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 1 ................................................................................................................. 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 2 ................................................................................................................. 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 3 ................................................................................................................. 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 4 ................................................................................................................. 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 5 ...............................................................................................................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 6 ...............................................................................................................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 7 ...............................................................................................................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 ..................................................................................................................... 1
List of protocols of analysis used in the studies .............................................................. 1

viii


LIST OF FIGURES
Figure 2.1 Total fisheries and aquaculture production 2020 (FAO, 2022). .................. 23
Figure 2.2 World fisheries and aquaculture production, utilization and trade .............. 24
Figure 2.3 World production of striped catfish (thousand tons) (FAO, 2022). ............. 24
Figure 2.4 Farming area and production of striped catfish in Mekong Delta (2015-2021;
VASEP, 2022) .............................................................................................. 24
Figure 2.5 Export value of striped catfish in the period 2015-2021 (VASEP, 2022). .. 26
Figure 2.6. Stress response in response to temperature elevation.. .............................. 30
Figure 2.7 E. hirta L. (Igwe et al., 2016); Phyllanthus amarus (Abeng, 2017); Mimosa
pudica (Goyal, 2014); Psidium guajava (Deepa et al., 2017); Azadirachta
indica (Neelakantan et al., 2011). ................................................................ 66
Figure 2.8 Antioxidant activities of different A. indica extracts and their bioactive
compounds. .................................................................................................. 80
Figure 3.1 Plasma glucose concentrations (mg/100 mL) of P. hypophthalmus fed

extract-based diets in a 60-day experiment. ................................................. 87
Figure 3.2 The pepsin (A) and gastric amylase (B) activities (U min/mg protein) of P.
hypophthalmus fed extract-based diets in a 60-day experiment.
..... 90
Figure 3.3 The intestinal amylase (A), trypsin (B) and chymotrypsin (C) activities (U
min/mg protein) of P. hypophthalmus fed extract-based diets in a 60-day
experiment.. .................................................................................................. 92
Figure 4.1 Pepsin (A) and gastric amylase (B) activities of P. hypophthalmus fingerlings
under various temperatures for 42 days. .................................................... 110
Figure 4.2 Intestinal amylase (A), trypsin (B), and chymotrypsin (C) activities of P.
hypophthalmus fingerlings under various temperatures for 42 days.. ....... 111
Figure 4.4 LPO-gill (A) and LPO-liver (B) of P. hypophthalmus fingerlings under
various temperatures in 42 days. ............................................................... 112
Figure 4.3 Growth performance (WG, DWG, and SGR) and survival rates of P.
hypophthalmus fingerlings under various temperatures for 42 days.
.................................................................................................................... 112
Figure 4.5 LPO-brain (A) and LPO-muscle (B) of P. hypophthalmus fingerlings
under various temperatures in 42 days. ...................................................... 113
Figure 4.7 CAT-gill (A) and CAT-liver (B) of P. hypophthalmus fingerlings under
various temperatures in 42 days.. ............................................................... 115
Figure 5.1 RBCs (A), Hb (B), and Hct (C) of P. hypophthalmus fingerlings under various
salinity in 14 days. Asterisk (*) present significant differences (p<0.05)
among salinity levels whereas different lowercase letters (a, b) indicate
significant differences among feed groups (p<0.05). ................................ 130
Figure 5.2 MCV (A), MCH (B), and MCHC (C) of P. hypophthalmus fingerlings under
various ........................................................................................................ 131
ix


Figure 5. 3 Glucose concentration (A), osmolality (B) of P. hypophthalmus fingerlings

under various salinity in 14 days. Asterisk (*) presents significant differences
(p<0.05) among salinity levels ................................................................... 132
Figure 5.4 LPO- liver (A), LPO-gill (B), LPO-muscle (C) and LPO-brain (D) of P.
hypophthalmus fingerlings under various salinity in 42 days. Asterisk (*)
present significant differences (p<0.05) among salinity levels whereas
different lowercase letters (a, b) indicate significant differences among feed
groups (p<0.05). ......................................................................................... 137
Figure 5. 5 CAT-liver (A), CAT-gill (B), CAT- brain (C) and CAT- muscle (D) of P.
hypophthalmus fingerlings under various salinity in 42 days. Asterisk (*)
present significant differences (p<0.05) among salinity levels whereas
different lowercase letters (a, b) indicate significant differences among feed
groups (p<0.05). ......................................................................................... 139
Figure 6.1 RBCs, Hb, and Hct of P. hypophthalmus fingerlings exposed to different
nitrite concentrations for 14 days. .............................................................. 134
Figure 6.2 MCV (A), MCH (B), and MCHC (C) of P. hypophthalmus fingerlings
exposed to different nitrite concentrations for 14 days. ............................. 135
Figure 6.3 Pepsin and gastric amylase activities of P. hypophthalmus fingerlings
exposed to different nitrite concentrations for 42 days. ............................. 136
Figure 6.4 Intestinal amylase, trypsin, and chymotrypsin activities of P. hypophthalmus
fingerlings exposed to different nitrite concentrations for 42 days.. ......... 137
Figure 6.5 DWG, WG, and survival rate of P. hypophthalmus fingerlings exposed to
different nitrite concentrations for 42 days. ............................................... 142

x


LIST OF TABLES
Table 2.1 Effects of salinity on fish’s physiology under culture ................................. 41
Table 2.2 Effects of some herbal extracts on hematological parameters of fish
under culture ................................................................................................ 51

Table 2.3 Effects of some herbal extracts on digestive enzyme activities of fish
under culture ................................................................................................ 54
Table 2.4 Main oxygen reactive species and their performance Garcez et al. (2004) . 56
Table 2.5 Antioxidant defense system of cell enzymes and biological mechanism
(Shi and Noguchi, (2001). ............................................................................ 56
Table 2.6 Main groups of plant compounds with antimicrobial activity
(Cowan, 1999) .............................................................................................. 61
Table 2.7 Effects of some herbal extracts on oxidative stress indices of fish
under culture ................................................................................................ 64
Table 2.8 Lists of plant extract (from World Flora Online,
............................................................... 65
Table 2.9 Identification information of five plant extracts used in the experiment
(Dao et al., 2020). ......................................................................................... 66
Table 2.10 Phytochemical screening of Mimosa pudica ............................................... 68
Table 2.11 Bioactive metabolites of Mimosa pudica (Rizwan et al., 2022) ................. 68
Table 2.12 Phytochemical screening of Psidium guajava ........................................... 70
Table 2. 13 Phytochemical screening of different species of Psidium guajava ............ 72
Table 2.14 Bioactive metabolites of Psidium guajava .................................................. 72
Table 2.15 Bioactive metabolites of P. amarus (Patel et al., 2011) ............................. 76
Table 2.16 Phytochemical screening of Euphorbia hirta (Asha et al., 2014) ............... 78
Table 3.1 Experimental feed ingredients and formulation ........................................... 84
Table 3.2 Haematological parameters of P. hypophthalmus fed extract-based diets in a
60-day experiment ........................................................................................ 89
Table 3.3 Growth performance of P. hypophthalmus fed plant extract-based diets
in a 60-day experiment ................................................................................. 91
Table 4.1 Experimental feed ingredients and formulation .........................................100
Table 4.2 RBCs (106 cells/mm3) of P. hypophthalmus fingerlings under various
temperatures in ...........................................................................................105
Table 4.3 Hb (mg/100 mL) of P. hypophthalmus fingerlings under various
temperatures in 14 days..............................................................................106

Table 4.4 Hct (%) of P. hypophthalmus fingerlings under various temperatures in
14 days .......................................................................................................107
Table 4.5 MCV (µm3) of P. hypophthalmus fingerlings under various temperatures
in 14 days ...................................................................................................107
Table 4.6 MCH (pg) of P. hypophthalmus fingerlings under various temperatures
in 14 days ...................................................................................................108
xi


Table 4.7 MCHC (%) of P. hypophthalmus fingerlings under various temperatures
in 14 days ...................................................................................................108
Table 4.8 Glucose concentration (mg/100 mL) of P. hypophthalmus fingerlings
under various temperatures in 14 days .......................................................109
Table 5.1 Pepsin of P. hypophthalmus fingerlings exposed to different salinities
for 42 days. .................................................................................................134
Table 5.2 Gastric amylase of P. hypophthalmus fingerlings exposed to different
salinities for 42 days. .................................................................................134
Table 5.4 Chymotrypsin of P. hypophthalmus fingerlings exposed to different
salinities for 42 days. .................................................................................136
Table 6.1 Glucose concentration (mg/100 mL) of P. hypophthalmus fingerlings
exposed to different nitrite concentrations for 14 days ……………… 133
Table 6.2 LPO-gill (nmol MDA/g) of P. hypophthalmus fingerlings exposed to
different nitrite concentrations for 42 days ................................................138
Table 6.3 LPO-brain (nmol MDA/g) of P. hypophthalmus exposed to different
nitrite concentrations for 42 days ...............................................................139
Table 6.4 LPO-muscle (nmol MDA/g) of P. hypophthalmus exposed to different
nitrite concentrations for 42 days ...............................................................139
Table 6.5 CAT-liver (U/min/mg protein) of P. hypophthalmus exposed to different
nitrite concentrations for 42 days ...............................................................140
Table 6.6 CAT-brain (U/min/mg protein) of P. hypophthalmus exposed to different

nitrite concentrations for 42 days ...............................................................141
Table 6.7 CAT-muscle (U/min/mg protein) of P. hypophthalmus exposed to different
nitrite concentrations for 42 days ...............................................................141
Table 7.1 Physiological parameters of striped catfish fed extract-based diets
at 31°C and 35°C compared to 27°C .........................................................151
Table 7.2 Physiological parameters of striped catfish fed extract-based diets
at 10‰ and 20‰ compared to 0‰ ...........................................................152
Table 7.3 Physiological parameters of striped catfish fed extract-based diets at
0.08 mM and 0.8 mM compared 0 mM .....................................................154

xii


LIST OF ABBREVIATION
CAT

Catalase

DWG

Daily weight gain

FAO

Food and Agriculture Organization

FCR

Feed conversion ratio


Fig.

Figure

GW

Gained weight

Hb

Haemoglobin

Hct

Haematocrit

LC50 96 h

Lethal concentration in 96 hours

LPO

Lipid peroxidation

MCH

Mean corpuscular haemoglobin

MCHC


Mean corpuscular haemoglobin concentration

MCV

Mean corpuscular volume

MRD

Mekong River Delta

Mt

Metric ton

MIC

Minimum inhibitory concentration

NO2-

Nitrite

RBCs

Red blood cells

SEM

Standard error of mean


SGR

Specific growth rate

SR

Survival rate

WG

Weight gain

WBCs

Number of white blood cells (leukocytes)

Ai

Azadirachta indica

Eh

Euphorbia hirta

Mp

Mimosa pudica

Pa


Phyllanthus amarus

Pg

Psidium guajava

xiii


ABSTRACT
Striped catfish (Pangasianodon hypophthalmus) is an important species of
aquaculture in the Mekong River Delta (MRD) of Vietnam. However, it has been
constrained by several obstacles, among them which are climate change and diseases.
Plant extracts as a dietary supplement is regarded as the easiest and most efficient
strategy to improve antioxidant activity while contributing to the stress mitigation. The
study aimed to evaluate the effect of selected plant extracts medicated in feed on
physiological haematological parameters, digestive enzyme activities and growth and
stress responses of striped catfish exposed to environmental stressors (salinity,
temperature and NO2-). The final aim would be a selection of plant extracts that have a
positive effect on fish to reduce the use of antibiotics and avoid water environmental
pollution. This doctoral dissertation was, therefore structured into four separate
experiments.
First, five plant extracts, 0.4% or 2% Euphorbia hirta (Eh), 0.2% or 1%
Phyllanthus amarus (Pa), 0.4% or 2% Mimosa pudica (Mp), 0.2% or 1% Psidium
guajava (Pg), and 0.4% or 2% Azadirachta indica (Ai), were investigated on
haematology, digestive enzyme activities and growth throughout the duration of 60
days. These extracts were identified based on the promising and applicable findings
regarding the immunity and antioxidant capacity of striped catfish reported (Nhu et al.,
2019; Dao et al., 2020). P. hypophthalmus fingerlings' haematological indices and
digestive enzyme activities were modified after sixty days of oral administration with

Pg 0.2% or Pa 0.2% extracts, resulting in improved growth performance.
Second, the effects of Psidium guajava L. (0.2%) - Pg0.2 and Phyllanthus amarus
(0.5%) – Pa0.5 on haematology, thermal stress tolerance, enzymatic activities, and
growth of striped catfish subjected to temperatures of 27°C, 31°C, and 35°C for 42 days
were examined. Although haematological indicators were most significant at 35°C, they
were not significantly different from results noted at 31°C on day 14 post-temperature
challenge. The glucose concentration elevated on the third post-temperature challenge
day subsequently decreased and remained constant at 35°C until the end of the trial,
which was not significantly different compared to those at 27°C. After 42 days, the
Pg0.2 and mix diets substantially lowered lipid peroxidation and increased catalase in
the gills and liver. Digestive enzymes (trypsin, chymotrypsin, amylases, and pepsin)
were accelerated by the Pg0.2 and mix treatments, and enzymatic activity improved
from 31°C to 35°C. Overall, fish maintained at 31°C presented the most favorable
growth performance, followed by those reared at 35°C, and there was no significant
difference in survival rates among these treatments. Assuming the Mekong Delta's
average water temperature remains below 35°C, feeding diets incorporating Pg0.2 or
Mix (Pg0.2+Pa0.5) extracts strengthen fish health via haematology and oxidative stress
resistance.
xiv


Third, the haematology, digestion, oxidative stress, and growth of striped catfish
subjected to three salinity levels (0, 10, and 20‰) in formulated extract-based diets (P.
guajava, P. amarus, and a mixture of these two extracs) were investigated. The
haematological parameters recovered after three days of exposure to 10‰ and were
stable for 14 days; however, this was not observed in 20‰. At 0-10‰, higher digestive
enzyme activities (trypsin, chymotrypsin, amylase, and pepsin) was noticed, while a
substantial reduction was observed at 20‰ in extended exposures (day 42). On day 42
of high salinity exposure, LPO levels in muscle, liver, brain, and gills were considerably
higher at 20‰ than lower tested salinities. Fish can thrive normally up to a salinity of

10‰, and no serious damage to fish organs was identified throughout all salinity levels
and sampling durations. This preliminary study revealed that the striped catfish
responded more strongly to increased salinity exposure, suggesting that this species
might serve as a model bio-indicator in coastal farming.
Fourth, fish was susceptible to nitrite exposure and reveal alterations to biological
indicators and stress response when randomly administered to the aforementioned
extract-based diets for 42 days at (0, 0.08, and 0.8 mM) nitrite concentrations. After 24
hours of exposure to 0.8 mM nitrite, the acquired haematological markers decreased
substantially. Almost all digestive enzyme activities in fish exposed to 0.8 mM nitrite
decreased from 7 to 42 days after exposure. Nitrite concentration at 0.8 mM resulted in
alterations that impaired the antioxidant system (reduced CAT activity) and enhanced
oxidative damage in lipids (LPO). Meanwhile, as a result of the high nitrite
concentration, the gills were the most severely injured organs. Eventually, 0.8 mM
nitrite concentrations damaged the antioxidant system and produced stress in striped
catfish fingerlings.
The study's findings convey a scientific foundation for assessing the effects of
climate change on the aquaculture sector in general, and striped catfish farming in
particular. The findings of the study also assist farmers to comprehend the positive and
negative impacts of temperature, salinity, and nitrite on striped catfish, simultaneously,
assisting farmers to incorporate the positive impacts of preserving environmental
elements at optimal levels to promote metabolic activities, as well as beneficial effects
on growth to enhance farming production. Furthermore, farmers can augment the
dietary intake of striped catfish fingerlings with essential antioxidant components found
in plant extracts such as P. guajava 0.2% or combine P. amarus 0.5% to improve health,
contribute to the growth and mitigate stress under the current climate change scenario.

xv


TĨM TẮT

Cá tra (Pangasianodon hypophthalmus) là đối tượng ni quan trọng của vùng
Đồng bằng sông Cửu Long (ĐBSCL), Việt Nam. Tuy nhiên, ngành nuôi cá tra hiện này
đang đối mặt với một số trở ngại, trong đó có thể kể đến là biến đổi khí hậu và dịch bệnh
trên cá nuôi. Chất chiết thực vật hiện nay được bổ sung vào chế độ ăn của cá tra được
đánh giá là phương pháp dễ dàng và hiệu quả nhất để cải thiện hoạt động chống oxy hóa
đồng thời góp phần giảm stress trên cá nuôi. Nghiên cứu được thực hiện nhằm đánh giá
ảnh hưởng của một số chiết xuất thực vật được bổ sung vào chế độ ăn của cá tra thơng
qua một số chỉ tiêu sinh lý máu, hoạt tính của enzyme tiêu hóa, tăng trưởng và giảm
stress trên cá tra khi tiếp xúc với các tác nhân gây stress từ môi trường (độ mặn, nhiệt
độ và nitrite). Mục tiêu chính yếu nhằm sàng lọc các chất chiết thực vật có tác động tích
cực đến sức khỏe cá tra ni để hạn chế tối đa việc sử dụng kháng sinh và tránh ô nhiễm
môi trường nuôi. Luận án tiến sĩ này bao gồm thành bốn thí nghiệm được thực hiện riêng
biệt.
Thí nghiệm 1, năm loại chiết xuất thực vật khác nhau bao gồm cỏ sữa (Euphorbia
hirta) (Eh) 0,4%, 2%; diệp hạ châu (Phyllanthus amarus) (Pa) 0,2%, 1%; mắc cỡ
(Mimosa pudica) (Mp) 0,4%; ổi (Psidium guajava) (Pg) 0,2%, 1%, và sầu đâu
(Azadirachta indica) (Ai) 0,4%; 2%, đã được nghiên cứu về sinh lý máu, hoạt tính của
enzyme tiêu hóa và tăng trưởng trên cá tra trong 60 ngày. Các chất chiết thực vật này
được chọn dựa trên những kết quả nghiên cứu trước đó về khả năng miễn dịch và chống
oxy hóa tốt trên cá tra (Nhu et al., 2019; Dao et al., 2020). Các chỉ tiêu sinh lý máu và
hoạt tính của enzyme tiêu hóa của cá tra được cải thiện sau 60 ngày bổ sung chất chiết
Pg 0,2% hoặc Pa 0,2% vào chế độ ăn, dẫn đến tăng trưởng được cải thiện.
Thí nghiệm 2, ảnh hưởng của chất chiết ổi (0,2%) – Pg0,2 và diệp hạ châu (0,5%)
– Pa0,5 qua các chỉ tiêu sinh lý máu, khả năng đáp ứng stress, hoạt tính enzyme tiêu hóa
và tăng trưởng của cá tra ở nhiệt độ 27°C, 31°C và 35°C trong 42 ngày đã được thực
hiện. Mặc dù các chỉ tiêu sinh lý máu thay đổi có ý nghĩa thống kê ở 35°C, nhưng khác
biệt khơng có ý nghĩa thống kê so với kết quả được ghi nhận ở 31°C sau 14 ngày dưới
tác động của nhiệt độ. Nồng độ glucose tăng cao vào ngày thứ ba dưới ảnh hưởng của
nhiệt độ sau đó giảm xuống và duy trì nồng độ glucose ở 35°C cho đến khi kết thúc thí
nghiệm, khác biệt không đáng kể so với nồng độ ở 27°C. Sau 42 ngày, chế độ ăn có bổ

sung Pg0,2 và hỗn hợp chất chiết (Pg0,2+Pa0,5) làm giảm đáng kể quá trình oxy hóa
lipid và tăng catalase trong mang và gan. Các enzyme tiêu hóa (trypsin, chymotrypsin,
amylase và pepsin) được tăng cường ở chế độ ăn có bổ sung Pg0,2 và hỗn hợp, đồng
thời hoạt tính của enzyme được cải thiện ở nhiệt độ từ 31°C đến 35°C. Nhìn chung, cá
ni ở nhiệt độ 31°C có tăng trưởng tốt nhất nhất, kế đế đến là ở nhiệt độ 35°C và khác
biệt khơng có ý nghĩa thống kê về tỷ lệ sống giữa hai mức nhiệt độ này. Nếu nhiệt độ
trung bình của ĐBSCL dưới 35°C, và chế độ ăn có bổ sung Pg0,2 hoặc hỗn hợp

xvi


(Pg0,2+Pa0,5) giúp tăng cường sức khỏe của cá thông qua các chỉ tiêu sinh lý máu và
khả năng chống stress oxy hóa ở cá.
Thí nghiệm 3, các chỉ tiêu sinh lý máu, enzyme tiêu hóa, oxy hóa stress và tăng
trưởng của cá tra với mức độ mặn (0, 10 và 20 ‰) và chế độ ăn có bổ sung chất chiết từ
ổi, diệp hạ châu và hỗn hợp của hai chất chiết này) đã được thực hiện. Các chỉ tiêu sinh
lý máu phục hồi sau ba ngày tiếp xúc với độ mặn 10‰ và duy trì trong 14 ngày; tuy
nhiên, điều này không được ghi nhận ở độ mặn 20‰. Ở khoảng độ mặn 0-10‰, hoạt
tính của enzyme tiêu hóa (trypsin, chymotrypsin, amylase và pepsin) cao hơn, trong khi
đó, hoạt tính enzyme tiêu hóa giảm đáng kể ở độ mặn 20‰ khi tiếp xúc trong thời gian
dài (ngày 42). Vào ngày thứ 42 tiếp xúc với độ mặn cao, nồng độ LPO trong cơ, gan,
não và mang của cá tra cao hơn ở độ mặn 20‰ so với độ mặn thấp hơn. Cá có thể phát
triển bình thường ở độ mặn 10‰ và các cơ quan của cá không bị ảnh hưởng đáng kể
trong tất cả thời gian lấy mẫu. Nghiên cứu sơ bộ này cho thấy rằng cá tra có ảnh hưởng
càng lớn khi tiếp xúc với độ mặn tăng lên quá cao (20%), điều này cho thấy rằng lồi
này có thực hiện ni ở các vùng nước lợ ven biển.
Thứ nghiệm 4, cá tra được tiếp xúc với các nồng độ nitrite khác nhau và thể hiện
những thay đổi thông qua các chỉ tiêu sinh lý máu và khả năng đáp ứng stress khi được
bổ sung vào chế độ ăn các loại chất chiết xuất đã đề cặp ở các thí nghiệm trên trong 42
ngày với các nồng độ nitrite khác nhau (0; 0,08 và 0,8 mM). Sau 24 giờ tiếp xúc với

nitrite ở nồng độ 0,8 mM, các chỉ tiêu sinh lý máu đã giảm đáng kể. Hầu như tất cả hoạt
tính của enzyme tiêu hóa ở cá tiếp xúc với 0,8 mM nitrite đều giảm từ giai đoạn 7 đến
42 ngày sau khi tiếp xúc với. Nồng độ nitrite 0,8 mM gây ra những thay đổi làm suy yếu
hệ thống chống oxy hóa (giảm hoạt tính CAT) và tăng cường oxy hóa lipid (LPO). Trong
khi đó, do nồng độ nitrite cao, mang là cơ quan bị ảnh hưởng nặng nề nhất. Chính vì
vậy, nồng độ nitrite 0,8 mM đã ảnh hưởng đáng kể đến hệ thống chống oxy hóa và gây
ra stress trên cá tra giai đoạn giống.
Tóm lại, kết quả của nghiên cứu là cơ sở khoa học góp phần đánh giá ảnh hưởng
của điều kiện biến đổi khí hậu lên ngành ni trồng thủy sản nói chung và nghề ni cá
tra nói riêng. Kết quả nghiên cứu cũng giúp người nuôi nhận biết được ảnh hưởng có lợi
và bất lợi của nhiệt độ, độ mặn và nitrite lên cá tra. Đồng thời, giúp người ni ứng dụng
ảnh hưởng tốt của việc duy trì các yếu tố mơi trường ở mức thích hợp nhằm tăng cường
hoạt động trao đổi chất, ảnh hưởng tốt đến tăng trưởng có thể tăng năng suất ni. Bên
cạnh đó, nhờ vào các thành phần chống oxy hóa hữu ích có trong chất chiết thực vật như
ổi và diệp hạ châu, người ni có thể ứng dụng bổ sung vào chế độ ăn của cá tra giai
đoạn giống nhằm tăng cường sức khỏe và giảm stress, góp phần vào tăng trưởng cho cá
ni trong điều kiện biến đổi khí hậu hiện nay.

xvii


CHAPTER 1
INTRODUCTION
1.1 General introduction
The development of striped catfish (Pangasianodon hypophthalmus) aquaculture
in particular for the Mekong River Delta (MRD), Viet Nam has been challenged with
various issues, of which climate change and disease are the most concern of the fish
farmers and researchers. This region is most likely to be affected by global climate
change. In years ago, the average temperature in Viet Nam increased approximately 0.50.7°C; and the annual average temperature of Viet Nam will increase by 2.3°C by the
end of the 21st century (MONRE, 2009); this will greatly affect the sustainable

development not only fisheries but also aquaculture including striped catfish. As fish is
poikilothermic species, therefore temperature is the main factor that cause affects
directly or indirectly to fish life. Changing temperature can be negative effects to fish
by increasing or reducing metabolic rates (Galloway and Kieffer, 2003), swimming
performance (Hocutt, 1973), impairing immune functions (Hurst, 2007), consequence
in reducing the ability to capture prey, increasing susceptibility to disease and enhancing
mortality (Donaldson et al., 2008). Temperature shock can also impede predator
avoidance (Ward and Bonar, 2003), alter rates of recovery from exercise (Suski et al.,
2006), and disrupt homeostasis (Vanlandeghem et al., 2010).
The MRD is also one of three extensive low topographical regions in Viet Nam
that have been predicted to be impacted by sea-level rises this century (Parry et al.,
2007). If sea-level rise occurs as predicted, the striped catfish industry in Viet Nam is
likely to be severely impacted because the region is flat and low lying with a maximum
elevation of less than 4.0 m above mean sea level. If predictions of future climate change
of this century are realized, a 1m increase in mean sea level will result in approximately
1,000 km2 of cultivated land and farming area in Viet Nam becoming salt marshland,
and 15,000 - 20,000 km2 of the MRD is likely to be flooded by seawater (i.e. up to 30‰)
(Parry et al., 2007). Increasing salinization of freshwater areas will, as a consequence,
impact the large local freshwater aquaculture industry, in particular the striped catfish
culture, as a result of changes to both soil and local freshwater resources (Sebesvari et
al., 2011).
In addition, intensive ponds with overfeeding and wastes may lead to the
decomposition processes of organic matters. Toxic gases are significantly generated in
hypoxic and high-temperature conditions such as ammonia, nitrite, nitrate, carbon
dioxide and hydrogen sulfite. Especially, nitrite is a naturally occurring component of
the nitrogen cycle, which under some circumstances can reach high levels where it can
present a problem, because of well-documented toxicity to animals (Lewis and Morris,
1986), typically for freshwater species. Methaemoglobin formation from reaction of
nitrite with haemoglobin (Hb) is the main reason for the exceptional low arterial O2, but
18



a decrease in blood O2 affinity also contributes (Jensen et al., 1987). Fish can
accommodate relatively high metHb levels at rest, but the decrease in blood O2 content
will limit the scope for activity. All these restrictions have made it difficult for fish
farmers to translate the benefits of greater production yield associated with intensive
production methods into commercial profits.
Antibiotics and chemotherapeutics used to control these diseases can result in the
development of drug-resistant bacteria, environmental pollution and residues in fish
(Jian and Wu, 2004). The use of antibiotics to control disease in fish was found to be
significantly high in Vietnamese striped catfish farming; a total of 17 different antibiotic
compounds belonging to 10 different antibiotic classes were used (Rico et al., 2013).
Several therapeutic medicines are widely used in aquaculture to prevent or cure disease
outbreaks to minimize economic losses caused by sanitary deficiencies. Antimicrobials
and other veterinary drugs are given on a regular basis as feed additives or in baths and
injections as prophylactics or growth boosters. Nonetheless, the use of pharmaceutical
products is becoming more restricted because to the various negative consequences on
the environment and human health (Mckenzie et al., 2012). Vaccination is also being
considered as a possible therapy for disease outbreaks in aquaculture. Commercial
vaccinations, on the other hand, are prohibitively expensive for broad usage by fish
farmers, and a single vaccine is effective against just one type of disease (Harikrishnan
et al., 2011). As a result, researchers have increased their efforts to use natural
ingredients in the development of alternative nutritional supplements that improve the
growth performance, health, and immune system of farmed fish from less expensive
sources without producing toxicity (Pandey et al., 2012).
Nowadays, more environmental-friendly prophylactic and preventive solutions are
required, and natural bio-active products are being searched for to enhance the immune
system and health status of cultivated animals. Plant compounds such as phenolics,
polyphenols, alkaloids, quinones, terpenoids, lectins, and polypeptides have been shown
to be very effective alternatives to antibiotics and other synthetic compounds (Citarasu,

2010). Phytochemicals have been shown to have antioxidant, antibacterial, antifungal,
antidiabetic, anti-inflammatory, antiarthritic, and radioprotective properties (Nair et al.,
2005). Study on the use of plant extracts in aquatic animals has been becoming a global
concern as it is a trend in organic aquaculture production and reduction use of antibiotic.
However, despite a high diversity of wild plants distributed in the different eco-regions
of Viet Nam and the interest of a large proportion of aquaculture farmers to use
alternatives to antibiotics, the actual use of natural derived products in aquaculture is not
yet very popular in the country, due to a lack of knowledge regarding the existence of
such bio-active products and the demonstration of their efficiency on fish. The selected
5 types of plant extracts and concentrations in the study were inherited from the results
of screening research on the effects of 20 types of plants (Nhu et al., 2019). The
emphasized research has exclusively concentrated on certain findings on immunity,
19


whereas the physiological aspects of the striped catfish (hematology, digestive enzymes,
stress, etc.) need to be addressed. Additionally, the effects of extracts (Euphorbia hirta,
Phyllanthus amarus, Mimosa pudica, Psidium guajava (Pg), and Azadirachta indica)
on the physiology of striped catfish have not been comprehensively investigated, and
they ought to be regarded as abundantly and relatively cost-effective active components
beneficial for dietary incorporation. Our findings should be particularly helpful for
researchers in both scientific and practical fields, since they provide numerous
biological insights and highlight the various plant-based bioactive substances available
for improving fish health. In fact, the state of drugs, chemical substances and plant
extracts used in striped catfish farming indicated a scarcity of useful information
regarding herbal-derived commercial items utilized by farmers. As a result, there is an
urgent need to offer farmer training courses to update their understanding of active
constituents, proper use, and administratively applied doses of plant extract products.
1.2 The objectives of the dissertation
Overall objectives: The final aim of the study is to choose the plants that have a

positive effect on fish contributing to the production of biological safety products and
environmental sustainability. Then it can also recommend for fish farmers to use herbs
in the striped catfish farming to reduce the use of antibiotics and avoid water
environmental pollution.
Specific objectives: The objectives include evaluation the effect of selected plant
extracts medicated in feed on (1) physiological hematological parameters; (2) digestive
enzyme activities; and (3) growth and stress responses of striped catfish exposed to
environmental stressors (salinity, temperature and NO2-).
1.3 The main contents of the dissertation
- Study on the effect of dietary supplementation of 5 plant extracts (Euphorbia
hirta, Phyllanthus amarus, Mimosa pudica, Azadirachta indica and Psidium guajava)
on hematology physiology, digestive enzymes activities and growth performance of
striped catfish fingerlings.
- Study on the effect of dietary supplementation of P. amarus and P. guajava on
hematology physiology, digestive enzymes activities, oxidative stress and growth
performance of striped catfish exposed to elevated temperatures
- Study on the effect of dietary supplementation of P. amarus and P. guajava on
hematology physiology, digestive enzymes activities, oxidative stress and growth
performance of striped catfish exposed to sublethal salinities
- Study on the effect of dietary supplementation of P. amarus and P. guajava on
hematology physiology, digestive enzyme activities, oxidative stress and growth
performance exposed to various nitrite concentrations

20


1.4 The hypotheses of the dissertation
- The five selected plant extracts displayed different dose-dependent effects, and
the supplementation of the extract-based diet improved hematological parameters and
digestive enzyme activities and growth performance of striped catfish.

- Elevated temperature did not negatively disturbances on hematological
parameters, digestive enzymes as well as oxidative stress with the supplemented P
amarus and P. guajava based diets consequently, resulted in rapid growth performance
of striped catfish.
- Chronic exposures of striped catfish to higher salinity resulted in fish gradually
acclimated to the elevated salinity in the habitat and without disturbing physiological
functions and growth performance in P. amarus and P. guajava dietary.
- With the supplementation of two mentioned extracts, striped catfish has high
tolerance of high nitrite concentration, leading to more significant effects to
physiological parameters and growth performance compared to those in the basal diet.
1.5 New findings of the dissertation
In five plant extract-based diets (Euphorbia hirta (Eh); Phyllanthus amarus (Pa);
Mimosa pudica; Psidium guajava (Pg), and Azadirachta indica (Ai)) augmented with
plant extracts at various doses and feeding durations, the diet including Pg 0.2 or Pa
0.2% extracts in 60 days imposed the potential to modify hematology, enzymatic
activity, and P. hypophthalmus growth. As a result, the preference of fish for various
plant extract-based diets should be evaluated, as these findings could indicate a high use
and consumption of aquafeed, which could lead to enhanced growth performance of
fish.
Through hematology profile, digestive enzyme activity, and oxidative stress
biomarkers, the Pg0.2 and Mix diets promote the health of P. hypophthalmus. The use
of Pg0.2 or Mix is indicated to promote fish health and stress reduction. Several
haematological parameters (RBCs, Hct, Hb, glucose) were unaffected by high
temperatures (35°C) until day 7 and oxidative stress indicators (LPO, CAT in gill and
liver) were unaltered till day 14. Following this, the fish recovered and became
accustomed to the experimental conditions.
In terms of salinity experiment, the liver and gills are two internal organs that are
subjected to severe oxidative damage as a result of increased salinity. Although the
extent of modifications occurs at the maximum salinity (20‰) and exposure days,
alterations were insignificant up to 10‰. This species may be suitable for farming in the

low-salt brackish zone in the future. Furthermore, the Pg0.2-administered food,
followed by Mixture (Pg0.2:Pa0.5), offered the most efficient impact in preserving the
fish's normal physiological function in terms of haematology, digestion, and stress
mitigation.
21


Striped catfish can be negatively affected by nitrite-induced stress at 0.8mM. A
deleterious nitrite concentration of 0.8 mM might reduce RBCs, Hb, and Hct while
increasing plasma glucose concentration in fish plasma, leads to adverse effects after 24
hours of exposure. Nitrite exposure, on the other hand, may result in a decrease in
digestive enzyme activities in fish at intervals of 7-42 days of high nitrite exposure,
resulting in poor growth performance. These modifications were observed in fish
exposed to 0.8mM and became more severe with increased exposure and nitrite
concentrations.
As a result, regardless of the case of short-term exposure, nitrite concentrations of
0.8 mM or more should be avoided in production systems in order to prevent the fish
from stress and enhance growth performance. The Pg0.2 and Mix diets improve the
health of P. hypophthalmus by modifying hematology, digestive enzyme activity, and
oxidative stress biomarkers.
1.6 Significant contributions of the dissertation
Scientific contributions:The dissertation also revealed that P. amarus and P.
guajava improve catfish health through a variety of physiological parameters. The
administration of small quantities of the extract to the daily diet promotes the growth
performance of the striped catfish under adverse environmental conditions.
Practical contributions:The results of the study (1) provide promising solutions
based on plant extracts that meet further development and can be applied on a large scale
effectively in the future; and (2) offer solutions to significantly reduce the use of
antibiotics or other chemicals in aquaculture contributing to environmental
sustainability. Furthermore, (3) the acquired knowledge from the research may be

utilized for education purposes, as a source of reference, and as a base for the
development of ongoing studies on plant extracts in other aquatic species.
1.7 Structure of the dissertation
The four primary contents of the dissertation are illustrated as follows:

Content 1

Euphorbia
hirta L.

Mimosa
pudica

Phyllanthus
amarus

Psidium
guajava

Azadirachta
indica
o

27 C
o

Content 2

Content 3


Phyllanthus
amarus

Phyllanthus
amarus

Psidium
guajava

Psidium
guajava

31 C
o

35 C

0‰
10‰
20‰
0% LC 96h
50

Content 4

10% LC 96h
Phyllanthus
amarus

50


Psidium
guajava

22

20% LC 96h
50


CHAPTER 2
LITERATURE REVIEW
2.1 The status and importance of aquaculture and fisheries
Fisheries and aquaculture significantly contribute to the domestic economy,
particularly in developing countries. In 2020, the sector's sale value was estimated at
$424 billion, with 225 countries participating in global markets. However, due to the
Covid-19 pandemic, this value declined by 7%. FAO (2022) reported a record 214
metric tons of fish and algae production in 2020, with 178 million aquatic animals and
36 million algae. Aquaculture experienced a 2.7% growth, below the 4.5% annual
average growth over the previous decade. (FAO, 2022).

Figure 2.1 Total fisheries and aquaculture production 2020 (FAO, 2022).

The international commerce of aquatic products has encountered a variety of
challenges as a result of the COVID-19 outbreak. The value of exports of aquatic
products worldwide is also projected to have decreased by 7.0%, reaching US$ 151
billion in 2020. This followed a 2.1% drop in 2019 compared to the peak attained in
2018. Trade volumes are anticipated to decline by 10.1% in 2020, with declines
documented in all regions. As fishing and aquaculture began and international markets
reopened, trade recovered strongly in 2021. The overall value of global aquatic product

exports increased by 12% in 2021 compared to 2020. Meanwhile, trade volume growth

23