EVALUATION OF THE NUTRITIONAL REQUIREMENTS … · EVALUATION OF THE NUTRITIONAL REQUIREMENTS OF REDCLAW CRAYFISH, Cherax quadricarinatus ... in particular feed formulation. To better

Queensland University of Technology

School of Natural Resource Sciences

EVALUATION OF THE NUTRITIONAL

REQUIREMENTS OF REDCLAW

CRAYFISH,

Cherax quadricarinatus

Ana Pavasovic

Bachelor of Agricultural Science

Honours (I) - Applied Science

Submitted in fulfilment of the requirements for

the degree of Doctor of Philosophy

2008

any given phenomenon (thesis) contains within itself a contradictory aspect

(antithesis). Tension between the two is resolved only through a movement to a new

situation (a synthesis), whose own instability leads to the whole process starting

again. *

George F. Hegel (1770-1831)

*Jeremy Stangroom (2006). Philosophy. pp.39. ABC Books, Sydney

i

Abstract

Aquaculture represents a sustainable alternative to natural fisheries for provision of high

quality, animal protein. Crustaceans make a significant contribution to global aquaculture

production, of which decapods are the most economically important group. Among freshwater

crayfish, the genus Cherax includes several species that have emerged as important culture

species. A suite of favourable biological attributes, including fast growth and an omnivorous

feeding habit, have contributed to establishment of successful culture of Cherax

quadricarinatus (redclaw) in many countries. Aspects of redclaw production, however, remain

relatively undeveloped, in particular feed formulation. To better understand the digestive

processes and nutritional requirements of redclaw, this study examined the relationship

between diet composition and digestive enzyme activity, growth performance and diet

digestibility coefficients.

The extent to which redclaw can efficiently utilise complex polysaccharides, such as cellulose,

has been speculated on by authors who reported endogenous cellulase activity in this

species. I evaluated the use of insoluble -cellulose by redclaw, demonstrated that high

dietary levels (30%) can significantly reduce the specific activity of selected digestive

enzymes (amylase and cellulase), while also lowering apparent digestibility coefficients.

Inclusion of -cellulose above 12% also significantly reduced survival rate, specific growth

rate and feeding efficiency in this organism which corresponds with low tolerance for insoluble

fibre by other decapods. Even though redclaw possess endogenous cellulases, they appear

to have only a limited capacity to utilise insoluble fibre in their diets.

Further, I assessed the impact of different nutrient profiles on digestive enzyme activity,

growth and tail muscle composition in redclaw. Purified diets containing varying levels of

dietary protein significantly affected activity of digestive enzymes (protease, amylase and

cellulase) and the composition of the tail muscle tissue. Redclaw have a relatively low protein

requirement, which was reflected here, as little significant difference was observed in growth

rates and the feed conversion ratio was only significantly affected by the lowest protein diet.

Manipulation of the non-protein energy component in purified diets (protein to lipid ratio) had

no effect on growth performance indices in redclaw. Digestive enzyme activity (protease) was

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however, strongly influenced by both the amount of protein and lipid in the diet and a

significant correlation was observed between protease activity and growth performance

indices. The findings here, provide preliminary data for consideration of digestive enzymes

such as proteases as potential growth indicators for freshwater crayfish. These enzymes are

already recognised as reliable biological indicators for comparison of digestive efficiency and

potential growth rate in fish. The relationship between diet composition and digestive enzyme

expression observed here, stress the need for further empirical evaluation of specific

ingredients in artificial diets for redclaw.

A range of single cell, plant and animal-based, agricultural products were assessed for their

potential use in diets formulated for redclaw. Analysis of dietary supplements revealed that

apparent digestibility of crude protein was generally higher for diets containing plant-based

ingredients. A similar outcome was observed for digestibility coefficients of test ingredients.

Ingredient type also had a significant effect on digestive enzyme activity. Importantly, a

significant correlation was observed for enzyme activity and apparent digestibility coefficients.

It appears that redclaw have the capacity to utilise nutrients from a broad range of dietary

ingredients successfully including animal, single cell and in particular, plant matter in their

diet.

Taken together, the results presented here demonstrate that digestive enzyme activities in

redclaw are significantly influenced by diet composition. I show clearly that the ability of

redclaw to utilise various nutrients (measured as digestibility coefficients) is highly correlated

with digestive enzyme activity. Finally, protease activity demonstrated a potential for use as

an indicator of redclaw growth performance. The data presented here will contribute to

development of better and cheaper feed formulations for use in redclaw aquaculture and have

broader applications to freshwater crustacean culture. In particular, the potential for use of

plant-based ingredients in aqua-feeds for redclaw will contribute to a more economically and

environmentally sustainable redclaw culture.

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Keywords

aquaculture, Cherax quadricarinatus, redclaw, freshwater crayfish, crustaceans, crustacean

nutrition, digestive enzymes, protease, amylase, cellulase, lipase, cellulose, dietary fibre,

protein, protein/lipid ratio, lipid, carbohydrates, digestibility.

iv

List of publications/submitted manuscripts

1. Ana Pavasovic, Neil A. Richardson, Peter B. Mather & Alex J. Anderson (2006)

Influence of insoluble dietary cellulose on digestive enzyme activity, feed digestibility

and survival in the redclaw crayfish, Cherax quadricarinatus (von Martens).

Aquaculture Research 37, 25-32.

2. Ana Pavasovic, Alex J. Anderson, Peter B. Mather & Neil A. Richardson (2007)

Influence of dietary protein on digestive enzyme activity, growth and tail muscle

composition in redclaw crayfish, Cherax quadricarinatus (von Martens). Aquaculture

Research 38, 644 - 652.

3. Ana Pavasovic, Neil A. Richardson, Alex J. Anderson & Peter B. Mather. Influence of

dietary protein and lipid levels on digestive enzyme activity in redclaw crayfish,

Cherax quadricarinatus (von Martens) (in prep.)

4. Ana Pavasovic, Alex J. Anderson, Peter B. Mather & Neil A. Richardson (2007) Effect

of a variety of animal, plant and single cell-based feed ingredients on diet digestibility

and digestive enzyme activity in redclaw crayfish, Cherax quadricarinatus (Von

Martens 1868). Aquaculture 272, 564-572.

v

Table of Contents

Abstract ii

Keywords iv

List of publications/submitted manuscripts v

Table of contents vi

List of tables and figures x

Statement of original authorship xii

Acknowledgments xiii

Chapter 1.

INTRODUCTION

1.1 Description of research problems investigated 1

1.2 Overall objectives of the study 3

1.3 Specific aims of the study 3

1.4 Account of research progress linking the research papers 3

1.5 References 6

Chapter 2.

LITERATURE REVIEW

2.1 Introduction 10

2.1.1 Aquaculture 10

2.1.2 Crustacean aquaculture 11

2.1.3 Aqua-feeds 12

2.2 General nutritional requirements of crustaceans 14

2.2.1 Natural feeding habit of crayfish 14

2.2.2 Macronutrient requirements 15

2.3 Digestibility 20

2.4 Digestive enzymes 23

2.5 Redclaw aquaculture 24

2.5.1 Redclaw in culture 24

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2.5.2 Culture technologies and nutrition 25

2.6 Conclusion 27

2.7 References 29

Chapter 3.

INFLUENCE OF INSOLUBLE DIETARY CELLULOSE ON DIGESTIVE ENZYME

ACTIVITY, FEED DIGESTIBILITY AND SURVIVAL IN THE REDCLAW CRAYFISH,

Cherax quadricarinatus (VON MARTENS)

Statement of joint authorship 44

3.1 Abstract 46

3.2 Introduction 47

3.3 Materials and methods 49

3.3.1 Digestibility trial 49

3.3.2 Feeding trial 52

3.3.3 Statistical analysis 53

3.4 Results 54

3.4.1 Digestibility trial 54


3.5 Discussion 56

3.6 Acknowledgments 59

3.7 References 60

Tables and figures 64

Chapter 4.

INFLUENCE OF DIETARY PROTEIN ON DIGESTIVE ENZYME ACTIVITY, GROWTH

AND TAIL MUSCLE COMPOSITION IN REDCLAW CRAYFISH, Cherax quadricarinatus

(VON MARTENS)


4.1 Abstract 71

4.2 Introduction 72


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4.3.1 Diet formulation 75

4.3.2 Feeding trials 75

4.3.3 Digestive enzymes analyses 76

4.3.4 Analysis of tail muscle 78

4.3.5 Statistical analyses 78

4.4 Results 79

4.4.1 Feeding trials 79

4.4.2 Digestive enzymes analyses 79

4.4.3 Tail muscle analysis 79

4.5 Discussion 81


4.7 References 86


Chapter 5.

INFLUENCE OF DIETARY PROTEIN AND LIPID LEVELS ON DIGESTIVE ENZYME

ACTIVITY AND GROWTH IN REDCLAW CRAYFISH, Cherax quadricarinatus (VON

MARTENS)


5.1 Abstract 98

5.2 Introduction 99


5.3.1 Diet formulation and feeding trial 101

5.3.2 Digestive enzyme analyses 101


5.4 Results 103


5.4.2 Digestive enzyme analysis 103

5.5 Discussion 105


5.7 References 110

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Chapter 6.

EFFECT OF A VARIETY OF ANIMAL, PLANT AND SINGLE CELL-BASED FEED

INGREDIENTS ON DIET DIGESTIBILITY AND DIGESTIVE ENZYME ACTIVITY IN

REDCLAW CRAYFISH, Cherax quadricarinatus (VON MARTENS 1868)


6.1 Abstract 124

6.2 Introduction 125


6.3.1 Experimental animals and laboratory facility 128

6.3.2 Diets and digestibility determinations 128

6.3.3 Enzymatic determinations 129


6.4 Results 131

6.4.1 Digestibility determinations 131

6.4.2 Enzymatic determinations 131

6.5 Discussion 133


6.7 References 138


Chapter 7.

GENERAL DISCUSSION

7.1 Discussion 152

7.2 References 158

Appendices

Appendix A Digestive enzyme assays 164

Appendix B Manuscript 1. (PDF re-print of the journal article)

Appendix C Manuscript 2. (PDF re-print of the journal article)

Appendix D Manuscript 3. (PDF re-print of the journal article)

ix

List of tables and figures

page

Chapter 3.

Table 1. Ingredient and proximate composition of the reference diet used in the digestive

enzyme and digestibility determinations. 64

Table 2. Ingredient and proximate composition of the six treatment diets used in the analysis

of survival, feed conversion efficiency and growth rate. 65

Table 3. Digestibility coefficients for apparent dry matter digestibility (ADMD) and apparent

protein digestibility (APD). 66

Table 4. Specific enzyme activity levels recorded from gastric fluid (GF) and midgut gland

(MG) under different dietary treatments. 67

Table 5. Specific growth rate (SGR), feed conversion ratio (FCR) and survival rate (SR)

estimated for animals fed diets containing different levels of -cellulose or Fullers earth. 68

Chapter 4.

Table 1. Diet ingredients and proximate composition of the four experimental diets used in

the feeding trial. 92

Table 2. Growth parameters, including specific growth rate (SGR), survival, feed conversion

ratio (FCR), crude protein (CP), dry matter (DM) and ash content in tail muscle for redclaw fed

diets containing different levels of crude protein. 93

Table 3. Specific enzyme activity levels from the midgut gland (MG) of redclaw fed diets

containing different levels of crude protein. 94

Figure 1. Correlation between the dietary protein level and crude protein (CP) in tail muscle

tissue of redclaw, C. quadricarinatus. 95

Chapter 5.

Table 1. List of ingredients and proximate composition of the six experimental diets used in

the growth trial. 117

Table 2. Growth performance parameters expressed as specific growth rate (SGR), survival

and feed conversion ratio (FCR) for C. quadricarinatus fed diets containing different crude

x

protein and lipid ratios. 118

Table 3. Summary of analysis of covariance for the effects of dietary protein and lipid levels

on the growth performance parameters and digestive enzyme activity observed for

C. quadricarinatus used in the growth trial. 119

Table 4. Specific enzyme activity levels extracted from the midgut gland of C. quadricarinatus

fed experimental diets. 120

Table 5. Correlation matrix of specific enzyme activity and growth performance parameters

for C. quadricarinatus. 121

Chapter 6.

Table 1. List of ingredients and proximate composition of experimental diets. 146

Table 2. Proximate composition of the test ingredients used in the digestibility trial on

C. quadricarinatus. 147

Table 3. Apparent digestibility coefficients for dry matter (ADMD), crude protein (ACPD) and

gross energy (AGED) of the test diets for C. quadricarinatus. 148

Table 4. Apparent digestibility coefficients for dry matter (ADMD), crude protein (ACPD) and

gross energy (AGED) of the test ingredients for C. quadricarinatus. 149

Table 5. Digestive enzyme activity in MG extracts from C. quadricarinatus fed different

experimental diets. 150

Table 6. Correlation matrix of specific enzyme activity and apparent digestibility coefficients

for C. quadricarinatus. 151

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Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for

an award at this or any other higher education institution. To the best of my knowledge and

belief, the thesis contains no material previously published or written by another person

except where due reference is made. Chapters presented as published or submitted

manuscripts (with multiple authors), acknowledge the contribution of co-authors at the start of

each chapter.

Signature: .

Date: ..

x i i

Acknowledgments

I would like to thank my supervisors Dr. Neil Richardson, Associate Professor Peter Mather

and Dr. Alex Anderson for their guidance and encouragement during this project. I also wish

to thank staff at the School of Natural Resource Sciences, in particular Mr. Vincent Chand,

Ms. Wathsala Kumar and Mr. Mark Crase for their technical assistance.

Many thanks must also go to fellow colleagues within the school for providing abundant

advice on matters regarding experimental design, data analysis, as well as extensive

evaluation of the coffee brewing protocols, in particular Mark Schutze and Alex Wilson. To

Lily, Marko and Milan, I am forever grateful; your have given me strength and encouragement

to complete this project. Finally, to my partner in life, Peter - ti i ja moemo sve!

x i i i

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Chapter 1.

INTRODUCTION

1.1 Description of research problems investigated

Redclaw, Cherax quadricarinatus, (Von Martens 1868) is a decapod crustacean (Decapoda;

Parastacidae) endemic to freshwater river systems and lakes of northern Australia (Jones et al.,

1998) and parts of Papua New Guinea (Holthuis, 1986). In recent years, this species has become

an important aquaculture commodity, and is now cultured successfully both in Australia and

overseas (Muzinic et al., 2004). Nevertheless, there is limited literature that addresses the

nutritional requirements of redclaw, and nutrition in general in this species, remains poorly

understood.

Studies that have investigated the nutritional requirements of redclaw have focused mainly on

assessing optimum dietary protein levels (Webster et al., 1994; Cortes-Jacinto et al., 2003;

Thompson et al., 2004; Cortes-Jacinto et al., 2005; Thompson et al., 2005; Thompson et al.,

2006). Typically, these studies have employed a dose-response analysis to determine the

optimum nutrient requirement based on data from growth trials. Results of such studies indicate

that redclaw require relatively low levels of dietary protein (22-28%), depending on rearing

conditions (Thompson et al., 2004) and the protein source used (Thompson et al., 2006). While

type of protein used (plant or animal-based) is increasingly considered to be a major factor when

assessing optimum protein levels (Muzinic et al., 2004; Thompson et al., 2006), more research is

required to elucidate the best protein sources necessary for optimum growth in this animal.

Information relating to the use of carbohydrates in redclaw diets is also limited and there is no

comprehensive evaluation of the optimum amount or type of carbohydrates best suited to this

species. Interestingly, freshwater crayfish, among other crustaceans, are reported to hydrolyse

complex carbohydrates (Yokoe & Yasumasu, 1964) based on their digestive enzyme profile. For

example, redclaw in particular, demonstrate the potential to break down complex

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polysaccharides, such as cellulose, due to the presence of endogenous cellulase activity in the

gut (Byrne et al., 1999; Xue et al., 1999; Figueiredo et al., 2001; Figueiredo & Anderson, 2003;

Crawford et al., 2004). Despite this significant finding, no studies have evaluated empirically the

relationship between digestive enzymes and digestibility of this ingredient in redclaw.

Data on digestive enzyme profiles and general feeding habit of redclaw has been used as a basis

from which to evaluate the suitability of various plant and animal-based ingredients as potential

substitutes for the expensive fish meal component in redclaw diets. Digestibility studies by

Campana-Torres et al., (2005 & 2006) indicate that redclaw have the potential to utilise some

plant-based ingredients effectively. Lopez-Lopez et al., (2005) observed significant changes in

activity levels of digestive enzymes in response to different dietary ingredients. Neither of these

studies, however, provided a clear definition of the relationship between digestive enzymes and

digestibility coefficients.

Investigating the profile and activity of digestive enzymes under different dietary treatments will

be essential to provide information on the nutritional requirements for this species. Evidence

exists to show that growth of aquatic animals can be limited by the capacity of their digestive

system to break down and assimilate specific nutrients (Houlihan et al., 1988). Typically, a strong

correlation is observed between the activity levels of specific digestive enzymes and an animals

dietary preference (Lundstedt et al., 2004). There is also evidence to suggest that digestive

enzymes may set physiological limits on growth rates in fish (Lemieux et al., 1999). As a

consequence, it has been argued that information on digestive enzyme activities may be used to

predict a species ability to utilise a particular nutrient (Hofer & Kock, 1989; Kuzmina, 1996;

Lundstedt et al., 2004).

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1.2 Overall objectives of the study

The overall objective of this study was to expand on current knowledge about redclaw nutritional

requirements. The focus was to contribute to development of a better understanding of this

species ability to utilise plant and animal-based feed ingredients by assessing the relationship

between specific digestive enzyme activity, growth parameters and apparent digestibility

coefficients. Therefore the specific aims were to:

1.3 Specific aims of the study

1. To evaluate the impact of -cellulose in diets formulated for redclaw, on: (1) activity of

digestive enzymes; (2) feed digestibility and (3) survival, feed conversion ratio and

specific growth rate.

2. To evaluate the effects of different levels of dietary protein on: (1) activity of digestive

enzymes and (2) growth and tail muscle composition.

3. To evaluate the effects of various protein and lipid ratios on: (1) activity of digestive

enzymes and (2) growth parameters.

4. To evaluate the suitability of a range of ingredients for inclusion in redclaw diets by

determining: (1) digestive enzyme activities and (2) apparent digestibility coefficients.

1.4 Account of research progress linking the research papers

This thesis has been structured according to the guidelines set out by the Queensland University

of Technology (QUT) to provide a clear and coherent guide to the research progression. The

literature review provides the setting for the research by detailing the current state of aquaculture

production, and in particular focusing on current redclaw aquaculture trends. The emphasis of this

extended review is the current understanding of crustacean nutrition, in particular the dietary

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requirements of freshwater crayfish. References cited in the literature review, of necessity,

replicate some literature cited in subsequent chapters.

Four papers that arose from this study are presented as individual chapters. The first paper

(chapter 3) investigated the effects of varying levels of complex carbohydrates (-cellulose) fed to

redclaw. Determinations were made of digestive enzyme activities, feed digestibility, survival,

feed conversion ratio and specific growth rate. From these findings it was concluded that, despite

the presence of cellulase in the digestive system, there is no apparent nutritive benefits from

including insoluble cellulose in diets for this species. Distinct changes in digestive enzyme

activities were observed however, when nutrient profiles of formulated feeds were altered.

Following from paper 1, the second major paper (chapter 4) further investigated the impact of

different nutrient profiles on digestive enzyme activity, growth and tail muscle composition in

redclaw. Specifically, the effect of formulated feeds containing different levels of protein was

evaluated. Findings from this study demonstrate that there is a strong correlation between dietary

protein levels, digestive enzyme activity and crude protein content in the tail muscle.

The third paper (chapter 5) was designed to complement paper 2 by increasing the complexity of

the test diets. Different ratios of protein to lipid in the diet appeared to have little effect on growth

performance, but digestive enzyme activity (protease) was strongly influenced by the amount of

protein and lipid in the diet, and significantly correlated with growth performance indices.

The fourth paper (chapter 6) investigated the potential of different dietary ingredients for use by

redclaw. Following on from results in the previous three studies, experimental diets were

supplemented with inclusion (30%) of a broad range of single cell, plant and animal-based

ingredients. Overall apparent digestibility of crude protein was significantly higher for diets and

ingredients containing plant-based ingredients. A significant correlation was observed for enzyme

activity and apparent digestibility coefficients. This demonstrates that redclaw have the capacity

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to digest nutrients from a broad range of dietary ingredients successfully, including animal, single

cell and in particular, plant matter in their diet.

The final section provides an overarching discussion of the significance of the findings, problems

encountered and potential future directions. The reference section includes all literary resources

used in the thesis including those in the published or submitted manuscripts. References used

specifically in the research papers accompany their respective manuscripts for completeness.

The appendix section contains PDF copies of the papers that have been published.

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1.5 References

Byrne, K.A., Lehnert S.A., Johnson, S.E., Moore, S.S. 1999. Isolation of a cDNA encoding a

putative cellulase in the redclaw crayfish Cherax quadricarinatus. Gene 239, 317-324.

Campana-Torres, A., Martinez-Cordova, L.R., Villarreal-Colmenares, H., Civera-Cerecedo, R.,

2005. In vivo dry matter and protein digestibility of three-plant derived and four animal-derived

feedstuffs and diets for juvenile Australian redclaw, Cherax quadricarinatus. Aquaculture 250,

748-754.

Campana-Torres, A., Martinez-Cordova, L.R., Villarreal-Colmenares, H., Civera-Cerecedo, R.,

2006. Carbohydrate and lipid digestibility of animal and vegetal ingredient and diets for juvenile

Australian redclaw crayfish, Cherax quadricarinatus. Aquaculture Nutrition 12, 103-109.

Cortes-Jacinto, E., Villarreal-Colmenares, H., Civera-Cerecedo, R., Martinez-Cordova, R., 2003.

Effect of dietary protein level on growth and survival of juvenile freshwater crayfish Cherax

quadricarinatus (Decapoda: Parastacidae). Aquaculture Nutrition 9, 207-213.

Cortes-Jacinto, E., Villarreal-Colmenares, H., Cruz-Suarez, L.E., Civera-Cerecedo, R., Nolasco-

Soria, H., Hernandez-Llamas, A., 2005. Effect of different dietary protein and lipid levels on

growth and survival of juvenile Australian redclaw crayfish, Cherax quadricarinatus (von Martens).

Aquaculture Nutrition 11, 283-291.

Crawford, A.C., Kricker, J.A., Anderson, A.J., Richardson, N.A., Mather, P.B., 2004. Structure and

function of -cellulase gene in redclaw crayfish, Cherax quadricarinatus. Gene 340, 267-274.

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Figueiredo, M.S.R.B., Kricker, J.A., Anderson, A.J., 2001. Digestive enzyme activities in the

alimentary tract of redclaw crayfish: Cherax quadricarinatus (Decapoda: Parastacidae). Journal of

Crustacean Biology 21, 334-344.

Figueiredo, M.S.R.B., Anderson, A.J., 2003. Ontogenetic changes in digestive proteases and

carbohydrases from the Australian freshwater crayfish, redclaw Cherax quadricarinatus

(Crustacea, Decapods, Parastacidae). Aquaculture Research 34, 1235-1239.

Hofer, R., Kock, G., 1989. Method for quantitative determination of digestive enzymes in fish

larvae. Polish Archives of Hydrobiology 36, 439-441.

Holthuis, L.B., 1986. The freshwater crayfish of New Guinea. Freshwater Crayfish 6, 48-58.

Houlihan, D.F., Hall, S.J., Gray, C., Noble, B.S., 1988. Growth rates and protein turnover in

Atlantic cod, Gadus morhua. Canadian Journal of Fisheries and Aquatic Sciences 45, 951-964.

Jones, C.M., McPhee, C.P., Ruscoe, I.M., 1998. Breeding redclaw Management and selection

of broodstock. Department of Primary Industries, Queensland, Australia, pp. 1-31.

Kuzmina, V.V., 1996. Influence of age on digestive enzyme activity in some freshwater teleosts.

Aquaculture 148, 25-37.

Lemieux, H., Blier, P., Dutil, J.D., 1999. Do digestive enzymes set a physiological limit on growth

rate and food conversion efficiency in the Atlantic cod (Gadus morhua)? Fish Physiology and

Biochemistry 20, 293-303.

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Lopez-Lopez, S., Nolasco, H., Villarreal-Colmenares, H., Civera-Cerecedes, R., 2005. Digestive

enzyme response to supplemental ingredients in practical diets for juvenile freshwater crayfish

Cherax quadricarinatus. Aquaculture Nutrition 11, 79-85.

Lundstedt, L.M., Melo, J.F.B., Moraes, G., 2004. Digestive enzymes and metabolic profile of

Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition.

Comparative Biochemistry and Physiology Part B 137, 331-339.

Muzinic, L.A., Thompson, K.R., Morris, A., Webster, C.D., Rouse, D.B., Manomaitis, L., 2004.

Partial and total replacement of fish meal with soybean meal and brewers grains with yeast in

practical diets for Australian redclaw crayfish Cherax quadricarinatus. Aquaculture 230, 359-376.

Thompson, K.R., Muzinic, L.A., Engler, L.S., Morton, S.R., Webster, C.D., 2004. Effects of

feeding practical diets containing various protein levels on growth, survival, body composition and

processing traits of Australian redclaw crayfish (Cherax quadricarinatus) and on pond water

quality. Aquaculture Research 35, 659-668.

Thompson, K.R., Muzinic, L.A., Engler, L.S., Webster, C.D., 2005. Evaluation of practical diets

containing different protein levels, with or without fish meal, for juvenile Australian redclaw

crayfish (Cherax quadricarinatus). Aquaculture 244, 241-249.

Thompson, K.R., Metts, L.S., Muzinic, L.A., Dasgupta, S., Webster, C.D., 2006. Effects of feeding

practical diets containing different protein levels, with or without fish meal, on growth, survival,

body composition and processing traits of male and female Australian redclaw crayfish (Cherax

quadricarinatus) grown in ponds. Aquaculture Nutrition 12, 227-238.

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Webster, C.D., Goodgame-Tiu, L.S., Tidwell, J.H., 1994. Evaluation of practical feed formulations

with different protein levels for juvenile redclaw crayfish (Cherax quadricarinatus). Transactions of

the Kentucky Academy of Science 55, 108-112.

Xue, X.M., Anderson, A.J., Richardson, N.A., Xue, G P., Mather, P.B., 1999. Characterisation of

cellulase activity in the digestive system of the redclaw crayfish (Cherax quadricarinatus).

Aquaculture180, 373-386.

Yokoe, Y., Yasumasu, I., 1964. The distribution of cellulase in invertebrates. Comparative

Biochemistry and Physiology 13, 323-338.

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Chapter 2.

LITERATURE REVIEW

2.1 Introduction

2.1.1 Aquaculture

Capture fisheries and aquaculture production represent an important source of food across the

globe. Production from capture fisheries and aquaculture provided 15.5% of total animal protein

supplies worldwide in 2003, although the contribution of fish to total animal protein intake is

thought to be higher than 20% in view of the unrecorded contribution from subsistence fisheries

(FAO, 2006). Global capture fisheries production reached 95.6 million tonnes in 2000, then

declined to 90.5 million tonnes by 2003 (FAO, 2006). An increase in total capture fisheries

production was observed in 2004, where 95.0 million tonnes were harvested, however, the

preliminary data by FAO (2006) indicates that global capture fisheries production was down to

93.8 million tonnes in 2005.

As described above, world capture fisheries production has experienced relatively minor

oscillations in production output, primarily as a result of changes in the oceanographic conditions

determined by the El Nio Southern Oscillation, and the subsequent collapse of the main

anchoveta fisheries in the southeast Pacific (FAO, 2006). While world capture fisheries

production remained steady, global population pressures have resulted in significant increases in

the number of marine fish stocks that are fully exploited (40-50% of the total number of stocks)

and the number that are overexploited, depleted or recovering from over-fishing (9-38%), (FAO,

2000). Furthermore, fully exploited and overexploited stocks are producing catches that have

either reached or are very close to their maximum sustainable limits with little room or potential

for further expansion (FAO, 2000).

By contrast, aquaculture has continued to increase rapidly and is currently the fastest growing

food producing sector in the world (FAO, 2002; Tacon, 2003). Global aquaculture production has

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grown at 9.2% per year since 1970, while capture fisheries and terrestrial farmed meat production

systems have grown at only 1.4% and 2.8% respectively, per year over the same period (Tacon,

2003). The contribution from aquaculture to total fisheries production has experienced

considerable growth, increasing from 5.3% in 1970 to 32.2% by weight in 2000 (Tacon, 2003).

Global aquaculture production in 2000 mostly came from fin fish (50.4% of total production),

followed by molluscs, aquatic plants and crustaceans. While crustacean contribution to global

aquaculture production in terms of weight has been comparatively small (3.6%), the same

commodity achieved the highest value per unit weight (16.6%) in 2000 (Tacon, 2003).

2.1.2 Crustacean aquaculture

Crustaceans are an important aquaculture commodity. Crustacean aquaculture includes the

production of a diverse range of species including marine and freshwater prawns, lobsters, crabs

and crayfish. Marine prawns (or shrimp) are the principal culture species and have accounted for

66% of global crustacean aquaculture production in 2000 (Tacon, 2003). Other cultured

crustaceans, which are of significance, include freshwater species such as crayfish that

contributed 23% to total crustacean aquaculture production (Tacon, 2003).

Marine prawn culture has grown into one of the largest and most important crustacean

aquaculture crops worldwide, the significance of which is reflected in production increases of

250% between 1985 and 1995 (Treece, 2000). One of the main cultivated marine prawn species

is the giant tiger prawn (Penaeus monodon). In 2000, P. monodon was ranked first by value at

US$ 4,046,751,000, while being rated 20th in terms of global aquaculture production by weight

(Tacon, 2003).

Prawn culture activity increased in the early 1990's and this expansion was apparently influenced

by a number of economic factors such as relatively high market value and high demand (Bautista,

1986; Treece, 2000). Simultaneously, new innovative culture technologies were being developed

including commercialisation of prawn-hatchery techniques, improvements in overall pond

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management practices and the development of high-performance feeds (Farfante & Kensley,

1997; Rosenberry, 1998). Worldwide increase in demand for crustaceans has led to

intensification of culture practices. Currently, prawns are produced in extensive, semi-intensive

and intensive culture systems. The most technologically advanced culture systems are intensive

and produce high yields in the range of 3,000 kg/ha/crop to 10,000 kg/ha/crop in prawn culture

(Treece, 2000).

With increases in culture intensity there has been a concomitant increase in stocking density. At

high stocking densities, only minimal nutritional contribution comes from natural foods found in

the pond (Treece, 2000). Thus a nutritionally complete artificial diet must be provided to the stock

in order to maintain optimum performance (Bautista, 1986; Yeh & Rouse, 1994). Both semi-

intensive and intensive culture systems are highly dependent on quality artificial diets which

generally constitute the major cost of the production system.

2.1.3 Aqua-feeds

Availability of nutritionally adequate and cost effective artificial diets (aqua-feeds) limits expansion

of many aquaculture ventures (Wee, 1992). Feeds and feeding systems can contribute up to 70%

to total production costs in fish and prawn farms (Wee, 1992; Thompson et al., 2005), with the

most expensive component of any artificial pelleted feed, being protein (Thompson et al., 2005).

Most cultured species require 25-55% protein in their diet, depending on whether the species is

herbivorous, omnivorous or carnivorous (Lovell, 1989; NRC, 1993). The major protein source

incorporated in most aquaculture diets is fish meal (Lovell, 1989) and commercial formulated

diets may contain 25-60% of this ingredient (Wee, 1992; Amaya et al., 2007).

Currently, nutrition research in aquaculture species has focussed on opportunities for reducing

the fish meal component in aqua-feeds due to its high cost. World supplies of fish meal,

composed of whole caught fish or fisheries waste, are static and are also vulnerable to

fluctuations in supply; 50% of fish meal is produced by a single fishery in Peru where output is

- 13 -

heavily influenced by the periodical El Nio effect (CSIRO, 1998). Increasing competition for

limited supplies of fish meal will inevitably cause the cost of pelleted feeds to increase,

significantly.

High quality aqua-feeds used in intensive culture situations are often associated with water

pollution. For example, in recent years it has been demonstrated that the use of suboptimal

prawn farming methods has damaged many natural estuaries and bay systems (Lawrence,

1996). Cho et al. (1994) suggested that production at intensive levels will not be possible in the

future without carefully considering the environmental effects of feeds and feed management.

Lee and Lawrence, (1997) also suggest that new environmental regulations will provide greater

impetus for expanding aquatic feed digestibility research in the future. Feed digestibility (nutrient

availability to the animal) has received significant attention recently due to increased need for low

polluting feeds (Cho et al., 1994). Implementation of strict environmental regulations on effluents

in addition to very high treatment costs have focused research towards highly assimilable aqua-

feeds that result in lower nitrogen and phosphate waste outputs. Establishing the correct

digestibility data will be important for feed formulation to reduce the amount of environmental

testing required to certify a feed as low polluting (Cho et al., 1994).

One of the most common pollutants from aquaculture activities is solids (clay and other soil

particles often suspended in rearing water). Suspended solids together with other organics,

uneaten feed, and faeces, can result in low oxygen and/or elevated ammonia levels in effluent

water (Treece, 2000). A number of researchers have proposed different strategies to decrease

pollutant levels in effluent discharge for the benefit of estuarine and coastal ecosystems affected

by aquaculture (Rosas et al., 2000). Examples of these strategies reviewed by Rosas et al.

(2000) include: diets with reduced protein content, optimisation of amino acid profiles, an

optimum protein/energy ratio in the food, and developing a better understanding of nutritional

physiology and biochemistry. Better understanding of the optimum levels of other components in

the diet (lipids and carbohydrates) and their relation to the protein/energy ratio can also be

- 14 -

utilised to reduce production cost of feeds and their relative impact as pollutants (Rosas et al.,

2000).

As aquaculture production practices become more intensive, food given to cultured animals must

not only include all the necessary nutrients to supply good growth, but must also be attractive

and palatable to ensure rapid consumption and minimal environmental impacts. If aquaculture is

to continue to expand, cost-effective diets based on agricultural ingredients need to be developed

urgently (Allan & Rowland, 1998). To develop such diets, however, requires data on the

nutritional requirements and digestive strategies of target species with potential for aquaculture.

2.2 General nutritional requirements of crustaceans

2.2.1 Natural feeding habit of crayfish

Crustaceans have exploited a wide range of habitats within the aquatic environment, and this

ecological diversity is paralleled by the diversity of the types of food commonly eaten by

crustaceans (Dall & Moriarty, 1983). The range of food consumed in the wild is generally used as

the starting or reference point when determining the nutritional requirements of any potential

aquaculture species.

Freshwater crayfish consume a wide range of food items in the wild (Thomas, 1970), primarily

due to the structure of their mouthparts and the ability to hold and grasp food items with their

walking legs (Nystrom, 2002). Natural foods often consumed include: detritus, algae,

macrophytes, invertebrates, fish, and fish eggs (Westman et al., 1986; Saffran & Barton, 1993;

Collier et al., 1997; Guan & Wiles, 1998). In general, freshwater crustaceans such as crayfish and

freshwater prawns, tend to consume more carbohydrate rich foods while marine crustaceans

(prawns and lobsters) tend to be more carnivorous and favour more protein in their diets (Nose,

1964; Lee et al., 1980).

- 15 -

Juvenile crayfish are capable of filter-feeding and scraping algae from surfaces (Budd et al.,

1978), and they also appear to be non-selective in feeding behaviour (Figueiredo & Anderson,

2003). As they grow, crayfish generally become better adapted to processing detritus (Parkyn et

al., 1997), and consequently change to a more selective feeding behaviour, consuming a variety

of decayed plant matter (Momot et al., 1978; Figueiredo & Anderson, 2003). Moreover, available

data indicate that an ontogenetic diet shift may occur in some crayfish species with age as food of

animal origin is observed in the gut of young crayfish more often than in adults (Nystrom, 2002).

Although freshwater crayfish are often classified as herbivores or detritivores, based on stomach

content analysis from wild caught crayfish, they also consume considerable amounts of animal-

based food. This suggests that an omnivorous feeding habit may more closely reflect the

polytrophic feeding habit of most crayfish species (Hill & Lodge, 1994; Vogt, 2002). Such data on

feeding habits and food preferences in the wild can serve as a guide for identifying potential feed

ingredient sources for use by crustacean feed manufacturers and farmers (Tacon & Akiyama,

1997).

2.2.2 Macronutrient requirements

Proteins are the principal macronutrients which limit life sustaining processes (e.g. reproduction

and growth) in crustaceans. Proteins are large, complex molecules, which range in size, function

and their constituent amino acids (Akiyama et al., 1992). In crustaceans, requirement for protein

is influenced by a number of factors such as the animals physiological stage, size, dietary

characteristics (digestibility) and various abiotic factors (Guillaume, 1997).

Careful assessment of optimal protein requirements is essential to ensure that no protein

catabolism occurs to meet energy requirements, at the expense of somatic growth (Sedgwick,

1979). Provision of too much protein in the diet will result in only a limited amount being used to

synthesise new protein while the remainder will be transformed to energy and/or waste (Akiyama

- 16 -

et al., 1992). The development of aqua-feeds with optimum protein levels will reduce the under or

over supply of protein to culture animals.

Extensive research on nutrition in marine prawns has established the significance of quantifying

dietary protein levels in order to reduce production costs (Shiau, 1998). Protein requirements in

crustaceans are similar to those observed in many species of finfish (Tacon & Cowey, 1985) and

overall are considered to be high (Ward et al., 2003). Generally, the required level of dietary

protein in cultured crustacean diets vary from 30% to 61%, depending on species (Shiau, 1998;

Smith et al., 2005). For example, Panulirus ornatus utilise 53-61% protein in their diet (Smith et

al., 2003, 2005), while Penaeus japonicus and Penaeus penicillatus also have high protein

requirements ranging from 50-55% (Guillaume, 1997). Species such as Penaeus aztecus and

Macrobrachium rosenbergii require only 25-30% protein in their diet (Guillaume, 1997). A number

of studies have evaluated the protein requirements of freshwater crayfish; C. quadricarinatus

(Webster et al., 1994; Keefe & Rouse, 1999; Cortes-Jacinto et al., 2003; Thompson et al., 2004,

2005, 2006), C. destructor (Jones et al.,1996a,b,c), C. tenuimanus (Morrissy, 1989), Astacus

astacus (Ackefors et al., 1992) and Procambarus clarkii (Hubbard et al.,1986). Wide inter-

species differences in optimum protein requirement have been observed and have been

interpreted to result from evolution and adaptation to specific feeding habits (Guillaume, 1997).

For example, protein requirements are lowest in herbivorous crustacean species such as P.

vannamei and highest in typically carnivorous species such as P. japonicus (Kanazawa, 1990).

Recommended optimum dietary protein levels may also vary depending on the type of culture

system employed.

In culture systems which are not completely dependent on artificial feeds, such as in extensive

culture, dietary protein levels in formulated feeds can be reduced (Akiyama et al., 1992). Morrissy

(1989) recommended that if C. tenuimanus cultured in ponds have access to natural food sources

they do not require completely specified artificial diets. By contrast, semi-intensive and intensive

culture systems will depend highly on supplementation with aqua-feeds. This has been

- 17 -

demonstrated in a number of crayfish species. In more intensive systems such as closed

recirculating (tank-culture) systems, Reigh et al. (1990) recommended that dietary protein

requirements for red swamp crayfish (P. clarkii) and white river crayfish (P. astacus astacus)

need to be between 25-35%. Under similar conditions, Keefe and Rouse (1999) reported that, C.

quadricarinatus juveniles demonstrate best growth when fed 33% protein, although they

suggested that protein requirements can be as low as 28%. Interestingly, Cortes-Jacinto et al.

(2004) indicated that 25.6% crude protein in the diet produces an optimum growth and survival

response in pre-adult C. quadricarinatus under cell (battery) culture conditions. Comparisons

between studies are very difficult however, because of differences in experimental conditions,

such as feeding rates and energy content of the diets.

The general protein requirements of prawns and other crustaceans have been researched

extensively (Akiyama et al., 1992). Proteins are one of the major components in crustacean diets

in terms of cost and volume (Akiyama et al., 1992). Traditionally, cultivation of crayfish, like

redclaw, has relied heavily on the use of feeds which were formulated for species that may have

relatively high requirements for dietary protein, such as prawns (Cortes-Jacinto et al., 2003).

Intensification of crayfish culture has highlighted the need for establishing and evaluating a

balanced and species specific diet with the lowest protein levels (Guillaume, 1997; Tacon &

Akiyama, 1997) which still produce good growth rates.

Carbohydrates generally represent the cheapest available source of energy in animal feeds

(Shiau, 1997) however, they have been poorly researched in crustacean nutrition. Primarily,

because unlike proteins and lipids, carbohydrates are not considered to be essential nutrients in

the diet and deficiencies do not cause disease (Ali, 1996). The role of carbohydrates is, however,

significant for glycogen storage, chitin synthesis, and the formation of steroids and fatty acids in

prawns (Ali, 1996).

- 18 -

Utilisation of carbohydrates by aquatic organisms varies with species, although it is less efficient

than that of terrestrial domesticated animals (Shiau, 1998). Carbohydrates structure usually

determines the degree of utilisation by animals. Simple sugars, in general appear to be poorly

utilised by crustaceans. A number of studies have demonstrated that prawns can utilise

disaccharide and polysaccharide better than monosaccharide sugars (Andrews et al., 1972;

Abdel Rahman et al., 1979; Pascual et al., 1983; Shiau & Peng, 1992; Ali, 1993). Studies

investigating the influence of protein, carbohydrate and lipid on weight gain in P. setiferus, using

glucose and starch as the carbohydrate source indicated that addition of glucose to diets resulted

in depressed growth whereas supplemental starch did not cause a reduction in weight gain.

Studies by Shiau and Peng (1992) demonstrated that P. monodon could better utilise cornstarch

for growth, than glucose. Shiau and Peng (1992) went on to investigate utilisation of different

carbohydrate sources and the possible sparing of dietary protein with carbohydrate in P.

monodon reared in seawater. Carbohydrate content of diets ranged from 20% to 30%. Results

indicated that prawns fed starch and dextrin showed significantly higher weight gain, a better feed

efficiency ratio, protein efficiency ratio and survival than those fed glucose. Required dietary

protein level for P. monodon is lower if starch, instead of glucose or dextrin, is used as the

carbohydrate source (Shiau, 1997). This is because starch has a better protein-sparing effect

than does dextrin or glucose.

Carbohydrates are used in diets in aquaculture principally to spare protein. Ali (1996)

demonstrated that in juvenile Penaeus indicus, highest growth and lowest food conversion ratios

were obtained on diets which contained 53.4% carbohydrate and 21.9% protein, respectively.

Further analyses in the same study demonstrated that prawns fed a high carbohydrate and low

protein diet did not show any reduction in body nitrogen (crude protein). This study, in addition to

a number of other studies conducted on prawns, demonstrated a protein sparing effect by

carbohydrates (Andrews et al., 1972).

- 19 -

The reason for poor utilisation of glucose by certain species of crustaceans is not yet fully

understood (Shiau, 1997). Some explanations offered include the possibility that absorption of

glucose occurs at a higher rate across the digestive tract (Shiau, 1997). Furuichi and Yone (1982)

suggested that rapid absorption of free glucose results in a considerable amount of glucose

entering the body tissue before the activities of carbohydrate metabolising enzymes are

sufficiently elevated. Although this phenomenon has only been tested in carp and trout it is

thought that a similar process may also operate in crustaceans (Shiau, 1997).

An alternative explanation suggests that poor growth performance of prawns fed glucose

containing diets is due to inhibition of amino acid absorption in the intestine due to the presence

of glucose there (Alvarado & Robinson, 1979). Hokazeno et al. (1979) reported that presence of

10 mM of glucose reduced uptake of L-lysine from 26.64% to 12.34% and from 23.24% to 5.4%

in the mid-intestine and the posterior intestine, respectively, of rainbow trout. This interaction has

not been studied however, in crustaceans (Shiau, 1997).

Shiau (1997) reported that complex carbohydrates such as fibre can have individual and diverse

effects which depend principally on type and composition of fibre. Interestingly, a number of

studies reported that the addition of cellulose to the diet did not improve growth in some prawn

species although it did help to improve food conversion ratios (Ali, 1993). It has been suggested

that dietary cellulose aids efficient food conversion and better survival, although it should not

exceed 10% inclusion level in feeds formulated for prawns (Ali, 1993). Overall, there is very

limited understanding, about the physiological role that dietary fibre plays in crustaceans as well

as limited understanding of differences in response to dietary fibre among species (Shiau, 1997).

Triacylglycerols and fatty acids represent an important source of nutrients for crustaceans

(DAbramo, 1997). Essential fatty acids composed of linoleic (n-6) and linolenic (n-3) families of

polyunsaturated fatty acid are not synthesised de novo by crustaceans and as such are

considered essential nutrients in diets (Kayama et al., 1980). Work by Castell (1983) and

- 20 -

Chummugam et al. (1983) demonstrated that body tissue in marine crustaceans contains

proportionally higher levels of highly unsaturated fatty acids and polyunsaturated fatty acids of the

linolenic family than that of freshwater crustaceans. In parallel, body tissue in freshwater

crustaceans has higher levels of fatty acids belonging to the linoleic family (DAbramo, 1997).

To date, nutritional studies on the best dietary levels of one oil or mixtures of oils indicate that the

best growth (weight gain) response is achieved at inclusion levels of 5-8% for a number of

crustacean species (DAbramo, 1997). DAbramo (1997) observed however, that growth is

influenced ultimately by a variety of factors and high dietary levels (>10%) of oils in isolation are

usually associated with a significant reduction in growth rate. The main aspect of lipid nutrition to

consider is that the actual level of dietary lipids that elicits the most efficient growth response

(protein deposition) depends on a combination of factors that include other available sources of

energy, appropriate provision of essential fatty acids, both quantity and quality, and whether

protein requirements have been satisfied (DAbramo, 1997).

A unique requirement of crustaceans for phospholipids and sterols in their diet has also been

demonstrated. The principal roles of these nutrients are thought to be as cell constituents,

metabolic precursors of steroid hormones and for moulting hormones (Teshima, 1997). As is the

case with other nutrients, however, the exact dietary requirements for these compounds in

crustaceans have yet to be established.

2.3 Digestibility

The nutritive value of any feed ingredient will depend primarily on an animals capacity to digest

and absorb it. Although the nutrient profile of an ingredient may appear sufficient, if it is not

digested, absorbed or utilised by the animal, it is of little nutritive value (Akiyama et al., 1992;

Guillaume & Chaubert, 2001). Digestibility relates to the digestive balance of ingested nutrients

- 21 -

that are absorbed by the animal, and it involves mechanical breakdown, solubilisation and

absorption of nutrients (Akiyama et al., 1992; Guillaume & Chaubert, 2001).

Digestibility of feedstuffs is generally influenced by relative ratios of both major and minor

nutrients as well as the presence of inhibitory components (Lee & Lawrence, 1997). For example,

nutritive value of proteins or protein quality is based on the amino acid composition of the protein

(Li et al., 2000); in particular this is reflected in the concentration of essential amino acids and

their bioavailability. Nutritive value of protein can be assessed by establishing one or more of the

following factors: protein digestibility, amino acid availability, protein efficiency ratio, net protein

utilisation, percent protein deposition and essential amino acid index (Li et al., 2000). There are

problems however, in determining the exact digestibility values of nutrients such as proteins, and

this is generally caused by species differences, and differences in experimental methodology and

diet composition (Li et al., 2000). A reduction in protein digestibility of soybean meal and fish

meal after heating is a well known industrial example of processing techniques that affect protein

digestibility (Li et al., 2000).

Many plant ingredients have been evaluated as protein sources in prawn feeds, however, their

nutritional value may be significantly reduced due to presence of antinutritional factors (Jobling et

al., 2001). These include a variety of compounds such as protease inhibitors, phyto-oestrogens,

lectins, antivitamins, phytates, saponins, various oligosaccharides and antigentic proteins (Jobling

et al., 2001). Kaushik et al. (1995) reported that the main deleterious effects of antinutritional

factors manifest as reduced feed intake and lower nutrient bioavailability in addition to depressed

growth in animals. There are a number of techniques used to reduce, remove or inactivate

antinutritional factors. These include various extraction and processing techniques (Jobling et al.,

2001). For example, dehulling and extrusion of some plant ingredients, such as lupins and other

legumes (where antinutritional factors are present in seed hulls) is considered to reduce

antinutritional factor levels (Jobling et al., 2001), such treatments have also been shown to

reduce negative effects on feed intake, nutrient utilisation and growth (Robaina et al., 1995).

- 22 -

Feed digestibility is currently used as the best, empirical method for measuring nutrient

availability in a feed (Shneider & Flatt, 1975). Digestibility can be determined using direct or

indirect methods. The direct or total collection method relies on total recovery and biochemical

analyses of feed presented, uneaten feed and faeces separately (Lee & Lawrence, 1997).

Although this method is theoretically the best, the difficulty in separating faeces from uneaten

food in the aquatic environment causes it to be used infrequently (Lee & Lawrence, 1997). An

indirect or marker method, has been utilised by most investigators, employs the use of an inert

marker such as chromic oxide or yetterbium (Akiyama et al., 1989). Use of markers mean that

only a representative sample of total faeces is needed and accurate estimation of dietary intake is

not required (Jones & De Silva, 1998).

Freshwater crustaceans are considered to have higher apparent feed digestibilities for energy

and for crude protein using feeds with high carbohydrate content (eg. wheat flour or rice bran)

than do equivalent marine crustaceans. Reigh et al. (1990) found that, based on apparent dry

matter (ADM) coefficients, high-starch feedstuffs were most (>87%) digestible and high-fibre

feedstuffs were least (35%) digestible in P. clarkii. High digestibility values for ingredients of plant

origin indicate the potential for development of nutritionally balanced crayfish specific feeds

utilising cheaper plant ingredients.

Careful assessment of feed digestibility is also important for waste reduction. Most aquaculture

waste is derived from diets, therefore reduction of waste outputs should be considered via

improvements in diet formulation and feeding strategies. One method for reducing waste is to

develop diets which produce less solid waste by eliminating poorly digestible ingredients such as

whole grain or grain by products used as binders and fillers in the feed formulae (Cho & Bureau,

2001). Solid waste in aqua-feeds can be further reduced by selecting ingredients that improve

apparent digestibility and the nutrient balance of the diet. For example, nitrogen waste can be

minimised through reduction of digestible protein to digestible energy (DP/DE) ratio of the diet

(Cho & Bureau, 2001).

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2.4 Digestive enzymes

Appropriate digestive enzymes are of fundamental importance as they allow species to extract

nutrients from specific dietary elements (reviewed by Lee et al., 1984). The need for research on

mechanisms of food digestion and the impact of diet on growth and survival has long been

recognised in crustaceans (Guzman et al., 2001). The activity of digestive enzymes has been

investigated in many crustacean species and it has been demonstrated that activity is affected by

ontogeny (Lovett & Felder, 1990; Figueiredo & Anderson, 2003), moulting (Fernandez et al.,

1997; Vega-Villasante et al., 1999) and diet composition (Kumulu & Jones, 1995; Guzman et al.,

2001; Pavasovic et al., 2004).

In crayfish, enzymatic protein content generally amounts to 50mg ml-1 and comprise a variety of

digestive enzymes including proteinases, lipases and carbohydrases including cellulases (Zwilling

& Neurath, 1981; Brown, 1995). Proteinases present in the gut of crustaceans include trypsin,

astacin, chympotrypsin, and exopeptidases (e.g. carboxypeptidase and aminopeptidase)

(Guillaume, 1997; Navarrete del Toro et al., 2006). Research has demonstrated that crustacean

trypsin will digest undenatured protein while vertebrate trypsin will not (Dall & Moriarty, 1983).

Similarly, astacin activity is confined to crustaceans and does not occur in vertebrates (Guillaume,

1997; Vogt, 2002).

Carbohydrases have been widely reported in crustaceans (Gamboa-Delgado et al., 2003), with

amylase being the most common carbohydrase in marine and freshwater crayfish species. Starch

and glycogen are hydrolysed primarily by -amylase and -glucosidase, with complementary

action by sucrase-isomaltase and -dextrinase (Gaxiola et al., 2005). Evidence also exists to

suggest that some crab species can hydrolyse complex carbohydrates (cellulose and

hemicellulose), due to presence of enzymes such as licheninase, laminarinase and xylanase, in

their digestive juice (Linton & Greenaway, 2004). Significantly, Xue et al. (1999) have

demonstrated the presence of endogenous cellulase enzymes in C. quadricarinatus. These

results are of particular interest to aquaculture, as they indicate potential for inclusion of cheaper

- 24 -

and more abundant plant-derived polysaccharides into aqua-feeds for this species. Limited lipase

activity has been reported in prawns (Andrew & Sick, 1972). By contrast, crayfish produce

considerable amounts of highly efficient fat emulsifiers that assist in the digestion of lipids (Vogt,

2002).

As described previously analysis of digestive enzymes helps identify specific dietary elements

that have the potential to meet the nutritional requirements of crustacean species exploited for

aquaculture. In the next section, the nutritional requirements and feeding strategies of redclaw will

be considered with reference to aquaculture of this species.

2.5 Redclaw aquaculture

2.5.1 Redclaw in culture

There are a number of freshwater crayfish species endemic to Australia, but the most promising

for aquaculture production all belong to the Cherax genus. Three species in the Cherax genus

with high potential for culture include redclaw (Cherax quadricarinatus), yabby (or yabbie)

(Cherax destructor) and marron (Cherax tenuimanus). Redclaw is currently considered to hold

most promise as a high value aquaculture species, both in Australia and overseas.

Redclaw, are endemic to rivers of northwest Queensland and Northern Territory in Australia and

the southern parts of Papua New Guinea (Lawrence & Jones, 2002). This species has proven

well suited to culture because of a number of physical, biological and commercial attributes. For

example, redclaw are relatively non-aggressive, when compared with other members of the

Cherax genus, they tolerate crowded conditions and are non-burrowing (Masser & Rouse, 1997).

In addition, they are capable of spawning multiple times (3 to 5 times) per year (Meade & Watts,

1995).

Redclaw can tolerate a broad range of environmental conditions. Specifically, they are highly

tolerant of variation in water temperature (5 - 42oC) (Ackefors, 1994), although the optimum

- 25 -

growth is achieved at water temperatures of 25 - 30oC (Austin, 1995). Redclaw are also tolerant

of fluctuations in dissolved oxygen and salinity. Lethal minimum for dissolved oxygen is 0.5 mg/L

(5 mg/L for best growth), while preferred salinity range is 0-12 ppt (Frost, 1975; Jones, 1995).

Redclaw are physically hardy and have a simple life cycle. Consequently, they require relatively

simple production technology and simple foods, contributing to lower production costs (Webster

et al., 1994; Jones et al., 1998). These favourable characteristics make the redclaw crayfish

particularly suited for aquaculture production. Expansion of commercial culture of redclaw, has

until recently, been restricted primarily by limited available scientific information, and particularly

on issues related to supplying appropriate nutrition in captivity (Austin, 1992).

2.5.2 Culture technologies and nutrition

In the past two decades, redclaw culture has received considerable interest with the focus on

production technology, husbandry and nutritional requirements (Morrissy et al., 1995; Jones &

Ruscoe, 1996; Lawrence et al., 1998). Consequently, a significant redclaw culture industry has

developed in a number of overseas countries (Muzinic et al., 2004).

At present, redclaw are cultured commercially utilising one of the three main grow out

technologies; extensive (Lawrence & Morrissy, 2000), semi-intensive and intensive production

(Romero, 1997). Extensive and semi-intensive production systems are the most common culture

methods used. Semi-intensive culture can produce 1500-2000 kg ha-1 in 6-7 months at stocking

densities of 3-4 juveniles m-2 (Lawrence & Jones, 2002). Intensive production in pond conditions

has been reported to yield up to 4000 kg ha-1 in 6 months at stocking densities of 7 juveniles m-2

(Romero, 1997).

Increased interest and growing demand for freshwater crayfish, has been accompanied by a

need to develop better semi-intensive and intensive culture methods (Manor et al., 2002). A

principal advantage of culture intensification is that intensive culture technologies facilitate greater

- 26 -

control over production parameters (Lee & Wickins, 1992) as well as producing increased yields

per volume of water (Manor et al., 2002). This in turn allows for optimum growing conditions to be

maintained all year round, resulting in hatching or harvesting of more than a single cohort per

year (Savolainen et al., 2003).

In general, higher stocking densities can often be achieved in intensive culture systems. There

are reports of recirculating systems being utilised to maintain, spawn and culture redclaw in

temperate regions (Keefe & Rouse, 1999), while intensive farming of individually held crayfish

(battery or cell system) has also been reported to be viable at a commercial scale (Mattei, 1995;

Wickins & Lee, 2002). Battery farms allow for stocking densities of 100-200 juveniles m-2 and

yield >5000 kg ha-1yr-1 (Wickins & Lee, 2002). Manor et al. (2002) demonstrated that grow out in

separate cell systems can dramatically improve yields, by as much as two orders of magnitude

with good survival rates, in comparison with open cultures systems.

Intensive and semi-intensive culture of freshwater crayfish provides the advantage of being able

to maintain environmental parameters for best production. Under intensive culture conditions,

only minimal nutritional contribution comes from natural food organisms found in the system

(Treece, 2000). In particular, animals cultivated under individual cell conditions do not normally

have access to any natural food sources. As a consequence, a nutritionally complete artificial diet

must be provided to the stock in order to maintain optimum production (Yeh & Rouse, 1994;

Anson & Rouse, 1996).

Early research on nutrition and culture of crayfish recognised the need for development of diets

specific for the organism. Pioneering work by Reigh et al. (1990) on red swamp crayfish (P.

clarkii) and Morrissy (1989) on marron (C. tenuimanus), recommended supplemental feeding of

outdoor pond-raised crayfish in order to prevent late-season production declines in ponds with

high population densities. Consequently, a number of studies addressed the nutritive

requirements of crayfish in order to develop an aqua-feed suited for intensive and semi-intensive

- 27 -

culture. Comparisons between these studies however, are difficult as tested nutrient levels were

highly variable while species studied were diverse, resulting in limited or incomplete data (Jover

et al., 1999). For example, dietary protein and fat levels varied significantly across different

studies (Tarshis, 1978; Huner & Meyers, 1979; Hubbard et al., 1986; Ackefors et al., 1992), while

very few studies investigated utilisation of dietary carbohydrates by crayfish (Reigh et al., 1990,

Jover et al., 1999).

Currently, the redclaw culture industry relies on use of selected penaeid prawn feeds (Garcia-

Ulloa et al., 2003). This practice is inappropriate because of fundamental differences between

prawn and crayfish physiology, natural feed sources and general nutritive requirements.

Moreover, redclaw may have potential to utilise many cheap plant-based ingredients since they

possess endogenous cellulases which allow for digestion of cellulose and related plant

carbohydrates (Xue et al., 1999). Future profitability for the industry will lie in developing artificial

feeds that are cheaper than equivalent marine prawn feeds often used to support aquaculture of

this species. To achieve this objective, a clearer understanding of the nutritional requirements,

feeding habits and digestive strategies of redclaw are required.

2.6 Conclusion

Redclaw crayfish are an excellent candidate for aquaculture production. They have been found

to possess a host of favourable physical, biological and commercial attributes that have resulted

in development of an expanding culture industry in Australia and overseas. Nutritional

requirements in this species, however, are poorly defined. Nevertheless, recent studies indicate

that redclaw may have good potential to utilise diets significantly cheaper than those consumed

by some marine crustacean species widely exploited for aquaculture.

Observations of natural feeding habits, digestive strategies and dietary macronutrient

requirements, will allow a better nutritive profile for the redclaw crayfish to be developed.

Investigations to date have already indicated that dietary protein levels required by this species

- 28 -

are generally lower than those required by marine prawns. Understanding the requirements for

carbohydrates, may result in opportunities to reduce protein levels in formulated feeds, further.

Digestibility trials evaluate the nutritional value of an ingredient and provide important information

to supplement growth measurements. Determining the digestibility coefficients of diets and

specific dietary ingredients for redclaw can allow formulation of lowest cost diets which meet the

nutritional requirements of this species in aquaculture systems. Selection of highly digestible

dietary ingredients has the additional benefit of reducing effluent derived from uneaten or poorly

digested aqua-feeds.

Understanding an organisms digestive potential in terms of its digestive tract and accompanying

digestive enzymes is an important strategy for optimising formulation of species specific diets.

Enzyme activity will vary with modifications to feeds and observing this relationship allows more

accurate definition of an animals capacity to utilise nutrients present in its feed.

As described in this review, literature addressing the nutritional requirements of redclaw crayfish

is limited. A general aim of the current project is to investigate the parameters outlined in this

section and obtain data that will improve our understanding of redclaw nutrition. Data from these

studies will provide a basis to help formulate nutritionally adequate, lowest cost, low polluting

diets to support future development of redclaw aquaculture.

- 29 -

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EVALUATION OF THE NUTRITIONAL REQUIREMENTS … · EVALUATION OF THE NUTRITIONAL REQUIREMENTS OF REDCLAW CRAYFISH, Cherax quadricarinatus ... in particular feed formulation. To better

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