Doctoral dissertation of aquaculture: Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831)

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The objectives of dissertation: The objectives of this dissertation were to investigate the effects of nitrite, high concentrations of carbon dioxide and elevated temperatures to physiological parameters and growth of the air-breathing C. ornata during sub-lethal and chronic exposures of these factors in isolation and combination in order to provide a better physiological understanding, particularly recommendations and solutions for minimizing impacts of nitrite toxicity and its combination with other environmental elements in aquaculture ponds under global climate change

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Nội dung Text: Doctoral dissertation of aquaculture: Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831)

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, Gray 1831) DOCTORAL DISSERTATION OF AQUACULTURE Can Tho, 2018
MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, Gray 1831) Major: Aquaculture Major code: 9 62 03 01 DOCTORAL DISSERTATION OF AQUACULTURE Supervisor Prof. Dr. NGUYEN THANH PHUONG Can Tho, 2018
Data sheet Title: Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831) Subtitle: PhD Dissertation Author: Le Thi Hong Gam, PhD student code: P0613005 Major: Aquaculture, Major code: 9 62 62 03 01 Affiliation: Department of Nutrition and Aquatic Products Processing, College of Aquaculture and Fisheries, Can Tho University, Vietnam Publication year 2018 Cited as: Le Thi Hong Gam, 2018. Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (Chitala ornata, Gray 1831). Doctoral Dissertation. College of Aquaculture and Fisheries, Can Tho University, Vietnam. Keywords: Climate change, air-breathing fish, clown knifefish, nitrite, temperature, hypercapnia, methaemoglobin reductase activity, acid-base balance, ion exchange Supervisors: Prof. Dr. Nguyen Thanh Phuong, College of Aquaculture and Fisheries, Can Tho University, Viet Nam. Assoc. Prof. Dr. Mark Bayley, Zoophysiology, Department of Bioscience, Aarhus University, Denmark. Assoc. Prof. Dr. Do Thi Thanh Huong, Department of Nutrition and Aquatic Products Processing, College of Aquaculture and Fisheries, Can Tho University, Viet Nam. Assoc. Prof. Dr. Frank Bo Jensen, Department of Biology, University of Southern Denmark, Odense, Denmark. i
Result commitment I commit that this dissertation was investigated based on all the results of my study. All showed data and results in the dissertation were honest and have never been published before. The iAQUA project can completely use these data and results. Can Tho, 18th Nov, 2018 ii
Acknowledgements Foremost, my sincere thanks go to my principal supervisors Nguyen Thanh Phuong and Do Thi Thanh Huong, who have given me the opportunity of studying, enthusiastic guidances and detailed revisions for my thesis as well as positive encouragements in any situation throughout my academic research process. I would also like to express my deep gratitude to Mark Bayley, who has supported, inspired, built my passion in doing researches, also revised the manuscripts and shared the life experiences for me being more mature. My deep thanks also give to Frank Bo Jensen, who has taught me the techniques related to my main research about nitrite toxicity and helped me in manuscript revisions as well as showed me Odense city in my trip to Denmark. The invaluable supports from all of them from the first day I stepped into the iAQUA project have brought to what I have today. Also, I would like to give my thanks to Tobias Wang, the staff and students in Zoophysiology Section, Department of Bioscience, Aarhus University for my stays in Denmark. I sincerely thank for the positive supports from Christian Damsgaard, who showed me the knowledge related to acid-base regulation. My thanks also go to Roy, John, Elin and Louise for showing Aarhus city and inviting me to their home. My great thanks give to the staff and students in Department of Nutrition and Aquatic Products Processing, where my project was investigated. I greatly thank to Nguyen Quoc Thinh, Le Thi Minh Thuy and Tran Minh Phu, who shared me their experiences about studying PhD, writing and publishing the articles. I would like to give my thanks to my fellow friends in iAQUA project: Nguyen Thi Kim Ha, Le My Phuong, Phan Vinh Thinh, and Dang Diem Tuong for their friendships and what we enjoyed together from the working environment as well as all the funs outside the campus, especially Phan Vinh Thinh, who has cared me from meals and movements during my intensively experimental works and the trips in Denmark. I also appreciate the positive co-operations from Master students such as Nguyen Thi Thuy Vu and Tran Trong Nhan; Bachelor students: Ma Thanh Quoc Tri, Pham Quoc Boong, Dinh Phuc Tai, Ly Thi Ngoc Huynh, Nguyen Ngoc Mai and Dao Dang Hoang Ngan during our academic activities. iii
I would like to thank my family and my friends for their love and spiritual supports during my study. My special thanks give to my sister Le Thi Hong Dao, who has supported me about both financial and mental sides from Bachelor’s degree up to date. She has always been my side for sharing happiness and sadness throughout my whole life. Last but not least, thanks to all the sacrifired clown knifefish in my project. iv
Table of contents Data sheet ............................................................................................................................i Result commitment ........................................................................................................... ii Acknowledgements .......................................................................................................... iii Table of contents ................................................................................................................ v List of figures ..................................................................................................................... x List of tables .................................................................................................................... xii List of abbreviation .........................................................................................................xiv Summary .........................................................................................................................xvi Tóm tắt ......................................................................................................................... xviii Chapter 1 ............................................................................................................................ 1 INTRODUCTION ............................................................................................................. 1 1.1 Introduction .............................................................................................................. 1 1.2 The objectives of dissertation ................................................................................... 3 1.3 The main projects of dissertation ............................................................................. 3 1.4 The hypotheses of dissertation ................................................................................. 3 1.5 New findings of the dissertation ............................................................................... 4 1.6 Significant contributions of the dissertation ............................................................. 5 References .......................................................................................................................... 5 Chapter 2 ............................................................................................................................ 7 LITERATURE REVIEW................................................................................................... 7 2.1 The status and importance of aquaculture and fisheries ........................................... 7 2.2 Climate changes and impacts on aquaculture and fisheries ..................................... 9 2.3 The status of farming clown knifefish (C. ornata) in MD ..................................... 10 2.4 Background about effects of some key environmental parameters on physiological processes and growth in aquaculture ............................................................................ 11 2.4.1 Temperature........................................................................................................ 11 2.4.2 Nitrite (NO2-) ...................................................................................................... 14 2.4.3 Hypercapnia (elevated level of carbon dioxide) and acid-base balance ............ 18 References ........................................................................................................................ 20 Chapter 3 (Paper 1) .......................................................................................................... 29 v
EXTREME NITRITE TOLERANCE IN THE CLOWN KNIFEFISH CHITALA ORNATA IS LINKED TO UP-REGULATION OF METHAEMOGLOBIN REDUCTASE ACTIVITY .............................................................................................. 29 3.1 Introduction ............................................................................................................ 30 3.2. Materials and methods........................................................................................... 32 3.2.1 Experimental animals ......................................................................................... 32 3.2.2 Determination of acute nitrite toxicity (96 h LC50) ............................................ 32 3.2.3 Sub-lethal exposures and blood sampling .......................................................... 33 3.2.4 Analysis of haemoglobin derivatives ................................................................. 34 3.2.5 Plasma ion and protein analysis ......................................................................... 34 3.2.6 Measurements of whole body water content ...................................................... 35 3.2.7 Methaemoglobin reductase activity.................................................................... 35 3.2.8 Statistics.............................................................................................................. 36 3.3. Results ................................................................................................................... 36 3.4. Discussion ............................................................................................................. 45 3.4.1 Nitrite tolerance .................................................................................................. 45 3.4.2 MetHb reductase activity.................................................................................... 46 3.4.3 Plasma ions ......................................................................................................... 47 3.5. Conclusions ........................................................................................................... 49 References ........................................................................................................................ 49 Chapter 4 (PAPER 2) ....................................................................................................... 54 EFFECTS OF NITRITE EXPOSURE ON HAEMATOLOGICAL PARAMETERS AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, GRAY 1831) .................. 54 4.1 Introduction ............................................................................................................ 55 4.2 Materials and methods............................................................................................ 56 4.2.1 Effects of nitrite on haematological parameters in C. ornata ............................ 56 4.2.2 Effects of nitrite on growth of C. ornata ............................................................ 57 4.2.3 Data analysis....................................................................................................... 57 4.3 Results and discussion ............................................................................................ 58 4.3.1 Effects of nitrite on haematological paramters in C. ornata .............................. 58 4.3.2 Effects of nitrite on growth parameters in clown knifefish C. ornata ............... 62 4.4 Conclusions ............................................................................................................ 64 References ........................................................................................................................ 64 vi
Chapter 5 (PAPER 3) ....................................................................................................... 69 THE EFFECTS OF ELEVATED ENVIRONMENTAL CO2 ON NITRITE UPTAKE IN THE AIR-BREATHING CLOWN KNIFEFISH CHITALA ORNATA .................. 69 5.1 Introduction ............................................................................................................ 71 5.2 Materials and methods............................................................................................ 73 5.2.1 Animal holding ................................................................................................... 73 5.2.2 Experimental protocols....................................................................................... 74 5.2.3 Analytical procedures ......................................................................................... 74 5.2.4 Statistics.............................................................................................................. 76 5.3 Results .................................................................................................................... 76 5.3.1 Acid-base parameters and plasma ions .............................................................. 76 5.3.2 Nitrite uptake and levels of Hb derivatives ........................................................ 81 5.4 Discussion .............................................................................................................. 85 5.5 Conclusions ............................................................................................................ 88 References ........................................................................................................................ 88 Chapter 6 (Manuscript 1) ................................................................................................. 93 THE COMBINED EFFECTS OF NITRITE AND ELEVATED ENVIRONMENTAL CO2 ON HAEMATOLOGICAL PARAMETERS IN SMALL-SIZED CLOWN KNIFEFISH (CHITALA ORNATA) ................................................................................. 93 6.1 Introduction ............................................................................................................ 94 6.2 Materials and methods............................................................................................ 95 6.2.1 Animal handling and experimental protocols .................................................... 95 6.2.2 Statistics.............................................................................................................. 96 6.3 Results .................................................................................................................... 97 6.3.1 Combined effects of nitrite and carbon dioxide on haematological parameters in small-sized C. ornata .................................................................................................. 97 6.3.2 Combined effects of nitrite and carbon dioxide on acid-base parameters and plasma ions in small-sized C. ornata ........................................................................ 103 6.4 Discussion ............................................................................................................ 107 6.5 Conclusions .......................................................................................................... 111 References ...................................................................................................................... 111 Chapter 7 (Manuscript 2) ............................................................................................... 115 EFFECTS OF DIFFERENT TEMPERATURES ON HAEMATOLOGICAL PARAMETERS IN CLOWN KNIFEFISH (CHITALA ORNATA) ............................... 115 vii
7.1 Introduction .......................................................................................................... 116 7.2. Materials and methods......................................................................................... 117 7.2.1 Experimental animals ....................................................................................... 117 7.2.2 Determination of temperature limits in the clown knifefish ............................ 118 7.2.3 Effect of different levels of temperature on haematological parameters ......... 118 7.3. Results ................................................................................................................. 120 7.3.1 Temperature tolerance in C. ornata ................................................................. 120 7.3.2 Effects of different temperatures on physiological parameters in small-sized C. ornata ........................................................................................................................ 121 7.3.3 Effects of different temperatures on physiological parameters in large-sized C. ornata ........................................................................................................................ 127 7.4 Discussion ............................................................................................................ 134 7.5 Conclusions .......................................................................................................... 136 References ...................................................................................................................... 136 Chapter 8 (Manuscript 3) ............................................................................................... 141 EFFECTS OF NITRITE AT DIFFERENT TEMPERATURES ON HAEMATOLOGICAL PARAMETERS AND GROWTH IN CLOWN KNIFEFISH CHITALA ORNATA ....................................................................................................... 141 8.1. Introduction ......................................................................................................... 142 8.2. Materials and methods......................................................................................... 143 8.2.1 Experimental animals and general experimental design .................................. 143 8.2.2 Determination of acute nitrite toxicity (96 h LC50) at 30ºC and 33ºC in C. ornata ........................................................................................................................ 144 8.2.3 Sub-lethal nitrite exposures at different temperatures and blood sampling in C. ornata ........................................................................................................................ 144 8.2.4 Analysis of haemoglobin derivatives ............................................................... 146 8.2.5 Effects of nitrite at different temperatures on growth and digestive enzyme activities in C. ornata ................................................................................................ 146 8.2.6 Calculations ...................................................................................................... 147 8.2.7. Statistics........................................................................................................... 147 8.3 Results .................................................................................................................. 148 8.4. Discussion ........................................................................................................... 159 8.4.1 Values of 96 h LC50 for nitrite at different temperatures in C. ornata ............. 159 8.4.2 Effects of nitrite at different temperatures in C. ornata ................................... 161 viii
8.4.3 Effects of nitrite at different temperatures on growth and digestive enzyme activity in C. ornata................................................................................................... 163 8.5 Conclusions .......................................................................................................... 165 References ...................................................................................................................... 165 Chapter 9 ........................................................................................................................ 173 A SURVEY ON SOME ENVIRONMENTAL PARAMETERS IN CLOWN KNIFEFISH (Chitala ornata, Gray 1831) PONDS ....................................................... 173 9.1 Introduction .......................................................................................................... 174 9.2. Materials and methods......................................................................................... 174 9.2.1 Materials ........................................................................................................... 174 9.2.2 Methods ............................................................................................................ 174 9.3 Results and discussion .......................................................................................... 175 9.4 Conclusions .......................................................................................................... 177 References ...................................................................................................................... 177 Chapter 10 ...................................................................................................................... 178 GENERAL DISCUSSIONS .......................................................................................... 178 10.1 Effects of nitrite exposure to physiological functions in C. ornata ................... 178 10.2 Effects of nitrite exposure on growth in C. ornata............................................. 179 10.3 Effects of elevated temperatures to physiogical parameters in C. ornata .......... 180 10.4 Combined effects of hypercapnia and nitrite on nitrite uptake and acid-base regulation in C. ornata ............................................................................................... 180 References ...................................................................................................................... 181 Chapter 11 ...................................................................................................................... 185 CONCLUSIONS AND RECOMMENDATIONS ........................................................ 185 11.1 Conclusions ........................................................................................................ 185 11.2 Recommendations .............................................................................................. 186 11.2.1 Recommendations for intensive farming systems .......................................... 186 11.2.2 Recommendations for further studies............................................................. 186 List of appendices .......................................................................................................... 187 Appendix 3.2.1. Information in the C. ornata culture ponds ..................................... 187 Appendix 9.3: Determing the values of 96h LC50 for nitrite at 27, 30 and 33ºC in C. ornata (SPSS analysis) ............................................................................................... 188 List of pictures about experimental setup, blood sampling and devices of analysis used in the studies................................................................................................................... 189 ix
List of figures Figure 3.3.1 Mortality of C. ornata (8-10g) by a function of nitrite concentration . 37 Figure 3.3.2 Extinction coefficients for the four haemoglobin species at wavelengths from 480 to 700 nm and spectrum from a fish exposed to 1 mM nitrite for 2 day and the fitted curve ............................................................... 38 Figure 3.3.3 Plasma NO2-, plasma NO3-, percentage metHb, percentage HbNO, functional Hb and total plasma nitrite and nitrate after exposure to nitrite .............................................................................................................. 41 Figure 3.3.4 Plasma chloride, plasma sodium, plasma HCO3-, plasma osmolality, blood lactate after exposure to nitrite ................................................... 43 Figure 3.3.5 Plasma protein and whole body water content after exposure to nitrite .............................................................................................................. 44 Figure 3.3.6 Davenport diagram, blood PCO2, pHe after exposure to nitrite ........... 45 Figure 3.3.7 Rate constant (k, min-1) for erythrocyte metHb decline via metHb reductase in fish exposed to nitrite ....................................................... 45 Figure 4.3.1 Haematological paramters in C. ornata after 14 days exposed to nitrite. .............................................................................................................. 61 Figure 4.3.2 Growth paramters in C. ornata after 90 days exposed to nitrite .......... 64 Figure 5.3.1.1 Time-dependent changes in pHe, plasma bicarbonate, plasma Cl-, PCO2, plasma Na+, and plasma osmolality during exposure to nitrite and hypercapnia ..........................................................................................77 Figure 5.3.1.2 Davenport diagram showing changes in acid-base status during exposure to nitrite and hypercapnia ................................................................... .81 Figure 5.3.2 Time-dependent changes in plasma NO2-, metHb percentage, HbNO percentage, functional Hb, plasma NO3-, and the sum of plasma nitrite and nitrate during exposure to nitrite and hypercapnia ....................…84 Figure 6.3.1 Plasma NO2-, metHb, HbNO (C), functional Hb, plasma NO3- (E), and total nitrite and nitrate after exposure to nitrite and carbon dioxide…100 Figure 6.3.2.1 pHe, plasma HCO3-, PCO2, plasma Na+ and osmolality after exposure to nitrite and carbon dioxide................................................................... 104 x
Figure 6.3.2.2 Davenport diagram presenting the changes in acid-base status after exposure to nitrite and carbon dioxide ............................................... 107 Figure 7.3.2.1 Plasma Na+, plasma osmolality plasma glucose, plasma K+ in small- sized C. ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ................................................................................ 125 Figure 7.3.2.2 pHe, blood PCO2, plasma HCO3-, plasma Cl- in small-sized C. ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC .................................................................................................... 127 Figure 7.3.3.1 Plasma Na+, plasma osmolality, plasma glucose, plasma K+ in large- sized C. ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ................................................................................ 132 Figure 7.3.3.2 pHe, PCO2, plasma HCO3-, plasma Cl- in large-sized C. ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................................................................ 133 Figure 8.3.1 Mortality (96 h LC50 for nitrite) of C. ornata (8-10 g) at three different temperatures: 27ºC, 30ºC, and 33ºC .................................................. 149 Figure 8.3.2 Plasma NO2-, metHb, HbNO, functional Hb, plasma NO3-, and total NO2- and NO3- after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................ 153 Figure 8.3.3 Plasma Na+, plasma osmolality, plasma Cl-, plasma HCO3- after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................................................................ 154 Figure 8.3.4 Davenport diagram presenting the changes in acid-base status, blood PCO2, and pHe after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................ 156 Figure 8.3.5 Survival rate and FCR after 90 days exposed to nitrite at 27ºC (control), 30ºC, 33ºC, 1 mM nitrite at 27ºC, 1 mM nitrite at 30ºC, 1 mM nitrite at 33ºC .................................................................................................... 157 Figure 9.3 Temperature, pH, PCO2, PO2, NO2- (E), NO3- (F) in the water at the C. ornata ponds ...................................................................................... 176 xi
List of tables Table 3.3.1 Hct, Hb and MCHC after exposure to nitrite ..................................... 40 Table 4.3.1 RBCs and WBCs after 14 days exposed to nitrite ............................. 59 Table 4.3.2 Initial weight (W0), weight at day 90 (W90), WG, SGR, and DWG after 90 days exposed to nitrite................................................................... 63 Table 5.3.1.1 Plasma K+, plasma glucose during exposure to nitrite and hypercapnia ............................................................................................................ 79 Table 5.3.1.2 Hct, Hb and MCHC during exposure to nitrite and hypercapnia ....... 83 Table 6.3.1.1 RBCs and WBCs after exposure to nitrite and carbon dioxide.......... 98 Table 6.3.1.2 Hct, Hb and MCHC after exposure to nitrite and carbon dioxide ..... 102 Table 6.3.2 Plasma potassium and plasma glucose after exposure to nitrite and carbon dioxide .................................................................................... 106 Table 7.3.2.1 RBCs and WBCs in small-sized C. ornata after exposed to five different temperatures 24ºC; 27ºC; 30ºC; 33ºC 36ºC ....................................... 122 Table 7.3.2.2 Hct, Hb and MCHC in large-sized after exposed at five different temperatures 24ºC; 27ºC; 30ºC; 33ºC; 36ºC ...................................... 124 Table 7.3.3.1 RBCs and WBCs in large-sized C. ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 129 Table 7.3.3.2 Hct, Hb and MCHC in large-sized after exposed at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 131 Table 8.3.1 RBCs and WBCs after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................ 150 Table 8.3.2 Hct, Hb and MCHC after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 151 Table 8.3.3 Plasma glucose and potassium after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 155 Table 8.3.4 Initial weight (W0), final weight (W90), GW, DWG, and SGR after 30, 60, and 90 days exposed to 27ºC (control), 30ºC, 33ºC, 1 mM nitrite at 27ºC, 1 mM nitrite at 30ºC, 1 mM nitrite at 33ºC .............................. 158 Table 8.3.5 Activities of digestive enzymes: pepsine (in stomach), trypsine (in intestine), chymotrysine (in intestine), α-Amylase (in stomach and xii
intestine) after 90 days exposed to nitrite at 27ºC (control), 30ºC, 33ºC, 1 mM nitrite at 27ºC, 1 mM nitrite at 30ºC, 1 mM nitrite at 33ºC ..... 159 Table 8.4.1 The values of 96h LC50 for nitrite in some fish species ..................... 160 xiii
List of abbreviation [CO2]total total plasma CO2 concentration 96 h LC50 Lethal concentration in 96 hours Cl- Chloride CO2 Carbon dioxide DARD Department of Agriculture and Rulral Development DeoxyHb Deoxygenated haemoglobin DWG Daily weight gain FAO Food and Agriculture Organization FCR Feed conversion ratio Fig. Figure GW Gained weight Hb Haemoglobin HbNO Haemoglobin nitrosyl HCO3- Bircabonate Hct Haematocrit IPCC Intergovernmental Panel on Climate Change K+ Potassium MCHC Mean corpuscular haemoglobin concentration MD Mekong Delta metHb Methaemoglobin Na+ Sodium NH3+ Ammonia NO2- Nitrite NO3- Nitrate OxyHb Oxygenated haemoglobin PCO2 Partial pressure of carbon dioxide pHe Extracellular pH xiv
PO2 Partial pressure of oxygen RBCs Number of red blood cells (erythrocytes) SEM Standard error of mean SGR Specific growth rate SR Survival rate WBCs Number of white blood cells (leukocytes) αCO2 CO2 solubility in trout plasma βNB Non-bicarbonate buffer effect xv
Summary This dissertation investigated the isolated and combined effects of environmental factors such as nitrite, temperature and hypercapnia (high concentration of carbon dioxide) on physiological parameters, growth and digestive enzyme activity in clown knifefish (Chitala ornata) in Mekong Delta, Vietnam. This air- breathing species, which is one of the most popular species has been culturing in the South East Asia with high protein quality and ornamental purposes, typically high environmental resistance under intensive culturing systems. The current situation of climate change has been seriously affecting almost all fields of living organisms including: human, plants, animals, particularly aquatic animals – pokilothermic species. Therefore, the studies in the dissertation about changes of aquatic environment related to fish health and growth, including physiological, biochemical processes in fish have been one of the pressing and necessary issues in order to provide a better physiological understanding as well as recommendations and solutions for minimizing nitrite toxicity and its combination with other environmental elements in aquaculture ponds under global climate change at the present. We discovered that C. ornata has become the most tolerant air-breathing species of nitrite with 96 h LC50 of 7.82 mM at 27ºC. Behind the effective mechanism of denitrification coverting nitrite to nitrate in sub-lethal nitrite exposure, this is also the first study to show that up-regulation methaemoglobin reductase activity in metHb reduction in fish increased almost 5 folds (the rate constant from 0.01 in controls to 0.046 min-1 after 6 days of nitrite exposure for converting metHb to functional Hb). Interestingly, C. ornata had an incomplete acid-base regulation with 50% of extracellular pH compensated during 96 h exposed to 21 mmHg PCO2 by plasma bicarbonate accumulation while it is considered that the air- breathing species with the reduced surface area of gills may cause limitations on transepithelial ion exchange, leading to low capacity of pH regulation. Morever, in combined exposure of acclimated hypercapnia and nitrite, acid-base regulation mainly resulted in chloride-mediated (reduced Cl- influx via the branchial HCO3- /Cl- exchanger) reduced significantly the nitrite uptake across the gill during 96 h. xvi
In addition, C. ornata had rather high temperature tolerance among various tropical species with upper and lower limits of temperature (41ºC and 12ºC, respectively). There were no significant impacts of various temperatures (24ºC, 27ºC, 30ºC and 33ºC) to physiological parameters in both 2 sizes of C. ornata (small-sized and large-sized), but the appearance of mortality after 2 days exposed to 36ºC in commercial fish accompanied with the sudden declines in extracellular pH, haematocrit and haemoglobin concentration may resulted from insufficient oxygen carrying in the blood. In three different temperatures of 27ºC, 30ºC, 33ºC, C. ornata had the highest nitrite tolerance at 30ºC with 96 h LC50 of 8.12 mM, where the values of 96 h LC50 at 27ºC and 33ºC were 7.82 mM and 6.75 mM, respectively. After 2 weeks in nitrite exposures at 5 different temperatures (24ºC, 27ºC, 30ºC, 33ºC, 36ºC), the significant decrease in methaemoglobin via the recovery in functional haemoglobin to 80-85% of total haemoglobin despite of the peak of methaemoglobin of 55% after 2 days exposed to 36ºC. Also nitrite exposure at elevated temperatures caused significant effects to acid-base regulation compared to this at low temperature, e.g. the significant rises of PCO2 and reduction in extracellular pH at the first day. However, extracellular pH was recovered more than 50% for all groups with accumulation of plasma bicarbonate via a HCO3-/Cl- exchanger after 14 days. In addition, we found that long-term exposure of nitrite significantly affected growth parameters. The treatment of 30ºC had the highest survival rate and the lowest FCR compared to other treatments (27ºC, 30ºC, 33ºC, 1 mM nitrite at 27ºC, 1 mM nitrite at 30ºC, and 1 mM nitrite at 33ºC). The activities of digestive enzyme were influenced by nitrite and temperature, where chymotrypsine in intestine in the group of isolated temperature reached the highest values 30ºC compared to this in other groups after 90 days culturing. Key words: Chitala ornata, growth, hypercapnia, metHb reductase, nitrite, physiological processes, temperature. xvii
Tóm tắt Luận án này được thực hiện để tìm hiểu ảnh hưởng đơn lẻ và kết hợp của một số yếu tố môi trường như nitrit, nhiệt độ và hypercapnia (nồng độ carbon dioxide cao trong nước) lên các chỉ tiểu sinh lý máu, tăng trưởng và hoạt động của enzyme tiêu hóa trên cá thát (Chitala ornata) ở đồng bằng sông Cửu Long, Việt Nam. Loài cá hô hấp khí trời này là một trong nhưng loài được nuôi phổ biến nhất ở vùng Đông Nam Á với chất lượng thịt cao và có giá trị làm cảnh, đặc biệt là khả năng chịu đựng môi trường cao trong hệ thống nuôi thâm canh. Tình trạng biến đổi khí hậu (sự tăng nhiệt độ đã và đang ảnh hưởng nghiêm trong đến tất cả các sinh vật sống bao gồm con người, cây trồng, các loài động vật, đặc biệt là động vật thủy sản, loài chịu ảnh hưởng trực tiếp từ sự thay đổi nhiệt độ môi trường. Vì vậy, những nghiên cứu trong luận án này về sự thay đổi của môi trường nước liên quan đến sức khỏe và sinh trưởng của cá bao gồm các quá trình sinh lý, hóa sinh của cá là một trong các vấn đề cấp thiết để cung cấp những kiến thức sinh lý tốt hơn cũng như là các đề xuất và giải pháp nhằm hạn chế tối thiểu tính độc của nitrite và ảnh hưởng kết hợp của nó với các yếu tố môi trường khác trong ao nuôi thủy sản dưới tác động biến đổi khí hậu ngày nay. Nghiên cứu đã phát hiện ra cá thát lát còm là loài cá hô hấp khí trời có khả năng chịu đựng nitrit cao nhất hiện nay với giá trị LC50 96 h là 7.82 mM ở 27ºC. Bên cạnh cơ chế giải độc nitrit là quá trình nitrat hóa bên trong cơ thể cá chuyển đổi nitrit thành nitrat khi tiếp xúc với nồng độ nitrit bán cấp tính, đây cũng là nghiên cứu đầu tiên thể hiện sự tăng hoạt động của enzyme khử nitrit methaemoglobin reductase gấp 5 lần (hằng số hoạt động của enzyme này tăng từ 0.01 ở nghiệm thức đối chứng lên 0.046 min-1 sau 6 ngày tiếp xúc 2.5 mM nitrite). Quá trình cân bằng acid-base ở cá thát lát còm cũng khá hiệu quả với 50 % giá trị pH ngoại bào được đền bù sau 96 h tiếp xúc 21 mmHg CO2 nhờ vào sự tích lũy đáng kể của ion HCO3- trong huyết tương trong khi các loài hô hấp khí trời với sự tiêu giảm diện tích mang có thể làm hạn chế quá trình trao đổi ion qua lớp biểu mô, dẫn tới khả năng điều hòa pH ngoại bào thấp. Hơn nữa, trong sự tiếp xúc kết của hypercapnia và nitrit, quá trình cân bằng acid-base chủ yếu từ cơ chế trao đổi ion chloride gián tiếp (giảm ion Cl- qua sự trao đổi HCO3-/Cl-) đã làm giảm đáng kể lương nitrit qua mang cá suốt 96 h tiếp xúc. xviii