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 smallsized 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 largesized 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 airbreathing 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 airbreathing 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-/Clexchanger 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.
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