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OPTIMIZATION OF GROWTH CONDITIONS OF LOCAL LEECHEES FOR HIRUDIN PRODUCTION
ZULHISYAM BIN ABDUL KARI @ ABDULLAH MASTER OF SCIENCE
2011
Optimization of Growth Conditions of Local Leeches for Hirudin Production
by
Zulhisyam Bin Abdul Kari @ Abdullah
A thesis submitted in fulfillment of the requirements for the degree of Master of Science (Agricultural Biotechnology)
Faculty of Agro Industry and Natural Resources UNIVERSITI MALAYSIA KELANTAN
2011
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A special dedication to my beloved parents and respected supervisor and co-supervisor
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THESIS DECLARATION
I hereby certify that the work embodied in this thesis is the result of the original research and has not been submitted for a higher degree to any other University or Institution.
I agree that my thesis is to be made immediately available as hardcopy or on-line open access (full text).
I agree that my thesis is to be made available as hardcopy or on- line (full text) for a period approved by the Post Graduate Committee.
Dated from ____________ until _____________
Contains confidential information under the Official Secret Act 1972
Contains restricted information as specified by the organization where research was done
I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.
1. The thesis is the property of Universiti Malaysia Kelantan.
2. The library of Universiti Malaysia Kelantan has the right to make copies for the purpose of research only.
3. The library has the right to make copies of the thesis for academic exchange.
SIGNATURE SIGNATURE OF SUPERVISOR
IC / PASSPORT NO. NAME OF SUPERVISOR
Date: Date:
iii OPEN ACCESS
EMBARGOES
CONFIDENTIAL
RESTRICTED
ACKNOWLEDGMENTS
I wish to express my sincere gratitude and appreciation to my supervisors Professor Datuk Dr. Ahmad Anwar Ismail and Professor Dr. Hj. Ibrahim Che Omar for their invaluable guidance, encouragement, help, support and patient throughout the period of this study. I am grateful to Professor Dr. Awang Soh Mamat, Hj. Rosly Bin Hassan and En. Suhaimi Omar for their indispensable advice and suggestion. A millions thanks also to Addi Che Din and Raja Zainal Abidin Bin Raja Ahmad from PT Dynamic Consultant (KT 0224558 – W) for providing me the stock (leeches) over the whole duration of this research.
Acknowledgement is also extended to the Faculty of Agro Industry and Natural Resources of Universiti Malaysia Kelantan, for providing me the use of lab facilities. Thanks are also due to all technical staff in the department including lab assistants for their assistance and help during the course of my research. My sincere thanks are also extended to Universiti Malaysia Kelantan for the Graduate Research Assistantship (GRA) scheme and Short Term Research Grant (SGJP) under the vote R/SGJP/A03.00/00193A/001/2010/000031 for financial support. Last but not least, I am pleased to have all friends, my roommates, postgraduate students in Faculty Agro Industry and Natural Resources who have had helped and encouraged me to accomplish this research.
Invaluable thanks are also due to En. Jaffar Sulong from Faculty of Entrepreneurship and Business Universiti Malaysia Kelantan for the statistical analysis and also for advice and help.
To my best friends, Zack, Zairi, Due Ya and Julat I will always remember their support, encouragement, help and understanding during my stay in the campus.
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Not least, I am most grateful to both my parents; Abah and Ummi for their endless love, prayers, care and financial support throughout my study. Finally, not forgeting my gratitude to my siblings, Abe Ju, Ha, Kak Na, Pok Teh, Kak Chik, Pok Chik, Pok Dik and Mok Dik for their encouragement and support. Without the encouragement of my family, I will never be where I am now accomplishing this challenging task.
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TABLE OF CONTENTS
PAGE DEDICATION
THESIS DECLARATION
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF EQUATIONS
LIST OF ABBREVIATIONS / NOTATIONS / GLOSSARY OF TERMS
ABSTRAK ABSTRACT
CHAPTER 1 INTRODUCTION
1.1 Background of the study
1.2 Significance of the study 1.3 Objectives
CHAPTER 2 LITERATURE REVIEW 2.1 Taxonomy
2.2 Morphology
2.2.1 External anatomy 2.2.2 Suckers
2.2.3 Internal anatomy
2.3 Habitat and factors that affect leech survivorship
2.4 Leech mating
2.5 Leech feeding and metabolic changes
2.6 Reproduction
2.6.1 Cocoons
2.7 The importance of leeches in trophic webs 2.7.1 Rule of leeches in thropic webs
2.7.2 Uses in medicine
2.7.3 Hirudin protein in medicine 2.8 First biochemical and structural studies
ii iii iv vi ix x xii xiii xv xvii
1 3 6
7 8 8 10 12 13 16 16 18 19 20 20 21 22 22 vi
2.9 Purification of hirudin 2.9.1 Extract from leeches
2.9.2 Protein detection and separation 2.9.3 Protein quantification assay 2.9.4 Protein purification CHAPTER 3 MATERIALS AND METHODS
3.1 Effect of light intensity, temperature, and diet on reproductive performance
3.1.1 Origin of broodstock
3.1.2 Experimental design 3.1.3 Data collection
3.1.3.1 Cocoon deposition number 3.1.3.2 Hatching number and hatching rate
3.1.3.3 Broodstock mortality
3.1.3.4 Cocoon size and wet weight
3.1.4 Statistical analyses
3.2 Effect of diet on juvenile growth 3.2.1 Experimental design
3.2.2 Experimental method
3.2.2.1 Juvenile growth 3.2.2.2 Juvenile survivorship 3.2.3 Statistical analyses
3.3 Study on leech crude extract as an anti- coagulant agent 3.3.1 Sample preparation
3.3.2 Preparation of leech crude extracts
3.4 Detection and purification of hirudin protein 3.4.1 Materials and chemicals
3.4.2 Preparation of leech crude extract
3.4.3 Protein quantification
3.4.4 Sephadex G-75 Gel Filtration of leech crude extract
3.4.5 Protein activity
3.4.6 Protein profiling CHAPTER 4 RESULTS
4.1 Effect of different temperatures, light intensities and diet on the reproductive performance of leeches
4.1.1 Cocoon deposition number 4.1.2 Hatching number
4.1.3 Hatching rate
4.1.4 Mortality of broodstock leeches 4.1.5 Cocoon size
4.1.6 Cocoon wet weight
23 23 24 25 27
28 28 29 30 30 30 31 31 31 32 32 33 33 33 34 34 34 34 36 36 36 37 37 39 40
41 43 44 45 46 47 48 vii
4.2 Effect of diet on juvenile growth
4.2.1 Growth parameters of juvenile influenced by diet 4.2.2 Influence of diet on juvenile survivorship
4.3 Anticoagulant property of crude extract from local leeches
4.4 Detection and purification of hirudin protein
4.4.1 Protein quantification from different body parts of local
leeches.
4.4.2 Sephadex G-75 Gel Filtration from different parts of body of leeches
4.4.2.1 Protein separation from leech head
4.4.2.2 Protein separation from the whole body of leeches 4.4.2.3 Protein separation from headless body
4.4.3 Hirudin protein profiling
4.4.4 Protein profiling
CHAPTER 5 DISCUSSIONS
5.1 Effect of different temperatures, light intensities and diet on the reproductive performance of leeches
5.2 Pre-test on leech crude extract as an anti-coagulant agent 5.3 Detection and purification of hirudin protein from local leeches
CHAPTER 6 CONCLUSION
REFERENCES APPENDICES
LIST OF PUBLICATIONS LIST OF AWARDS
49 49 50 50 51 51 52 52 54 56 57 58
61 72 77 84 86 95 127 129
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LIST OF TABLES
NO. PAGE
3.1 Different conditions tested on growth and reproductive efficiency of leeches
3.2 Ingredients of the juvenile commercial product ‘A’ experimental
diet.
4.1 Comparison of reproductive parameters of the local leeches under different culture conditions.
4.2 Effect of diet on the survivorship and growth performance of juvenile local leeches
4.3 Time taken for blood clotting and contraction under three treatment regimes
4.4 Protein concentration of local leech crude extract from a different parts of leeches body
4.5 Protein concentration of different peaks from Sephadex G-75 Gel Filtration crude extract from leech head
4.6 Time taken for blood clotting and contraction under three different peaks. Sample obtained from leech head
29 33 42 50 51 52
54 58
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LIST OF FIGURES
NO. PAGE
1.1 Body of local leech
2.1a The upper part of the body leech 2.1b The lower part of the body leech
2.2 The posterior sucker acts as adhesive foot for the mobility 2.3 Anterior sucker with triradiate opening
2.4 Leech dissection
2.5 Reproduction process of leeches (Mason et al., 2004) 2.6 Physical appearance of cocoons
4.1 Changes in the cocoon number deposition under different culture conditions
4.2 Changes in the hatching number under different culture conditions 4.3 Changes in the hatching rate (%) under different culture conditions 4.4 Changes in the mortality rate (%) under different culture conditions 4.5 Changes in the cocoon length (mm) under different culture
conditions
4.6 Changes in the cocoon diameter (mm) under different culture conditions
4.7 Changes in the cocoon weight (g) under different cultured conditions
4.8 Purification of protein obtained from leech head
4.9 Purification of protein obtained from the whole body of leeches 4.10 Purification of protein obtained from the body of leeches
3 9 10 11 12 13 18 20 43 44 45 46 47 48 49 53 55 57 x
4.11 Electrophoretic patterns (12% gel) on crude extract from leech head 4.12 Electrophoretic patterns (12% gel) on purification by Sephadex
G-75 Gel Filtration from leech head
5.1 Individual cocoon produced by an adult local leech laid on top of the soil
5.2 The juvenile enclosed in the cocoon 5.3a A dry cocoon structure
5.3b A dry cocoon laid on soil after 2 weeks deposition
5.4 A burrowing hole made by leeches to enable them to hide in the soil 5.5 Rabbit blood treated with leech crude extract
5.6 Pure rabbit blood only (control)
5.7 Rabbit blood treated with distilled water
5.8 Scheme of the hirudin interaction with the active site of thrombin (Markwardt, 1992)
5.9 Sephadex G-75 Gel Filtration profiles of leech crude extract from different parts of body
59 60 62 65 66 67 68 74 74 75 76 82
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LIST OF EQUATIONS
NO.
3.1 Average hatching number 3.2 Average hatching rate 3.3 Percentage weight gain 3.4 Specific growth rate
PAGE 30 30 33 33
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LIST OF ABBREVIATIONS / NOTATIONS / GLOSSARY OF TERMS
JAKIM cm BC kDa
°C
%
SDS APS TEMED Nm GF UV m lx
± L FT1 FT2
≤
Jabatan Kemajuan Islam Malaysia
Department of Wildlife and National Parks Centimetre
Before Christ Kilo Dalton Degree Celcius Percent
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Sodium dodecyl sulfate Ammonium persulfate Tetramethylethylenediamine Nanometer
Gel Filtration Ultraviolet Meter Lux
Plus-minus Length
Food Treatment 1 Food Treatment 2 Less and same than
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SPSS SGR WG ANOVA rpm h min BSA
ml
w/v
mg g µl A595
O.D M Rf v/v Mr DNA mA V ATU PCR SEC sq. cm µg Kg
Statistical Program for Social Sciences Specific Growth Rate
Weight Gain
Analysis of Variance Revolution per minute Hours
Minutes
Bovine Serum Albumin Mililitre
Weight per Volume
Miligram Mililitre Gram Weight per Volume
Microlitre
Absorbance at 595 Optical Density Mole
Relative mobility Volume per Volume Molecular Mass
Deoxyribonucleic Acid Miliampere
Volt
Anti Thrombin Unit
Polymerase Chain Reaction Size Exclusion Chromatography Square centimeter
Microgram Kilogram
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Pengoptimuman Terhadap Keadaan Pembesaran Lintah Tempatan Untuk Pengeluaran Hirudin
ABSTRAK
Lintah (Filum: Annelida, Kelas: Hirudinea) terdapat di serata tempat di seluruh dunia dalam pelbagai habitat, seperti air tawar, laut, gurun, dan oasis (Gouda, 2006). Dalam kajian ini, kesan terhadap kadar pengamatan cahaya, suhu dan diet yang berlainan ke atas pembiakan lintah tempatan telah dikenalpasti dengan lapan keadaan ternakan yang berbeza. Selepas 3 bulan lintah diternak, bilangan kepompong yang terhasil adalah sangat berbeza (p=0.00) mengikut keadaan ternakan yang berbeza. Purata bilangan penetasan kepompong juga berbeza (p ≤ 0.05) di mana keadaan ternakan 1 mempunyai nilai yang paling tinggi iaitu sebanyak 6.23±0.25. Bagi purata kadar penetasan kepompong pula, tidak ada perbezaan telah dikenalpasti di antara semua keadaan ternakan (p=0.354). Keadaan ternakan 5 menunjukkan kadar kematian yang paling tinggi dengan nilai purata dan sisihan piawai sebanyak 52±13.86%. Saiz di antara kepompong juga tidak menunjukkan perbezaan di mana keadaan ternakan 1 mempunyai saiz kepompong yang paling besar jika dibandingkan dengan keadaan-keadaan ternakan lain dengan nilai purata dan sisihan piawai sebanyak 22.19±0.92 mm (panjang) dan 13.26±0.07 mm (ukur lilit). Berat kepompong juga berbeza dengan keadaan ternakan yang berbeza (p ≤ 0.05) di mana keadaan ternakan 1 menghasilkan kepompong yang paling berat dengan nilai 1.26±0.11g jika dibandingkan dengan keadaan ternakan 5 di mana ia menghasilkan kepompong yang paling ringan dengan nilai purata dan sisihan piawai sebanyak 0.22±0.38g. Selepas 2 bulan juvenil diternak, berat akhir adalah sangat berbeza mengikut rawatan, dimana juvenil yang diberi makan darah belut (FT1) memberikan berat akhir yang paling tinggi (0.8893±0.012g). Peratus kenaikan berat (WG) dan spesifik kadar tumbesaran (SGR) juvenil yang diberi makan produk komersial ‘A’ (FT2) adalah paling rendah dengan nilai purata dan sisihan piawai ialah masing-masing 769.41±11.54% dan 3.6±0.02%. Juvenil yang diberi makan darah belut menunjukkan kadar hidup yang paling tinggi (93.33±5.77%). Hirudin adalah nama generik dalam kelompok peptide dan ianya dijumpai di dalam semua kelenjar liur lintah (Wallis, 1996). Dalam kajian ini, pengekstratan lintah dilakukan dengan menggunakan kaedah konvensional dan larutan ekstrak ini akan diuji terhadap darah arnab. Keputusan menunjukkan darah arnab yang dirawat dengan larutan ekstrak dari lintah tempatan memberikan masa yang paling lama untuk darah menjadi beku (15.33±0.6 minit) dan darah yang telah beku tidak mengecut sehingga 77.67±2.52 minit jika dibandingkan dengan rawatan pertama dan rawatan kedua
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dimana darah akan menjadi beku dalam masa yang singkat masing-masing ialah 0.59±0.01 dan 2.83±0.3 minit dan darah yang beku tidak mengecut masing-masing sehingga 1.17±0.29 dan 3.17±0.29 minit. Kajian awal ini menunjukkan bahawa ekstrak dari lintah tempatan mengandungi agen anti-pembekuan darah untuk mencegah darah daripada menjadi beku.
Lintah tempatan yang mengandungi agen anti-pembekuan sudah lama diketahui. Penulinan protein hirudin, profil protein dan kuantifikasi protein dari bahagian yang berbeza dari lintah tempatan telah dilaporkan. Keputusan kajian menunjukkan larutan ekstrak lintah dari bahagian kepala memberikan nilai yang paling tinggi pada kepekatan protein iaitu sebanyak 69.33 µg/ml jika dibandingkan dengan larutan ekstrak lintah dari bahagian badan tanpa kepala dan seluruh badan ialah masing-masing sebanyak 26.67 µg/ml dan 36.67 µg/ml.
Larutan ekstrak dari bahagian kepala akan digunakan untuk proses penulinan dengan menggunakan kromatografi gel permeasi Sephadex-G 75. Fraksi yang mengandungi sejumlah besar protein dengan nilai O.D yang melebihi 0.2 pada 595nm akan diuji untuk aktiviti anti- pembekuan darah. Keputusan menunjukkan Puncak III (Nombor Fraksi= 23, Ketumpatan Optik (O.D) = 0.256, kepekatan protein = 82.00 µg/ml) memberikan masa yang paling lama untuk darah menjadi beku iaitu 123.33±15.28 minit dan darah yang telah beku tidak mengecut sehingga 380.00±17.32 minit jika dibandingkan dengan Puncak II (Nombor Fraksi= 21, Ketumpatan Optik (O.D) = 0.293, kepekatan protein = 94.33 µg/ml) dan Puncak V (Nombor Fraksi= 36, Ketumpatan Optik (O.D) = 0.214, kepekatan protein = 68.00 µg/ml ) dimana darah akan menjadi beku masing-masing pada 13.33±2.89 dan 23.33±2.89 minit dan darah yang beku akan mengecut sehingga masing-masing pada to 24.33±0.58 dan 41.67±2.89 minit. Larutan extrak lintah yang mempunyai masa yang paling lama utuk darah menjadi beku akan dianalisis untuk profil protein dengan menggunakan SDS-PAGE. Keputusan menunjukkan berat protein hirudin sekitar 11.36 kDa. Daripada profil penulinan dengan menggunakan Sephadex G-75 ke atas ekstrak lintah daripada bahagian badan lintah yang berlainan ia juga menunjukkan bahawa anti pembekuan darah (protein hirudin) hanya terdapat pada bahagian kepala sahaja.
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Optimization of Growth Conditions of Local Leeches for Hirudin Production
ABSTRACT
Leeches (Phylum: Annelida, Class: Hirudinea) are widely distributed all over the world in various habitats, such as freshwater, seas, desert, and oases (Gouda, 2006). In this study, the effect of light intensity, temperature and diet on the reproductive efficiency of local leeches was examined with eight different conditions. After 3 months of culture, the number of cocoons produced was very significantly different among the different conditions (p=0.00). The average number of hatchings per cocoon was significantly different (p ≤ 0.05) where Condition 1 gave the highest number (6.23±0.25), but hatching rate was not (p=0.354).
The Condition 5 produced the highest mortality of parent leeches (52±13.86%). The sizes of the cocoons were not significantly different among the treatments, with the Condition 1 having the largest cocoon of 22.19±0.92 mm and 13.26±0.07 mm according to their length and diameter, respectively. The wet weight of cocoons was significantly different (p ≤ 0.05) with the Condition 1 producing the heaviest cocoons of 1.26±0.11g compared to Condition 5 producing the lightest cocoons of 0.22±0.38 g. The effect of diet (FT1: fresh eel blood and FT2: juvenile commercial product ‘A’) on the growth and survivorship of the juvenile leeches was also studied. After 2 months of culture, the final body weight was significantly different among the treatments, with juveniles in the FT1 (fresh eel blood) gave the highest final body weight (0.8893±0.012g). Percentage weight gain (WG) and specific growth rate (SGR) of the juveniles in the treatment FT2 (juvenile commercial product ‘A’) were lowest with mean and standard deviation of 769.41±11.54% and 3.6±0.02%, respectively. Juveniles in the FT2 (fresh eel blood) treatment had the highest survival rate (93.33±5.77%). Hirudin is the generic name for a family of closely related homologous peptides that are all found in the cranial salivary glands of leech (Wallis, 1996). In this study, the leech crude extract was prepared by using conventional method and tested to fresh rabbit blood. The results showed that the rabbit blood treated with leech crude extract gave the longest time of blood to clot and the clotted blood was did not contract up to 77.67±2.52 minutes when compared with treatments 1 (Control) and treatment 2 (Rabbit blood + distilled water) where the blood clot in 0.59±0.01 and 2.83±0.3 minutes and the clotted blood did not contract up to 1.17±0.29 and 3.17±0.29 minutes, respectively. These results revealed that crude extract of leeches contain an anti-coagulant agent to prevent blood from clotting. It has long been known that local leeches contain a substance with anticoagulant properties. The purification of hirudin, protein profiling and protein quantification from different parts of leeches are reported. The result shows the sample from leech head gave the highest result in protein concentration with value of 69.33 µg/ml compared with either the sample from the whole body and headless body which was 36.67 µg/ml and 26.67 µg/ml, respectively. Sample from leech head was purified
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by gel permeation chromatography on Sephadex G-75. Fractions which contain significant amounts of protein with absorbance value more than 0.2 at 595 nm were tested for their anti- coagulant activity. The result shows that the Peak III (Fraction Number= 23, Optical Density (O.D) = 0.256, protein concentration = 82.00 µg/ml) gave the longest time for blood to clot (123.33±15.28 minutes) and the clotted blood did not contract up to 380.00±17.32 minutes compared with Peak II (Fraction Number= 21, Optical Density (O.D) = 0.293, protein concentration = 94.33 µg/ml) and Peak V (Fraction Number= 36, Optical Density (O.D) = 0.214, protein concentration = 68.00 µg/ml ) where the blood took 13.33±2.89 and 23.33±2.89 minutes to clot and the clotted blood did not contract up to 24.33±0.58 and 41.67±2.89 minutes, respectively. Crude protein and eluted fractions which contain significant amounts of protein with the longest time of blood to clot were subjected to SDS- PAGE analysis. The result shows that the molecular weight of hirudin was about 11.36 kDa.
From the purification made the profiles of leech crude extract from different parts of body indicate that anti-coagulant agent (hirudin protein) was only found in leech head.
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CHAPTER 1
INTRODUCTION
1.1 Background of the study
Leeches (Phylum: Annelida, Class: Hirudinea) are widely distributed all over the world in a diversity of habitats, such as freshwater, seas, deserts, and oases (Gouda, 2006).
They can be predators, vectors of parasites, as well as prey of other aquatic animals (Saywer, 1986a; Keim, 1993). In recent years, some leech populations have declined dramatically due to over-exploitation for fishing bait and medicinal purposes (particularly in Europe and Asia), and due to pollution (Sawyer, 1981; Elliot & Tullett, 1984; Wells &
Coombes, 1987; Petrauskiene, 2003; Trontelj & Utevsky, 2005). Leech therapy is one of the most important and widely practiced methods of regimental therapy used for local evacuation of morbid tumours (Ibn Sina, 1998). The therapy is known from the time of extreme antiquity, more than 2,500 years ago and is still alive. This fact testifies to its efficiency in various health problems. In Malaysia, the entrepreneurs used leeches as trading animal and the stock are exported to meet the demand in countries such as Korea, Ukraine, China and Spain. Normally, the leech products are exported in the dried form approved and certified by the Department of Fisheries, Ministry of Agriculture and Agro Based Industry, Malaysia. However, there are also a handful of unscrupulous entrepreneurs who used leeches for personal gain by deceiving the public in traditional medicine. This action on their part will aggravate the leech farming industry here and apprehension amongst the local folks on the viability of the industry for new entrepreneurs who want to venture in this field. In some cases, traditional medicinal practitioners use leeches for therapeutic healing treatment locally called ‘bekam’ and it is quickly gaining acceptance amongst the local folks which is now becoming a thriving business (Appendix A) and some of them make this treatment their
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main business even though this therapy is quite recent for people in this country. Even though this may be so the response is very encouraging where there have been some entrepreneurs opening up new branches to serve their customer.
Leeches have been used for centuries in the treatment of a variety of diseases, including thromboembolic disorders and inflammatory states. With the advent of modern medicine, leeching fell into disrepute, yet interest in the medicinal use of the leech persisted.
Leeches have been used successfully to restore the flow of blood in microcirculation after plastic surgery (Mutimer et al., 1897; Henderson et al., 1983).
Leeches is a sanguivorous (haemopagic), freshwater leech, with a wide distribution in Southeast Asia, such as in southern China, the Philippines, Thailand, Vietnam, and Malaysia. In Malaysia, this leeches are known as ‘Lintah Kerbau’ (406th Medical Lab. Special Report, 1968). There has been increasing collection of this species for medical purposes in the 20th century (Steiner et al., 1990; Electricwala et al., 1993; Singhal & Davies, 1996). The leech industry in this country was previously operated by the Department of Wildlife Protection and National Parks (PERHILITAN) and since 22 January 2009, the Department of Fisheries Malaysia has taken over the operation (Department of Fisheries Malaysia, 2009).
The Fisheries Department have since identified the leech species used for commercial purposes. The taxonomic status of the local leech are as follows: Order: Hirudinea; Species:
Hirudinaria manillensis; Local name: Buffalo leech or “Lintah kerbau” (Figure 1.1).
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Figure 1.1: Body of local leech (age: two years and three months)
However, it is not known or proven conclusively that the locally named Buffalo Leech is not of H. manillensis although this has been confirmed by Department of Fisheries Malaysia. In addition, local taxonomists have not been able to identify those species used for medicinal purposes (Hj. Rosly Hassan, personal communication1, April 2, 2010).
1.2 Significance of the study
Leech are forbidden (Haram) for consumption as a food, but it is recommended (Sunnah) for cosmetic purposes and as medical products (Ustaz Mohd Amri Abdullah, personal communication2, June 10, 2009). Nowadays, the breeding of leeches for medical purposes has bright commercial potential and of late many entrepreneurs have embarked on the farming of leeches. Besides that, the Malaysian National Fatwa Council of Islamic Affairs which met on 31 March 2007 has come out with an edict that the usage of worms and leech should be encouraged.
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Scale:
1cm Anterior sucker
Segment
Posterior sucker
1cm
Today, there are many products which have been derived from leeches for pharmaceutical and medicinal purposes in and around also the world. Without realizing, many of our own entrepreneurs have also made their work and some have already exported their products to overseas markets which augur well for this new industry. To meet the demand for clinical use, in Chinese traditional medicine and other scientific research, there has been growing interest in culturing and breeding of leeches in many countries (Yang, 1996; Trontelj & Utevsky, 2005). Several factors determine leech distribution in freshwater environments such as availability of food organisms; nature of the substrate; depth of water;
presence of water currents; size and nature of the body of water; hardness and pH;
temperature of the water; dissolved oxygen; siltation and turbidity; and salinity (Sawyer, 1986b). However, no study has been attempted to investigate the factors affecting growth and production of leeches in this country. Particularly lacking is the effect of water, temperature, dissolved oxygen, pH, and light intensity on growth performance of these leeches bred in a farm as well as their feeding requirements. Understanding the growth conditions, reproduction and development required could help maximize the quality of leech farming and those for medical purposes.
Leech farming was seen as a solution to the growing demand for the production of leeches throughout the world. Modern leech therapy differs from that of an ancient one because now wild leeches are not used. The leeches are grown at special leech farms where they are subjected to severe quarantine requirements. Also, to exclude infection on to the patient a leech is used only once and thrown away. In this country, large-sized leeches are usually used for breeding and production while small-sized leeches (2-3 weeks juveniles) are used for “bekam” therapy. As it is known, until now the leech stock is always insufficient and this problem sometimes caused many entrepreneurs to discontinue their business for a
4
temporary period due to lack of leeches. If this shortage of leech supply continue there may be a negative effect on the economy as well as on the entrepreneurs. In order to address this problem of uncertainly and shortage of supply of local leeches optimization of their growing condition is strongly needed.
With the rapidly expanding leech based industries the request for supply of leeches has increased tremendously. The present source of obtaining leeches from the wild are fast depleting and far in between and as such some interested parties have taken the initiative to organize training and workshop on leech rearing and breeding and a few of them offer contract farming package. To date, there are various culturing methods that have been introduced but more have come out with a standard practice method of leech farming on a viable commercial basis involving all components in the breeding and rearing of local leeches. Among the constraints, there has been very little research done to date on ways to proceed with large scale production of leeches on a sustainable basis and couple with the lack of scientific information to support the industry.
In this country, there is no proper standard or guide on how leech farming should be carried out and this problem has led to the difficulty for entrepreneurs to handle breeding on large scale besides the fact that a few of them did not succeed in the leech industry as the leeches failed to reproduce well. This problem arose because the entrepreneurs are not aware of the basic practice that should be included in the breeding of leeches and the uncertainties in leech growth condition cause an ineffective product in leech farming.
Therefore, this study was carried out to investigate the optimum growing condition for culturing local leeches for hirudin production.
It is common knowledge that when the leech ceases to feed, the host bleeds for a long time. It has long been known that leeches contain a substance with anticoagulant properties namely hirudin protein. Numerous studies with experimental animal models have been performed that corroborated the antithrombotic efficacy of hirudin (Kaiser, 1991).
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However, in Malaysia work on gathering more information on hirudin protein is still far and between. In this study, the detection and quantification of protein extracted from different body parts, followed by protein purification and profiling for this local leeches were carried out.
1.3 Objectives
This research was undertaken with the following objectives:
1. To optimize the growth conditions in breeding of leeches inclusive of reproduction, growth and development of the leeches.
2. To quantify the protein found in different body parts of local leeches.
To purify the hirudin protein obtained and to determine the protein pro
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