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
2000
2500
3000
3500
4000
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
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
68
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).
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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).
71
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
80
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
110
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
132
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
153
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
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(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).
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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.
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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.