Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus)

Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY PHAM NGOC NHU EFFECTS OF DIETARY SUPPLEMENTATION WITH SELECTED PLANT EXTRACTS ON THE PHYSIOLOGICAL PARAMETERS OF STRIPED CATFISH (Pangasianodon hypophthalmus) DOCTORAL DISSERTATION MAJOR: AQUACULTURE (9620301) 2023 MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY PHAM NGOC NHU Ph.D student ID: P0618001 EFFECTS OF DIETARY SUPPLEMENTATION WITH SELECTED PLANT EXTRACTS ON THE PHYSIOLOGICAL PARAMETERS OF STRIPED CATFISH (Pangasianodon hypophthalmus) DOCTORAL DISSERTATION MAJOR: AQUACULTURE (9620301) SUPERVISOR Prof Dr Do Thi Thanh Huong Assoc Prof Dr Bui Thi Bich Hang 2023 DATA SHEET Title: Effects of dietary supplementation with selected plant extracts on the physiological parameters of striped catfish (Pangasianodon hypophthalmus) Subtitle: Ph.D Dissertation Author: Pham Ngoc Nhu, Ph.D student ID: P0618001 Affiliation: Faculty of Aquatic Biology and Environmental Science, College of Aquaculture and Fisheries, Can Tho University, Vietnam Publication year 2023 Cited as: Nhu, Pham Ngoc (2023) Effects of dietary supplementation with selected plant extracts on the physiological parameters of striped catfish (Pangasianodon hypophthalmus) Doctoral Dissertation College of Aquaculture and Fisheries, Can Tho University, Vietnam Keywords: Digestive enzyme activities, hematological parameters, growth, oxidative stress, Pangasianodon hypophthalmus, plant extracts Supervisors: Prof Dr Do Thi Thanh Huong, College of Aquaculture and Fisheries, Can Tho University, Viet Nam Assoc Prof Dr Bui Thi Bich Hang, College of Aquaculture and Fisheries, Can Tho University, Viet Nam i RESULT COMMITMENT I certify that this dissertation was thoroughly researched using all of the findings of my research The data and results presented in the dissertation were believable and had never been published These data and results are entirely applicable to the BioAquaActive project Can Tho, ………… , 2023 Supervisors Ph.D Student Prof Dr Do Thi Thanh Huong Assoc Prof Dr Bui Thi Bich Hang ii Pham Ngoc Nhu ACKNOWLEDGEMENTS First of all, I would like to express my sincere appreciation to Prof Dr Do Thi Thanh Huong, Assoc Prof Dr Bui Thi Bich Hang and Prof Dr Nguyen Thanh Phuong for offering me the opportunity to properly study the Ph.D program These teachers enthusiastically accompanied and addressed the concepts, reviewed the dissertation details, and provided me with encouragement throughout the study; and have always created the best conditions for me to accomplish the research thoroughly, not only during the study but also when I experienced problems or failures From the smallest attention to the valuable experiences that the professors have taught, it has always been a source of encouragement to promote motivation The fact that I am currently completing the dissertation and the source of the gathered knowledge is proof of the accomplishment of my efforts I would like to thank Prof Dr Patrick Kestemont for his encouragement, motivation, and assistance in accomplishing my responsibilities, not just in terms of knowledge but also in my experiences at Namur University, Belgium The far distance from home, family, relatives, and friends, as well as the experience of visiting a foreign country in terms of culture and language (I initially assumed that Belgian citizens spoke English rather than French), did not prevent me from being concerned On the other hand, Professor Patrick acknowledged all of my anxieties and always created the most supportive conditions for me to rapidly integrate and approach the learning experience and research appropriately I'd like to thank Patrick's team members (Robert, Emily, Enoha, Hossain, Mai, ) for their assistance in improving my analytical skills, as well as the full support of equipment and materials well as the retention of great experiences at Namur University Unfortunately, the Covid-19 pandemic outbreak while I was experiencing in Belgium, and my studies seemed abrupt Everything would probably be stuck if it weren't for the professor's encouragement and assistance in completing my responsibilities completely Once again, I would like to thank Professor Patrick and the members of the team for accompanying me in my beloved Belgium I’d like to thank my companions Dang Quang Hieu, Nguyen Tinh Em, and especially Ms Nguyen Thi Kim Ha for their experience in laboratory analysis techniques, chemicals, and materials at Can Tho University (CTU), as well as, their accompanying each other during the above experience at Namur University I would want to express my sincere appreciation to Dr Do Van Buoc for granting me the opportunity to access and participate in the AquaBioactive project, as well as to complete this dissertation Being with him for the initial two years of my study was a pleasure and an unforgettable experience, as well as an advisor who assisted me when I encountered my initial difficulties Furthermore, my heartfelt gratitude to Ms Hong Mong Huyen, who always promotes the spirit, relieves stress, and shares sorrows and iii pleasures, as well as experiences, during the period in Belgium, as well as at CTU's dormitory In addition, I would like to thank the physiology team at the College of Aquaculture and Fisheries (CAF), Can Tho University for their great assistance starting from the first day of implementing my study I'd like to thank Ms Le Thi Bach, who helped with the supply of plant extracts for research, as well as the staff of CAF as Assoc Prof Dr Tran Minh Phu and Dr Nguyen Le Anh Dao, aided me with experimental materials for my experiments I also appreciate the collaborative efforts of four female masters (Nguyen Thi Hoi, Tran Thi Phuong Hang, Vien Tuyen Anh, and Doan Anh Thu) who have accompanied together throughout my study; as well as the students in the Faculty of Aquatic Biology and Environmental Science, who have supported and shared the happiness and assistances to implement my experiments Finally, I'd want to express my gratitude to my family and friends for always loving and supporting me during my studies iv TABLE OF CONTENTS DATA SHEET .i RESULT COMMITMENT ii ACKNOWLEDGEMENTS iii LIST OF FIGURES ix LIST OF TABLES xi LIST OF ABBREVIATION xiii ABSTRACT xiv TÓM TẮT xvi CHAPTER 18 INTRODUCTION 18 1.1 General introduction 18 1.2 The objectives of the dissertation Error! Bookmark not defined 1.3 The main contents of the dissertation 20 1.4 The hypotheses of the dissertation 21 1.5 New findings of the dissertation 21 1.6 Significant contributions of the dissertation 22 CHAPTER 22 LITERATURE REVIEW 23 2.1 The status and importance of aquaculture and fisheries 23 2.2 Climate changes and impacts on aquaculture and fisheries 27 2.3 Mechanism of stress on fish 28 2.4 Effects of environmental factors on fish 30 2.4.1 Effect of temperature on fish 30 2.4.2 Effects of salinity on fish 37 2.4.3 Effects of nitrite on fish 43 2.5 Effects of plant extracts on fish 47 2.5.1 Effects of plant extracts on hematological parameters of fish 47 2.5.2 Effects of plant extracts on digestive enzymes activities and growth of fish 49 2.5.3 The effects of plant extracts on oxidative stress of fishes 60 2.5.4 Effects of plant extracts on striped catfish (P hypophthalmus) 62 a) Effects of Mimosa pudica on aquaculture species 67 b) Effects of Psidium guajava on aquaculture species 67 c) Effect of Phyllanthus amarus on fish 71 d) Effect of Euphorbia hirta on fish 77 e) Effect of Azadirachta indica on fish 79 CHAPTER 81 EFFECTS OF PLANT EXTRACTS ON SELECTED HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES, AND GROWTH PERFORMANCE v OF STRIPED CATFISH, Pangasianodon hypophthalmus (Sauvage, 1878) FINGERLINGS 81 Abstract 81 3.1 Introduction 81 3.2 Material and Method 83 3.2.1 Plant extract and feed preparation 83 3.2.2 Experimental fish acclimation, facilities, and feeding management 84 3.2.3 Haematological and biochemical parameters 85 3.2.4 Digestive enzyme activities 86 3.2.5 Growth performance and survival rate 86 3.2.6 Statistical analysis 87 3.3 Results 87 3.3.1 Effects of plant extracts on haematological parameters 87 3.3.2 Effects of plant extract on digestive enzyme activities 90 3.3.3 Effects of plant extracts on growth performance and the survival rate 91 3.4 Discussion 92 3.5 Conclusions 96 CHAPTER 97 EFFECTS OF Psidium guajava AND Phyllanthus amarus EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO HIGH-TEMPERATURE STRESS 97 4.1 Introduction 98 4.2 Materials and methods 100 4.2.1 Plant extracts and diet preparation .100 4.2.2 Experimental fish acclimation, facilities, and feeding trial 101 4.2.3 Haematological and biochemical parameters .102 4.2.4 Oxidative stress assays .103 4.2.5 Statistical analysis 104 4.3 Results 105 4.3.1 Effect of plant extract on P hypophthalmus haematological parameters 105 4.3.2 Effect of plant extract on digestive enzyme activities 109 4.3.3 Effect of plant extract on growth performance of P hypophthalmus 111 4.3.4 Effect of plant extract on P hypophthalmus oxidative stress 113 4.4 Discussion .115 4.5 Conclusions 122 Acknowledgments .122 CHAPTER .123 vi EFFECTS OF GUAVA (Psidium guajava L.) AND BHUMI AMLA (Phyllanthus amarus Chum et Thonn.) EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO SUBLETHAL SALINITIES .123 Abstract 123 5.1 Introduction 123 5.2 Material and Method .125 5.2.1 Plant extract and feed preparation 125 5.2.2 Experimental fish acclimation, facilities, and feeding management 126 5.2.3 Haematological and biochemical parameters .127 5.2.4 Digestive enzyme activities 127 5.2.5 Oxidative stress biomarkers .128 5.2.6 Growth performance and survival rate .129 5.2.7 Statistical analysis 129 5.3 Results 129 5.3.1 Effects of plant extracts on haematological parameters .129 5.3.2 Effects of plant extracts on digestive enzyme activities 133 5.3.3 Effects of plant extracts on oxidative stress .136 5.3.4 Effects of plant extracts on growth performance and the survival rate 140 5.4 Discussion 140 5.5 Conclusions 144 CHAPTER .126 EFFECTS OF Psidium guajava AND Phyllanthus amarus EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO NITRITE-INDUCED TOXICITY .126 Abstract 126 6.1 Introduction 126 6.2 Material and Method .128 6.2.1 Plant extract and feed preparation 128 6.2.2 Experimental fish acclimation, facilities, and feeding management 128 6.2.3 Haematological and biochemical parameters .130 6.2.4 Digestive enzyme activities 130 6.2.5 Oxidative stress biomarkers .131 6.2.6 Growth performance and survival rate .132 6.2.7 Statistical analysis 132 6.3 Results 132 6.3.1 Effects of plant extracts on haematological parameters .132 vii 6.3.2 Effects of plant extracts on digestive enzyme activities 136 6.3.3 Effects of plant extracts on oxidative stress .136 6.3.4 Effects of plant extracts on growth performance and the survival rate 142 6.4 Discussion 142 6.5 Conclusions 146 CHAPTER .147 GENERAL DISCUSSION 147 7.1 Effects of five selected extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings 147 7.2 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings exposed to hightemperature stress 150 7.3 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings exposed to sublethal salinities .152 7.4 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings exposed to nitriteinduced toxicity 153 CONCLUSIONS AND RECOMMENDATIONS 156 8.1 Conclusion .156 8.2 Recommendations for further studies 157 APPENDIX List of protocols of analysis used in the studies viii Te, B Q., Thanh, L X., Quang, N D., and Ngoc, L H (2006) Report on the results of research on preparations (VTS1-C and VTS1-T) extracted from herbal extracts for disease prevention and treatment for black tiger shrimp and catfish Research Institute For Aquaculture No.1 (In Vietnamese) Tengjaroenkul, B., Smith, B J., Caceci, T., & Smith, S A (2000) Distribution of intestinal enzyme activities along the intestinal tract of cultured Nile tilapia, Oreochromis niloticus L Aquaculture, 182(3-4), 317-327 Thangam, Y (2014) Effect of nitrite toxicity in carbohydrate metabolism to freshwater fish Cirrhinus mrigala IOSR Journal of Pharmacology and Biosciences, 9(5), 03-11 Thiangthum, S., Dejaegher, B., Goodarzi, M., Tistaert, C., Gordien, A Y., Hoai, N N., & Vander Heyden, Y (2012) Potentially antioxidant compounds indicated from Mallotus and Phyllanthus species fingerprints Journal of Chromatography B, 910, 114-121 Thinh, P V., Phuong, N T., Huong, D T T., & Phuc, N T H (2014) The effects of temperature on physiological parameters and growth rate of catfish (Pangasianodon hypophthalmus) Can Tho University Journal of Science, 1, 292-301 Thompson, J F., Morris, C J., & Smith, I K (1969) New naturally occurring amino acids Annual Review of Biochemistry, 38(1), 137-158 Tijskens, L M M., Greiner, R., Biekman, E S A., & Konietzny, U (2001) Modeling the effect of temperature and pH on activity of enzymes: the case of phytases Biotechnology and bioengineering, 72(3), 323-330 Tobin, D., & Wright, P J (2011) Temperature effects on female maturation in a temperate marine fish Journal of Experimental Marine Biology and Ecology, 403(1-2), 9-13 Tobin, D., & Wright, P J (2011) Temperature effects on female maturation in a temperate marine fish Journal of Experimental Marine Biology and Ecology, 403(1-2), 9-13 Todd, J H., Engstrom, D., Jacobson, S., & McLarney, W O (1972) Introduction to environmental ethology: a preliminary comparison of sublethal thermal and oil stresses on the social behavior of lobsters, and fishes from a fresh-water and a marine ecosystem progress report (no coo-3567-1; whoi-72-42) Woods Hole Oceanographic Institution, Mass Torrissen, K R (1991) Genetic variation in growth rate of Atlantic salmon with different trypsin-like isozyme patterns Aquaculture, 93(4), 299-312 Trevizan, L N F., Nascimento, K F., Santos, J A., Kassuya, C A L., Cardoso, C A L., Carmo Vieira, M., & Formagio, A S N (2016) Anti-inflammatory, antioxidant and anti-Mycobacterium tuberculosis activity of viridiflorol: The major constituent of Allophylus edulis (A St.-Hil., A Juss & Cambess.) Radlk Journal of ethnopharmacology, 192, 510-515 Triet, T H., Tinh, B T., Hau, L V., Huong, T V., & Binh, N Q (2019) Development and potential use of an Edwardsiella ictaluri wzz mutant as a live attenuated vaccine against enteric septicemia in Pangasius hypophthalmus (Tra catfish) Fish & Shellfish Immunology, 87, 87-95 Tseng, H C., Grendell, J H., & Rothman, S S (1982) Food, duodenal extracts, and enzyme secretion by the pancreas American Journal of Physiology-Gastrointestinal and Liver Physiology, 243(4), G304-G312 Tsurumi, S., & Asahi, Y (1985) Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin‐and light‐induced opening of the pulvinules Physiologia Plantarum, 64(2), 207-211 Tunna, T S., Zaidul, I S M., Ahmed, Q U., Ghafoor, K., Al-Juhaimi, F Y., Uddin, M S., & Ferdous, S (2015) Analyses and profiling of extract and fractions of neglected weed Mimosa pudica Linn traditionally used in Southeast Asia to treat diabetes South African Journal of Botany, 99, 144-152 Turkez, H., Çelik, K., & Toğar, B (2014) Effects of copaene, a tricyclic sesquiterpene, on human lymphocytes cells in vitro Cytotechnology, 66(4), 597-603 Ueda, M., & Yamamura, S (1999a) Leaf-opening substance of Mimosa pudica L.; chemical studies on the other leaf movement of mimosa Tetrahedron letters, 40(2), 353-356 Ukwueze, S E., Osadebe, P O., & Okoye, F B (2015) A new antibacterial benzophenone glycoside from Psidium guajava (Linn.) leaves Natural product research, 29(18), 1728-1734 UNFCCC - United Nations Framework Convention on Climate Change (2003) Vietnam initial national communication Socialist Republic of Viet Nam Ministry of Natural Resources and Environ ment, Ha Noi USEPA [United States Environmental Protection Agency] (2012) Climate change indicators in the United States, 2012 Technical documentation USEPA 188 Usha, R (2011) Effect of salinity changes on haematological parameters of the tiger shark Pangasius hypophthalmus Journal of Ecobiology, 29(4), 283 Ushie, O., Neji, P., Etim, E., & Nsor, G (2013) Phytochemical screening and antimicrobial activities of Phyllanthus amarus stem bark extracts International Journal of Modern Biology and Medicines, 3(3), 101112 Uys, W., & Hecht, T (1987) Assays on the digestive enzymes of sharptooth catfish, Clarias gariepinus (Pisces: Clariidae) Aquaculture, 63(1-4), 301-313 Uysal, K., Çolak, Y., & Kavasoğlu, M (2019) Effects of Water Temperature on Catalase Activity and Malondialdehyde Level in Muscle, Mouth, and Intestine Tissues of Doctor Fish (Garra rufa) Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 23(1), 191-194 Valdeira, A S., Darvishi, E., Woldemichael, G M., Beutler, J A., Gustafson, K R., & Salvador, J A (2019) Madecassic acid derivatives as potential anticancer agents: synthesis and cytotoxic evaluation Journal of natural products, 82(8), 2094-2105 Valko, M., Leibfritz, D., Moncol, J., Cronin, M T., Mazur, M., & Telser, J (2007) Free radicals and antioxidants in normal physiological functions and human disease The international journal of biochemistry & cell biology, 39(1), 44-84 Valko, M., Leibfritz, D., Moncol, J., Cronin, M T., Mazur, M., & Telser, J (2007) Free radicals and antioxidants in normal physiological functions and human disease The international Journal of Biochemistry & Cell Biology, 39(1), 44-84 Van Acker, S A., de Groot, M J., van den Berg, D J., Tromp, M N., Donne-Op den Kelder, G., van der Vijgh, W J and Bast, A (1996) A quantum chemical explanation of the antioxidant activity of flavonoids Chem Res Toxicol 9, 1305-1312 Van de Laar, F A., Lucassen, P L., Akkermans, R P., Van de Lisdonk, E H., & De Grauw, W J (2006) Alpha‐ glucosidase inhibitors for people with impaired glucose tolerance or impaired fasting blood glucose Cochrane Database of Systematic Reviews, (4) Van Dyk, J C., Cochrane, M J., & Wagenaar, G M (2012) Liver histopathology of the sharptooth catfish Clarias gariepinus as a biomarker of aquatic pollution Chemosphere, 87(4), 301-311 Van Rijn, J A., & Reina, R D (2010) Distribution of leukocytes as indicators of stress in the Australian swellshark, Cephaloscyllium laticeps Fish & shellfish immunology, 29(3), 534-538 Van Weerd, J H., & Komen, J (1998) The effects of chronic stress on growth in fish: a critical appraisal Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 120(1), 107-112 Vanlandeghem, M M., Wahl, D H., & Suski, C D (2010) Physiological responses of largemouth bass to acute temperature and oxygen stressors Fisheries Management and Ecology, 17(5), 414-425 Vargas‐Chacoff, L., Calvo, Á., Ruiz‐Jarabo, I., Villarroel, F., Muñoz, J L., Tinoco, A B., & Mancera, J M (2011) Growth performance, osmoregulatory and metabolic modifications in red porgy fry, Pagrus pagrus, under different environmental salinities and stocking densities Aquaculture Research, 42(9), 1269-1278 Vargas-Chacoff, L., Saavedra, E., Oyarzún, R., Martínez-Monto, E., Pontigo, J P., đez, A., & Bertrán, C (2015) Effects on the metabolism, growth, digestive capacity and osmoregulation of juvenile of SubAntarctic Notothenioid fish Eleginops maclovinus acclimated at different salinities Fish physiology and biochemistry, 41(6), 1369-1381 Varsamos, S., Nebel, C., & Charmantier, G (2005) Ontogeny of osmoregulation in postembryonic fish: a review Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 141(4), 401429 Vasep [Vietnam Association of Seafood Exporters and Producers] - https://vasep.com.vn/san-pham-xuat-khau/catra/nguyen-lieu/doanh-nghiep-ca-tra-se-bung-tinh-sau-chuoi-ngay-am-dam-24210.html Will the striped catfish industry "wake up" after a series of unfavorable period? Accessed on 2/10/2022 Vasylkiv, O Y., Kubrak, O I., Storey, K B., & Lushchak, V I (2011) Catalase activity as a potential vital biomarker of fish intoxication by the herbicide aminotriazole Pesticide biochemistry and physiology, 101(1), 1-5 Vinagre, C., Madeira, D., Narciso, L., Cabral, H N., & Diniz, M (2012) Effect of temperature on oxidative stress in fish: lipid peroxidation and catalase activity in the muscle of juvenile seabass, Dicentrarchus labrax Ecological Indicators, 23, 274-279 189 Vinson, J A., Al Kharrat, H., & Andreoli, L (2005) Effect of Aloe vera preparations on the human bioavailability of vitamins C and E Phytomedicine, 12(10), 760-765 Vo, T K., Ta, Q T H., Chu, Q T., Nguyen, T T., & Vo, V G (2020) Anti-hepatocellular-cancer activity exerted by β-Sitosterol and β-Sitosterol-glucoside from Indigofera zollingeriana Miq Molecules, 25(13), 3021 Volkoff, H., & Rønnestad, I (2020) Effects of temperature on feeding and digestive processes in fish Temperature, 7(4), 307-320 Voslářová, E., Pištěková, V., Svobodova, Z., & Bedáňová, I (2008) Nitrite toxicity to Danio rerio: Effects of subchronic exposure on fish growth Acta Veterinaria Brno, 77(3), 455-460 vwsa.org.vn (2017) Overview of water sources for domestic and industrial use in the Mekong Delta http://vwsa.org.vn/vn/article/2272/tong-quan-nguon-nuoc-phuc-vu-sinh-hoat-va-cong-nghiep-vung-dongbang-song-cuu-long.html Accessed on 2/10/2022 Wang, C Y., Li, Z B., Sun, Y Z., Chen, Q., Li, W J., Huang, Y C., & Lu, J (2018) Effects of Chinese herbal medicines mixture on growth performance digestive enzyme activities immune response of juvenile Japanese seabass, Lateolabrax japonicus Aquaculture Nutrition, 24(2), 683-693 Wang, F., Yang, H., Gao, F., & Liu, G (2008) Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 151(4), 491-498 Wang, J Q., Lui, H., Po, H., & Fan, L (1997) Influence of salinity on food consumption, growth and energy conversion efficiency of common carp (Cyprinus carpio) fingerlings Aquaculture, 148(2-3), 115-124 Wang, L., Wu, H., Yang, F., & Dong, W (2019) The protective effects of myricetin against cardiovascular disease Journal of nutritional science and vitaminology, 65(6), 470-476 Ward, D L., & Bonar, S A (2003) Effects of cold water on susceptibility of age-0 flannelmouth sucker to predation by rainbow trout The Southwestern Naturalist, 43-46 Ware, A D (2020) The complete blood count and white blood cell differential In Contemporary Practice in Clinical Chemistry (pp 429-444) Academic Press Ware, A D (2020) The complete blood count and white blood cell differential In Contemporary Practice in Clinical Chemistry (pp 429-444) Academic Press Wedemeyer, G A (1981) Methods for determining the tolerance of fishes to environmental stressors Stress and fish, 247-275 Wendelaar Bonga, S E (1997) The stress response in fish Physiological reviews, 77(3), 591-625 Wendelaar-Bonga, S E (2011) Hormonal response to stress In: Farrel, A.P (Ed.), Encyclopedia of Fish: Fish Physiology From Genome to Environment Academic Press, pp 1515-1523 Wenzel, U (2013) Flavonoids as drugs at the small intestinal level Current opinion in pharmacology, 13(6), 864868 Winkaler, E U., Santos, T R., Machado-Neto, J G., & Martinez, C B (2007) Acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish Prochilodus lineatus Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 145(2), 236-244 Wootton, R (2011) Growth: Environmental Effects, in: Farrell, A.P (Ed.), Encyclopedia of fish physiology: From genome to environment Academic Press, pp 1629-1635 Worthington, T M., & Manual, W E (1982) Enzymes and related biochemicals Biochemical Products Division, Worthington Diagnostic System, Freehold, NJ, USA, 215-226 Wu, X., Zhang, G., Hu, X., Pan, J., Liao, Y., & Ding, H (2019) Inhibitory effect of epicatechin gallate on protein glycation Food Research International, 122, 230-240 Wu, X., Zhang, G., Hu, X., Pan, J., Liao, Y., & Ding, H (2019) Inhibitory effect of epicatechin gallate on protein glycation Food Research International, 122, 230-240 Wu, Y R., Gong, Q F., Fang, H., Liang, W W., Chen, M., & He, R J (2013) Effect of Sophora flavescens on non-specific immune response of tilapia (GIFT Oreochromis niloticus) and disease resistance against Streptococcus agalactiae Fish & Shellfish Immunology, 34(1), 220-227 Wuertz, S., Schulze, S G E., Eberhardt, U., Schulz, C., & Schroeder, J P (2013) Acute and chronic nitrite toxicity in juvenile pike-perch (Sander lucioperca) and its compensation by chloride Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 157(4), 352-360 190 Wurts, W A (1995) Using salt to reduce handling stress in channel catfish World Aquaculture-Baton Rouge, 26, 80-90 Xie, J., Liu, B., Zhou, Q., Su, Y., He, Y., Pan, L., & Xu, P (2008) Effects of anthraquinone extract from rhubarb Rheum officinale Bail on the crowding stress response and growth of common carp Cyprinus carpio var Jian Aquaculture, 281(1-4), 5-11 Xie, Y W., Xu, H X., Dong, H., Fiscus, R R., & But, P P (2007) Role of nitric oxide in the vasorelaxant and hypotensive effects of extracts and purified tannins from Geum japonicum Journal of ethnopharmacology, 109(1), 128-133 Xiong, D M., Xie, C X., Zhang, H J., & Liu, H P (2011) Digestive enzymes along digestive tract of a carnivorous fish Glyptosternum maculatum (Sisoridae, Siluriformes) Journal of Animal Physiology and Animal Nutrition, 95(1), 56-64 Xu, L., Ran, C., He, S., Zhang, J., Hu, J., Yang, Y., & Zhou, Z (2015) Effects of dietary yeast nucleotides on growth, non-specific immunity, intestine growth and intestinal microbiota of juvenile hybrid tilapia Oreochromis niloticus♀× Oreochromis aureus♂ Animal Nutrition, 1(3), 244-251 Xu, Z., Zhang, H., Guo, M., Fang, D., Mei, J., & Xie, J (2022) Analysis of acute nitrite exposure on physiological stress response, oxidative stress, gill tissue morphology and immune response of large yellow croaker (Larimichthys crocea) Animals, 12(14), 1791 Yadava, R N., & Yadav, S (2001) A novel bufadienolide from the seeds of Mimosa pudica Linn Asian Journal of Chemistry, 13(3), 1157-1160.145 Yaghoubi, M., Mozanzadeh, M T., Marammazi, J G., Safari, O., & Gisbert, E (2016) Dietary replacement of fish meal by soy products (soybean meal and isolated soy protein) in silvery-black porgy juveniles (Sparidentex hasta) Aquaculture, 464, 50-59 Yagi, K (1984) Increased serum lipid peroxides initiate atherogenesis BioEssays, 1(2), 58-60 Yang, H., Jung, E M., Ahn, C., Lee, G S., Lee, S Y., Kim, S H., & Jeung, E B (2015) Elemol from Chamaecyparis obtusa ameliorates 2, 4-dinitrochlorobenzene-induced atopic dermatitis International journal of molecular medicine, 36(2), 463-472 Yang, W., Tu, H., Tang, K., Huang, H., Ou, S., & Wu, J (2021) Reynoutrin improves ischemic heart failure in rats via targeting S100A1 Frontiers in Pharmacology, 12 Yao, X., Jiao, S., Qin, M., Hu, W., Yi, B., & Liu, D (2020) Vanillic acid alleviates acute myocardial hypoxia/reoxygenation injury by inhibiting oxidative stress Oxidative medicine and cellular longevity, 2020 Yeo, S K., Ali, A Y., Hayward, O A., Turnham, D., Jackson, T., Bowen, I D., & Clarkson, R (2016) β‐ Bisabolene, a sesquiterpene from the essential oil extract of opoponax (Commiphora guidottii), exhibits cytotoxicity in breast cancer cell lines Phytotherapy Research, 30(3), 418-425 Yfera, M., Nguyen, M V., Navarro-Guillén, C., Moyano, F J., Jordal, A E., Espe, M., & Rønnestad, I (2019) Effect of increased rearing temperature on digestive function in cobia early juvenile Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 230, 71-80 Yılmaz, S., & Ergün, S (2018) Trans-cinnamic acid application for rainbow trout (Oncorhynchus mykiss): I Effects on haematological, serum biochemical, non-specific immune and head kidney gene expression responses Fish & shellfish immunology, 78, 140-157 Ylmaz, S., Acar, ĩ., Kesbiỗ, O S., Gültepe, N., & Ergün, S (2015) Effects of dietary allspice, Pimenta dioica powder on physiological responses of Oreochromis mossambicus under low pH stress SpringerPlus, 4(1), 1-9 Yoo, H J., & Jwa, S K (2018) Inhibitory effects of β-caryophyllene on Streptococcus mutans biofilm Archives of Oral Biology, 88, 42-46 Yuan, K., Lü, J L., & Yin, M W (2006) Chemical constituents of C-glycosylflavones from Mimosa pudica Yao xue xue bao= Acta Pharmaceutica Sinica, 41(5), 435-438 Yuan, K., Lu, J L., Jia, A., & Zhu, J X (2007) Two new C-glycosylflavones from Mimosa pudica Chinese Chemical Letters, 18(10), 1231-1234 Yuan, X., Yang, H., Wang, L., Zhou, Y., & Gabr, H R (2010) Effects of salinity on energy budget in pond-cultured sea cucumber Apostichopus japonicus (Selenka)(Echinodermata: Holothuroidea) Aquaculture, 306(1-4), 348-351 Zahin, M., Ahmad, I., & Aqil, F (2017) Antioxidant and antimutagenic potential of Psidium guajava leaf 191 extracts Drug and chemical toxicology, 40(2), 146-153 Zahran, E., Risha, E., AbdelHamid, F., Mahgoub, H A., & Ibrahim, T (2014) Effects of dietary Astragalus polysaccharides (APS) on growth performance, immunological parameters, digestive enzymes, and intestinal morphology of Nile tilapia (Oreochromis niloticus) Fish & Shellfish Immunology, 38(1), 149157 Zakaria, M B., & Mohd, M A (1994) Traditional Malay Medicinal Plants, Fajar Bakti Sdn Bhd., Kuala Lumpur, Malaysia Zaki, M A., Labib, E M., Nour, A M., Tonsy, H D., & Mahmoud, S H (2012) Effect some medicinal plants diets on mono sex Nile tilapia (Oreochromis niloticus), growth performance, feed utilization and physiological parameters APCBEE Procedia, 4, 220-227 Zarejabad, A M., Sudagar, M., Pouralimotlagh, S., & Bastami, K D (2010) Effects of rearing temperature on hematological and biochemical parameters of great sturgeon (Huso huso Linnaeus, 1758) juvenile Comparative clinical pathology, 19(4), 367-371 Zaware, B., Chaudhari, S., & Shinde, M (2014) An overview of Mimosa pudica Linn Chemistry and pharmacological profile Res J Pharm Biol Chem Sci, 5, 754-761 Zhang, B C., Li, Z., Xu, W., Xiang, C H., & Ma, Y F (2018) Luteolin alleviates NLRP3 inflammasome activation and directs macrophage polarization in lipopolysaccharide-stimulated RAW264 cells American Journal of Translational Research, 10(1), 265 Zhang, B., Deng, Z., Ramdath, D D., Tang, Y., Chen, P X., Liu, R., & Tsao, R (2015) Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects on αglucosidase and pancreatic lipase Food Chemistry, 172, 862-872 Zhang, J., Yuan, K., Zhou, W L., Zhou, J., & Yang, P (2011) Studies on the active components and antioxidant activities of the extracts of Mimosa pudica Linn from southern China Pharmacognosy magazine, 7(25), 35 Zhang, W J., Chen, B T., Wang, C Y., Zhu, Q H., & Mo, Z X (2003) Mechanism of quercetin as an antidiarrheal agent Di jun yi da xue xue bao= Academic Journal of the First Medical College of PLA, 23(10), 1029-1031 Zhang, X., Yang, F., Zhang, X., Xu, Y., Liao, T., Song, S., & Wang, J (2008) Induction of hepatic enzymes and oxidative stress in Chinese rare minnow (Gobiocypris rarus) exposed to waterborne hexabromocyclododecane (HBCDD) Aquatic Toxicology, 86(1), 4-11 Zhang, Y., Wu, Q., Zhang, L., Wang, Q., Yang, Z., Liu, J., & Feng, L (2019) Caffeic acid reduces A53T αsynuclein by activating JNK/Bcl-2-mediated autophagy in vitro and improves behaviour and protects dopaminergic neurons in a mouse model of Parkinson's disease Pharmacological research, 150, 104538 Zhao, F., Wang, Y., Zhang, L., Zhuang, P., & Liu, J (2013) Survival, growth, food conversion efficiency and plasma osmolality of juvenile Siganus guttatus (Bloch, 1787): experimental analyses of salinity effects Fish physiology and biochemistry, 39(4), 1025-1030 Zhao, Y., Chen, R., Wang, Y., & Yang, Y (2018) α-Pinene inhibits human prostate cancer growth in a mouse xenograft model Chemotherapy, 63(1), 1-7 Zheng, J., Li, Q., He, L., Weng, H., Su, D., Liu, X., & Wang, D (2020) Protocatechuic acid inhibits vulnerable atherosclerotic lesion progression in older Apoe−/− mice The Journal of Nutrition, 150(5), 1167-1177 Zheng, J., Mao, Y., Su, Y., & Wang, J (2016) Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus Fish & shellfish immunology, 58, 239-252 Zheng, Q., Han, C., Zhong, Y., Wen, R., & Zhong, M (2017) Effects of dietary supplementation with green tea waste on growth, digestive enzymes and lipid metabolism of juvenile hybrid tilapia, Oreochromis niloticus × O aureus Fish physiology and biochemistry, 43(2), 361-371 Zheng, Z L., Tan, J Y., Liu, H Y., Zhou, X H., Xiang, X., & Wang, K Y (2009) Evaluation of oregano essential oil (Origanum heracleoticum L.) on growth, antioxidant effect and resistance against Aeromonas hydrophila in channel catfish (Ictalurus punctatus) Aquaculture, 292(3-4), 214-218 Zhou, Z., Hu, F., Li, W., Yang, X., Hallerman, E., & Huang, Z (2021) Effects of salinity on growth, hematological parameters, gill microstructure and transcriptome of fat greenling Hexagrammos otakii Aquaculture, 531, 735945 192 Zhu, L S., Shao, B., Song, Y., Xie, H., Wang, J., Wang, J H., & Hou, X X (2011) DNA damage and effects on antioxidative enzymes in zebra fish (Danio rerio) induced by atrazine Toxicology mechanisms and Methods, 21(1), 31-36 Ziaei, A., Ramezani, M., Wright, L., Paetz, C., Schneider, B., & Amirghofran, Z (2011) Identification of spathulenol in Salvia mirzayanii and the immunomodulatory effects Phytotherapy Research, 25(4), 557562 Zijlstra, W G., Buursma, A., & Zwart, A (1983) Molar absorptivities of human hemoglobin in the visible spectral range Journal of Applied Physiology, 54(5), 1287-1291 193 APPENDIX List of protocols of analysis used in the studies DIGESTIVE ENZYMES ACTIVITY Samples are homogenized 1:10 (w:v) in ice-cold 50 mM phosphate buffer pH 6.9 Buffer pH 6.9: KH2PO4 20mmol (M: 136.09):1360.9 mg NaCl 6mmol (M: 58.44): 175.32 mg Dissolve in 500 mL distilled water and adjust pH of 6.9 The homogenate was centrifuged at 4200 × g for 30 at 4◦C and the supernatant (postmitochondrial fraction – PMF) was kept at −80◦C for digestive enzyme activities assays CHYMOTRYPSIN (Worthington, 1982) *Solutions: Buffer pH 7.8: Tris HCl (Tris (Hydroxy methyl) aminomethane 99%, for biochemistry) 80mM: 0.9688g CaCl2 100mM: 1.11g Dissolve in 100 mL distilled water and adjust pH of 7.8 (with HCl) Substrate: Methanol 50%: 63mL Methanol + 50mL H2O BTEE (N-Benzoyl-tyrosine-ethyl ester, Sigma B6125): 16.8 mg/50mL Methanol 50% (add substrate in the same day) Incubate Buffer 7.8 and Substrate at 25oC and before analyzing and maintain throughout the analysis *Assay: Test Blank Buffer pH 7.8 700 µL 700 µL BTEE 700 µL 700 µL Sample X µL - H2O - 10 µL Measure by spectrophotometer at 256 nm wavelength about minutes by cuvette UV (Kinetics program) 𝐂𝐡𝐲𝐦𝐨𝐭𝐫𝐲𝐩𝐬𝐢𝐧 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲: 𝐦𝐔/𝐦𝐋 (( ∆𝐎𝐃 )∗(𝐓𝐨𝐭𝐚𝐥 𝐯𝐨𝐥𝐮𝐦𝐞)∗𝟏𝟎𝟎𝟎∗𝟏𝟎𝟎𝟎 𝐦𝐢𝐧 𝟗𝟔𝟒∗𝟏𝟎−𝟔 ∗𝟏𝟎𝟎𝟎∗𝐕𝐬𝐚𝐦𝐩𝐥𝐞 / protein Total volume: 1.4 mL +Vsample Vsample: (10, 15, 20 µL) α-AMYLASE (Bernfeld, 1951) *Solutions: Buffer pH 6.9: NaH2PO4.H2O 20 mM (M=137.99): 2.76g NaCl 6mM: 0.351g Dissolve in 1L distilled water and adjust pH of 6.9 (2 weeks) Starch 1%(Amidon) (48h): Dilute 100 mg Amidon in 10 mL Buffer pH 6.9 Boil slightly to dissolve solution and incubate at 25oC about 4–5mins before analysis NaOH 2M: g in distilled water up to 100 mL Substrate (2 weeks): Dilute 1g of 3,5 - dinitro salicylic acid in 50mL distilled water (put half of water) Add 30g Sodium potassium tartrate tetrahydrate (NaKC4H4O6) Add 20mL NaOH 2M Add distilled water up to total volume of 100mL Standard solution: Maltose 5µM: Dissolve 180 mg Maltose dilutes with 100 mL distilled water Calibration curve: Prepare the glass tube Maltose concentration (µM) Maltose 5µM (µL) H2O (µL) - 200 2.5 40 80 100 160 160 120 100 40 200 - (*Do the calibration curve at the same time with blank, in advance) *Assay: Prepare the glass tube: Test Blank Standard Sample X µL H2O (100-X) µL 100 µL Incubate at 250C in mins Starch 1% 100 µL 100 µL Incubate at 250C in mins Substrate 200 µL 200 µL 200 µL Incubate at 100 C in mins H2O mL mL mL Measure by spectrophotometer at 540 nm wavelength Standard: not incubate at 25oC, only incubate in 100oC α-amylase activity: mU/min/mg protein= (x*1000/(Vsample*(5 mins))/protein Vsample: 10, 50, 100 µL x: maltose, hydrolysis of maltose is based on calibration curve y = ax + b, where y is the sample's DO (y = ax + b) TRYPSIN (Tseng et al., 1982; Holm et al., 1988) *Solutions: Buffer pH 8.2, 50mM: Tris HCl (Tris (Hydroxy methyl) aminomethane 99%, for biochemistry) (M: 121.14g): 0.6057g CaCl2 (M: 110.98g/mol 20 mM): 0.222g Dissolve in 100 mL distilled water and adjust pH of 8.2 (with HCl) Substrate: BAPNA (Nα-Benzoyl-DL-Arginine P-nitroanilide, Sigma B4875): 10.87 mg BAPNA/250 µL DMSO (add substrate in the same day and keep in the dark) For (final): 1mM = 0.0217g/500µl 1.5mM = 0.0326g/500µl 1.75mM = 0.03805g/500µl Incubate Buffer 8.2 and Substate at 25oC and before analyzing and maintain throughout the analysis *Assay: Prepare the 2.5-mL cuvette Test Blank Buffer pH 8.2 1mL mL BAPNA 10 µL - Sample X µL - Measure by spectrophotometer at 256 nm wavelength about minutes by cuvette UV (Kinetics program) Calculation: unit of trypsin = activity which hydrolyzes 1µmol of BAPNA/min U/L = (ΔOD/t) * (Vt/Ve) * (1/ε * 1) t = reading time (4.83 mins) Vt = total volume Ve = Vsample: (10, 15, 20 µL) and ε: 8200 M-1L Cm-1 molar extinction coefficient U/mL = (ΔOD/t) * (Vt/ (Ve*ε*10-3)) Simplified calculations: For 10µl of homogenate: U/mL = (ΔOD/t) * 12.4390 20µl: U/mL = (ΔOD/t) * 6.2805 50µl: U/mL = (ΔOD/t) * 2.5854 5µl: U/mL = (ΔOD/t) * 24.7561 100µl: U/mL = (ΔOD/t) * 1.345 PEPSIN (Worthington T M., 1982) *Solutions: TCA 5% (M: 163.39g/mol): 5g/100 mL distilled water HCl 1N (37% 12N) : 20 mL HCl/ 240 mL H2O Substrate: Bovine hemoglobin: 2.6g/100 mL (Dissolve Hb in water at 60°C for mins; 80 mL Hb filtered + 6mL HCl 1M) (storage between 0-4°C) (*Take long time to filter the Hb) *Assay: Prepare the 2-mL Eppendorf: Test Blank test Blank Substrate 500 µL Incubate at 37 C in mins Sample 100 µL Incubate at 37 C in 10 mins TCA 5% 1000 µL Sample - 500 µL 500 µL - 100 µL H2O 1000 µL 100 µL 1000 µL - Centrifuge 4000, 4oC, 10 mins and measure by spectrophotometer at 280 nm wavelength (cuvette UV) (*stop reaction first with TCA before adding 100 µL of sample to the blank test) Calculation: unit of Pepsin = activity which hydrolyzes 1µmol of tyrosine/min at 37°C/mL of sample U/L = (ODm/t) * (Vt/Ve) * (1/ε*1) ODm = ODsample -ODBlank test t = reading time (10 mins) Vt = 1mL, Ve = Vsample: 100 µL and ε: 1250 M-1L Cm-1 molar extinction coefficient U/mL = ODm * (1/1250*10 min*100 µL) OXIDATIVE STRESS MARKERS Samples are homogenized 1:5 (w:v) in ice-cold 50 mM phosphate buffer pH 7.5 200µL of homogenate were taken for lipid peroxidation The rest of the homogenate was centrifuged at 10 000 × g for 10 at ◦C and the supernatant (postmitochondrial fraction – PMF) was kept at −80◦C for antioxidant enzyme activities assays http://serge.engi.tripod.com/MolBio/Buffer_cal.htmL (make 50 mM phosphate buffer pH 7.5) LIPID PEROXIDATION (Fatima et al., 2000) *Solutions: Thiobarbituric acid (TBA, Sigma) 0.67%: 67mg/1mL Dimethyl sulfoxide (DMSO) → + mL H2O, avoid light Trichloroacetic acid (TCA, sigma) 5%: 5g/100 mL H2O, keep in ice Standard solution: TMOP 500µM: 4.167 µL Malondialdehyde (MDA)/1mL ethanol + 49 mL H2O Calibration curve: Prepare the Eppendorf MDA concentration (µL) Dilution 500 100 mL Standard solution +4 mL H2O 50 mL S1 +2 mL H2O 10 mL S2 +4 mL H2O mL S3+2 mL H2O 2.5 mL S4 +2 mL H2O 1.25 mL S5 +2 mL H2O 0.625 mL S6 +2 mL H2O H2O *Assay: Test Sample TCA 5% TBA 0.67 % 200 µL (prepare in homogenization step, spin down) 200 µL (put in the sample Eppendorf) Keep in ice in 15 mins 200 µL Centrifuge at 2200 × g at 4◦C for 10 mins Withdraw the supernatant, put into the glass tube, boiled in 10 mins Refresh at room temperature, withdraw 200 µL into the 96-well plate and measure by spectrophotometer at 535 nm wavelength A calibration curve with increasing MDA concentrations allowed the calculation of LPO expressed as nmol MDA equiv g−1 tissue CATALASE (Goth, 1991) *Solutions: Buffer pH 7.4, 60 mmol/l Na2HPO4: 11 g KH2PO4: g Dissolve in 1L distilled water and adjust pH of 7.4 (with HCl) Hydrogen Peroxide (H2O2 30%) 0.065 M 6.639 mL of H2O2 (30%) dilute to a final volume of 1L of buffer 7.4 Ammonium Molybdate ((NH4)6 Mo7O24 4H2O) 32.4 mmol/l Dissolve 40.04 g of Ammonium Molybdate in 1L of distilled water *Assay: All solutions are incubated in 37°C Sample, Blank 1, Blank 2, and blank test tubes are prepared then pipette into test tubes Test (μl) Blank (μl) Blank (μl) Blank (μl) Sample 50 H2O2 30% 1000 1000 1000 Buffer 7.4 50 1050 Ammonium Molybdate 1000 1000 1000 Sample 100 Tubes are incubated for 60 second at 37°C Ammonium Molybdate 1000 Measure by spectrophotometer at 405 nm wavelength against blank Calculation: unit of CAT = activity which hydrolyzes µmole of hydrogen peroxide/l minute under 37°C U/mL = (A(Blank 1) - A(Sample))/ A(Blank 2) - A(Blank 3))*271 Blank = Control, Blank = Standard, Blank = Reagent Blank, and 271 = Coefficient CAT activity is linear up to 100 U/mL If the CAT activity exceeds 100 U/mL, the samples should be diluted with phosphate buffer (2- to 10-fold) PROTEIN (Bradford, 1976) *Solutions: Bradford 1mL + 4mL H2O, avoid light Standard solution: BSA 1mg/1mL H2O: Calibration curve: Prepare the Eppendorf BSA concentration (mL) Dilution 0.5 100 µL BSA +100 µL H2O 0.25 25 µL BSA +75 µL H2O 0.1 10 µL BSA +90 µL H2O 0.05 µL BSA +95 µL H2O 0.025 2.5 µL BSA +97.5 µL H2O H2O *Assay: Prepare the 96-well plate Test Sample Bradford µL 200 µL Keep in dark in mins Measure by spectrophotometer at 595 nm wavelength A calibration curve with increasing BSA concentrations allowed the calculation of protein If the absorbance of the test sample is outside of the absorbance range for the standards, then the assay must be repeated with a more appropriate dilution SOME PHOTOS OF THE EXPERIMENTS Fish fingerling Blood sampling Sampling preparation Fish and blood sample Hemoglobin (Drabkin method) Hematocrit (Larsen and Snieszko, 1961) Blood sample Centrifuge 6000 rpm; mins; C Intestine and stomach Plasma Experimental tanks