effect of lactobacillus acidophilus and mannan ... · ketahanan ikan patin berjalur juvenil...

EFFECT OF Lactobacillus acidophilus AND

MANNAN OLIGOSACCHARIDE ON GROWTH

PERFORMANCE, DIGESTIVE ENZYME

ACTIVITIES, INTESTINAL MORPHOLOGY,

HAEMATOLOGY AND RESISTANCE OF

STRIPED CATFISH (Pangasianodon

hypophthalmus, Sauvage, 1878) JUVENILES

AGAINST Aeromonas hydrophila

MST. NAHID AKTER

UNIVERSITI SAINS MALAYSIA

2015

i

EFFECT OF Lactobacillus acidophilus AND

MANNAN OLIGOSACCHARIDE ON GROWTH

PERFORMANCE, DIGESTIVE ENZYME

ACTIVITIES, INTESTINAL MORPHOLOGY,

HAEMATOLOGY AND RESISTANCE OF

STRIPED CATFISH (Pangasianodon

hypophthalmus, Sauvage, 1878) JUVENILES


by

MST. NAHID AKTER

Thesis submitted in fulfillment of the

requirements for the degree of

Doctor of Philosophy

December 2015

ii

KESAN Lactobacillus acidophilus DAN MANNAN

OLIGOSAKARIDA TERHADAP PRESTASI

PERTUMBUHAN, MORFOLOGI USUS, AKTIVITI ENZIM

PENGHADAMAN, HEMATOLOGI DAN

KETAHANAN IKAN PATIN BERJALUR JUVENIL

(Pangasianodon hypophthalmus, Sauvage, 1878) TERHADAP

Aeromonas hydrophila

Disember 2015

Oleh

Tesis yang diserahkan untuk

memenuhi keperluan bagi

Ijazah Doktor Falsafah

MST. NAHID AKTER

ii

ACKNOWLEDGMENT

All praises and gratitude are due to Almighty Allah, the great, gracious,

merciful and supreme ruler of the universe, who enabled me to complete the

research work successfully for the degree of Doctor of Philosophy (Ph.D) in

Universiti Sains Malaysia (USM), Malaysia.

I would like to express my deepest sense of gratitude to my honorable

supervisor, Professor Roshada Hashim and Professor Siti Azizah Mohd Nor for

choosing me as a Ph.D candidate, their scholastic guidance, innovative suggestions,

constructive criticism, great patience in going through the research and the

manuscript, continuous encouragement throughout the research and study in USM.

I am very much impressed with laboratory staff Anna Mary Denis, who had

lent her hand whenever needed and friends, especially Kabir, Latif, Ali, Mehedi,

Munir, Rajib, Amalia, Jana, Hui Lay, Nadia, Azira and Ann, the teachers and staff in

the School of Biological Sciences and IPS for their friendly manners and continuous

assistance in the laboratories during my study period at USM.

I would like to express my sense of gratitude to my beloved parents, brother,

sister-in-law, nephew, niece and other relatives for their enduring sacrifice, blessings,

inspiration, cooperation, forbearance and endless love throughout my life to

complete my higher study.

Finally, I am grateful to Organization for Women in Science in the

Developing World (OWSDW) and Postgraduate Research Grant Scheme in USM for

providing the scholarship and funds with which the completion of my Ph.D work is

possible.

iii

TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES xiv

LIST OF FIGURES xix

LIST OF PLATES xxv

LIST OF ABBREVIATIONS xxvii

ABSTRAK xxx

ABSTRACT xxxii

CHAPTER 1 INTRODUCTION

1.1 Pangasianodon hypophthalmus 1

1.2 Problem Statement 5

1.3 Purposes of the Study 6

CHAPTER 2 LITERATURE REVIEW

2.1 Aquaculture 7

2.2 World Aquaculture 7

2.3 Taxonomy, Distribution and Morphology of Pangasianodon hypophthalmus 8

2.4 Diseases of Pangasianodon hypophthalmus Farming 11

2.5 Probiotic 13

2.5.1 Characteristics of Probiotic 14

2.5.2 Types of Probiotics 15

2.5.3 Lactic Acid Bacteria 16

2.5.4 Mechanisms of Action 17

2.5.4.1 Competition for Adhesion Sites / Competitive Exclusion 18

iv

2.5.4.2 Colonization 19

2.5.4.3 Production of Antimicrobial Substances / Antibacterial Activity 20

2.5.4.4 Sources and Competition for Nutrients 22

2.5.5 Beneficial Effects of Probiotics 23

2.5.5.1 Effect of Probiotics on Intestinal Ecosystem 23

2.5.5.2 Effect of Probiotics on Digestive Enzymes 23

2.5.5.3 Effect of Probiotics on Intestinal Morphology 24

2.5.5.4 Effect of Probiotics on Haematological Parameters 25

2.5.5.5 Effect of Probiotics on Immune Response 26

2.5.5.6 Effect of Probiotics on Histopathology of Liver 28

2.5.5.7 Effect of Probiotics on Growth and Feed Utilization 28

2.6 Prebiotics 29

2.6.1 Mannan Oligosaccharide (MOS) 30

2.6.2 Modes of Action of Prebiotics 31

2.6.2.1 Stimulation of Selective Beneficial Microflora 32

2.6.2.2 Effect on Intestinal Morphology 34

2.6.2.3 Improvement of Haematologcal Parameters 35

2.6.2.4 Improvement of Immune Functions and Disease Resistance 36

2.6.2.5 Effect of Prebiotics on Histopathology of Liver 37

2.6.2.6 Effect on Fish Growth and Feed Utilization 38

CHAPTER 3 MATERIALS AND METHODS

3.1 Introduction 39

3.2 Water Quality Parameters 39

3.3 Proximate Composition Analysis 40

3.4 Determination of Growth, Survival and Body Indices 41

v

3.5 Nutrient Digestibility Analysis 42

3.6 Enzyme Assay 43

3.6.1 Preparation of Intestine for Digestive Enzyme Assays 43

3.6.2 Amylase Activity 43

3.6.3 Protease Activity 44

3.6.4 Lipase Activity 44

3.6.5 Total Protein Content in Intestinal Enzyme Extract 45

3.7 Analysis of Intestinal Microflora 46

3.8 Intestinal Morphology Analysis 47

3.9 Haematological Parameters 48

3.9.1 Erythrocyte Sedimentation Rate (mm h-1

) 49

3.9.2 Packed Cell Volume / Haematocrit (%) 49

3.9.3 Total Red Blood Cell Count (Erythrocyte / RBC x106 mm

-3) 50

3.9.4 Haemoglobin Concentration and Red Blood Cell Indices 50

3.9.5 Total White Blood Cells Count (Leukocyte / WBC x104 mm

-3) 51

3.9.6 Differential Leukocyte Count (%) 52

3.10 Determination of Immune Indices 52

3.10.1 Total Immunoglobulin Content (Ig) 52

3.10.2 Lysozyme Assay 53

CHAPTER 4 ANTIMICROBIAL ACTIVITY OF Lactobacillus acidophilus

AGAINST FRESHWATER BACTERIA Aeromonas hydrophila

4.1 Introduction 55

4.2 Materials and Methods 56

4.2.1 Isolation and Identification of Aeromonas hydrophila 56

4.2.1.1 Gram Staining Method 57

4.2.1.2 Molecular Identification Method 58

vi

4.2.2 Antimicrobial Activity of L. acidophilus Against A. hydrophila 63

4.2.2.1 Preparation of A. hydrophila 63

4.2.2.2 Culture of L. acidophilus 64

4.2.2.3 In-vitro Antimicrobial Activity of L. acidophilus Against A.

hydrophila

65

4.2.2.4 Effect of L. acidophilus on Survival of P. hypophthalmus

infected to A. hydrophila

66

4.2.3 Identification of Bioactive Compounds from L. acidophilus Cells

and Cell Free Extract

67

4.2.3.1 Sample Preparation 67

4.2.3.2 Gas Chromatograph-Mass Spectrophotometry (GC-MS) 68

4.2.4 Statistical Analysis 68

4.3 Results 69

4.3.1 In-vitro Antimicrobial Activity of L. acidophilus Against A.

hydrophila CT19

69

4.3.2 Effect of L. acidophilus on survival of P. hypophthalmus infected

to A. hydrophila

71

4.3.3 Bioactive Compounds Identified from L. acidophilus Cells and

Cell Free Extract

74

4.4 Discussion 77

CHAPTER 5 EFFECT OF Lactobacillus acidophilus

SUPPLEMENTATION ON GROWTH PERFORMANCE, DIGESTIVE

ENZYME ACTIVITIES, INTESTINAL MORPHOLOGY,

HAEMATOLOGY, IMMUNE PARAMETERS AND RESISTANCE OF

STRIPED CATFISH (Pangasianodon hypophthalmus, Sauvage, 1878)

JUVENILES AGAINST Aeromonas hydrophila

5.1 Introduction 79


vii

5.2.1 Experimental Fish and Culture Conditions 80

5.2.2 Preparation of Experimental Diets 81

5.2.3 Water Quality Parameters Analysis 84

5.2.4 Sampling Procedure 84

5.2.5 Evaluation of Growth Performance and Body Indices 86

5.2.6 Proximate Composition Analysis 86

5.2.7 Determination of Nutrient Digestibility 86

5.2.8 Digestive Enzyme Assays 86

5.2.9 Analysis of Intestinal Microflora 87

5.2.10 Evaluation of Intestinal Morphology 87

5.2.11 Haematological Indices 87

5.2.11.1 Erythrocyte Sedimentation Rate (mm h-1

) 87

5.2.11.2 Packed Cell Volume / Haematocrit (%) 87

5.2.11.3 Total Red Blood Cell Count (Erythrocyte / RBC x106 mm

-3) 88

5.2.11.4 Haemoglobin Concentration (g dL-1

) 88

5.2.11.5 Red Blood Cell Indices 88

5.2.11.6 Total White Blood Cell Count (Leukocyte / WBC x104 mm

-3) 88

5.2.11.7 Differential Leukocyte Counts (%) 88

5.2.12 Determination of Immune Parameters 88

5.2.12.1 Total Immunological Concentration (Ig) 89

5.2.12.2 Lysozyme Assay 89

5.2.13 Determination of Resistance to A. hydrophila Infection 89

5.2.13.1 Pathogenicity Test 89

5.2.13.2 Challenge Test 90

5.2.14 Histological Study of the Liver 91

viii


5.3 Results 92

5.3.1 Viability of L. acidophilus in Experimental Diets 92

5.3.2 Growth Performance, Survival and Body Indices 94

5.3.3 Proximate Composition of Muscle 96

5.3.4 Nutrient Digestibility Analysis 97

5.3.5 Digestive Enzyme Activities 98

5.3.6 Intestinal Microflora Analysis 100

5.3.7 Intestinal Morphology Analysis 101

5.3.8 Haematological Parameters 108

5.3.8.1 Pre-challenged Fish with A. hydrophila 108

5.3.8.2 Post-challenged Fish with A. hydrophila 110

5.3.8.2.1 Two-week Post-challenged 110

5.3.8.2.2 Three-week Post-challenged 112

5.3.9 Immune Indices 116

5.3.9.1 Total Immunoglobulin Content (Ig) 116

5.3.9.2 Lysozyme Activity 117

5.3.10 Determination of Resistance A. hydrophila Infection 118

5.3.10.1 Pathogenicity Test 118

5.3.10.2 Challenge Test 119

5.3.11 Histopathology of Liver 119

5.4 Discussion 125

5.5 Conclusion 140

CHAPTER 6 EFFECT OF DIETARY SUPPLEMENTATION OF

MANNAN OLIGOSACCHARIDE ON GROWTH PERFORMANCE,

DIGESTIVE ENZYME ACTIVITIES, INTESTINAL MORPHOLOGY,

HAEMATOLOGICAL PARAMETERS AND DISEASE RESISTANCE

ix

OF STRIPED CATFISH (Pangasianodon hypophthalmus, Sauvage, 1878)

JUVENILES AGAINST Aeromonas hydrophila

6.1 Introduction 141


6.2.1 Experimental Site 143

6.2.2 Fish and Rearing Conditions 143

6.2.3 Diet Preparation 144

6.2.4 Sample Collection 146

6.2.5 Water Quality Parameters 146

6.2.6 Determination of Growth Performance, Survival and Body Indices 148

6.2.7 Evaluation of Proximate Composition 148



6.2.10 Quantification of Intestinal Microflora 149

6.2.11 Histological Study of the Intestine 149



) 149

6.2.12.2 Packed Cell Volume (%) 149


-3) 149

6.2.12.4 Haemoglobin Content (g dL-1

) 150


6.2.12.6 Total White Blood Cell Count (Leukocyte/ WBC x104 mm

-3) 150

6.2.12.7 Leukocyte Types (%) 150

6.2.13 Evaluation of Immune Parameters 150



x

6.2.14 Evaluation of Resistance to A. hydrophila CT19 Infection 151



6.3 Results 152

6.3.1 Growth Performance and Body Indices 152

6.3.2 Muscle Proximate Composition 154



6.3.5 Intestinal Microflora Analysis 158



6.3.7.1 Pre-challenged Fish 165

6.3.7.2 Post-challenged Fish 167



6.3.8 Immune Parameters 173



6.3.9 Evaluation of Resistance to A. hydrophila Infection 175

6.3.10 Histopathology of Liver 175

6.4 Discussion 181

6.5 Conclusion 195

CHAPTER 7 EFFECT OF Lactobacillus acidophilus (LAB) AND

MANNAN OLIGOSACCHARIDE (MOS) ON GROWTH

PERFORMANCE AND HEALTH STATUS OF JUVENILE STRIPED

CATFISH (Pangasianodon hypophthalmus) FED DIETS CONTAINING

SOYBEAN MEAL

xi

7.1 Introduction 196


7.2.1 Experimental Diets 198

7.2.2 Experimental Fish and Feeding Trial 201

7.2.3 Collection of Sample 201

7.2.4 Water Quality Parameters 202

7.2.5 Proximate Composition 202

7.2.6 Growth Performance Assessment 204


7.2.8 Evaluation of Digestive Enzyme 204

7.2.9 Evaluation of Intestinal Microflora 204




) 205

7.2.11.2 Packed Cell Volume (%) 205


-3) 205

7.2.11.4 Haemoglobin Content (g dL-1

) 205


7.2.11.6 Total White Blood Cell Count (Leukocyte/ WBC x104 mm

-3) 206

7.2.11.7 Leukocyte Types (%) 206

7.2.12 Determination of Immune Indices 206



7.2.13 Challenge Assay 207


xii

7.2.15 Cost Effectiveness of using Probiotic and Prebiotic in SBM Diets 207


7.3 Results 208

7.3.1 Growth Performance and Body Indices 208

7.3.2 Muscle Composition 210



7.3.5 Intestinal Microflora 214

7.3.6 Intestinal Morphology 216


7.3.7.1 Pre-challenged Fish 222

7.3.7.2 Post-challenged Fish 224



7.3.8 Immune Parameters 230



7.3.9 Evaluation of Resistance to A. hydrophila Infection 232

7.3.10 Histopathological Changes of Liver 232

7.3.11 Cost Effectiveness of Supplementing Probiotic and Prebiotic in SBM

Diets

234

7.4 Discussion 239

7.5 Conclusion 250

CHAPTER 8 CONCLUSION AND RECOMMENDATIONS

8.1 Conclusion 251

xiii

8.2 Recommendations for Future Research 252

REFERENCES 253

APPENDICES 282

LIST OF PUBLICATION 311

xiv

LIST OF TABLES

Table No. Content PAGE

Table 2.1 Common probiotics used for human and animal consumption 16

Table 4.1 Primers used in the study 60

Table 4.2 Components used in PCR 60

Table 4.3 PCR profiles used 61

Table 4.4 Identification of strain using BLAST analysis 63

Table 4.5 Inhibition zone diameter (mm) of cells and cell free extract of

Lactobacillus acidophilus against Aeromonas hydrophila

69

Table 4.6 Survival of immunized and non immunized P. hypophthalmus

after infected with A. hydrophila

71

Table 4.7 Bioactive compounds identified from the Lactobacillus

acidophilus cells (LABP) and cell free extract (LABS) by GC-

MS

75

Table 5.1 Ingredients used and proximate composition of control and

varying concentrations of Lactobacillus acidophilus

supplemented diets (g kg-1

)

83

Table 5.2 Water quality parameters of striped catfish (Pangasianodon

hypophthalmus) reared in experimental tanks

85

Table 5.3 Viability of Lactobacillus acidophilus in experimental diets (log

CFU/g) at a temperature of -20oC for 90 days of storage

93

Table 5.4 Growth performances and feed utilization of striped catfish,

Pangasianodon hypophthalmus, juveniles fed diets containing

varying levels of Lactobacillus acidophilus supplemented diets

for 12 weeks

95

xv

Table 5.5 Body muscle composition of striped catfish, Pangasianodon

hypophthalmus, fed diets with various levels of Lactobacillus

acidophilus for 12 weeks

96

Table 5.6 Apparent protein digestibility (APD%) and apparent dry matter

digestibility (ADM%) in juvenile striped catfish, Pangasianodon

hypophthalmus, after feeding with Lactobacillus acidophilus

supplemented diets

97

Table 5.7 Effect of varying concentrations of Lactobacillus acidophilus on

specific digestive enzyme activities (Umg-1

protein) of juvenile

striped catfish, Pangasianodon hypophthalmus

99

Table 5.8 Haematological parameters of juvenile striped catfish,

Pangasianodon hypophthalmus, fed diets containing varying

levels of Lactobacillus acidophilus and control diets after 12

weeks

109

Table 5.9 Haematological parameters of striped catfish, Pangasianodon

hypophthalmus, juveniles fed diets containing varying levels of

Lactobacillus acidophilus challenged with Aeromonas

hydrophila after 2-week

111



levels of Lactobacillus acidophilus and control diets after a 3-

week challenged with Aeromonas hydrophila

113

Table 5.11 Pathogenicity test of Aeromonas hydrophila in striped catfish,

Pangasianodon hypophthalmus

118

Table 5.12 Effect of various concentrations of Lactobacillus acidophilus 120

xvi

supplementation on the post-challenged survival of juvenile

striped catfish, Pangasianodon hypophthalmus, with Aeromonas

hydrophila

Table 6.1 Ingredients used and proximate composition of the experimental

diets containing varying levels of mannan oligosaccharide

(MOS) (g kg-1

)

145

Table 6.2 Water quality parameters of striped catfish (Pangasianodon

hypophthalmus) juveniles reared in the experimental tanks

147

Table 6.3 Growth performance, feed utilization and body indices of

juvenile striped catfish, Pangasianodon. hypophthalmus, fed a

diet containing varing levels of mannan oligosaccharide (MOS)

for 12 weeks

153

Table 6.4 Proximate composition of body muscle of juvenile striped

catfish, Pangasianodon hypophthalmus, fed varying levels of

mannan oligosaccharide (MOS) for 12 weeks

154


digestibility (ADM %) in juvenile striped catfish,

Pangasianodon hypophthalmus, fed mannan oligosaccharide

(MOS) treated diets

155

Table 6.6 Effect of different levels of dietary mannan oligosaccharide

(MOS) supplementation on specific digestive enzyme activities

(U

mg-1

protein) in juvenile striped catfish Pangasianodon

hypophthalmus

157



166

xvii

levels of MOS and control diets for 12 weeks

Table 6.8 Hematological parameters of juvenile striped catfish,

Pangasianodon. hypophthalmus, fed diets containing varying

levels of MOS and control diets at 2-week post challenged with


168

Table 6.9 Hematological parameters of juvenile striped catfish,


levels of MOS and control diets after 3-week infection with


170

Table 6.10 Effect of various concentrations of MOS supplementation on the

post-challenged survival of juvenile striped catfish,

Pangasianodon hypophthalmus, with Aeromonas hydrophila

176

Table 7.1 Ingredients used and proximate composition of experimental

diets (g kg-1

)

200

Table 7.2 Water quality parameters in the experimental tanks during

feeding trial

203

Table 7.3 Growth performances and feed utilization of juvenile striped

catfish, Pangasianodon hypophthalmus, fed on various

experimental diets for a 12-week period

209

Table 7.4 Body muscle composition of juvenile striped catfish,

Pangasianodon hypophthalmus, fed on experimental diets for a

12-week period

210


digestibility (ADM%) of juvenile striped catfish, Pangasianodon

hypophthalmus, after 12 weeks feeding with experimental diets

211

xviii

Table 7.6 Effect of varying experimental diets on the specific digestive

enzyme activities (U mg-1

protein) in juvenile striped catfish,

Pangasianodon hypophthalmus

213

Table 7.7 Haematological parameters of striped catfish juveniles

(Pangasianodon hypophthalmus) fed with LAB or MOS

supplemented fish meal or fish meal replaced with soybean meal

for a 12-week period

223


Pangasianodon hypophthalmus, fed with FM and FM replaced

with SBM supplemented with LAB and MOS after 2-week

challenged with Aeromonas hydrophila CT19

225

Table 7.9 Hematological parameters of juvenile striped catfish

Pangasianodon hypophthalmus, fed with FM and FM replaced

with SBM supplemented with LAB and MOS after 3-week

infection with Aeromonas hydrophila

227

Table 7.10 Effect of various experimental diets on the post-challenged

survival of juvenile striped catfish, Pangasianodon

hypophthalmus, with Aeromonas hydrophila

233

Table 7.11 Cost benefit analysis of experimental diets used after 12 weeks

238

xix

LIST OF FIGURES

Figure No. Content PAGE

Figure 4.1 AM indicated Aeromonas hydrophila cells; LABP,

Lactobacillus acidophilus cells; LABS, Lactobacillus

acidophilus cell free extract injected striped catfish survival

after 2-week of the challenged.

71

Figure 5.1 Total lactic acid bacteria and total bacteria count in the intestine

of striped catfish, Pangasianodon hypophthalmus, juvenile fed

various concentrations of Lactobacillus acidophilus for a 12-

week period.

100

Figure 5.2 Light microscopy of the anterior and the posterior intestine of

striped catfish, Pangasianodon hypophthalmus, shows A) Villi

length, B) Villi width and C) Crypt depth after 12 weeks

feeding with experimental diets.

103

Figure 5.3 Light microscopy of the anterior intestine of striped catfish,

Pangasianodon hypophthalmus, fed A) control, B) 103, C) 10

5,

D) 107, and E) 10

9 CFU/g of L. acidophilus supplemented diets

after 12 weeks feeding trial.

104

Figure 5.4 Light microscopy of the posterior intestine of striped catfish,

Pangasianodon hypophthalmus, fed A) control, B) 103, C) 10

5,

D) 107, and E) 10

9 CFU/g of L. acidophilus supplemented diets

after 12 weeks feeding trial.

105

Figure 5.5 The length of the microvilli of the posterior intestine of striped

catfish, Pangasianodon hypophthalmus, after 12 weeks feeding

with Lactobacillus acidophilus supplemented diets.

106

xx

Figure 5.6 Transmission electron microscopy (TEM) from the posterior

intestine of striped catfish, Pangasianodon hypophthalmus, fed

A) control, B) 103, C) 10

5, D) 10

7, and E) 10

9 CFU/g of L.

acidophilus supplemented diet after 12 weeks feeding trial.

107

Figure 5.7 A) RBC, B) WBC, C) Lymphocyte and D) Granulocyte count

in pre- and 3-week post-challenged striped catfish,

Pangasianodon hypophthalmus.

115

Figure 5.8 Immunoglobulin content of pre- and post-challenged striped

catfish, Pangasianodon hypophthalmas, feeding with various

concentrations of Lactobacillus acidophilus supplemented

diets.

116

Figure 5.9 Lysozyme activity of striped catfish, Pangasianodon

hypophthalmas, fed with Lactobacillus acidophilus

supplemented diets in pre- and post-challenged with

Aeromonas hydrophila.

117

Figure 5.10 Liver stained with haematoxylin and eosin, bar 200 µm; sample

collection at pre-challenged striped catfish, Pangasianodon

hypophthalmus, with Aeromonas hydrophila A) Control, B)

103, C) 10

5, D) 10

7, E) 10

9 CFU/g of Lactobacillus acidophilus

supplemented diets.

122


collection at 2-week post infected striped catfish,

Pangasianodon hypophthalmus, with Aeromonas hydrophila.

123


collection at 3-week post-challenged striped catfish,

124

xxi


Figure 6.1 Total lactic acid bacteria and total bacteria count in the intestine

of juvenile striped catfish (Pangasianodon hypophthalmus) fed

mannan oligosaccharide (MOS) for 12 weeks.

158

Figure 6.2 Light microscopy of the anterior and posterior intestine of

striped catfish, Pangasianodon hypophthalmus, shows (A) Villi

length, (B) Villi width and (C) Crypt depth after 12 weeks


161

Figure 6.3 Light microscopy of the anterior A) and posterior B) intestine

of striped catfish, Pangsianodon hypophthalmus, shows villus

length, villus width, villus crypt junction and crypt depth after

12 weeks feeding with various MOS supplemented diets.

162

Figure 6.4 The length of microvilli of the posterior intestine of juvenile

striped catfish, Pangsianodon hypophthalmus, after 12 weeks


163

Figure 6.5 TEM micrograph from the posterior intestine of juvenile striped

catfish, Pangsianodon hypophthalmus, fed either the control or

MOS supplemented diets.

164

Figure 6.6 A) RBC, B) WBC, C) Lymphocyte and D) Granulocyte count

in pre- and 2-week post-challenged striped catfish,


172

Figure 6.7 Total immunoglobulin (Ig) content of juvenile striped catfish,

Pangasianodon hypophthalmus, fed MOS supplemented diets

in pre- and post-challenged with Aeromonas hydrophila.

173

Figure 6.8 Lysozyme activity of pre- and post-challenged striped actfish, 174

xxii


fed with varying levels of MOS supplemented diets.



hypophthalmus, fed on A) Control, B) 0.2% MOS, C) 0.4%

MOS, D) 0.6% MOS, E) 0.8% MOS diets.

178




179




180

Figure 7.1 The bacterial abundance in the intestine of juvenile striped

catfish (Pangasianodon hypophthalmus) fed with supplemented

fish meal and fish meal replaced with soybean meal diets for 12

weeks.

215

Figure 7.2 Light microscopy of the anterior and posterior intestine of

striped catfish, Pangasianodon hypophthalmus, shows A) Villi

length, B) Villi width and C) Crypt depth after 12 weeks


217

Figure 7.3 Light microscopy of the anterior intestine of striped catfish,

Pangasianodon hypophthalmus, fed A) FM, B) FM+LAB, C)

FM+MOS, D) FM+SBM, E) FM+SBM+LAB, and F)

FM+SBM+MOS diets after 12 weeks feeding trial.

218

Figure 7.4 Light microscopy of the posterior intestine of striped catfish, 219

xxiii

Pangasianodon hypophthalmus, fed A) FM, B) FM+LAB, C)

FM+MOS, D) FM+SBM, E) FM+SBM+LAB, and F)

FM+SBM+MOS diets after 12 weeks feeding trial.

Figure 7.5 The length of the microvilli of the posterior intestine of striped

catfish, Pangasianodon hypophthalmus, after 12 weeks feeding

with various experimental diets.

220

Figure 7.6 TEM micrograph from the posterior intestine of juvenile striped

catfish, Pangasianodon hypophthalmus, fed on various

experimental diets A) FM, B) FM+LAB, C) FM+MOS, D)

FM+SBM, E) FM+SBM+LAB, and F) FM+SBM+MOS.

221

Figure 7.7 A) Hb, B) RBC, C) WBC and D) Granulocyte count in pre- and

2-week post-challenged striped catfish, Pangasianodon

hypophthalmus.

229

Figure 7.8 Total immunoglobulin (Ig) content of striped catfish,

Pangasianodon hypophthalmus, fed FM and FM replaced with

SBM supplemented with Lactobacillus acidophilus and MOS

in pre- and post-challenged with Aeromonas hydrophila.

230

Figure 7.9 Lysozyme activity of pre- and post-challenged striped catfish,


fed with various FM and FM replaced with SBM supplemented

with Lactobacillus acidophilus and MOS diets.

231



hypophthalmus, with Aeromonas hydrophila A) FM, B)

FM+LAB, C) FM+MOS, D) FM+SBM, E) FM+SBM+LAB,

235

xxiv

and F) FM+SBM+MOS of diet.



Pangasianodon hypophthalmus with Aeromonas hydrophila.

236


collection at 3-week post-challenged striped catfish with


237

xxv

LIST OF PLATES

Plate No. Content PAGE

Plate 2.1 Pangasianodon hypophthalmus juvenile used in this study. 9

Plate 2.2 Disease is the end result of the host due to interaction and

interrelationship of above 3 factors (Source: Francis-Floyd, R.,

1997).

11

Plate 2.3 Inhibition of enteric bacteria and enhancement of barrier

function by probiotic bacteria. Schematic representation of the

crosstalk between probiotic bacteria and the intestinal mucosa.

21

Plate 2.4 The cell wall structure of yeast (Saccharomyces cerevisiae). 31

Plate 3.1 Blood sampling of juvenile striped catfish (Pangasianodon

hypophthalmus).

49

Plate 4.1 A) Aeromonas hydrophila isolated from infected striped catfish,

Pangasianodon hypophthalmus kidney and liver; B) Pure

colonies of Aeromonas hydrophila reisolated from infected

kidney of striped catfish.

57

Plate 4.2 Gram staining of the isolated bacteria (pink to red stain

indicated gram-negative bacteria).

58

Plate 4.3 Agarose gel (1%) stained ethdium bromide (EtBr). Lane 1

shows a 1kb DNA ladder, lane 2 shows control and lane 3

shows PCR for Aeromonas hydrophila.

63

Plate 4.4 The commercial Lactobacillus acidophilus grown on MRS agar

plate.

65

Plate 4.5 Zone of antimicrobial inhibition of Lactobacillus acidophilus 70

xxvi

against Aeromonas hydrophila CT19 using the well diffusion

method, A) indicates cells of Lactobacillus acidophilus; B) cell

free extract of Lactobacillus acidophilus, and C) control.

Plate 4.6 Clinical signs of infected Pangasianodon hypophthalmus with


72

Plate 4.7 Immunized striped catfish with A) Lactobacillus acidophilus

cells (LABP) and B) Lactobacillus acidophilus cell free extract

(LABS) followed challenged with Aeromonas hydrophila cells

showed a recovery from severe infection.

73

Plate 5.1 Intraperitoneal injection of P. hypophthalmus with A.

hydrophila.

90

Plate 5.2 Challenge assay performed in disease lab where continuous

water flow with aeration was provided. Water was treated using

UV light before being discharged.

91

xxvii

LIST OF ABBREVIATIONS

ADM Apparent dry matter digestibility

AEP Acini of the exocrine pancreas

AM Aeromonas hydrophila

ANOVA One-way analysis of variance

AOAC Association of official analytical chemists

APD Apparent protein digestibility

BD Bile duct

BLAST Basic local alignment search tool

CAC Centroacinar cells

CFU Colony forming unit

CMC Carboxy methyl cellulose

CN Central nucleoli

DN Disappearance of nucleoli

DNA Deoxyribonucleic acid

EP Epithelium

ESR Erythrocyte sedimentation rate

EtBr Ethidium bromide

FAO Food and agriculture organization

FM Fish meal

FOS Fructo oligosaccharides

FCR Food conversion ratio

GC Goblet cells

GC-MS Gas Chromatography-Mass spectrometry

GE Gross energy

xxviii

GI Gastrointestinal

GOS Galacto oligosaccharides

H Hepatocyte

Hb Haemoglobin

HSI Hepatosomatic index

Ig Immunoglobulin

IPF Intraperitoneal fat

L Lumen

LAB Lactobacillus acidophilus

LABP Lactobacillus acidophilus pellet

LABS Lactobacillus acidophilus supernatant

LC Leukocyte count

LP Lamina propia

MCH Mean corpuscular haemoglobin

MCHC Mean corpuscular haemoglobin concentration

MCV Mean corpuscular volume

MMC Melanomacrophage centre

MOS Mannan oligosaccharide

MRS De Man, Rogosa and Sharp agar

MV Microvilli

NFE Nitrogen free extract

N Necrosis

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PCV Packed cell volume

xxix

PER Protein efficiency ratio

PEG Polyethylene glycol

PK Pyknotic nuclei

PN Periphery nucleoli

PV Portal vein

PVA Polyvinyl alcohol

RBC Red blood cell

SBM Soybean meal

SCFAs Short chain fatty acids

scFOS Short-chain fructo oligosaccharides

SGR Specific growth rate

SPSS Statistical package for social science

SR Survival rate

TEM Transmission electron microscopy

TSA Tryptic soy agar

TSB Tryptic soy broth

VSI Viscerosomatic index

V Vacuolization

WG Weight gain

WBC White blood cell

WHO World health organization

xxx

KESAN Lactobacillus acidophilus DAN MANNAN OLIGOSAKARIDA

TERHADAP PRESTASI PERTUMBUHAN, MORFOLOGI USUS,

AKTIVITI ENZIM PENGHADAMAN, HEMATOLOGI DAN

KETAHANAN IKAN PATIN BERJALUR JUVENIL (Pangasianodon

hypophthalmus, Sauvage, 1878) TERHADAP Aeromonas hydrophila

ABSTRAK

Penyakit dianggap sebagai salah satu faktor yang paling penting

membimbangkan dalam akuakultur ikan patin berjalur (Pangasianodon

hypophthalmus, Sauvage, 1878). Oleh itu untuk itu beberapa siri ujikaji telah

dijalankan untuk mengenalpasti tahap yang paling sesuai bagi probiotik

Lactobacillus acidophilus (LAB) dan prebiotik mannan oligosakarida (MOS), yang

dapat meningkatkan pertumbuhan dan tahap kesihatan ikan patin berjalur juvenil.

kumpulan tiga replikat ikan patin berjalur juvenil (21.69±0.18g) telah diberi

makan diet 0 (kawalan), 103, 105, 10

7 dan 10

9 CFU/g LAB, dua kali sehari pada

kadar 2.5% daripada berat badan ikan, selama 12 minggu. Prestasi pertumbuhan dan

tahap kesihatan ikan berdasarkan parameter hematologi dan immunologi dalam

kumpulan ikan yang diberi makan diet yang mengandungi LAB pada kadar 105

CFU/g dan keatas menunjukkan peningkatan yang signifikan. Dalam kajian yang

lain, apabila ikan patin juvenil (20.41±1.64g) diberi makan lima diet berbeza terdiri

daripada 0 (kawalan), 0.2%, 0.4%, 0.6% dan 0.8% MOS, prestasi pertumbuhan dan

tahap kesihatan meningkat secara signifikan pada diet yang mengandung 0.6% MOS

berbanding kumpulan lain. Kesan suplemen LAB atau MOS terhadap diet

penghadaman dan kadar pertumbuhan dan kesihatan ikan (20.50±0.28g) telah dikaji

dengan menggantikan 45% protin daripada serbuk ikan (FM) dengan serbuk kacang

xxxi

soya (SBM). Walaupun ikan yang diberi suplemen LAB atau MOS mengandungi

SBM tidak meningkatkan prestasi pertumbuhan dan penghadaman ikan, namun

status kesihatan ikan patin berjalur juvenil menunjukkan peningkatan yang ketara

terhadap parameter hematologi, jumlah kandungan immunoglobulin dan aktiviti

lysozyme. Berdasarkan kepada semua kajian yang dijalankan, boleh dirumuskan

bahawa diet yang disuplemen dengan 105

CFU/g LAB dan 0.6% MOS adalah

mencukupi untuk meningkatkan tahap pertumbuhan dan kesihatan ikan patin berjalur

juvenil apabila didedahkan kepada jangkitan A. hydrophill. Disamping itu, suplemen

105 CFU LAB or 0.6 % MOS pada diet yang mengandungi 55 % protin daripada FM

dan 45 % protin daripada SBM dapat meningkatkan tahap kesihatan tanpa memberi

kesan buruk terhadap pertumbuhan dan penghadaman ikan patin berjalur juvenil.

xxxii

EFFECT OF Lactobacillus acidophilus AND MANNAN OLIGOSACCHARIDE

ON GROWTH PERFORMANCE, DIGESTIVE ENZYME ACTIVITIES,

INTESTINAL MORPHOLOGY, HAEMATOLOGY AND RESISTANCE OF

STRIPED CATFISH (Pangasianodon hypophthalmus, Sauvage, 1878) ES


ABSTRACT

Disease has been considered one of the most significant concern for the

sustainable development of striped catfish, Pangasianodon hypophthalmus,

therefore, a series of experiments were carried out to determine the most suitable

level of probiotic Lactobacillus acidophilus (LAB) and prebiotic mannaan

oligosaccharide (MOS), respectively, that can promote growth and improve the

health status of e striped catfish. Triplicate groups of e striped catfish (initial weight

21.69±0.18 g) were fed twice daily at 2.5% of body weight, with 0 (control), 103,

105, 10

7 and 10

9 CFU/g LAB diets, for 12 weeks. Results revealed that the growth

performance and health status based on haematology and immunological parameters

were significantly improved when fish were fed the diet supplemented with

105CFU/g and above LAB. In another study under similar experimental conditions,

when striped catfish (20.41±1.64g) were fed with five experimental diets containing

0 (Control), 0.2%, 0.4%, 0.6% and 0.8% MOS, respectively, the growth performance

and health status were significantly improved for 0.6% MOS diet fed group than the

control fed group. The effect of LAB or MOS supplementation on diet digestibility

and subsequently the growth and health status, were evaluated by feeding fish

(20.50±0.28 g) with test diets in which 45% of fish meal protein was replaced with

soybean meal protein. Although feeding fish with the LAB or MOS supplemented in

xxxiii

the diets containing SBM did not improve the growth performance and nutrient

digestibility, the health status of striped catfish was significantly improved by

increasing the haematological parameters, total immunoglobulin content and

lysozyme activity. Based on the results of all the studies, it can be concluded

that supplementation with 105 CFU/g of LAB and 0.6% MOS is sufficient to

improve growth and health status of e striped catfish when they were exposed to A.

hydrophila CT19. Further, supplementation of a soybean based diet containing 55%

protein from FM and 45% protein from soybean meal with 105 CFU LAB or 0.6 %

MOS, improves the health status of P. hypopthalmus es without compromising

growth and nutrient digestibility.

1

CHAPTER 1

INTRODUCTION

1.1 Pangasianodon hypophthalmus

Pangasianodon hypophthalmus, is currently considered to be one of the most

important species among commonly cultured freshwater fish. In 2010, global

aquaculture production of striped catfish (P. hypophthalmus) exceeded 1.40 million

tonnes. Vietnam is by far the world‘s largest producer of striped catfish; in 2010, it

produced 1.14 million tonnes, with an estimated value of US$1.40 billion, and

exported the fish to 136 countries (De Silva & Phuong, 2011). Pangasianodon

hypophthalmus which is known as the striped catfish (Sauvage, 1878) (formerly as

Pangasius sutchi or Pangasius hypophthalmus) (Slembrouck et al., 2009) played a

great role in the rapid growth of aquaculture. This species is flourishing as a

significant new source of white fish aquaculture products on the global fish market

with several desirable characteristics including its adaptability for intensive culture,

acceptance of low input sustainable feeds, adaptability to impaired water quality,

and widespread consumer acceptance. This species has a great economic

significance in the Southeast Asia region, including Vietnam. It lives in the main

water basins of the Mekong and Chao Phraya rivers and has been widely introduced

into other cities rivers and ponds for aquaculture. This species is native to tropical

weather and suited to water with a 6.5-7.5 pH (Riede, 2004) range, and a temperature

range of 22-26 °C (72-79 °F) (Riehl & Baensch, 1996). They desire large bodies of

water similar to the deep waters of their native Mekong river basin. Freshwater

farming in Vietnam, particularly in the Mekong delta, has shifted from being a rural

activity, providing local people with animal proteins, to becoming an aquaculture

enterprise. Raised in floating cages in rivers or ponds, striped catfish supply the

2

domestic Vietnamese market and its export market, which is expanding rapidly

(Orban et al., 2008) and has become an iconic success story of aquaculture

production in Vietnam. Although other Asian countries are producing

Pangasianodon as a food fish, none have reached a competitive position in the

global market.

The intensification process of the culture practices increases the risk of

prevalence of stress related disease outbreaks (Bondad-Reantaso et al., 2005), which

are responsible for huge fish losses. Therefore, aquaculture industry considers the

disease occurrence as the state of restriction to aquaculture production at which

adversely affects economical development (Ibrahem et al., 2010). Several pathogens

including fungi, endo and ecto parasites as well as bacteria have been shown to be

associated with diseases in various fish species. Stress factors that are responsible for

disease outbreaks includes confined environmental degradation, waste released from

agricultural activities, low quality of seed, high stocking density and inadequate care

that make the stock vulnerable to infectious pathogens (Phuong et al., 2007).

For a long time, administration of antibiotics was used as the most common

method for dealing with the occurrence of bacterial infections in aquaculture (Li &

Gatlin, 2005). The indiscriminate application of these antibiotics as a remedial

method for controlling bacterial pathogens has been responsible for the development

of resistance bacteria, which has greatly reduced the effectiveness of the treatment

options and may be responsible for long term adverse effects in the aquaculture

environment (Defoirdt et al., 2007; Villamil et al., 2014) including accumulation in

fish body tissue, immuno suppression and destruction of beneficial microbial flora

(Smith et al., 2003; Sapkota et al., 2008). Among them, the development of

antibiotic resistant bacteria (Gómez-Gil et al., 2000; Kolndadacha et al., 2011) has

3

attracted the greatest concern globally. The use of antibiotics in aquaculture has been

reduced over time to fulfil the demands of food safety standards. In fact, in Vietnam

the list of banned or restricted antibiotics and chemicals used in aquaculture is

closely monitored. Vaccination can be used as a method for controlling many fish

diseases instead of antibiotic treatments, but for many diseases vaccines are

unavailable or are in the early stages of development (Yousefian & Amiri, 2009).

Furthermore, the use of antibiotics or vaccines in fish culture is laborious and

expensive. Disease prevention is thus preferable as it is more beneficial than

treatment.

Restrictions or bans on the use of antibiotics as feed additives (Mazlum et al.,

2011) in aquaculture has sparked the growth of alternative strategies in developing

eco-friendly aquaculture for the promision of health and the enhancement of disease

control. Therefore, the modern aquaculture industry seeks alternative prophylactics

which may help in maintaining a healthy environment, resulting in higher production

and better profits (Sáenz de Rodrigáñez et al., 2009). In recent years, considerable

attention has been given to the modification of intestinal microflora through the use

of non-nutrient dietary components, particularly probiotics and prebiotics (termed

biotics), to boost the growth, immune system and disease resistance capability of fish

(Sakai, 1999; Irianto & Austin, 2002; Gatesoupe, 2005; Kesarcodi-Watson et al.,

2008; Wang et al., 2008; Merrifield et al., 2010a). Though the objectives of using

these dietary supplements are similar, the manner in which they modify the intestinal

microbial community differ.

Probiotic is regarded as ―one or more microorganisms which have beneficial

effects for the host, and is able to exist in the digestive tract due to its tolerance to

acid and bile salts‖ (Irianto & Austin, 2002). Though using probiotics in aquaculture

4

is relatively new, the interest in them has increased tremendously due to their

potential in disease control (Wang et al., 2008). Lactic acid bacteria, particularly

Lactobacillus acidophilus, is considered to be one of the most utilized probiotic

strains (Faramarzi et al., 2011). This group of bacteria produces lactic acid as their

main metabolic product during carbohydrate fermentation benefits the host animal

by modifying the growth of beneficial microorganisms which ultimately outcompete

potentially hazardous bacteria in the intestinal ecosystem and reinforce the natural

defense mechanisms (Panigrahi et al., 2004).

Another comparatively new but effective approach to disease control in

aquaculture is the use of prebiotics. Prebiotics are primarily defined as those non

digestible food ingredients that beneficially affect the host by selectively stimulating

the growth of and/or activating the metabolism of one or a limited number of health-

promoting bacteria in the intestinal tract, thus improving the host‘s intestinal balance

(Gibson & Roberfroid, 1995). Several well established prebiotic oligosaccharides

used in aquaculture include mannan-oligosaccharides (MOS), fructo-

oligosaccharides (FOS), short-chain fructo-oligosaccharides (scFOS) and galacto-

oligosaccharides (GOS). Among the established prebiotics, MOS is one of the

preferable prebiotics used as the dietary supplement for fish and crustacean species

(Sang & Fotedar, 2010), which is also used in this current research. Modes of action

of MOS differ from other oligosaccharide candidates, such as fructo

oligosaccharides and transgalacto oligosaccharides as it functions in an indirect

manner within the gastrointestinal tract as opposed to changing the natural intestinal

microbiota directly (Flickinger et al., 2003). MOS has some important characteristics

which make it more attractive as an animal feed additive. Earlier research has stated

5

that heat treatment does not change the ability of MOS to perform normally, which

make it possible to include MOS in pelleted diets (Hooge, 2004).

1.2 Problem Statement

Disease in striped catfish is one of the major consequence of the

intensification of aquaculture and may eventually become a limiting factor to the

economics of a successful and sustainable development of striped catfish farming

industry (Crumlish et al., 2010). Among the infectious diseases, bacterial agents

have been responsible for the major epizootics that affect striped catfish farming.

The rate of loss due to bacterial diseases in striped catfish (such as hemorrhagic

symptoms caused by pathogenic Aeromonas hydrophila) has been estimated to be as

high as 50% compared to others (Thinh et al., 2004; Phuong et al., 2007). Billions of

dollars have been lost annually because of disease outbreaks, which has been

recognized as a major threat to the sustainability of the aquaculture industry. In the

sustainable development of striped catfish farming, outbreaks of disease and the

inappropriate use of drugs and chemicals (antibiotics) have been determined as one

of the major challenges. As an alternative strategy to antibiotics, the fast growing

fish industry urgently needs sustainable approaches for disease control (Gatesoupe,

2005). Among the sustainable approaches available, the use of probiotics and

prebiotics have recently attracted extensive attention in aquaculture. Despite

numerous studies of L. acidophilus and MOS in many cultured fish species, limited

information is available on the effects of L. acidophilus and MOS on the growth and

health status of striped catfish led to conduct this study.

6

1.3 Purposes of the Study

The principal objective of this research was to determine the feasibility of

using the probiotic Lactobacillus acidophilus (LAB) and prebiotic mannan

oligosaccharide (MOS) to produce healthy striped catfish juveniles and evaluate their

influence on the digestibility of a plant based practical diet for striped catfish P.

hypophthalmus juveniles by comparing with a fish meal based diet.

Specific Objectives

To determine the influence of dietary probiotic LAB and prebiotic MOS on

the growth performance, digestive enzyme activities, intestinal morphology,

haematology, immunological response and resistance in striped catfish (P.

hypopthalmus) juveniles against A. hydrophila.

To determine the effectiveness of selected best level of LAB and MOS

respectively, in improving of growth, digestibility, digestive enzyme

activities, intestinal morphology, microflora and resistance against A.

hydrophila fed a practical diet replacing fishmeal with soybean meal.

7

CHAPTER 2

LITERATURE REVIEW

2.1 Aquaculture

Aquaculture refers to the farming of marine or freshwater organisms,

especially food fish, shellfish or aquatic plants, under controlled conditions.

Generally, aquaculture is an important economic sector in many developing

countries, as it contributes significantly towards the improvement in the livelihoods

of the poor either through an improved food supply and/or through employment and

increased income.

2.2 World Aquaculture

Currently the world is facing multiple but interlinked challenges due to the

impacts of the ongoing economic crisis caused by tremendous climate change. At the

same time, another most significant concern is the necessity to meet the food and

nutrition needs of the gradually expanding world population. The fisheries and

aquaculture sector offers great opportunities to fulfil food and nutrition security, to

alleviate poverty, generate economic growth and to ensure proper use of resources.

In general, global fish production has grown faster than world population

growth. Therefore, aquaculture has remained one of the fastest food production

sectors. According to FAO global food fish aquaculture production had increased by

5.8 percent to 70.5 million tonnes in 2013 with the production of farmed aquatic

plants which were mostly seaweeds estimated at 26.1 million tonnes. Besides, China

alone had contributed 43.5 million tonnes of food fish and 13.5 million tonnes of

aquatic algae in 2013 (FAO, 2014). ―Food fish‖ refers to those types of food which

8

are used as food for human consumption, including finfishes, crustaceans, molluscs,

amphibians, freshwater turtles and other aquatic animals such as sea cucumbers, sea

urchins, sea squirts and edible jellyfish.

Aquaculture production over the last fifteen years has expanded

tremendously to augment supplies from natural fisheries. According to FAO reports,

the contribution of aquaculture to the world fisheries and aquaculture production in

2012 outpaced the 50% of the total capture fisheries which is now the world's

leading source of aquatic products (FAO, 2014). Freshwater species, striped catfish

plays a significant role to this rapid growth of aquaculture which is flourishing as

valuable new sources of white fish aquaculture products in the global fish market

with positive attributes including the adaptability to intensive culture, acceptability

of low input sustainable feeds, adaptability to impaired water quality and the

widespread of consumer acceptance. Since the dramatic augment of the

Pangasianodon in the world aquaculture markets, Vietnam has become an iconic

success story and remains the world's largest producer of striped catfish; in 2010, it

produced 1.14 million tonnes, with an estimated value of US$1.40 billion, and

exported the fish to 136 countries (De Silva & Phuong, 2011). Although other

tropical Asian countries also produce Pangasianodon spp. as food fish, yet none has

reached a competitive position in the world market.

2.3 Taxonomy, Distribution and Morphology of Pangasianodon hypophthalmus

Sutchi catfish, Pangasianodon hypophthalmus, was formerly known as the

iridescent shark, Pangasius hypophthalmus. It is also known as the ikan patin in

Malaysia, Thai pangus or striped catfish in Bangladesh, the Siamese shark or swai in

Thailand and ca tra in Vietnam (Plate 2.1).

9

Plate 2.1 Pangasianodon hypophthalmus juvenile used in this study.

The scientific classification of Pangasianodon hypophthalmus is given below

(Sauvage, 1878)

Kingdom: Animalia

Phylum: Chordata

Super-class: Osteichthyes

Class: Actinopterigii (ray-finned fishes)

Sub-class: Neopterigii

Infra-class: Teleostei

Super-order: Ostariophysi

Order: Siluriformes (catfish)

Family: Pangasiidae (shark catfishes)

Genus: Pangasianodon

Species: Hypophthalmus

Scientific name: Pangasianodon hypophthalmus (Sauvage, 1878)

Synonym: Helicophagus hypophthalmus (Sauvage, 1878),

Pangasius hypophthalmus (Sauvage, 1878),

10

Pangasius sutchi, (Fowler, 1937)

The striped catfish (P. hypophthalmus), the native freshwater fish of the main

South-East Asia water basin, such as the Mekong delta river region of Vietnam,

Laos, Cambodia, Chao Phraya rivers in Thailand (Kabir, 2012) and Ayeyawady

basin of Myanmar (FAO, 2009). Basically, this species is a migratory fish that

moves upstream for breeding purpose during the rainy season when the water levels

are high and return to downstream for finding the rearing habitat when the water

levels recede (Rainboth, 1996). However, this fish has also been introduced for

farming in many countries in Southeast Asia including Malaysia (Phumee et al.,

2011), India, China, Indonesia (FAO, 2009) and Bangladesh (Ahmed et al., 2010).

Most of the fish available in the market have been mass-bred for the purpose, and

also cultivated for food in most of its native countries. It is omnivorous in nature,

feeding principally on plants, algae, zooplankton, insects, fruits, crustaceans, and

fish, and it prefers water with a 6.5-7.5 pH (Riede, 2004) and a temperature range of

22-26 °C (72-79 °F) (Riehl & Baensch, 1996). They have a preference to live large

water bodies similar to the deep waters of their native Mekong river basin.

The striped catfish is the member of the freshwater Siluriformes in the

Pangasidae family (Slembrouck et al., 2009). This fish has scale less skin with a

comparatively long and laterally flattened body. Its head is comparatively small,

whereas the mouth is quite broad with small sharp teeth on jaws, vomerine and

palatal bones. The eyes of this species are relatively large. Two pairs of barbells are

present, the upper pairs (maxillary barbel) are smaller compared to the lower pairs

(mandibulary barbel) (Robert & Vidthayanon, 1991; Rainboth, 1996). The fins are

generally dark grey or black in color. The dorsal fin consists of one hard fin ray

(spine) and six soft fin rays. An adipose fin is present in between the dorsal and

11

caudal fin and the caudal fin is isocoetoral. Generally, the body colour of this fish is

black to dark gray on the dorsal side, whereas it is light gray in the lateral side and

silvery in colour in the abdominal side (Kabir, 2012).

2.4 Diseases of Pangasianodon hypophthalmus Farming

For the last few years, fish health has become a major global problem to

aquaculturists. In an intensive fish culture, fish diseases are a common phenomena.

Any imbalance among the three important factors such as the existence of the

pathogen in fish body and the quality of environment and the general health status of

the fish, are the possible reasons of the outbreak of disease (Plate 2.2). Good fish

health can be maintained without the use of chemotherapeutic agent when the above

three conditions can be assured of its balance. The required water quality, the

appropriate stocking densities and a balanced diet are the key factors for maintaining

the good health status of fish.

Plate 2.2 Disease is the end result of the host due to interaction and interrelationship

of above 3 factors (Source: Francis-Floyd, 1997).

12

The continuous decline of fish in the wild stocks tend to increase the

necessity to develop aquaculture in order to assure the supply of high quality protein

to fulfil the world's growing population demand. Tolerance to high stocking

densities, rapid growth, high production as well as good palatability and high market

value are some important characteristics, which should be taken into consideration

for pond aquaculture (Phuong & Oanh, 2009).

As the disease outbreak of aquaculture species increases with the increasing

farming intensity (Bondad-Reantaso et al., 2005), aquaculturists have set two most

important goals, the rapid growth and high disease resistance of aquaculture species.

Fish diseases are commonly caused by pathogenic microorganisms such as protozoa,

fungus, bacteria and virus which are common in water during the culture period.

Among the various pathogenic microorganisms, bacterial agents, particularly

Aeromonas hydrophila, has been observed as being responsible for the major

epizootics affecting the striped catfish farming (Subagja et al., 1999). Therefore, the

conventional techniques of disease management which are commonly applied in

aquaculture are the prophylactic and therapeutic control of bacterial diseases based

on the use of chemical disinfectants and the oral administration of antimicrobial

drugs (Verschuere et al., 2000). However, due to apply the conventional disease

management techniques, microbial populations are facing several problems and the

most significant problem is the development of antibiotic resistant bacteria (Aoki et

al, 1985; Gómez-Gil et al., 2000). Besides, this can cause a potential hazard to

public health as well as to the environment because of the imbalance of intestinal

microflora due to the accumulation of excess antibiotic in the fish body (Esiobu et

al., 2002). Therefore, many researchers have proposed several alternative approaches

to develop environmental friendly aquaculture, such as the use of probiotics and

13

prebiotics as biological control agents in place of the use of chemotherapeiutents in

aquaculture (Robertson et al., 2000; Gatesoupe, 2005; Merrifield et al., 2010a).

2.5 Probiotic

"Probiotic‖ term originate from the Greek words 'pro; and 'bios' which means

―for life‖ (Schrezenmeir & de Vrese, 2001) and this term carries different meanings

over the years. Dr. Elie Metchnikoff was the first person who clarified the useful role

played by some bacteria as observed among farmers who consumed milk containing

pathogen and that ―reliance on intestinal microbes for food makes it possible to take

steps to change the flora of our bodies and to replace harmful microbes by beneficial

microbes‖ (Metchnikof, 1907). However, the term probiotic was first introduced by

Lilly and Stillwell (1965) after modification of the original word ―probiotika.‖ This

term was used to illustrate the substances secreted by a microorganism which

stimulates the growth of another. It was also described as an agent which functions

as a contrary to antibiotics. In 1974, Parker (Parker, 1974) defined it as

―organisms and substances which have a beneficial effect on the host of animal by

contributing to its intestinal microbial balance.‖ Later in 1989, Fuller attempted to

improve Parker's definition by clarifying that it is ―a live microbial feed supplement

which beneficially affects the host animals by improving its intestinal microbial

balance‖ and mentioned that it would be useful in a range of extreme temperatures

and salinity changes (Fuller, 1989). Subsequently, it was recommended that

probiotics were ―monocultures or mixed cultures of microorganisms which can be

applied to humans or animals, that do good to the host by improving the properties

of indigenous microflora‖ (Havenaar & Huis, 1992). Because of high interest of

probiotics used in aquaculture, Moriarty (1998) suggested to extend the definition of

probiotics to ―living microbial additives that benefit the health of hydrobionts and

14

therefore increase productivity.‖ The following year, Gatesoupe (1999) defined

probiotics as ―in a certain way of microbial cells administered, so that they can reach

the gastrointestinal tract and be alive with the aim of improving health‖. The

inhibition of pathogens by using probiotics was carried out in the same year and this

led the definition to be expanded to―… live microbial supplementation can give

benefits to the host by improving its microbial balance‖ (Gram et al., 1999). An

expert with the Joint Food and Agriculture Organization of the United

Nations/World Health Organization (FAO/WHO) stated that probiotics are live

microorganisms which when consumed in adequate amounts confer a health benefits

for the host (FAO, 2001).

Recently, it has been established that probiotic organisms have shown

antimicrobial activity by altering the intestinal microflora thereby secreting a wide

range of antibacterial substances such as bacteriocins and organic acids and

competing with pathogens for adhesion to the intestine, competing for nutrients

which are necessary for pathogen to survive. They are also capable of modulating the

immune system. Because of these criteria, when viable probiotics are administered at

certain concentrations, they will favorably affect the health of the host (Myers,

2007). In addition, nowadays probiotics are defined commonly as ―friendly or

healthy bacteria,‖ (Wang et al., 2008).

2.5.1 Characteristics of Probiotic

According to Fuller (1989) a good probiotic should have the following

characteristics:

Should be a strain which is capable of exerting a beneficial effect on

the host animal, e.g. increased growth or resistance to disease.

15

Should not have any side effect; should neither be pathogenic nor

toxic, not only with regard to the host species but also with regard to aquatic

animals in general and human consumers.

Should be viable under normal storage conditions and able to survive

during industrial process.

Should be capable of surviving and metabolizing in the gut

environment, e.g. resistant to bile and low pH due to organic acids

enrichment.

Possess high ability to multiply in the intestine.

Should have strong adhesive capability with the digestive tract of the

host.

Should have antagonistic characteristics against one or more

pathogenic microorganisms.

2.5.2 Types of Probiotics

There are various types of microorganisms that have been shown probiotic

characteristics and commonly used for both human and animal consumption is

presented in Table 2.1. Among them two most important types of probiotics are

Lactobacilli and Bifidobacteri (Robertson et al., 2000). Besides, various types of

yeast species, particularly, under the genus of Saccharomyces have been considered

as significant probiotic candidate for human and animal uses (Holzapfel et al., 1998).

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Table 2.1 Common probiotics used for human and animal consumption

Lactobacillus species Bifidobacterium species Other species

Lactobacillus acidophilus Bifidobacterium bifidum Bacillus cereus

L. rhamnosus B. breve Enterococcus faecium

L. casei B. infantis Lactococcus lactis

L. bulgaricus B. longum L. cremoris

L. gasseri B. adolescentis Streptococcus thermophilus

L. crispatus B. lacis Saccharomyces cerevisiae

L. plantarum B. animalis S. boulardii

L. salivarius

L. buchneri

L. johnsonii

L. reuteri

L. fermentum

2.5.3 Lactic Acid Bacteria

Considering the above probiotic characteristics, lactic acid bacteria are

claimed to be the most utilized probiotic (Robertson et al., 2000). These probiotic

bacteria are characterized as gram positive, non-sporulating cocci or rod shaped and

produced lactic acid as their main metabolic product during carbohydrate

fermentation. Previous study claimed, lactic acid bacteria as the normal microflora

in the gastrointestinal (GI) tract of healthy animals such as mammals and aquaculture

species (Ringø, 2008; Aly et al., 2008; Wang, 2011) and have been considered as

being safe for feeding fish (El-Ezabi et al., 2011). These probiotic bacteria are

provided benefit to the host animal by modifying the growth of beneficial

microorganism that can contribute inhibitory compounds such as lactic acid,

hydrogen peroxide, diacetyl, acetaldehyde and bacteriocin, which would ultimately

outcompete the potentially hazardous bacteria from the intestinal ecosystem and

17

would reinforce the natural defence mechanisms of organisms (Ringø & Gatesoupe,

1998; Gatesoupe, 1999; Balcázar et al., 2007a). Among the lactic acid bacteria,

Lactobacillus acidophilus has been considered one of the important probiotic

because of its inhibitory effect against numerous pathogenic microorganism (Al-

Dohail, 2010; Talpur et al., 2014).

2.5.4 Mechanisms of Action

Probiotics can influence the decline of the prevalence and reduce the duration

of diseases by stimulating the resistance to colonization and inhibitory effects against

pathogens. Both in-vitro and in-vivo tests after using several probiotic strains,

exhibited the inhibition of pathogenic bacteria through various mechanisms. For a

few years, many research were conducted on the beneficial effects of probiotics. It is

difficult to understand the real mechanism of actions of probiotics, and only some

explanations are available.

The possible modes of action of using probiotic include:

i) Competition for adhesion site/competitive exclusion of pathogenic bacteria

(Gómez-Gil et al., 2000; Vine et al., 2004; Bagheri et al., 2008).

ii) Colonization (Bagheri et al., 2008).

iii) Production of antimicrobial substances/antibacterial activity (De

Keersmaecker et al., 2006; Aly et al., 2008; Vila et al., 2010; Enany et al.,

2012).

iv) Source and competition for nutrients (Prieur et al., 1990; Verschuere et

al., 2000; Bagheri et al., 2008).

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2.5.4.1 Competition for Adhesion Sites / Competitive Exclusion

Competition for adhesion sites and colonization on the intestine and other

tissue surfaces are the most common and significant mechanism of probiotics action

to combat against harmful pathogens (Ringø et al., 2007). This is referred to as

competitive exclusion of pathogenic bacteria (Ohashi & Ushida, 2009). The

appropriate adhesion of pathogenic bacteria to the enteric mucus and intestinal wall

surface is very important for any pathogen to cause damage to the host animal

(Olsson et al., 1992; Vine et al., 2004). The physical blocking of pathogenic bacteria

colonization by probiotic bacteria from their favorite adhesion site, such as intestinal

villus, goblet cells and colonic crypts is referred to as competitive exclusion

(Chichlowski et al., 2007). The adhesion of the probiotics on the epithelial cells is

normally performed in two ways. One is non-specific depending on the physico-

chemical factors and another is specific which is based on the adhesion of the

probiotics on the surface of the adherent bacteria and receptor molecules (Salminen

et al., 1996). One of the most important mechanisms of the intestinal microflora is to

resist the adhesion of pathogenic bacteria to the host intestine, thereby creating of a

physiologically negative environment, with regard to pH, redox potential, and

hydrogen sulfide enrichment (Fons et al., 2000). However, by maintaining good

husbandry practices and environmental conditions, the composition of microbial

communities can be altered, which ultimately will be able to excite the proliferation

of selected bacterial species. It is well established that the presence/existence of

microflora in the intestinal tract of aquatic animals can be altered by supplying

beneficial microorganisms through feeding, which ultimately will reduce the

availability of opportunist pathogens (Deven et al., 2009). In fact, there are some

evidence that lactic acid bacteria, particularly L. lactis and L. plantarum can

19

successfully reduce the adhesion of pathogenic bacteria A. hydrophila, A.

salmonicida and V. anguillarum in intestinal mucus of fish (Balcázar et al., 2008).

2.5.4.2 Colonization

Probiotics execute their activity through colonization and the secretion of

many growth-promoting substrate in the host intestine (Bagheri et al., 2008).

Probiotics colonization of the gastrointestinal tract of host animals is probable only

after birth, and prior to a very competitive indigenous microflora that has been

installed. After being installed, only the supplement of high levels of probiotic can

activate its temporary and artificial dominance. After the intake had stopped, the

population of probiotics in the gastrointestinal tract of mature animals show a rapid

decrease within days (Fuller, 1992). A microorganism is capable to colonize in the

gastrointestinal tract whenever it can stay there for a long time by possessing a

higher multiplication rate than its expulsion rate (Conway, 1997). The attraction of

bacteria to the mucosal surface association within the mucous gel or attachment to

epithelial cells is the main reason of this colonization. The adhesion and colonization

of the mucosal surfaces by probiotics are possible protective mechanisms against

pathogenic organisms through a competition for nutrients and the binding sites

(Westerdahl et al., 1991), or modulation of immune (Salminen et al., 1998). Several

factors responsible for the colonization of microorganisms are as follows:

(i) Host related factors: Thses include body temperature, redox potential levels,

enzymes, and genetic resistance. For instance, bacteria can enter the host body

through the mouth, either being mixed with water or food particles, and while

passing along the alimentary tract, some of them are able to adhere as part of a

resident microflora. Among the rest of the microflora some are destroyed throughout

20

the digestive process while passing through the intestine, and the rest is eliminated

via the faeces. Another factor which may be able to suppress bacterial growth is the

production of any antimicrobial substances by the host.

ii) Microbe-related factors: These are the effects of antagonistic microorganims,

bacteriocins, lysozymes, proteases, formation of hydrogen peroxide, ammonia, and

sudden changes of pH values due to production of organic acids (Gram et al., 1999;

De Vrese & Marteau, 2007; Vila et al., 2010). It is well recognized that lactic acid

bacteria produce substances, for example bacteriocins, that are capable to inhibit the

growth of some pathogenic microorganisms.

2.5.4.3 Production of Antimicrobial Substances / Antibacterial Activity

Bacterial antagonism is a familiar event in nature; therefore, the interactions

of microbes play a significant role to maintain the equilibrium between useful and

potentially pathogenic micro-organisms (Balcázar et al., 2004). Probiotics secret

certain inhibitory substances such as bacteriocins, organic acids (lactic, acetic and

butyric acid) and H2O2 (De Keersmaecker et al., 2006; Vila et al., 2010) which are

able to show antagonistic activity against the pathogenic microorganisms, and

consequently will prevent their propagation in the host bodies. It was previously

reported that bacteriocins acidophilin, lactocidin and acidolin are secreted by one of

the most important probiotic strain L. acidophilus, whereas L. plantarum produces

lactolin (Vila et al., 2010). They are also able to show antimicrobial activity by

lowering the pH of the intestine (De Keersmeacker et al., 2006), the agglutination of

pathogenic microorganisms, binds with toxic metabolites (Fonden et al., 2000;

Oatley et al., 2000; Haskard et al., 2001), and the production of mucus (Mattar et al.,

2002; De Vrese & Marteau, 2007). Several researches have reported that probiotics

21

especially Lactobacillus sp., Bifidobacterium sp. and Bacillus sp. have been able to

secrete a wide range of chemical substances which have shown an inhibitory effect

on pathogenic bacteria (Oscáris et al., 1999; Gotteland et al., 2006; Vila et al.,

2010). There is evidence that the probiotic strain of L. salivarius sub sp. salivarius

UCC118 secrete a peptide, which has shown the inhibition capability against a broad

range of pathogens, including Staphylococcus, Enterococcus, Listeria, Bacillus, and

Salmonella species (Flynn et al., 2002).

Plate 2.3 Inhibition of enteric bacteria and enhancement of barrier function by

probiotic bacteria. Schematic representation of the crosstalk between

probiotic bacteria and the intestinal mucosa. Antimicrobial activities of

probiotics include the (1) production of bacteriocins/defensins, (2)

competitive inhibition with pathogenic bacteria, (3) inhibition of

bacterial adherence or translocation, and (4) reduction of luminal pH.

Probiotic bacteria can also enhance the intestinal barrier function by (5)

increasing mucus production. [The picture taken from

www.interscience.wiley.com.]

22

2.5.4.4 Sources and Competition for Nutrients

Intestinal microorganisms play a crucial role in nutrition as well as the well-

being of several animals (Floach et al., 1970) including fish (Sugita et al., 1989).

The ability of intestinal microorganism to synthesize vitamins, essential growth

factors (fatty acid, amino acid) and digestive enzymes are well documented

(Teshima & Kashiwada, 1967; Clements, 1997). Microorganisms particularly

anaerobic bacteria play a significant role in carp (Cyprinus carpio) nutrition by

contributing fatty acids (Clements, 1997) which are the end products of anaerobic

fermentation (Smith et al., 1996). There is also evidence that the useful microflora

(aerobic, anaerobic) can serve as a source of food by providing vitamins, such as

B12 and essential amino acids, which play a significant role in the host‘s nutrition

(Sugita et al., 1991a, 1991b).

In fact, probiotics improve the health or well-being of the host by adhering to

the mucus membrane, epithelial cells, gastrointestinal tract as well as other tissues

(Gatesoupe, 1999; Farzanfar, 2006). Several researches have been conducted to

evaluate the in-vitro and in-vivo attachment ability of many beneficial

microorganisms and their results recommended that the some potential probiotic

have ability to displace the pathogens by competing for necessary nutrients, space,

etc. (Verschuere et al., 2000). They can cause the unavailability of nutrients and

energy sources for essential growth of some pathogenic bacteria, by effectively

utilizing those nutrients which would otherwise can be consumed by pathogenic

microbes.

23

2.5.5 Beneficial Effects of Probiotics

2.5.5.1 Effect of Probiotics on Intestinal Ecosystem

The gastrointestinal (GI) tract of fish considered are as harbours of a complex

microbial community, especially two distinct groups, i.e. allochthonous (exogenous)

and autochthonous (indigenous) (Nayak, 2010). Autochthonous microorganisms are

capable of playing significant roles within the GI tract such as to contributing to the

development/maturation of the intestine and immune system (Bates et al., 2006;

Picchietti et al., 2007; Nayak, 2010), and managing resistance to infectious

pathogens (Birkbeck & Ringø, 2005; Ringo et al., 2007). They are also able to

hinder the colonization of bacteria through several modes of action including

competition for food and space as well as being receptors at mucosal surfaces, and

secreting antimicrobial compounds (Nayak, 2010). Thereby, probiotics provide the

beneficial effects on the host by improving its intestinal ecosystem (Julio & Marie-

José, 2011). However, very few studies have been conducted to evaluate the effects

of probiotics supplementation on the intestinal autochthonous microflora of fish

(Nayak, 2010; Merrifield et al., 2010a). Previous researches only focused on

cultivation-based techniques to evaluate intestinal microbial population which

noticeably only permit the investigation of culturable bacteria, while the non-

culturable bacteria, which may account a great effect in the intestine of fish,

remained unclear (Nayak, 2010; Merrifield et al., 2010a; Zhou et al., 2009).

2.5.5.2 Effect of Probiotics on Digestive Enzymes

It is well documented that digestive organs are greatly influenced by the

composition of food and are responsible for instantaneous changes in the activities

of the digestive enzymes (Bolasina et al., 2006; Shan et al., 2008), which is finally

24

linked with fish health and growth. In fact, the efficiency of feed utilization in the

fish is greatly influenced by the presence of digestive enzymes. Protease, amylase,

and lipase are three different types of enzyme which are responsible for the

breakdown of protein, carbohydrate and fat respectively. There is evidence that

dietary probiotics are able to increase the digestion of protein, starch and fat by

increasing digestive enzyme activities in Nile tilapia, which ultimately increase the

growth and feed utilization (Essa et al., 2010). Elevated enzyme activities (protease,

amylase and lipase) were also noticed in Labeo rohita after being fed with a

combination of probiotics (Lactococcus lactis, Bacillus subtilis and Saccharomyces

cerevisiae) (Mohapatra et al., 2012). Microorganisms and their secreted enzymes

play a significant role on digestion process (Munilla-Moran & Stark, 1990) by

raising the total enzyme activity of the intestine (Ziaei-Nejad et al., 2006; Wang,

2007). The enzymes secreted by probiotic cells are known as exogenous enzymes

which can help to synthesize endogenous enzymes (Mohapatra et al., 2012). These

exogenous enzymes are able to tolerate a wide range of pH compared to endogenous

enzymes, which can delay the digestion period (Essa et al., 2010) thereby assuring

the maximum utilization of nutrients. However, it is complicated to separate

between exogenous enzymes secreted by the probiotics and endogenous enzymes

synthesized by fish (Essa et al., 2010). The exoenzymes produced by probionts are

able to increase the digestive utilization of feed (Mohapatra et al. 2012). Probiotics

not only have a beneficial impact on the digestive systems, but also in the

assimilation of food components (Irianto & Austin, 2002).

2.5.5.3 Effect of Probiotics on Intestinal Morphology

It was reported that probiotics could establish and execute their activity in the

intestine of animals considered as a harbour of the diverse population of pathogenic,

effect of lactobacillus acidophilus and mannan ... · ketahanan ikan patin berjalur juvenil...

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