VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE PHETPHAILIN SAISOMBUT EFFECTS OF MORINGA (MORINGA OLEIFERA) ON GROWTH AND ITS ANTIMICROBIAL ACTIVITIES AGAINST PATHOGENIC BACTERIA INFECTING GRASS CARP Major: Aquaculture Code: 62 03 01 Supervisor: Dr Nguyen Ngoc Tuan AGRICULTURAL UNIVERSITY PRESS - 2021 HỌC VIỆN NÔNG NGHIỆP VIỆT NAM PHETPHAILIN SAISOMBUT NGHIÊN CỨU ẢNH HƯỞNG CỦA CÂY CHÙM NGÂY (MORINGA OLEIFERA) LÊN SINH TRƯỞNG VÀ KHẢ NĂNG ỨC CHẾ MỘT SỐ LOẠI VI KHUẨN GÂY BỆNH Ở CÁ TRẮM CỎ Ngà nh: Nuôi trồng thủy sản Mã số : 62 03 01 Người hướng dẫn: TS Nguyễn Ngọc Tuấn NHÀ XUẤT BẢN HỌC VIỆN NÔNG NGHIỆP - 2021 STATEMENT I confirm that I wrote my master thesis titled: “Effects of Moringa (Moringa oleifera) on growth and its antimicrobial activities against pathogenic bacteria infecting grass carp” case study in Viet Nam It done by myself and there were used only sources that are listed in the part of reference The result of my research and follow-up during my internship, ensure reliability, accuracy and honesty The data and results in the thesis are true and never been published in any previous research Documents for analysis, commentary and evaluation collected from different sources are clearly traced and cited In addition, the topic also uses some comments, reviews as well as data of the author or some other organization and organization are also shown in the references Any help from individuals, agencies and organizations is thanks All invalid copies, violating the regulations of writing essays or fraud, I will be entirely responsible In Ha Noi on date /2021 Supervisor Author Dr Nguyen Ngoc Tuan Phetphailin SAISOMBUT Certified by faculty of Fisheries, VNUA i ACKNOWLEDGEMEN First of all, I would like to express my sincere gratitude to the Viet Nam National University Agricultrue which offered me the opportunity to get financial support for my education I would like to express my sincere gratitude to Dr Nguyen Ngoc Tuan enthusiastically guided, edited, spent a lot of effort and time and created conditions for me during the process of studying and implementing the thesis I would like to express my sincere gratitude to staff of the Faculty of Fisheries, Vietnam National University of Agriculture Especially Dr Kim Van Van Dean of the Faculty, Dr Truong Dinh Hoai vice-dean and teacher, created favorable conditions and enthusiastically helped me in the process of studying, implementing the thesis and completing the thesis I would like to express my special thanks to Dr Doan Thi Nhinh, Dr Le Viet Dung and the students of the Laboratory, Department of Aquatic Diseases, Department of Fisheries for their help and support during the implementation of this experiment I cannot forget that the Faculty of Agriculture, Laos National University always helped and monitored my studies during my study abroad in Vietnam Thank you Prof Thonglom PHOMMAVONG vice-dean of Agriculture and thanks Dr Phouvin PHOUSAVANH Head of Livestock and Fisheries Department, special thanks are deserved to all staff of Faculty of Agriculture and also all staff of Department of Livestock and Fisheries in Laos for all kind of help I got from them I was very lucky realize my experimental work there Finally I thank a lot to my family for giving me my life and for supporting, helping and never-ending encouragement during me stay in Vietnam ii LỜI CẢM ƠN Trong suốt thời gian học tập, nghiên cứu hồn thành luận văn, tơi nhận hướng dẫn, bảo tận tình, giúp đỡ, động viên thầy cô giáo, cán bộ, nhân viên, đồng nghiệp, bạn bè gia đình Nhân dịp hồn thành luận văn, cho phép tơi bày tỏ lịng kính trọng biết ơn sâu sắc TS Nguyễn Ngọc Tuấn tận tình hướng dẫn, chỉnh sửa, dành nhiều công sức, thời gian tạo điều kiện cho tơi suốt q trình học tập thực đề tài Tơi xin bày tỏ lịng biết ơn chân thành tới tập thể lãnh đạo, cán viên Khoa Thủy sản, Học viện Nông nghiệp Việt Nam Đặc biệt TS Kim Văn Vạn trưởng khoa Thủy Sản, TS Trương Đình Hồi phó khoa Thủy Sản giáo viên khoa tạo điều kiện, tận tình giúp đỡ tơi q trình học tập, thực đề tài hồn thành luận văn Tơi xin chân thành cảm ơn ThS Đoàn Thị Nhinh, TS Lê Việt Dũng bạn sinh viên phịng thí nghiệm, khoa thủy sản giúp đỡ hỗ trợ suốt q trình thực thí nghiệm đề tài Tôi quên Khoa Nông nghiêp, Đại học Quốc gia Lào giúp đỡ, theo dõi việc học tập thời gian du học Việt Nam Cảm ơn PGS Thonglom PHOMMAVONG phó trưởng khoa Nông nghiệp cảm ơn Dr Phouvin PHOUSAVANH trưởng môn chăn nuôi thủy sản tập thể giáo viên cán viên lo theo dõi việc học tập cổ vũ Cuối tơi xin chân thành cảm ơn gia đình, người thân, bạn bè, đồng nghiệp tạo điều kiện thuận lợi giúp đỡ mặt, động viên khuyến khích tơi hồn thành luận văn Hà Nội, ngày tháng năm 2021 Tác giả luận văn Phetphailin SAISOMBUT iii ຄຳຂອບໃຈ ຜ່ານຈາກການສຶກສາຄົ້ນຄວ້າບົດວິທະຍານິພົນ ຂອງຂ້າພະເຈົ້າໃນຄັ້ງນີ້ ແມ່ນມີຄວາມສຳຄັນ ມີຄວາມໝາຍອັນລ້ຳຄ່າ ແລະ ເຊິ່ງເປັນບົດຮຽນ, ປະສົບການອັນຍິ່ງໃຫຍ່ໃນບົດວິທະຍານິພົນຄັ້ງນີ້ ຈົນສາມາດໄດ້ຮັບຜົນສຳເລັດຕາມຄາດໝາຍຢ່າງສົມບູນ ເຊິ່ງຄວາມສຳເລັດດັ່ງກ່າວນີ້ ຊ່ວຍເຫຼືອ ແລະ ກໍ່ເກີດມາຈາກການຊຸກຍູ້, ການຊ່ວຍເຫຼືອຈາກຫຼາຍພາກສ່ວນ ສະນັ້ນ, ຂ້າພະເຈົ້າຈຶ່ງຖືເອົາໂອກາດນີ້ ສະແດງຄວາມຮູ້ບຸນຄຸນອັນສູງສົ່ງ ແລະ ສະແດງຄວາມຂອບໃຈມານຳ ຂໍຂອບໃຈມາຍັງ ສະຖາບັນກະສິກຳຫວຽດນາມ, ຄະນະປະມົງ ທີ່ໃຫ້ສະຖານທີ່ການສຶກສາ ແລະ ການຄົ້ນຄວ້າວິໄຈໃນຄັ້ງນີ້ ຂໍຂອບໃຈ ແລະ ຮູ້ບຸນຄຸນມາຍັງອາຈານທີ່ປຶກສາ ທ່ານ ປອ NGUYEN NGOC TUAN ທີ່ໄດ້ເອົາໃຈໃສ່ ຊີ້ນຳຕະຫຼອດຈົນປັບປຸງແກ້ໄຂ ດ້ວຍຄວາມເອົາໃຈໃສ່ຢ່າງດີ ໃຫ້ຄຳປຶກສາ, ຂໍ້ບົກພ່ອງຕ່າງໆ ແຕ່ຕົ້ນຈົນເຖິງສຸດທ້າຍ ຂອງຄວາມສຳເລັດໃນການຂຽນບົດນິພົນທີ່ຄົບຖ້ວນສົມບູນຢ່າງຈ ົບງາມ ພ້ອມດຽວກັນນັ້ນ, ຂໍສະແດງຄວາມຂອບໃຈມາຍັງ ອຈ ທຸກໆທ່ານ ພາກວິຊາການປະມົງ ທີ່ໄດ້ສິດສອນວິຊາຄວາມຮູ້ຕ່າງໆໃຫ້ແກ່ຂ້າພະເຈົ້າ ໂດຍສະເພາະແມ່ນ ທ່ານ ປອ KIM VAN VAN ຫົວໜ້າພາກວິຊາ, ທ່ານ ປອ TRUONG DINH HOAI ຮອງພາກວິຊາ ແລະ ອຈ ທຸກໆທ່ານທີ່ໄດ້ແນະນຳ ແລະ ສີດສອນຕະຫຼອດມາ ສະແດງຄວາມຮູ້ບຸນຄຸນມາຍັງ ທ່ານນາງ ປອ DOAN THI NHINH ທີ່ຄອຍໃຫ້ຄຳປຶກສາ,ກວດແກ້ບົດ, ຊີ້ນຳວຽກງານການທົດລອງ ຕັ້ງແຕ່ຕົ້ນຈົນຈົບການທົດລອງ; ຂອບໃຈມາຍັງ ທ່ານ ປອ Le Viet iv Dung ທີ່ຊ່ວຍເຫລືອດ້ານສະຖານທີ່ການເຮັດງານທົດລອງຄັ້ງນີ້; ຂອບໃຈມາຍັງບັນດານ້ອງໆນັກສຶກສາ ປະຈຳຢູ່ຫ້ອງທົດລອງທີ່ໃຫ້ຄວາມຊ່ວຍເຫຼືອດ້ານວຽກງານການວ ິໃຈ ຂ້າພະເຈົ້າບໍ່ສາມາດລືມໄດ້ຄະນະກະເສດສາດ, ມະຫາວິທະຍາໄລແຫ່ງຊາດ ທີ່ໄດ້ຄອຍຕິດການຮຽນການສຶກສາຂອງຂ້າພະເຈົ້າ ແລະ ຊ່ວຍເຫຼືອ ໃຫ້ຄຳປຶກສາໃນທຸກໆດ້ານ ພ້ອມດຽວກັນນັ້ນ ຂອບໃຈມາຍັງ ທ່ານ ຮສ ທອງຫຼອມ ພົມມະວົງ ຮອງຄະນະບໍດີ, ທ່ານ ປອ.ພູວິນ ພູສະຫວັນ ລວມໄປເຖິງ ພະນັກງານ ຄູອາຈານ ພາກວິຊາລ້ຽງສັດ-ການປະມົງ ໝົດທຸກຄົນ ທີ່ຄອຍເປັນຫ່ວງເປັນໄຍ, ຕິດຕາມການຮຽນການສຶກສາຂອງຂ້າພະເຈົ້າ ພ້ອມທັງເປັນກຳລັງໃຈໃຫ້ແກ່ຂ້າພະເຈົ້າຕະຫຼອດມາ ສຸດທ້າຍ ຂໍສະແດງຄວາມຮູ້ບຸນຄຸນມາຍັງ ອ້າຍເອື້ຶອຍນ້ອງ, ທີ່ຄອຍເບິ່ງແຍງ, ຍາດພີ່ນ້ອງ, ພໍ່ແມ່, ໝູ່ເພື່ອນ ຊຸກຍູ້ຊ່ວຍເຫຼືອຂ້າພະເຈົ້າຕະຫຼອດມາ; ພ້ອມດຽວກັນນັ້ນບໍ່ອາດລືມໄດ້ ຄອບຄົວຂອງຂ້າພະເຈົ້າ ຜູ້ທີ່ຄອຍໃຫ້ກຳລັງໃຈ, ປຶກສາຫາລື, ສະໜັບສະໜູນທຸກດ້ານບໍ່ວ່າຈະເປັນດ້ານຊັບສິນ ແລະ ດ້ານຈິດໃຈ ໃນໄລຍະທີ່ຂ້າພະເຈົ້າໄດ້ສຶກສາຢູ່ທີ່ປະເທດຫວຽດນາມໃນໄລຍະສາ ມປີ v CONTENT Statement i Acknowledgemen ii Lời cảm ơn iii ຄຳຂອບໃຈ iv Content vi List of acronyms and abbreviations ix Table of contents x Figure of contents xi Thesis abstract xii Trích yếu luận văn xiv ບົດຄັດຫຍໍ້ xvi I Introduction II Literature review 2.1 Origin of grass carp 2.1.1 Classification of grass carp 2.1.2 Morphological characteristics 2.1.3 Habitat 2.1.4 Nutritional characteristics 2.1.5 Growth characteristics 2.1.6 Biochemical characteristics of grass carp meat 2.1.7 The situation of grass carp farming in the world 2.1.8 Status of grass carp farming in Vietnam 2.2 Some diseases on grass carp 2.2.1 Disease caused by virus on grass carp 2.2.2 Bacterial disease 2.2.3 Diseases caused by parasites 2.3 Antibiotics and antibiotic resistance in aquaculture 2.3.1 Antibiotics used in aquaculture 2.3.2 Harmful effects of overuse of antibiotics 10 vi 2.3.3 Measures to limit antibiotic use 11 2.4 Effects of some herbs 12 2.5 Moringa (Moringa oleifera) 13 2.5.1 Taxonomy of Moringa 13 2.5.2 Nutritional value and benefits 14 2.5.3 Important substances in moringa leaves 15 2.6 Duckweed 18 2.6.1 Important elements 19 2.6.2 Factors affecting duckweed growth 19 III Materials and methods 21 3.1 Location of the study 21 3.2 Materials of the study 21 3.2.1 Feed, fish and bacteria 21 3.2.2 Materials of research 21 3.3 Methodologies and experimental design 22 3.3.1 Experiment 1: Evaluation of the antibacterial ability of moringa to some pathogenic bacteria 22 3.3.2 LD50 determination of A hydrophila 24 3.3.3 Experiment 2: Evaluation of the effect of moringa leaves on the growth of grass carp 25 3.3.4 Experiment 3: Evaluation of the effect of moringa leaves on disease resistance in grass carp 27 3.4 Statistical analysis 28 IV Results and discussions 29 4.1 Results on screening antibacterial activity of extracts from moringa on common pathogenic bacteria infecting grass carp 29 4.2 Determination of virulence of a Hydrophila infecting grass carp 31 4.3 Evaluation of the effect of moringa leaves on the growth of herbivorous grass carp 33 4.3.1 Environmental factors in the growth experimental 33 4.3.2 The effect of moringa leaves on the growth of herbivorous grass carp 35 vii 4.4 Effects of moringa on disease resistance of grass carp 37 4.4.1 Temperature 37 4.4.2 Evaluation of disease resistance of fish after use moringa as feed 38 V Conclusions and suggestions 44 5.1 Conclusions 44 5.2 Suggestions 44 References 45 Appendices 53 viii Figure 4.10 Colonies morphology of A hydrophila on the Rhimler-Shotts agar Bacteria A hydrophila was grows well on conventional cultures, colonies of A hydrophila were round, slightly convex, color was smooth and milky (Figure 4.9) and were exactly the same as described previously By the authors Shotts Jr & Rimler (1973), on the Rhimler-Shotts agar, colonies were round, convex, smooth, pale yellow The colonies with size 1-1.5 mm Checked the basic indicators also showed that the bacteria were short rod-shaped, round with two ends, Gram-negative, motile, 1-2 µm in size Figure 4.11 Gram stain A hydrophila from infected fish The fish suspected of being infected with A hydrophila, the main clinical symptoms and gross lesions observed include: fish anorexia, lethargic swimming, haemorrhage of skin, gill cover, base of fin, bleeding in the liver and intestines, the abdomen contains a lot of yellow fluid, the anus is red and swollen Some 42 signs of the disease such as an enlarged spleen and kidneys The results in this study showed that all dead fish showed typical symptoms of A hydrophila infection, such as haemorrhage at the fins and gills, haemorrhage of skin which were similar to the description by Bui (2002) The results from isolation after infection also confirmed the cause of mortality No other bacteria were found from these fish samples 43 V CONCLUSIONS AND SUGGESTIONS 5.1 CONCLUSIONS After 70 days of experiment, grass carp fed on moringa leaves grew best, higher than dried form and all the other feed in the experiment Although fish fed on dried moringa leave showed lower weight gain as compared to that from fresh moringa leave, it was still better than fed on fresh duckweed and industrial feed Thus, fresh moringa leaf is a good feed for grass carp, and dried moringa leaves were recommended for storage The extracts from moringa in this study showed potential on inhibit to infectious bacteria which cause high mortality in aquaculture The extract from fresh moringa leaf showed results of antibacterial activity on all tested bacteria including F columnare, A hydrophila Whereas, extract from moringa kernel showed strong antibacterial activity to F columnare only The results from the challenge test showed the fish fed fresh moringa leaves showed the lowest mortality rate after 14 days post infection In another word, grass carp in fresh moringa leaf group resisted to A hydrophilla better than all the other groups Therefore, fresh moringa leaves are recommended to use for grass carp in order to enhance not only growth performance, but also for disease resistance 5.2 SUGGESTIONS 1) Future research should continue to exam products from moringa with other pathogenic bacteria in grass carp, especially to F columnare 2) It is recommended to carry out future experiment in a large environment such as hapa-net or in a earthen pond in order to access closer to practice conditions and avoid other problems with changes of water quality in small close tanks 3) Further study could be focused to immune responds of fish, in which fish blood parameters need to be collected and analyzed 44 REFERENCES Abalaka, M., Daniyan, S., Oyeleke, S & Adeyemo, S (2012) The antibacterial evaluation of Moringa oleifera leaf extracts on selected bacterial pathogens Journal of Microbiology research 2(2): 1-4 Abbas, T E (2013) The use of Moringa oleifera in poultry diets Turkish Journal of Veterinary and Animal Science 37(5): 492-496 Adams, B M., Bertrand, K N., Brown, M L & Auger, D (2011) Genetic structure of grass carp populations in the Missouri and Mississippi River basins, USA Afuang, W., Siddhuraju, P & Becker, K (2003) Comparative nutritional evaluation of raw, methanol extracted residues and methanol extracts of moringa 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Proceedings of the 49th Kasetsart University Annual Conference, Kasetsart University, Thailand, 1-4 February, 2011 Volume Subject: Fisheries Zeng, L W G R & Zhiwei, Q Z (2009) Factors Affecting Textural Characteristics of Dorsal Muscle of Crisp Grass Carp Journal of South China University of Technology (Natural Science Edition) 2009(4): 28 http://en.cnki.com.cn/Article_en/CJFDTotal-HNLG200904028.htm Zhao, H., Xia, J., Zhang, X., He, X., Li, L., Tang, R., Chi, W & Li, D (2018) Diet affects muscle quality and growth traits of grass carp (Ctenopharyngodon idellus): a comparison between grass and artificial feed Frontiers in physiology 9: 283 Zhu, Z., Ruan, Z., Li, B., Meng, M & Zeng, Q (2013) Quality loss assessment of crisp grass carp (Ctenopharyngodon idellus C et V) fillets during ice storage Journal of Food Processing and Preservation 37(3): 254-261 https://doi.org/https://doi.org/10.1111/j.1745-4549.2011.00643.x 52 APPENDICES Appendix1 Some pictures of implementation of thesis Feed used in the experiments Stocking fish Wash duckweed 53 Dried moringa leaves Test water quality during farming perform LD50 and infection Dissecting fish in the laboratory Colonies and microscopic morphology were isolated after infection 54 55 56