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


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

Prof. Dr. Do Thi Thanh Huong
Assoc. Prof. Dr. Bui Thi Bich Hang

Ph.D. Student

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


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.


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.............................
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


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 HIGHTEMPERATURE 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


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


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


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 (20152021; 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


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


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,
/>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


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



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


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 posttemperature challenge. The glucose concentration elevated on the third posttemperature 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.


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.


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 thông 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


(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 loài 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.