“EFFECT OF CAROTENOIDS ON
GROWTH, IMMUNE RESPONSE AND DISEASE
RESISTANCE IN GIANT FRESHWATER PRAWN,
MACROBRACHIUM ROSENBERGII”
ALI HOSSEINPOOR ZELATY M. F. SC.
DEPARTMENT OF AQUACULTURE
COLLEGE OF FISHERIES, MANGALORE
KARNATAKA VETERINARY, ANIMAL AND
FISHERIES SCIENCES UNIVERSITY, BIDAR
MAY, 2014
“EFFECT OF CAROTENOIDS ON
GROWTH, IMMUNE RESPONSE AND DISEASE
RESISTANCE IN GIANT FRESHWATER PRAWN,
MACROBRACHIUM ROSENBERGII”
Thesis submitted to the Karnataka Veterinary, Animal and
Fisheries Sciences University, Bidar in partial fulfillment of the
requirements for the
Degree of
DOCTOR OF PHILOSOPHY
IN
AQUACULTURE
By
ALI HOSSEINPOOR ZELATY M. F. SC.
DEPARTMENT OF AQUACULTURE
COLLEGE OF FISHERIES, MANGALORE
KARNATAKA VETERINARY, ANIMAL AND
FISHERIES SCIENCES UNIVERSITY, BIDAR
MAY, 2014
KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR 585401
DEPARTMENT OF AQUACULTURE
COLLEGE OF FISHERIES, MANGALORE
CERTIFICATE
This is to certify that the thesis entitled “EFFECT OF CAROTENOIDS ON
GROWTH, IMMUNE RESPONSE AND DISEASE RESISTANCE IN GAINT
FRESH WATER PRAWN MACHROBRACHIUM ROSENBERGII’submitted by
Mr. Ali Hosseinpoor Zelathy, I.D. NO. DFK 1101 in the partial fulfillment of the
requirement for the award of Doctoral of Fisheries Science in Aquaculture of
Karnataka Veterinary, Animal and Fisheries Sciences university, Bidar is a
record of bonafide research work carried out by him during the period of his study in
this university under my guidance and supervision and the thesis has not previously
formed the basis for the award of any degree, diploma , fellowship or other similar
titles.
Mangalore
(Dr. H. SHIVANAND MURTHY)
(Prof and HOD)
May, 2014
APPROVED BY:
Chairman: 1.
(Dr. H. SHIVANAND MURTHY)
Nominated Ext. member: 2. --------------------------------------------(Prof. Y K Khillare)
Members : 3.
( DR. N. BASAVARAJA)
4.
( DR. K.S. RAMESH)
5.
(DR. S. BENAKAPPA)
6.
(SHRI KRISHNA BHAT)
DEDICATED TO
BIGGEST LEADER OF THE ISLAMIC REVOLUTION
OF IRAN “AYATOLLAH KHOMEINI”
SUPREME LEADER OF THE ISLAMIC REPUBLIC OF
IRAN
“AYATOLLAH SEYED ALI KHAMENEI”
MARTYRS OF ISLAM ESPECIALLY MARTYRS OF
THE
ISLAMIC REPUBLIC OF IRAN,
MY LOVELY WIFE, SON, DAUGHTER, SON-IN LAW,
DEAR GRANDSON” AMIR REZA NAZARKARDEH”,
PARENTS, BROTHERS AND SISTERS.
ACKNOWLEDGEMENT
I express my deep sense of gratitude and heartfelt thanks to my major advisor Dr. H.
Shivananda Murthy Professor and head of Department of college for fisheries,
College of Fisheries, Mangalore, for his able guidance, pertinent comments, kind
counsel and support throughout the period of study. It has been my great pleasure and
privilege to work under his guidance.
I extend my sincere thanks to my advisory committee members Dr. N. Basavaraja,
Dr. K.S. Ramesh, Dr. S. Benakappa and Shri Krishna Bhat for their valuable
suggestions and kind support during the course of this study.
I am very much thankful to Dr. K. M. Shankar Dean, College of Fisheries,
Mangalore for providing me with all the necessary facilities for the conduct of the
research work.
It is hard to forget the unconditional help and support render by Dr. Indrani
Karunasagar, Dr. E. G. Jayaraj, Shri Iqlas Ahmed, Dr. Ganapathi Naik, Dr. V.
G. Bhat, Dr. S. K. Girisha, Mrs. Prafulla Shetty, Abhiman, Mr. Naveen Kumar
B.T, Mr. Adnan Amin, Mr. Faisal Rashid Sofi and Mr. Shashi Meshram during
the research period.
I express my sincere gratitude to all the staff members of the department of
Aquaculture.
I warmly acknowledgement the technical, practical and friendly help received from
Ashokanna, Devadassanna and sandesh.
I am very much thankful to all the staff in the academic unit who rendered a helping
hand whenever I approached them.
I thankfully acknowledge the assistance provided by the librarian and library staff of
College of fisheries, Mangalore.
Finally, I must acknowledge the valuable help, co-operation and moral support
provided by my dear wife, son, daughter, son-in-law, dear father, mother, brother and
all friends throughout my study.
I am conscious my indebtedness to each and every individual who helped me in
many ways in the preparation of this thesis special my dear sister seyedeh hoda
Ahmadian.
Mangalore,
May, 2014
(ALI HOSSEINPOOR ZELATY)
CONTENTS
SI.NO.
Title
Page No
1
I.
Introduction
II.
REVIEW OF LITRATURE
2.1
2.2
Macrobrachium
rosenbergii:
its
freshwater aquaculture
Biology of Macrobrachium rosenbergii
2.3
Nutrient requirements
12
2.3.1
Protein and amino acid requirement
12
2.3.2
Lipid and free fatty acid requirement
12
2.3.3
Carbohydrate requirement
13
2.3.4
Mineral requirement
13
2.3.5
Vitamin requirement
13
2.3.6
Some of the important other additives
14
2.3.6.1
Probiotics
14
2.3.6.2
Feed attractants
15
2.3.6.3
Antibiotics
15
2.3.6.4
Pigments
15
2.4
Crustacean immunity
16
2.4.1
Some of the most important bio- defense system in crustaceans
16
2.4.1.1
Cellular immunity of crustaceans
17
9
significance
in
9
10
2.4.1.1.1 Hemocytes
17
2.4.1.1.2 Hyaline cells
17
8
2.4.1.1.3 Semigranular cells
18
2.4.1.1.4 Granular cells
18
2.4.1.1.5 Phagocytosis
20
2.4.1.1.6 Nodule formation
20
2.4.1.1.7 Encapsulation
21
2.4.1.2
22
Humoral immunity
2.4.1.2.1 Agglutinins
23
2.4.1.2.2 Lectins
23
2.4.1.2.3 Prophenoloxidase activating system (propo system)
24
2.5
Bacterial diseases in M. rosenbergii
28
2.6
Immunostimulants
30
2.6.1
Use of immunostimulants as a strategy to protect cultured
31
invertebrates against diseases
2.6.2
Immunostimulants for crustacean (shellfish)
31
2.6.3
The mechanisms of defense system in crustacean with
33
immunostimulants
2.7
Carotenoids
36
2.7.1
Classification of carotenoids
37
2.7.2
Structure of selected carotenoids
38
2.7.3
Normal Carotenoids sources used in aquaculture
42
2.7.3.1
Animal based natural carotenoids
42
2.7.3.2
Plant based carotenoids
43
2.7.3.3
Carotenoid used in experiments
44
9
2.7.3.3.1 Marigold oleoresin (Tagetes erecta)
44
2.7.3.3.2 Xanthophylls (oxygenated carotenoids): Lutein, Zeaxanthin
45
and Meso-zeaxanthin
2.7.3.3.3 Diacetate of lutein-mesozeaxanthin
47
2.7.4
Carotenoids absorbtion and transport
48
2.7.5
Metabolism And Deposition Of Carotenoids
50
2.7.6
Function Of Carotenods
52
2.7.6.1
Pigmentation Functions
52
2.7.6.2.
Provitamin A
53
2.7.6.3
Reproduction
54
2.7.6.4
Stress tolerance
55
2.7.6.5
Diet supplementation
56
2.7.6 .6
Growth
57
2.7.6.7
Survival
62
2.7.6.8
Proximate composition
63
2.7.6.9
Immune system
65
2.7.6.9.1 Prophenoloxidase activity
65
2.7.6.9.2 Superoxide anion production (Respiratory burst)
68
2.7.6.9.3 Total Haemocyte count
69
2.7.6.9.4 Total Heamolymph protein
71
2.7.6.9.5 Antioxidant activity
72
2.7.6.10
Disease resistance
73
III.
MATERIAL AND METHODS
74
3.1
Feed ingredients, formulation and analysis
74
10
3.2
Proximate composition of the feed ingredients
74
3.3
Formulation and preparation of experimental diets
75
3.3.1
Experiment, 1 (Marigold oleoresin)
75
3.3.2
Experiment, 2 (Diacetate of lutein-mesozeaxanthin)
75
3.4
Experimental animals
76
3.5
Experimental Design
76
3.6
Experimental set up
76
3.7
Stocking and rearing
77
3.8
Feeding
77
3.9
Water sampling
78
3.10
Prawn sampling
78
3.11
Growth Studies
78
3.11.1
Specific growth rate (SGR)
78
3.11.2
Feed conversion ratio (FCR)
79
3.11.3
Protein efficiency ratio (PER)
79
3.11.4
Survival Rate
79
3.11.5
Feed Efficiency Ratio
79
3.11.6
Weight gain (WG)
80
3.11.7
per Day Growth (g)
80
3.11.8
Percentage of mean weight gain (PMWG)
80
3.11.9
Condition factor (CF)
80
3.11.10
Daily Growth Rate (DGR)
80
3.11.11
Daily Growth Index (DGI)
81
11
3.11.12
Growth Coefficient (GC)
81
3.12
Biochemical composition
81
3.13
Anticoagulant Preparation
81
3.14
Hemolymph sample collection
81
3.15
Immune parameters of Macrobrachium rosenbergii
82
3.15.1
Super oxide anion production assay (NBT assay)
82
3.15.2
Pro-phenoloxidase assay (PPO)
82
3.15.3
Haemocyte count
82
3.15.4
Total Hemolymph protein
83
3.16
Histological studies
83
3.17
Experimental infection of Aeromonas hydrophila
83
3.17.1
Aeromonas hydrophila inoculum
83
3.17.2
Preparation of injection
84
3.17.3
LD50 of A. hydrophila
84
3.17.4
Challenge study
84
3.17.5
Confirmation of prawn mortality
84
3.17.6
Relative percent survival (RPS)
86
3.18
Statistical analysis
85
IV.
EXPERIMENTAL RESULTS
86
EXPERIMENT- 1
86
Proximate composition of the feed ingredients and formulated
86
4.1
feeds
12
4.1.1
Feed ingredients
86
4.1.2
Experimental diets
86
4.2
Carotenoid content of the feed
87
4.3
Water quality parameters
87
4.3.1
Temperature
87
4.3.2
pH
87
4.3.3
Dissolved oxygen
87
4.3.4
Free carbon dioxide
87
4.3.5
Total alkalinity
87
4.3.6
Ammonia-Nitrogen
88
4.4
Growth studies
88
4.4.1
Specific Growth Ratio
88
4.4.2
Food Conversion Ratio
88
4.4.3
Protein Efficiency Ratio
88
4.4.4
Survival Ratio
89
4.4.5
Feed Efficiency Ratio
89
4.4.6
Weight Gain
89
4.4.7
Per Day Growth
89
4.4.8
Percentage Weight Growth
89
4.4.9
Condition Factor
89
4.4.10
Daily Growth Ratio
89
4.4.11
Daily Growth Index
90
4.4.12
Growth Coefficient
90
13
4.5
Biochemical composition
90
4.5.1
Moisture
90
4.5. 2
Protein
90
4.5.3
Fat
90
4.5.4
Ash
90
4.5.5
Nitrogen Free Extract
90
4.6
Resistance of M. rosenbergii to A. hydrophila infection
91
4.7
Immune parameters of M. rosenbergii
91
4.7.1
Prophenoloxidase Assay (PPO)
91
4.7.2
Superoxide onion production (NBT)
91
4.7.3
Total heamolymph protein
92
4.7.4
Total heamocyte count
92
EXPERIMENT- 2
92
4.8
Experimental diets
92
4.9
Carotenoid content of the feed
92
4.10
Water quality parameters
93
4.10.1
Temperature
93
4.10.2
pH
93
4.10.3
Dissolved oxygen
93
4.10.4
Free carbon dioxide
93
4.10.5
Total alkalinity
93
4.10.6
Ammonia-Nitrogen
93
4.11
Growth studies
94
14
4.11.1
Specific Growth Ratio
94
4.11.2
Food Conversion Ratio
94
4.11.3
Protein Efficiency Ratio
94
4.11.4
Survival Ratio
95
4.11.5
Feed Efficiency Ratio
95
4.11.6
Weight Gain
95
4.11.7
Per Day Growth
95
4.11.8
Percentage Weight Growth
95
4.11.9
Condition Factor
95
4.11.10
Daily Growth Rate
95
4.11.11
Daily Growth Index
96
4.11.12
Growth Coefficient
96
4.12
Biochemical composition
96
4.12.1
Moisture
96
4.12. 2
Protein
96
4.12.3
Fat
96
4.12.4
Ash
96
4.12.5
Nitrogen Free Extract
96
4.13
Resistance of M. rosenbergii to A. hydrophila infection
97
4.14
Immune parameters of M. rosenbergii
97
4.14.1
Prophenoloxidase Assay (PPO)
97
4.14.2
Superoxide onion production (NBT)
97
4.14.3
Total heamolymph protein
98
15
4.14.4
Total heamocyte count
98
V.
DISCUSSION
99
5.1
Proximate composition of ingredients and experimental diets
99
5.2
Proximate composition of prawn muscle
99
5.3
Effect of carotenoids on water quality
100
5.3.1
Temperature
100
5.3.2
pH
101
5.3.3
Dissolved oxygen (DO)
101
5.3.4
Free carbon dioxide
102
5.3.5
Total Alkalinity
103
5.3.6
Ammonia – Nitrogen
103
5.4
Effect of carotenoids on growth parameters of M. rosenbergii
104
5.4.1
Specific Growth Rate (SGR)
106
5.4.2
Feed Conversion Ratio (FCR)
106
5.4.3
Protein Efficiency Ratio (PER)
107
5.4.4
Effect of different carotenoids on survival of prawn
107
5.5
Resistance of M. rosenbergii to Aeromonas hydrophila
108
infection
5.6
Immune parameters of M. rosenbergii
109
5.6.1
Effect of carotenoids on prophenoloxidase system (PPO)
111
5.6.2
Effect of carotenoids on Nitroblue tetrazolium (NBT)
112
5.6.3
Effect of carotenoids on Total Haemocyte count
114
5.6.4
Effect of carotenoids on total heamolymph protein
115
16
VI.
VII.
VIII
117
SUMMARY
122
REFERENCES
ABSTRACT
184
17
LIST OF TABLE
Table1. Hemocytes types and known biological functions (Soderhall and Cerenius, 1992).
Table2. Summary of the key groups of compounds reported to stimulate the crustacean
immune sytem
Experiment 1
Table1.Proportion of various ingredients used in the preparation of different test diets
(Experiment 1)
Table 2. Proximate composition of ingredients used for the preparation of experimental
diets (% on dry weight basis) for experiment (1 and 2)
Table 3. Proximate composition of experimental diets (% on dry weight basis)
Table 4. Mean values of air and water temperature recorded during the experimental
period
Table 5. Values of pH recorded on different sampling days during the experimental period
Table 6. Fluctuation of dissolved oxygen (mg/l) level of water recorded on different
sampling days
Table 7. Fluctuation of carbon dioxide (mg/l) level of water recorded on different
sampling days
Table 8. Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on
different sampling days
Table 9. Fluctuation of total alkalinity (mg/l) level of water recorded on different
sampling days
Table 10. Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
Table 11. Analysis of variance for weight (g)of M. rosenbergii in different treatments and
control group
Table12. Duncan’s Multiple Range Test
Table 13. Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
Table 14a .Survival, Weight gain, SGR, FCR, PER of M. rosenbergii in different
18
treatments during the first experimental period
Table 14b. Growth parameters in freshwater prawn M. rosenbergii fed supplemented diets
with different levels of marigold oleoresin
Table 14b-1. Analysis of variance for specific growth rate of M. rosenbergii in different
treatments and control group
Table 14b-2. Duncan’s Multiple Range Test
Table 14b-3. Analysis of variance for feed conversion rate of M. rosenbergii in different
treatments and control group
Table 14b-4. Duncan’s Multiple Range Test
Table 14b-5. Analysis of variance for protein efficiency rate of M. rosenbergii in different
treatments and control group
Table 14b-6. Duncan’s Multiple Range Test
Table 14b-7. Analysis of variance for survival rate of M. rosenbergii in different
treatments and control group
Table 14b-8. Analysis of variance for feed efficiency rate of M. rosenbergii in different
treatments and control group
Table 14b-9. Duncan’s Multiple Range Test
Table 14b-10. Analysis of variance for weight gain (g) of M. rosenbergii in different
treatments and control group
Table 14b-11. Duncan’s Multiple Range Test
Table 14b-12. Analysis of variance for per day growth of M. rosenbergii in different
treatments and control group
Table 14b-13. Duncan’s Multiple Range Test
Table 14b-14. Analysis of variance for percentage of means weight growth of M.
rosenbergii in different treatments and control group
Table 14b-15. Duncan’s Multiple Range Test
Table 14b-16. Analysis of variance for condition factor of M. rosenbergii in different
treatments and control group
Table14 b-17. Duncan’s Multiple Range Test
19
Table 14b-18. Analysis of variance for daily growth rate of M. rosenbergii in different
treatments and control group
Table 14b-19. Duncan’s Multiple Range Test
Table 14b-20. Analysis of variance for daily growth index of M. rosenbergii in different
treatments and control group
Table 14b-21. Duncan’s Multiple Range Test
Table 14b-22. Analysis of variance for growth coefficient of M. rosenbergii in different
treatments and control group
Table 14b-23. Duncan’s Multiple Range Test
Table 15. Proximate composition of prawn meat taken from different treatments (% on
dry weight basis) 1st exp.
Table 16. Mortality and relative percent survival (RPS) of M. rosenbergii recorded in
different treatments and control group after challenged against with A. hydrophila
Table 17. Analysis of variance for mortality of M. rosenbergii recorded in different
treatments and control group after challenged against A. hydrophila infection
Table 18. Duncan’s Multiple Range Test
Table 19. The prophenol oxidase activity, super oxide anion production, total
haemolymph protein and total heamocytes count recorded in different treatments and
control group after feeding trials
Table 20. Analysis of variance for mean ppo activity of M. rosenbergii in different
treatments and control group
Table 21. Duncan’s Multiple Range Test
Table 22. Analysis of variance for mean superoxide anion production activity of M.
rosenbergii in different treatments and control group
Table 23. Duncan’s Multiple Range Test
Table 24. Analyses of variance for mean Total Heamolymph Protein activity of M.
rosenbergii in different treatments and control group
Table 25. Duncan’s Multiple Range Test
20
Table 26. Analysis of variance for mean Total heamocytes Count activity of M.
rosenbergii in different treatments and control group
Table 27. Duncan’s Multiple Range Test
Experiment 2
Table 28. Proportion of various ingredients used in the preparation of different test diets
Table 29. Proximate composition of experimental diets (% on dry weight basis)
Table 30. Mean values of air and water temperature recorded during the experimental
period
Table 31. Values of pH recorded on different sampling days during the experimental
period
Table 32. Fluctuation of dissolved oxygen (mg/l) level of water recorded on different
sampling days
Table 33. Fluctuation of carbon dioxide (mg/l) level of water recorded on different
sampling days
Table 34. Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on
different sampling days
Table 35. Fluctuation of total alkalinity (mg/l) level of water recorded on different
sampling days
Table 36. Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
Table 37. Analyses of variance for final mean weight (g) of M. rosenbergii in different
treatments and control group
Table 38. Duncan’s Multiple Range Test
Table 39. Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
Table 40a. Survival, Weight gain, SGR, FCR, PER of M. rosenbergii in different
treatments during the first experimental period
Table 40b. Growth parameters in freshwater prawn M. rosenbergii fed supplemented diets
with different levels of Diacetete of Lutein-Mesozeaxanthin
Table 40b-1. Analysis of variance for specific growth rate of M. rosenbergii in different
21
treatments and control group
Table 40b-2. Duncan’s Multiple Range Test
Table 40b-3. Analysis of variance for feed conversion rate of M. rosenbergii in different
treatments and control group
Table 40b-4. Analysis of variance for protein efficiency rate of M. rosenbergii in different
treatments and control group
Table 40b-5. Analysis of variance for survival rate of M. rosenbergii in different
treatments and control group
Table 40b-6. Analysis of variance for feed efficiency rate of M. rosenbergii in different
treatments and control group
Table 40b-7. Analysis of variance for weight gain (g) of M. rosenbergii in different
treatments and control group
Table 40b-8. Duncan’s Multiple Range Test
Table 40b-9. Analysis of variance for per day growth of M. rosenbergii in different
treatments and control group
Table 40b-10. Duncan’s Multiple Range Test
Table 40b-11. Analysis of variance for percentage of mean weight gain of M. rosenbergii
in different treatments and control group
Table 40b-12. Duncan’s Multiple Range Test
Table 40b-13. Analysis of variance for condition factor of M. rosenbergii in different
treatments and control group
Table 40b-14. Analysis of variance for daily growth rate of M. rosenbergii in different
treatments and control group
Table 4ob-15. Duncan’s Multiple Range Test
Table 40b-16. Analysis of variance for daily growth index of M. rosenbergii in different
treatments and control group
Table 40b-17. Duncan’s Multiple Range Test
Table 40b-18. Analysis of variance for growth coefficient of M. rosenbergii in different
treatments and control group
22
Table 40b-19. Duncan’s Multiple Range Test
Table 41. Proximate composition of prawn meat taken from different treatments (dry
weight basis)
Table 42. Mortality and relative percent survival (RPS) of M. rosenbergii recorded in
different treatments and control group after challenged against with A. hydrophila
Table 43. Mortality of M. rosenbergii recorded in different treatments and control group
after challenged against A. hydrophila infection
Table 44. Duncan’s Multiple Range Test
Table 45. The prophenol oxidase activity, super oxide anion production, total
haemolymph protein and total heamocytes count recorded in different treatments and
control group after feeding trials
Table 46. Analysis of variance for mean ppo activity of M. rosenbergii in different
treatments and control group
Table 47. Duncan’s Multiple Range Test
Table 48. Analysis of variance for mean superoxide anion production activity of M.
rosenbergii in different treatments and control group
Table 49. Duncan’s Multiple Range Test
Table 50. Analysis of variance for mean Total Heamolymph Protein activity of M.
rosenbergii in different treatments and control group
Table 51. Duncan’s Multiple Range Test
Table 52. Analysis of variance for mean Total heamocytes Count activity of M.
rosenbergii in different treatments and control group
Table 53. Duncan’s Multiple Range Test
23
LIST OF FIGURES
Fig.1. Life cycle of M. rosenbergii
Fig. 2. Flow diagram of the crustacean host defense system.
Fig.3. Chemical structure of lutein, zeaxanthin, and meso-zeaxanthin.
Experiment 1
Fig. 1 Mean values of air and water temperature recorded during the experimental period
Fig.2 Values of pH recorded on different sampling days during the experimental period
Fig. 3 Fluctuation of dissolved oxygen (mg/l) level of water recorded on different sampling days
Fig. 4 Fluctuation of carbon dioxide (mg/l) level of water recorded on different sampling days
Fig. 5 Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on different
sampling days
Fig. 6 Fluctuation of total alkalinity (mg/l) level of water recorded on different sampling days
Fig .7 a Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment (Increment)
Fig. 7b Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
Fig.8 Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
Fig. 9 Specific growth Rate of M. rosenbergii in different treatments and control group
Fig. 10 Feed conversion Rate of M. rosenbergii in different treatments and control group
Fig.11 protein efficiency Rate of M. rosenbergii in different treatments and control group
Fig. 12 Survival Rate of M. rosenbergii in different treatments and control group
Fig. 13 Feed efficiency Rate of M. rosenbergii in different treatments and control group
Fig. 14. Weight gain of freshwater prawn recorded in different tanks during the experiment
Fig. 15 per day growth of M. rosenbergii in different treatments and control group
24
Fig. 16 percentage of mean weight growth 0f M. rosenbergii in different treatments and control
group
Fig. 17 condition factor of M. rosenbergii in different treatments and control group
Fig. 18 Daily growth rate of M. rosenbergii in different treatments and control group
Fig. 19 Daily growth index of M. rosenbergii in different treatments and control group
Fig. 20 Growth coefficient of M. rosenbergii in different treatments and control group
Fig. 21 Percentage of mortality recorded in A.hydrophila infected treatments and control group
Fig. 22 The ppo activity recorded in different treatments and control group after feeding trials
Fig. 23 The superoxide anion activity recorded in different treatments and control group after
feeding trials
Fig. 24 The total heamolymph protein recorded in different treatments and control group after
feeding trials
Fig 25. The total heamocytes count recorded in different treatments and control group after
feeding trials
Experiment 2
Fig. 26 Mean values of air and water temperature recorded during the experimental period
Fig. 27 Values of pH recorded on different sampling days during the experimental period
Fig. 28 Fluctuation of dissolved oxygen (mg/l) level of water recorded on different sampling
days
Fig. 29. Fluctuation of carbon dioxide (mg/l) level of water recorded on different sampling days
Fig. 30 Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on different
sampling days
Fig. 31 Fluctuation of total alkalinity (mg/l) level of water recorded on different sampling days
Fig. 32a Details of weight (cm) of freshwater prawn recorded in different tanks during the
experiment (Increment)
Fig. 32b Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
25
Fig. 33 Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
Fig. 34 Specific growth Rate 0f M. rosenbergii in different treatments and control group
Fig. 35 Feed conversion Rate 0f Macrobrachium rosenbergii in different treatments and control
group
Fig. 36 protein efficiency Rate 0f M. rosenbergii in different treatments and control group
Fig. 37 Survival Rate 0f M. rosenbergii in different treatments and control group
Fig. 38 Feed Efficiency Rate 0f M. rosenbergii in different treatments and control group
Fig. 39 Weight gain of freshwater prawn recorded in different tanks during the experiment
Fig. 40 per day growth 0f M. rosenbergii in different treatments and control group
Fig. 41 percentage of mean weight growth 0f M. rosenbergii in different treatments and control
group
Fig. 42 condition factor 0f Macrobrachium rosenbergii in different treatments and control group
Fig. 43 Daily growth rate 0f M. rosenbergii in different treatments and control group
Fig. 44 Daily growth index of M. rosenbergii in different treatments and control group
Fig. 45 Growth coefficient of M. rosenbergii in different treatments and control group
Fig. 46 Percentage of mortality recorded in A. hydrophila infected treatments and control group
Fig. 47 The ppo activity recorded in different treatments and control group after feeding trials
Fig. 48 The superoxide anion recorded in different treatments and control group after feeding
trials
Fig. 49 The T.H.P recorded in different treatments and control group after feeding trials
Fig. 50 The T.H.C recorded in different treatments and control group after feeding trials
26
I. INTRODUCTION
Giant freshwater prawn, Macrobrachium rosenbergii is a native prawn of Thailand and
other Southeast Asian countries including Vietnam, Kampuchea, Malaysia, Myanmar,
Bangladesh, India, Sri Lanka and The Philippines. However, productions of M. rosenbergii are
also reported from regions outside its native habitat such as Israel, Japan, Taiwan and some
countries in Africa, Latin America and the Caribbean (New, 1990). M. rosenbergii is an
important cultured palaemonid prawn all over the world and its culture is an economically
important activity in many of the developing countries of South-East Asia. Global production of
this prawn exceeded 200000 metric tons (mt) in 2008 (FAO, 2008) with potential scope for
growth. In India too, a spurt in freshwater prawn farming activities has been seen in recent years.
Considering its high export market, the giant freshwater prawn enjoys immense potential for
culture in India. About 4 million hectares of impounded freshwater bodies in India offer great
potential for freshwater prawn culture. In India its culture was expanded to compensate the
heavy economic losses due to the epidemic white spot syndrome (WSS) in penaeid shrimp
farming, hypothesizing the resistance of the giant freshwater prawn to WSS (Sahul Hameed et
al., 2000). Generally, M. rosenbergii is considered to be moderately disease-resistant in
comparison to penaeid shrimp. However, the increasing demand for this species in both domestic
and international markets has led to a remarkable increase in the number of large-scale culture
systems with high stocking density and intensive feeding. With the rapid development in
hatchery production of post larvae (PL) and the number of prawn grow-out farms, good
husbandry and environmental managerial practices have often been neglected. Subsequently,
microbial pathogens gained easy entry, as the prawns are stressed and weakened under adverse
27
environmental conditions resulting disease outbreaks (Tonguthai, 1995). Moreover, poor
quarantine in many prawn hatcheries and farms in West Indies, China, Taiwan, India, and
Thailand have witnessed the epizootics of M. rosenbergii nodavirus (MrNV) (Cheng W, Chen
JC., 1998, 2000). In India, juveniles and adults of M. rosenbergii have been suffered a major
setback due to occurrence of appendage deformity syndrome (ADS) (Sahoo PK, 2005) and
several disease outbreaks have occurred due to bacterial pathogens (Chen SC, 2001 and
Phatarpekar P V, 2002) such as Vibrio spp., Aeromonas spp., and Pseudomonas spp., and
Lactococcus garviae infection (Cheng W, 2000) which caused high mortalities in hatcheries
(Sung HH. 2000, Tonguthai K. 1995, Delves-Broughton 1976). Among these Aeromonas spp.
is considered to be the major threat to the commercial cultivation of M. rosenbergii aquaculture
in Taiwan (Sung HH. 2000) and Brazil (Lombardi JV. 1991) including India (Chand RK. 2008,
Shankar R. 2011, Sahoo PK. 2007, Lalitha KV, 2006).White tail disease is the major disease
outbreak in M. rosenbergii hatcheries and farms in the recent years (Murthy HS, 2008; Sahul
Hameed et al., 2009). To prevent and control prawn diseases large quantities of antibiotics and
vaccines were used. Application of the antibiotics develops drug-resistance bacteria while
vaccines are specific for pathogen, thus novel strategies to control Aeromonas spp. are needed
for prawn culture. Several studies have been reported that the application of herbal, or other
immunostimulants in fish and shrimp farming for enhancing immune response and reduction of
disease impacts (Chand RK. 2008, Shankar R. 2011, Liu B. 2010, Balasubramanian G. 2008,
Harikrishnan R. 2011, Harikrishnan R, 2011). However, use of immunostimulants can be
thought of, as one of the means to increase the innate immune system of the cultured species to
avoid frequent chances of infection there by reducing the risk of damage caused by the outbreak
to the industry. Immunostimulants are chemical compounds and activate white blood cells
28
(leukocytes) and hence render animals more resistance to infections by virus, bacteria, fungi and
parasites. Immunostimulants are used to prevent the fish and crustaceans from diseases by
enhancing the immune system as well as to combat immunosuppressive conditions. Prawns
possess very primitive defense system and therefore nonspecific immune system plays a vital
role in prevention of infection (Anderson and Jency, 1992). There is evidence of beneficial
effect of dietary carotenoid supplementation on immunological response (Estermann, 1994).
Recently, the immunostimulant effect of carotenoids and a positive correlation between immune
defenses and concentration of carotenoids in the hemolymph was demonstrated in the crustacean
Gammarus pulex, suggesting an important role of carotenoids in invertebrate immunity. Aure´lie
Babin (et al., 2010) found that dietary carotenoids had a clearly and broad immunostimulating
effects, enhancing phenoloxidase activity and resistance to a bacterial infection. When immune
challenged, gammarids fed with carotenoids did not pay an additional survival cost because of
autoreactivity, despite their intensified immune activity. Therefore, dietary carotenoids improved
gammarids’ immunity without inducing additional self-harming. This underlines the importance
of carotenoids both in the regulation and the evolution of immunity in G. pulex. Amar et al.
(2001) indicated that carotenoid supplemented diets had beneficial effect on biodefense
mechanisms in rainbow trout and found the serum complement activity in astaxanthinsupplement fed groups were
significantly higher than control fish. Moreover, the
supplemented astaxanthin fed groups also exhibited better nonspecific cytotoxicity for the
peripheral blood lymphocytes and higher phagocytic activity compared with control.
Astaxanthin elevated humoral factors such as serum complement and lysozyme activity, as
well as cellular factors such as phagocytosis and nonspecific cytotoxicity (Amar et al., 2001).
Linan-Cabello et al. (2003) sampled the captive and wild female white shrimp (Litopenaeus
29
vannamei) at two maturation stages, II and IV. The carotenoid concentration and range of
activity were significantly higher in wild than in captive shrimp, particularly at maturation
s t a ge IV . In the s t u d y b y S u p a m a t t a y a e t a l . (2005) of black tiger shrimp (Penaeus
monodon), the concentration of β-carotene did not affect the immune parameters
(total
hemocyte count, phenoloxidase activity, and bacterial clearance ability) but enhanced the
resistance to white spot syndrome virus. One alternative solution to the disease problem is to
enhance the animal’s antioxidant capacity, which would consequently increase its resistance
against stress (Pan et al. 2003, Chien & Shiau 2005) and microbial infection. Carotenoids as an
antioxidant, inactivates free radicals produced from normal cellular activity and various stressors
(Chew 1995). The health of stressed aquatic organisms is linked to the overproduction of
reactive oxygen species (Di Guilio,Washburn,Wenning,Winston & Jewell1989), which is a
precursor to the occurrence of disease. Moreover, antioxidants help protect cell membranes
against the damage from excessive production of reactive oxygen species (Mourente, Di ́azSalvago, Bell & Tocher 2002). The antioxidant system of M. rosenbergii helps in eliminating
reactive oxygen species (ROS), which are produced under oxidative damage (Halliwell &
Gutteridge 1989) when they are exposed to stressors. Activity
of
superoxide
dismutase
(SOD) in heamolymph of captive shrimp at both stages of maturation was significantly
greater than that of wild organisms. SOD is part of a group of enzymes that catalyze the
metabolism of superoxide radicals, and therefore, is considered as an indicator of oxidative
stress ( Nii and Muscatine, 1997 ). Wild shrimp with access to greater quantity and diversity of
dietary carotenoids h a d l o w e r e n z y m a t i c antioxidant activity (SOD) (Linan-Cabello et
al., 2 0 0 3 ). These suggested that low carotenoid and retinoid concentrations in captive
specimens might have induced an increase in SOD activity to neutralize oxidative stress-
30
induced damage, such as lipid peroxidation of polyunsaturated fatty acids, and damage to
other key biomolecules in the gonad development of penaeids. According to Miki (1991),
carotenoid compounds, such as astaxanthin, quench superoxide radicals approximately 100
times more than α-tocopherol. Other carotenoids that could act in the hemolymph and tissues
( Harrison, 1990) h a d b e e n d e s c r i b e d a s potential a n t i o x i d a n t s (Miki et al., 1994).
These antioxidants could play a key role in preventing oxidative stress by protecting key
molecules during critical processes, such as gonad maturation ( Linan-Cabello et al., 2 0 0 3 ).
Astaxanthin (AX), a naturally occurring carotenoid pigment, is a powerful biological antioxidant.
The enhancement of antioxidant capacity by dietary AX and, consequently, the improvement in
recovery against stress demonstrated that AX is a semiessential nutrient for the black tiger
shrimp Penaeus monodon (Fabricius 1798) (Chien et al. 2003). Antioxidant capacity and
immune response can be assessed by measuring hemolymph antioxidant enzymes such as
superoxide superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione
reductase (GR), and by determining the total hemocyte count (THC). SOD is involved in
protective mechanisms within tissue injury following oxidative processes and phagocytosis (Bell
& Smith 1993). GPx, on the other hand, protects cells from excessive levels of hydrogen
peroxide (H2O2) and intracellular lipid peroxides (Robertson & Harmon 2007), whereas GR
catalyzes the reduction of glutathione to yield reduced glutathione, which is readily oxidized by
ROS (Stohs and Bagchi 1995). Carotenoids can influence of growth and survival of fish and
shellfishes, but The effects of carotenoids on growth and survival rate of aquatic
organisms have been controversial, because several studies reporting a positive influence
whereas others did not find any effect. Torrissen (1984) reported that an improved growth of
red tilapia (Oreochromis niloticus) was found by supplementing commercial starter diets with
31
astaxanthin or canthaxanthin, and no significant differences were observed between the
astaxanthin and canthaxanthin supplemented diets. Corresponding results were found for the
Kuruma shrimp (Penaeus japonicus) showing a positive relationship between carotenoid
concentration and survival in the shrimp, but survival fell to 50% after 3 months on a
carotenoid-free diet (Chien and Jeng, 1992). Supplementation of β- carotene and canthaxanthin
to the diets of Indian major carps (Labeo rohita and Cirrhina mrigala) resulted in their better
survival and growth compared to those f e d on conventional diets without carotenoids
(Goswami 1993) . Similarly, Negere-Sadargues
et
al. (1993) found a higher survival for
Penaeus japonicus receiving astaxanthin-canthaxanthin supplementation (50/50), but no
difference was observed in the growth and molting. A study of the interaction between
astaxanthin and vitamin A supplementation on growth and survival of Atlantic salmon (Salmo
salar L) during the first 135 days feeding resulted in significantly improved growth and
survival when astaxanthin was supplemented to the experimental diet. It also had a provitamin A function, whereas vitamin A supplementation alone did not support growth and
survival (Christiansen et al., 1994). Dall (1995) also investigated the effect of carotenoids and
retinoids as growth factors in penaeid shrimp (Penaeus semisulcatus ) . The results showed
that no retinoids could be detected in the eggs, the naupliar stages or Protozoea I, but free
astaxanthin was metabolized exponentially , falling from 19 µg/g in the eggs to 4 µg/g
in Protozoea. This suggested that retinoids are not essential in early development but
carotenoid could be a substitute. Thong rod et al. (1995) indicated that growth and
survival were positively correlated to d i e t a r y a s t a x a n t h i n supplementation of up to
300 mg/kg diet. Petit et al (1997) reported that the supplements of synthetic astaxanthin
(60 mg/kg diet) modified the exuviations frequency, shortened the molting cycle, and
32
hastened Penaeus japonicus postlarval development during a 20-day test. Supamattaya et
al. (2005) found that the black tiger shrimp (Penaeus monodon) fed 125 to 300 mg of
the Dunaliella extract per kg diet for 8 weeks showed higher weight gain and survival
compared to the control diet without Dunaliella extract , indicating that 125 mg β-carotene per
kg diet was a sufficient requirement of Penaeus monodon. Niu et al. (2009) fed four diets
containing four supplemented levels of astaxanthin (0, 100, 200, and 400 mg/kg) to the
postlarval Litopenaeus vannamei for 30 days. The shrimp fed diets containing 100, 200, and
400 mg astaxanthin per kg diet showed higher weight gains compared to the control, but no
significant differences were found across the three astaxanthin supplemented groups in
weight gain. However, survival of shrimp in the 0 and 100 mg/kg diet treatments was
significantly lower than that of shrimp in the 200 and 400 mg/kg diet treatments. This
suggests that the content of astaxanthin in the diet should
be
supplemented at levels
between 100 and 200 mg/kg di et . The adult Japanese shrimp, Penaeus japonicus, fed pigment
supplemented meal, showed no significant modification of the growth rate at the end of the
experimental period (Negre-Sadargues et al., 1993). An improved weight gain was however
obtained in prawns receiving either β-carotene or astaxanthin supplemented diet (Chien and
Jeng, 1992). A series of feeding experiments on the prawn P. japonicus duri ng larval and
postlarval periods suggested that dietary carotenoids could influence the early d e v e l o p m e n t
o f this species. Results attained with Atlantic salmon fry and juveniles, together with reports
for shrimp and sea urchins (Petit et al., 1997) also suggested that carotenoids might have a
positive effect on growth, mainly during the first developmental stages. Thus, different factors
like the developmental status, the nature and amount of the pigment added to the food, and the
duration of feeding conditions, must be taken into account to understand these different
33
results. With this background the present study was undertaken to evaluate the effect of
carotenoides on growth, survival and immune response of giant freshwater prawn,
Macrobrachium rosenbergii and disease resistance against Aeromonas hydrophila infection with
the following objectives.
1. To evaluate the effect of dietary supplementation of carotenoides on growth and survival
of giant freshwater prawn, Macrobrachiumrosenbergii.
2. To study the effect of dietary supplementation of carotenoides on biochemical
composition of giant freshwater prawn, Macrobrachiumrosenbergii.
3. To investigate the effect of dietary supplementation of carotenoides on immune response
and disease resistance in giant freshwater prawn, Macrobrachiumrosenbergii.
34
II. REVIEW OF LIETRATURE
2.1 Macrobrachium rosenbergii: its significance in freshwater aquaculture
M. rosenbergii, commonly known as freshwater prawn or scampi, is the largest species in
the genus and the most widely cultured freshwater prawn species all over the world. As a
candidate species, M. rosenbergii has many advantages, of fast growth rate, compatibility for
polyculture, high protein content and disease resistance (Murthy, 1998). The species is native to
Southeast Asia, South Pacific, Northern Oceania and Western Pacific islands and it has been
transported to many parts of the world including South America and China (New, 1982, 2002).
Because of the domestic and international market demand, culture of the species has expanded
rapidly not only within Asia but also in regions far remote from the natural distribution and is
now cultured in at least 43 counties across five continents (FAO, 2000). Since 1995, there has
been a rapid increase in global production of freshwater prawn. A large amount of the
production has taken place in China, India and in Bangladesh (New, 2005). In India, the Marine
Product Export Development Authority (MPEDA) has been promoting freshwater prawn
farming in order to diversify the Indian export basket (Bojan et al., 2007). Since 1999, the area
under scampi culture has expanded considerably and it has reached more than 33,000 ha and is
still expanding, mostly in the landlocked states of Punjab, Haryana and Himachal Pradesh
(Zacharia, 2007). Murthy et al. (2006) reported scampi farming activities in Bihar, while
Dixitulu (2007) noted the operations in Tripura, Chandigarh, Madhya Pradesh and Rajasthan.
Among the states, Andhra Pradesh is the centre of scampi culture in India.
35
2.2 Biology of M. rosenbergii
M. rosenbergii (De Man, 1879) is a decapods crustacean in the family Palaemonidae
(New, 2002) (Plate 3). Some taxonomists recognize two sub-species of M. rosenbergii based on
morphological differences: a Western form, M. rosenbergii dacqueti (Sunier, 1925), that
distributed in western Asia (east coast of India, Bay of Bengal, Gulf of Thailand, Malaysia and
the northern Indonesian islands of Sumatra, Java and Kalimantan), and an Eastern form M.
rosenbergii rosenbergii (De Man, 1879), which is native to the eastern Asia-Pacific, occurring in
the Philippines, the Indonesian islands of Sulawesi and in Papua New Guinea and northern
Australia (Holthuis, 2000). They mostly inhabit inland freshwater areas including rivers, lakes,
irrigation ditches, canals and ponds as well as in estuarine areas. Most species require
brackishwater in the initial stages of their life cycle, while some complete their cycle in inland
saline and freshwater lakes. Generally, M. rosenbergii prefer rivers with extremely turbid
conditions. The life cycle of the species consists of four distinct phases namely eggs, larvae,
postlarvae (PL) and adults (Plate 4). Most scientists accept that the larvae go through 11 distinct
stages before metamorphosis (Uno and Soo, 1969). However, from stage VI onwards their size
is variable, which has led some workers, notably Ling (1969) to describe only eight stages. As
the eggs hatch, within one or two nights, the larvae (free-swimming zoae) are dispersed by rapid
movements of the abdominal appendages of the parent. The larvae are planktonic and swim
actively tail first, ventral side uppermost (i.e. upside down). On completion of their larval life,
prawns metamorphose into postlarvae (PL), where they resemble miniature adult prawns and
swim as normal (dorsal side uppermost) way. The most sticking characteristic of PL is they
exhibit good tolerance to a wide range of salinity. The body of postlarval and adult prawns
consists of twenty segments known as somites, the cephalothorax (head) and the abdomen
36
(‘tail’). The head is divided into 14 segments, while the tail is very clearly divided segments,
each bearing a pair of appendages known as pleopods or swimmerets. In addition, circulatory
system, body defense system provides vital information for understanding the disease
development processes. Like other invertebrates, freshwater prawn lack a true adaptive immune
system (Xianle and Yanping, 2003) but have developed effective mechanisms for detecting and
eliminating pathogens, which depend entirely on non-specific immune system such as prophenol
oxidase (proPO) system, phagocytosis, encapsulation, nodule formation and mediation of
cytotoxicity (Soderhall K. and Cerenius, 1992). In addition, prawns are also blessed with a hard
cuticle which acts as a structural and chemical barrier against various pathogens. However, lack
of true memory, sensitive to pathogens during molting period, shorter life cycle and faster
metabolism of these animals results in easy pathogen access and disease outbreaks (Xianle and
Yanping, 2003). Fig.1. Life cycle of M.rosenbergii
37
2.3 Nutrient requirements
2.3.1 Protein and amino acid requirement
Diets with about 35-40 % protein and gross energy level of about 3.2 kcal/g diet and
protein: energy ratio of about 125-130 mg protein/kcal is suitable for growth of M. rosenbergii
in clear water systems that do not have any supply of natural foods. Bloodstock reared in ponds
having natural food (benthic micro and macro fauna) requires about 30 % protein in the diet.
Many commercial feeds for grow-out contain 24-32 % crude protein. Protein/starch ratio of 1:1
is known to be effective for better feed efficiency and growth rate. The prawn requires the same
ten essential amino acids as that of crustacean and fish species, but quantitative requirements
have not been determined. The amino acid composition of the prawn muscle is used to provide
guidance values in feed formulation (Mukhopadhyay et al, 2005).
2.3.2 Lipid and free fatty acid requirement
Freshwater prawn uses dietary carbohydrate efficiently as energy source, protein sparing
by lipids is not considered to be crucial. The dietary lipid level in prawn diets can be as low as
5 % provided the lipid source contains sufficient levels of essential fatty acids. There is a
dietary requirement for highly unsaturated fatty acids (HUFA) although in very small quantities.
Both n-3 and n-6 HUFAs at dietary levels of 0.075 % are known to increase weight gain and feed
efficiency remarkably. Further both 18:2 n-6 and 18:3 n-3 are also required. M. rosenbergii, like
other crustaceans, is unable to synthesize cholesterol due to the absence of the enzyme 3hydroxy-3 methylgluta'yl CoA reductase.
38
2.3.3 Carbohydrate requirement
The comparatively high specific activity of amylase found for M. rosenbergii supports
the fact that the species efficiently utilizes carbohydrates as a source of energy. During fasting,
energy metabolism in the prawn is dominated by carbohydrates, followed by lipids and proteins.
Complex polysaccharides including starch and dextrin are more effectively utilized than simple
sugars. Dietary glucosamine (an amino sugar and intermediary between glucose and chitin)
facilitates molting followed by enhanced growth. Dietary protein is efficiently utilized at dietary
lipid-carbohydrate ratio of 1:3-1:4. The prawns are also known to utilize as high as 30 % dietary
fiber (Mukhopadhyay e tal, 2005).
2.3.4 Mineral requirement
Information on quantitative mineral requirement of freshwater prawn is limited. Dietary
supply of calcium seems to improve growth of freshwater prawn performance of the prawns
better when calcium was provided at 3% level in soft water (calcium concentration at 5
ppm).Even when the calcium concentration was higher at 74 ppm, performance improved when
calcium was provided at 1.8%. The optimum level of zinc at 50-90 mg/kg diet. Growth and feed
conversion efficiency declined at higher dietary doses (>90mg/kg) of zinc (Mukhopadhyay et al.,
2005).
2.3.5 Vitamin requirement
Vitamin requirements of M. rosenbergii are probably similar to other crustaceans and fish
species. The prawn requires 60-150 mg vitamin C/kg diet. Levels of 60 mg ascorbic acid and 300
mg tocopherol per kg diet are considered sufficient for proper production and offspring viability in
prawn broodstock. However, feeding female prawn with higher levels of both these vitamin (each
39
around 900 mg/kg) might improve larval quality including higher tolerance to ammonia stress. It
has been reported that vitamin E at 200 mg/kg modulate some of the antioxidants defense system by
decreasing lipid peroxidation in the hepatopancreas (Mukhopadhyay et al, 2005).
2.3.6 Some of the important other additives
2.3.6.1 Probiotics
The term, probiotic, simply means “for life”, originating from the Greek words “Pro” and
“bios” (Gismondo et al., 1999). The most widely quoted definition was made by Fuller (1989).
He defined a probiotic as “a live microbial feed supplement which beneficially affects the host
animal by improving its intestinal balance. Multiple ways exist in which probiotics could be
beneficial and these could act either singly or in combination for a single probiotic. These
include: inhibition of a pathogen via production of antagonistic compounds, competition for
attachment sites, competition for nutrients, alteration of enzymatic activity of pathogens,
immunostimulatory functions, and nutritional benefits such as improving feed digestibility and
feed utilization (Fuller, 1989; Fooks et al, 1999; Bomb&etal, 2002). Some of the commercially
trade produced probiotics include the spore forming Bacillus spp. and yeasts. Bacillus spp hold
added interest in probiotics as they can be kept in the spore form and therefore stored
indefinitely on the shelf (Hong et al, 2005). The yeast, Saccharomyces cerevisiae, also has
been commonly studied whereby immunostimulatory activity was demonstrated and production
of inhibitory substances shown (Castaglmolo et al, 1999; Pahan et al, 2003; Van der Aa Kuhle et
al, 2005).
40
2.3.6.2 Feed attractants
The use of feed attractants in manufactured aqua feeds has received considerable
attention in the recent years. The reason behind their use has been to improve dietary food
intake and at the same time by promoting quicker food intake. The time interval between feed
offered and intake by the animal is minimized in water and thereby the leaching of water
soluble nutrients. Further, attractants provide additional nutrients for protein and energy
metabolism so that aqua feeds are ingested with minimum wastage and maximum feed
efficiency, which also helps to reduce water pollution. The commonly used feed attractants are
reviewed below. Pavadi and Murthy 2004 reported that aquasavor as dietary feed attractant
enhances growth, survival and feed utilization.
2.3.6.3 Antibiotics
Sykhoverhov (1967) reported that 20000 units of Terramycin given every three days
raised growth by 9.5 % in Cyprinus carpio. "Nitrovin" was found to be a better growth promoter
than 17a-MT in Bpinephalus salmoides (Chua and Teng, 1980). Virginamycin at 40 ppm was
found to be optimum in Pangasius sutchi (Pathmasothy, 1987). Stafac-20 (having 2%
virginamycin) incorporated at 80 ppm and 20 ppm levels gave the highest SGR in rohu and
common carp fingerlings respectively (Keshavanath et at, 1991), whereas 60 ppm and 100 ppm
gave superior growth in catla and rohu fry respectively (Manojkumar, 1994).
2.3.6.4 Pigments
Four main groups of pigments account for the coloration of mammals, birds, fish and
invertebrates of economic importance. These are porphyrins, pteridines, melanins and
carotenoids (Hudon, 1994). Porphyrins are of primary importance mainly in the coloration of
avian eggshell (Kennedy and Vevers, 1976; Lang and Wells, 1987). Pteridines are responsible
41
for many of the bright yellows and reds in fish, amphibians and reptiles (Nixon, 1985; Ziegler,
1965); these pigments are water soluble and are produced endogenously (Hudon, 1994). Melanin
gives all the blacks, grays and browns to vertebrates and many invertebrates, and also several of
their reds and yellows. Melanins are heterogeneous polymers made up of metabolites of tyrosine
(Hudon, 1994). Carotenoids are pigments naturally occurring in a number of fruits and
vegetables. They are synthesized by all photosynthetic organisms and many nonphotosynthetic
bacteria and fungi. There are 2 main classes of naturally occurring carotenoids: (1) carotenes
such as β-carotene and α-carotene, which are hydrocarbons, are either linear or cyclized at one or
both ends of the molecule, and (2) xanthophylls, the oxygenated derivatives of carotenes. All
xanthophylls produced by higher plants, such as violaxanthin, antheraxanthin, zeaxanthin,
neoxanthin, and lutein, are also synthesized by green algae (Eonseon and others 2003). Latter
will discuss about carotenoids in detail.
2.4. Crustacean immunity
2.4.1 Some of the most important bio-defense system in crustaceans
Like other invertebrates crustaceans possess only innate immunity which is also efficient
to defend against pathogens. It involves both cellular and humoral factors. The cellular response
is mediated by hemocytes and the humoral immune response involves constitutive and inducible
extracellular molecules. Hemocytic reactions involve direct cellular interaction between
circulating hemocytes and bacteria and these typically occur within minutes after infection.
Specific cellular defense mechanisms include phagocytosis, nodulation and encapsulation
(Sodehrall and Cerenius, 1992) and humoral defense mechanism involves several protein
molecules which are stored in hemocytes and are released during defense action which eliminate
42
foreign substance. Crustacean immunity is mainly featured by the presence of several kinds of
hemocytes.
2.4.1.1. Cellular immunity of crustaceans
2.4.1.1.1 Hemocytes
Hemocytes are important in removing foreign entities that gain entry into crustacean
body. The haemocytes of these animals are not different from other animals. There are different
types of hemocytes found in crustaceans which have characteristic morphology. The difference
can be distinguished by morphological criteria and by employing different staining techniques.
They are functionally differentiated into semigranular, granular and hyaline cells (Soderhall and
Cerenius, 1992). Despite biochemical characterization progress in handling and isolating
hemocytes, the establishment of cell lineage and the process of hemopoiesis remain largely
unresolved (Bachere et al., 2000). In the absence of development of molecular markers for each
cell types, the functional assignment of particular function for each cell type has been termed as
only tentative (Soderhall and Cerenius, 1992). Most of the hemocytes have granules at the submicron level, visualization of the granules with the light microscope, particularly with phase
contrast, does however serve to distinguish between cell types.
2.4.1.1.2 Hyaline cells
Hyaline cells are characterized by the absence of granules although with cytoplasmic
inclusions (Martin and Graves, 1985). In M.rosenbergii the hyline cells comprises 70% of the
circulating hemocytes .They are readily identified by their fusiform or spindle shape (31 μm- 35
μm length and width, respectively ). In most cells, the elongated nucleus is located centrally, but
other cells show an eccentric location and therefore have only one tapered end. The typical
fusiform hyline hemocytes are easily identified under TEM. They readily attach and spread on
43
glass surface and are capable of phagocytosis and they initiate coagulation .They readily attach
and spread on glass Surfaces and are capable of phagocytosis and they initiate coagulation, (Soderhall
and Smith, 1986; Tsing et al., 1989).
2.4.1.1.3 Semigranular cells
Small granule hemocytes (SGH) comprise approximately 26% of the total circulating
cells in Penaeus paulensis, 54% in M. rosenbergii, and 60% in M. acanthurus. These ovoid or
fusiform cells (9-18 μm length 3 6-8 μm width) have a small nucleus (3-4 μm) compared with
that of the HH (Rogerio et al., 1998).These cells are intermediate between hyalinocytes and
granulocytes. The semigranular cells contain variable number of small granules. These cells are
unstable in- vitro and require careful, delicate handling. They get rapidly lysed in the presence of
microbial polysaccharides like β-1, 3-glucans or lipopolysaccharide (Bachere et al., 2000). It is
the major cell type involved in encapsulation reaction. According to Rogerio et al., (1998)
reported that palaemonids species , the Semigranular cells was the predominet hemocytes type in
circulation, where as in Penaeus paulensis the hyaline cells was the most abundant.
2.4.1.1.4 Granular cells
The granular hemocytes represent 29% in M. rosenbergii and 20% in Macrobrachium
acanthurus and 33% in the P. paulensis of the total circulating hemocytes. These are round or
ovoid shape cells (9-18 μm length × 7-10 μm width) have a relatively small nucleus (3-4 μm)
(Rogerio et al., 1998). According to Lorena et al., reported that, granular hemocytes comprises
of 20% of suspended cells and having a size of 25 μm x 9 μm in M. rosenbergii. The granular
cells are filled with comparatively large granules when compared with semigranular cells. They
are repository of number of molecules which are involved in immune reactions like the
prophenoloxidase (PPO), serine protease zymogens, and cell adhesion molecules and anti
microbial peptides (Johansson and Soderhall, 1985; Chisholm and Smith, 1992). Microbial
44
polysaccharide does not directly induce exocytosis of granular cells but this involves two
endogenous proteins namely peroxinectin (Thornqvist et al., 1997) and the β-1, 3-glucan binding
protein (Barracco et al., 1991) induce degranulation of granular cells. The sites of generation of
these cells are not clearly understood. According to general agreement, circulating hemocytes of
most crustaceans do not divide and thus old cells must be replenished continuously.
Table 2. Hemocytes types and known biological functions (Soderhall and Cerenius, 1992).
HEMOCYTE TYPE
PHAGOCYTOSIS
ENCAPSULATION
CYTOTOXICITY
PROPPO
ACTIVITNG
SYSREM
HYLINE CELLS
Yes
SEMIGRANULAR Limited
No
Not detected
No
Yes
Yes
Yes
Very limited
Yes
Yes
CELLS
GRANULAR
No
CELLS
45
2.4.1.1.5 Phagocytosis
Phagocytosis is the immediate mode of cellular reaction. The defense system exhibits
phagocytosis when a foreign body enters the host after crossing the physiochemical barrier of the
cuticle. Phagocytosis has been observed in isolated and separate hemocytes of crab Carcinus
maenas (Soderhall et al., 1986). The phogocytic cell number in crustacean haemocytes vary from
2 to 28%. Chemotactic and opsonisins properties of the hemocytes have not been very well
understood yet in crustaceans. Phagocytosis of invaders, cell debris and waste products is an
important function of hemocytes. All hemocyte types are capable of phagocytosis but
hyalinocytes and semigranulocytes are more actively phagocytic than granulocytes. There are
evidences that hyalinocytes and semigranulocytes are phagocytically active against Gramnegative bacteria. It is established that hyalinocytes and semigranulocytes are phagocytic in
crayfish. It has also been demonstrated that granulocytes are the primary phagocytic cells in
shrimp, lobsters and crabs (Hose et al., 1989). Efficient phagocytosis of bacteria depends on
various factors present in the heamolymph plasma (Albores et al., 1998). There are indirect
evidences that support the concept that PPO system provides factors that behaves as opsonins
(Smith and Soderhall, 1983). Peroxinectine a cell adhesion factor is released during
degranulation process and activated by the PPO system (Soderhall and Cerenius, 1992) and β-1,
3-glucan binding protein have been shown to opsonise yeast (Thornqvist et al., 1994).
2.4.1.1.6 Nodule formation
Nodule formation is known to occur in several invertebrates including the crustaceans
when microbial invasion is far in excess of the phagocytic capabilities of the host. Microbes
become entrapped in several layers of hemocytes and it occurs when the number of
microorganisms is more than the number that can be removed by phagocytosis. Nodules
46
represent aggregates of hemocytes entrapped in a sticky extracellular material and these
aggregates are often melanized by the activity of the enzyme phenoloxidase (Smith and Ratcliffe,
1980 a, b). The first phase is initiated by the contact between granular hemocytes and bacteria
and this results in degranulation and release of a „sticky coagulum‟ within a minute of exposure
to bacteria, this happens in the presence of endogenous protein namely peroxinectin and β-1, 3binding protein. Shortly afterwards, PPO activation leads to melanin deposition around the
bacteria (Soderhall and Smith, 1986). The second phase is marked by the attachment and
spreading of the hemocyte which forms an outer sheath around the developing nodule. Normally
the microbes in the nodule are rapidly removed from the circulation and they are localized in
hemocyte clumps, usually present in the gills and also in sinuses between the hepatopancreatic
tubules. At the same time phagocytic activity also takes place in other locations (Ratcliffe and
Rowly, 1979). The regulation and the molecular mechanism involved covering both these
mechanisms remain largely undefined. Several cell specific responses have been found
suggesting a receptor based recognition of the foreign invaders (Baines and Downer, 1992).
2.4.1.1.7 Encapsulation
Encapsulation is similar to nodule formation, but occurs as a defense reaction against
larger invaders such as fungi, nematodes, parasitoid eggs, or larvae (Gunnarsson and Lackie,
1985; Hoffmann et al., 1996; Koizumi et al., 1997; 1999; Gillespie et al., 1997). When a parasite
is too large to be engulfed or phagocytosed. It is encapsulated by hemocytes and thus it is seated
of from circulation. Encapsulation is more readily and easily observed defense mechanism, it
occurs when the size of the pathogen to be engulfed is too large in size; several hemocytes will
then collaborate by sealing off the foreign particles from circulation. The cells become flattened
and melanised (Ratcliffe et al., 1985). Semigranular cells are the first cell type to react to foreign
47
molecules. It is demonstrated that peroxinectin a multifunctional protein released by hemocytes
upon degranulation promotes encapsulation and nodulation (Johansson, et al., 1999). It is
demonstrated that the 76 KDa proteins can function as an opsonin for semigranular cells during
their encapsulation process. For haemocytes to encapsulate need to recognize the foreign targets
as non self and change from non adhesion cells to adhesive fattened cells that adhere to the
foreign targets one another (Pech and Strand, 1995). Phenoloxidase activation in encapsulated
material results in melanization. Foster and Stehr (1994) have demonstrated in insect Manduca
sexta that specific induction of activity and localization of FAD glucose dehydrogenase (GLD)
(EC1.199.10), during invasion and encapsulation they hypothesized that GLD participates in
strengthening and in killing responses of melanin molecules. By the following mechanism during
encapsulation, quinones generated by phenoloxidase may serve as electron acceptor from
FADH2 generated by FADGLD enzyme, and get reduced by a single electron reduction to
semiquinones radicals that may further react with oxygen to produce superoxide anions radicals.
In the presence of low oxygen levels, semiquinone radicals could react either with other
molecules such as lipids, carbohydrates or proteins to result in a cross linked matrix or react with
other semiquinones to generate complex phenols and quinones (Bekman and Siedow, 1985).
2.4.1.2 Humoral immunity
It is intricate and not proper to distinguish cellular defense reactions from humoral (cell
independent) defense reactions because hemocytes are known to be very labile and release
substance by exocytosis into the plasma. Humoral factors include large number of lectins, lysine
and agglutinins. Nevertheless, there are active substance(s) originating from the hemolymph
which constitute humoral immunity. However, it is the combination of both the defense reactions
which gives good protection to the crustaceans. There are many substances present in the plasma
48
which assist in the immunity of the host in variety of ways. Equivalent factors may also reside in
plasma of invertebrates, although in many cases they may have been derived at some time from
the circulating blood cells.
2.4.1.2.1 Agglutinins
Agglutinins are molecules that induce the aggregation of small particles (bacteria,
erythrocytes etc.,) in the blood. Agglutinins assist in defense by impairing the growth of
infection agents, preventing their spread around the body and facilitating their ingestion by the
phagocytes. Agglutinins in several crustaceans have been characterized as heat-labile and
calcium-dependent substances. For most agglutinins, there must be no less than two binding sites
on the molecules, and these sites are often specific for particular sugars. Addition of excess of
the appropriate sugars to the reaction mixture inhibits the agglutinating action. Agglutinins
displaying such specificity are termed as lectins (Yeaton, 1981). Prawns are susceptible to many
natural bacterial infections. Vazquez et al. (1997) showed bacterial agglutinin in M. rosenbergii
to agglutinate Bacillus cereus and Aeromonas sp. Agglutination activity was also detected in the
heamolymph of M. rosenbergii (Cheng et al.,2005).
2.4.1.2.2 Lectins
Lectins are known to bring about agglutination of foreign cells, containing carbohydrate
in their membrane. Lectins for their abilities of recognizing the carbohydrate membrane surface
and represent very primitive immune responses. Lectins with diverse physiological roles are
widely distributed in various organisms; plants, vertebrates, invertebrates and microorganisms.
Lectins are ubiquitous and were first detected amongst arthropods as both humoral and
membrane associated components (Vasta, 1991). Lectins are defined as class of proteins or
glycoproteins often without catalytic activity that can bind carbohydrate and they can selectively
49
agglutinate vertebrate erythrocytes, malignant cells, and microorganisms (Barondes, 1981). In
crustaceans, lectins are known to have at least two roles firstly, they can bring about
agglutination of the foreign cells and secondly can help in adhesion of hemocytes to foreign cells
and thus function as opsonins. According to one hypothesis, these types of carbohydrate-binding
proteins, which recognize surface structures and common for different pathogens, represent a
primitive immune response. As for crustaceans, there are now a number of reports regarding the
purification and characterization of lectin molecules and most of them belong to the C-type
lectins, a calcium-dependent lectin family (Marques and Barracco, 2000). Lectins with no
requirement for calcium are present in some crustacean species such as Jasus verreauxi (Imai et
al., 1994), Penaeus indicus (Mashewari et al., 1977), P. japonicus (Kondo et al., 1998),
Litopenaeus schmitti (Cominetti et al.,2002), and P. paulinensis (Marques and Barracco, 2000).
The lectin of the freshwater prawn M. rosenbergii recognizes N- and O-acetylated groups in
carbohydrates from Aeromonas and Bacillus cereus (Vasta, 1992).
2.4.1.2.3 Prophenoloxidase activating system (propo system)
It has been recognized that defense reactions in many invertebrates are often
accompanied by melanization. In arthropods, melanin synthesis is involved in the process of
sclerotization and wound healing of the cuticle and also to defend (nodule formation and /or
encapsulations) against invading microorganisms entering the hemocoel (Soderhall, 1982;
Ratcliffe et al., 1985; Sugumaran, 2000). The enzyme involved in melanin formation is
phenoloxidase (PO; monophenol, L-dopa:oxygen oxidoreductase; EC 1.14.18.1 ) and has been
detected in the hemolymph blood or coelom of both protostomes and deutereostomes, as well as
in the cuticle of arthropods ( Soderhall and Cerenius, 1998) . PO is a bifunctional copper
containing enzyme, which catalyses both the ά-hydroxylation of monophenols and the oxidation
50
of phenols to quinones (Sugumaran, 2000). Thus, this enzyme is able to convert tyrosine to
DOPA, as well as DOPA to DOPA-quinone, followed by several intermediate steps that lead to
the synthesis of melanin, a brown pigment. PO is the terminal enzyme of the so-called
proposystem, a non-self recognition system present in arthropods and other invertebrates
(Soderhall, 1982; Ashida, 1990; Soderhall et al., 1986). Phylogenetically, propobelongs to one of
four well-supported subfamilies within: (i) the arylphorin subfamily (a storage protein in insects
without copper binding function), (ii) the hemocyanins of branchiopod crustaceans (copper
binding proteins involved in oxygen transport), (iii) the hemocyanins of chelicerates and (iv) the
prophenoloxidase of insects and crustaceans (copper binding proteins involved in immune
responses). However, hemocyanin is found in chelicerates and crustaceans but not in insects. The
active
form
of
proPO,
phenoloxidase
(PO;
monophenol,
dihydroxyphenylalanine:
oxidoreductase; EC 1.14.18.1), known as tyrosinase, catalyzes two successive reactions namely,
hydroxylation of a monophenol to ά-diphenol (monophenoloxidase activity) and the oxidation of
the ά-diphenol to ά -quinone (diphenoloxidase activity) (Söderhäll and Cerenius, 1998; Decker
and Tuczek, 2000). Production of ά -quinones by PO is an initial step in the biochemical cascade
of melanin biosynthesis. It is also important in cuticular sclerotization, wound healing, and
encapsulation of foreign materials (Lai-Fook, 1966; Sugumaran, 1991; Söderhäll and Cerenius,
1998). Arthropod POs exists as an inactive zymogen under normal physiological conditions and
gets activated by proteolytic cleavage except for a recently discovered propofrom the insect,
wasp Pimpla hypochondriaca, which was active without any proteolytic cleavage (Parkison et
al., 2001). The activation of this propocascade is exerted by extremely low quantities (pg/l) of
microbial cell wall components lipopolysaccharides LPS, β-1,3-glucans or peptidoglycans (PG)
and results in the production of the melanin pigment, which can often be seen as dark spots in the
51
cuticle of arthropods (Soderhall, 1982; Sugumaran and Kanost, 1993). During the formation of
melanin, toxic metabolites are formed which have fungistatic activity (Soderhall et al., 1990;
Nappi and Vass, 1993). Several components or associated factors of the proposystem have been
found to play several important roles in the defense reactions of the freshwater crayfish,
Pacifastacus leniusculus (Soderhall and Cerenius, 1998; Soderhall et al., 1996). Biochemical
studies on shrimp proposystem has been carried out in Penaeus californiensis (Vargas-Albores et
al., 1993a, 1996), P. paulensis (Perazzolo and Barracco, 1997), P. stylirostris (Le Moullac et al.,
1997) and P. monodon (Sritunyalucksana et al., 1999b ). In the penaeid shrimp, enzymes of the
proposystem are localized in the semigranular and granular cells (Vargas-Albores et al., 1993a;
Perazzolo and Barracco, 1997). After the first primary structure of propowas determined in
crayfish (Aspán et al., 1995), great numbers of proposequences were (13) reported from various
invertebrates. In crayfish, propois synthesized and localized in granules of the blood cells, and
released into plasma by an exocytosis triggered by the α -1,3-glucan binding protein. Recent
study shows that P. monodon propomRNA is expressed only in the hemocytes (Sritunyalucksana
et al., 1999a). The characteristics of propoin arthropods have been purified and cloned. Recently
cloned propoof P. monodon was (Sritunyalucksana et al., 1999a) analysed for its complete
sequence with the BLAST algorithm showed that the P. monodon propodeduced amino acid
sequence has highest similarity to crayfish propo74%. Significant similarity was also found in
other insect proPOs. The shrimp propohas a 3002 bp cDNA and contains an open reading frame
of 2121 bp encoding a putative polypeptide with 688 amino acids and with a molecular mass of
78.7 kDa. The active form with a molecular mass of 107 kDa was produced after hydrolysis with
a commercial proteinase preparation. The conversion of inactive propoto active PO is by a serine
protease named the prophenoloxidase activating enzyme ppA, which has been isolated and
52
purified from several arthropods; from the cuticle of Bombyx mori (Satoh et al., 1999 and
Manduca sexta (Jiang et al., 1998), from a crayfish hemocyte lysate (Aspan et al., 1990, from
Drosophila pupae) and from plasma of Holotrichia diomphalia (Lee et al., 1998a, b). Shrimp
propohas been shown to be activated by commercial trypsine proteinases in vitro (Perazzolo and
Barracco, 1997; Sung et al., 1998), but no endogeneous enzyme has been reported so far. The
common feature of arthropod ppA enzymes are of serine proteases and possess clip-like domains
(Jiang et al., 1998; Satoh et al., 1999). It was found in crayfish by (Aspan et al. 1990) that just
ppA is sufficient for the activation of crayfish proPO, but in two insects; Hyalophora cecropia
ppA and an additional unknown factor are required for the activation of the proposystem.
(Andersson et al., 1989 and Lee et al.,. 1998a, b) However, the mechanism by which this
proteinase converts propoto active enzyme is still unclear. PRPs are the triggering molecules of
the proposystem, since they bind microbial components and then they encourage activation of
proteinases in the proposystem. Finally propois prototypically transformed to phenoloxidase by
an endogenous trypsine like serine proteinase, the so called prophenoloxidase activating enzyme
(ppA). So far, four ppAs and one cofactor has been characterized and cloned from four different
animals; a beetle, Holotrichia diomphalia (Lee et al., 1998a; b), a tobacco hornworm, M. sexta
(Jiang et al., 1998), and a crayfish, P. leniusculus. Their primary structure demonstrates that they
all exist as zymogens of typical serine proteinases and are similar to Drosophila serine
proteinases involved in the organization of the developing embryo (Chasan and Anderson, 1989;
Jin and Anderson, 1990; Jiang and Kanost, 2000). With the activation of the proposystem, other
proteins will also gain their biological activity and can participate in cellular defense. One such
molecule is a cell adhesion protein, peroxinectin (Johansson and Söderhäll, 1988; 1989; 1993;
1995). The proposystem has to be controlled and regulated to avoid the deleterious effects of
53
active components of the system, and in particular PO, which can produce highly toxic
intermediates. Several proteinase inhibitors for preventing over-activation of ppA (Hergenhahn
et al., 1987; Aspán et al., 1990a) and a phenoloxidase inhibitor (POI), which can directly inhibit
the activity of phenoloxidase (Daquing et al., 1995; 1999; Sugumaran and Nellaiappan, 2000)
have been reported from several arthropod species.
2.5 Bacterial diseases in M. rosenbergii
A wide range of bacteria have been isolated from rearing water, eggs, larvae, postlarvae
and/or adults of M. rosenbergii. Predominately, gram-negative bacteria such as Aeromonas spp.,
Pseudomonas spp., Vibrio spp. and the gram positive Bacillus spp. and non spore formers (NSF)
are dominant in hatchery rearing system (Kennedy et al., 2006). These bacteria are common in
water and some species may take advantage of ecological changes occurring in hatcheries and
grow-out ponds. A number of genera are chitinoclastic, eroding surface of the exoskeleton and
resulting in shell necrosis (“shell disease”, “black spot” or “brown spot”), a condition
characterized by black or brown spots on the carapace (e.g., Pseudomonas, Vibrio, Benekea,
Leucothrix) (Sindermann, 1977; Sung et al., 2000). Such infections are typically of a secondary
nature, occurring after physical injury to the exoskeleton. The filamentous bacterium Leucothrix
has associated with “appendage necrosis” and epibiotic fouling diseases (Sindermann, 1977;
Brock, 1988). Members of the genera Aeromonas and Vibrio, in particular are often pathogenic
to crustaceans under hatchery conditions, such as Vibrio harveyi associated with luminescence
disease causing high losses in M. rosenbergii hatcheries (Tonguthai, 1995). Recently, the L.
gavieae has been reported to cause muscle necrosis and mass mortality in pond cultured M.
rosenbergii during the summer months (Cheng and Chen, 2002). In addition, L. lactis subspecies
lactis also causes white muscle disease in farmed M. rosenbergii (Wang et al., 2008). The
54
Aeromonas species belonging to Aeromonadaceae family were first isolated 100 years ago from
water and diseased animal (Martin et al., 2005). They are naturally occurring inhabitants of
aquatic environments, mainly freshwater, marine water and estuarine water. They cause diseases
of aquatic animals and opportunistic infections in humans (Graevenitz et al., 1968). Aeromonas
is a Gram negative, facultative anaerobic, oxidase and catalase positive, rod shaped bacteria,
which include Aeromonas, Oceaniomonas, Oceanisphaera, and Toiumonas (incertae sedis)
(Martin-carnahan and Joseph. 2005). A. hydrophila are motile that can grow at 35-37⁰C.
Although Aeromonas spp. are not generally considered to be a major threat to the commercial
cultivation of the giant freshwater prawn (M. rosenbergii), they have sometimes been linked to
outbreaks of disease in this species (New, 1995). For example, epizootic reports from Taiwan
indicated that in 1993 the most common bacterial disease of M. rosenbergii affected their shell,
and the bacteria isolated from infected tissues were identified as species in the genera Vibrio,
Pseudomonas, and Aeromonas. Similarly, in Taiwan during the past few years (1995 to 1997),
diseases that occurred mostly during the summer months (June to September) were attributed to
infections by bacterial pathogens that included Aeromonas. Bacterial diseases associated with
Aeromonas and other genera of chitinolytic bacteria (e.g., Pseudomonas, Vibrio, Beneckea, and
Leucothrix) have also been reported in Brazilian prawn hatcheries (Lombardi and Labao, 1991a,
b), where they led to “black-spot” bacterial necrosis and gill obstruction, while Brady and Lasso
(1992) reported a predominance of Aeromonas spp., Bacillus spp., and Pseudomonas spp. among
bacteria isolated from the hemolymph of prawn lesions. Most significantly, (Hung-Hung
Sung.,2000 ) noted , both Aeromonas and Pseudomonas have been isolated from the
hepatopancreas (HP) of healthy-looking prawns, and all of these Aeromonas and Pseudomonas
isolates were able to produce five extracellular products (ECP): protease, gelatinase, chitinase,
55
lipase, and hemolysin (Sung and Hong, 1997). Not only is it unusual to find any bacteria at all in
the hepatopancreatic lumena of a healthy decapods crustacean (Johnson, 1980, 1983), but also all
five of these ECPs are usually considered to be potential virulence factors for fish pathogens
(Allan and Stevenson, 1981; Kanai and Wakabayashi, 1984; Kodama et al., 1985; Sakai, 1985;
Thune et al., 1986; Santos et al., 1988; Ellis, 1991). Several reports have indicated that
pathogenic bacteria grown under specific environmental conditions can cause disease and death
in cultured animals (Arp, 1988; Riquelme et al., 1995; Prayitno and Latchford, 1995). Since
Aeromonas spp. are generally present in the hepatopancreas (1.2–1.6 × 106 cell-forming units/g
of HP) of apparently healthy prawns (Sung and Hong, 1997) and they do not cause disease at low
concentrations (103 cells per prawn), it seems that in giant freshwater prawns Aeromonas spp.
may function as opportunistic pathogens and only cause disease in prawns that are already
weakened. These possibilities must be studied further.
56
2.6 Immunostimulants
2.6.1. Use of immunostimulants as a strategy to protect cultured invertebrates against
diseases
There is a growing need to control, prevent or minimize the devastating effects of disease
in crustacean culture, without recourse to toxic chemicals or antibiotics. In keeping with
approaches to disease control in finfish and shellfish, interest is developing in compounds that
confer protection and/or enhance immune reactivity to likely pathogens in them. Because the
application of antibiotics or other chemicals to culture ponds is expensive and undesirable as it
risks contamination of both the environment and the final product (Brown JH, et al., 1991; Grant
A, et al., 1998), as well as causing mortality or impaired growth in juvenile stock (Stuck KC, et
al., 1992; Swastika IBM, et al. 1992). The repeated application of antibiotics, in the long term,
is also encouraging the spread of drug resistant pathogens (Brown JH. Et al., 1989; Juwana S. et
al., 1990; Aoki T. et al., 1990; Karunasagar I, et al., 1994 and Smith P, et al., 1994). Moreover,
chemical disinfection may be incompatible with geographical location of the shellfish farm or
with the physical requirements of the stock. As Bache`re (et al., 1995) have discussed, there is a
very great need to maximise the immunocompetence of the stock whilst minimising the use of
therapeutic chemicals.
2.6.2 Immunostimulants for crustacean (shellfish)
The selection of suitable compounds with potential immunostimulating properties
presents a bewildering task and research funds have rarely been generous enough for appropriate
screening programmes using ‘model’ species, as is the case with any potential novel drugs for
humans. It is therefore perfectly reasonable that clues might be obtained from information
available about non-specific ‘immune stimulants’ under test as ‘adjuvants’ for higher vertebrates,
57
or alternatively, to identify likely candidates from amongst the various reagents that have been
experimentally found to induce immune reactivity of invertebrate defences in vitro. Clearly the
over-riding criteria for suitable shellfish immunostimulants are cost, ease of administration,
efficacy and low toxicity for the host. Immunostimulants receiving most attention and claims for
success in promoting survival of crustaceans against experimental exposure to infectious microorganisms comprise five main types: (i) live bacteria; (ii) killed bacteria (bacterins or bacterial
antigen); (iii) glucans; (iv) peptidoglycans; and (v) lipopolysaccharides (LPS) (Table 1).(
Teunissen OSP, et al., 1998; Alabi AO, et al., 2000).
58
Table 1. Summary of the key groups of compounds reported to stimulate the crustacean
immune
syt
2.6.3 The mechanisms of defense system in crustacean with immunostimulants
Many aspects of the crustacean immune system are now comparatively well characterised
and detailed reviews have been published (Smith VJ, et al., 1992; So¨derha¨ll K, et al., 1998;
Holmblad T, et al., 1999 and Sritunyalucksana K, et al., 2000).
Much of the early work was
conducted on crab, crayfish and lobster but subsequent research on shrimps and prawn has
shown that the basic mechanisms also operate within the penaeid and sergestid shrimps
(Bache`re E, et al., 1995; Sritunyalucksana K, et al., 1999; Sritunyalucksana K, et al., 1999 and
59
Sritunyalucksana K, et al., 2001).
A generalized scheme for decapods is given in Fig. 3. As
indicated on this figure, the circulating haemocytes play extremely important roles not only by
direct sequestration and killing of infectious agents but also by synthesis and exocytosis of a
battery of bioactive molecules (Smith VJ, et al., 1992 and Smith VJ, et al., 2001). Essentially,
the haemocytes execute inflammatory-type reactions such as phagocytosis, haemocyte clumping,
production of reactive oxygen metabolites and the release of microbicidal proteins (Fig. 3).
There appears to be partitioning of these between the different cell types, although there may be
species differences in the way this occurs. For example, in crabs, phagocytosis and production of
reactive oxygen are mainly executed by the hyaline haemocytes, although in other species,
semigranular cells may also be phagocytic. p. japonicus is unusual in that the hyaline cells
appear not to be phagocytic; this function being provided by the granular haemocytes (Bache`re
E, et al., 1995 and Itami T, et al., 1998). By contrast, all three cell types display at least some
phagocytic activity in the freshwater prawn, M.rosenbergii (Sung HH, 2000).
60
Fig. 3. Flow diagram of the crustacean host defense system.
Certainly, full immune reactivity is always achieved through co-operation and interaction
between haemocyte types or their products. A key protein is peroxinectin and this is present in
granulocytes of both Dendrobranchiata (shrimps and prawns) and Pleocyemata (crayfish and
crabs) (Sritunyalucksana K, et al., 1999 and Sritunyalucksana K, et al., 2001).
Likewise, the
fundamental role of phenoloxidase seems to be similar in the immune system of P. monodon
(Bache`re E, et al., 1995; Sritunyalucksana K, et al., 1999) and other decapods. Further, the class
of antibacterial peptides known as penaeids has so far only been found in species of penaeid
shrimp and so would seem to be exclusive to this group (Destoumieux D, et al., 2000; Munoz M,
et al., 2002 and Destoumieux D, et al., 1997). It is clear therefore, that although the crustacean
immune mechanism is generally conserved across the phylum, specific differences between
distantly related species do occur. Under non-challenge (i.e. uninfected) conditions, most
immuno-reactive factors (e.g peroxinectin, antibacterial peptides, clotting components) are stored
within the haemocytes, usually in an inactive state. Their release and ‘activation’ occurs through
regulated exocytosis following stimulation by the presence of non-self molecules in the
haemolymph. The ‘clumping’ (i.e. encapsulating) behavior of the haemocytes is influenced by
some of the proteins exocytose from the cells. Importantly, this has a dual effect; it serves to
contain the spread of any infective particles that may have gained entry to the hemocoel and it
also localizes the response, so that induction of the biochemical pathways that underpin
immunity does not result in massive intravascular inflammation. The biochemical and molecular
signals involved in these events are gradually being elucidated and many of these are associated
with the prophenoloxidase (proPO) activating system (Fig.3). This is a cascade of enzymes and
other proteins located in the granular and semigranular haemocytes of decapods that is activated
61
by the signature carbohydrate constituents of microbial cell walls, through pattern recognition
binding molecules. Activation and results in the generation of a number of potent bioactive
products which assist phagocytosis, cell to cell adhesion and the formation of melanin deposits
(So¨derha¨ll K, et al., 1998 and So¨derha¨ll K, et al., 1986). Without doubt, the proposystem is a
dominant part of the crustacean defence system exerting effects on cell behavior, liberation
and/or activation of functionally important molecules and ‘neutralisation’ of infective agents. Its
multiplicity of effects, direct or indirect, not surprisingly has made it one of the central parts of
the crustacean immune system to be targeted for up-regulation by externally administered
‘stimulants’. This has probably been fuelled by the early observations of the biochemical
character and functional properties of the proposystem that led workers to consider it as an
ancestral form of a vertebrate defense pathway, possibly, an invertebrate ‘equivalent’ or
forerunner of the alternate pathway of vertebrate complement (So¨derha¨ll K. et al., 1982 and
Ashida M, et al., 1984).
2.7 Carotenoids
Carotenoids are a class of 800 natural fat-soluble pigments found principally in plants,
algae, fungi, animals, photosynthetic bacteria and some non-photosynthetic bacteria. Only plants,
bacteria, fungi and algae can synthesize carotenoids; animals cannot biosynthesize them thus,
they must be obtained from the diet (Schiedt, 1998). In the animal kingdom, carotenoids are the
most widely occurring pigments after melanin. They play a critical role in the photosynthetic
process and they carry out a protective function against damage by light and oxygen.
Carotenoids
also
play
other
important
functions
as
pro-vitamin
A,
antioxidants,
immunoregulators and Growth, and they are mobilized from muscle to ovaries which suggest a
function in reproduction (Shahidi et al., 1998; Nakano et al., 1999; Bell et al., 2000). It has also
62
observed that fishes with a high level of carotenoids are more resistant to bacterial and fungal
diseases (Shahidi et al., 1998). The majority of carotenoids are derived from a 40-carbon polyene
chain, which could be considered as the backbone of the molecule. This chain may be terminated
by cyclic end-groups (rings) and may be complemented with oxygen containing functional
groups.
2.7.1 Classification of carotenoids
Carotenoids are isoprenoid polyenes formed by joining of eight C5 isoprene units in a
regular head to tail manner except in the center of the molecule, where the order is tail to tail and
molecule is symmetrical (Gross 1991).
Isoprene
The carotenoids can be divided into two major classes depending on the degree of
substitution (Gross 1991). The first class is the highly unsaturated carotene hydrocarbons, which
contain no oxygen. The example being lycopene and β-carotene. The second class is the
oxygenated derivatives of carotenes called xanthophylls. Xanthophylls contain one or more
oxygenated group substituants on the terminal rings (Haard 1992). The examples being
astaxanthin, lutein, zeaxanthin . In addition to structural differences, carotenes and xanthophylls
also differ in their diversity and distribution (Latsch 1990). Generally carotenes have greater
distribution in plants than in animals, while xanthophylls are more widely distributed both in
plants and animals (Shahidi et al 1998). It is generally accepted that animals are unable to
synthesize carotenoids de novo, but are able to modify dietary plant carotenoids (Buchecker
1982). Thus the distribution of carotenoids in animal sources is primarily the result of specific
63
dietary habits, absorption and metabolic transformation (Torrison 2000). In animals the
astaxanthin is the most widely distributed xanthophylls, followed by lutein and zeaxanthin
(Haard 1992).
Katayama et al. (1973) proposed that aquatic animals can be divided into three classes
based on their biosynthetic capabilities:
Group I. Red carp type: Animals that can convert lutein, zeaxanthin or intermediates to
astaxanthin, but β-carotene is not the majo precursor of astaxanthin. They can store astaxanthin
in the diet directly to their body. Goldfish, red carp and fancy red carp belongs to this group.
Group II. Prawn type: Animals that can convert β-carotene and zeaxanthin to astaxanthin.
Generally crustaceans belong to this group.
Group III. Sea bream type: Animals that cannot convert β-carotene, lutein or zeaxanthin to
astaxanthin but can transfer pigments from diet to their body tissue pigment, as free form or
esterified. Sea bream and red sea bream are the examples of this group.
2.7.2 Structure of selected carotenoids
Below some structures of common carotenoids. These are showing the chemical diversity
and presence of the different types of carotenoids. http://www.food-info.net/uk/caro/stru.htm
Astaxanthin
present in : salmon, shrimp, lobster, flamingo feathers, algae
64
Canthaxanthin
present in : salmon, shrimp, cantharel and other mushrooms, algae, flamingo feathers
α-carotene
Present in carrots, most green plants
β-carotene
Present in carrots and most other plants
ε-carotene
65
Present in most green plants
γ-carotene
present in many plants, often with β-carotene
7, 8-didehydroastaxanthin
Present in: salmon and crustaceans
lutein
Present in many green plants including Marigold oleoresin
Lycopene
66
Present in many plants, especially in tomato
neurosporene
Present in many plants, intermediate compound between carotene and lycopene
phytoene
Present in many plants
rhodopin
Present in many red bacteria
spirilloxanthin
Present in many red bacteria
67
zeaxanthin
Present in many plants, especially in maize
2.7.3 Normal carotenoids sources used in aquculture
Carotenoids, both synthetic and naturally occurring products, are available or are being
developed for use in aquaculture. The most common are synthetically produced astaxanthin (
3 , 3'-dihydroxy-13 , 13-carotene- 4 , 4 '-dione ) , canthaxanthin ( 13 , 13-carotene-4 , 4 '-dione ) ,
and natural materials such as krill , Spirulina , crustacean-meals , marigold , Capsicum , and
other xanthophyll-containing
vegetable meals. Carotenoids derived from natural sources
contain mixture of several carotenoids like α-carotene, β-carotene, zeaxanthin, lutein,
cryptoxanthin, etc. whereas synthetic processes provide only specific carotenoids like β-carotene.
Contrary to this, synthetic processes involve petrochemical solvents, leading to residue problems.
Further, synthetic carotenoids are expensive and it has limitation to be used in aquaculture feed
formulation depending upon species. If used in excess synthetic carotenoids lead to deteriorating
effect on the environment. Natural carotenoids are categorized into two groups as plant and
animal based carotenoids.
2.7.3.1 Animal based natural carotenoids
The commercial natural astaxanthin production utilizes by products of crustacean such as
the Atlantic krill, crayfish meal, crab meal, etc., and some microorganisms. These are rich
sources of carotenoid astaxanthin and are used in aquaculture feed formulation as additive.
68
Among microorganisms, yeast Phaffiar hodozyma is probably the most important as it contains
astaxanthin as its main carotenoid (Andrewes and Star, 1976), constituting approximately 85% of
total pigments (Shahidi et al., 1998). Johnson et al. (1980) reported that Phaffia rhodozyma also
serves as a good source of proteins and lipids. The inclusion of this carotenoid source, aside from
its positive effect on fish pigmentation, enhances liver function and defensive potential against
oxidative stress (Nakano et al., 1995, 1999). Crustacean processing discards (shrimp, krill and
crabs) are also potential carotenoid sources. Crustaceans discards constitute and attractive
ingredient for industrialization, since around 70% of the raw weights of the catch are processing
discards (Wilkie, 1972; Simpson and Haard, 1985) with high carotenoid content and its use
reduces the environmental problem caused by the large amounts of wastes (Torrissen and
Naevdal, 1984; Shahidi and Synowiecki, 1991; Shahidi, 1995). Astaxanthin is the predominant
carotenoid (Sahidi et al., 1994; Higuera-Ciaparaet al., 2006) in crustacean by-products that also
include significant proportions of mineral salts (15-35%), proteins (25-50%) and chitin (25-35%)
(Lee and Peniston, 1982). Crustacean by-products have been successfully used for coloration of
integument and flesh in feeds of fish with high economic importance (Satio and Regier, 1971;
Spinelli et al., 1974; Torrissen and Naevdal, 1984; Coral et al., 1997).
2.7.3.2 Plant based carotenoids
Plants also have potential as carotenoid sources. Feed ingredients such as yellow corn,
corn gluten meal and alfalfa are also used as sources of carotenoids in aquaculture feed
formulation. Other carotenoids rich ingredients used are marigold (Tagetes erecta) meal and red
peppers (Capsicum sp.) extract. Experiments with red pepper have given good results, although a
lower efficacy was found in comparison to commercially available astaxanthin (Carter et al.,
1994; Yanar et al., 1997). Marigold, rich in lutein, might be an interesting dietary alternative
69
given its efficacy with egg and skin coloration of poultry. However, plant based carotenoids are
mainly derived from the micro algal pigment. For example if the culture conditions such as
nitrogen depletion, high light intensity and temperature are kept optimum, the algae,
Haematococcus pluvialis, Chlorella vulgaris, Dunaliella salina and Arthospira maxima will
accumulate secondary carotenoids and their biomass can be used as a coloring ingredient in
aquaculture.
The freshwater micro algae, H. pluvialis have been commercially exploited for
aquaculture primarily due to its rapid growth and high astaxanthin content (Sommer et al., 1991,
1992; Choubert and Heinrich, 1993). It is the primary source of pigmentation in ornamental or
tropical fish, responsible for various species related yellow, red and others colors. These are
obtained through carotenoids containing organisms in the aquatic food chain. The biflagellate
algae, D. salina is a source of β-carotene and used as natural food coloring agent in aquaculture
feed industry. Under appropriate culture conditions, some strains of D. salina were reported to
accumulate up to 10% carotenoids consisting mostly of β-carotene (Ben-Amotz et al., 1982;
Ben- Amotz and Avron, 1983; Borowitzka and Borowitzka, 1983). It is an inexpensive and best
source of natural mixed carotenoids. The discovery of commercial production of natural βcarotene from Dunaliella is currently a substantial and growing industry.
2.7.3.3 Carotenoids used in experiments
2.7.3.3.1 Marigold oleoresin (Tagetes erecta)
Carotenoids are responsible for the yellow, orange and red pigments in a large variety of
plants and animal kingdoms, including carrots, red tomatoes, paprika, annatto, saffron, palm oil,
corn kernels, Marigold petals and red salmon, trout, shrimps, crabs, lobsters (Gonza´lez de
Mejı´a, Loarca-Pina, & Ramos-Gomez, 1997; Hendry, 1996). Marigold is an ornamental plant
70
belonging to the Asteraceae (Compositae) family and a stout branching annual with large yellow
to orange flower heads which prefers a warm, low humidity climate and is as easily cultivated
ornamental and medical plant, originating from Latin America, widespread all over the world
with numerous species (Piccagliy, Marotti, & Grandi, 1998; Zorn, Breithaupt, Takenberg,
Schwack, & Berger, 2003). Its flower petals are an excellent and most important source of
carotenoids, the yellow carotenoids such as carotenes (and β-carotene) and xanthophylls (lutein,
zeaxanthin) as well as red carotenoids such as capsanthin, canthaxanthin and astaxanthin
(Handelman, 2001). Lutein is one of the major constituent of green vegetable, orange fruits and
egg yolk, where it exists in its free or nonesterified form. The most important source (more than
20-times higher amount) is flower petals of Marigold, where lutein is chemically bound to
various types of fatty acids such as lauric, mystric and palmitic acids. Upon saponification of the
Marigold extract, the lutein fatty acid esters are converted to free lutein (Khachik, 1995).
Commercially, lutein isolated from Marigold flowers (Tagetes erecta) was first used in chicken
feed to provide a yellow colour to the skin of broilers and yolks of layers. Its field of application
has been as an excellent antioxidant enlarged on nutritional, cosmetic and pharmaceutical
industry. It is reported in the literature that, the risk of chronic disease, such as heart disease,
cancer and age-related eye diseases might be significantly reduced by diets rich in lutein
(Khachik, 1995; Madhavi & Kagan, 2002; Rodriguez, Torres-Cardona, & Diaz, 2001).
2.7.3.3.2 Xanthophylls (oxygenated carotenoids): Lutein, Zeaxanthin and Meso-zeaxanthin
Xanthophylls (oxygenated carotenoids) are used as additives for poultry (e.g. chicken),
crustaceous (e.g. shrimp) and fish (e.g. salmon) feeds to provide bright colors in egg yolks, skin,
and fatty tissues due to its pigmenting properties (Bernhard, Broz, Hengartner, Kreienbuhl, &
Schiedt, 1997; Bletner, Mitchell, & Tugwell, 1966; Hencken, 1992; Levi, 2001). Among the
71
xanthophylls, lutein, and zeaxanthin are two of the most abundant oxygenated carotenoids found
in the diet (IOM, 2000). These two oxygenated carotenoids are present in high amounts in green
leafy vegetables (Khachik et al., 1995; Omaye et al., 1997; Calvo, 2005), and in chicken egg
yolk (Handelman et al., 1999; Sies and Stahl, 2003). Lutein and zeaxanthin are found in the free
form in spinach, kale, and broccoli and as esters (fatty acid esters) in mango, orange, papaya, red
paprika, algae, and yellow corn (van het Hof et al., 1999; Sajilata et al., 2008). In many foods,
particularly vegetables and fruits, lutein occurs with the isomeric xanthophyll zeaxanthin.
Chemically, lutein and zeaxanthin (Fig. 1) contain two cyclic end groups (α, β and and α-ionone
ring) and the basic C40 isoprenoid structure common to all carotenoids. The polyene chain double
bonds present in lutein could exist in a cis or trans configuration and thus can be in a large
number of possible mono-cis and poly-cis isomers. However, the majority of carotenoids are in
the all-trans configurations (Rice-Evans et al., 1997; IOM, 2000). Structurally, lutein and
zeaxanthin have identical chemical formulas and are isomers, but they are not stereoisomers. The
chemical formula of lutein and zeaxanthin is C40H56O2 and the molecular weight is 568.88. The
main difference between them is in the location of a double bond in one of the end rings (Fig. 1).
The minute structural differences are responsible for variations in the biological activities of
these compounds (carotenoids). Multiple conjugate double bonds exist in carotenoids, which
confer specific biological characteristics on the family of carotenoids. Furthermore, the
orientation of carotenoids depends on the molecular structure. While lutein is present as a single
stereoisomer, zeaxanthin occurs as a mixture of three isomers (Sajilata et al., 2008), two of
which are referred to as zeaxanthin and meso-zeaxanthin, respectively (Bone et al., 1993). Mesozeaxanthin is a unique member of the xanthophyll family of carotenoids and along with lutein
[(3R,30R,60R)-b,e-carotene-3,30-diol)] (L) and zeaxanthin [(3R,30R)-b,b-carotene-3.30diol] (Z)
72
are members of the xanthophylls class of carotenoids(Bone et al., 1985, 1993; Handelman et al.,
1991; Snodderly et al., 1991). The presence of meso-zeaxanthin was reported in shrimp
carapace, fish skin, and turtle fat, where all three isomers of zeaxanthin were found (Maoka et
al., 1986). Although meso-zeaxanthin is considered a rare isomer, it is present in significant
quantities in commercially produced chickens and eggs in Mexico where it is commonly added
to the feed to achieve desirable coloration in these products (Bone et al., 2007).
Fig.1. Chemical structure of lutein, zeaxanthin, and meso-zeaxanthin. Zeaxanthin, 3, 3́dihydroxy-β,β-carotene is synonymously referred to as 3R,3́R zeaxanthin, while mesozeaxanthin, 3,3́ -dihydroxy-β,β-carotene is referred as 3R,3́S mesozeaxanthin.
2.7.3.3.3 Diacetate of lutein-mesozeaxanthin
The Diacetate of lutein-mesozeaxanthin is a paste like compound. It contains the
acetylated forms of lutein and meso-zeaxanthin. Diacetate, a salt or ester containing two acetate
groups ( CH3 COO- + CH3 COO- ),whereas acetate , a salt or ester of acetic acid (CH3 COOH),
on the other hand, acetate are usually seen derivatives of acetic acid and acetate is also the ion
formed when acetic acid losses it acidic hydrogen:
H+ + CH3 COO73
CH3 COOH -----------
2.7.4 Carotenoids absorbtion and transport
Carotenoids are hydrophobic compounds that are not easily solubilized in the aqueous
environment of the gastrointestinal tract of fish; therefore, digestion, absorption and transport
processes are associated to lipids (Castenmiller and West, 1998). The intestinal absorption of
carotenoids involves several steps, including disruption of the matrix, dispersion in lipid
emulsions and solubilisation into mixed bile salt micelles, before being carried to the enterocyte
brush border were the absorption takes place (Furr and Clark, 1997; Tyssandier et al., 2001). In
salmonids, approximately 35% of dietary astaxanthin is absorbed (Torrissenet al., 1989;
Storebakken and No, 1992; Ytrestøylet al., 2005) mainly along the proximal intestine (Torrisen,
1986; Al-Khalifa and Simpson, 1988; Torrisen, 1989; Hardy et al., 1990; White et al., 2002),
taking approximately 18 to 30 hours (March et al., 1990; Choubert et al., 1994). In comparison
with other fish nutrients, absorption of carotenoids is considered slow. Furthermore, many
authors suggest that the intestinal absorption from micelles is a passive diffusion process
(Choubert et al., 1994; Parker, 1996; Castemiller and West, 1998; Van den Berg, 1999).
Carotenoids are absorbed without prior metabolic conversion, except for xanthophylls esters,
hydrolyzed before absorption, by a nonspecific bile salt dependent lipase, since no esters are
found in plasma or white muscle of salmonids (Schiedt, 1998; White et al., 2003). Salmonids
preferentially absorb more polar carotenoids, particularly astaxanthin rather than canthaxanthin,
zeaxanthin or carotenes (Schiedt et al., 1985; Guillou et al., 1992; Foss et al., 1984). The
unesterified or esterified carotenoids forms also seem to influence absorption. Many studies have
led to contradictory results, with authors claiming that the free form is better absorbed than the
ester form (Schiedt et al., 1985; Foss et al., 1987; Storebakken et al., 1987; Choubert and
74
Heinrich, 1993), while other report that both forms are equally absorbed (Barbosa et al., 1999;
Bowen et al., 2002). Japanese red sea bream seems to absorb synthetic astaxanthin dipalmitate
more efficiently than unesterified astaxanthin as reflected by skin pigmentation results (Ito et al.,
1986). Most of astaxanthin within the epidermal tissue is in the mono-esterified form, meaning
that one of the hydroxyl groups is esterified to a fatty acid; whereas, carotenoid and protein
complexes, called carotenoproteins and carotenolipoproteins, predominate in the exoskeleton
(Howell and Matthews, 1991). Carotenoids are synthesized through the isoprenoid pathway
which also produces such diverse compounds as essential fatty acids, steroids, sterols, vitamins
A, D, E, and K. Within the various classes of natural pigments, the carotenoids are the most
widespread and structurally diverse pigmenting agents. They are responsible, in combination
with proteins, for many of the brilliant yellow to red colors in plants and the wide range of blue,
green, purple, brown, and reddish colors of fish and crustaceans. The general distribution and
metabolic pathways of carotenoids has been extensively detailed previously (Matsuno and Hirao,
1989) (Fig.1).
75
Fig. 1. Metabolic pathway of carotenoids in shrimp (Latscha, 1991).
2.7.5 Metabolism and deposition of carotenoids
In fish, reductive and oxidative metabolic transformations play an important role
(Schiedt, 1998). Carotenoid metabolism is suggested to take place in the organs where their
metabolites are found (Storebakken and No 1992), such as the liver (Hardy et al., 1990;
Metusalach et al., 1996) or in the intestine (Aas et al., 1999). In salmonids, approximately 50%
of dietary astaxanthin absorbed may be metabolized (Torrissen et al., 1989; Storebakken and No,
1992; Ytrestøylet al., 2005). Early studies established a classification based on carotenoids
metabolic capacity of fish (Tanaka, 1978): A first type of fish cannot oxidize the ionone ring and,
therefore, the specific oxygenated derivatives have to be included in their diet. A second type of
fish, such as gold fish (Carassius auratus) and the fancy red carp (Cyprinus carpio) are able to
76
oxidize 4 and 4´ positions of the ionone ring, hence being able to convert zeaxanthin and lutein
to astaxanthin (Matsuno and Tsushima, 2001). Salmonid species are capable of reducing, but not
oxidizing dietary carotenoids to their own tissue-specific molecules. These reductive metabolic
reactions involve the stepwise removal of the keto group at 4 and 4´ positions of the ionone ring
(Matsuno and Tsushima, 2001). The skin of this group of fish presents predominantly
astaxanthin esters, when fed astaxanthin either free or esterified (Bjerkeng et al., 2000). In a
study carried out with Arctic charr, aside from mono- and di-ester astaxanthin, small amounts of
unesterified astaxanthin and yellow xanthophylls (iodaxanthin, tunaxanthin, lutein and
zeaxanthin) were found, all of them expected metabolites of astaxanthin (Bjerkeng et al., 2000).
When canthaxanthin was included in salmonid diets, β-carotene prevailed in the skin, followed
by echinone and finally canthaxanthin; in Arctic charr skin the presence of isocryptoxanthin was
also reported (Bjerkeng et al., 1990; Metusalach et al., 1996). All these carotenoids are reductive
metabolites of canthaxanthin, β-carotene being the end product. Japanese red sea bream fed diets
supplemented with β-carotene and canthaxanthin, showed a decrease in skin carotenoid level,
however when fed zeaxanthin or lutein a certain increase was shown, although not comparable to
levels achieved when fed an esterified astaxanthin source (Nakazoe et al., 1984). Japanese red
sea bream is capable of reducing but not oxidizing dietary carotenoids. The increase in skin
carotenoid concentration observed with zeaxanthin and lutein supplementation could be due to
the transfer of these carotenoids unchanged or perhaps due to a reductive metabolic process of
both lutein and zeaxanthin to tunaxanthin; in yellowtail this transformation has also been
suggested (Miki et al., 1985).
77
2.7.6 Function of carotenoids
Carotenoid pigmentation of fish is affected by their dietary pigment source, dosage
level, duration of feeding, and dietary composition ( Bjerkeng , 2000 ) . Although most
researches to date have focused on the effect of carotenoid deficiency on aquatic animal
coloration , there are growing evidences suggest that carotenoids have additional functions such
as : they are vitamin A precursors ( Lificm - Cabello et al. , 2002 ; Furuita et al. , 2003 ) ; they
can affect reproduction performance ( Liñά-Cabello et al. , 2003) ; they can influence growth
and survival ( Supamattaya et al. , 2005 ; Niu et al. , 2009 ) ; they can enhance immune
system
( Amar et al. , 2001; Linan-Cabello et al. , 2003 ; Supamattaya et al. ,2005) ; they
are potent antioxidants ( Meyers , 1994 ) , and they can enhance stress tolerance ( Chien et al.
, 2003 ; Supamattaya et al. ,2005; Niu et al. ,2009 ) .
2.7.6.1 Pigmentation Functions
Studies have shown that there are many sources of dietary carotenoids that can be used
for the coloration of cultured fish. Carrot and hibiscus (Shahreza, 1994), astaxanthin and
canthaxanthin (Torrissen, 1986; Ito et al., 1986; Storbakken et al., 1987; Choubert &
Storbakken, 1989; Choubert & Heinrich, 1993; Lim, 1999; Barbosa et al., 1999; Baker et al.,
2002) and microalgae (Harpaz et al., 1998; Law, 2000, Gouveia et al., 2003) can be used in fish
diets to enhance the colours of fishes. Synthetic astaxanthin has long been used in aquaculture to
enhance the flesh colour of cultured rainbow trout and salmon (Torrissen, 1984, 1986 and 1989;
Choubert & Storbakken, 1989; Choubert & Heinrich, 1993; Barbosa et al., 1999; Baker et al.,
2002).
78
2.7.6.2. Provitamin A
One of the most important physiological functions of carotenoids is their action as
vitamin A precursors in animals. Almost all animals are able to enzymatically convert
certain plant carotenoids into vitamin A (Gross, 1991). Carotenoids are structurally related to
retinol and β-carotene, the main source of vitamin A for animals. Lificm-Cabello et al.
(2002) suggested that dietary carotenoids were the sole biological precursors of retinoids in
crustaceans. The importance of carotenoids as bioactive molecules is primarily as precursors
to retinoids that are involved in the activation of hormonal nuclear receptors. The presence of
retinoic acid receptors in crustaceans and the finding of retinoids in their neuroendocrine
complex and in reproductive tissue, as well as the enhancement of the ovarian development
in shrimp suggest important roles for retinoids in shrimp physiology. Matsuno (et al., 1986)
reported that dihydroxy-carotenoids , such as astaxanthin , zeaxanthin , lutein , and
tunaxanthin were bioconverted into vitamin A alcohol in Nile tilapia ( Oreochromis niloticus
). Schiedt et al. ( 1985) found astaxanthin was converted to vitamin Al and A2 in rainbow
trout ( Oncorhynchus mykiss ) when fed a diet deficient in vitamin A. Vitamin A plays a
central role in many essential biological processes , including growth promotion ,
reproduction , and bone development as well as in vision. Vitamin A is known to be
involved in fetal development and in regulating the proliferation and differentiation of many
cells, whereas vitamin A deficiency changes the differentiation of epithelial cells (Zile, 1998).
Japanese flounder , Paralichthys olivaceus , fed a diet supplemented with vitamin A have
longer egg production period ( 2. 5 months ) when compared to the control group ( 1. 5
months) , and the percentage of normal larvae in the control group was significantly lower
than that in the vitamin A supplemented group ( Furuita et al. ,2003 ) .
79
2.7.6.3 Reproduction
In Penaeus shrimp, most of the body carotenoid is esterified astaxanthin, but in
the mature ovaries the astaxanthin is mostly unsterilized, suggesting that free astaxanthin
has an important function in egg production ( Dall et al. ,1995). Linan-Cabello et al. (
2003 ) found the total carotenoid content in the digestive gland and ovary increased
significantly from maturation stage II to IV ( P < 0. 05 ) in both wild and captive shrimp.
This increase in carotenoid concentration could represent the ability of carotenoids to bind
vitellin into a lipo-glyco-carotene-protein complex , by which the macromolecule
accumulates in the oocyte cytoplasm as a source of food for the embryo ( Chang ,
1993 ) . Linan-Cabello et al. ( 2003 ) also found a tendency for the enrichment of retinoids
in the ovary and digestive gland of captive shrimp ( L. vannamei ) during the stage II to
IV of maturation , and a significantly low concentration ( P <0. 05 ) of retinal in the
digestive gland of captive shrimp at stage II of gonadic maturation. The results suggest
that lower carotenoids and vitamin A in the ovary and
digestive gland could be a
biochemical indicator of insufficient diet or reproductive exhaustion in captive females.
Other authors reported a role for dietary retinoids or carotenoid derivatives in promoting
m a t u r a t i o n , rate of reproduction, fecundity, and embryogenesis of crustaceans (Morrissey,
1990). The presence of retinal in ovarian tissue and the increase in concentration in the
digestive gland and ovary of wild and captive L. vannamei at level IV gonadic maturation
suggest that dietary carotenoids serve not only as a source of pigmentation , but also as
precursors of retinoids , molecules whose functionality in the transcription of gene and
cellular differentiation is highly bioactive and specialized ( Durica et al. , 1999 ) . These
activities are of great importance during oocyte differentiation , embryonic development ,
80
and metamorphosis of crustaceans , which may enhance the reproductive capacity of shrimp
( Linan-Cabello et al. ,2003) . Many fish owe their bright yellow, orange, and red color
patterns to the presence of carotenoids concentrated in the integumentary chromatophores
(Good win, 1984). During sexual maturation in salmonids, carotenoids are mobilized from the
muscle and transported to the integument and ovaries, suggesting that carotenoids have a
function in reproduction ( Kitahara, 1983). Craik (1985) concluded that 1 to 3 mg of
carotenoids per gram of salmonid eggs was associated with a hatching percentage greater
than 60%, whereas lower carotenoid levels resulted in reduced percentage of hatching t o
less than 50%. Egg
quality
has
also been substantially improved by supplementing
carotenoids , specifically astaxanthin , in the diets of red sea bream ( Pagrus major ) and
yellowtail ( Seriola quinqueradiata ) ( Verakunpiriya et al. , 1997 ) . Krill meal has already
been proven to be an effective ingredient in fish feeds and has been used as a pigment
enhancer in yellowtail ( Seriola quinqueradiata ) and red sea bream ( Pagrus major ) (
Shimizu et al. ,1990 ) .
2.7.6.4 Stress tolerance
Enhancements of resistance in penaeid shrimp postlarval to oxygen depletion (DO )
stress (Chien et al. , 1999 ; Niu et al. ,2009 ) , salinity stress (Chien et al. , 2003) , thermal
stress (Chien et al. , 2003 ) , and ammonia stress (Pan et al. ,2003) were found to be
associated with an increase in dietary astaxanthin. In the study of Niu et al. (2009), the shrimp
fed the diets containing 400 and 200 mg astaxanthin per kg diet showed no significant
difference in survival during 9 days stress test. Shrimp fed the diet without astaxanthin
supplementation exhibited poor resistance to low DO stress compared with the shrimp on
the other diets. Similar r e s u l t s w e r e r e p o r t e d
81
by C h i n e (e t a l . , 1999) on juvenile
P. monodon fed a diet without astaxanthin supplementation compared with those fed 360
mg/kg astaxanthin. Craik (1985) postulated that oxygen-containing carotenoids such as
astaxanthin, in which oxygen was attached at the center of the hydrocarbon chain, might act
as an intracellular oxygen reserve for respiration in salmonid eggs subjected to oxygen stress.
Astaxanthin might serve as an intracellular oxygen supply for shrimp, allowing survival under
the hypoxic conditions in the pond bottom (Chien and Jeng, 1992). Darachai et al. (1998)
concluded that astaxanthin helped to prolong the life of the postlarval shrimps subsequent to
acute environment stress. High astaxanthin content in shrimp , about 90% of its total pigments
( Ishikawa et al. ,1966 ) , might have contributed to their high tolerance to low DO and lower
demand for oxygen , and presence of astaxanthin could become critical when the animal was
under physiological stress caused by abiotic changes ( Chien et al. , 2003)
2.7.6.5 Diet supplementation
Carotenoids supply to the diet of shrimps has been studied in P. japonicus, P. monodon,
and L. vannamei. The ingredients of vegetable origin that have been used in the diet are red yeast
(Phaffiarhodozyma); microalgae, D.a salina (Chien and Jeng, 1992), Chnoospora minima
(Menasveta et al., 1993), Spirulina sp. And S. pacifica (Liao et al., 1993; Chien and Shiau,
1998), H. pluvialis (Chien and Shiau, 1998), and Isochrysis galbana (Pan et al., 2001); wheat,
corn and alfalfa (Meyers and Latscha, 1997); marigold Tagetes erecta (Vernon-Carter et al.,
1996; Arredondo-Figueroa et al., 1999); paprika Capsicum annuum (Vernon-Carter et al., 1996;
Arredondo-Figueroa et al., 2004). A marked increase of carotenoid content in the carapace has
been observed when organisms were fed with plant pigment source diets. Arredondo-Figueroa et
al. (1999) studied the effects of various dietary unesterified Aztec marigold carotenoid
concentrations on the pigmentation of the Pacific white shrimp. Four pigmented diets containing
82
the unesterified marigold extracts at concentrations of 50, 100, 200, and 350 ppm, a reference
diet containing 200 ppm Carophyll pink, and a non-pigmented control diet were fed to the
shrimp during 35 days. Abdominal muscle and exoskeleton pigmentation was influenced by the
diet pigment concentration and by the duration of the feeding test. The degree of abdomen
pigmentation achieved by the 200 ppm Carophyll diet was equaled by the 200 ppm unesterified
marigold diet, whereas the exoskeleton pigmentation was superior with the Carophyll-containing
diet than with any diet containing up to 350 ppm unesterified marigold pigment.
2.7.6.6 Growth
The effects of carotenoids on growth and survival rate of aquatic organisms have been
controversial, because several studies reporting a positive influence whereas others did not find
any effect. Eduardo Aguirre-Hinojosa (et al., 2012) have been indicated that juvenile of L.
vannamei fed diets supplemented with xanthophylls (75% Zeaxanthin, 15% lutein) industrially
extracted from marigold (Tagetes erecta) improved in survival shrimps fed treatment diets
compared to those fed the control diet but there were no significant differences in growth
between experimental groups fed different diets. Similar to observation in P. japonicus and
P. monodon (Boonyaratpalin et al., 2001; pan et al., 2001). However, carotenoid supplemented
diets had positive effects on growth in P. japonicus and P. monodon (Yamada et al., 1990; pan
and chen, 2004). Zhang, J. (et al., 2013) have been reported that after 56 days of culture, shrimp
(L. vannamei) fed astaxanthin (125 and 150 mg kg-1) diets had higher (P< 0.05) weight gain
(WG), final wet body weight (FBW), SGR and had significantly lower FCR than of shrimp Fed
control feed. Vijay Kumar (et. al., 2009) has been suggested that the M. rosenbergii were fed
with dietary incorporation of astaxanthin (50,100 and 200 mg kg-1) had significant increased on
mean weight gain and SGR. The growth rate improved and moulting cycle shortened in
83
M.rosenbergii post larvae during 20 days rearing when fed with supplemented of astaxanthin
(petit el al., 1997). White shrimp (L. Vannamei) were fed with three different treatments
(synthetic astaxanthin, lutein and astananthin derives from marigold extract), results have shown
higher growth, lower FCR and higher survival on marigold treatments only as compared to other
treatments
and
control
group
(Rohriguez
et
al.,
2008)
and
/
or
www.google.co.in.Patents/U57383788). Maricela Flores (et al., 2007) have been studied the
effect of dietary astaxanthin supplemented at 0, 40, 80 or 150 mg astaxanthin kg
–1
on growth
and survival, they have found Daily Growth Coefficient (D GC%) was significantly higher in
shrimp (L. vannamei) fed with the 80 mg astaxanthin kg -1 diet than in shrimp fed with the other
diets (p <0.05). However, there was no significant in survival rate within all groups. Three
different concentrations (0.5, 1.0 and 2.0%) of D.Salina incorporated in the diet and fed to
shrimp, which showed that the growth of P. monodon was similar among the three concentration
but when compared to the control, 1.0% of diet showed better growth (M. madhumathi, 2011).
Similarly, Boonyaratpalin (et al., 2001) reported a higher survival rate and growth in P.
Japanicus Fed with astaxathin-supplemented diets than supplemented of β-caroten or algal meal.
Diaz, A.C. (et al., 2011) have been suggested that red shrimp (pleoticus muelleri) were fed two
different carotenoids (astaxanthin and β-caroten) there was no evidence supporting a possible
influence of these pigments on growth and survival by spectroscopy UV/ visible method.
Mustafa Gocer (et al., 2006) have been evaluated the effect of red pepper (6.6%), marigold
flower (2.4%) and 100 mg/kg synthetic astaxanthin on P. semisulcatus, which results showed
that dietary carotenoid sources did not significantly affect the growth of the shrimps (P>0.05) but
there were significantly difference among the treatment and control group in trems of survival
rate. Supamattaya, K. (et al., 2005) have been investigated the effect of D. Salina extract
84
(containing β- caroten with different levels of 125, 200 and 300 mg kg -1) on growth of shrimp
(p. monodon) with small size (1-2 g body weight) for 8 weeks, results showed weight gain and
survival of shrimp fed diet supplemented with 125 and 300 mg β- caronten / kg diet was
significantly higher than the shrimp fed control diet. However, it was not significantly different
in FCR among each treatment. Harpaz, S. ; Schm albac, E. A. (1986) stated that dietary plant
matter, which can also serve as a pigment source, had a beneficial effect on crustacean moulting
and growth. Chien and cheng have been reported a higher survival rate of shrimps (P. japonicus)
Fed astaxanthin-supplemented diets compared to shrimps fed supplemented of beta caroten or
algal meal. Dall (1995) has been noted instead of retinoids (vitamin A), astaxanthin was
suggested to be essential growth factor in the early development of bear shrimp (P.
semisulcatus), Thongrod (et al., 1995) have also been demonstrated that a significant positive
correlation was found between supplementation of dietary astaxanthin and growth of tiger
shrimp post-larvae. Gupta (et al., 2012) have investigated the effect of H. pluvialis (at the levels
of 1, 2, 3 and 4%) on Macrobrachium dayanum, which results revealed that prawns fed with 4%
achieved best growth performance in (WG, SGR, FCR) while feed with 1.0% H. Pluvialis
showed lower growth efficiency. Chiu (et. al., 2014) have been evaluated the effect of lycogen
TM
(a natural occurring source of carotenoids from photobacterium Rhodobacter sphaeroides
WL-APD 911) on
red tilapia (Oreochromis mossambicus ), and observed that dietary
supplementation with 1.0% of lycogen did not cause changes in body length but significantly
increased muscle weight, SGR, FCR and weight gain. The effect of marigold (Tagetes erecta) as
natural carotenoid source on growth and survival of gold fish with average initial weight of 1.8 g
for a rearing period of 63 days were evaluated by Alma, A. (et. al., 2013), that results indicated
no significant difference was observed in the survival rate, growth or feed utilization of the
85
fishes. Amar Edgar, C. (et al., 2012) have demonstrates the effect of synthetic (β-carotene,
astaxanthin and canthaxanthin) and natural carotenoids (Tagetes erecta, D. salina,
phaffiarhodozyma and c. annuum) on rainbow trout fry weighting 0.11 kg for 6 weeks, results
indicated no significant differences in growth performance (SGR, WG, Mean final weight, Total
feed consumed, Feeding gain ratio) among the groups. These results further validated their
previous finding both with natural and synthetic sources (Amar, E.C. et al., 2001, 2004).
Similarly, Rehulka (2000) and yanar (et al., 2007) found no effect of synthetic and ntural
carotenoids on growth in salmonids. Ezhil, J. (et al., 2008) have been reported that swordtails,
Xiphophorus helleri, fed marigold petal meal (at arte of 0, 3, 4, 6, 8, 15 g / 100g basal diet) for
28 days, could not significant increase on growth performance compared to the control group.
50. Hakan Murat (et al., 2007) have been investigated the effect of various natural carotenoid
sources (1.6%, 2.4% and 3.2% of marigold Flower and 4.4%, 6.6% and 8.8% of red pepper and
synthetic astaxanthin on rainbow trout, results exhibited that there were no significant
differences in weight gains of the fish on days 20 (P<0.05) but growth differed significantly
among the groups on days 40 and 60 (P<0.05). However, an addition level of 2.4% or higher
marigold flower and 6.6% or higher red pepper into the diet had negative effects on growth
performance (P<0.05). Several studies have shown (Olvera-Novoa, M. A, 1990, BoonyarAtpalin,
M. 1989; Ergun, S. 2000) that the use of high levels of plant material in diets of fish especially
carnivorous fish retards their growth. The main reason for this has been considered due to
contain high level cellulose the plant. Yasemen yanar (et al., 2007) have been assessed the effect
of carotenoid sources (1.8% marigold flower, 5% red pepper and 70 mg kg-1 commercial
astaxanthin
on rainbow trout with average initial weight 120.57 g for 60 days, results
demonstrated that growth was not affected by carotenoid sources among fish groups. Olsen, R.E.
86
(et al., 2006) have been studied that the Altantic Salmon were fed with two xanthophylls
carotenoid (astaxanthin and lutein), has no signiciant effect or growth performance such as FCR,
SGR, WG and condition factor. Rodriguez (et. al., 2008) and / or www.google.co.in.patents/US
7383788 reported that rainbow trout fed with astaxanthin derived from marigolds a weight gain
of more than 46% was obtained compared to the control and FCR was also better in the
treatment groups with an overall average of 1.61 than to 1.75 in the control group. Rainbow trout
were fed two different sources of carotenaids (astaxanthin derived from marigold extract and
synthetic astaxanthin), results showed that fishes had much more dietary supplementation of
astaxanthin from natural source which consequently enhanced growth as compared to synthetic
astaxanthin groups (Rohriguez et al., 2008). Niu et al., (2011); Amar et al., (2001) and segner et
al., (1981) have been reported that the carotenoids could enhance nutrient utilization and might
ultimately improve growth, play an important role in the intermediary metabolism of aquatic
animals.
2.7.6.7 Survival
Several studies on carotenoids (especially astaxanthin as a one of the most important
crotenoids) on aquatic animals including crustaceans have been shown different survival effect,
which variations survival effect may be related to stress level, species, life stage, nutritional
history, sources of carotenoids, rearing period condition and many other factors (Chin- Hung Pan
et al., 2004). There was no significant difference between treatments on survival rate of M.
rosenbergii which were fed at different levels, of dietary astaxanthin (Vijay Kumar et al., 2009).
However, several studies with penaeids have reported increased survival in astaxanthin fed
groups (yamada et al., 1990; chien and jeng 1992 Negere-sadrgues et al., 1993 and pen et al.,
2001). There was no significant difference between shrimp (P. monodon) fed with different
87
carotenoids (β-caroten or astaxanthin) in survival rates ( Boonyarataplin, M. et al., 2001). Flores,
M. (et al., 2007) have been reported that survival rate in juvenile shrimp (L.vannamei) were fed
with 80 mg kg-1 astaxnthin improved compared to other diet 0, 40 or 150mg kg-1) when shrimps
acclimated to low-salinity water. Kuo-Hsun Chin (et al., 2014) have been demonstrated that
there was no significant difference between treatment and control groups in terms of survival rate
sea water red tilapia (o. mossambicus) were fed lycogenTM (a commercial carotenoid product
from the probiotic photobacterium Rodobacter spaeeroides WL-ADD 911). Menasveta (1995)
has shown that the shrimp (P. monodon) were fed 100 to 200 mg kg-1 dietary astaxanthin had
improved resistance to bacterial and viral infection. Survival rate of shrimp (L. vannamei) fed
with 125 and 150 mg kg-1 of dietary supplementation of astaxanthin after feeding trial for 65
days, had no significant difference (P>0.05) among experimental groups (Zhang, j. et al., 2013).
Chin-Hung Pan (et al., 2004) have stated that shrimp (P. monodon) fed with supplemented
dietary of astaxanthin at 80 mg kg-1 resulted in higher survival rate than the control group. White
shrimp (L. vannamei) post larvae (PL 17) were fed during 11 days with H1-Zeaxanthin (or
Zeaxanthin short chain like diacetate) had a noticeable improvement in their survival rate,
(Hinojosa et al., 2008; www.google.com/patents US20080107768). Pre-juvenile (0.115 g) white
shrimp (L. vannamei) fed dietary supplementation of Hi-Zeaxanthin or Zeaxanthin short chain
like diactate during 7 weeks, the average weight and survival rate was significantly higher than
control group. (Hinojosa et al., 2008; www.google.com/patents /US20080107768). Survival rate
of juvenile (2.5 g) white shrimp (L.vannamei) were fed supplemented diet of Hi-Zeaxanthin (or
Zeaxanthin short chanin like diacetate) within 30 days was significantly higher as compared to
the control group (Hijonosa et al., 2008; www. google.com/patents/us20080107768). Yadama
(et al., 1990) have been observed 91% survival rate when M. japonicus fed a diet supplemented
88
with 100 mg kg-1 synthetic astaxanthin for 4-8 weeks in comparison to a 57 % survival rate
among the controls. P. japonicus were fed by various of carotenoid sources (astaxanthin, βcaroten and D. salina) at a rate of (50, 100 and 200 mg kg-1, finding indicated that shrimps fed
the astaxanthin diet had a higher rate of survival rates than those of fed the β-carotine or algal
meal diets. A positive correlation between survival rates and pigmentation concentration in
shrimp tissue showed that pigment may play a role in improving the survival of shrimps (YewHU Chien, Shu-Ching Jeng, 1992).
2.7.6.8 Proximate composition
Eduardo Aguirre-Hinojosa et al. (2012) have noted that supplementation of experimental
diets with (Tagetes erecta) extract (Marigold oleoresin) on L. vannamei did not significantly alter
the proximate composition of the practical diet because very small amounts were required to
attain the desired concentrations in the feeds (less than 0.5% of the total ingredients).The same
results were reported by Arredondo-Figueroa (et al., 2003) and flores (et al., 2007). This is
important in determining the commercial application of Tagetes erecta supplements. There were
no significant differences in proximate composition (crude protein, crude fat, moisture and ash)
of rainbow trout with initial weight of 135 g after 60 days of feeding with supplementation of
astaxanthin diets (Juan et al., 2013). Dietary carotenoid sources (1.8% marigold flowers, 5% red
pepper and 70 mg kg-1 astaxnthin) on rainbow trout weighing 120.57 g for 60 days did not
significantly affect fatty acid composition of the fish fillets (Yasemen et al., 2007). proximate
composition of P. semisulcatus were fed by different dietary carotenoid sources (red pepper-RP,
Manigold flowers-MF and synthetic astaxanthin-SA at the levels of 6.6%, 2.4% and 100 mg kg-1
respectively), showed that shrimps fed RP and SA had a greater protein increase in shrimp
muscle than MF (p<0.05), however lipid, ash and water content (moistures) were similar in all
89
diet groups (p>0.05), (Mustafa Gocer et al, 2006). Bagre, P. et al., (2011) had conducted studies
to analyze and compare the proximate composition of formulated feeds impregnated with natural
carotenoids Tagetes erecta (Marigold oleoresin), Hibiscus rosaseiensis, Rosa centifolia in 4, 6, 8
and 10% with basic feed formulated by Pearson square method. Among the different diets 6 and
10% of Tagetes erecta showed highest crude protein content 38.41% followed by 38.34% in 10
% of H. rosaseiensi and 37.35% of R. centifolia feed additive. The crude lipid was the highest
17.5% in basic feed, 17.11% in 4% Tagetes feed,17.01% in 4% H. rosaseiensi and 11.10% in 4%
Rosa feed. There was the highest NFE 31.59% in 10% H. rosaseiensi feed followed by 31.26%
in Tagetes and 31.39% in 4% Rosa feed. The total ash content was 12.4% in 10% of Tagetes
feed, the content of crude fiber was 8.46% in 10% H. rosaseiensi and the highest moisture
content was 6.77% in Rosa feed. They have resulted that on the basis of nutrient content 6 and
8% of Tagetes erecta (Marigold oleoresin) additives feed are quite good for the growth of the
fishes. 6. Proximate composition of marine ornamental fish sources such as carrot (Ducus
carota), Marigold petal (Tagetes erecta), China rose petal, China rose (H. rosasiensis) and Rose
petal (R. chinensis) at a rate of 15g / 100g feed, showed that there were no significant differences
among all treatment groups. However, the amount of protein carbohydrates and moisture in
Marigold group were greater than R. chinensis but the lipid of Marigold petal was less than R.
chinensis by contrast protein, carbohydrate and moisture in Marigold lesser than carrot and
Hibiscus, while its lipid level higher than them (Ramamoorthy, K. et al., 2010). Kuo-Hsun chinu
et a1, (2014) reported that in general, dietary carotenoids have been associated with skin
coloration or pigmentation, and few studies have addressed the effects of dietary carotenoids on
growth via compositional changes and they suggests that significant changes in muscle
proximate composition of seawater red tilapia (o. mossambicus × o. niloticus) after lycogenTM in
90
(a natural occurring source of carotenoid product extracted from the photobacterium
Rhodobacter sphaeroides wL- ADP 911) supplementation. Therefore, the LygogenTM in
enhanced growth may have resulted from another physiological mechanism, e.g., the
improvement of the immune response.
2.7.6.9 Immune system
2.7.6.9.1 Prophenoloxidase activity
Carotenoids are biologically active pigments of obligate dietary origin for animals
(Moller et al., 2002); Hill et al., 2002). Apart from their positive effect on body condition e,g.
Higuera-ciapara et al., (2006), these pigments stimulate both innate and adaptive components of
vertebra rate immunity, improving the efficiency of immune responses (Blount et al., 2003; Mc
Graw and Ardia 2003, 2007). Caretenoids are also potent antioxidants (Bendich and Olson 1989;
Moller et al., 2000), which help endogenous enzymes (e.g., catalase, superoxide dismutase)
detoxify radicals produced by immune activity (Ewen et al. 2006; Constantini 2008). Different
studies have reported that dietary carotenoids were immediately beneficial, either through their
antitoxin properties (Chien et al., 2003; chien and shiau 2005; Schleder et al., 2008) or through
their immune stimulant power (Flores et al., 2007) reducing stress-induced immunodepression
(Khansari et al., 1990) or improving the gammarids general body condition (Flores et al., 2007).
Aureli Babin et al. (2010) have reported that a positive correlation between immune defense and
concentration of carotenoids in the hemolymph was demonstrated in the crustacean and they
found that dietary carotenoids had clear and broad immune stimulating effect, enhancing
phenoloxidase activity and resistance to bacterial infection. Invertebrate immune response relies
on non-specific cellular and humoral effectors acting in tight synergy (Siva-Jothy et al., 2005).
One important humoral responses is the prophenoloxidase (proPO) enzymatic cascade based on
91
the activity of the phenoloxidase (po) enzyme (Chase et al., 2000). After a step wise proteolytic
activation, the po enzyme is responsible for the production of melanin, along with the release of
quinine-derived metabolites and reactive oxygen species. These chemicals have a cytotoxic
oxidative effect on pathogens, as wells on the basic host cell components (Nappi and Vass, 1993,
1998; Nappi and Ottaviani 2000). Hence, the activation of the Po cascade proves to be beneficial
and detrimental for the host at the same time. As immunity is essential, mechanisms minimizing
the cost of autoreactivity have to be evolved. PPo activity has been employed as one of the
important immune parameters to investigate the status of immune system, immune modulation
and disease resistance in crustacean (Smith et al., 2003) and also (Cheng et al., 2004; Baruah and
Pani Prasad 2001) have suggested an enhancement in PPo activity which is believed thought to
enhance the immune ability of animals. Phenoloxidase (Po), the key enzyme in the synthesis of
melanine, occurs in heamolymph as an inactive proenyme prophenoloxidase (propo). Propois
activated to form PO (phenol oxidase) when it reacts with zymosan (carbohydrates from yeast
cell walls,) bacterial lipopolysacchride (LPS), urea, calcium ions, trypsin, or heat (soderhall et
al., 1986). Al bores (et al., 1993) stated that phenoloxidase (po) is the terminal enzyme in the
proposystem of the orthropod defence system and act as both recognition and effector
component, by promoting cell-to-cell communication and subsequently eliminating pathogens.
The active material formed during the activation of propostimulate several cellular defense
reaction, including phagocytosis, nodule formation, hemocyte locomotion, non- self recognition
and other immune reactions (Ourth et al., 1993). In the study done by Harikrishnan et al., (2012)
on prawn (M. rosenbergii) with 0.1% and 1.0% doses of Withania somnifera have suggested, PO
seems to act as promoter in M. rosebergii by enhancing the pigmentation, increasing o2(respiratory
burst) production and SOD activity, and then protecting from A. hydrophila
92
infection. Chang et al. (2013) reported that the PO activities of prawns (M. rosenbergii) were
significantly enhanced by ECE- supplemented diets at doses of 2.0 and 3.0 g Kg-1 fed for 3-12
days of feeding. However, no significant differences in PO activity per granulocyte was observed
in prawn fed ECE-containing diets for 12 days. (Water hyacinth) Eichhornia crassipes extracts
promote PO activity due to increase po activity at SGCs + GCs and after 12 days, the po activity
still remains because of the increased GC count. Harikrishnan (et al., 2012) Found that the innate
immune parameters such as phenoloxidase activity, superoxide anion level (respiratory burst),
total hemocyte count and superoxide dismutase as one of the indicators of antioxidant activity
were significantly enhanced in prawn (M. rosenbergii) fed with 0.1% and 1.0% doses of W.
somnifera supplemented diet against Aeromonas hydrophila as compared to the control. Vijay
Kumar (et al., 2009) have indicated phenoloxidase (PO) activity was which significantly higher
(P <0.05) in prawns (M. rosenbergii) were fed supplemented diets compare to the control group.
Boonyarataphin et al., (2001) have showed that were not found significantly different among the
shrimp (P. monodon) fed with different carotenoids (D. salina as source of β-caroten and
astaxanthin) on the immune response in terms of phenoloxidase activity, number of haemocytes
in the circulating system and resistance to infectious disease with challenged V. harveyi and
WSSV.
2.7.6.9.2 Superoxide anion production (Respiratory burst)
Reaction oxygen species (ROs) like superoxide anion (o2-), hydrogen peroxide (H2O2),
and hydroxyl radicals (OH-) are produced during phagocytosis. This phenomenon as a
respiratory burst plays an important role in microbial activity (Song and Hsieh, 1994). The
generation of oxygen has been reported in hemocyte of tiger shrimp, (P. monodon) (Song and
Hsieh, 1994), L. stylirostris (Bacher et al., 1995) and white shrimp, L. vannanei (Munoz et al.,
93
2000). The production of oxygen has been reported as an accurate method to measure the
effectiveness of potential immune stimulants (Song, YL. et al., 1994 and Munoz, M. et al.,
2000). These facts suggest that increase in the superoxide anion is considered to be beneficially
protecting disease with respect to increased immunity. An increase in the superoxide anion
production against pathogens is to be beneficial after exposing shrimp to immunestimulants. 10.
Nitroblue tertrazolium (NBT) staining has been used for the qualitative and quantitative analyses
of oxygen generated by hemocyte which is the first product of respiratory burst (Holmblad and
Soderhall, 1999).
Lee et al., (1998) indicated that the production of superoxide radicals
decreased in hypoxic L. Stylirostris due to a decrease in the THC, suggesting that NADPH
oxidase is responsible for the production of superoxide and was not affected by hypoxic
conditions. Harikrishnan (et al., 2012) indicated that the o2- levels and SOD activity against A.
hydrophila significantly increased in M. rosenbergii fed 0.1% and 1.0% doses of withania
somnifera supplemented diets 1-4 weeks. Chin-Chyuan Chang (et al., 2013) have suggested that
the RBs (Respiratory burst or superoxide anion production) and SOD, GPx activity significantly
increased in prawn fed ECE-containing diets for 12 days, indicating that the ECE (Eichhornia
crassipes extracts) may promote regulation of the ROL system in prawns. Dietary ECE
administration promoted o2- production of single haemocytes in the initial 9 days and then was
down regulated ad 12 days, but increased o2- (superoxide anion) production due to THC is the
duration of the experiment. Citarasu et al., (2006) have suggested that herbal supplementation
diets seem to act as a promoter for increasing
propoactivity
and o2- production in black tiger
shrimp, P. monodon against WSSV infection. Amar et al., (2004) reported that the production of
o2- by head kidney leukocytes of rainbow trout in the supplemented groups fed with natural
94
sources of carotenoids (micro algae D. salina) and red yeast phaffiarhodozyma at 100 and 200mg
kg-1) did not increase significantly over that of the control.
2.7.6.9.3 Total Haemocyte count
Haemocytes of crustaceans play important role in regulating functions and immune defense
mechanisms (Martin, et al., 1991 and Ratchi, et al., 1985). In decapod crustaceans, hemocytes
are involved in phagocytosis, which eliminates microbes or foreign particles (Bachere et al,
1995, Johansson, 1995). Hemocytes are associated with proteins like prohenoloxidase (Propo)
which is involved in encapsulation, melanization and cytotoxicity as a non-self recognition
system (Johansson and soderhall, 1989). Total Haemocyte Count varies in crustaceans in
response to infections, environmental factors and moult cycle (Bachere 2000; Johansson, et al.,
2000, Moulac & Haffner 2000). Simth, (1980 and 1992) has demonstrated that the haemocyte
count varies among crustacean species and is known to be affected by a variety of factors such as
infection agents and environmental stress. Rodrigues and Moullac (2000) noted that in
crustaceans an increase in THC has been related to disease resistance and Moullac and Haffiner,
(2000); Chand et al., (2006) stated that the higher THC could be provided enhancement of
immune capability during periods of higher activity or increased environmental bacterial loads.
A decrease in THC has been reported when shrimps were exposed to different environmental
stress factors and it was correlated with a higher susceptibility to viral and bacterial disease
(Moullac, et al., 1998; Song, et al., 2003, Liu, et al., 2004). It was reported that a reduction in
THC might be an indication of suppressed resistance in M. rosenbergii (Yeh et al., 2006).
Isagani. et al., (2009) reported that the significant increase in THC showed the efficacy of
injected AX at different dosages (0.67 and 1.34 nmol g-1 BW-1) in enhancing the non specific
immune response of adult M. rosenbergii. Chang et al., (2013) observed that THCs, HCs, and
95
GCs, significantly increased in prawns M. rosenbergii Fed ECE- supplemented diets at levels
1.0, 2.0 and 3.0 kg-1 for 12 days, but no significant difference in SGCs, of prawns among the
treatment groups was seen during the experimental period. These results suggested that ECE
added to the diet can induce proliferation of haemocytes in HPTs, promote mobilization of
mature GCs and increase THC resulting from increased HCs and GCs. Harikrishnan, et al.,
(2012) observed that the hemocyte counts significantly increased in M. rosenbergii fed with
0.1% and 1.0% Withania somnifera supplementation diets from weeks 1-4 against A. hydrophila.
Total Haemocyte count in prawns (M. rosenbergii) fed with 50,100 and 200 mg kg-1 of dietary
astaxanthin for 28 days, showed a significant increase than those in control group an increase in
the number of circulating haremocytes might be due to the higher haemocyte mobilization
indicating a better immune status in that group (Vijay Kumar, et al., 2009). Shrimp (L.
vannamei) were fed 80 mg astaxanthin kg-1 diet had highest THC than in shrimp fed with other
diets (0, 40 or 150 mg kg-1) (Flores, M. et al., 2007). However, shrimp fed with the diet
supplemented with, 150 mg astaxanthin kg-1 had the lowest values in growth, survival, OC and
THC, and this suggests that this group was under additional stress because of pigmentation
saturation (Herring 1973; Castillo, Negre-sadrgues & lenel 1982). There was no significant
difference between treatment groups in total hemocyte count and phenoloxidase activity of small
size (1-2 g body weight) of shrimp (P. monodon) fed with D. salina for 2 months. (Supamattya,
et al., 2005).
2.7.6.9.4 Total Heamolymph protein
Vijay Kumar et al., (2009) have demonstrated there was a significant increased (P<0.05)
in total serum protein content of prawns (M. rosenbergrii) were fed with 50,100 and 200 mg kg-1
astaxanthin compared to control group, who justified the increase in protein content may be due
96
to the increase in defense molecules in prawns fed with higher doses of astaxanthin and may
indicate a better immune status in these animals. Vargas-Albores et al., (1993); Hall at al., (1995)
stated that several immune molecules have been identified and purified in crustaceans such as the
lipopolysacchride binding protein, β-glucan binding protein and peptidolycan binding protein,
which exposure to any of the immunoestimutony substances is expected to rise one or more of
these defense proteins in particular and total protein levels in the hymolymph in general. The
highest haemocyanin concentration was found in L.vannamei that were fed with the 80 mg
astaxanthin kg-1 diet (Maricela et al., 2007; Pascual et al., 2003) reported that in L.vannamei 6097% of the protein in heamolymph was HC (haemocyanin). Supplementing astaxathin at 80 mg
kg-1 diet increased heamolymph HC in juveniles maintained in low-salinity water (3gL-1),
improving the hyper OC (Osmoregulatory capacity) of shrimps in a dilute medium.
2.7.6.9.5. Antioxidant activity
Bell, et al., (1993) have reported that the Reactive Oxygen Intermediates (ROIs) are
released during RBS (respiratory burst or superoxide onion production) of phagocytosis, which
represent a defense mechanisms against microbial infection. However, the excessive
accumulation of ROIs is extremely toxic to host cell. In a normal physiological state, harmful
effect of ROIs are effectively neutralized by the antioxidant defense system of organisms, which
in general comprises enzymes like superoxide dismutase (SODs) catalase, various peroxides and
small antioxidant molecules like ascorbate, polyunsaturated fatty acids and sugars. Therefore, the
activity of an antioxidant is usually considered a defense response in living system. Different
dosages of astaxanthin injected in M. rosendergii showed no significant effects on the capacity
of certain antioxidant indicators (superoxide dismutase, glutathione peroxide and glutathione
reductase), (Isagani, et al., 2009), which implies that L.garvieae infection suppressed the activity
97
of the haemolymph antionxidant system of infected M. rosenbergii. Improved resistance in
aquatic organisms to physical stress has been proved through dietary supplementation of
astaxanthin due to its antioxidant properties (Chien & Hunter 2003). Pan, et al., (2003), have
demonstrated dietary astaxanthin could enhance the antioxidant defense capability in P.
monodon Juvenile and provided protection against V. damsela challenge.
2.7.6.10 Disease resistance
Sung, H. H. et al., (1997) have stated that Aeromonas spp. are generally present in the
hepatopancreas (1.2 1.6 × 106 cell - forming units / g of HP) of apparently healthy prawns and
they do not cause disease at low concentrations (103 cells per prawns), it seems that in giant
freshwater prawns Aeromonas spp. may function as opportunistic pathogens and only cause
disease in prawns that are already weakened. Also, they have reported that results from the
challenge experiments showed giant freshwater prawn M.resenbegii are susceptible to
Aeromonas spp. (both strains, A. veronii and A. caviae), however the A. veronii strain was more
virulent than A. caviae strain. Hung-Hung Sung et al., (2000) also, they have reported that result
from the challenge experiments showed giant freshwater prawn M.resenbegii are susceptible to
Aeromonas spp. ( both strains, A. veronii and A. caviae), however the A. veronii strain was more
virulent than A. caviae strain. Harikrishnan, R. et al., (2012) have demonstrated that, the survival
rate in prawns M. rosenbergii fed with 0.1% 1.0% supplementation diets (Withnia somnifera)
with 60% and 70% was higher than to the other group treated 0.01% and control. The mortality
was high in control and 0.01% supplementation diets with 85% and 55% against A. hydrophila.
Similar results were reported in other studies (Chand, et al., 2008; Shankar, et al., 2011,
Balasubramanian, et al., 2008, Chand, et al., 2006 and Chiu, et al., 2010). Chang et al., (2013)
have indicated that dietary administration of water hyacinth (Eichhornia crassipes) extracts
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(ECE) at 2.0 and 3.0 g kg-1 for 12 days significantly increased survival rates of the prawn M.
rosenbergii against L. garvieae, suggesting that ECE administration can enhance prawn
resistance to L. garvieae, and can be related to the dosage of supplementation.
99
III. MATERIALS AND METHODS
The study was conducted in fiber reinforced plastic tanks (120 l capacity) in indoor
recirculatory system at the fish farm of the College of Fisheries, Mangalore to evaluate the
effect of Marigold oleoresin and Diacetate of lutein-mesozeaxanthin on growth, survival
immune responses and disease resistance challenged to Aeromonas hydrophila in freshwater
prawn, Macrobrachium rosenbergii. The study was carried out in triplicate tanks for each
treatment for a period of 60 days
3.1 Feed ingredients, formulation and analysis
The ingredients used in the formulation of different experimental diets were fishmeal,
rice bran, groundnut oil cake, wheat flour, soya beans meal, shrimp meal and vitamin and
mineral premix. The basal diet was supplemented with Marigold oleoresin and Diacetate of
lutein-mesozeaxanthin. All the ingredients were purchased from the local market except
Diacetate of lutein-mesozeaxanthin, which were procured from M/S AVASTHAGEN Pvt. Ltd.
Bangalore. All the ingredients except Diacetate of lutein-mesozeaxanthin were ground and
sieved to get particles of uniform size. The sieved ingredients were packed in high density
polythene bags and stored at room temperature. Vitamin and mineral premix (Agrimin Forte)
were procured from VET CARE.
3.2 Proximate composition of the feed ingredients
All the feed ingredients were analyzed for proximate composition prior to formulation of
the test diets employing standard methods (AOAC, 1975). Moisture content was estimated by
heating samples at 105 0C for 30 min and then cooling and weighing to a constant weight.
Crude protein was analyzed using Kjeltec system (Tecater 1002 distilling Unit), fat content by
Soxtech system (Tecater 1043 Extraction Unit), fibre content by using Fibretech system (Tecater
100
1017 Hot Extractor). Carbohydrate content was calculated as nitrogen free extract (NFE) by the
difference method (Hastings, 1976) as given below.
NFE = 100 – (% moisture + % crude protein + % crude fat + % crude fibre + % ash).
The ash content was determined by first drying the sample and then heating it in a muffle
furnace at 550 ± 10 0C for 6 h.
3.3 Formulation and preparation of experimental diets
3.3.1 Experiment, 1 (Marigold oleoresin)
Three test diets namely T1, T2 and T3 having 35% protein content were formulated using
the square method (Hardy, 1980). Diet T1 had 60 mg/kg Marigold oleoresin, T2 had 120 mg/kg
Marigold oleoresin and T3 had 180 mg/kg Marigold oleoresin, and diet without Marigold
oleoresin supplementation served as a control (T0).
3.3.2 Experiment, 2 (Diacetate of lutein-mesozeaxanthin)
Three test diets namely T1, T2 and T3 with 35% protein content were formulated using
the square method (Hardy, 1980). Diet T1 had 60 mg/kg Diacetate of lutein-mesozeaxanthin, T2
had 120 mg/kg Diacetate of lutein-mesozeaxanthin and T3 had 180 mg/kg Diacetate of luteinmesozeaxanthin, and diet without Diacetate of lutein-mesozeaxanthin supplementation served as
a control (F0).
The required quantities of ingredients were weighed accurately, mixed and hand kneaded
to required consistency with just sufficient quantity of water (1: 0.8) to get smooth dough. The
dough so obtained was cooked under steam in a pressure cooker at 105 0C for 20 to 30 min. The
cooked feed was cooled to room temperature rapidly by spreading in an enamel tray. Then
required dose of Diacetate of lutein-mesozeaxanthin and vitamin-mineral premix were added,
mixed and blended. The dough was extruded through a pelletizer. Pellets of size 1.2 mm were
101
dried in a hot air oven at 60 0C till the moisture content was reduced to less than 10%. Diets
were packed separately in high density polythene bags, labeled and stored in a wooden shelf at
room temperature for further use.
3.4 Experimental animals
The post larvae (PL-20) produced in the Prawn Hatchery at the College of Fisheries was
reared in cement tanks and juveniles were utilized for experiments. The juveniles were
acclimatized to the experimental condition by feeding with dry pelleted basal diet.
3.5 Experimental Design
The complete randomized design (CRD) was followed for both the experiments.
Experiments were conducted in triplicate tanks.
3.6 Experimental set up
An indoor closed recirculatory system consisting of 12 fiberglass reinforced plastic
(FRP) (dia. 66 cm) tanks each of 120 l capacity and arranged in a 2 tier system, which was
developed and installed in the Field Laboratory of the College of Fisheries, Mangalore (Murthy,
1997) was used for the study.
Three stage bio-filtering units comprised of four inter connected cement tanks. Two
bigger rectangular tanks each of 1000 l capacity, two smaller with 750 l and 250 l capacity each
and one circular plastic overhead tank of 2000 l were kept outside the field laboratory. These
tanks were covered with black PVC sheet to avoid direct sunlight and subsequent algal growth.
The tanks were connected to one another in such a way that waste water collected from outlets
of the 12 experimental tanks was carried by a drain pipe and falls into first rectangular tank. The
first bio-filter tank was filled with graded sand and gravel. The water gets filtered in the graded
layers of sand and gravel and enters from the bottom of first tank to the top of second
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rectangular tank. The second bio-filter tank was filled with dry oyster shells for biological
filtration. Water passes through this tank and enters third tank which was filled with clam shells
for efficient bio-filtration. The filtered crystal clear water then enters fourth tank which serves as
temporary storage tank.
The water was pumped from this tank to an overhead tank. The water flows from over
head tank to each of the experimental tanks by gravity. During experimental period each tank
was supplemented and allowed to overflow at a volume of 0.8 liter/min. Separate control valve
is provided for each tank to regulate water flow. A 1 HP air blower was continuously used to
aerate all the 12 experimental tanks. All the experimental tanks were covered with net to avoid
jumping of prawns from experimental tanks. Equal numbers (3) of hideouts were provided in
each tank to avoid cannibalism during culture period.
3.7 Stocking and rearing
Uniform sized PL of Macrobrachium rosenbergii with an average range of weight (0.85
to 0.89 g) for first experiment and (0.32 to 0.37 g) for second experiments were stocked at the
rate of 50 numbers / tanks respectively. The experiment was carried out for a period of 60 days
with an exchange of water once in two days in the tank. Fecal matter and uneaten food was
removed daily in the morning hours.
3.8 Feeding
Prawns were fed at the rate of 5% of their body weight till the end of the experiment. The
feed was broadcasted over the surface of water twice daily in the morning and evening. After
each sampling the quantity of feed given was re-adjusted based on the increased weight of
prawn.
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3.9 Water sampling
Water samples were collected for determining its quality every fortnight. Water quality
parameters were maintained within the normal range throughout the experimental period. Water
samples collected on each sampling day were analyzed for pH, temperature, dissolved oxygen,
free carbon dioxide, NH3-N and total alkalinity. Digital pocket pH meter (Hanna) was used to
record pH. Atmospheric temperature and water temperature were recorded by using
thermometer. Dissolved oxygen was estimated by Winkler’s method. Total alkalinity, NH3 and
free carbon dioxide were determined by following standard methods (APHA, 1995).
3.10 Prawn sampling
The prawns were sampled fortnightly to assess the growth. Length was measured from
tip of rostrum to tip of telson by using fiberglass measuring scale fixed on wooden frame.
Weight was measured on electronic balance (Essae, India).
3.11 Growth Studies
3.11.1 Specific growth rate (SGR)
The specific growth rate was calculated by using the following formula, (Hevroy et al
2005).
ln final weight (g) – ln Initial weight (g)
SGR = ------------------------------------------------------ X 100
No.
of days
The calculated value gives the average percentage increase in weight per day over the
experimental period.
104
3.11.2 Feed conversion ratio (FCR)
Feed conversion ratio (FCR) was calculated by using the following formula, (De Silva &
Anderson 1995).
Total dry feed offered (g)
FCR =
---------------------------------------Total wet weight gain (g)
3.11.3 Protein efficiency ratio (PER)
The protein efficiency ratio of the formulated diet used in experiments was estimated by
taking into consideration the weight gain of prawn (live weight) / per gram of crude protein
consumed on dry weight basis and calculated as
Increment of body weight (g)
PER = --------------------------------------------------Protein intake (g)
3.11.4 Survival Rate
Survival was calculated as the difference between the number of live animals at the
beginning and the end of the experiment and expressed in percentage (%), (Ai et al 2006).
3.11.5 Feed Efficiency Ratio
Feed efficiency ratio was calculated by using the following formula.
Feed Efficiency Ratio =Mean Weight Gain / Total Feed Given
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3.11.6 Weight gain (WG)
Weight gain (WG %) was calculated by using the following formula, (Tacon 1990).
WG % = [(Final mean weight – Initial mean weight) / (Initial mean weight] x 100
3.11.7 per Day Growth (g)
Per day growth (g) was calculated by using the following formula, (Tacon 1990).
Per Day Growth (g) = mean weight gain (g)/number of days.
3.11.8 Percentage of mean weight gain (PMWG)
Percentage of mean weight gain (PMWG) was calculated by using the following
formula.
Percentage of mean weight gain (PMWG) = ((Wf (g)-Wi (g))/Wi (g) ×100 (Bekcan et al
2006)
3.11.9 Condition factor (CF)
Condition factor (CF) was calculated by using the following formula, (Ai et al 2006).
Condition factor (CF) = W×100/L3
3.11.10 Daily Growth Rate (DGR)
Daily growth rate (DGR) was calculated by using the following formula, (De Silva &
Anderson 1995).
Daily growth rate (DGR) = {100× (final weight (g)-initial weight (g)/(days × initial
weight (g));
106
3.11.11 Daily Growth Index (DGI)
Daily growth index (DGI) was calculated by using the following formula, (De Silva & Anderson
1995).
Daily growth index (DGI) = {100×(Wf 1/3 –Wi 1/3 )/days}
3.11.12 Growth Coefficient (GC)
Growth coefficient (GC) was calculated by using the following formula, (De Silva &
Anderson 1995).
Growth Coefficient (GC) = {100×(Wf 1/3 - Wi 1/3 )/Σθ}
Where Wf = Final weight, Wi = Initial weight, Σθ= sum of average daily temperature in °c
3.12 Biochemical composition
Proximate composition of prawn muscle was estimated soon after completion of the
experiment. All the prawns were peeled and deveined. Whole meat of the prawn was dried at 60
0
C for 48 hrs to obtain the dry matter. The dry matter was powdered in mortar and used for
further analysis. The samples were analyzed for crude protein, crude fat, total ash and
carbohydrate (NFE) employing standard methods as explained earlier.
3.13 Anticoagulant Preparation
Anticoagulant was freshly prepared for collection of hemolymph samples of all treated
animals for immune assay. Sodium citrate (100 mM), Sucrose (250 mM) and Tris-Hcl (10 mM)
at pH 7.6, preparation was used as an anticoagulant.
3.14 Hemolymph sample collection
After completion of feeding trials hemolymph were collected from ventral sinus using
1ml disposable insulin syringe having needle size 0.30 x 8 mm. Hemolymph was collected in
107
sterile Eppendorff tube in (1:1) ratio of hemolymph and pre-cooled anticoagulant. These
samples were used for immune assay.
3.15 Immune parameters of Macrobrachium rosenbergii
3.15.1 Super oxide anion production assay (NBT assay)
The NBT assay was performed as described by Song and Hsieh (1994) using Ubottomed microtitre plates (Tarson, India). 100µl of HBSS was added to first two wells as blank.
100µL of hemolymph was added into separate wells and incubated for 1h at 37 0C for adherence
of cells. Then, the supernatant was removed and 100 µL NBT solution was added to each well
and incubated for 30 min at 37 0C. NBT solution was removed after 30 min and 100 µL absolute
methanol was added. Absolute methanol was removed and cells were washed gently three times
with 120µL of 70 % methanol. Methanol was removed and 120µL KOH and 140µl DMSO
were added to dissolve cytoplasmic formazan. The optical density was measured at 620 nm in
ELISA reader (Bio-Tek, USA).
3.15.2 Pro-phenoloxidase assay (PPO)
The PPO activity was measured by spectrophotometry
according to the formation of
dopachorome produced from L-dihydroxyphenylalanine (L-DOPA) (Herandez-Lopez et al.,
1996). 10 µL hemolymph supernatant was evenly mixed with 200µL phosphate buffer (0.1mol
L-1, pH 6.0) and 10 µL L-DOPA (0.1mol L-1) in U- bottomed microtitre plates (Tarson, India).
The optical density at 450 nm was measured using ELISA reader (Bio - Tek, USA).
3.15.3 Haemocyte count
Haemocyte was counted using nebular haemocytometer (B. S. 748 ROHEM, India)
3.15.4 Total Hemolymph protein
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Total hemolymph protein was determined by GeNeiTM protein analysis kit (by Lowry’s
method) using bovine serum albumin (BSA) as a standard (Lowry et al., 1951).
3.16 Histological studies
At the end of the experiment, the prawns from each treatment groups were selected and
dissected out to get the gut and hepatopancrease. This organ was kept in 10% formalin for 24
hrs. These organs were fixed whole. In the case of larger sized organs, the tissue blocks from the
respective organs were excised and fixed and during cassetting each tissue block was cut into
smaller pieces and cassetted separately. The fixed tissues were processed using an automatic
tissue processor (Shandon, Citadel 1000, England) and embedded in paraffin wax (Shandon,
Histocenter 2, England). Sections were cut at 5-6 µm thickness (Weswox Dptk MT-1090A,
India) and stained with haematoxylin and eosin. All the histological procedures followed were
as described by Bullock (1989). Slides were documented photographically by light microscope
(Olympus CX 41, Japan).
3.17 Experimental infection of Aeromonas hydrophila
3.17.1 Aeromonas hydrophila inoculum
The one day old culture of Aeromonas hydrophila (AH-40) in 1.5% (w/v) Tryptic Soya
broth was taken from the laboratory of Department of Microbiology, College of Fisheries,
Mangalore and used for challenge study. This culture was inoculated in 100 ml TSB flask and
incubated at 37 0C (60 rpm) for 24 hrs. This was used for preparation of injection.
3.17.2 Preparation of injection
Cultured A. hydrophila was centrifuged at 10000 rpm for 10 min. Supernatant was
discarded and pellets resuspended in PBS solution. Serial dilutions were prepared from 10 1 to
1010. This was used for injecting the experimental animals. Sterile PBS solution was injected as
109
a negative control during experimental study. Prawns were injected by 1ml sterile disposable
insulin syringe having needle size 0.30 x 8 mm.
3.17.3 LD50 of A. hydrophila
A. hydrophila isolate was tested for pathogenecity in juvenile of prawn maintained in
aquarium tanks (20L) with aeration. Prawn were injected with 0.1 ml each, A. hydrophila
inoculate ranging from 102 to 1010 CFU/ml. Ten prawns were used for each dose. Mock injection
was given to control groups with sterile PBS. Mortalities were recorded daily for 10 days and
the lethal dose 50% (LD50) were calculated according to Reed and Muench (1938).
3.17.4 Challenge study
The juveniles were challenged intramuscularly with a 24 hour old culture of A.
hydrophila (20 µL of 3.25 x 106 CFU/ml). The injection was given between the first and second
pleura. The susceptibility was tested for 10 days. Ten prawns per treatment in duplicate were
challenged after 60 days feeding trial. Challenged prawns were maintained in plastic tubs (20 l
capacity) with aeration for 24 hrs. and were covered with net to avoid jumping of prawns.
Appearance of gross clinical lesions and mortality pattern if any, were observed during the
study.
3.17.5 Confirmation of prawn mortality
The cause of mortality was further confirmed by re-isolating the organism from
moribund or dead prawn hepatopancreas on Rimler Shot’s (RS) medium (Himedia, India).
Dissected hepatopancreas were placed in sterile test tube. 5ml PBS (Phosphate Buffered Saline)
was added to the test tube and homogenized using glass rod to obtain a solution. Serial dilutions
were prepared using PBS solution. Aliquot of each diluted homogenized solution was inoculated
in RS plate (0.1 ml/plate) in duplication for each dilution. Plates were incubated for 24 hrs at 37
110
0
C in order to determine Aeromonas hydrophila colonies. After 24 hrs incubation yellowish
color colonies were developed, this confirmed the mortality of prawn due to Aeromonas
hydrophila infection.
3.17.6 Relative percent survival (RPS)
Relative percent survival (RPS) was calculated according to Amend (1981).
RPS = [1-(% mortality of treatment group / % mortality in control] × 100
3.18 Statistical analysis
Mean growth, survival, total hemolymph protein, hemocyte, prophenol oxidase activity,
super oxide anion production assay and disease resistance of prawn achieved in response to
different formulated test diets was analyzed statistically by using one way analysis of variance
(ANOVA) followed by Duncan’s multiple range test were done by using SPSS software (16.0
version).
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IV. EXPERIMENTAL RESULTS
To study the effect of carotenoids (Marigold oleoresin and Diacetate of luteinmesozeaxanthin at the levels of 60, 120 and 180 mg/kg) on immune response, growth
performance, survival and disease resistance of giant freshwater prawn, M. rosenbergii, and three
experiments were conducted in a closed recirculatory system over a period of 60 days. The
results obtained are presented below.
Experiment 1
4.1 Proximate composition of the feed ingredients and formulated feeds
4.1.1 Feed ingredients
Proximate composition of the ingredients used in formulation of the pelleted feed is
presented in Table 2. Moisture content was highest in soya flour (8.34 %) and lowest in fish meal
(5.14 %). Crude protein ranged from 62.28 % in fish meal to 6.90 % in rice bran. Crude fat
content were recorded 9.53 % in fish meal, 6.89 % in ground nut oil cake, 4.98 % in rice bran,
1.73% in wheat flour, 19.62 in soya flour and 3.08 in shrimp meal. Dry matter content was
highest in fish meal (94.86 %) and lowest in soya flour (91.66 %). Ash content was highest in
fish meal (18.82 %) and minimum in wheat flour (1.28%). Fiber content was highest in rice bran
(28.62 %) and lowest in fish meal (0.72 %). Carbohydrate content (nitrogen free extract) of
ingredients fluctuated from 77.84 % in wheat flour to 3.51 % in fish meal.
4.1.2 Experimental diets
Moisture content of diet ranged from 6.30 % in T2 to 7.10 % in T3. Diet T1 and T0
recorded the moisture content of 6.60 % and 6.83 % respectively. The values of crude protein
were 34.75 % in T1, 34.69% in T3, 34.83 % in T2 and 34.98 % in T0. Crude fat content was
highest in T0 (7.68 %), followed by T2 (7.65 %), T1 (7.61 %) and T3 (7.44 %) (Table 3).
112
4.2 Carotenoid content of the feed
Carotenoid of the diets ranged from 0 mg/kg in T0, 60 mg/kg in T1, 120 mg/kg in T2 and
180 mg/kg in T3 (Table 1).
4.3 Water quality parameters
4.3.1 Temperature
Table 4 and Fig 1 show the fluctuation in air and water temperature recorded at 15 days
interval during the experimental period. Temperature of air and water ranged from 28 0C to 29.5
0
C and 27.0 0C to 28.5 0C respectively.
4.3.2 pH
The pH recorded during the study period, ranged from 7.4 to 8.7 (Table 5 and Fig 2).
4.3.3 Dissolved oxygen
The dissolved oxygen content recorded on different sampling days is tabulated in Table 6
and average values are presented in Fig 3. The average values of dissolved oxygen were 6.90 to
8.10 mg/l in T0, 6.90 to 8.10 mg/l in T1, 7.00 to 8.10 mg/l in T2 and 6.90 to 8.10 mg/l in T3.
4.3.4 Free carbon dioxide
The values of free carbon dioxide recorded over the experimental period are shown in
Table 7 and Fig 4 .The average values ranged from 0.32 to 1.30 mg/l.
4.3.5 Total alkalinity
The total alkalinity values recorded in the different tanks during the experimental period
are presented in Table 9 and the average values are presented in Fig 6. The minimum mean total
alkalinity recorded was 73.70 mg/l of CaCO3 in treatment T2, while the maximum mean was
84.73 mg/l of CaCO3 in treatment T3.
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4.3.6 Ammonia-Nitrogen
The total ammonia-nitrogen levels estimated during the experimental period is presented
in Table 8 and the mean values presented in Fig 5. The average value ranged between 0.01 to
0.20 μg at N/l.
4.4 Growth studies
The experiment was carried out to investigate the effect of carotenoid on the growth,
survival and immune response of M. rosenbergii, fed with three test diets (T1, T2 and T3). All
the three feeds were evaluated in three replicate groups. The increase in length and weight of
prawn under different treatments and control during experimental period were recorded in Table
10 and Fig 7 Table 13 and Fig 8 respectively. It was observed that the best growth of prawn, M.
rosenbergii in terms of weight was recorded in treatment T2 followed by the treatment T3, T1 and
T0. The average weight recorded after 60 days of culture in treatment T2 was 4.59 g, followed by
4.28 g in T3, 3.99 g in T1 and 3.91 g in T0. The final average length was found highest in T2 (5.31
cm) followed by T1 (5.20 cm), T0 (5.14 cm) and T3 (5.10 cm).
4.4.1 Specific Growth Ratio
Specific Growth Rate (% / day) of prawn was highest in T2 (2.77), followed by T3 (2.64),
T1 (2.53) and T0 (2.49). Specific Growth Rate was given in Table 14b-2.
4.4.2 Food Conversion Ratio
The best Food Conversion Ratio (FCR) was recorded in T2 (1.86) and maximum in
T0(2.19). Data are presented in Table 14b-4.
4.4.3 Protein Efficiency Ratio
Protein Efficiency Ratio calculated (Table 14b-6) was in the range of 1.29 to 1.53. The
PER was highest in T2 (1.53) followed by T3 (1.41), T1 (1.30) and T0 (1.29).
114
4.4.4 Survival Ratio
The rate of survival of prawn recorded in different treatments during the experimental
period is shown in Fig 12. The highest average survival rate of 83.32 % was recorded in T 2
followed by T3 (78 %), T1 (75.32 %) and T0 (73.32 %).
4.4.5 Feed Efficiency Ratio
The best Feed Efficiency Ratio (FER) was recorded in T2 (0.535) followed by T3(0.495),
T1(0.456) and T0(0.454). Data of FER was shown in Table 14b-9.
4.4.6 Weight Gain
Weight gain of prawn was highest in T2 (3.72), followed by T3 (3.40), T1 (3.12) and T0
(3.04). Weight gains are presented in Table 14b-11.
4.4.7 Per Day Growth
Per day growth calculated (Table 14b-13) was in the range of 0.050 to 0.062. The PDG
was highest in T2 (0.062) followed by T3 (0.054), T1 (0.052) and T0 (0.050).
4.4.8 Percentage Growth
The percentage weight growth of prawn recorded in different treatments during the
experimental period was given in Table 14b-15. The highest average percentage growth of 4.31
% was recorded in T2 followed by T3 (3.88 %), T1 (3.58 %) and T0 (3.48 %).
4.4.9 Condition Factor
Condition factor of prawn was highest in T3 (3.21), followed by T2 (3.06), T0 (2.87) and
T1 (2.83). Data of condition factor are presented in Table 14b-17.
4.4.10 Daily Growth Ratio
Daily growth rate calculated (Table 14b-19) was in the range of 5.79 to 7.13. The DGR
was highest in T2 (7.13) followed by T3 (6.47), T1 (5.97) and T0 (5.79).
115
4.4.11 Daily Growth Index
Daily growth index was highest in T2 (1.17), followed by T3 (1.11), T1 (1.05)
and T0 (1.03). Data of daily growth index are given in Table 14b-21.
4.4.12 Growth Coefficient
The growth coefficient of prawn recorded in different treatments during the
experimental period is shown in Table 14b-23. The average growth coefficient was
highest (2.54) and recorded in T2 followed by T3 (2.40), T1 (2.27) and T0 (2.22).
4.5 Biochemical composition
The result of biochemical composition of prawn muscle at the end of the
experiment is given in Table 15.
4.5.1 Moisture
Maximum moisture content was recorded in T2 (76.76%) and minimum in T3
(75.92 %). The values were 76.64 % in T1 and 76.24 % in T0.
4.5. 2 Protein
Protein content of prawn muscle was maximum in T3 (37.8 %) followed by T1
(37.2%), T0 (36.81 %) and T2 (36.50 %).
4.5.3 Fat
Crude fat was maximum in T0 (2.67 %) and minimum in T3 (2.25 %).
Intermediate values of 2.55 % in T2 and 2.45 % in T1.
4.5.4 Ash
Ash levels were 7.2% in T2, 5.7 % in T1, 5.6 % in T0 and 5.3 % in T3.
4.5.5 Nitrogen Free Extract The highest NFE was recorded in T3 (1.95 %), followed
by T1 (1.83%), T0 (1.58 %) and T2 (1.46 %).
116
4.6 Resistance of M. rosenbergii to A. hydrophila infection
The mortality and relative percentage of survival of prawn recorded in different
treatments and control groups after challenged with A. hydrophila disease are presented in Table
16, 18 and Fig 21. Highest mortality 75 % was recorded in T0 followed by 50 % in T1, 45 % in T2
and 36 % in T3 respectively. Highest relative percentage survival of 53.34 % was recorded in T3
followed by 40 % in T2, 33.34 % in T1. Mortality of prawn recorded in different treatments is
significantly higher (p< 0.05) than that of control groups are presented (Tables 17 and 18). The
mortality was confirmed by plating of isolates from hepatopancreas.
4.7 Immune parameters of M. rosenbergii
4.7.1 Prophenoloxidase Assay (PPO)
Phenoloxidase activity of prawn fed 60, 120 and 180 mg/kg (T1, T2 and T3) of
carotenoid containing diets was higher than that of prawn fed control diet are presented in Table
19, 20, 21 and fig 22. Highest phenoloxidase activity of 1.45 was recorded in T3 followed by
1.32 in T2, 1.08 in T1 and 0.68 in T0 respectively. Phenoloxidase activities of prawn recorded in
different treatments were significantly (P< 0.01) higher than that of control groups.
4.7.2 Superoxide onion production (NBT)
Superoxide onion production or respiratory burst of prawn fed 60, 120 and 180 mg/kg
(T1, T2 and T3) of carotenoid containing diets was higher than that of prawn fed control diet and
are presented in Table 19, 22, 23 and fig 23. The highest superoxide onion production of 1.173
was recorded in T3 followed by 0.53 in T2, 0.11 in T1 and 0.05 in T0 respectively. Superoxide
onion production of prawn recorded in different treatments were significantly (P< 0.05) higher
than that of control groups.
4.7.3 Total heamolymph protein
117
Total heamolymph protein of prawn fed 60 mg/kg. 120 mg/kg and 180 mg/kg (T1, T2 and
T3) of carotenoid containing diets was higher than that of prawn fed control diet and are
presented in Table 19, 24, 25 and Fig 24. The highest total heamolymph protein of 14.20 mg/ml
was recorded in T3 followed by 11.90 mg/ml in T2, 11.65 mg/ml in T1 and 9.25 mg/ml in T0
respectively. Total heamolymph protein of prawn recorded in different treatments were
significantly (P> 0.05) higher than that of control groups.
4.7.4 Total heamocyte count
Total heamocyte count of prawn fed 60 mg/kg, 120 mg/kg and 180 mg/kg (T1, T2 and T3)
of carotenoid containing diets was higher than that of prawn fed control diet and are presented
(Table 19, 26, 27 and fig 25.The highest total heamocyte count of 14.06 was recorded in T 3
followed by 10.42 in T2, 8.08 in T1 and 6.75 in T0 respectively. Total heamocyte count of prawn
recorded in different treatments were significantly (P< 0.01) higher than that of control groups.
Experiment 2
4.8 Experimental diets
Moisture content of diet ranged from 6.50 % in T0 to 7.24 % in T1. Diet T2 and T3
recorded the moisture content of 6.85 % and 7.15 % respectively. The values of crude protein
were 35.20 % in T2, 35.09% in T0, 34.95 % in T1 and 34.87 % in T3. Crude fat content was
highest in T3 (7.76 %), followed by T1 (7.65 %), T0 (7.52 %) and T2 (7.40 %) (Table 29).
4.9 Carotenoid content of the feed
Carotenoid of the diets ranged from 0 mg/kg in T0, 60 mg/kg in T1, 120 mg/kg in T2 and
180 mg/kg in T3 (Table 28).
4.10 Water quality parameters
118
4.10.1 Temperature
Table 30 and Fig 26 show the fluctuation in air and water temperature recorded at 15
days interval during the experimental period. Temperature of air and water ranged from 28.5 0C
to 29.5 0C and 27.5 0C to 29.00C respectively.
4.10.2 pH
The pH recorded during the study period, ranged from 7.8 to 9.1 (Table 31 and Fig 27).
4.10.3 Dissolved oxygen
The dissolved oxygen content recorded on different sampling days is tabulated in Table
32 and average values are presented in Fig 28. The average values of dissolved oxygen were 6.80
to 8.80 mg/l in T0, 6.79 to 8.45 mg/l in T1, 6.69 to 8.65 mg/l in T2 and 7.10 to 8.60 mg/l in T3.
4.10.4 Free carbon dioxide
The values of free carbon dioxide recorded over the experimental period are shown in
Table 33 and Fig 29 .The average values ranged from 0.40 to 1.40 mg/l.
4.10.5 Total alkalinity
The total alkalinity values recorded in the different tanks during the experimental period
are presented in Table 35 and the average values are presented in Fig 31. The minimum mean
total alkalinity recorded was 72.43 mg/l of CaCO3 in treatment T2, while the maximum mean
was 82.10 mg/l of CaCO3 in treatment T3.
4.10.6 Ammonia-Nitrogen
The total ammonia-nitrogen levels estimated during the experimental period is presented
in Table 34 and the mean values presented in Fig 30. The average value ranged between 0.01 to
0.10 μg at N/l.
119
4.11 Growth studies
The experiment was carried out to investigate the effect of carotenoid on the growth,
survival and immune response of M. rosenbergii, fed with three test diets (T1, T2 and T3). All
the three diets were evaluated in three replicated groups. The increase in length and weight of
prawn under different treatments and control during experimental period were recorded in Table
36 and Fig 32a and Table 39 and Fig 33 respectively. It was observed that the best growth of
prawn, M. rosenbergii in terms of weight was recorded in treatment T3 followed by the treatment
T2, T1 and T0. The average weight recorded after 60 days of culture in treatment T3 was 1.585 g,
followed by 1.518 g in T2, 1.42 g in T1 and 1.22 g in T0. The final average length was found
highest in T3 (4.46 cm) followed by T2 (4.27 cm), T1 (4.09 cm) and T0 (4.00 cm).
4.11.1 Specific Growth Rate
Specific Growth Rate (% / day) of prawn was highest in T3 (2.52), followed by T2 (2.43),
T1 (2.34) and T0 (2.09). Specific Growth Rate was given in Table 40b-2.
4.11.2 Food Conversion Ratio
The best Food Conversion Ratio (FCR) was recorded in T3 (2.26) and maximum in T0
(2.73). Data are presented in Fig 35.
4.11.3 Protein Efficiency Ratio
Protein Efficiency Ratio calculated (Fig 36) was in the range of 1.043 to 1.259. The PER
was highest in T3 (1.259) followed by T2 (1.226), T1 (1.159) and T0 (1.043).
4.11.4 Survival Ratio
The rate of survival of prawn recorded in different treatments during the experimental
period is shown in Fig 37. The highest average survival rate of 80 % was recorded in T3 followed
by T2 (77.32 %), T1 (74%) and T0 (69.32 %).
120
4.11.5 Feed Efficiency Ratio
The best Feed Efficiency Ratio (FER) was recorded in T3 (0.44) followed by T2 (0.429),
T1 (0.405) and T0 (0.365). Data of FER was shown in Fig 38.
4.11.6 Weight Gain
Weight gain of prawn was highest in T3 (1.23), followed by T2 (1.17), T1 (1.07) and T0
(0.883). Weight gains are presented in Table 40b-8.
4.11.7 Per Day Growth
Per day growth calculated (Table 40b-10) was in the range of 0.014 to 0.020. The PDG
was highest in T3 (0.020) followed by T2 (0.019), T1 (0.017) and T0 (0.014).
4.11.8 Percentage Growth
The percentage growth of prawn recorded in different treatments during the experimental
period was given in Table 40b-12. The highest average percentage growth of 355.43 % was
recorded in T3 followed by T2 (339.06 %), T1 (308.72 %) and T0 (253.34 %).
4.11.9 Condition Factor
Condition factor of prawn was highest in T1 (2.075), followed by T2 (1.949), T0 (1.914)
and T3 (1.786). Data of condition factor are presented in Fig 42.
4.11.10 Daily Growth Rate
Daily growth rate calculated (Table 40b-15) was in the range of 4.22 to 5.92 The DGR
was highest in T3 (5.92) followed by T2 (5.65), T1 (5.14) and T0 (4.22).
4.11.11 Daily Growth Index
Daily growth index was highest in T3 (0.769), followed by T2 (0.743), T1 (0.700) and T0
(0.613). Data of daily growth index are given in Table 40b-17.
4.11.12 Growth Coefficient
121
The growth coefficient of prawn recorded in different treatments during the experimental
period is shown in Table 40b-19. The average growth coefficient was highest (1.625) in T3
followed by T2 (1.57), T1 (1.47) and T0 (1.295).
4.12 Biochemical composition
The result of biochemical composition of prawn muscle at the end of the experiment was
given in Table 41.
4.12.1 Moisture
Maximum moisture content was recorded in T0 (80.86%) and minimum in T3 (78.46 %).
The values were 78.80 % in T2 and 78.59 % in T1.
4.12. 2 Protein
Protein content of prawn muscle was maximum in T2 (22.95 %) followed by T1 (22.73
%), T3 (22.42 %) and T0 (21.80 %).
4.12.3 Fat
Crude fat was maximum in T0 (1.8 %) and minimum in T3 (1.3 %). Intermediate values
of 1.7 % in T2 and 1.5 % in T1.
4.12.4 Ash
Ash levels were 5.8 % in T2, 5.3 % in T0, 5.2 % in T3 and 4.9 % in T1.
4.12.5 Nitrogen Free Extract
The highest NFE was recorded in T1 (1.6 %), followed by T2 (1.5 %), T3 (1.4 %) and T0
(1.3 %).
122
4.13 Resistance of M. rosenbergii to A. hydrophila infection
The mortality and relative percentage of survival of prawn recorded in different
treatments and control groups after challenged with A. hydrophila disease are presented in Table
42, 44 and Fig 46. Highest mortality 75 % was recorded in T0 followed by 30 % in T1, 50 % in T2
and 70 % in T3 respectively. Highest relative percentage survival of 60 % was recorded in T 1
followed by 33.34 % in T2, 6.67 % in T3. Mortality of prawn recorded in different treatments is
significantly higher (p< 0.05) than that of control groups (Tables 42 and 44). The mortality was
confirmed by plating of isolates from hepatopancreas.
4.14 Immune parameters of M. rosenbergii
4.14.1 Prophenoloxidase Assay (PPO)
Phenoloxidase activity of prawn fed 60, 120 and 180 mg/kg (T1, T2 and T3) of
carotenoid containing diets was higher than that of prawn fed control diet (Table 45, 46, 47 and
Fig 47). Highest phenoloxidase activity of 1.38 was recorded in T1 followed by 0.57 in T2, 0.54
in T3 and 0.27 in T0 respectively. Phenoloxidase activities of prawn recorded in different
treatments were significantly (P> 0.05) higher than that of control groups.
4.14.2 Superoxide onion production (NBT)
Superoxide onion production or respiratory burst of prawn fed 60, 120 and 180 mg/kg
(T1, T2 and T3) of carotenoid containing diets was higher than that of prawn fed control diet and
are presented in Tables 45, 48, 49 and Fig 48. The highest superoxide onion production of 0.6
was recorded in T1 followed by 0.14 in T2, 0.11 in T3 and 0.09 in T0 respectively. Superoxide
onion production of prawn recorded in different treatments were significantly (P< 0.01) higher
than that of control groups.
123
4.14.3 Total heamolymph protein
Total heamolymph protein of prawn fed 60 mg/kg. 120 mg/kg and 180 mg/kg (T1, T2 and
T3) of carotenoid containing diets was higher than that of prawn fed control diet and are
presented in Tables 45, 50, 51 and Fig 49. The highest total heamolymph protein of 12.8 mg/ml
was recorded in T1 followed by 11.4 mg/ml in T2, 11.2 mg/ml in T3 and 9.36 mg/ml in T0
respectively. Total heamolymph protein of prawn recorded in different treatments were
significantly (P< 0.01) higher than that of control groups.
4.14.4 Total heamocyte count
Total heamocyte count of prawn fed 60 mg/kg, 120 mg/kg and 180 mg/kg (T1, T2 and T3)
of carotenoid containing diets was higher than that of prawn fed control diet (Tables 45, 52, 53
and Fig 50).The highest total heamocyte count of 14.83 was recorded in T1 followed by 9.63 in
T2, 8.22 in T3 and 6.2 in T0 respectively. Total heamocyte count of prawn recorded in different
treatments were significantly (P< 0.01) higher than that of control groups.
124
Experiment 1
Table 1. Proportion of various ingredients used in the preparation of different test diets
(Experiment 1)
Treatments
T0
T1
T2
T3
Fish
Meal
g/kg
200.00
200.00
200.00
200.00
Groun Shrimp
d nut meal
oil cake g/kg
g/kg
300.00
100.00
300.00
100.00
300.00
100.00
300.00
100.00
Rice
bran
g/kg
100.00
99.94
99.88
99.82
Wheat
flour
g/kg
Soya
flour
g/kg
100.00
100.00
100.00
100.00
150.00
150.00
150.00
150.00
Vitamin
Mineral
mixture
g/kg
50.00
50.00
50.00
50.00
Marigold
Oleoresin
mg/kg
0.00
60.00
120.00
180.00
Table 2. Proximate composition of ingredients used for the preparation of experimental
diets (% on dry weight basis) for experiment (1 and 2)
Ingredients
Fish meal
Rice bran
Shrimp
meal
Groundnut
oilcake
Wheat
flour
Soya flour
Moisture Dry
(%)
matter
(%)
5.14
94.86
6.83
93.17
8.11
91.89
Crude
protein
(%)
62.28
6.90
62.07
Crude
fat
(%)
9.53
4.98
3.08
Crude
fibre
(%)
0.72
28.62
1.69
Ash
(%)
NFE
(%)
Energy
Kcal /Kg
18.82
15.60
17.10
3.51
37.07
7.95
4523.90
2337.32
4082.44
7.01
92.99
34.28
6.89
3.02
10.80
38.00
4087.34
6.50
93.50
11.23
1.73
1.42
1.28
77.84
3905.10
8.34
91.66
39.67
19.62
6.50
5.24
20.63
4891.00
Table 3. Proximate composition of experimental diets (% on dry weight basis)
Treatments
Moisture
(%)
6.83
T0
6.60
T1
6.30
T2
7.10
T3
*Means of three replicates
Dry
matter
(%)
93.17
93.40
93.70
92.90
Crude
protein
(%)
34.98
34.75
34.83
34.69
Crude
fat
(%)
7.68
7.61
7.65
7.44
125
Crude
fibre
(%)
26.12
25.42
26.05
25.15
Ash
(%)
NFE
(%)
15.15
15.44
15.27
15.48
9.24
10.18
9.90
10.14
Energy
Kcal /Kg
3050.40
3085.54
3065.58
3047.60
Table 4. Mean values of air and water temperature recorded during the experimental
period
Air temperature (0C)
Days after stocking
0
15
30
45
60
Water temperature(0C)
28.0
29.5
29.0
28.5
29.5
27.5
27.5
28.5
27.0
28.5
Fig. 1 Mean values of air and water temperature recorded during the experimental period
30
Temperature( oc )
29.5
29
28.5
28
27.5
27
26.5
26
25.5
1
2
3
4
Weeks
Air temp.
Water temp.
126
5
6
Table 5. Values of pH recorded on different sampling days during the experimental period
No of days
Treatment
Replication
T0
R1
R2
R3
Mean±SE
T1
T2
T3
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
0
days
7.5
7.6
7.6
7.56
±0.03
15
days
8.0
8.2
7.9
8.03
±0.08
30
days
8.2
8.0
8.2
8.13
±0.06
8.0
8.2
8.1
8.1
±0.05
8.2
8.1
8.1
8.13±
0.03
45
days
8.3
8.2
8.4
8.3
±0.05
8.4
8.1
8.3
8.26
±0.08
8.3
8.2
8.1
8.2
±0.05
60
days
8.6
8.7
8.6
8.53±
0.03
8.5
8.7
8.5
8.56
±0.06
8.5
8.6
8.7
8.6
±0.05
7.6
7.5
7.4
7.50
±0.05
7.5
7.5
7.6
7.53
±0.03
8.1
8.2
7.9
8.06
±0.08
8.0
7.9
8.1
8.00
±0.05
7.4
7.5
7.6
7.50
±0.05
8.2
8.1
7.9
8.06
±0.08
8.2
8.2
8.0
8.13±
0.06
8.4
8.2
8.1
8.23
±0.08
8.6
8.6
8.7
8.63
±0.03
Fig.2 Values of pH recorded on different sampling days during the experimental period
127
PH
8.8
8.6
8.4
8.2
8
7.8
7.6
7.4
7.2
7
6.8
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 6 . Fluctuation of dissolved oxygen (mg/l) level of water recorded on different
sampling days
No of days
Treatment
Replication
0
days
15
days
30
days
45
days
60
days
T0
R1
R2
R3
Mean±SE
7.8
7.9
8.1
7.93±
0.08
8.0
7.9
8.1
8.0
±0.05
7.8
8.0
8.1
7.96
±0.08
7.9
8.1
7.8
7.93
±0.08
7.4
7.7
7.0
7.39±
0.20
7.3
7.6
7.1
7.33
±0.14
7.2
7.5
7.0
7.23
±0.14
7.4
7.7
7.2
7.43
±0.14
7.5
6.9
7.4
7.26±
0.18
7.6
6.9
7.5
7.33
±0.21
7.5
7.3
7.2
7.33
±0.08
7.1
6.9
7.4
7.13
±0.14
7.0
7.0
8.0
7.33±
0.33
7.1
7.4
7.3
7.26
±0.08
7.4
7.6
7.5
7.5
±0.05
7.3
7.6
7.5
7.46
±0.08
7.8
7.6
7.5
7.63
±0.08
7.8
7.4
7.5
7.54
±0.12
7.7
7.3
7.6
7.53
±0.12
7.5
7.4
7.9
7.6
±0.15
T1
T2
T3
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
128
Fig. 3 Fluctuation of dissolved oxygen (mg/l) level of water recorded on different sampling
days
Dissolved oxygen (mg/l)
8.2
8
7.8
7.6
7.4
7.2
7
6.8
6.6
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 7. Fluctuation of carbon dioxide (mg/l) level of water recorded on different sampling
days
No of days
Treatment
Replication
0
days
15
days
30
days
45
days
60
days
T0
R1
R2
R3
Mean±SE
0.89
0.95
0.82
0.88
±0.03
0.81
0.80
0.83
0.81
±0.00
0.82
0.81
0.80
0.81
±0.00
0.86
0.83
0.85
0.56
0.49
0.32
0.45
±0.07
0.55
0.50
0.44
0.49
±0.03
0.56
0.47
0.39
0.47
±0.04
0.54
0.55
0.53
0.58
0.32
0.48
0.46
±0.07
0.56
0.48
0.47
0.50
±0.02
0.57
0.49
0.51
0.52
±0.02
0.49
0.57
0.58
0.88
0.90
1.30
1.02
±0.13
0.86
0.79
0.82
0.82
±0.02
0.83
0.92
0.94
0.89
±0.02
0.78
0.95
0.91
0.90
0.78
1.00
0.89
±0.06
0.90
0.76
0.96
0.87
±0.05
0.91
0.89
0.90
0.90
±0.00
0.79
0.89
0.92
T1
T2
T3
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
R1
R2
R3
129
Mean±SE
0.84
±0.00
0.54
±0.00
0.54
±0.02
0.88
±0.05
0.86
±0.02
Fig. 4 Fluctuation of carbon dioxide (mg/l) level of water recorded on different sampling
days
Carbon dioxide (ppm)
1.2
1
0.8
0.6
0.4
0.2
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 8. Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on different
sampling days
No of days
Treatment
Replication
R1
T0
T1
T2
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
0
days
0.03
15
days
0.10
30
days
0.07
45
days
0.15
60
days
0.11
0.01
0.01
0.01
±0.00
0.02
0.01
0.02
0.01
±0.00
0.02
0.01
0.03
0.02
±0.00
0.07
0.06
0.07
±0.01
0.04
0.05
0.07
0.05
±0.00
0.06
0.08
0.07
0.07
±0.00
0.03
0.04
0.04
±0.01
0.06
0.04
0.05
0.05
±0.00
0.05
0.09
0.04
0.06
±0.01
0.20
0.03
0.12
±0.05
0.14
0.21
0.16
0.17
±0.02
0.09
0.18
0.07
0.11
±0.03
0.07
0.17
0.12±
0.03
0.10
0.12
0.13
0.11
±0.00
0.11
0.12
0.09
0.10
±0.00
130
R1
R2
R3
Mean±SE
T3
0.02
0.01
0.01
0.01
±0.00
0.10
0.05
0.07
0.07
±0.01
0.08
0.07
0.05
0.06
±0.00
0.06
0.15
0.08
0.09
±0.02
0.11
0.10
0.09
0.10
±0.00
Fig. 5 Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on different
sampling days
0.18
0.16
Ammonia ( )
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 9. Fluctuation of total alkalinity (mg/l) level of water recorded on different sampling
days
No of days
Treatment
Replication
0
days
T0
R1
R2
R3
Mean±SE
73.4
73.1
75.4
73.79
± 0.72
R1
R2
R3
Mean±SE
74.2
73.5
75.6
74.43
±0.61
R1
73.8
T1
131
15
days
30
days
76.3
78.9
78.4
77.6
76.4
75.8
77.03 77.43
± 0.68 ± 0.89
75.9
77.8
78.1
76.1
74.7
75.3
76.23 76.40
±0.99 ±0.73
77.4
78.3
45
days
60
days
83.9
84.4
80.7
86.9
80.5
82.9
81.70 84.73
± 1.10 ± 1.17
82.4
83.9
81.9
85.2
83.3
84.1
82.53 84.40
±0.40 ±0.40
80.2
82.9
R2
R3
Mean±SE
T2
73.1
74.2
73.70
±0.32
74.0
73.7
73.6
73.76
±0.12
R1
R2
R3
Mean±SE
T3
78.0
76.1
77.16
±0.56
75.7
76.4
77.1
76.40
±0.40
77.7
76.0
77.33
±0.68
76.0
77.3
78.1
77.13
±0.61
83.0
81.7
81.63
±0.80
81.5
82.0
80.5
81.33
±0.44
86.1
83.4
84.13
±0.99
81.9
83.1
84.7
83.23
±0.81
Total Alkalinity (mg/l)
Fig. 6 Fluctuation of total alkalinity (mg/l) level of water recorded on different sampling
days
86
84
82
80
78
76
74
72
70
68
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 10.
Details of weight (g) of freshwater prawn recorded in different tanks during
the experiment
Treatments
T0
T1
No. of
tanks
1
2
3
Avg.
SE±
1
2
3
Avg.
SE±
0
0.86
0.89
0.87
0.87
0.00
0.88
0.87
0.86
0.87
0.00
15
1.50
1.56
1.53
1.53
0.01
1.59
1.54
1.52
1.55
0.02
132
No. of days
30
2.28
2.38
2.37
2.34
0.03
2.44
2.31
2.33
2.36
0.04
45
3.13
3.25
3.23
3.20
0.03
3.37
3.24
3.22
3.27
0.04
60
3.85
3.96
3.93
3.91
0.03
4.07
3.98
3.93
3.99
0.04
T2
1
2
3
Avg.
SE±
1
2
3
Avg.
SE±
T3
0.87
0.89
0.85
0.87
0.01
0.86
0.88
0.89
0.87
0.00
1.59
1.64
1.56
1.59
0.02
1.55
1.57
1.58
1.56
0.00
2.56
2.59
2.49
2.54
0.02
2.44
2.45
2.46
2.45
0.00
3.54
3.58
3.46
3.52
0.03
3.37
3.41
3.42
3.40
0.01
4.59
4.70
4.50
4.59
0.05
4.22
4.36
4.27
4.28
0.04
Weight (g)
Fig . 7 a Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 11. Analysis of variance for weight (g) of M. rosenbergii in different treatments and
control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.8674
3
Within Groups 0.046667 8
Total
*P<0.01
MS
F
0.289133 49.56571
0.005833
0.914067 11
133
P-value
F crit
1.64E-05* 4.066181
Table12. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
N
1
1 (T0)
3
3.9133a
2 (T1)
3
3.9933a
4 (T 3)
3
3 (T 2)
3
2
3
4.2833b
4.5967c
Sig.
0.235
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant difference.
Fig. 7b Details of final mean (g) of freshwater prawn recorded in different tanks during the
experiment
4.8
4.59 c
4.6
weight (g)
4.4
4.24 b
4.2
4
3.91 a
3.99 a
3.8
3.6
3.4
T0
T1
T2
T3
Treatments
Table 13. Details of length (cm) of freshwater prawn recorded in different tanks during
the experiment
Treatments
T0
No.
tanks
1
2
3
of No. of days
0
3.55
3.88
3.84
15
4.18
4.21
4.19
134
30
4.51
4.54
4.53
45
4.72
4.76
4.74
60
5.10
5.15
5.17
T1
T2
T3
Avg.
SE±
1
2
3
Avg.
SE±
1
2
3
Avg.
SE±
3.75
0.1
3.85
3.79
3.62
3.75
0.06
3.81
3.89
3.52
3.74
0.11
4.19
0.00
4.33
4.32
4.20
4.28
0.04
4.31
4.34
4.34
4.33
0.01
4.52
0.00
4.55
4.50
4.45
4.50
0.02
4.59
4.55
4.60
4.58
0.01
4.74
0.01
4.77
4.76
4.75
4.76
0.00
4.78
4.79
4.81
4.79
0.00
1
2
3
Avg.
SE±
3.59
3.86
3.87
3.77
0.09
4.29
4.30
4.35
4.31
0.01
4.55
4.51
4.58
4.54
4.72
4.73
4.75
4.73
0.00
0.02
5.14
0.02
5.23
5.21
5.17
5.20
0.01
5.31
5.36
5.27
5.31
0.02
5.09
5.13
5.10
5.10
0.01
Fig.8 Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
6
Length (cm)
5
4
3
2
1
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 14a. Survival, Weight gain, SGR, FCR, PER of M. rosenbergii in different treatments
during the first experiment.
135
Treatments Replication
T0
T1
1
2
3
Avg
±SE
1
2
3
Avg
±SE
T2
T3
Avg
±SE
1
2
3
Avg
±SE
Survival
No.
%
37
35
38
36.66
2.45
38
36
39
37.66
2.54
42
40
43
41.66
2.94
42
38
37
39
2.71
74.00
70.00
76.00
73.32
2.02
76.00
72.00
78.00
75.32
2.16
84.00
80.00
86.00
83.37
5.92
84.00
76.00
74.00
78.00
4.23
SGR
FCR
PER
Weight
gain(g)
2.49
2.48
2.51
2.50
0.00
2.55
2.53
2.53
2.53
0.00
2.77
2.77
2.77
2.77
0.00
2.65
2.66
2.61
2.65
0.00
2.20
2.25
2.14
2.19
0.03
2.18
2.17
2.20
2.19
0.00
1.86
1.87
1.85
1.86
0.00
1.96
2.00
2.08
2.01
0.03
1.289
1.267
1.332
1.301
0.01
1.308
1.311
1.295
1.304
0.00
1.529
1.525
1.542
1.533
0.00
1.456
1.423
1.368
1.421
0.00
2.99
3.07
3.06
3.04
0.02
3.19
3.11
3.07
3.12
0.03
3.72
3.81
3.65
3.72
0.04
3.36
3.48
3.38
3.41
0.03
Table 14b. Growth parameters in freshwater prawn M. rosenbergii fed supplemented diets
with different levels of marigold oleoresin
136
Growth Indices
T0
(Control)
T1
(60 mg kg-1)
T2
(12o mg kg-1)
T3
(180 mg kg-1)
Initial Weight (g)
0.87±0.00
0.87±.00
0.87±0.01
0.87±0.00
Final Weight (g)
3.91±0.04
3.99±0.04
4.59±0.05
4.28±0.04
Mean Weight Gain (g)
(MWG)
3.04±0.02
3.12±0.03
3.72±0.04
3.41±0.03
Per Day Growth(g)
(PDG)
0.050
0.052
0.062
0.056
Feed Efficiency Rate
(FER)
0.455±0.00
0.456±0.00
0.536±0.00
0.497±0.00
Feed Conversion Ratio
(FCR)
2.19±0.03
2.19±0.00
1.86±0.00
2.01±0.03
Specific Growth Rate (%)
(SGR)
2.50±0.00
2.53±0.00
2.77±0.00
2.65±0.01
Protein Efficiency Rate
(PER)
1.301±0.01
1.304±0.00
1.533±0.00
1.421±0.02
Survival Rate (%)
(SR)
73.32
75.32
83.32
78.00
Percentage of Mean Weight
Gain (%) (PMWG)
349.42
358.62
427.58
391.95
Condition Factor
(CF)
2.87
2.83
3.06
3.19
Daily Growth Rate
(DGR)
5.82
5.97
7.12
6.53
Daily Growth Index
(DGI)
1.035
1.053
1.178
1.115
Growth Coefficient (%)
(GC)
2.233
2.273
2.543
2.406
137
Table 14b-1. Analysis of variance for specific growth rate of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.136467 3
Within Groups 0.002133 8
Total
*P>0.05
0.1386
MS
F
P-value
1.37E0.045489 170.5833 07*
0.000267
F crit
4.066181
11
Table 14b-2 . Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
2.5a
2 (T1)
3
4 (T3)
3
3 (T2)
3
Sig.
2
3
4
2.53b
2.6400c
2.7700d
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicates significant difference.
Fig. 9 Specific growth Rate of M. rosenbergii in different treatments and control group
138
2.77 d
2.8
Specific Growth Rate
2.75
2.7
2.64 c
2.65
2.6
2.55
2.53 b
2.5 a
2.5
2.45
2.4
2.35
T0
T1
T2
T3
Treatments
Table 14b-3. Analysis of variance for feed conversion ratio of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.228067 3
Within Groups 0.0142
8
Total
*P>0.05
MS
F
P-value
0.076022 42.82942 2.84E-05* 4.066181
0.001775
0.242267 11
Table 14b-4. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
3 (T2)
3
1.8600a
4 (T3)
3
2 (T1)
3
2.1833c
1 (T0)
3
2.1967c
Sig.
F crit
2
3
2.0133b
1.000
1.000
0.708
Means for groups in homogeneous subsets are displayed.
139
Different superscripts indicate significant differences
Fig. 10 Feed conversion Ratio of M. rosenbergii in different treatments and control group
2.3
Feed Conversion Ratio
2.2
2.19 c
2.19 c
2.1
2.01 b
2
1.86 a
1.9
1.8
1.7
1.6
T0
T1
T2
T3
Treatments
Table 14b-5. Analysis of variance for protein efficiency ratio of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.11072 3
Within Groups 0.006441 8
Total
MS
F
P-value F crit
2.2E0.036907 45.83713 05*
4.066181
0.000805
0.117161 11
*P>0.05
Table 14b-6. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
1.2960a
2 (T1)
3
1.3047a
4 (T3)
3
2
1.4157b
140
3
3 (T2)
1.5320c
3
Sig.
0.718
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant difference
Fig.11 Protein efficiency Ratio of M. rosenbergii in different treatments and control group
1.6
1.533 c
Protein Efficiency Ratio
1.55
1.5
1.421 b
1.45
1.4
1.35
1.301 a
1.304 a
T0
T1
1.3
1.25
1.2
1.15
T2
T3
Treatments
Table 14b-7. Analysis of variance for survival rate of M. rosenbergii in different treatments
and control group
ANOVA
Source
of
Variation
SS
Between
Groups
169
Within Groups 112
Total
*P>0.05
281
df
MS
F
P-value
F crit
3
8
56.33333 4.02381 0.051202* 4.066181
14
11
Fig. 12. Survival Rate of M. rosenbergii in different treatments and control group
141
86
83.32 a
84
Survival Rate (%0)
82
80
78 a
78
75.32 a
76
74
73.32 a
72
70
68
T0
T1
T2
T3
Treatments
Table 14b-8. Analysis of variance for feed efficiency ratio of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.013229 3
Within Groups 0.000798 8
Total
MS
0.00441
9.98E-05
F
P-value
2.52E44.20691 05*
0.014027 11
*P>0.05
Table 14b-9. Duncan’s Multiple Range Test
VAR00001
Duncan
142
F crit
4.066181
Subset for alpha = 0.05
Treatments
N
1
2
1 (T0)
3
0.4543a
2 T(T1)
3
0.4567a
4 (T3)
3
3 (T2)
3
3
0.4950b
0.5357c
Sig.
0.782
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant difference
Fig. 13. Feed efficiency Ratio of M. rosenbergii in different treatments and control group
0.56
0.536 c
Feed Efficiency Ratio
0.54
0.52
0.497 b
0.5
0.48
0.46
0.455 a
0.456 a
T0
T1
0.44
0.42
0.4
T2
T3
Treatments
Table 14b-10. Analysis of variance for weight gain (g) of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.869692 3
Within Groups 0.0324
8
Total
*P>0.05
MS
F
P-value
4.04E0.289897 71.57956 06*
0.00405
0.902092 11
143
F crit
4.066181
Table 14b-11. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
2
1 (T0)
3
3.0400a
2 (T1)
3
3.1233a
4 (T3)
3
3 (T2)
3
3
3.4067b
3.7267c
Sig.
0.147
1.000
1.000
Means for groups in homogeneous subsets are displayed
Different superscript indicate significant difference
Fig. 14. Weight gain of freshwater prawn recorded in different tanks during the
experiment
3.72 c
4
3.5
3.04 a
3.12 a
T0
T1
3.41 b
Weight Gain (g)
3
2.5
2
1.5
1
0.5
0
T2
T3
Treatments
Table 14b-12 . Analysis of variance for per day growth of M. rosenbergii in different
treatments and control group
ANOVA
Source
Variation
Between
Groups
of
SS
df
0.000236 3
MS
F
7.86E-05
27.94112 0.000137* 4.066181
144
P-value
F crit
Within Groups
Total
*P<0.01
2.25E-05 8
2.81E-06
0.000258 11
Table 14b-13. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
0.0506a
2 (T1)
3
0.0520a
4 (T3)
3
3 (T2)
3
Sig.
2
3
0.0520b
0.0545b
0.0621c
0.347
0.101
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant difference
Fig. 15. Per day growth of M. rosenbergii in different treatments and control group
0.07
Per Day Growth (G)
0.06
0.062 c
0.0506 a
0.052 ab
T0
T1
0.0568 b
0.05
0.04
0.03
0.02
0.01
0
T2
T3
Treatments
Table 14b-14 . Analysis of variance for percentage of means weight growth of M.
rosenbergii in different treatments and control group
ANOVA
Source
of SS
df
MS
F
P-value
F crit
145
Variation
Between
Groups
Within Groups
Total
*P>0.05
12586.49 3
235.7065 8
12822.2
4195.497 142.3973
29.46331
2.79E07*
4.066181
11
Table 14b-15. Duncan’s Multiple Range Test
VAR00001
Duncan
Treatments
N
Subset for alpha = 0.05
1
1 (T0)
3
2
3
4
3.4811E2
a
2 (T1)
3
3.5898E2
b
4 (T3)
3
3.8864E2
c
3 (T2)
3
4.3173E2
d
Sig.
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicates significant difference
Percentage of Mean Weight Gain (%)
Fig. 16. Percentage of mean weight growth 0f M. rosenbergii in different treatments and
control group
427.58 d
450
400
350
349.42 a
358.62 b
T0
T1
391.95 c
300
250
200
150
100
50
0
T2
Treatments
146
T3
Table 14b-16. Analysis of variance for condition factor of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.275025 3
Within Groups 0.003067 8
Total
*P>0.05
MS
F
P-value
F crit
0.091675 239.1522 3.62E-08*
0.000383
4.066181
0.278092 11
Table14 b-17. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
2 (T1)
3
2.8300a
1 (T0)
3
3 (T2)
3
4 (T3)
3
Sig.
2
3
4
2.8767b
3.0600c
3.2100d
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicates significant difference.
Fig. 17 condition factor of M. rosenbergii in different treatments and control group
147
3.3
3.21 d
Condition Factor
3.2
3.06 c
3.1
3
2.9
2.87 b
2.83 a
2.8
2.7
2.6
T0
T1
T2
T3
Treatments
Table 14b-18. Analysis of variance for daily growth rate of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
3.222833 3
Within Groups 0.052267 8
Total
3.2751
MS
F
P-value
F crit
1.074278 164.4303 1.59E-07*
0.006533
4.066181
11
*P>0.05
Table 14b-19. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1 (T0)
3
5.80a
2 (T1)
3
4 (T3)
3
3 (T2)
3
Sig.
2
3
4
5.9767b
6.4733c
7.1333d
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
148
Different superscript indicates significant difference.
Fig. 18 Daily growth rate of M. rosenbergii in different treatments and control group
8
7.13 d
Daily growth Rate
7
6
5.80 a
5.97 b
T0
T1
6.47 c
5
4
3
2
1
0
T2
T3
Treatments
Table 14b-20. Analysis of variance for daily growth index of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.039332 3
Within Groups 0.000956 8
Total
*P>0.05
MS
F
P-value
0.013111 109.7134 7.73E-07*
0.000119
F crit
4.066181
0.040288 11
Table 14b-21. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1 (T0)
3
1.0320a
2 (T1)
3
4 (T3)
3
2
3
1.0530b
1.1130c
149
4
3 (T2)
1.1790d
3
Sig.
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicates significant difference.
Fig. 19. Daily growth index of M. rosenbergii in different treatments and control group
1.2
1.179 d
Daily Growth Index
1.15
1.113 c
1.1
1.05
1.032 a
1.053 b
1
0.95
T0
T1
T2
T3
Treatments
Table 14b-22. Analysis of variance for growth coefficient of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.183694 3
Within Groups 0.004198 8
Total
*P>0.05
MS
F
P-value
F crit
0.061231 116.6865 6.08E-07*
0.000525
4.066181
0.187892 11
Table 14b-23. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments N
1
2
150
3
4
1 (T0)
3
2 (T1)
3
4 (T3)
3
3 (T2)
3
2.2273a
2.2727b
2.4023c
2.5450d
Sig.
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicates significant difference
Growth Coefficient
Fig. 20 Growth coefficient of M. rosenbergii in different treatments and control group
2.6
2.55
2.5
2.45
2.4
2.35
2.3
2.25
2.2
2.15
2.1
2.05
2.545 d
2.402 c
2.227 a
T0
2.272 b
T1
T2
T3
Treatments
Table 15. Proximate composition of prawn meat taken from different treatments ( % on
dry weight basis) 1st exp.
Treatments
Moisture
(%)
Dry
Crude
Crude fat Ash (%)
matter
protein
(%)
(%)
(%)
T0
76.24
23.76
36.81
2.67
5.6
T1
76.64
23.36
37.2
2.45
5.7
T2
76.76
23.24
36.5
2.55
7.2
T3
75.92
24.08
37.8
2.25
5.3
*Means of three replicates. Moisture and dry matter on wet weight basis
NFE (%)
1.58
1.83
1.46
1.95
Table 16. Mortality and relative percent survival (RPS) of M. rosenbergii recorded in
different treatments and control group after challenged against with A. hydrophila
151
Treatments
Replications
No. of dead
Mortality
(%)
RPS (%)
T0
1
2
Avg
1
2
Avg
1
2
Avg
1
2
Avg
7
8
7.5
6
4
5
5
4
4.5
3
4
3.5
70
80
75
60
40
50
50
40
45
30
40
36
---------------------------------14.29
50
33.34
28.58
50
40
57.15
50
53.34
T1
T2
T3
Table 17. Analysis of variance for mortality of M. rosenbergii recorded in different
treatments and control group after challenged against A. hydrophila infection.
ANOVA
Source
of
Variation
Between Groups
Within Groups
Total
*p<0.05
SS
1737.5
350
2087.5
df
3
4
7
MS
F
P-value
F crit
*
579.1667 6.619048 0.049664 6.591382
87.5
Table 18. Duncan’s Multiple Range Test
VAR00001
152
Duncan
Subset for alpha = 0.05
Treatments
N
1
4 (T3)
2
35.0000a
3 (T2)
2
45.0000a
2 (T1)
1 (T0)
2
2
50.0000a
2
50.0000b
75.0000b
Sig.
0.190
0.056
Means for groups in homogeneous subsets are displayed.
Different superscripts indicate significant differences
Fig. 21. Percentage of mortality recorded in A.hydrophila infected treatments and control
group
80
75 b
70
Mortality (%)
60
50 ab
45 a
50
35 a
40
30
20
10
0
T0
T1
T2
T3
Treatments
Table 19. The prophenol oxidase activity, super oxide anion production, total haemolymph
protein and total heamocytes count recorded in different treatments and control group
after feeding trials
treatments
Replication
prophenol
oxidase
activity
super oxide
anion
production
T0
1
2
0.72
0.65
0.06
0.05
153
total
haemolymph
protein
(mg/ml)
9.3
9.2
total
heamocytes
count
X 106 /ml
6
7.5
T1
T2
T3
Avg
SE±
1
2
Avg
SE±
1
2
Avg
SE±
1
2
Avg
SE±
0.68
0.035
1.104
1.056
1.08
0.024
1.266
1.387
1.32
0.060
1.5
1.4
1.45
0.05
0.05
0.005
1.112
1.234
0.11
0.061
0.93
0.138
0.53
0.396
0.119
0.113
1.173
0.003
9.25
0.05
11.5
11.8
11.65
0.115
11.9
11.9
11.9
0.00
14.2
14.2
14.2
0.00
6.75
0.75
7.8
8.37
8.08
0.285
9.7
11.15
10.42
0.725
14.25
13.87
14.06
0.19
Table 20. Analysis of variance for mean ppo activity of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
MS
F
P-value
F crit
*
Between
0.682843 3
0.227614 57.18058 0.000966 6.591382
154
Groups
Within Groups
Total
*P<0.01
0.015923 4
0.003981
0.698766 7
Table 21. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1
2
0.6850a
2
2
3
2
1.3265c
4
2
1.4500c
Sig.
2
3
1.0800b
1.000
1.000
0.122
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant difference.
Fig. 22. The ppo activity recorded in different treatments and control group after feeding
trials
Optical Density of ppo activity
1.6
1.45 c
1.32 c
1.4
1.08 b
1.2
1
0.68 a
0.8
0.6
0.4
0.2
0
T0
T1
T2
T3
Treatments
Table 22 . Analysis of variance for mean superoxide anion production activity of M.
rosenbergii in different treatments and control group
155
ANOVA
Source
of
Variation
SS
df
Between
Groups
1.59169 3
Within Groups 0.321142 4
Total
*p<0.05
MS
F
P-value
F crit
0.530563 6.608458 0.049792* 6.591382
0.080286
1.912832 7
Table 23. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1
2
0.0550a
2
2
0.1160a
3
2
0.5340a
4
2
Sig.
2
0.5340b
1.1730b
0.172
0.087
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant differences
Fig. 23. The superoxide anion activity recorded in different treatments and control group
after feeding trials
156
1.4
1.173 b
Optical Density of NBT
1.2
1
0.8
0.53 ab
0.6
0.4
0.2
0.11 a
0.05 a
0
T0
T1
T2
T3
Treatments
Table 24. Analyses of variance for mean Total Heamolymph Protein activity of M.
rosenbergii in different treatments and control group
ANOVA
Source
of
Variation
SS
Between
Groups
24.57
Within Groups 0.05
Total
24.62
df
MS
F
P-value
F crit
3
4
8.19
0.0125
655.2
7.73E-06*
6.591382
7
*P>0.05
Table 25 . Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1
2
9.2500a
2
2
11.6500b
3
2
11.9000b
4
2
2
3
14.2000c
Sig.
1.000
0.089
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant differences
157
Fig. 24 The total heamolymph protein recorded in different treatments and control group
after feeding trials
Total Heamolymph Protein
16
14.20 c
14
12
10
11.65 b
11.90 b
T1
T2
9.25 a
8
6
4
2
0
T0
T3
Treatments
Table 26. Analysis of variance for mean Total heamocytes Count activity of M. rosenbergii
in different treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
61.5567 3
Within Groups 2.4109 4
Total
*P<0.01
MS
F
P-value
F crit
20.5189 34.04355 0.00263* 6.591382
0.602725
63.9676 7
Table 27. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1
2
6.7500a
2
2
8.0850a
3
2
4
2
2
3
10.4250b
14.0600c
158
Sig.
0.161
1.000
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant difference
Fig 25. The total heamocytes count recorded in different treatments and control group
after feeding trials
16
14.06 c
Total Heamocytes Count
14
12
10.42 b
10
8.08 a
8
6.75 a
6
4
2
0
T0
T1
T2
T3
Treatments
Experiment 2
Table 28. Proportion of various ingredients used in the preparation of different test diets
Treatments
T0
T1
T2
T3
Fish
Meal
g/kg
200.00
200.00
200.00
200.00
Ground
nut oil
cake
g/kg
300.00
300.00
300.00
300.00
Shrimp
meal
g/kg
Rice
bran
g/kg
Wheat
flour
g/kg
Soya
flour
g/kg
100.00
100.00
100.00
100.00
100.00
99.94
99.88
99.82
150.00
150.00
150.00
150.00
100.00
100.00
100.00
100.00
Vitamin
Mineral
mixture
g/kg
50.00
50.00
50.00
50.00
Diacetate of
luteinmesozeaxanthin
mg/kg
0.00
60.00
120.00
180.00
Table 29. Proximate composition of experimental diets (% on dry weight basis)
Treatments
Moisture
(%)
Dry
matter
Crude
protein
Crude
fat
159
Crude
fiber
Ash
(%)
NFE
(%)
Energy
Kcal / Kg
T0
6.50
T1
7.24
T2
6.85
T3
7.15
*Means of three replicates
(%)
(%)
(%)
(%)
93.50
92.76
93.15
92.85
35.09
34.95
35.20
34.87
7.52
7.65
7.40
7.76
26.28
27.05
26.15
27.45
15.05
16.10
15.70
15.65
9.56
7.01
8.70
6.12
3054.32
2956.70
3014.80
2926.96
Table 30. Mean values of air and water temperature recorded during the experimental
period
Days after stocking
Air temperature (0C)
Water temperature(0C)
0
15
30
45
60
29.0
28.5
29.0
29.5
29.5
28.5
27.5
28.5
29.0
28.5
Fig. 26 Mean values of air and water temperature recorded during the experimental period
160
Temperature (oc )
30
29.5
29
28.5
28
27.5
27
26.5
1
2
3
4
5
Weeks
Air temp.
Water temp.
Table 31. Values of pH recorded on different sampling days during the experimental
period
Treatment
Replication
T0
R1
R2
R3
Mean±SE
0
days
8.5
7.9
8.2
8.2
± 0.17
No of days
15 days
30 days
8.4
8.6
8.4
8.46
±0.06
161
8.8
8.8
8.6
8.73
±0.06
45 days
60 days
8.3
8.7
8.6
8.53
±0.06
8.6
9.1
9.0
8.9
± 0.15
T1
T2
T3
R1
R2
R3
Mean±SE
8.3
8.4
7.8
8.16
±0.18
8.5
8.6
8.3
8.46 ±0.08
8.7
8.8
8.6
8.7
±0.05
8.4
8.6
8.6
8.53 ±0.06
8.8
9.0
9.1
8.96
±0.08
R1
R2
R3
Mean±SE
8.2
8.0
8.4
8.20
±0.11
8
8.3
8.6
8.43 ±0.08
8.5
8.6
8.5
8.53
±0.03
8.6
8.7
8.2
8.50 ±0.15
8.9
9.1
8.7
8.90
±0.11
R1
R2
R3
Mean±SE
8.3
7.9
8.1
8.10
±0.11
8.2
8.1
8.3
8.20 ±0.05
8.6
8.4
8.6
8.53
±0.06
8.7
8.5
8.4
8.53 ±0.08
9.0
8.9
8.7
8.86
±0.08
PH
Fig. 27. Values of pH recorded on different sampling days during the experimental period
9.2
9
8.8
8.6
8.4
8.2
8
7.8
7.6
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 32. Fluctuation of dissolved oxygen (mg/l) level of water recorded on different
sampling days
No of days
Treatment
Replication
R1
0
days
6.80
15
days
6.9
30
days
8.4
45
days
7.47
60
days
8.6
R2
7.65
7.8
8.1
8.8
7.8
162
T0
R3
Mean±SE
7.50
7.32±
0.26
6.79
6.81
7.75
7.11
±0.31
7.69
6.90
7.57
7.38
±0.24
7.60
7.35
7.10
7.35
±o.14
R1
R2
R3
Mean±SE
T1
R1
R2
R3
Mean±SE
T2
R1
R2
R3
Mean±SE
T3
7.5
7.4±0.
26
6.85
7.40
7.60
7.28
±0.22
7.80
7.40
7.50
7.56
±0.12
7.50
7.49
7.38
7.45
±0.03
7.78
8.09±
0.18
8.30
8.00
7.65
7.98
±0.18
8.10
7.90
7.85
7.95
±0.07
8.20
7.95
8.00
8.05
±0.07
8.2
8.16±
0.38
7.81
8.65
8.15
8.20
±0.24
8.60
8.25
8.00
8.28
±0.17
8.45
8.35
7.85
8.21
±0.18
8.2
8.2±0.
23
8.30
8.45
8.20
8.31
±0.07
8.65
8.10
8.10
8.28
±0.18
8.60
8.20
7.90
8.23
±0.20
Dissolved oxygen (mg/l)
Fig. 28 Fluctuation of dissolved oxygen (mg/l) level of water recorded on different sampling
days.
8.4
8.2
8
7.8
7.6
7.4
7.2
7
6.8
6.6
6.4
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 33. Fluctuation of carbon dioxide (mg/l) level of water recorded on different
sampling days.
Treatment
Replication
No of days
163
R1
R2
R3
Mean±SE
T0
R1
R2
R3
Mean±SE
T1
R1
R2
R3
Mean±SE
T2
R1
R2
R3
Mean±SE
T3
0
days
15
days
30
days
45
days
60
days
0.8
0.5
0.4
0.56
±0.12
0.6
0.4
0.7
0.56
±0.08
0.5
0.6
0.8
0.63
±0.08
0.4
0.7
0.8
0.63
±0.12
0.9
0.7
0.6
0.73
±0.08
0.8
0.6
0.6
0.66
±0.06
0.7
0.8
0.8
0.76
±0.03
0.6
0.8
0.9
0.76
±0.08
0.8
0.5
1.1
0.80
±0.17
0.9
0.4
0.9
0.73
±0.16
0.7
0.9
1.1
0.9
±0.11
0.7
0.9
1.1
0.9
±0.11
0.9
0.7
1.0
0.86
±0.08
1.0
0.6
1.0
0.86
±0.13
0.8
1.0
1.0
0.93
±0.06
0.9
1.0
1.1
1.0
±0.05
1.2
1.1
1.4
1.23
±0.08
1.2
0.9
1.3
1.13
±0.12
0.9
1.2
1.1
1.06
±0.08
1.1
1.2
1.4
1.23
±0.08
Fig. 29. Fluctuation of carbon dioxide (mg/l) level of water recorded on different sampling
days
Carbon dioxide (mg/l)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1
2
3
4
Weeks
T0
T1
T2
T3
164
5
Table 34. Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on
different sampling days
No of days
Treatment
T0
T1
Replication
0
days
R1
0.01
0.01
0.02
0.07
0.07
R2
R3
Mean±SE
0.02
0.01
0.01
±0.00
0.02
0.01
0.02
0.01
±0.00
0.02
0.02
0.07
0.03
0.03
±0.01
0.03
0.05
0.04
0.04
±0.00
0.06
0.05
0.04
0.04
0.05
±0.00
0.04
0.06
0.02
0.04
±0.01
0.04
0.07
0.05
0.03
0.05
±0.01
0.06
0.05
0.04
0.05
±0.00
0.05
0.07
0.09
0.10
0.08
±0.00
0.08
0.06
0.07
0.07
±0.00
0.07
0.09
0.01
0.01
±0.00
0.03
0.02
0.01
0.02
±0.00
0.03
0.04
±0.00
0.07
0.04
0.02
0.04
±0.01
0.01
0.04
±0.01
0.06
0.04
0.03
0.04
±0.00
0.05
0.05
±0.00
0.05
0.07
0.04
0.05
±0.00
0.08
0.08
±0.00
0.09
0.10
0.06
0.08
±0.01
R1
R2
R3
Mean±SE
R1
R2
T2
T3
R3
Mean±SE
R1
R2
R3
Mean±SE
15 days 30 days 45 days 60 days
Fig. 30. Fluctuation of ammonia levels (µg-at NH3-N/l) level of water recorded on different
sampling days
165
)
Amoonia (µg-at NH3-N/l
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 35. Fluctuation of total alkalinity (mg/l) level of water recorded on different sampling
days.
No of days
Treatment
Replication
0
days
15
days
30
days
45
days
60
days
T0
R1
R2
R3
Mean±SE
75.4
72.6
70.5
72.8
±1.41
78.6
75.4
77.5
77.16
±0.93
79.2
74.2
79.3
77.56
±1.68
81.2
78.9
82.1
80.73
±0.95
83.79
83.79
80.6
82.72
±1.06
R1
R2
R3
Mean±SE
73.6
72.1
75.7
73.8
±1.04
74.4
70.8
72.1
72.43
±1.05
75.2
73.1
71.0
73.10
±1.21
76.9
74.9
77.3
76.36
±0.74
78.5
75.0
74.7
76.06
±1.21
76.8
77.7
74.9
76.46
±0.82
77.6
76.2
79.7
77.83
±1.01
79.3
77.1
76.2
77.53
±0.92
78.4
79.9
76.0
78.10
±1.13
80.3
79.1
81.5
80.30
±0.69
82.3
79.5
80.2
80.66
±0.84
80.1
81.6
77.1
79.60
±1.32
81.4
82.6
82.0
82.00
±0.34
81.7
82.5
80.7
81.63
±0.52
83.6
82.5
80.2
82.10
±1.00
T1
T2
T3
R1
R2
R3
Mean±SE
R1
R2
R3
Mean±SE
166
Fig. 31. Fluctuation of total alkalinity (mg/l) level of water recorded on different sampling
days
84
Total Alkalinity (mg/l)
82
80
78
76
74
72
70
68
66
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 36. Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
Treatments
T0
T1
T2
T3
No. of
tanks
1
2
3
Avg.
SE±
0
0.338
0.344
0.362
0.348
1
2
3
Avg.
SE±
0.00
0.329
0.360
0.352
0.347
0.00
1
2
3
Avg.
SE±
1
2
3
Avg.
0.379
0.327
0.338
0.348
0.01
0.336
0.342
0.365
0.347
15
0.537
0.554
0.677
0.589
0.10
No. of days
30
0.742
0.695
0.786
0.741
0.02
0.710
0.561
0.697
0.656
0.04
0.937
0.916
0.895
0.916
0.01
0.699
0.641
0.736
0.692
0.02
0.695
0.745
0.714
0.718
0.959
0.954
0.988
0.967
0.01
0.996
1.048
1.085
1.043
167
45
0.979
0.997
0.982
0.986
0.00
60
1.093
1.275
1.325
1.225
0.07
1.100
1.045
1.260
1.135
0.06
1.210
1.249
1.390
1.283
0.05
1.103
1.295
1.515
1.304
1.265
1.450
1.545
1.420
0.08
1.414
1.520
1.620
1.518
0.05
1.433
1.612
1.710
1.585
SE±
0.00
0.01
0.01
0.11
0.08
Fig. 32a Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment (Increment).
1.8
1.6
Weight (g)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 37. Analyses of variance for final mean weight (g) of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
MS
F
P-value
F crit
Between
Groups
0.213543 3
0.071181 4.345339 0.042896* 4.066181
Within Groups 0.131048 8
0.016381
Total
0.344591 11
*P<0.05
Table 38. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Trearment
N
1
2
1 (T0)
3
1.2310a
2 (T1)
3
1.4200a
168
1.4200b
3 (T2)
3
1.5180b
4 (T3)
3
1.5850b
Sig.
0.108
0.168b
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant differences
Fig. 32b Details of weight (g) of freshwater prawn recorded in different tanks during the
experiment
1.8
1.6
Weight (g)
1.4
1.420 ab
1.518 b
1.585 b
1.231 a
1.2
1
0.8
0.6
0.4
0.2
0
T0
T1
T2
T3
Treatments
Table 39. Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
Treatments
T0
T1
T2
No. of
tanks
1
2
3
Avg.
SE±
1
2
3
Avg.
SE±
1
2
3
Avg.
0
3.10
2.93
3.10
3.04
0.05
3.07
2.59
3.09
2.91
0.16
2.94
2.93
2.99
2.95
15
3.20
3.24
3.30
3.24
0.02
3.26
3.10
3.28
3.21
0.05
3.24
3.37
3.26
3.29
169
No. of days
30
3.52
3.42
3.50
3.48
0.03
3.64
3.19
3.50
3.44
0.13
3.36
3.60
3.94
3.63
45
3.75
3.60
3.81
3.72
0.06
3.89
3.39
3.96
3.74
0.17
3.88
3.80
4.25
3.97
60
4.00
3.88
4.12
4.00
0.06
4.25
3.77
4.27
4.09
0.16
4.15
4.13
4.53
4.27
SE±
0.01
2.86
2.86
3.15
2.95
0.09
1
2
3
Avg.
SE±
T3
0.04
0.16
0.13
0.13
3.38
3.27
3.40
3.35
0.04
3.66
3.74
3.97
3.79
0.09
3.95
4.02
4.27
4.08
0.09
4.50
4.30
4.58
4.46
0.08
Length (cm)
Fig. 33 Details of length (cm) of freshwater prawn recorded in different tanks during the
experiment
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
1
2
3
4
5
Weeks
T0
T1
T2
T3
Table 40a. Survival, Weight gain, SGR, FCR, PER of M. rosenbergii in different treatments
during the first experimental period
Treatments Replication
T0
T1
1
2
3
Avg
±SE
1
2
3
Avg
±SE
Survival
No.
%
35
33
36
34.66
2.15
39
32
40
37
2.52
70.00
66.00
72.00
69.32
2.71
78.00
64.00
80.00
74.00
2.11
SGR
FCR
PER
Weight
gain(g)
1.95
2.18
2.16
2.09
0.07
2.24
2.32
2.46
2.39
0.06
3.05
2.52
2.64
2.73
0.16
2.76
2.46
2.25
2.46
0.14
0.934
1.131
1.079
1.043
0.05
1.034
1.161
1.266
1.159
0.06
0.755
0.931
0.963
0.877
0.06
0.936
1.09
1.193
1.073
0.07
170
T2
T3
1
2
3
Avg
±SE
1
2
3
Avg
±SE
37
41
38
38.66
2.68
43
39
38
40
2.76
74.00
82.00
76.00
77.32
4.10
86.00
78.00
76.00
80.00
4.12
2.19
2.55
2.57
2.45
0.12
2.41
2.58
2.57
2.53
0.05
2.66
2.12
2.25
2.32
0.16
2.28
2.24
2.29
2.26
0.01
1.071
1.344
1.267
1.226
0.08
1.249
1.273
1.244
1.259
0.00
1.035
1.193
1.282
1.170
0.07
1.097
1.270
1.345
1.238
0.07
Table 40b. Growth parameters in freshwater prawn M. rosenbergii fed supplemented diets
with different levels of Diacetete of Lutein-Mesozeaxanthin
Growth Indices
T0
(Control)
T1
(60 mg kg-1)
T3
(180 mg kg-1)
0.347±0.00
T2
(12o mg kg1)
0.348±0.00
Initial Weight (g)
0.348±0.00
Final Weigh (g)
1.225±0.07
1.420±0.08
1.518±0.05
1.585±0.08
171
0.347±0.00
Mean Weight Gain (g)
(MWG)
0.877±0.06
1.073±0.07
1.170±0.07
1.238±0.07
Per Day Growth(g)
(PDG)
0.0146
0.0178
0.0195
0.020
Feed Efficiency Rate
(FER)
0.365±0.02
0.405±0.02
0.429±0.02
0.440±0.00
Feed Conversion Ratio
(FCR)
2.73±0.16
2.46±0.14
2.32±0.16
2.26±0.01
Specific Growth Rate (%)
(SGR)
2.09±0.07
2.39±.06
2.45±0.12
2.53±0.05
Protein Efficiency Rate
(PER)
1.043±0.05
1.159±0.06
1.226±0.08
1.259±0.00
Survival Rate (%)
(SR)
69.32
74.00
77.32
80.00
Percentage of Mean Weight
Gain (%) (PMWG)
252.01
309.22
336.2
356.77
Condition Factor
(CF)
1.91
2.07
1.94
1.78
Daily Growth Rate
(DGR)
4.20
5.15
5.60
5.94
Daily Growth Index
(DGI)
0.61
0.70
0.74
0.77
Growth Coefficient (%)
(GC)
1.288
1.482
1.570
1.630
Table 40b-1. Analysis of variance for specific growth rate of M. rosenbergii in different
treatments and control group
ANOVA
Source
Variation
Between
Groups
of
SS
df
0.302033 3
MS
F
P-value
F crit
0.100678 4.824814 0.033387* 4.066181
172
Within Groups
Total
*P<0.05
0.166933 8
0.020867
0.468967 11
Table 40b-2. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1 (T0)
3
2.0967a
2 (T1)
3
2.3400a
3 (T2)
3
2.4367b
4 (T3)
3
2.5200b
2
2.3400b
Sig.
0.073
0.181
Means for groups in homogeneous subsets are displayed.
Fig. 34. Specific growth Rate 0f M. rosenbergii in different treatments and control group
3
Specifi Growth Rate
2.5
2.34 ab
2.43 b
2.52 b
T1
T2
T3
2.09 a
2
1.5
1
0.5
0
T0
Treatments
Table 40b-3. Analysis of variance for feed conversion ratio of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
MS
F
P-value
F crit
*
Between
0.381467 3
0.127156 2.280135 0.156283 4.066181
173
Groups
Within Groups
Total
*P>0.05
0.446133 8
0.8276
0.055767
11
Fig. 35 Feed conversion Ratio 0f Macrobrachium rosenbergii in different treatments and
control group.
3
2.73 a
2.46 a
Feed Conversion Ratio
2.5
2.32 a
2.26 a
T2
T3
2
1.5
1
0.5
0
T0
T1
Treatments
Table 40b-4. Analysis of variance for protein efficiency ratio of M. rosenbergii in different
treatments and control group
ANOVA
174
Source
of
Variation
SS
df
Between
Groups
0.077145 3
Within Groups 0.087944 8
Total
*P>0.05
MS
F
P-value
F crit
0.025715 2.339213 0.149717* 4.066181
0.010993
0.165089 11
Fig. 36 protein efficiency Ratio of M. rosenbergii in different treatments and control group
1.4
Protein Efficiency Ratio
1.2
1.159 a
1.226 a
1.259 a
T2
T3
1.043 a
1
0.8
0.6
0.4
0.2
0
T0
T1
Treatments
175
Table 40b-5. Analysis of variance for survival ratio of M. rosenbergii in different treatments
and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
190.3333 3
Within Groups 261.3333 8
Total
*P>0.05
MS
F
P-value
F crit
63.44444 1.942177 0.201404* 4.066181
32.66667
451.6667 11
Fig. 37 Survival Ratio of M. rosenbergii in different treatments and control group.
82
80.00 a
80
77.32 a
78
Survival Rateio(%)
76
74.00 a
74
72
70
69.32 a
68
66
64
62
T0
T1
T2
Treatments
176
T3
Table 40b-6. Analysis of variance for feed efficiency ratio of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.009456 3
Within Groups 0.010813 8
Total
*P>0.05
MS
F
P-value
F crit
0.003152 2.331833 0.150518* 4.066181
0.001352
0.020269 11
Fig. 38 Feed Efficiency Ratio of M. rosenbergii in different treatments and control group.
0.5
Feed Efficiency Ratio
0.45
0.4
0.405 a
0.429 a
0.44 a
T2
T3
0.365 a
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
T0
T1
Treatments
177
Table 40b-7. Analysis of variance for weight gain (g) of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.213727 3
Within Groups 0.122197 8
Total
*P<0.05
MS
F
P-value
F crit
0.071242 4.66411 0.036252* 4.066181
0.015275
0.335924 11
Table 40b-8. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
0.8830a
2 (T1)
3
1.0730a
3 (T2)
3
1.1700b
4 (T3)
3
1.2373b
Sig.
0.096
2
1.0730b
0.157
Means for groups in homogeneous subsets are displayed.
Fig. 39 Weight gain of freshwater prawn recorded in different tanks during the
experiment.
178
1.4
Weight gain (g)
1.2
1
1.17 b
1.073 ab
1.237 b
0.883 a
0.8
0.6
0.4
0.2
0
T0
T1
T2
T3
Treatments
Table 40b-9. Analysis of variance for per day growth of M.
treatments and control group.
ANOVA
Source
Variation
of
Between Groups
Within Groups
Total
*P<0.05
rosenbergii in different
SS
5.92E05
3.39E05
9.31E05
df
3
8
MS
1.97E05
4.24E06
F
P-value
F crit
4.646963 0.036575* 4.066181
11
Table 40b-10 . Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
0.0147a
2 (T1)
3
0.0178a
3 (T2)
3
0.0194b
4 (T3)
3
0.0206b
Sig.
0.096
2
0.0178b
0.158
Means for groups in homogeneous subsets are displayed.
179
Fig. 40 Per day growth of M. rosenbergii in different treatments and control group
0.025
Per Day Growth (g)
0.02
0.015
0.0194 b
0.0178 ab
0.0206 b
0.0147 a
0.01
0.005
0
T0
T1
T2
T3
Treatments
Table 40b-11. Analysis of variance for percentage of mean weight gain of M. rosenbergii in
different treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
18157.59 3
Within Groups 10789.36 8
Total
*P<0.05
MS
F
P-value
F crit
6052.531 4.487779 0.039755* 4.066181
1348.67
28946.95 11
Table 40b-12. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
253.3400a
2 (T1)
3
308.7267a
3 (T2)
3
339.0633b
4 (T3)
3
355.4367b
180
2
308.7267b
Sig.
0.102
0.173
Means for groups in homogeneous subsets are displayed.
Percentage of mean weight gain (%)
Fig. 41 Percentage of mean weight growth of M. rosenbergii in different treatments and
control group.
400
350
300
339.06 ab
308.72 ab
355.43 b
253.34 a
250
200
150
100
50
0
T0
T1
T2
T3
Treatments
Table 40b-13. Analysis of variance for condition factor of M. rosenbergii in different
treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.155005 3
Within Groups 0.890404 8
Total
*P>0.05
MS
F
P-value
F crit
0.051668 0.464223 0.715136* 4.066181
0.111301
1.045409 11
Fig. 42. condition factor of M. rosenbergii in different treatments and control group.
181
2.075 a
2.1
2.05
Condition factor
2
1.95
1.949 a
1.914 a
1.9
1.85
1.786 a
1.8
1.75
1.7
1.65
1.6
T0
T1
T2
T3
Treatments
Table 40b-14. Analysis of variance for daily growth ratio of M. rosenbergii in different
treatments and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
5.042025 3
Within Groups 2.9884
8
Total
MS
F
P-value
F crit
1.680675 4.499197 0.039516* 4.066181
0.37355
8.030425 11
*P<0.05
Table 4ob-15. Duncan’s Multiple Range Test
VAR00001
182
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
3
4.2200a
2 (T1)
3
5.1400a
3 (T2)
3
5.6500b
4 (T3)
3
5.9200b
Sig.
0.102
2
5.1400b
0.172
Means for groups in homogeneous subsets are displayed.
Fig. 43 Daily growth ratio of M. rosenbergii in different treatments and control group.
7
Daily Growth ratio
6
5
5.92 b
5.65 b
5.14 ab
4.22 a
4
3
2
1
0
T0
T1
T2
T3
Treatments
Table 40b-16. Analysis of variance for daily growth index of M. rosenbergii in different
treatments and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.042194 3
Within Groups 0.023119 8
Total
*p<0.05
MS
F
P-value
F crit
0.014065 4.866947 0.032683* 4.066181
0.00289
0.065313 11
183
Table 40b-17. Duncan’s Multiple Range Test
VAR00001
Duncan
Treatment
s
N
Subset for alpha = 0.05
1 (T0)
3
0.6130a
2 (T1)
3
0.7000a
3 (T2)
3
0.7430b
4 (T3)
3
0.7693b
1
2
0.7000b
Sig.
0.083
0.168
Means for groups in homogeneous subsets are displayed.
Fig. 44 Daily growth index of M. rosenbergii in different treatments and control group.
0.9
Daily Growth Index
0.8
0.7
0.700 ab
0.743 b
0.769 b
T2
T3
0.61 a
0.6
0.5
0.4
0.3
0.2
0.1
0
T0
T1
Treatments
Table 40b-18. Analysis of variance for growth coefficient of M. rosenbergii in different
treatments and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.187703 3
Within Groups 0.102914 8
MS
F
P-value
F crit
0.062568 4.863683 0.032737* 4.066181
0.012864
184
Total
*P<0.05
0.290617 11
Table 40b-19. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments N
1
2
1 (T0)
3
1.2957a
2 (T1)
3
1.4793a
3 (T2)
3
1.5700b
4 (T3)
3
1.6253b
1.4793b
Sig.
0.083
0.169
Means for groups in homogeneous subsets are displayed.
Fig. 45 Growth coefficient of M. rosenbergii in different treatments and control group.
1.8
Growth Coefficient
1.6
1.4
1.479 ab
1.625 b
1.570 b
1.295 a
1.2
1
0.8
0.6
0.4
0.2
0
T0
T1
T2
T3
Treatments
Table 41. Proximate composition of prawn meat taken from different treatments (dry
weight basis).
Treatment
Moisture
(%)
T0
80.86
Dry
matter
(%)
19.14
Crude
protein
(%)
21.80
185
Crude fat
(%)
Ash (%)
NFE (%)
1.8
5.3
1.3
T1
78.59
21.41
22.73
1.5
4.9
T2
78.80
21.20
22.95
1.7
5.8
T3
78.46
21.54
22.42
1.3
5.2
*Means of three replicates. Moisture and dry matter on wet weight basis
1.6
1.5
1.4
Table 42. Mortality and relative percent survival (RPS) of M. rosenbergii recorded in
different treatments and control group after challenged against with A. hydrophila
Treatments
Replications
No. of dead
Mortality
(%)
RPS (%)
T0
1
2
Avg
1
2
Avg
1
2
Avg
1
2
Avg
8
7
7.5
2
4
3
5
5
5
8
6
7
80
70
75
20
40
30
50
50
50
80
60
70
---------------------------------75
42.86
60
37.5
28.58
33.34
0
14.29
6.67
T1
T2
T3
Table 43. Mortality of M. rosenbergii recorded in different treatments and control group
after challenged against A. hydrophila infection
ANOVA
Source
Variation
of
SS
df
MS
F
186
P-value
F crit
Between
Groups
Within Groups
Total
*P<0.05
2537.5
450
3
4
2987.5
7
845.8333 7.518519 0.040341* 6.591382
112.5
Table 44. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
2 (T1)
2
30.0000a
3 (T2)
2
50.0000a
4 (T3)
2
70.0000b
1 (T0)
2
75.0000b
Sig.
0.132
2
50.0000b
0.082
Means for groups in homogeneous subsets are displayed.
Fig. 46. Percentage of mortality recorded in A. hydrophila infected treatments and control
group
80
75 b
70 b
70
Mortality (%)
60
50 ab
50
40
30 a
30
20
10
0
T0
T1
T2
Treatments
187
T3
Table 45. The prophenol oxidase activity, super oxide anion production, total haemolymph
protein and total heamocytes count recorded in different treatments and control group
after feeding trials
treatments
Replication
prophenol
oxidase
activity
super oxide
anion
production
T0
1
2
Avg
SE±
1
2
Avg
SE±
1
2
Avg
SE±
1
2
Avg
SE±
0.292
0.256
0.27
0.018
1.34
1.435
1.38
0.047
0.567
0.575
0.57
0.004
0.539
0.560
0.54
0.10
0.097
0.083
0.09
0.007
0.54
0.662
0.6
0.061
0.158
0.13
0.14
0.014
0.108
0.127
0.11
0.009
T1
T2
T3
188
total
haemolymph
protein
(mg/ml)
9.45
9.27
9.36
0.09
12.6
13
12.8
0.2
11.3
11.1
11.45
0.1
11.5
11.3
11.2
0.1
total
heamocytes
count
X 106 /ml
5.58
6.82
6.2
0.62
15.36
14.3
14.83
0.53
9.26
10.01
9.63
0.375
8.14
8.3
8.22
0.08
Table 46. Analysis of variance for mean ppo activity of M. rosenbergii in different
treatments and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
1.386685 3
Within Groups 0.005413 4
Total
*P>0.05
MS
F
0.462228 341.5691
0.001353
P-value
2.83E05*
F crit
6.591382
1.392098 7
Table 47. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments N
1
2
3
0.2740a
1 (T0)
2
4 (T3)
2
0.5495b
3 (T2)
2
0.5710b
2 (T1)
2
1.3875c
Sig.
1.000
0.590
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate which there are significant difference
Fig. 47. The ppo activity recorded in different treatments and control group after feeding
trials
189
1.6
1.38 c
1.4
ppo activity
1.2
1
0.8
0.6
0.4
0.57 b
0.54 b
T2
T3
0.27 a
0.2
0
T0
T1
Treatments
Table 48. Analysis of variance for mean superoxide anion production activity of M.
rosenbergii in different treatments and control group
ANOVA
Source
of
Variation
SS
df
Between
Groups
0.354058 3
Within Groups 0.008113 4
Total
*P<0.01
MS
F
P-value
0.118019 58.19141 0.000934* 6.591382
0.002028
0.362171 7
Table 49. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatments
N
1
1 (T0)
2
0.0900a
4 (T3)
2
0.1175a
3 (T2)
2
0.1440a
2 (T1)
2
Sig.
F crit
2
0.6010b
0.303
190
1.000
Means for groups in homogeneous subsets are displayed.
Fig. 48 The superoxide anion recorded in different treatments and control group after
feeding trials
superoxide anion production
0.7
0.6 b
0.6
0.5
0.4
0.3
0.2
0.1
0.14 a
0.09 a
0.11 a
0
T0
T1
T2
T3
Treatments
Table 50. Analysis of variance for mean Total Heamolymph Protein activity of M.
rosenbergii in different treatments and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
11.9704 3
Within Groups 0.1362 4
Total
*P<0.01
MS
F
P-value
F crit
3.990133 117.1845 0.000236* 6.591382
0.03405
12.1066 7
Table 51. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1 (T0)
2
9.3600a
4 (T3)
2
11.2000b
3 (T2)
2
11.4000b
191
2
3
2 (T1)
12.8000c
2
Sig.
1.000
.339
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant differences
Fig. 49 The T.H.P recorded in different treatments and control group after feeding trials
Total Heamolymph Protein
14
12.8 c
12
10
11.4 b
11.2 b
T2
T3
9.36 a
8
6
4
2
0
T0
T1
Treatments
Table 52. Analysis of variance for mean Total heamocytes Count activity of M. rosenbergii
in different treatments and control group.
ANOVA
Source
of
Variation
SS
df
Between
Groups
81.51944 3
Within Groups 1.62465 4
Total
*P<0.01
MS
F
P-value
F crit
27.17315 66.90215 0.000711* 6.591382
0.406163
83.14409 7
Table 53. Duncan’s Multiple Range Test
VAR00001
Duncan
Subset for alpha = 0.05
Treatment
s
N
1
1 (T0)
2
6.2000a
192
2
3
4 (T3)
2
8.2200b
3 (T2)
2
9.6350b
2 (T1)
2
14.8300c
Sig.
1.000
0.091
1.000
Means for groups in homogeneous subsets are displayed.
Different superscript indicate significant differences
Fig. 50 The T.H.C recorded in different treatments and control group after feeding trials
14.83 c
16
Tota Heamocytes Count
14
12
9.63 b
10
8
8.22 b
6.20 a
6
4
2
0
T0
T1
T2
Treatments
193
T3
V. DISCUSSION
5.1 Proximate composition of ingredients and experimental diets
Proximate composition of all ingredients was analyzed before formulation of
experimental diets. The level of protein in fishmeal used for the preparation of test diets
was 62.28%. National Research Council (1997) considers fish meal with protein content
above 50% and above as of good quality. Soya flour had the highest fat content (19.62
%) while fiber content was highest in rice bran (28.62 %). Fish meal recorded the highest
ash (18.20 %) content. Wheat flour had highest NFE content (77.84 %) followed by
groundnut cake (38.00 %). In the present study, protein content of the diets formulated
for freshwater prawn, Macrobrachium rosenbergii ranged from 34.69 to 34.98 % and
falls in the range of reported dietary requirement for prawns (30 to 40 %) (Bhenana and
Mathews 1995). Feed used in the present study could be considered as optimum protein
level for freshwater prawn, Macrobrachium rosenbergii.
5.2 Proximate composition of prawn muscle
Carcass composition is known to be influenced by many factors such as age, sex,
maturity and feeding conditions. Among these factors, the type and nature of feed
ingested are considered to be the most important (Parove, 1976; Reimers and Meske,
1977; Srikar et al., 1979). But in the present study different levels of feed additives
(caroteniods such as marigold oleoresin and diacetate of lutein meso-zeaxanthin) did not
affect significantly on biochemical composition of prawn muscle. Similar results have
been reported by other workers. Eduardo Aguirre-Hinojosa et al., (2012) have noted that
supplementation of experimental diets with Tagetes erecta extract (Marigold oleoresin)
on Litopenaeus vannamei did not significantly alter the proximate composition of the
194
practical diet because very small amounts were required to attain the desired
concentrations in the feeds (less than 0.5% of the total ingredients). The same results
were reported by Arredondo-Figueroa et al., (2003) and Flores et al., (2007). This is
important in determining the commercial application of Tagetes erecta supplements.
There were no significant differences in proximate composition (crude protein, crude fat,
moisture and ash) of rainbow trout with initial weight of 135 g after 60 days of feeding
with supplementation of astaxanthin diets (Juan et al., 2013). Dietary carotenoid sources
(1.8% marigold flowers, 5% red pepper and 70 mg kg-1 astaxnthin) on rainbow trout
weighing 120.57 g for 60 days did not significantly affect fatty acid composition of the
fish fillets (Yasemen et al., 2013).
5.3 Effect of carotenoids on water quality
5.3.1 Temperature
Temperature is one of the most important environmental variables for aquatic
organisms, because it influences the oxygen content of water. Every aquatic animal has
got its own temperature tolerance limit. Increase in temperature accelerates growth up to
certain level, beyond which it is lethal. Optimum temperature for metabolism varies from
species to species. It influences the survival as well as growth of any organisms
depending on climate, sunlight and depth of water (Banerjee, 1972a). Food intake
increase with increasing temperature up to optimum level as the maintenance energy
requirement increases (Cho and Slinger, 1980). M. rosenbergii can tolerate temperature
range from 18 0C to 32 0C, provided temperature fluctuation is not sudden and longer
duration (Faramanfarmain and Moor, 1980). The water temperature ranged from 27.0 0C
to 28.5 0C in experiment 1 and 27.5 0C to 29.00C in experiment These ranges were within
195
the acceptable limits for growth of freshwater prawn M. rosenbergii reported by
Ravishankar (1983), Jayaram (1998), Ramachandra Naik et al. (2000), Srinivasa (2000)
and Prakash Pavadi (2004).
5.3.2 pH
The pH is an expression of hydrogen ion concentration in water which serves as an
indicator of acidity and alkalinity. It is an important parameter to consider because it
affects metabolism and other physiological processes of aquatic animals. The acute
toxicity of pH on decapod crustaceans has been studied in several species of crayfish
(Morgan and McMahon, 1982; France, 1984; Distefano et al., 1991) and tiger shrimp
Penaeus monodon (Allan and Maguire, 1992). Low pH water has been reported to cause
retarded growth in P. monodon (Allan and Maguire, 1992), disturbed ion regulation in
crayfish and tiger shrimp (Morgan and McMahon, 1982; Allan and Maguire, 1992) and
acid–base imbalance in crayfish and freshwater prawn (Morgan and McMahon, 1982;
Chen and Lee, 1997). However, not much is known regarding the chronic effect of pH on
M. rosenbergii. Ideal range of pH for culture of freshwater prawn, M. rosenbergii is
between 7.0 and 8.5 (Hiseh et al., 1989). In the present investigation, the values of pH
ranged from 7.4 to 8.7 in experiment-1 and 7.8 to 9.1 in experiment-2. The pH values
recorded during the course of investigation fall within the desirable limits for M.
rosenbergii.
5.3.3 Dissolved oxygen (DO)
Dissolved oxygen (DO) is a major limiting factor in aquaculture. Dissolved
oxygen in ponds is very crucial for aquatic life and survival of fishes (Boyd, 1982). For
the best growth of M. rosenbergii the optimum level of dissolved oxygen is 4.65 ppm
196
(TVA, 1978). Avault (1987) reported that dissolved oxygen levels down to 1.0 mg/l can
be tolerated by M. rosenbergii in ponds. Banerjea (1972a) opinied that oxygen
concentration of above 5mg/l is an indication of productivity and below that level the
water is unproductive. Hypoxia or low DO is usually less than 2.8 mg/1 (Diaz and
Rosenberg, 1995). The effect of hypoxia on growth, survival, feeding, molting, behavior,
osmoregulatory capacity, and immune response of penaeid shrimps has been well
documented by some authors (Clark, 1986; Renaud, 1986; Allan and Maguire, 1991;
Charmantier et al., 1994; Moullac et al., 1998; Wannamaker and Rice, 2000; McGraw et
al., 2001; Wu et al., 2002; Pérez-Rostro et al., 2004; Mugnier and Soyez, 2005). It has
been reported that the lethal DO levels ranged from 0.2 to 1.27 mg/1 for a number of
penaeid shrimps. Allan and Maguire (1991) reported that 96-h LC50 of DO for juvenile
Penaeus monodon was estimated to be 0.9 mg/1. Egusa (1961) found that the lethal DO
level for Marsupenaeus japonicus lie between 0.5 and 1.0 mg/1. Martínez et al. (1998)
reported that 48-h LC50 of DO for postlarvae Litopenaeus setiferus was 1.27 mg/1 and
1.16 mg/1 for juveniles at 72-h LC50. Chen and Nan (1992) found that the lethal DO
level for Fenneropenaeus chinensis weighing from 0.31 to 10.54 g was 0.74 mg/1 on the
average. Wu et al. (2002) reported that 8-h LC50 of DO for juvenile Metapenaeus ensis
was 0.77 mg/1. Pérez-Rostro et al. (2004) indicated that the DO level of 0.2 mg/1 was
lethal for the shrimp Litopenaeus vannamei after 1 h of exposure, and Hopkins et al.
(1991) found that the lethal DO level for L. vannamei was about 1 mg/1. The recorded
dissolved oxygen in the present study ranged from 6.9 to 8.1 mg/l in experiment 1 and
6.79 to 8.6 in experiment 2. The recorded levels in the present experiment could be
considered suitable for optimum growth of M. rosenbergii.
197
5.3.4 Free carbon dioxide
The main source of carbon dioxide in pond water is through absorption from the
atmosphere, decomposition of organic matter and respiration of aquatic animals. Aquatic
animals are affected by carbon dioxide depending on the oxygen level of water. If there
is sufficient oxygen in pond water fish can survive at carbon dioxide levels as high as 60
ppm (Hart, 1944). It has been reported that carbon dioxide provides the inorganic carbon
essential for photosynthesis, there by indicate as a decisive factor in organic production
(Swingle, 1961; Murthy and Devaraj, 1991). The recorded free carbon dioxide in the
present study ranged from 0.32 to 1.30 mg/l in experiment 1 and 0.40 to 1.40 mg/l in
experiment 2 considered as acceptable for optimum growth of M. rosenbergii.
5.3.5 Total Alkalinity
Total alkalinity is the total concentration of bases in water expressed in mg/l of
equallient calcium carbonate (Boyd, 1982). It refers to buffering capacity of water.
Natural water that contain 40 mg/l or more total alkalinity consider more productive than
waters of lower alkalinity (Moyele, 1945 and Mair, 1966). The alkalinities in the range of
20-120 mg/l have a little effect on fish production (Banerjea, 1972). The experiments
carried out in indoor circulatory system, the total alkalinity values fluctuated between
73.1 to 86.9 in experiment 1 and 70.5 to 83.79 mg/l. The recorded total alkalinity values
in the present investigation were within the acceptable range for culture of M.
rosenbergii and comparable to those values reported by Jayaram (1998), Ramachandra
Naik et al. (2001), Srinivas (2000) and Prakash Pavadi (2004).
5.3.6 Ammonia – Nitrogen
198
The major end product of protein catabolism is ammonia which is excreted
primarily as un-ionized ammonia by fish. Nitrogenous substances present in the feaces
and uneaten feed reach water and get mineralized rapidly releaseing ammonia in to
water. Boyd (1982) opined that the amount of ammonia reaching pond water through fish
metabolite is proportional to the feeding rate. Unionized ammonia at 100 to 600 µg at
NH3-N/l has been reported to reduce growth of M. rosenbergii by 30% (Wickins, 1976;
Armstrong et al., 1978). In the present investigation ammonia was within the acceptable
range for culture of M. rosenbergii and comparable to that reported by other workers
(Ramachandra Naik et al. 2000; Srinivasa 2000 and Prakash Pavadi 2004).
5.4 Effect of carotenoids on growth parameters of M. rosenbergii
The effects of carotenoids on growth and survival rate of aquatic organisms have
been controversial because several studies have reported a positive influence whereas
some others did not find any effect. Vijay Kumar et al., (2009) have been suggested that
the M. rosenbergii fed with dietary incorporation of astaxanthin (50,100 and 200 mg kg1
) had significant impact on mean weight gain and SGR. The growth rate improved and
moulting cycle shortened in M.rosenbergii post larvae during 20 days rearing when fed
with supplemented of astaxanthin (Petit et al., 1997). Zhang, J. (et al., 2013) have
reported that after 56 days of culture, (Litopeneaus. vannamei) fed astaxanthin (125 and
150 mg kg-1) diets had higher (P< 0.05) weight gain (WG), final wet body weight
(FBW), SGR and had significantly lower FCR than of shrimp Fed control feed. White
shrimp (L. Vannamei) were fed with three different treatments (synthetic astaxanthin,
lutein and astananthin derives from marigold extract), results have shown higher growth,
lower FCR and higher survival on marigold treatments only as compared to other
199
treatments
and
control
group
(Rohriguez
et
al.,
2008
and
/
or
www.google.co.in.Patents/U57383788). Supamattaya, et al., (2005) have investigated the
effect or D. Salina extract (containing β- caroten with different levels of 125, 200 and
300 mg kg -1) on growth of shrimp (P. monodon) with small size (1-2 g body weight) for
8 weeks. Results showed weight gain and survival of shrimp fed diet supplemented with
125 and 300 mg β- caronten / kg diet was significantly higher than the shrimp fed control
diet. However, it was not significantly different in FCR among each treatment. Gupta et
al. (2012) have investigated the effects of H. pluvialis (at the levels of 1, 2, 3 and 4%) on
Macrobrachium dayanum, results revealed that prawns fed with 4% achieved best
growth performance in (WG, SGR, FCR) while feed with 1.0% H. Pluvialis showed
lower growth efficiency. (Niu et al. 2011; Amar et al., 2001 and segner et al., 1981) have
opined that the carotenoids could enhance nutrient utilization and might ultimately
improve growth and play an important role in the intermediary metabolism of aquatic
animals. Kuo- Hsun Chiu, et al. (2014) have evaluated the effect of lycogen TM (a natural
occurring source of carotenoids from photobacterium Rhodobacter sphaeroides WLAPD 911) on red tilapia (Oreochromis mossambicus), and observed that dietary
supplementation with 1.0% of Lycogen did not cause changes in body length but
significantly increased muscle weight, SGR, FCR and weight gain. Amar Edgar, C. et
al., (2012) have demonstrated the effect of synthetic β-carotene, astaxanthin and
canthaxanthin and natural carotenoids (Tagetes erecta, D. salina, phaffiar hodozyma and
c. annuum) in rainbow trout fry weighting 0.11 kg for 6 weeks and results showed no
significant differences in growth performance (SGR, WG, Mean final weight, Total feed
consumed, Feed to gain ratio) among the groups. These results further validated their
200
previous findings both with natural and synthetic sources (Amar, E.C. et al., 2001, 2004).
Similarly, Rehulka (2000) and Yanar et al., (2007) found no effect of synthetic and
natural carotenoids on growth in salmonids. Olsen, R.E. et al., (2006) studied Altantic
Salmon impact of two xanthophylls carotenoid (astaxanthin and lutein) and observed no
signiciant effect on
growth performance such as SGR, WG and condition factor.
Similarily significant effect of marigold (Tagetes erecta) as natural carotenoid source on
growth and survival of gold fish survival rate, growth or feed utilization of the fishes was
observed by Alma, et al., (2013). In the present study, higher final weight was recorded
with the diet having 120 mg kg -1 of marigold oleoresin in the first experiment and there
were no significannt differences among the treatments in the prawn fed Diactate of lutein
meso-zeaxanthin but higher final weight was recorded in T3 which significantly higher
than control groups.
5.4.1 Specific Growth Rate (SGR)
Specific growth rate (SGR % / day) can be taken as an index of growth in the
evaluation of diets. Higher SGR indicates better utilization and efficient conversion of
the feed by prawn (Srinivas, 2000). Vijay Kumar et al., (2009) observed higher SGR in
M. rosenbergii fed astaxanthin incorporated diets than in control. Shrimp (L. vannamei)
fed astaxanthin (125 and 150 mg kg-1) diets had higher (P< 0.05) SGR than that of
shrimp fed control diet Zhang, et al., (2013). Gupta et al., (2012) have investigated the
effect of H. pluvialis (at the levels of 1, 2, 3 and 4%) on Macrobrachium dayanum, and
observed that prawns fed 4% achieved best growth performance in (WG, SGR, FCR)
while feed with 1.0% H. Pluvialis showed lower growth efficiency. In the present study,
higher SGR was recorded with the diet having 120 mg kg
201
-1
of marigold oleoresin in the
first experiment but there were no significannt differences among the treatments in the
prawn fed Diactate of lutein meso-zeaxanthin (Experiment 2), although T2 and T3
significantly higher than control group.
5.4.2 Feed Conversion Ratio (FCR)
Better feed conversion rate or efficiency may be due to increased digestive
enzyme activity (Srinivas, 2000). The high rate of feed intake does not always bring out
an effective growth rate, there by leading to higher FCR (Shigueno et al., 1972). Zhang,
J. et al., (2013) reported that (L. vannamei) fed astaxanthin (125 and 150 mg kg-1)
supplemented diets had significantly lower FCR than that of shrimp fed control feed.
White shrimp (L. Vannamei) were fed with three different treatments (synthetic
astaxanthin, lutein and astananthin derives from marigold extract), results have shown
higher growth, lower FCR and higher survival on marigold treatments only as compared
to other treatments and control group (Rohriguez et al., 2008 and / or
www.google.co.in.Patents/U57383788). Gupta et al., (2012) have investigated the effect
of H. pluvialis (at the levels of 1, 2, 3 and 4%) on Macrobrachium dayanum, and
observed that prawns fed with 4% achieved best growth performance in (WG, SGR,
FCR) while that with 1.0% H. Pluvialis showed lower growth efficiency. In the present
study, lower FCR was recorded with the diet having 120 mg kg
-1
of marigold oleoresin
in the first experiment, however no significant difference between control and treatments
was observed in the second experiment.
5.4.3 Protein Efficiency Ratio (PER)
Protein efficiency ratio gives the idea of the efficiency with which animals are
able to utilize protein and at the same time indicates the quality and level of protein in the
202
diet. PER can be determined by the gain in weight of prawn and the amount of feed
supplied with protein content of feed. Higher values depict efficient utilization of protein
and it changes with the level of protein in the diet. PER increase with a protein level up
to an optimum utilization of the nutrition and decreased thereafter. PER values provide
better indication of the nutritional status of fish with respect to dietary protein than FCR
(Jauncey, 1982). In the present study, higher PER was recorded with the diet having 120
mg kg
-1
of marigold oleoresin in the first experiment and in the second experiment,
lower PER was recorded in control group, but no significant differences between control
and treatments were observed.
5.4.4 Effect of different carotenoids on survival of prawn
In the present study, no significant difference in the survival rate of M.
rosenbergii was observed after 2 months feeding between diets containing marigold
oleoresin in the first experiment and diacetate of lutein meso-zeaxanthin in the second
experiment at 60, 120 and 180 mg kg
-1
and control groups. Similar results of no
significant differences in survival have been observed by the other workers
Boonyarataplin, et al., (2001) in shrimp (P. monodon) after feeding with different
carotenoids (β-caroten or astaxanthin). Similarly Vijay Kumar et al., (2009) did not
observe significant difference between treatments in survival rate of M. rosenbergii
which were fed different levels, of dietary astaxanthin Survival rate of shrimp (L.
vannamei) fed with 125 and 150 mg kg-1 of dietary supplementation of astaxanthin after
feeding trial for 65 days, had no significant difference (P>0.05) among all experimental
groups (Zhang, j. et al., 2013). Chin et al., (2014) have demonstrated that there was no
significant difference between treatment and control groups in terms of survival rate of
203
red tilapia (O. mossambicus) in sea water fed LycogenTM (a commercial carotenoid
product from the probiotic photobacterium Rodobacter spaeeroides WL-ADD 911).
5.5 Resistance of M. rosenbergii to Aeromonas hydrophila infection
Harikrishnan et al., (2012) have demonstrated that enriched with at 0.1% and 1%
(Withnia somnifera). M.rosenbergi fed with and was higher 60% and 70% compared to
that of groups treated 0.01% and control (0%). The mortality was high in control and
0.01% supplementation diets with 85% and 55% against A. hydrophila. The similar
results were reported in other studies in prawns against diets or pathogen (Chand, et al.,
2008; Shankar, et al., 2011, Balasubramanian, et al., 2008, Chand, et al., 2006 and Chiu,
et al., 2010). Chang et al., (2013) observed that dietary administration of water hyacinth
(Eichhornia crassipes) extracts (ECE) at 2.0 and 3.0 g kg-1 for 12 days significantly
increased survival rates of the prawn M. rosenbergii against L. garvieae, suggesting that
ECE administration can enhance prawn resistance to L. garvieae, and can be related to
the dosage of supplementation. Isagani P Angeles Jr. et al., (2009) have been reported
that, carotenoids help to strengthen the immune system of M. rosenbergii under normal
or stress conditions, Furthur, reported that that injected astaxanthin (AX) at 1.34 nmol g1
BW-1 had significant positive effect on the survival of M. rosenbergii, with a survival
improvement of 150% at 4 days and 700% at 6 and 7 days compared with challenge
control. They have demonstrated that AX improved health status of M. rosenbengii in
terms of resistance against L. garvieae. This supports the study of PS
an et al., (2003), in which significantly lower survival (36.5 ± 3.2%) in control
black tiger prawn, P. monodon, than that of fed Ax (45±2.8%) after 48-h bath challenge
with Vibrio damsella. In the present study, addition of marigold oleoresin and diacetate
204
of lutein meso-zeaxanthin enhanced the resistance against
Aeromonas hydrophila
infection.
5.6 Immune parameters of M. rosenbergii
The immune system of crustacean is primarily related to their blood or
hemolymph and to its circulating cells or hemocytes. In M.rosenbergii three types of
hemocytic cells have been identified (Vazqnez et al., 1997). Each has distinctive
morphological features and physiological functions (Moullac et al., 1997 and Johansson
et al., 2000). The blood cells (hemocytes) are mainly responsible for the cellular immune
reactions, such as the phagocytosis of invading microorganisms, their immobilization in
nodular aggregation the encapsulation of large foreign bodies, and healing that is
accompanied by an immediate clotting of the haemolyph as well as release of the
prophenoloxidase (PO) system (Bauchau, 1981; Hose et al., 1990). In crustacea,
melanization occurs when the cellular defence reactions are initiated (So”derha”ll and
Smith, 1986 and Ratcliffe et al., 1985). PO, the key enzyme in the synthesis of melanin,
occurs in haemolymph as an inactive pro-enzyme prophenoloxidase (proPO). Propois
activated to form PO when it reacts with zymosan (carbohydrates from yeast cell walls),
bacterial lipopolysaccharide (LPS), urea, calcium ions, trypsin, or heat, chitin, β-1, 3glucan, ( Soderhall et al., 1986,1984: Shi-Hang Wang et al.,2005: Cheng et al., 2003).
Results from several experiments have demonstrated that apart from their role in
melanization, components of the putative propoactivating system stimulate several
cellular defence reactions, including phagocytosis, nodule formation, encapsulation and
haemocyte locomotion (Johansson et al., 1989 and So”derha”ll and Hall,1984). This
205
proposystem is confined to the semigranular and granular cells (So¨derha¨ll and Smith,
1986).
The activation of this propocascade is exerted by extremely low quantities of
(po/l) of microbial cell wall components lipopolysaccharides LPS, β-1,3-glucans or
peptidoglycans and results in the production of the melanin pigment, which can often be
seen as dark spots in the cuticle of arthropods (Soderhall, 1982; Sugumaran and Kanost,
1993). During the formation of melanin, toxic metabolites are formed which have
fungistatic activity (Soderhall 1990; Nappi and Vass, 1993). Several components or
associated factors of the proposystem have been found to play several important roles in
the defence reactions of the freshwater crayfish, Pacifastacus leniusculus (Soderhall and
Cerenius, 1998; Soderhall et al., 1996). Biochemical studies on shrimp proposystem has
been carried out in Penaeus californiensis (Vargas-Albores et al., 1993a, 1996 ), P.
paulensis (Perazzolo and Barracco, 1997), P. stylirostris (Le Moullac et al., 1997) and P.
monodon (Sritunyalucksana et al., 1999b ). In the penaeid shrimp, enzymes of the
proposystem are localized in the semigranular and granular cells (Vargas-Albores et al.,
1993a; Perazzolo and Barracco, 1997).
5.6.1 Effect of carotenoids on prophenoloxidase system (PPO)
One of the hallmarks of the crustacean immune system is the prophenoloxidase
enzyme cascade. Despite the primitive phylogenetic nature of crustacea, they possess the
complex and effective mechanism like proposystem for eliminating pathogens. Recent in
vitro researches have shown that the Phenoloxidase (PO) activating system and
206
associated factors of proposystem are important activators in crustacean immunity.
Prophenoloxidase system acts as a major recognition and defense pathway in crustaceans
(Johansson et al., 1992). Prophenoloxidase system is activated by several microbial
polysaccharides including ß-1, 3-1, 6-glucan (Soderhall, 1986) and peptidoglycan
(Ashida et al., 1984) or lipopolysaccharide (Soderhall et al., 1984) from bacterial cell
wall. The enzyme is a part of complex system of proteinases, pattern recognition proteins
and protein inhibitors constituting the so called proposystem. The activation of
proposystem results in the production of various proteins including PO, which
participating in melanisation around the parasite, coagulation, opsonisation of foreign
materials and direct microbial killing (Soderhall and Hall, 1984). Activation of the
proposystem which is measured in terms of the PO activity has been used by some
investigators to measure immunostimulation in shrimp (Sung et al., 1994; Devaraja et
al., 1998; Scholz et al., 1999). Therefore in this study, similar method was followed. The
present research studied the effect of marigold oleoresin in the first experiment and
diacetate of lutein meso-zeaxanthin in second experiment at 60, 120 and 180 mg kg-1 on
propoactivation system in M.rosenbergii. Results showed that PO activity is higher in the
carotenoid fed groups compared with control. The similar results were obtained on prawn
(M. rosenbergii) fed with 0.1% and 1.0% doses of Withania somnifera which had
showed increase in PO activity (Ramasamy Harikrishnan et al., 2012). Chin-Chyuan
Chang (et al., 2013) reported that the PO activities of prawns (M. rosenbergii) were
significantly enhanced by ECE- supplemented diets at doses of 2.0 and 3.0 g Kg-1 for 312 days of feeding. Vijay Kumar et al., (2009) have indicated phenoloxidase (PO)
activity was significantly higher (P <0.05) in prawns (M. rosenbergii) fed supplemented
207
diets compared to the control group. Aureli Babin et al., (2010) have reported, a positive
correlation between immune defense and concentration of carotenoids in the hemolymph
in the crustacean. In addition, they also found that dietary carotenoids had clear and
broad immune stimulating effect, enhancing phenoloxidase activity and resistance to a
bacterial infection. PO activity has been employed as one of the important immune
parameters to investigate the status of immune system, immune modulation and disease
resistance in crustacean (Smith et al., 2003) and also Cheng (et al., 2004; Baruah and
Pani Prasad 2001) have reported an enhancement in PO activity which is thought to
enhance the immune ability of animals.
5.6.2 Effect of carotenoids on Nitroblue tetrazolium (NBT)
Secombes et al. (1990) reported that, first product released during the NBT assay
is 02−concentration and it is widely accepted as an accurate parameter quantifying the
intensity of a respiratory burst. This fact suggested that increase in the superoxide anion
is considered to be beneficially protecting disease with respect to increased immunity.
An increase in the superoxide anion production against pathogens is to be beneficial after
exposing shrimp to immunestimulants. Nitroblue tetrazolium (NBT) staining has been
used for both qualitative and quantitative analyses of superoxide anion generated by
haemocytes (Holmblad and Soderhall, 1999). The oxygen-dependent defence mechanism
of mammalian phagocytic cells is involved in the generation of reactive oxygen
intermediates (ROIs) that are powerful microbicidal agents (Babior, 1984). Reactive
oxygen species (ROIs) like superoxide anion (o2-), hydrogen peroxide (H2O2), and
hydroxyl radicals (OH-) are produced during phagocytosis. This phenomenon as a
respiratory burst plays an important role in microcidal activity (song and Hsieh, 1994).
208
The generation of o2- has been reported in hemocyte of tiger shrimp, (P. monodon) (Song
and Hsieh, 1994), L. stylirostris (Bacher et al., 1995) and white shrimp, L. vannanei
(Munoz et al., 2000). Chin-Chyuan Chang et al., (2013) have suggested that the RBs
(Respiratory burst or superoxide anion production) and SOD (superoxide dismutase),
GPx activity significantly increased in prawn fed ECE-containing diets for 12 days,
indicating that the ECE (Eichhornia crassipes extracts) may promote regulation of the
ROL system in prawns. Dietary ECE administration promoted o2- production of single
haemocytes in the initial 9 days and then was down regulated ad 12 days, but increased
o2- (superoxide anion) production due to THC is the duration of the experiment.
Citarasu, et al., (2006) reported that herbal supplementation diets seem to act as a
promoter for increasing PO activity and o2- production in black tiger shrimp, P. monodon
against WSSV infection. Harikrishnan et al. (2012) indicated that the o2- levels
(superoxide anion production) and SOD activity against A. hydrophila significantly
increased in M. rosenbergii fed 0.1% and 1.0% doses of Withania somnifera
supplemented diets 1-4 weeks. Amar et al. (2004) reported that the production of o2(superoxide anion or respiratory burst) by head kidney leukocytes of rainbow trout in the
supplemented groups which fed with natural sources of carotenoids (micro algae D.
salina) and red yeast phaffiarhodozyma at 100 and 200mg kg-1) did not increase
significantly over that of the control. In the present study, M. rosenbergii fed with a diet
containing marigold oleoresin in the first experiment and diacetate of lutein mesozeaxanthin in the second experiment at 60, 120 and 180 mg kg-1 had showed a significant
increase in respiratory burst activity as compared to control. Therefore, it is evident that
209
these Carotenoids added to the diet can trigger the respiratory burst indicating an increase
in immune ability.
5.6.3 Effect of carotenoids on Total Haemocyte count
In the decapods crustaceans, circulating haemocytes are associated with cellular
defense (Johansson et al., 2000). It was known that THC is a useful indicator of shrimp
health (Chang et al., 1999). THC varies in crustaceans in response to infections disease,
environmental factors and moult cycle (Bachere 2000; Johansson, Keyser, Stritunyalucks
and Soderhall 2000, le Moulac & Haffner 2000). Simth, (1980; 1992) has demonstrated
that the haemocyte count varies among crustacean species and is known to be affected by
a variety of factors such as infection agents and environmental stress. Rodrigues and
Moullac (2000) noted that in crustaceans an increase in THC has been related to disease
resistance and also Moullac and Haffiner, (2000) Chand et al., (2006) stated that the
higher THC could be provided enhancement of immune capability during periods of
higher activity or increased environmental bacterial loads. Total Haemocyte Count in
prawns (M. rosenbergii) fed with 50,100 and 200 mg kg-1 of dietary astaxanthin for 28
days, showed a significant increase than those of in control group in which the increase
in the number of circulating haremocytes might be due to the higher haemocyte
mobilization indicating a better immune status in that group (Vijay Kumar, et al., 2009).
Isagani, et al., (2009) reported that the significant increase in THC showed the efficacy
of injected AX at different dosages (0.67 and 1.34 nmol g-1 BW-1) in enhancing the non
specific immune response of adult M. rosenbergii. Chang et al., (2013) observed that
THCs, HCs, and GCs, significantly increased in prawns M. rosenbergii fed ECEsupplemented diets at 1.0, 2.0 and 3.0 kg-1 for 12 days, but no significant difference in
210
SGCs, of prawns among the treatment groups was seen. These results suggests that ECE
(Eichhornia crassipes extracts) added to the diet can induce proliferation of haemocytes
in HPTs, promote mobilization of mature GCs and increase THC resulting from
increased HCs and GCs. Shrimp (L. vannamei) were fed 80 mg astaxanthin kg-1 diet had
highest THC than in shrimp fed with other diets (0, 40 or 150 mg kg-1) (Flores, M. et al.,
2007). However, shrimp fed with the diet supplemented with, 150 mg astaxanthin kg-1
had the lowest values in growth, survival, OC (Osmoregulatory capacity) and THC, and
this suggested that this group was under additional stress because of pigmentation
saturation (Herring 1973; Castillo, Negre-Sadrgues, et al., 1982). Harikrishnan, et al.,
(2012) observed that the hemocyte counts significantly increased in M. rosenbergii fed
with 0.1% and 1.0% Withania somnifera supplementation diets from weeks 1-4 against
A. hydrophila. In the present study, M. rosenbergii fed marigold oleoresin in the first
experiment and diacetate of lutein meso-zeaxanthin in second experiment at 60, 120 and
180 mg kg-1 significantly increased in THC, when compared to control group.
5.6.4 Effect of carotenoids on total heamolymph protein
Vargas-Albores et al., (1993) and Hall et al., (1995) stated that several immune
molecules have been identified and purified in crustaceans such as the lipopolysacchride
binding protein, β-glucan binding protein and peptidoglycan binding protein, exposure to
any of the immunoestimutony substances is expected to rise one or more of these defense
proteins in particular and to total protein levels in the hymolymph in general. The highest
haemocyanin concentrations was found in L.vannamei that were fed with the 80 mg
astaxanthin kg-1 diet (Maricela et al., 2007), Pascual et al., (2003) reported that in
L.vannamei 60-97% of the protein in heamolymph was HC (haemocyanin). Vijay Kumar
211
et al., (2009) have been demonstrated that there was a significant increased (P<0.05) in
total serum protein content of prawns (M. rosenbergrii) fed with 50,100 and 200 mg kg-1
astaxanthin compared to control group, who justified the increase in protein content may
be due to the increase in defense molecules in prawns fed with higher doses of
astaxanthin and may indicate a better immune status in these animals. In the present
study, M. rosenbergii fed marigold oleoresin in the first experiment and diacetate of
lutein meso-zeaxanthin in second experiment at 60, 120 and 180 mg kg-1 significantly
increased in total heamolymph protein, when compared to control group.
212
VI. SUMMARY
Macrobrachium rosenbergii is one of the most economically important cultured
palaemonid shrimp and it is now cultured on a large scale in many parts of the world, including
India. There is tremendous scope for development of freshwater prawn culture in India.
Experiment, 1 (Marigold oleoresin)

The experiment was conducted to evaluate the effect of marigold oleoresin at 60 mg /kg,
120 mg/kg and 180 m g/kg levels against Aeromonas hydrophila infection of freshwater
prawn, M. rosenbergi.

Uniform sized post larvae of freshwater prawn with an average weight of (0.85 to 0.89 g)
were stocked at a rate of 50 numbers/tank.

Prior to start of experiment, the post larvae were acclimatized to dry pellet diet it the
closed circulatory system which consists of 12 circular fiber glass tanks of 120 1
capacity.

The formulated feeds (35% protein) were designated as T0 (control) T1 (60 mg /kg), T2
(120 mg/kg) and T3 (180 m g/kg) of nucleotide inclusion.

Post larvae were fed at the rate of 5% of their body weight till the end of the
experiment.Continuous aeration is given.

Growth assessment of prawns and water sampling was carried out every fortnight.
Effect of marigold oleoresin on growth and survival of prawn

The growth and survival study of prawn was carried out in the indoor recirulatary
system for a period of 60 days.

The average weight recorded after 60 days of culture in treatment T2 was 4.59 g
followed by 4.28 g in T2, 3.99 g in T1 and 3.91g in T0.
213

The final average length was highest in T2 (5.31 cm) followed by T1 (5.20 cm),
T0 (5.14 cm) and T3 (5.10 cm)

The highest average survival rate of 83.37 % was recorded in T2 followed by T3
(78.00%), T1 (75.32 %) and T0 (73.32 %).

There were significant difference in growth but no significant in survival of prawn
was observed among treatments.
Resistance of M. Rosenbergii to Aeromonas hydrophila infection

Highest mortality of 75 % was recorded in T0 followed by 50% in T1, 45% in T2, and
36% in T3 respectively.

Highest relative percentage survival of 53.34% was recorded in T3 followed by 40% in T2
33.34% in T1.
Immune parameters of M. Rosenbergii

Highest phenoloxidase activity of 1.45 was recorded in T3 followed by 1.32 in T2, 1.08 in
T1 and 0.68 in T0 respectively.

Phenoloxidase activities of prawn recorded in different treatments were significantly
(P<0.05) higher than that of control groups.

The highest super oxide anion production of 1,173 was recorded in T3 followed by 0.53
in T2, 0.11 in T1 and 0.05 in T0 respectively.

The highest total haemocyte count of 14.2 was recorded in T3 followed by 11.9 in T2,
11.65 in T1 and 9.25 in T0 respectively.

The highest total heamolymph protein of 14.06 mg/ml was recorded in T3 followed by
10.46 mg/ml in T2, 8.08 mg/ml in T1 and 6.75 mg/ml in T0 respectively.
214
Experiment, 2 (Diacetate of lutein-mesozeaxanthin)
Effect of nucleotide on growth and survival of prawn

It was observed that the best growth of prawns in terms of weight was recorded in
treatment T3 followed by the treatment T2, T1 and T0. The average weight recorded after
60 days of culture in treatment T3 was 1.585 g, followed by 1.518 g in T2, 1.420 g in T1
and 1.225 g in T0.

The final average length of prawn also followed the same trend as that of weight. The
final average length was highest in T3 (4.46 cm) followed by T2 (4.27cm ), T1 (4.09cm)
and T0 (4.00cm)

The highest average survival rate of 80.00 % was recorded in T3 followed by T2
(77.32%), T1 (74.00%) and T0 (69.32 %).
Resistance of M. Rosenbergii to Aeromonas hydrophila infection

Highest mortality of 75% was recorded in T0 followed by 70% in T3, 50% in T2 and 30%
in T1 respectively.

Highest relative percentage survival of 60.00% was recorded in T1 followed by 33.34%
in T2 6.67% in T3.
Immune parameters of M. Rosenbergii

Highest phenoloxidase activity of 1.38 was recorded in T1 followed by 0.57 in T2
0.54 in T3 and 0.27 in T0 respectively.

Phenoloxidase activities of prawn recorded in different treatments were significantly
(P<0.05) higher than that of control groups.
215

The highest super oxide anion production of 0. 6 was recorded in T1 followed by 0.14
in T2, 0.11 in T3 and 0.09 in T0 respectively.

The highest total haemocyte count of 12.8 was recorded in T1 followed by 11.45 in T2
11.2 in T3 and 9.36 in T0 respectively.

The highest serum protein in 14.83 mg/ml was recorded in T1 followed by 9.63
mg/ml in T2, 8.22 mg/ml in T3 and 6.20 mg/ml in T0 respectively.
ectively.
Conclusion

Based on the results obtained in the experiment, it can be concluded that M. rosenbergii
fed a diet containing carotenoids at 60mg/ kg, 120mg/kg and 18.0mg/kg could enhance
growth but not increased survival in freshwater prawn, M. rosenbergii.

M. Rosenbergii fed a diet containing carotenoids at 60mg/ kg, 120mg/kg and 18.0mg/kg
were found to significantly (P<0.05) increase the disease resistance against Aeromonas
hydrophila infection and better resistance was observed between 180 mg/kg of marigold
in first experiment and 60 mg/kg of
Diacetate of lutein-mesozeaxanthin in second
experiment.

M. rosenbergii fed a diet containing carotenoids at 60mg/ kg, 120mg/kg and 18.0mg/kg
found to increased its immune ability by significantly (p<0.05) increasing its
phenoloxidase acivity (PPO), respiratory burst (NBT), total heamolymph protein and
total haemocytes count.

Based on the results obtained in the experiment, it can be concluded that the
incorporation of marigold oleoresin at 180 mg/kg and Diacetate of lutein-mesozeaxanthin
216
at 60 mg/kg have been found effective in promoting immune response, growth and
survival of freshwater prawn, M.rosenbergii.

carotenoids has immunostimulent properties and it can be supplemented in the diets as an
immunostimulant to increase the immune ability of prawn and improve production in
prawn farming.

The maximum amount of Marigold oleoresin (180 mg/kg) and minimum amount of
Diacetate of lutein- meso zeaxanthin(60 mg/kg) had higher effect on immune parameter
which may be showed that Diacetate of lutein- meso zeaxanthin more potent than
Marigold oleoresin.

About the Diacetate of lutein- meso zeaxanthin, as per my knowledge nobody has been
done in the world on any types of aquatic animals up to now, and this experiment was
done for first time, it shows that is necessary other experiment on different types of fish
and shellfish has to be done.
217
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ABSRACT
Experiments were conducted on M. rosenbergii to evaluate the effect of carotenoids on growth,
survival, immune response and disease resistance against of A. hydrophila. M. rosenbergii were
fed with dietary supplementation of carotenoids namely Marigold oleoresin and Diacetate of
lutein-mesozeaxanthin for 60 days in triplicate groups. In the first experiment, M. rosenbergii
(0.87 ± 0.01 g) fed on Marigold oleoresin at the rate of 60, 120 and 180 mg/kg. After 60 days of
feeding the best growth of M. rosenbergii in terms of weight was recorded in treatment T2
followed by T3, T1 and T0, and significant difference (P<0.01) between T2, T3 when compared to T1
and T0 was observed, but there were no significant difference (P>0.05) between them with regard
to survival rate. The highest ppo activity, superoxide anion production, total heamolymph protein
and total haemocyte count was recorded in T1 followed by T2, T3 and T0 as well as significant
difference between the treatments and control groups was observed, also significant difference
between them in terms of disease resistance was noticed. In the second experiment, diacetate of
lutein-mesozeaxanthin at levels of 60, 120 and 180 mg/kg were fed to M. rosenbergii (0.34 ± 0.01
g) . Significant difference (P<0.05) between treatments and control group in terms of growth were
observed but there were no significant differences (P>0.05) among all groups on the basis of
survival rate. All immune parameters and relative percent survival (RPS) significantly were higher
in treated groups than control. Based on the above results it can be concluded that 120 and 180
mg/kg of marigold oleoresin and 180 and 60 mg/kg of diacetate of lutein-mesozeaxanthin could
improve growth, immune responses and disease resistance against of A. hydrophila in M.
rosenbergii respectively.
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