skin colour changes in ornamental koi (cyprinus

SKIN COLOUR CHANGES IN ORNAMENTAL KOI

(Cyprinus carpio) FED DIFFERENT DIETARY CAROTENOID SOURCES

TAN PHAIK SHIANG

UNIVERSITI SAINS MALAYSIA 2006

SKIN COLOUR CHANGES IN ORNAMENTAL KOI (Cyprinus carpio) FED DIFFERENT DIETARY

CAROTENOID SOURCES

by

TAN PHAIK SHIANG

Thesis submitted in fulfillment of the requirements for the degree

of Master of Science

March 2006

viii

TABLE OF CONTENTS PRELIMINARIES Page ACKNOWLEDGEMENTS

i

ABSTRAK ii ABSTRACT v TABLE OF CONTENTS viii LIST OF TABLES xii LIST OF FIGURES xiv LIST OF PLATES xv CHAPTER 1 INTRODUCTION 1 CHAPTER 2 LITERATURE REVIEW 4 2.1 Carotenoids 4

2.1.1 Structure of carotenoids 8

2.1.2 Carotenoid sources 13

2.1.3 Function of carotenoids 19

2.1.3.1 Biological Functions 19

2.1.3.2 Pigmentation Functions 22

2.2 Koi 27

2.2.1 History of Koi 27

2.2.2 Physiology of Koi 30

2.2.3 Koi Keeping 32

2.2.4 Kohaku – Colour and pattern 33

2.3 Chromatophores 36

2.3.1 Shapes of Chromatophores 38

ix

2.3.2 Koi Coloration 39

2.3.3 Colour enhancement 40

CHAPTER 3 METHODOLOGY 42 3.1 Scales Chromatophore analysis 42

3.2 Proximate Analysis 42

3.3 Carotenoid Sources 43

3.3.1 Astaxanthin source @ Carophyll® Pink 8% 43

3.3.2 Astaxanthin source @ NatuRoseTM 1.5% 45

3.3.3 Canthaxanthin source @ Lucanthin® Red 10% 46

3.3.4 Canthaxanthin source @ Carophyll® Red 10%

47

3.3.5 Natural Carotenoids Source @ Spirulina PacificaTM 48

3.3.6 Palm Carotenoid Source @ CarosolTM 3% 49

3.3.7 Palm Carotenoid Source @ CarominTM 10% & 20% 49

3.4 Experimental Diets 51

3.4.1 Experiment I – Efficacy of Various Dietary Carotenoid Sources for Small Koi

52

3.4.2 Experiment II – Effect of Different Dietary Levels of Carotenoid Sources for Large Koi

54

3.4.3 Experiment III – Effect of Feeding Period on Pigmentation in Koi Fed Various Dietary Carotenoid Sources

57

3.5 Fish and experimental design 59

3.5.1 Experiment I – Efficacy of Various Dietary Carotenoid Sources for Small Koi

59


62

3.5.3 Experiment III – Effect of Feeding Period on Pigmentation in Koi Fed Various Dietary Carotenoid Source

64

3.6 Sample Collection 67

3.6.1 Experiment I – Efficacy of Various Dietary Carotenoid

Sources for Small Koi 67

x


67

3.6.3 Experiment III – Effect of Feeding Period on Pigmentation in Koi Fed Various Dietary Carotenoid Sources

68

3.7 Growth Parameters and Feed Efficiency Calculation 69

3.8 Condition Index 70

3.9 Colour measurement and analysis 70

3.10 Total carotenoid analysis 73

3.11 High Performance Liquid Chromatography - HPLC system and conditions 74

3.12 Statistical Analysis 75

CHAPTER 4 RESULTS 77 4.1 Chromatophores Observation 77

4.2 Experiment I – Efficacy of Various Dietary Carotenoid Sources for Small Koi

80

4.2.1 Growth Parameters and Feed Utilization Efficiency 80

4.2.2 Condition Index 84

4.2.3 Total Carotenoids in Koi Tissues 85

4.2.4 Astaxanthin, β-Carotene and Canthaxanthin Concentrations 88

4.2.5 Chromatophores Observation 91

4.3 Experiment II – Effect of Different Dietary Levels of Carotenoid Sources for Large Koi

95


4.3.2 Quantitative Measurement of Skin Colour Changes 98

4.3.3 Visual Observation of Skin Colour Changes 101

4.3.4 Total Carotenoids in Koi Blood and Gonads 106


4.4 Experiment III – Effect of Feeding Period on Pigmentation in Koi Fed Various Dietary Carotenoid Sources

112

xi


4.4.2 Visual Observation of Skin Colour Changes 113

4.4.3 Condition Index 115

4.4.4 Quantitative Measurement of Skin Colour Changes 117

4.4.5 Total Carotenoids in Koi Skin 118


CHAPTER 5 DISCUSSION 122

5.1 Chromatophores observation and analysis 122

5.2 Effect of carotenoid on growth rate and feed utilization efficiency

123

5.3 Effect of carotenoid on pigment deposition 126

5.4 Effect of carotenoid on skin coloration 132

5.5 Effect of background colour 134

CHAPTER 6 CONCLUSION 136 BIBLIOGRAPHY 138

APPENDICES 148

APPENDIX I – Proximate Analysis 149

APPENDIX II – Formulation for Mineral and Vitamin Premix 153

APPENDIX III - Growth Parameters and Feed Utilization Efficiency 154

APPENDIX IV - Total Carotenoids in Koi Tissues

158

APPENDIX V - The standard curve for Astaxanthin, β-carotene and Canthaxanthin analysis

163

APPENDIX VI - Colour parameters (L*, a*,b*)/(L*,c*,h*) in skin (mg kg-1)

of Koi (Cyprinus carpio) fed the experimental diets1 (Experiment III)

165

xii

LIST OF TABLES

Table 3.1 Technical characteristics of Carophyll® Pink 8% 44

Table 3.2 Specifications of NatuRoseTM 1.5% 45

Table 3.3 Technical characteristics of Lucanthin® Red 10% 46

Table 3.4 Specifications of Carophyll® Red 10% 47

Table 3.5 Technical characteristics of Spirulina 48

Table 3.6 Typical chemical properties of CarosolTM 3% 50

Table 3.7 Technical specifications of CarominTM 10% & 20% 50

Table 3.8 Feed composition and proximate analysis for diets (g 100g-1 dry matter) for Experiments I

53

Table 3.9 Feed composition and proximate analysis for diets (g 100g-1 dry matter) for Experiments II

56

Table 3.10 Feed composition and proximate analysis for diets (g 100g-1 dry matter) for Experiments III

58

Table 4.1 The evaluation of growth parameters and feed utilization efficiency for koi (Cyprinus carpio) in Experiment I

83

Table 4.2 Hepatosomatic Index (H.S.I) and Haematocrit Index of koi (Cyprinus carpio) fed various carotenoid sources for 10 weeks

84

Table 4.3 Astaxanthin, β-carotene and Canthaxanthin concentration (µg g-1) in various koi (Cyprinus carpio) tissues in Experiment I

90

Table 4.4 Visual observation of the morphology of chromatophores on the scales of koi (Cyprinus carpio) fed with different diets in Experiment I

92

Table 4.5 The evaluation of growth parameters and feed utilization efficiency for koi (Cyprinus carpio) in Experiment II

97

Table 4.6 Colour parameters (L*,c*,h*) of fish skin for koi (Cyprinus carpio) fed the experimental diets in Experiment II

100

Table 4.7 Astaxanthin, β-carotene and Canthaxanthin

concentration (µg g-1) in various koi (Cyprinus carpio) tissues in Experiment II

111

Table 4.8 The evaluation of growth parameters and feed utilization efficiency for koi (Cyprinus carpio) in Experiment III

112

Table 4.9 Hepatosomatic Index (H.S.I) and Haematocrit Index of koi (Cyprinus carpio) fed various carotenoid sources for 12 weeks in Experiment III

116

xiii

Table 4.10 Colour parameters (L*, a*, b*)/ (L*, c*, h*) and Total Carotenoids (TC) in skin (mg kg-1) for Koi (Cyprinus carpio) fed the experimental diets in Experiment III

118

Table 4.11 Carotenoid content (µg g-1) in carotenoid supplemented experimental diet and experimental koi (Cyprinus carpio) in Experiment III

121

xiv

LIST OF FIGURES

Figure 2.1 Postulated pathways of carotenoid conversions for red carp type fish modified from Simpson, 1982

7

Figure 2.2 β- carotene 8

Figure 2.3 Three stereoisomers of astaxanthin 11

Figure 2.4 Canthaxanthin 12

Figure 2.5 Stages comprising the melanophore index of Hogben and Slome

38

Figure 3.1 Flow chart for the processes involved in experiment I 61

Figure 3.2 Flow chart for the processes involved in experiment II 63

Figure 3.3 Flow chart for the processes involved in experiment III 66

Figure 3.4 The framework of the CIELAB (1976) colour space diagram.

72

Figure 3.5 The colour attributes in the CIELCH space (CIELCH, 1976) showing the relationship between Lightness, Chroma and hue

73

Figure 4.1 Weight gain (%) of experimental koi fed different diet with various dietary carotenoid sources in Experiment I

82

Figure 4.2 Total carotenoids in the skin of koi (Cyprinus carpio) fed with different diets measured at 490 nm wavelength in Experiment I

85

Figure 4.3 Total carotenoids in the muscle of koi (Cyprinus carpio) fed with different diets measured at 490 nm wavelength in Experiment I

86

Figure 4.4 Total carotenoids in the liver of koi (Cyprinus carpio) fed with different diets measured at 490 nm wavelength in Experiment I

86

Figure 4.5 Comparison of Specific Growth Rate, SGR (%/day) of experimental koi (Cyprinus carpio) fed different diets in Experiment II

95

Figure 4.6 Total carotenoids in the blood serum of koi fed different diet measured at 490 nm wavelength in Experiment II

107

Figure 4.7 Total carotenoids in the gonad of koi fed different diet measured at 490 nm wavelength in Experiment II

107

Figure 4.8 Total carotenoids in the skin of koi fed different diet measured at 490 nm wavelength in Experiment III a (D1, Control) b (D2, Carophyll Pink) c (D3, Carophyll Red) d (D4, Caromin)

120

xv

LIST OF PLATES

Plate 2.1 Kohaku 33

Plate 3.1 The high power microscope used for chromatophores observation

43

Plate 3.2 Experimental diets for Experiment I 55

Plate 3.3 Experimental diets for Experiment II 55

Plate 3.4 Experimental diets for Experiment III 57

Plate 3.5 Experimental aquarium used for Experiment I 60

Plate 3.6 Experimental tanks for Experiment II 65

Plate 3.7 Experimental system for Experiment III 65

Plate 3.8 One of the Experiment II koi with arrow pointing at the colour measurement region showing R-red, O-orange and W-white

72

Plate 3.9 Light absorption or UV-visible spectrophotometer 76

Plate 3.10 High Performance Liquid Chromatography (HPLC) 76

Plate 4.1ab Chromatophores found in black scales with silver background

78

Plate 4.2ab Chromatophores found in black scales with white background

78

Plate 4.3ab Chromatophores found in black scales with orange background

79

Plate 4.4ab Chromatophores found in red scales 79

Plate 4.5ab Chromatophores found in orange scales 79

Plate 4.6 Chromatophores on the scale of koi fed Diet 1 - Control, 0ppm

93

Plate 4.7 Chromatophores on the scale of koi fed Diet 2 - Carophyll Pink, 100ppm

93

Plate 4.8 Chromatophores on the scale of koi fed Diet 3 - Lucanthin Red, 100ppm

93

Plate 4.9 Chromatophores on the scale of koi fed Diet 4 - NatuRose, 100ppm

93

Plate 4.10 Chromatophores on the scale of koi fed Diet 5 - Spirulina, 100ppm

93

Plate 4.11 Chromatophores on the scale of koi fed Diet 6 - Carosol, 100ppm

93

xvi

Plate 4.12 Chromatophores on the scale of koi fed Diet 1 - Control, 0ppm

94

Plate 4.13 Chromatophores on the scale of koi fed Diet 2 - Carophyll Pink, 100ppm

94

Plate 4.14 Chromatophores on the scale of koi fed Diet 3 - Lucanthin Red, 100ppm

94

Plate 4.15 Chromatophores on the scale of koi fed Diet 4 - NatuRose, 100ppm

94

Plate 4.16 Chromatophores on the scale of koi fed Diet 5 - Spirulina, 100ppm

94

Plate 4.17 Chromatophores on the scale of koi fed Diet 6 - Carosol, 100ppm

94

Plate 4.18 Fish T1F1 on week 0 before fed Diet 1 (Control) 102

Plate 4.19 Fish T1F1 fed Diet 1 (Control) after 12 weeks 102





Plate 4.24 Fish T2F2 on week 0 before fed Diet 2 (Carophyll Pink 250ppm)

103

Plate 4.25 Fish T2F2 fed Diet 2 (Carophyll Pink 250ppm) after 12 weeks

103


103


103


103


103


103


103

Plate 4.32 Fish T4F1 on week 0 before fed Diet 4 (Spirulina 250ppm)

104

Plate 4.33 Fish T4F1 fed Diet 4(Spirulina 250ppm) after 12 weeks 104


104

xvii

Plate 4.35 Fish T4F2 fed Diet 4(Spirulina 250ppm) after 12 weeks 104


104

Plate 4.37 Fish T5F1 fed Diet 5 (Spirulina 500ppm) after 12 weeks 104


104



104


Plate 4.42 Fish T6F1 on week 0 before fed Diet 6 (Caromin 500ppm)

105

Plate 4.43 Fish T6F1 fed Diet 6 (Caromin 500ppm) after 12 weeks 105


105



105


Plate 4.48 Gonad inside koi before dissecting out 110

Plate 4.49 Comparison on koi gonad fed D1, D3, D5 and D6 110

Plate 4.50 Photo taken on pre week 114

Plate 4.51 Photo taken on week 0 114

Plate 4.52a Koi on week 0 before feed with Diet 1 (Control) 114

Plate 4.52b Koi fed with Diet 1 (Control) after week 12 114

Plate 4.53a Koi on week 0 before feed with Diet 2 (Carophyll Pink 250ppm)

114

Plate 4.53b Koi fed with Diet 2 (Carophyll Pink 250ppm) after week 12

114

Plate 4.54a Koi on week 0 before feed with Diet 3 (Carophyll Red 250ppm)

114

Plate 4.54b Koi fed with Diet 3 (Carophyll Red 250ppm) after week 12

114

Plate 4.55a Koi on week 0 before feed with Diet 4 (Caromin 250ppm)

114

Plate 4.55b Koi fed with Diet 4 (Caromin 250ppm) after week 12 114

i

ACKNOWLEDGEMENTS

First of all, I would like to extend my sincere appreciation and gratitude to

my supervisors, Associate Professor Dr. Ng Wing Keong and Professor Dr.

Boey Peng Lim. I really appreciate their invaluable guidance, patience,

kindness, understanding, encouragement and time throughout the period of my

postgraduate studies.

I would like to convey my appreciation and thanks to the School of

Biological Sciences and School of Chemistry for providing a conducive research

environment, USM for providing invaluable financial support through the

Graduate Assistance Scheme, and the Institute of Postgraduate Studies (IPS)

for providing assistance throughout the study.

My appreciation to Aquaculture Station for supplying the experimental

fishes Cyprinus carpio. My thanks also reach out to all laboratory assistants

especially Mr. Bahrim, Mr. Patchamuthu Ramasamy, Mr. Johari and Mr. Sobri

for their technical assistance and help.

Furthermore, I would like to thank all my friends especially Ms. Jillian Lim

Phaik Kin, Mr. Wang Yan, Mr. Chong Cheong Yew, Mr. Lee Kuan Shern and

Ms. Jacqueline Liew for their help, support and encouragement given to me

during my postgraduate studies. Last but not least; I would like to express my

most sincere and warmest gratitude to my family and friends for their love and

encouragement.

ii

PERUBAHAN WARNA KULIT PADA IKAN KOI HIASAN (Cyprinus carpio) YANG DIBERI MAKAN DENGAN PELBAGAI SUMBER DIET

BERKAROTENOID

ABSTRAK

Harga ikan koi meningkat sejajar dengan keamatan warna kulitnya.

Warna and pigmentasi ikan disebab oleh penyerapan dan penempatan

karotenoid. Ikan tidak berupaya untuk sintesis karotenoid sendiri, oleh itu,

karotenoid mesti dimasukkan ke dalam diet mereka.

Tiga eksperimen telah dijalankan untuk menyiasat perubahan warna kulit

ikan koi hiasan (Kohaku) dengan memberi diet yang diperkaya dengan

karotenoid daripada alga (Haematococcus pluvialis dan Spirulina) dan kelapa

sawit (dalam bentuk serbuk dan minyak). Diet yang ditambah dengan

karotenoid sintetik (astaxanthin dan kantaxanthin) dan satu diet kawalan yang

tidak ditambah sebarang karotenoid digunakan untuk perbandingan.

Dalam eksperimen pertama, ikan koi (12.89 + 0.02g) diberi diet yang

ditambah dengan sumber karotenoid sebanyak 100ppm. Eksperimen ini telah

dijalankan dengan sistem air yang statik dalam jangkamasa 10 minggu. Ikan

koi yang diberi diet karotenoid tambahan menunjukkan pewarnaan badan yang

lebih daripada ikan diet kawalan yang tiada penambahan karotenoid.

Parameter tumbesaran ikan koi dipengaruhi oleh sumber karotenoid. Ikan koi

yang diberi diet yang ditambah dengan spirulina atau Carosol masing-masing,

mendapat parameter tumbesaran yang lebih tinggi daripada ikan koi yang diberi

diet yang lain. Terdapat perbezaan yang signifikan (P< 0.05) dalam jumlah

karotenoid dan jenis karotenoid yang terdapat pada kulit, otot dan hati ikan koi.

iii

Jumlah karotenoid yang terdapat dalam kulit ikan yang diberi diet yang

ditambah dengan spirulina atau Carophyll Pink menunjukkan nilai tinggi yang

signifikan, 12.08mg kg-1 dan 11.76mg kg-1, masing-masing. Pemerhatian

morfologi kromatofor dalam sisik ikan koi yang diberi diet berlainan dijalankan

dengan mengguna mikroskop cahaya berkuasa tinggi.

Dalam eksperimen kedua, ikan koi (254.89 + 16.82g) diberi diet yang

ditambah dengan sumber karotenoid pada 250ppm atau 500ppm masing-

masing. Eksperimen ini telah dijalankan dalam sistem air statik untuk

jangkamasa 8 minggu. Parameter tumbesaran ikan koi tidak dipengaruhi oleh

penambahan karotenoid ke dalam diet mereka. Ikan koi yang diberi diet

karotenoid tambahan menunjukkan pewarnaan badan yang lebih baik daripada

ikan koi yang diberi diet kawalan. Parameter warna seperti L*, c* dan h* diukur

dengan menggunakan Minolta Colour Reader (CR-100) pada tiga bahagian

yang terdapat warna seperti putih, jingga dan merah. Berdasarkan skala warna

CIELCH (CIE, 1976), L* menunjukkan kecahayaan, C* menunjukkan Kroma

dan h* menunjukkan sudut warna. Ikan koi yang diberi diet yang ditambah

dengan 500ppm Carophyll Pink atau spirulina telah menambahkan kemerahan.

Sebahagian ikan koi yang diberi diet karotenoid tambahan 500ppm, bahagian

kepala menjadi kekuningan dan ini menujukkan bahawa karotenoid telah

melebihi batasan. Terdapat perbezaan signifikan (P< 0.05) dalam jumlah

karotenoid dan jenis karotenoid individu yang disimpan di dalam gonad dan

serum darah. Nilai jumlah karotenoid yang terdapat dalam serum darah dan

gonad ikan koi yang diberi diet 250ppm spirulina tambahan merupakan nilai

tertinggi.

iv

Dalam eksperimen ketiga, ikan koi (49.36 + 0.32g) diberi diet yang

ditambah dengan sumber karotenoid sebanyak 250ppm. Eksperimen ini telah

dijalankan dalam system air yang bergerak untuk jangkamasa selama 12

minggu. Parameter tumbesaran ikan koi tidak dipengaruhi oleh penambahan

karotenoid pada diet mereka. Tiada perbezaan signifikan (P> 0.05) pada

jumlah karotenoid dalam kulit semua ikan koi. Bagaimanapun, terdapat

perbezaan yang signifikan pada jenis karotenoid individu untuk ikan koi yang

diberi diet berlainan. Kandungan astaxantin dalam ikan koi diberi diet yang

ditambah dengan 250ppm Carophyll Pink adalah lebih tinggi daripada diet yang

ditambah dengan 250ppm Carophyll Red atau Caromin sepanjang tempoh

pemberian makanan. Parameter warna (L*, a*, b*, c* and h*) diukur pada

sebekah kiri bahagian dorsal. L* adalah kecahayaan, a* menunjukkan merah-

hijau chromaticity dan b* kuning-biru chromatociti (CIELAB, 1976). Terdapat

perbezaan yang signifikan (P<0.05) bagi nilai L* dan b* dalam kulit ikan koi.

Secara kesimpluan, diet yang ditambah dengan sebarang karotenoid boleh

mempengaruhi atau meninggikan parameter tumbesaran dan pewarnaan dalam

ikan koi. Pigmentasi ikan koi bergantung pada sumber karotenoid yang sesuai

(sintetik atau alga) dengan kepekatan yang sesuai (100-250 ppm) dan

jangkamasa pemeliharaan yang sesuai (8-12 minggu) adalah faktor yang

penting untuk pewarnaan dan parameter tumbesaran ikan koi yang lebih baik.

v

SKIN COLOUR CHANGES IN ORNAMENTAL KOI (Cyprinus carpio) FED DIFFERENT DIETARY CAROTENOID SOURCES

ABSTRACT

Koi value increases with intensity of skin colour. Fish coloration and

pigmentation is due to absorption and deposition of carotenoids. Fishes are

unable to synthesize their own carotenoids and therefore these must be

included in their diet.

Three trials were undertaken to investigate skin colour changes in

ornamental koi (Kohaku) by feeding a dietary carotenoid supplement of algal

(Haematococcus pluvialis or Spirulina) or palm carotenoid (powder and oil

form). Diets containing synthethic carotenoids (Astaxanthin or Canthaxanthin)

and a control diet with no carotenoids added were used for comparison.

In the first experiment, koi (12.89 + 0.02g) were fed diets supplemented

with carotenoid sources at a level of 100ppm. The experiment was conducted

in a static water system for a period of 10 weeks. Koi fed with diets

supplemented with carotenoids exhibited better body coloration than those fed

the control diet without added carotenoids. Growth performance of koi were

affected by the dietary carotenoids source. Koi fed diets supplemented with

spirulina or Carosol respectively, showed higher growth than koi fed other diets.

There were differences (P< 0.05) in skin total carotenoid and individual

carotenoid types in the skin, muscle and liver of koi. The total carotenoids on

the skin of koi fed with Spirulina or Carophyll Pink supplemented diets showed

significantly higher value, 12.08mg kg-1 and 11.76mg kg-1, respectively. Visual

vi

observations of the morphology of chromatophores on the scale of koi fed with

different diet were done under high power light microscope.

In the second experiment, koi (254.89 + 16.82g) were fed diets fortified

with carotenoids at a level of 250ppm or 500ppm, respectively. The experiment

was conducted in static water system for a period of 8 weeks. Growth

performances of koi were not affected by the addition of carotenoids to their

diet. Koi fed added dietary carotenoids exhibited better body coloration than koi

fed the control diet with no added carotenoids. Colour parameter such as L*, c*

and h* were measured using a Minolta Colour Reader (CR-100) at three

different colour regions (white, orange and red) on the koi body. Based on the

CIELCH colour scale (CIE, 1976), L* indicates Lightness, C* indicates chroma

and h* indicates the hue angle. Koi fed diets supplemented with 500ppm

Carophyll Pink or Spirulina increased their redness. Some of the head region of

koi fed carotenoids supplemented at 500ppm turned yellowish and this

indicated an overdose of dietary carotenoids. There were significant differences

(P< 0.05) in total carotenoid and individual carotenoid types deposited in koi

gonad and blood serum. The total carotenoids concentrations in blood serum

and gonad of the koi fed 250ppm Spirulina supplemented diet were the highest.

In the third experiment, koi (49.36 + 0.32g) were fed diets fortified with

carotenoid sources at a level of 250ppm in a water flow through system for a

period of 12 weeks. Growth performances of the koi were not affected by the

addition of carotenoids to their diet. There were no significant differences (P>

0.05) in total carotenoid on the skin of all the koi. However, there were

vii

significant differences (P< 0.05) in individual carotenoid types of koi fed different

diets. The astaxanthin content of koi fed diets supplemented with 250ppm

Carophyll Pink was significantly higher than diet supplemented with 250ppm

Carophyll Red or Caromin throughout the feeding trial. Colour parameter (L*,

a*, b*, c* and h*) were measured at the left dorsal regions. L* indicated

Lightness, a* indicates red-green chromaticity and b* indicates yellow-blue

chromaticity (CIELAB, 1976). There were significant differences (P<0.05) for L*

and b* of koi skin. In conclusion, certain dietary carotenoids may affect or

increase the growth performances and colouration in koi. Koi pigmentation

depends on proper carotenoid source (synthetic or algal) with suitable

carotenoid levels (100-250ppm) and appropriate feeding period (8-12 weeks)

which are the crucial factors for better colouration and growth performances of

koi.

1

CHAPTER 1 INTRODUCTION

The culture and export of ornamental fishes in Malaysia has expanded

rapidly in recent years. The requirement of feed has increased following the

high production of ornamental fishes. Nutrition is the most important factor in

keeping ornamental fishes healthy. In order to develop efficient and economical

feed formulas for aquaculture, basic information of nutrient requirement and

chemical composition of feed ingredients in relation to their acceptability and the

ability of fish to digest and utilise nutrients from various sources is required.

Thus, it is necessary to conduct research and development work to develop

effective diets which are of lower cost for the industry.

Differences in species, varieties, colours and patterns of ornamental

fishes play an important role in attracting home owners to keep these fishes in

their house. Ornamental fishes also derive the name as “life jewels” because of

its ornamental values. That means it must be beautiful and decorative as it

helps to beautify our living environment. In other words, coloration of

ornamental fish plays the most important role.

Coloration and hence, pigmentation plays an important role in both

ornamental and food fishes such as goldfish, koi, salmon, trout, sea bream and

prawns. Normally, the “quality” of the fish is based on the pigmentation. The

colour of fish might also bring some other meanings for certain people. For

example, red-coloured fishes such as koi and goldfish will be more attractive

2

and higher priced as the Chinese, Taiwanese and Japanese consider them

more auspicious.

Ornamental carp or koi is now available in many countries spanning

Europe, USA and the Far East. Availability of koi is maintained by local

producers in these countries, but trade of high quality koi plays a major role in

meeting a growing world-wide demand. European countries import large

quantities of koi, mainly from Israel, at an annual return of about US$ 10 million.

However, top-quality koi are imported mainly from Japan. Their price may

reach several thousand US dollar for each fish. Colours and patterns are the

major factors in making a high quality koi. Based on growing demand, it seems

that global koi production can still increase in both quantity and quality.

Koi is an economically important species of ornamental fish cultured in

Malaysia. A major challenge towards meeting the growing demand for koi is to

understand the genetic control of colour traits, but so far very few studies have

been carried out. Other ways to enhance the coloration of koi are hormone

stimulation, natural environment stimulation, feeding dietary pigment enriched

feed and injection of dyes. However, the most economical and practical way is

through feeding of pigment enriched feeds. Fishes are given special diets

containing additional pigments to trigger and enhance coloration of fishes and

this has lead to enhanced quality of the coloured ornamental koi.

The cost of each pigment and carotenoid source is different. Carophyll

Pink TM is the most effective pigment for colour enhancement tested so far. Due

3

to this reason, Carophyll Pink TM is also the most expensive among the various

commercially available pigment sources. Therefore, a lot of researches have

been carried out in food fish to replace Carophyll Pink with other cheaper

carotenoid sources but very few studies have been conducted in ornamental

fish such as koi. Even though small amounts of carotenoids are added in

commercial feeds, it is very expensive and sometimes can amount to 15% or

more of ornamental fish feed cost.

The palm-based carotenoid source is cheaper and is currently only used

in food applications. To date, no research has been done using the palm-based

carotenoid in fish diets for pigmentation purposes and this is the first study.

This study had been carried out to compare the efficacy of carotenoids

extracted from palm oil with that of established carotenoid sources currently

used in aquafeeds.

The present study has been designed to determine a proper dose-

response relationship regarding the pigmentation of koi. The aim was to benefit

feed formulators and the production of ornamental koi by improving koi colour

quality. Therefore, the objective for this study was to determine the effects of

different levels and various sources of carotenoid on the colour enhancement,

pigmentation efficiency and growth rate of Japanese koi (Cyprinus carpio L.).

4

CHAPTER 2 LITERATURE REVIEW

2.1 Carotenoids

According to the Britannica Encyclopaedia, carotenoids refer to any

group of non-nitrogenous and bio-chromes yellow, orange or red pigments that

are almost universally distributed in living organisms. Carotenoids can be

synthesized by bacteria, fungi, lower algae and green plants. They are most

conspicuous in the petals, pollen and fruit (e.g., carrots, tomatoes, sweet

potatoes and citrus fruits) of the flowering plants. Carotenoids can also be seen

in the autumn foliage of deciduous shrubs and trees. Carotenoids in the leaves

of green plants are serving as accessory pigments in photosynthesis. They trap

the solar energy and pass it to the primary photosynthetic pigment, chlorophyll.

Carotenoids play a major role in the biological colouration of animals.

Carotenoids are an important group of natural lipid soluble pigments that

are found in all families of the plant and animal kingdoms. There are about 600

different natural carotenoids that have been identified (Lorenz 1998a; Latscha,

1991). Carotenoids derive their names from the fact that they constitute the

major pigment in the carrot according to a trivial name system and an IUPAC

system (Bauernfeind & Klaui, 1981; Simpson, 1982). Carotenoids represent the

most widespread and structurally diverse pigmenting agents. Carotenoids and

their derivatives are important in animals as the basis of the visual pigments

responsible for light detection and colour discrimination (Britton, 1983).

Carotenoids are responsible for many of the brilliant yellow to red colours in

5

plants and animals, as well as the variety of bluish, greenish, purplish, brownish

and blackish colours seen in many fish and crustaceans (Latscha, 1990).

Carotenoids are structurally related to retinol and β-carotene, the main

sources of vitamin A for animals (Latscha, 1991). β-carotene can be converted

to two identical molecules of retinol and vitamin A (Boileau et al., 1999). The

beautiful yellow, orange and red colours found in the skin of ornamental fish are

the result of a group of carotenoid pigments, although colourless pteridines may

also be present (Chan et al., 1990). Fish, in the same way as other animals,

must ingest carotenoids as precursors for vitamin A, as well as depositing

pigments. Aquatic animals are unable to perform a de novo synthesis of

carotenoids in their body and therefore they must acquire these pigments from

their diet (Bagnara & Hadley, 1973; Simpson, 1982; Lorenz, 1998b; Latscha,

1991). In nature, fish gain their colour through their natural diets such as algae

because only plants and protists are able to synthesize carotenoids.

Bicyclic carotenoids like β–carotene or xanthophylls are ingested from

plants and may be converted to astaxanthin, the most commonly occurring

carotenoids in aquatic animals (Simpson et al., 1981; Goodwin, 1986;

Torrissen, 1989; Latscha, 1990). Based on this, carotenoid synthesised from

plants and algae can be added into diets for colour enhancement. Fish differ

greatly in their ability to ingest and transform various carotenoids. The

specificity of the carotenoid pattern of fish and the selectivity of carotenoids

uptake has been studied and it is known that they concentrate carotenoids in

their skin, ovary, liver, muscle, and other tissues (Bagnara & Hadley, 1973).

6

Dietary carotenoids are widely used to provide pigmentation of fish in

cases where the fish can no longer obtain it from natural feeds. Although the

usage of carotenoids in improving pigmentation is commonly adopted, the

proper type of carotenoids, the correct amounts and the feeding period are still

a research issue for different species of fishes in their various growing stage. In

intensive modern aquaculture systems, dietary carotenoids must be included as

part of the diets formulation, especially in diets for ornamental fishes.

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 major

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.

7

Simpson (1982) reported that a number of recent studies on pigmented

carp have shown the conversion to astaxanthin of β-carotene, of lutein, of

zeaxanthin and of isocrytoptoxanthin, echinenone, and canthaxanthin (Figure

2.1). Astaxanthin is mainly responsible for the bright reddish colour of

ornamental fishes. Figure 2.1 shows the carotenoid conversions that have

been postulated for red carp. Koi is listed under this group.

�� β-Carotene

Isocryptoxanthin

Lutein

Echinenone Zeaxanthin

�� α-Doradexanthin 4-Hydroxy-4’-keto- β-carotene

Canthaxanthin β-Doradexanthin

Phoenicoxanthin

Astaxanthin

8

Figure 2.1 Postulated pathways of carotenoid conversions for red carp type fish modified from Simpson, 1982.

2.1.1 Structure of carotenoids

According to Bagnara and Hadley (1973), the general group of

carotenoid pigments are divided into two main categories, the carotenes

(hydrocarbon class) and the xanthophylls (alcoholic, oxygenated class). The

carotenes have a chain of carbon atoms united by alternate single and double

bonds with an ionone ring at each end of the chain (Figure 2.2). The alternation

of single and double bonds is responsible for the colour of carotenoids. The

carotenes contain only carbon and hydrogen atom and the addition of oxygen

will form hydroxyl groups and gives xanthophylls.

Figure 2.2 β- carotene

Torrissen (1986, 1988 & 1989) found that astaxanthin and canthaxanthin

are two major carotenoids found in the flesh of salmonid: Atlantic salmon

(Salmo salar) and rainbow trout (Salmo gairdneri). These two carotenoids are

supplemented in feeds to achieve the desired coloration of the flesh to meet

consumer preferences and are the major commercially available carotenoid

source for aqua feeds formulation.

9

Astaxanthin

Astaxanthin, like other carotenoids, has been shown to have biological

and nutritional functions in fish (Torrissen, 1984; Torrissen et al. 1989b;

Torrissen & Christiansen, 1995a). Astaxanthin is the carotenoid responsible for

the pink-red pigmentation of wild fishes and shrimps. Astaxanthin is the main

carotenoid pigment of red and pink coloured aquatic animals (Simpson et al.

1981; Torrissen, 1986). According to the technical report of Aquasearch (2000),

astaxanthin has chemical features that result in the existence of several forms

of astaxanthin that can be found in nature, such as stereoisomers, geometric

isomers and free or esterified forms (Figure 2.3).

Stereoisomers - Astaxanthin has two chiral or asymmetric centres. These are

the carbons numbered 3 and 3’ on the two rings in the structure. Chemists

identify chiral centres as being either R or S. R and S are from the Latin words

rectus and sinister, meaning right and left. The two chiral centres in

astaxanthin, carbons 3 and 3’ can each exist either in the R or the S form.

Thus, there are a total of three stereoisomers: 3S, 3’S; 3R, 3’S or 3R, 3’R.

Geometric isomers - Carbon-carbon double bonds can have the atoms

attached to them arranged in different ways. If the two groups are attached on

the opposite side of the double bond, they are termed E. If the two groups are

attached on the same side of the double bond, they are termed Z. E and Z are

10

from the German words entgegen and zusammen, meaning together and

opposed. Older texts may refer to Z as “cis” and E as “trans”.

Free or esterified – Astaxanthin has two hydroxyl (OH) groups, one on each

terminal ring. These can be “free” (unreacted) hydroxyls, or can react with an

acid to form an ester. If one hydroxyl reacts with a fatty acid, the result is

termed as a mono-ester. If both hydroxyl groups are reacted with fatty acids,

the result is termed as a di-ester. Adding of a fatty acid to form an ester makes

the esterified end of the molecule more hydrophobic. The hydrophobicity

(difficulty in dissolving in water) of di-esters are higher than mono-ester followed

by the “free” form.

Difference between synthetic astaxanthin and natural astaxanthin

Synthetic astaxanthin is produced as the free (unesterified) xanthophylls

and as a 1:2:1 mixture of the three stereoisomers. Astaxanthin from natural

sources tends to occur predominantly as either the 3S, 3’S; or 3R, 3’R (all – E)

isomers, while the 3R, 3’S (meso) isomer is the most abundant in synthetic

astaxanthin.

Astaxanthin is deposited in different forms, level and tissues by different

fish species. Free astaxanthin is deposited mainly in the flesh, blood serum and

other internal organs, while esterified astaxanthin predominates in the skin,

teguments and eggs of salmon (Torrisen et al., 1989b).

11

Figure 2.3 Three stereoisomers of astaxanthin.

12

Canthaxanthin

Canthaxanthin is a naturally occurring carotenoid found in nature in

tissues of various bird species, fungal species Cantharellus cinnabarinus

(chanterelle), insects, fish, crustacean, fungi, algae and many other organisms.

Canthaxanthin is widely applied as a feed additive delivering red pigmentation

(Figure 2.4). Canthaxanthin is used in poultry pigmentation to impart a red

colour to egg yolks and to broiler skin. Used in conjunction with yellow

pigments, canthaxanthin increases yolk colour intensity to meet market

demands for golden-orange yolks (Roche). In the pigmentation of salmonid

fishes, canthaxanthin is supplied in the feed in order to impart a desirable

colouration to the flesh. Addition of this pigment to grow-out salmonid feeds

ensures that flesh products attain the colour according to consumer

expectations (Storebakken et al. 1987). As well as having vitamin A activity,

canthaxanthin is a well known free-radical scavenger.

Canthaxanthin

13

Figure 2.4 Canthaxanthin 2.1.2 Carotenoid Sources

Hoffman La Roche (Basel, Switzerland) started commercial production of

synthetic canthaxanthin under the trade name “Carophyll Red” in 1964 for

colouring food and feeds. The other manufacturer, BASF group, also produced

synthetic canthaxanthin under the trade name Lucanthin® Red. Beside this

synthetic canthaxanthin, free astaxanthin also been synthesized by Hoffman La

Roche under the trade name “Carophyll Pink”. Over 6000kg of carotenoid

pigments were used in salmonid culture at a cost over $1000 U.S. per kg for

synthesized product (Torrissen et al. 1989b).

Some of the commercial aquafeed producer is currently adding Carophyll

Pink into their dry-pelleted diets (Torrissen et al. 1989b). A group of scientific

researchers did several studies on Atlantic salmon and rainbow trout using

Carophyll Pink and Carophyll Red to improve flesh pigmentation (Choubert &

Storbakken, 1989; Bjerkeng & Berge, 2000; Baker et al., 2002). Based on the

results of studies on salmonids, astaxanthin seems to be absorbed and

deposited better than canthaxanthin. (Torrissen, 1986; Storbakken et al., 1986;

Storbakken et al., 1987; Foss et al., 1987b). Hatlen (1995) also tested the

pigmentation on Arctic charr using Carophyll Pink in different concentrations.

Harpaz et al. (1998) compared the effect of three different carotenoid

sources (dried algal cells Dunaliella salina, Carophyll Pink and alfalfa meal) on

14

growth and pigmentation of crayfish and found that crayfish fed feeds fortified

with carotenoids exhibited better coloration. Growth and survival of the crayfish

were not affected by carotenoids.

According to the NatuRose Technical Bulletin published by Cyanotech

Corporation, NatuRoseTM is a natural source of astaxanthin derived from a

unique strain of the microalga, Haematococcus pluvialis grown under controlled

conditions on the island of Hawaii. The largest percentage of carotenoid

fraction of NatuRose consists of astaxanthin, with about 15% of the remaining

fraction consisting of canthaxanthin, lutein and beta-carotene (Lorenz, 2000).

This is the algae first studied by the scientist Girod Chantrans two hundred

years ago and has recently received much attention due to its capability to

synthesize and accumulates large amounts of astaxanthin during and at the end

of the growth phase.

NatuRoseTM is used as a pigmentation enhancer and as a nutrition

source for a variety of freshwater and marine species and poultry animals

(Lorenz, 2000). The astaxanthin ester composition of Haematococcus algae

meal is similar to that of crustaceans (Lambertsen and Braekken, 1971, Maoka

et al., 1985, Foss et al., 1987a). The market price of alga products of

astaxanthin is about US$300 per kg and β-carotene is about US$600 per kg

(Borowitzka, 1994).

Turujman et al. (1997) indicated that farmed salmon should be fed a diet

containing natural astaxanthin to achieve the same astaxanthin profile as their

15

wild counterparts. This study showed that farmed salmon could be easily

distinguished from the wild salmon. The farmed salmon are fed synthetic

astaxanthin, which contains primarily the 3R-3'S isomer and unable to convert it

to the more common and natural 3S-3'S form. This study also concluded that

the 3S-3'S is the main form found in wild Pacific and Atlantic salmon species

and this is the same form found in H. pluvialis.

Scientific studies of Haematococcus microalgae by research institutions

and international feed companies have concluded that this microalgae is a safe

and effective natural alternative for pigmenting aquatic animals (Lorenz, 2000).

Miki (1991) found that the more stable esterified form of astaxanthin is believed

to be an adaptive feature to be able to store astaxanthin in tissues without

excessive oxidation. Esterified astaxanthin is the main form found in H.

pluvialis. Naturose has been successfully used as a source of pigments in

aquaculture in such species as shrimp, rainbow trout, coho and Atlantic salmon.

Scientific research done by Torrissen (1989) compared two different

pigment sources (Carophyll Pink and Haematococcus astaxanthin) also

indicated that the algal astaxanthin can be used as an alternative pigment

source for Atlantic salmon (Lorenz, 2000). Choubert & Heinrich, (1993) used H.

pluvialis in comparison with synthetic astaxanthin and canthaxanthin to test

pigmentation on rainbow trout, Oncorhynchus mykiss. Their results indicated

that physical colour measurements in fish fed with algal incorporation diets

showed that increased pigmentation of the trout muscle caused an increase in

Chroma (C*) and a reduction in hue (h*) and lightness (L*). Recent studies on

16

Haematococcus indicated that properly disrupted cells have similar

bioavailability as commercially formulated astaxanthin (Barbosa et al., 1999).

NatuRose has produced demonstrable results in the pigmentation of koi

and many varieties of ornamental fish (Lorenz, 2000). Koi breeders carried out

fish trials to determine the ability of the koi to assimilate xanthophylls from

Haematococcus algae and they found that koi fed with astaxanthin from

Haematococcus algal meal had a dark red coloration, whereas the control

group that had no astaxanthin had a pale orange skin coloration (Lorenz, 2000).

Based on the unpublished data, a group of fish breeder from Aquarium Center,

Inc. of Randallstown Maryland, Reef Propagations Inc. and fish farms in Hawaii

have added or mixed NatuRose into the ornamental fish feeding diets. A

significant improvement in colour and pigmentation could be seen in freshwater

and marine ornamental fish, such as tetras, cichlids, gouramis, goldfish, koi,

danios, swordtails, Rosy Bards, rainbow fish, discus and clown anemone fish

(Lorenz, 2000).

Spirulina is a multicellular, filamentous blue-green algae belonging to the

phylum Cyanophyceae (Torrissen et al. 1989b). Spirulina can be considered as

the most concentrated natural sources of nutrition known for both terrestrial and

aquatic animals. Wagener and Rebello (1987) stated that spirulina which

contain valuable ingredients of protein required in animal feed is an excellent

substitute for animal protein in human nutrition. Animal feed grade spirulina

powder is used as a feed ingredient for pigmentation of prawns, marine fish and

ornamental fish. Spirulina have relatively expensive market prize at around $17

17

to $25 per kg depends on the quality (Henson, 1990). The commercially

produced microalgae, Spirulina platensis or S. maxima are a remarkably rich

source of amino acids, carotenoids and vitamin (Henrikson, 1989 cited in

Henson, 1990).

Henson (1990) stated that Japanese fish farmers are discovering the

potential benefits of using spirulina algae as a feed supplement. They

discovered five key benefits of using spirulina, such as better growth rates,

increased survival rates, improvement in quality and enhanced coloration of the

cultured fish while reduced medication requirements and waste in the effluent

(wastewater treatment). They found that spirulina increases feed palatability

while providing essential nutrients.

Producers of fish fry starter feeds in Japan are including spirulina in their

premium feeds. Results show that the palatability and flavour of spirulina trigger

the feeding response of the fish fingerlings therefore allowing it to get the

nutrition it needs to survive. Taiyo Fisheries has conducted a sixty days feeding

trial with yellowtail fingerlings with results showing both an increase in growth

and decrease in mortality rates with a 37% increase in harvested tons (Kato,

1988). Fish fed with spirulina have improved flavour (Hirano, 1985; Suyama,

1984), flesh texture (Henson, 1990), firm flesh and bright skin colours (Mori,

1987). Japanese growers of ornamental koi carp, mackerel, yellowtail and sea

bream observed colour improvement with spirulina supplemented diets

(Matsuno, 1979). Fish with firm texture and sharp colours can command a

higher selling price.

18

Spirulina reduces toxicity of medications and may itself have anti-viral

properties (Henson, 1990). Many medications can cause kidney damage

resulting in reduced vitality or even death. Spirulina supplemented diets reduce

pollution of the environment by increasing feed utilization due to uneaten and

undigested feed. Moreover, spirulina also help to reduce wastewater pollutants,

and finally eliminating costly treatment systems. Overall, spirulina will increase

the effectiveness of existing systems.

Boonyaratpalin and Unprasert (1989) indicated that spirulina influence

the pigmentation of red tilapia. Likewise, spirulina added in the diet of

ornamental fish such as goldfish and fancy red carp enhanced pigmentation

(Miki et al., 1986; Borowitzka, 1994), whereas Choubert (1979) did not observe

pigmentation in rainbow trout fed diets containing spirulina. Gouveia et al.

(2003) compared various microalgal biomass carotenoid sources (Chlorella

vulgaris, H. pluvialis and Spirulina maxima) with synthetic astaxanthin

(Carophyll Pink) and the found that microalgal biomass, especially C. vulgaris,

may contribute to enhanced skin colour of koi and goldfish.

CarosolTM 3% is in a spray dried water dispersible form of natural mixed

carotenes which has been extracted and purified from fruits of the oil palm tree.

CarosolTM 3% contains natural alpha-carotene, beta-carotene, gamma-

carotene, lycopene and other carotenoids. It is a unique mixture in natural

proportions and commonly found in fruits and vegetables. Concentrated

CarominTM is an opaque reddish vegetable oil suspension of natural occurring

mixture of carotenoids, extracted and concentrated from fruits of the oil palm

19

tree. CarominTM has been used to formulate colour emulsions by food colorant

manufacturers worldwide. Besides being a natural food colorant, CarominTM

has vitamin A activity and confer protection against free radical damages to the

human cells. There is a lack of scientific studies on these two carotenoid

sources in aquatic animals. As far as we know, these two carotenoid sources

have not been evaluated for use as a pigmentation agent in fish feeds.

2.1.3 Function of carotenoids

2.1.3.1 Biological Functions

Czeczuga (1979) reported that fishes with higher level of carotenoids

were more resistant to bacterial and fungal diseases than fishes with low

carotenoid levels. Torrissen (1984) showed that carotenoids supplied in the diet

increased the growth rate in Atlantic salmon. This strongly indicated that

carotenoids have a biological function. Segner et al. (1989) found an improved

liver histology and performance in fish fed high astaxanthin levels in the diet.

Other carotenoids functions include (Torrissen 1989):

1) Fertilization hormone

2) Source of pigments for chromatophores

3) Function in respiration

4) Protection from light

5) Resistance to elevated temperature and ammonia

6) Provitamin A

20

Other than pigmentation, studies indicate that carotenoids also have a

biological function involved in growth and reproduction. Several researchers

have reported positive effects on growth for different fishes fed diets

supplemented with carotenoids, especially astaxanthin (Torrisen, 1984 and

1986; Christiansen et al., 1994). The mobilization of carotenoids from the flesh

to the skin and ovaries during maturation also shows that carotenoids may have

a function in reproduction. Torrissen and Torrissen (1985) detected a decrease

in the carotenoids content of the flesh of Atlantic salmon about six months prior

to spawning and proved the mobilization of carotenoids from the flesh to the

skin and ovaries during maturation. Torrissen (1984, 1989b) showed the

presence of astaxanthin and canthaxanthin in the plasma of feeding rainbow

trout, indicating that serum is the transport medium.

Astaxanthin has also been shown to increase egg survival and

percentage of fertilized eggs, to protect eggs against extreme conditions (Craik,

1985) and to stimulate growth (Torrissen, 1984). Craik (1985) assumed that the

colour of eggs provides an indication of the quality of eggs. It has been

suggested that carotenoids may have a respiratory function because the eggs

of species that undergo development in poorly oxygenated water have more

pigment than those which develop in well-oxygenated water and large eggs

tend to have higher carotenoid concentration than small eggs. It has been

proposed that astaxanthin has a positive effect on reproduction. Carotenoid

content has also been linked to the ability of the egg to tolerate harsh

environment conditions.

21

A number of studies published by Christiansen in Norway has examined

the effects of dietary astaxanthin supplementation on fertilization and egg

survival in Atlantic salmon (Christiansen & Torrissen, 1997), growth and survival

of Atlantic salmon juveniles (Christiansen & Torrissen, 1996), and first-feeding

fry (Christiansen et al., 1995a), antioxidant status and immunity in Atlantic

salmon (Christiansen et al., 1995b) and effects of astaxanthin and vitamin A on

growth and survival during the first feeding of Atlantic salmon (Christiansen et

al., 1994). A recent ground-breaking study (Christiansen & Torrissen, 1995b;

Christiansen et al., 1996) demonstrated that Atlantic salmon fry have a definitive

growth and survival requirement for astaxanthin in their diet. Fish fed diets with

astaxanthin below 5.3ppm were found to have marginal growth; those fed levels

above 5.3ppm had significantly higher lipid levels accompanied by lower

moisture levels. When fry were fed astaxanthin concentrations below 1ppm,

survival rates plummeted. More than 50% of the fry fed diets with less than

1.0ppm astaxanthin died during the experimental period and survival of those

groups receiving higher concentrations had survival rates greater than 90%.

Thus, Atlantic salmon have the distinction as being the first salmonid species for

which astaxanthin has been shown to be an essential vitamin, with absolute

minimum levels being about 5.1ppm. Higher astaxanthin levels of 13.7ppm in

the feed continued to improve the fish lipid levels. Their results also strongly

suggested a provitamin A function for astaxanthin over the same fry-feeding

period (Christiansen et al., 1994). Furthermore the results indicated astaxanthin

as a fertilization hormone and photo protective element (Christiansen &

Torrissen, 1997). Astaxanthin also has function in respiration and stress

protection from elevated temperatures or ammonia (Christiansen et al., 1995b).

22

2.1.3.2 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). Torrissen (1989) had shown evidence pointing to the

vital role of carotenoids in the physiology and health of plants and animals. This

study observed higher growth rates in Atlantic salmon. Astaxanthin has also

been incorporated into feeds for ornamental fishes.

Ito et al. (1986) tested the effects of feeding red sea bream (Pagrus

major) different astaxanthin sources. In the group fed free astaxanthin, the

carotenoid content of the skin improved for 1 month, but reached a saturation

point and did not increase further. In the group fed astaxanthin ester, the

carotenoids in the skin was significantly higher after the first and second

sampling periods. After two months, the group fed astaxanthin esters had 1.7-

fold higher astaxanthin content in the skin than the group fed free astaxanthin

23

(13.23 mg kg-1 compared to 7.94 mg kg-1, respectively). Thus, dietary

astaxanthin esters are more efficiently utilized than free astaxanthin for

deposition and coloration of skin of red sea bream.

The findings of Smith et al. (1992) on coho salmon suggested that

feeding low astaxanthin concentrations throughout the grow-out period from fry

to market size resulted in the most efficient use of pigment and produced

uniform pigmentation. In addition, an increased dietary dose has been shown

to have no significant effect on the variation in pigmentation in Atlantic salmon

(Torrissen et al., 1995), while an increase in the duration of feeding carotenoids

has been reported to reduce inter-fish variation in pigmentation in Atlantic

salmon (Torrissen et al., 1995), rainbow trout and Chinook salmon (March and

MacMillan, 1996).

Storebakken et al. (1987) also showed similar results in the study on

pigmentation of Atlantic salmon. The retention coefficients decreased as the

pigment dose in the diet increase and this phenomenon showed that there is an

optimal level of dietary carotenoids which maximised deposit accumulation of

carotenoid in the fish. The excess carotenoid given would be a waste and it will

increase the cost of production.

Storebakken et al. (1987) also reported that astaxanthin tends to be

better deposited than canthaxanthin in Atlantic salmon. Recently, published

data showed that canthaxanthin can be equally as effective as astaxanthin, if

not better, in pigmenting Atlantic salmon (Buttle et al., 2001). Given the

24

relatively lower market price for canthaxanthin, this has attracted enormous

interest. On the other hand, canthaxanthin has been perceived as being less

“natural” than astaxanthin, primarily because astaxanthin is by far the most

abundant carotenoid in the flesh of wild Atlantic salmon (Shahidi et al., 1998).

Stability of canthaxanthin through secondary processing (freezing/smoking) has

also been of concern (Sheehen et al., 1998).

Torrissen (1989) assumed that pink to red pigmentation in flesh could be

achieved by pigmentation of astaxanthin and canthaxanthin. The study of

Choubert and Storebakken (1989) on dose response to astaxanthin and

canthaxanthin pigmentation of rainbow trout fed various dietary carotenoids

showed that astaxanthin is more efficiently utilized and the flesh pigmentation

increased by increasing the dietary carotenoids concentration in different ratio

of different pigment.

Due to lack of scientific studies, most of the information on ornamental

fish, especially koi, is unpublished data, university project reports and

testimonies from hobbyists, aquaculturists and fish farmers.

Shahreza (1994) added hibiscus flower and carrot into goldfish practical

diet to test the skin colour enhancement and the result indicated that

carotenoids accumulated in the body of goldfish especially in the head region.

The study on the effect of Carophyll Pink on the skin coloration of Angelfish

(Pterophyllum scalare), Japanese carp (Cyprinus carpio) and goldfish

(Carassius auratus) also showed the same result (Lim, 1999). Paripatananont

skin colour changes in ornamental koi (cyprinus

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