1.6 0.002 0.177 0.693

Taurine 584 69 629.3 37 523.4 30 518.6 36 597.1 20 673.9 48 0.031 0.270 0.633 1 Values are means of four replicate tanks ± SE, with exception of treatment DEF30 where only three replicate tanks are considered. 2 Daily growth coefficient (%): (WF

(1/3) - WI (1/3) /days) × 100 with WI and WF: mean initial and final body weight (g), respectively.

3 Apparent feed intake: [(FG-FU) × (100 - Leaching%)] / ( N × 35 days) with FG: total feed gift (g DM), FU: total amount of uneaten feed (g DM), Leaching%: percentage of feed loss after 2 hours immersion in water (salinity 30 ppt), N: average number of shrimp per tank over 35 days. 4 Values are means of (n) samples ± SE. 5 Values are means ± SE of six shrimp hemolymph samples unless noted otherwise (in bracket). 6 ND: not detected.

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Table 5.4. Effect of Met and cystine on growth and metabolic parameters of juvenile P. monodon fed the experimental diets during 35 days Diets P-values

DEF30 DEF30+cyss DEF50 DEF50+cyss Met Cystine Met × Cystine

Performances 1 Mean SE Mean SE Mean SE Mean SE

Survival (%) 91.1 4.4 95 1.7 96.7 3.3 96.7 1.9 0.231 0.509 0.509 Final body weight (g) 5.1 0.1 5.7 0.2 5.4 0.1 5.7 0.3 0.606 0.055 0.608 Daily growth coefficient (%)2 0.62 0.04 0.84 0.04 0.73 0.04 0.84 0.05 0.236 0.002 0.226 Apparent feed intake (mg/shrimp/d) 3

245 18 269 4 253 16 284 25 0.539 0.148 0.832

Feed efficiency 0.2 0.01 0.25 0.02 0.23 0.01 0.24 0.01 0.57 0.03 0.132

Enzyme activites (U/mg protein) 4

BHMT 22 5 31 13 36 9 20 9 0.906 0.756 0.200 n=6 n=5 n=8 n=5 CBS 5 2 7 1 7 3 9 1 0.368 0.227 0.991 n=6 n=6 n=6 n=4

Free hemolymph amino acids (µmole/L) 5

Methionine 10.9 (5) 1.5 11.7 (5) 1.3 9.7 2.4 11.6 2.2 0.747 0.522 0.789 Homocysteine 3.6 0.6 4.4 1 4 0.8 3.6 0.7 0.778 0.835 0.480 Serine 36.8 4.8 36.3 4.5 49 4.7 31.2 (5) 5.9 0.48 0.079 0.096

Cysteine ND 6 ND ND ND

Cystine 2.2 c 0.2 3.7 (5) a 0.2 2.5 bc 0.2 2.9 b 0.2 0.334 0.000 0.009

Cysteic acid 5.1 0.7 5.1 0.9 4.4 1.4 2.2 0.5 0.069 0.246 0.268

Taurine 523.4 b 30.2 732.3 a 33.2 597.1 b 20.2 576.9 (5) b 42.1 0.211 0.007 0.002 1 Values are means of four replicate tanks ± SE, with exception of treatment DEF30 where only three replicate tanks are considered. 2 Daily growth coefficient (%): (WF

(1/3) - WI (1/3) /days) × 100 with WI and WF: mean initial and final body weight (g), respectively.

3 Apparent feed intake: [(FG-FU) × (100 - Leaching%)] / ( N × 35 days) with FG: total feed gift (g DM), FU: total amount of uneaten feed (g DM), Leaching%: percentage of feed loss after 2 hours immersion in water (salinity 30 ppt), N: average number of shrimp per tank over 35 days. 4 Values are means of (n) samples ± SE. 5 Values are means ± SE of six shrimp hemolymph samples unless noted otherwise (in bracket). Within a row, means without a common letter differ, P < 0.05. 6 ND: not detected.

5.6. Discussion

5.6.1. Both choline and cystine have a methionine-sparing effect on protein

accretion

Due to size-related differences in specific growth rate (SGR), with small animals having

a higher SGR than larger ones (Kaushik, 1998), some authors proposed to use daily

growth coefficients (DGC) in order to compare growth data between studies with fish

(Bureau et al., 2002) or crustaceans (Bureau et al., 2000). The present DGC (0.82-0.93

in Met adequate treatments) are similar to values found in other studies with shrimp fed

semi-purified diets, in P. monodon (max DGC of 0.76-0.86) (Chen et al., 1992;

Millamena et al., 1998, 1999) or M. japonicus (max DGC of 0.68-0.89) (Teshima et al.,

2001; Alam et al., 2005). The growth rate as seen here was also similar to that of

P.monodon (recalculated DGC of 0.75-1.18%) having, as in the present study, a

relatively high initial body weight (~3 g) and being fed a practical diet (Glencross et al.,

1999).

As anticipated from our previous study on the determination of Met requirements in

juvenile P. monodon (Richard et al., 2010a), the 30% SAA (Met and cystine) limitation

significantly decreased protein accretion. This reduction in protein accretion (-29%) due

to the SAA limitation as well as the daily N gains of the control group are similar to

data obtained in our previous study (Richard et al., 2010a). It remains however unclear

why N gain did not further decrease by the 20% extra deficiency (DEF50 vs. DEF30).

This contrasts with findings in other growing animals such as chicken (Sekiz et al.,

1975), pig (Chung and Baker, 1992b), or fish (Ruchimat et al., 1997; Simmons et al.,

1999; Kasper et al., 2000) where a severe Met deficiency induces a higher N loss than a

marginal Met deficiency.

Like vertebrates, shrimp have a choline requirement for maximal weight gain which

seems to vary among shrimp species, being eight times superior in P. monodon

compared to M. japonicus (0.47% vs 0.06%, respectively) (Shiau and Lo, 2001). Since

Met and choline share a common role as methyl donor, the requirement for Met is

influenced by that for choline and vice versa (Wu and Davis, 2005; Kasper et al., 2000).

The interactive effect of choline and Met on growth has been examined in only one

penaeid shrimp, M. japonicus (Michael et al., 2006). Using a 2x3 design with two Met

concentrations (0.30 and 1.65%) and three choline levels (0.03, 0.08 and 0.14%), the

foresaid authors found that choline in excess (0.14%) improved weight gain of M.

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japonicus only when fed the low-Met diet. Our results suggest a similar interaction

between choline and Met since extra choline significantly improved protein deposition

when added to both Met-limiting diets, but not when added to the Met-adequate control

diet. The latter observation not only validates that the basal Met supply (0.8%) was

appropriate for normal growth in P. monodon, but also suggests that the basal choline

supply (around 0.3%) fulfilled the requirements. This choline level is however lower

than the requirement value (0.47%) previously reported in P. monodon (Shiau and Lo,

2001), possibly because the authors used a low Met level (0.49 %) close to that of the

present 50% Met-limiting diet.

The dietary supplementation of cystine, at both limiting Met levels, increased the N gain

of the shrimp up to that obtained with the control diet. In other words, keeping the total

SAA level constant at 1.1% of the diet, while exchanging (on a weight basis) 30 or 50%

of Met by cystine, resulted in a similar protein accretion. Although the sparing effect of

cyst(e)ine on Met requirements for protein accretion has not been examined before in

shrimp, our data agree with studies in other animal species, showing that cystine can

replace around 50% of Met in growing pigs (Chung and Baker, 1992c), chick (Graber

and Baker, 1971; Sasse and Baker, 1974) or rainbow trout (Walton and Cowey, 1982b;

Cowey et al., 1992). The observation that the 1.1% SAA diets with cystine/Met ratio of

0.4, 0.9 and 1.6 led to equivalent nitrogen gain in juvenile P. monodon, as well as recent

results in fish which reported no reduction of protein accretion when feeding diets low

in Met but not in cyst(e)ine (e.g. Atlantic salmon) (Sveier et al., 2001; Espe et al.,

2008), underline the importance of estimating requirements for protein gain in terms of

total SAA rather than for Met alone.

5.6.2. The regulation of methionine metabolism by methionine, cystine, and

choline

In crustaceans, no study so far has examined the functionality of the pathways of Met

metabolism and the dietary regulation of remethylation (BHMT or MS) and

transsulfuration (CBS and cystathionase) enzyme activities, both being well

documented in mammalian vertebrates (Stipanuk, 2004). Our study is thus the first ever

to examine the ability of shrimp to regulate Met utilisation at the biochemical level.

Using the classical methodology developed for mammals (Mudd et al., 1965;

Finkelstein and Mudd, 1967; Lambert et al., 2002), we demonstrated the presence of

150

enzyme activity for both BHMT and CBS in P. monodon digestive gland, the major site

for digestive enzyme secretion and nutrient absorption. In rat, activity of BHMT is

principally detected in liver (Stipanuk, 2004) and CBS mostly in liver and pancreas

(Mudd et al., 1965). The specific BHMT activities in the present study varied between

13 and 42 U/mg protein (90 min reaction) and are within the range of those observed in

cattle (17 to 22 U/mg protein/60 min) (Lambert et al., 2002), but 2-8 fold lower than in

rat (Finkelstein et al., 1982a,b; Finkelstein et al., 1986) or chicken (Emmert et al.,

1996). Specific CBS activity (3 to 9 U/mg protein) was slightly below hepatic CBS

activity in growing cattle (15-20 U/mg protein/60 min) (Lambert et al., 2002) but more

than 10-fold lower than in rat liver (with or without Met supply) (Finkelstein and Mudd,

1967).

Our results show a significant effect of the dietary SAA content on BHMT

remethylation activity, being the lowest in shrimp fed the 30% SAA-limiting diet and

back to control levels with the 50% SAA-limiting diet. Whereas BHMT activity has

been reported to be unaffected by a reduction in dietary Met in pig (Emmert et al.,

1998), comparable quadratic BHMT responses have been observed in liver of cattle

(Lambert et al., 2002) and rat (Finkelstein et al., 1982b), following more extreme

variations in Met supply (from excessive to quasi-absent) than in our study (from

adequate to highly–limiting). Remethylation of Hcy to Met occurs by two pathways,

using either BHMT or MS as catalysing enzyme. Although the relative contribution of

both enzymes has been suggested to be equal in the rat liver (Finkelstein and Martin,

1984), recent data from broiler indicate that Hcy fluxes through BHMT and MS change

according to the dietary SAA level, with an almost equal contribution at adequate SAA

supply and a lower relative contribution of BHMT (compared to MS) at excess and

limiting SAA supply (Pillai et al., 2006a). The latter observation possibly explains the

reduced BHMT activity seen here with the 30% SAA-limiting compared to SAA-

adequate control diet. Regarding choline, several studies in mammals (Finkelstein et al.,

1982a; Emmert et al., 1998) and birds (Emmert et al., 1996) found that hepatic BHMT

activity increases by adding choline to diets either adequate or limiting in Met,

consistent with the use of choline-derived methyl groups for remethylation. In contrast,

BHMT activity in the current study tended (P = 0.126) to decrease in shrimp fed the

extra choline at the adequate or 30% limiting SAA supply. That BHMT activity may not

be as responsive to dietary choline as previously concluded has been recently

underlined in a study with broiler following similar observation as in our study (Dilger

et al., 2007). Moreover, analysis of both remethylation pathways in terms of Hcy fluxes

151

showed that excess choline in chicks (regardless of the dietary SAA level) decreases the

relative contribution of BHMT to remethylation while increasing that of MS (Pillai et

al., 2006a, b). Several hypotheses may explain the apparent inconsistencies in the effect

of choline on the regulation of Hcy remethylation (Pillai et al., 2006a). First, an excess

of methyl groups may inhibit BHMT due to an excess of dimethylglycine, a BHMT

reaction by-product (Finkelstein and Martin, 1984; Pillai et al., 2006b). Also, serine

may be used for the production of more 5-methylenetetrahydrofolates, increasing

remethylation through folate-dependent MS (Pillai et al., 2006a).

Following the addition of cystine in both SAA-limiting diets, shrimp did not respond by

increasing BHMT remethylation or decreasing CBS activity, which catalyses the first

step of transsulfuration. This was surprising as the effect of cysteine on hepatic Met

metabolism was earlier found to be related to dietary Met level, with a down regulation

of CBS only in rat fed a low Met diet with extra cysteine (Finkelstein et al., 1986,

1988). Based on these results, we expected the CBS activity to decrease with the

addition of dietary cystine, in line with the enhanced protein accretion observed in these

shrimps. Hence, the constant low CBS activity, irrespective of the cystine or Met

supply, seems to reflect a poor transsulfuration with Met being directed mostly towards

protein synthesis. However, metabolic fluxes were previously reported to be affected by

the catalytic constant rate, the enzyme concentration, or the substrate concentration

(Stipanuk and Dominy, 2006). This was already observed in rat liver in which the flow

through BHMT reaction increased while enzyme activity decreased (Finkelstein and

Martin, 1984). In our study, the absence of change in hemolymph concentrations of Met

and Hcy implies that the pathways controlling intracellular Met/Hcy metabolism were

effectively regulated in juvenile P. monodon. These observations emphasise the need to

undertake complementary studies on the effect of dietary SAA levels on the flux of Hcy

through remethylation and transsulfuration.

Apart from being needed for protein synthesis, Met and cyst(e)ine are precursors of

biologically important S-containing molecules such as glutathione or taurine (Stipanuk,

2004). Among its many physiological functions, taurine plays a major role in cellular

osmoregulation (Jacobsen and Smith, 1968), which is of particular importance in P.

monodon and other euryhaline animals living in intertidal ecosystems. In terrestrial

vertebrates, taurine is synthesized mostly via cysteine sulfinic acid, involving the

cysteine dioxygenase (CDO) and cysteinesulfinate decarboxylase (CSD) (Jacobsen and

Smith, 1968; Goto et al., 2001). However, the capacity for taurine synthesis appears to

be species-dependent as illustrated by the dietary taurine requirement in cat, due to a

152

lack of CSD (Knopf et al., 1978). Among teleost species, some debate remains

regarding the capacity of some marine fish to synthesise taurine (Kaushik and Luquet,

1977; Goto et al., 2001; Yokoyama et al, 2001; Gaylord et al., 2006; Espe et al., 2008;

Kim et al. 2008). This field of research has importance especially in the context of

replacing the fish and shrimp meal (rich in taurine) by plant protein sources (lacking

taurine) in the feeds. Literature on taurine synthesis capacity of crustacean species is

scarce. While the freshwater prawn Macrobrachium rosenbergii appears able to cover

its taurine requirement by biosynthesis (Smith et al., 1987), the enhanced growth of P.

monodon following the addition of taurine to purified diets (4-8 g/kg diet) suggests

limited taurine synthesis in P. monodon (Shiau and Chou, 1994). Nevertheless, growth

of the shrimp in our study fed diets devoid of crystalline taurine was not lower than that

of shrimp receiving semi-purified diets with 5g/kg diet of taurine as part of the

attractant blend (Richard et al., 2010a). Furthermore, the taurine concentrations in the

hemolymph of shrimp in this study were within the range of values reported before in P.

monodon reared at a salinity of 30 g/L (Fang et al., 1992), even with the 50% SAA-

limiting diet. Moreover, the increase in hemolymph taurine concentration following

cystine addition to the 30% Met-limiting diet suggests that P. monodon has a capacity to

regulate taurine synthesis in relation to dietary cystine levels. However, the role of

taurine and the regulatory pathways of taurine synthesis in P. monodon, and aquatic

crustacean species in general, need further investigation.

5.7. Conclusions

Our data demonstrate the sparing effect of choline and cystine on Met requirements for

protein accretion in juvenile P. monodon and suggest that growth requirements should

be expressed in terms of total SAA rather than Met alone. Our study shows for the first

time the existence and functionality of enzymes involved in Met metabolism (i.e.

BHMT and CBS) located in shrimp digestive gland. BHMT, but not CBS, which had an

overall low activity, was found to respond to the dietary SAA-deficiency. Choline and

cystine addition to the SAA-limiting diets did not stimulate remethylation by BHMT

nor did they downregulate CBS transsulfuration activity. However, the constant Met

and homocysteine concentrations in hemolymph, independent from dietary treatment,

suggest that the shrimp were able to maintain remethylation/transsulfuration

equilibrium. Finally, our data suggest the capacity of shrimp to synthesize taurine.

Further research is however needed to better understand the sparing effects of choline

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and cystine on Met requirements and to characterise the pathways regulating taurine

synthesis.

5.8. Acknowledgements

The authors acknowledge Frédéric Terrier and Peyo Aguirre for their help during diet

manufacturing and to Marie Jo Borthaire for assistance with the laboratory analyses.

Special thanks are due to Christian Ramamonjisoa (Aqualma facility) for his technical

assistance.

L.R. designed the study and did the data analysis. L.R., S.K. and I.G. contributed to the

drafting of the paper. C.V. and L.R. did the enzymatic analyses. A.S. did the AA

analyses. L.R. and V.R. contributed to the organisation of the experiment in

Madagascar. There are no contractual agreements for the presented data which might

cause conflicts of interest. The authors acknowledge UNIMA and institutional funds

from INRA for funding this study and ANRT (France) for the scholarship to L.R.

(CIFRE PhD Research Grant).

154

155

CHAPTER 6

General discussion

156

157

The experiments undertaken in this study aim at better defining protein and essential

amino acid requirements of black tiger shrimp, Penaeus monodon, in order to explore

the development of feeds less relying on fishmeal as the main protein source and to

assess the consequences on protein and sulphur amino acid metabolism. In this section,

the results presented in the previous chapters are further discussed and compared with

other species, especially regarding the metabolic response to a dietary amino acid

imbalance.

6.1. Protein requirements

6.1.1. The contribution of maintenance to the total protein requirement

For the first time, our work provides data on requirements for both maintenance (zero N

balance) and maximal protein accretion of juvenile P. monodon fed semi-purified diets,

using a factorial approach. We found a maintenance requirement of 4.5 g CP/kg body

weight (BW) per day (d) representing approximately 19% of the total crude protein

(CP) requirement for maximal N gain. In shrimp, the only available data to date on

protein requirements for maintenance are those of Kureshy and Davis (2002), who

found that juvenile and sub-adult whiteleg shrimp (L. vannamei) required 1.8–3.8 g CP

/kg BW/d and 1.5–2.1 g CP /kg BW/d, respectively. These values as well as the relative

contribution of CP requirements for maintenance to those for maximum growth (4 to

9% in juveniles and 6 to 10% in subadults) appear lower in L. vannamei than in our

study (about 19%). The present maintenance requirements are however within the range

of those reported in several finfish species (as discussed in chapter 2), for which the

proportion of CP spent for maintenance (i.e., 12-21%) also seems to vary according to

the studied fish species (Abboudi et al., 2006, Ozorio et al., 2009). Whether the

difference between P. monodon and L. vannamei is due to a physiological difference

between both species or to methodological issues warrants further consideration. The

apparently high contribution of dietary protein in maintaining N balance in P. monodon

also bears practical significance for the general approach to studies on protein supply

and utilisation. Another point, addressed in the study undertaken with shrimp grown in

cages (chapter 3), but which could not be evaluated since all feeding levels gave

positive weight gains, concerns the question whether and how maintenance

requirements of shrimp are affected by the dietary protein source.

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Table 6.1. Comparisons of CP requirements and performances between fish, terrestrial vertebrates and shrimp Requirements

Max SGR

Max DGC 1 % diet

g CP/kg BW/d

Max N retention

(%) 2

Max PER 3 FE 4 References

Terrestrial vertebrates

Rat 5.8 8.9 - - 36 2.1 0.5 Finke et al., 1989

Beef cattle - - 13 3 24 1.5 0.2 NRC, 1984

Chicken - - 14-18 7-37 - - - NRC, 1994

Turkey - - 14-28 14-40 - - - NRC, 1994

Piglet - - 16 - 23 - - Dourmad et al., 1999

Weaners - - 18 - 45 - - Dourmad et al., 1999

Growing pig - - 17 - 33 - - Dourmad et al., 1999

Growing pig 2.7-2.9

2.1-2.5 16-18 - - 2.5-2.9 0.4-0.5 Boomgaardt and Baker, 1973

Teleost fish

S.gairdneri 4.0 1.6 50 - 30 - 0.9 Murai et al., 1989

D.vulgaris 1.6 1.2 - 6 38 1.9 0.8 Ozorio et al., 2009

L.calcarifer 2.0 3.4 50-55 - 42 2.5 1.3 Williams et al., 2003

I.punctatus 2.1 1.7 - 9 45 2.6 0.9 Gatlin et al., 1986

O.mykiss 3.7 2.8 - 8 5 44 3.1 1.2 Fournier et al., 2002

P.maxima 4.2 2.9 - 8 5 46 2.6 1.2 Fournier et al., 2002

S.aurata 1.8 1.7 - 9 5 39 2.1 1.0 Fournier et al., 2002

D.labrax 2.5 2.1 - 9 5 38 2.4 0.9 Fournier et al., 2002

Crustaceans -

L.vannamei 4.6 2.3 - 43-46 48 2.2 0.7 Kureshy and Davis, 2002

L.vannamei 2.2 1.5 - 20-23 36 2.3 0.8 Kureshy and Davis, 2002

L.vannamei 5.3 2.5 30-40 - 49 2.4 1.2 Venero et al., 2007

P.monodon 1.8 0.9 6 28-34 7 20-24 16 6 0.8 6 0.3 6 Chapter 2

P.monodon 2.3 8 1.5 8 32 9 12 10 32 8 1. 7 8 0.7 8 Chapter 3 1 Calculated as (WF

1/3 –WI1/3)/days × 100 with WF and WI, the final and initial body weight (g)

2 N gain. N intake-1. N gain (g) is calculated as (WF × NF)-(WI × NI) with NF and NI the final and initial N content (g/100g fresh weight) 3 Weight gain. protein intake-1 4 Weight gain. feed intake-1 5 Recalculated as g/kg BW/day (originally expressed in g/kg BW0.75/day) 6 Values for shrimp fed the medium protein diet (chapter 2). 7 Value is calculated based on the nitrogen requirement estimated with the regression models. Mean shrimp body weight and daily feeding rate are assumed to be 5g and 7% biomass/day, respectively. 8 The values correspond to the performances of the shrimp reared in cages with the FM34 diet, fed at 75% of the normal feeding level. 9 The value is based on the N requirement estimate of chapter 3 (4.47% digestible N), corrected for a protein digestibility of 86.5% (mean value of the digestibility coefficient for protein of both diets) 10 Daily requirement estimated by plotting weight gain (g/kg BW/day) against N intake (g/kg BW/day) using a broken-line model (chapter 3).

159

6.1.2. Protein requirement for growth

The CP requirement (% diet DM) for maximal growth of P. monodon in our study

(Table 6.1) is estimated to be 28% and 34% using a broken-line and logistic model,

respectively (chapter 2). Using practical diets, the protein requirement is estimated at 32

% (chapter 3). These estimates are in line with available data on protein requirements of

different species of shrimp (chapter 1, table 1.4) and slightly below previous estimates

(36-40%) for P. monodon (Shiau et al., 1991). The similarity in requirement values

between both studies (chapter 2 and 3) confirms that CP requirement estimates, when

expressed in relative terms (% diet DM), are only marginally affected by differences in

experimental design: rearing systems (indoor tanks vs. outdoor cages), type of diets

(semi-purified vs. practical feed), animal size (2.4 vs. 4.5 g IBW), feeding protocol

(satiation vs. feed restriction). As previously pointed out, CP requirements (% DM)

appear considerably higher in fish and shrimp (> 30%) compared to terrestrial animals

(< 20%) (Table 6.1). Very early, Cowey and Luquet (1983) have put up a case for

expressing requirements in terms of daily intake, rather than as a dietary concentration,

as was also stressed by Bowen (1987) who even showed great similarities between fish

and terrestrial animals in protein required per unit body weight. But, as indicated in

Table 6.1, when expressed relative to body weight, our mean value for daily protein

requirement (19 CP/kg BW/day) appears to be twice that of fish (mean value from

Table 6.1 equal to 8 g/kg BW/day), but similar to that of terrestrial vertebrates (mean

value of 20 g/kg BW/day). Such discrepancy could be explained by several factors such

as difference in growth rate, feed intake or protein efficiency.

Expression of CP requirements as a function of body weight (g/kg per day) seems

highly affected by the body weight of the shrimp (Fig. 6.1). This is exemplified in our

study (Table 6.1), where CP requirements for maximal growth of P. monodon ranged

between 20 and 24 g CP/ kg BW/day (shrimp BW from 2.4 to 5.7g in 42 days)

compared to the value of 12 g CP/kg BW/day obtained in cages (shrimp BW from 4.5 to

14.5g in 49 days) using weight gain as the response criterion (chapter 3, data not

shown). Differences in relative instantaneous growth rates, being lower in bigger

animals, may also explain the higher daily protein requirement in juvenile L. vannamei

(~ 50 g CP/kg BW/d, SGR 4.6%) compared to sub-adult (~ 20 g CP/kg BW/d, SGR

2.2%) (Kureshy and Davis, 2002) when recalculated in the same manner as in our

experiments (Fig. 6.1). Thus, the differences in CP requirement between the two species

might be partly due to the lower growth rate of P. monodon in the

160

present studies compared to that of the juvenile L.vannamei in the study of Kureshy and

Davis (2002).

Fig. 6.1: Relation between growth rate and N requirement estimates for P. monodon from studies in tanks (chapter 2) or cages (chapter 3) and for L. vannamei (recalculated from Kureshy and Davis, 2002).

Also, based on the data of Kureshy and Davis (2002) and ours, the calculated

efficiencies of digestible protein utilisation for protein accretion between maintenance

and maximal growth are 38, 30, and 27% for juvenile L. vannamei, sub-adult

L.vannamei and juvenile P. monodon, respectively (Fig. 6.2). The requirement for

maximal growth appears to increase with growth rate (comparison between sub-adult

and juvenile L. vannamei) but not maintenance, leading to a relative decrease of the

maintenance to growth requirement ratio. Also, the type of diet does not seem to explain

the difference in requirements, as sub-adult L. vannamei and juvenile P. monodon

(having about the same growth rate) have similar efficiency (30 and 27%, respectively)

when fed a practical and semi-purified diet, respectively.

Fig. 6.2. Comparison of efficiency of digestible protein utilisation between juvenile and subadult L. vannamei and juvenile P. monodon.

Kureshy and Davis (2002):subadult L.vannamei fed 32% CP

y = 0.2973x - 0.4465

Kureshy and Davis (2002): juvenile L.vannamei fed 32% CP

y = 0.3838x - 0.8028

Juvenile P.monodonfed semi-purified diet (chapter 2)

y = 0.2695x - 1.0546

0

5

10

15

0 5 10 15 20 25 30 35

Digestible protein intake (g/kg BW/day)

Pro

tein

ga

in (

g/k

g B

W/d

ay)

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From literature values, total nitrogen retention in penaeid shrimp appears similar to that

in teleost fish (30-46%) or terrestrial animals (23-45%), although with a high variability

between studies (16-49%) (Table 6.1). Protein efficiency ratio (PER, weight

gain/protein intake) of the present shrimp (0.8-1.6) was lower than in other studies with

crustaceans (max PER of 2.4) and than the mean value for fish (2.5) or terrestrial

vertebrates (1.8) (Table 6.1). PER was higher (1.6) for shrimp reared with practical (in

cages or pond, chapter 3) compared to semi-purified diets (chapter 2), most likely due to

the difference in the N source (i.e., low/high level of crystalline AA). It can however not

be excluded that the present nitrogen retention values are underestimated due to

inherent difficulties in feed intake assessment. Although corrected for leaching, the

feeding schedule in the tank experiments (2 hours in the tank) and the probably only

partial recovery of uneaten feed in the cages may result in an overestimation of the real

feed consumption.

Using a logistic regression (chapter 2), the marginal efficiency of N utilisation (i.e.

between maintenance and maximum growth) was the highest (38%) at approximately

40% of the N intake required for maximal growth, similar to what is observed in pig

(Gahl et al., 1994). As N intake increased, efficiency of utilisation decreased

(‘diminishing returns’) to 6% at the intake level required for maximal growth. The

overall mean efficiency was 25%, a value similar to that obtained with the broken-line

model (24%) assuming constant marginal utilisation efficiency. The latter value appears

lower than efficiency values in studies with fish (between 30 and 46%), using a factorial

approach (Fournier et al., 2002; Abboudi et al., 2006). Therefore, the lower N utilisation

efficiency might partly explain the higher absolute N requirement observed in P.

monodon.

6.1.3. P. monodon regulates its transdeamination activity in response to

dietary protein supply

In fish, the relative high CP requirement has been reported to be a consequence of the

lack of control of amino acid catabolism (Rumsey, 1981, Walton and Cowey, 1982a,

Kaushik and Seiliez, 2010). To our knowledge, our data are the first to document in

penaeid shrimp or crustaceans in general the effect of the diet composition (protein level

or AA profile) on the regulation of enzymes (glutamate dehydrogenase, GDH and

alanine aminotransferase, ALAT) involved in transdeamination.

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Table 6.2. Comparison of enzyme activities in several species and tissues Species Organs ALAT 1 GDH References

Dig. gland 46-94 (2-4) ND Chapter 4

Muscle 187-203 (7-10) 7-13 (0.3-0.7) Chapter 4 P.monodon

Gills 124-167 (7-10) 4-14 (0.3-1.2) Chapter 4

Dig. gland - ~ 0.2 Li et al. (2009)

Muscle - ~ 9.5 Li et al. (2009) L.vannamei

Gills - ~ 1.5 Li et al. (2009)

Dig. gland - ~ ND-0.1 King et al.(1985)

Muscle - ~ 1-2.5 King et al.(1985) Decapod

crab Gills - ~ 0.5-1 King et al.(1985)

Crab Dig. gland 2-3.5 - Dewes et al. (2006)

Whole animal - (1.3) Batrel and Regnault (1985) Shrimp

Whole animal - 2 (0.03) Bidigare and King (1981)

Gills (10.1-10.2) - Greenway and Storey (1999) Oyster

Dig. gland (12.8-13.4) - Greenway and Storey (1999)

Turbot Liver - ~ 150-320 Gouillou-Coustans et al. (2002)

Liver 129-145 122-128 Peres and Oliva-Teles (2005)

Liver 512-780 98-156 Kirchner et al. (2003) Rainbow

trout Liver 580-668 (42-55) 94-112 (7-9) Martin et al. (2003)

Liver 711-812 90-112 Peres and Oliva-Teles (2006) European

seabass Liver 745-1075 90-105 Peres and Oliva-Teles (2007)

Gilthead

seabream Liver 721-875 143-212 (Gomez-Requeni et al., (2003)

Gilthead

seabream Liver 0.7-0.8 (35-53) 0.2-0.3 (11-18)

Gomez-Requeni et al.

(2004)

Liver 314 (22) 265 (19) Frick et al. (2008)

Muscle 3 (0.1) 17 (0.5) Frick et al. (2008) African

Lungfish Gills 24 (0.7) 20 (0.6) Frick et al. (2008)

Liver (32-40) (187-233) Pons et al. (1986)

Leg muscle (1-2) (5-6) Pons et al. (1986) Fowl chick

Breast muscle (0.5-1) (2-2.5) Pons et al. (1986)

Adult cat Liver (28-51) (28-41) Rogers et al. (1977)

Rat Liver 63-231 - Szepesi and Freedland (1967) 1 values are the range of specific activity (mU/mg protein) and in bracket the total activity when given (U/g tissue)

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The growth data as well as the observation that the levels of total free amino acids

(TFAA) in hemolymph and of ammonia N-excretion in the water were positively

correlated with the dietary CP concentration not only confirm proper diet intakes but

also suggest a physiological response to the variations in dietary protein (AA) supply

(chapter 4), a prerequisite for further enzyme analyses.

As mentioned in chapter 4, both GDH and ALAT activity have been measured in

crustacean species under different environmental conditions (Claybrook, 1983; King et

al., 1985; Regnault and Batrel, 1987; Chien et al., 2003). Our data confirm the

functionality of ALAT in the principal tissues of P. monodon involved in AA utilisation

(i.e. digestive gland, muscle and gills). In P. monodon, the digestive gland, however,

seems to play only a minor role in AA transdeamination. This is illustrated by i) the

undetectable GDH activity in this tissue, similar to recent findings in L. vannamei (Li et

al., 2009) and ii) the almost 10-fold lower ALAT specific activity in digestive gland

compared with vertebrate liver (Table 6.2). The major transdeamination activities in P.

monodon were found in muscle, similar to findings in decapod crab (King et al., 1985)

and L. vannamei, (Li et al., 2009) (Table 6.2).

Some caution is however necessary regarding the measured activity levels for both

enzymes. Intra-group variability was high (CV= 11-51% for example for total activity

in muscle using 5 to 6 individuals), reflecting individual variation between the shrimp,

either due to differences in feeding status (2h meal duration) or moulting stage (shrimp

from all stages, except for the moulting stage, were sampled). Also, the time of

sampling (4h post-feeding) possibly contributes to the low level of activity level since

TFAA concentrations in hemolymph already returned to basal concentrations 3h after

feeding.

Muscle transdeamination activity in P. monodon responded to changes in dietary

protein supply (chapter 4). Compared to shrimp fed the medium-protein diet, shrimp fed

the high-protein diet had a 26% higher and those fed the low-protein diet a 35% lower

muscle GDH activity. Muscle ALAT activity followed a comparable up and down

regulation, but less pronounced (+10% and -16%, respectively). The up-regulation of

GDH and ALAT following an increase in dietary protein intake has been documented

before in fish (Sanchez-Muros et al., 1998), but not the downregulation of their activity

with suboptimal protein levels in the diets (Cowey and Cho, 1993; Gouillou-Coustans et

al., 2002; Gomez-Requeni et al., 2003; Kirchner et al., 2003). Some of these studies

164

suggested that the absence of the adaptation of transdeamination activities to suboptimal

vs. optimal protein intakes might explain the high AA requirement of fish, similar to

observations in kittens (Rogers et al., 1977; Tews et al., 1984; Rogers and Morris,

2002).

It remains however uncertain to what extent the observed adaptations in enzyme activity

effectively regulate the utilisation of protein for growth. According to the logistic

regression (chapter 2 and Fig. 6.3), marginal efficiency of N utilisation for N gain

decreased from ~ 40% to ~ 10% in shrimp fed the low- vs. the medium-protein diet,

showing a high N utilization efficiency at low GDH/ALAT activity levels (Fig. 6.3.).

This suggests that the reduction in enzyme activity at low CP intakes may enable a more

efficient utilisation of N for body protein accretion than that observed at medium CP

intake, the former however remaining below that of shrimp fed the medium protein diet.

Fig. 6.3. Focus on the effect of changing N intake from medium to low on N gain, marginal efficiency of N utilisation (%) and changes in GDH activity.

Based on the linear regression between N intake and ammonia-N accumulation in the

water, 29% of the ingested N is excreted as ammonia-N (chapter 4, Fig. 4.2.) whereas

based on data on N intake and retention, we arrive at figures of ~ 80% total N loss

(chapter 2, table 2.4.). Apart from faecal N loss (10-20%), part of the N intake may be

lost in the form of intact AA through urine (Liou et al., 2005). Also, part of the

0.34

0.57

0.48

2.24

GDH (U/gabdominal muscle)

N gain(mg/shr/day)

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produced ammonia may be recycled into glucosamine and further acetylated into N-

acetylglucosamine, the major constituent of chitin. Shrimp are estimated to loose

approximately 3% of the body N at each ecdysis (Sarac et al., 1994). Although

modelling of metabolic N fluxes has never been realised in shrimp at the level of

organism, it could help in predicting the effect of the level and type of dietary protein on

N metabolism in shrimp, which requires however precise quantitative data at all levels

of N utilisation.

6.2. Amino acid requirements

6.2.1. The essential amino acid requirements are similar to previous estimates

A major issue with regard to the use of alternatives to fish meal in fish or crustacean

feeds is to meet the essential amino acid (EAA) requirements. In order to do so, our first

objective was to revisit the data on requirements for EAA established for post-larval P.

monodon by Millamena and coworkers a few decades back and this especially with

regard to lysine and methionine, both of which are generally recognised as the first-

limiting AA in a majority of terrestrial plant protein sources.

Using a factorial approach and semi-purified diets, requirements for lysine and

methionine were determined for maintenance and maximal growth (chapter 2). Our

study is the first to report data on maintenance requirements for lysine and methionine,

showing that their contribution to maintenance was similar and ranged between 14-16%

and 16-17%, respectively. The values for lysine are higher in P. monodon than in

vertebrates (4-6%) such as rainbow trout (Rodehutscord et al., 1997), rats (Benevenga et

al., 1994) or pigs (Heger et al., 2002), whereas the values for sulphur AA (10-12%) are

in the same range as for vertebrates.

Also, based on data obtained with practical diets (chapter 3), we could derive the EAA

profile (except tryptophan) for maximal growth. A comparison of the requirement

estimates is presented in Table 6.3. Comparisons of data obtained on lysine requirement

using two different experimental procedures indicate similar requirements for maximal

growth with either semi-purified (5.8-5.9% CP) or practical diets (5.7 % CP). The

values given in Table 6.3, expressed either as a percentage of the diet (dry matter, DM)

or as a percentage of the dietary protein agree with those reported for P. monodon post-

larvae (Millamena et al., 1996b, 1997, 1998, 1999). Only the estimation of methionine

requirement using practical diets was lower than that of Millamena and coworkers

166

(1996a) who found a requirement of 2.4% CP. The larger variation observed for the

methionine requirement is probably due to variations in cystine content of the

experimental diets (cf. paragraph 6.2.3). The choice of the regression model also

appears to affect the estimations for maximal growth requirement of lysine and

methionine. As already mentioned in chapter 2, the use of broken-line regression led to

the lowest estimates, while the non-linear model gave the highest estimates, similar to

observations in mammals (Fuller and Garthwaite, 1993; Baker, 1986) and fish (Hauler

and Carter, 2001b; Bodin et al., 2009).

Table 6.3. EAA requirement estimates in P. monodon obtained in chapters 2 and 3

Requirements

Maximal growth Maintenance

Chapter 3 Chapter 31 Chapter2 Chapter 2 g/kg BW g/kg BW

EAA % DM % CP 2 % DM % DP 3 % DM % CP /day % CP /day Arg 2.8 6.4 2.6 7 - - - - His 1.2 2.7 1.1 2.8 - - - - Ile 1.3 3 1.2 3.2 - - - - Leu 2.3 5.4 2 5.4 - - - - Lys 2.5 5.7 2.4 6.3 1.6-2.0 5.8-5.9 1.1-1.4 4.5 0.2 Met 0.7 1.6 0.6 1.7 0.8-1.0 2.9 0.6-0.7 2.4-2.5 0.1 Phe 1.3 3 1.1 3 - - - - Thr 1.2 2.8 1.1 2.9 - - - - Val 1.4 3.3 1.3 3.4 - - - - Dietary protein (%DM) 43.2 37.3 28-34

1 in terms of available EAA 2 calculated based on the dietary crude protein content (43.2%, mean of diets FM34 and FM16) 3 calculated based on the dietary digestible protein content (37.3%, mean of diets FM34 and FM16)

Fig. 6.4. Logistic regression of the Lysine intake vs. N gain and simultaneous marginal efficiency of N utilisation (chapter 2) in P. monodon (a) and rat (b) (Gahl et al., 1994).

37%

a) b)

40%

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The regression (logistic model) of N gain against lysine intakes shows a decrease in

lysine utilisation efficiency starting from a lysine intake level covering only ~ 40% of

that required for maximal growth, similar to findings in rat (Heger and Frydrych, 1985)

(Fig. 6.4.). Diminishing returns are worth considering depending on the targeted intake

level, either for maximal growth or for maximal efficiency (Susenbeth, 1995). These

data also confirm the importance to consider a large range of intakes for estimating

EAA utilisation efficiencies, as reconfirmed recently by Carter and Hauler (2011) in

Atlantic salmon.

Using a broken-line regression, the marginal efficiency of lysine utilisation for N gain

was 0.65. When recalculated, 248 mg of lysine are needed for 1 g protein gain.

Assuming a lysine content of 6.8% of body protein (Peñaflorida, 1989), this gives a

lysine retention efficiency of ~ 27%, far below the 60-70% marginal lysine retention

values reported in mammals (Heger et al., 2002) or fish (Carter and Hauler, 2011).

Efficiency of utilisation of EAA depends on many parameters such as maintenance, AA

catabolism and protein deposition. Our results indicate that a large proportion of lysine

is lost through other metabolic roads than protein deposition, part of which may be

attributed to inevitable lysine catabolism (Heger and Frydrych, 1985, Carter and Hauler,

2011).

6.2.2. The ideal protein concept does not apply at high protein intakes

The ideal protein is defined as one that provides the correct balance of EAA needed for

optimum performance with all EAA being equally limiting and being supplied in a

constant proportion of the protein (Boisen et al., 2000). As a consequence, EAA

requirements are expressed as a percentage of dietary protein, assuming that the

efficiency of utilisation of the first-limiting AA decreases with increasing protein levels

(Cowey and Cho, 1993). In this respect, the overall increase in AA catabolism at high

protein intake is assumed not to spare the limiting EAA, which requirement is believed

to increase concomitantly to the CP level in order to avoid an AA imbalance (Morris et

al., 1999).

In chapter 2, we applied a 30% reduction in dietary lysine or methionine supply using

the AA profile of shrimp as a reference (ideal) protein and this at three dietary protein

levels (low, medium, and high). The applied lysine limitation did not reduce protein

accretion at any of the tested protein levels, suggesting that lysine requirements are

below those of the reference protein used here (chapter 2). Regarding the Met

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deficiency, our data showed a significant interaction between dietary CP and Met levels

on protein accretion: while N gain was not affected by the Met limitation at low (10%)

or high (50%) dietary CP, the dietary Met imbalance in the 30% CP diet strongly

reduced N gain compared to the balanced 30% CP diet (from 2.2 to 1.5 mg/shrimp/day).

These data demonstrate that an imbalanced protein (limiting in Met and cystine),

supplied at high levels (HP diet) does not negatively affect performances of the shrimp,

indicating that EAA requirements should be expressed relative to dietary CP only at

protein intakes below that for maximal growth. AA imbalance phenomenon seems to be

species specific since similar results were observed in fish (Bodin et al., 2009, Carter

and Hauler, 2011), kittens (Strieker et al., 2006, 2007) or poultry (Urdaneta-Rincon et

al., 2005), but not in rats (Benevenga et al., 1968). For the latter, the AA imbalance (due

to an increase in CP supply without concomitant increase in the limiting EAA) has been

found to reduce feed intake and weight gain (Harper et al., 1970).

The interaction between dietary Met and CP levels was also evidenced in chapter 4,

where we examined the effect of an AA imbalance (Met-deficiency) at the three

different CP levels on the catabolic activity of enzymes involved in transdeamination.

When fed the low or high protein diets, the activities of ALAT and GDH were

unaffected by the relative Met-deficiency, with changes in their activity reflecting either

the conservation of AA for minimal N deposition or the elimination of excess dietary

AA (see also 6.1.3). In contrast, the AA imbalance in the diet adequate in protein but

limiting in Met affected AA catabolism, more specifically GDH oxidative activity in the

gills.

In summary, these results indicate that i) the ideal protein concept may be applied in P.

monodon only at protein intakes below or close to but not above those needed for

maximal growth, ii) given the observed interaction between dietary EAA and CP, EAA

requirements should be expressed as a percentage of the diet or more preferably as a

given intake level (g/kg BW/day) rather than as a percentage of dietary CP; iii) a trade-

off must be found between the use of imbalanced protein incorporated at level higher

than normally needed to guarantee adequate EAA supply, and the increase in N

excretion due to the overall excess in dietary N supply.

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6.2.3. Both cystine and choline can spare methionine

In the context of replacement of FM by plant protein sources, a point worthy of our

attention is that related to the supply of the relative amounts of sulphur amino acids

(SAA), methionine (Met) and cystine. Analysis of the data provided by Sauvant et al.

(2004) shows that almost all plant ingredients have higher Cystine to Met ratios (0.7 to

2) than FM (~0.3), underlining the importance to characterise the metabolic utilisation

of Met in relation to dietary cystine. Although Millamena et al. (1996a) expressed Met

requirement both as Met alone and as total SAA, no study in P. monodon or other

penaeid shrimp evaluated the effect of changes in dietary Cystine/Met ratios or, in other

words, to what extent cystine can spare the use of Met for growth.

The presented work evaluated in a first step the Met requirements for both maintenance

and growth (chapter 2), while keeping Cystine/Met ratios constant at 0.3. The estimated

growth requirements shown in Table 6.3 for Met (0.7-1.0 % DM) and total SAA (1.1%

DM) are close to those reported before for post-larval P. monodon (Millamena et al.,

1996a).

Also, marginal efficiency of Met utilisation for protein accretion was 1.35 using a

broken line regression, showing that ~118 mg of Met is necessary for 1 g of protein gain

(chapter 2). The lower contribution of Met than Lys to protein gain (~248 mg Lys/g

protein gain) can be ascribed to the differences in the body contents between both AA

and to differences in retention efficiency (AA gain/AA intake). Assuming that Met

represents 2.3% of body protein (Peñaflorida, 1989), the Met retention efficiency is

around 19.5% in the shrimp, compared with 27% for lysine. Also in mammals, the

retention of Met appears lower than that of Lys (Heger et al., 2002), which has been

attributed to the many metabolic functions of Met (as outlined in chapter 1) besides its

role in protein synthesis (Heger et al., 2002; Wu, 2009).

In chapter 5, we then evaluated the growth and metabolic responses to variations in the

Cystine/Met ratio (0.4, 0.9, 1.6) while keeping the amount of total SAA constant at a

dietary level of 1.1-1.2%. Our results demonstrated that at least 50% of the dietary Met

can be spared for growth by cystine and also by choline. While keeping the SAA at

1.1% (% DM) fluctuations in the cystine to Met ratios (0.4, 0.9 and 1.6) did not affect N

gains of the shrimp, confirming the importance of estimating requirements in terms of

total SAA rather than for Met alone, as suggested for salmonids (Sveier et al., 2001;

Espe et al., 2008).

170

On a metabolic level, cystine and choline addition to diets low in Met has been

proposed to promote Met incorporation into protein (Met sparing) by recycling Met by

homocysteine (Hcy) remethylation and inhibiting its irreversible transsulfuration to

cysteine. In order to study the metabolic regulation of the Met-sparing effect as

observed here, we assessed (chapter 5) the activity of remethylation (BHMT) and

transsulfuration (CBS) enzymes, known to respond to dietary changes in mammals.

Both enzymes were detected at a similar activity level in the digestive gland and

abdominal muscle, although with considerable variability (Table 6.4). The detection of

BHMT and CBS activity in muscle is surprising since both enzymes are mostly hepatic

in vertebrate species.

Table 6.4. Comparison of activity levels of BHMT and CBS in digestive gland and muscle (mean ± standard error) BHMT

(nmole product/mg protein/90 min)

CBS

(nmole product/mg protein/120 min)

Dietary treatments Digestive gland Abdominal muscle Digestive gland Abdominal muscle

CTL 42 ± 8 (6) 24 ± 6 (4) 5 ± 1 (5) 7 ± 2 (5)

CTL + CC 26 ± 5 (7) 65 ± 28 (4) 5 ± 1 (4) 12 ± 5 (5)

DEF30 22 ± 5 (6) 26 ± 4 (4) 5 ± 2 (6) 12 ± 4 (4)

DEF30 + CC 13 ± 4 (6) 14 ± 2 (3) 3 ± 1 (5) 15 ± 3 (3)

DEF 30 + Cyss 31 ± 13 (6) 16 ± 4 (6) 7 ± 3 (6) 7 ± 5 (6)

CC, choline chloride; Cyss, cystine

In the digestive gland, BHMT remethylation activity decreased in shrimp fed the diet

30% Met-limiting and increased back to control level in shrimp fed the 50% Met-

limiting diet. However, the positive response of choline on N gain of the shrimp was not

accompanied by an increase in BHMT activity (even a slight decrease), which contrasts

with findings in mammals (Finkelstein et al., 1982b). It is therefore believed that the

MS remethylation pathway probably contributed the most to the recycling of Met in the

present dietary conditions (Met deficiency and excess choline). Also, transsulfuration,

as measured here by the activity of CBS, did not respond to the dietary changes (Met,

Cystine or choline). Whether this is due to the low Met content (cystine was added to

diets deficient in Met) and thus an overall low transsulfuration activity or to a poor

regulation is not known. To elucidate this, it would be useful to test a diet with an

excessive amount of SAA (e.g., CTL + cystine diet). The observation that hemolymph

concentrations of Met and Hcy remained constant in our study, however, suggests that

the intracellular Hcy equilibrium between competing pathways was controlled in

juvenile P. monodon. Indeed, the metabolic fluxes are regulated by several parameters

171

like catalytic constant rate as well as enzyme or substrate concentration (Stipanuk and

Dominy, 2006). Therefore, metabolic fluxes rather than particular enzyme activity

should be studied in order to understand the mechanisms behind the sparing of

methionine. Furthermore, FAA response to dietary Met changes was recently found to

differ between plasma and tissues of fish (Espe et al., 2008). As a consequence,

measurement of FAA concentrations in the organs of shrimp involved in AA

metabolism (i.e., digestive gland and muscle) would be recommended to better

characterise the Hcy flux between tissues and hemolymph.

In chapter 5, we also considered the possible conversion of cystine into taurine, a major

osmoregulator in P. monodon (Claybrook, 1983). Conflicting literature exists regarding

the capacity of biosynthesis of taurine in marine fish (Goto et al., 2001; Yokoyama et

al., 2001; Gaylord et al., 2006; Espe et al., 2008; Kim et al., 2008) and shrimp (Shiau

and Chou, 1994). Measured 4 hours after feeding, the concentration of taurine in

hemolymph decreased only in shrimp fed the Met-limiting diet and was found to be

equal to that in control group when fed excess cystine, suggesting the capacity of P.

monodon to synthesize taurine.

It is of interest to underline here some methodological issues. First, individual

variability was high for BHMT and CBS activities, probably related to the same reasons

as previously mentioned for ALAT and GDH (paragraph 6.1.3), but also to the

separation techniques (manual preparation of the elution columns, differences in the

quantity of resine per column, density of the resine, elution duration, etc.) which

requires a better standardisation. Second, in light of the low response of some specific

free AA involved in Met metabolism to dietary changes at time of sampling (4 hours

after feeding), it is clear that more knowledge on the post-prandial pattern of changes in

FAA levels is needed in order to adjust the sampling time. A recent study in L.

vannamei (shrimp of 4.3g IBW, reared at 29°C) reported however the highest TFAA

concentration in tail muscle 4 hours after feeding when fed a diet with protein-bound

and crystalline AA (Fox et al., 2009), comforting our choice of sampling schedule.

Third, some specific issues (coelutions, unidentified peaks) with regard to the

identification (AccQ.Tag procedure) of the FAA in the hemolymph require further

improvements.

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6.2.4. Importance of available EAA in formulating feeds

There is general consensus to reduce fishmeal (FM) utilisation in feeds for farmed fish

and shrimp. In chapter 3, FM replacement was evaluated under practical conditions

(earthen ponds, over a complete production cycle from 1g to ~20g final BW) as

recommended by (Amaya et al., 2007). The low quality of plant proteins (e.g.

imbalanced EAA profile, lower EAA availability) often restricts their inclusion level in

shrimp feeds. This was illustrated in our study where only 25% of FM could be replaced

by a mixture of plant proteins (wheat gluten, corn gluten meal, sorghum, and rapeseed

meal) without affecting growth or feed utilisation. The increased incorporation of plant

protein highly decreased the availability of the EAA, especially of leucine (Table 6.5, -

26% in the 0% FM diet). Although our experiment was not designed to evaluate the

individual effect of ingredients, the inclusion of corn gluten meal in combination with

sorghum most likely explains the reduction in EAA availability (negative correlation, R²

= 0.74-0.89), in agreement with recent findings in L. vannamei (Yang et al., 2009;

Lemos et al., 2009). This highlights the importance of considering available rather than

crude nutrient quantities when formulating the diets (Table 6.5) and thus the need for

further studies on the availability of EAA from alternative protein sources in shrimp.

Table 6.5. Comparison of dietary composition of practical diets before and after correction for digestibility As fed Corrected for digestibility Proximate composition FM34 FM24 FM16 FM8 FM0 FM34 FM24 FM16 FM8 FM0 Dry matter (DM, %) 90.0 90.4 90.0 90.2 90.1 70.5 62.3 55.2 55.7 52.5 Protein (%DM) 42.1 42.9 44.2 44.5 44.2 38.6 35.5 36.0 34.9 33.1 Energy (kJ/g DM) 18.8 19.4 19.7 20.1 20.2 16.3 15.3 14.6 14.8 14.1 P/E (g/MJ) 22.4 22.1 22.4 22.1 21.9 23.6 23.2 24.7 23.6 23.4 EAA (% DM) Arg 4.0 3.3 3.4 3.0 3.1 3.8 3.1 3.1 2.7 2.7 His 1.6 1.4 1.5 1.3 1.4 1.5 1.3 1.3 1.1 1.2 Ile 1.8 1.7 1.7 1.6 1.8 1.7 1.5 1.5 1.3 1.5 Leu 2.9 3.6 3.5 4.3 4.8 2.8 2.9 2.8 3.1 3.3 Lys 3.6 3.0 2.9 2.7 2.6 3.5 2.9 2.7 2.5 2.4 Met 1.0 0.9 0.8 0.8 0.8 0.9 0.8 0.7 0.7 0.7 Phe 1.7 1.8 1.9 2.0 2.3 1.6 1.5 1.5 1.6 1.7 Thr 1.7 1.5 1.6 1.5 1.5 1.6 1.3 1.3 1.2 1.2 Val 1.9 1.9 1.9 1.7 1.9 1.8 1.6 1.6 1.4 1.5

While the digestible protein level was adequate in all diets (between 33.1 and 38.6%),

available Met contents might have been slightly limiting in diets where 50% or more of

the FM was replaced by plant protein sources (Table 6.5). Since the formulated

theoretical crude Met contents were above requirements, no extra Met was added to the

173

plant-based diets, contrary to lysine. As such, it is possible that the limited level of

available Met in diets FM16, FM8, or FM0 reduced the growth of the shrimp in the

pond trial, as seen using deliberate Met limitations in chapters 2 and 5. Unfortunately,

due to logistic issues (loss of whole shrimp samples from the cage trial), we could not

further validate the efficiency values obtained in chapter 2 on the utilisation of Met for

N gain using the practical diets. A further point is that we did not measure the levels of

available cyst(e)ine in the practical shrimp feeds, which is regretful regarding our

findings on Met-cystine interactions and also since the availability of cyst(e)ine appears

to highly fluctuate between ingredients (Baker, 2005). This is also seen in L. vannamei

for which availability of cystine was found to range from 81% for extruded soybean

meal to only 34% for corn gluten meal (Yang et al., 2009). Now that it is generally

recognised that it is preferable to use a mixture of several plant proteins rather than a

single protein source to improve dietary EAA profile (Gomes et al., 1989; Regost et al.,

1999; Fournier et al., 2004; Kaushik et al. 2004), better knowledge on nutrient and

especially the availability of cyst(e)ine and other AA will further improve the choice

and incorporation levels of plant ingredient for shrimp diets.

In the cage study, we found that the level of FM (34 or 16%) only slightly modified the

EAA requirement estimates for maximal growth (Chapter 3), similar to observations in

fish (Thu et al., 2007). Moreover, the similarity in weight gain of shrimp in the cage

trial fed diet FM16 (imbalanced available EAA supply) at a higher level and that of

shrimp fed diet FM34 (balanced protein) at a lower level, agrees with the finding that

feed allowance can be reduced when using a balanced protein source as seen in chapter

2 and in L. vannamei (Venero et al., 2007, 2008b). These observations are of practical

relevance for shrimp farming which mostly takes place in earthen ponds where natural

productivity may allow a further reduction of feed allowances by furnishing nutrients

(Venero et al., 2007). We also observed that the decrease in PER due to FM

replacement was more pronounced for the shrimp reared in cages (suspended into the

water column, without direct access to natural food) than for those in the ponds,

suggesting the positive complementary role of natural productivity in case of limiting

EAA supply, in line with findings that natural food may account for around 50% of P.

monodon gut content in semi-intensive ponds (Focken et al., 1998). Although not

measured in our experiments and difficult to do so, it would be useful to take full

account of the contributions of microbial and algal populations within the ponds on N

and EAA budgets.

174

175

CHAPTER 7

Conclusions and Perspectives

176

177

7.1. Conclusions

Studies undertaken as part of this thesis were initiated to get better understanding of

protein and amino acid utilisation in P. monodon in the general context of developing

feeds less relying on fish meal. Results of studies on protein and amino acid

requirements show that protein requirement of shrimp was 4.1-4.5 g/kg BW/day and

19.0-20.6 g/kg BW/day, respectively for maintenance and maximal growth. For the

two amino acids studied, lysine requirement was estimated to be 0.2 g/kg BW/day

and 1.1-1.4 g/kg BW/day (~ 2.0 % DM) and that of methionine at 0.1 g/kg BW/day

and 0.6-0.7 g/kg BW/day (~ 0.8 % DM), respectively for maintenance and maximal

nitrogen gain. We also found that the requirement for total sulphur amino acids

(methionine + cystine) could be met by a dietary supply at a level of 1.1 % DM.

Moreover, we obtained original data showing that cystine and choline can spare up to

50% of dietary methionine to maintain N gain at equivalent levels. The relative

contribution of dietary N or AA supply to maintenance is affected by growth rate and

growth stage of the shrimp. Also, the choice of the regression model can affect the

estimate of requirement for maximal growth.

At the metabolic level, in response to an increase or decrease in dietary protein level,

P. monodon can both up and down regulate transdeamination through ALAT and

GDH activities in the muscle. The metabolism of methionine, through the

remethylation and transsulfuration pathways, seems however poorly regulated at the

enzymatic level by dietary of Met, cystine, or choline supply.

At the practical farm level, 24% of fishmeal (instead of 34%) can be used in shrimp

diet formulation without reducing the performances of P. monodon. Given the

variability in amino acid availability among ingredients, it is important to integrate

values on AA availability from the ingredients in feed formulation to optimise

nitrogen utilisation and fishmeal replacement.

7.2. Perspectives

• Based on our results, it is recommended that due consideration is given to the

effect of growth rate in studies on requirements at different stages of the life

cycle within a shrimp species and between species. It is also worth employing

both practical and semi-purified diets in order to assess the effect of the type of

dietary protein on efficiency of utilisation of N.

178

• Given our results on the Met sparing effect of cystine and choline, the study of

Met metabolism deserves further consideration, especially with regard to Hcy

recycling by the MS regulated remethylation pathway, and also with taurine

synthesis.

• Analysis of EAA availability to shrimp is necessary to well characterise the

biological value of the different feed ingredients, through an assessment of the

EAA and N efficiency of utilisation.

• Determination of N fluxes through excretion, protein growth, and moulting are

necessary to better describe the effect of dietary N level and source on overall N

metabolism in shrimp. Due consideration should be given to the allocation of N

into exuvia, not only as part of N balance measurements but also as this can

contribute as a dietary N source under pond conditions.

• Modelling of N fluxes should also be applied at the pond level, by integrating

the potential effect of natural productivity on N utilisation in shrimp.

179

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Conséquences métaboliques du remplacement de la farine de poisson par des protéines végétales chez la crevette géante tigrée (Penaeus monodon) De part son profil équilibré en acides aminés essentiels (AAE), la farine de poisson (FP) est la source protéique principale utilisée dans l’alimentation des crevettes d’élevage. Cependant, compte tenu des enjeux du développement durable de la production aquacole, son utilisation doit être réduite, et remplacée par d’autres sources protéiques comme les protéines végétales (PV), qui sont souvent carencées en lysine et méthionine mais riches en cystine. Les conséquences d’un tel changement alimentaire sont peu connues chez la crevette tigrée Penaeus monodon. Pour les évaluer, nous avons utilisé des aliments semi-purifiés reflétant les carences/excès en AA des PV pour estimer les besoins en protéine, lysine et méthionine pour l’entretien et la croissance. Tout en confirmant les données antérieures sur les besoins pour la croissance des stades post-larves, nous avons pu préciser la contribution de l’apport en ces deux acides aminés pour l’entretien. Au niveau métabolique, la variation de l’apport protéique (10, 30, 50% protéine brute) et la carence en méthionine (-30% par rapport au besoin) entraînent une modification de l’activité des enzymes du catabolisme des AA, mais pas celle des voies de reméthylation et transsulfuration. En revanche, et pour la première fois chez la crevette, nos résultats démontrent une épargne de la méthionine par la cystine (et la choline), soulignant l’importance de l’apport en AA soufrés totaux (methionine + cystine). Nos résultats illustrent aussi l’importance qu’il convient d’accorder à la disponibilité des AAE dans les études de remplacement de la FP par un mélange de PV pour améliorer l’utilisation azotée chez la crevette P. monodon. Mots-Clés : crevette, P. monodon, farine de poisson, protéine végétale, protéine, lysine, méthionine, acides aminés soufrés, entretien, catabolisme, digestibilité.

Metabolic consequences of fishmeal replacement by plant proteins in black tiger shrimp (Penaeus monodon) Due to its well balanced essential amino acid (EAA) profile, fishmeal (FM) is the major protein source used in the formulation of aquafeed for cultured shrimp. To sustain farming systems, its incorporation, however, must be reduced and substituted by other protein sources less well nutritionally balanced, such as plant protein ( PP) which are often low in lysine and methionine but rich in cystine. The metabolic consequences of such a shift in dietary profile are not well known for the black tiger shrimp, Penaeus monodon. To describe these consequences, we used semi-purified diets limiting in lysine and methionine (to reflect PP profile) to determine juvenile requirements of protein, lysine and methionine for both maintenance and growth, applying a factorial approach. Our results confirm the previous data on growth requirement for post-larval stages of P. monodon while also providing new data on maintenance requirements. At the metabolic level, a variation in the dietary protein level (10, 30, 50 % crude protein) and methionine (adequate or 30% lower) resulted in a significant change in the activity of transdeaminating enzyme, but not those of remethylation and transsulfuration. Nevertheless, we found for the first time that methionine utilisation for body protein accretion can be spared by cystine and choline (up to 50%) in this species, illustrating the importance to consider total sulphur AA supply. Our data also show that full consideration should be given to AA availability in order to develop practical diets with low FM levels for P. monodon. Key words: shrimp, P. monodon, fishmeal, plant protein, protein, lysine, methionine, sulphur amino acid, maintenance, catabolism, digestibility.