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.
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 TABLE OF CONTENTS DATA SHEET .i RESULT COMMITMENT ii ACKNOWLEDGEMENTS iii LIST OF FIGURES ix LIST OF TABLES xi LIST OF ABBREVIATION xiii ABSTRACT xiv TÓM TẮT xvi CHAPTER 18 INTRODUCTION 18 1.1 General introduction 18 1.2 The objectives of the dissertation Error! Bookmark not defined 1.3 The main contents of the dissertation 20 1.4 The hypotheses of the dissertation 21 1.5 New findings of the dissertation 21 1.6 Significant contributions of the dissertation 22 CHAPTER 22 LITERATURE REVIEW 23 2.1 The status and importance of aquaculture and fisheries 23 2.2 Climate changes and impacts on aquaculture and fisheries 27 2.3 Mechanism of stress on fish 28 2.4 Effects of environmental factors on fish 30 2.4.1 Effect of temperature on fish 30 2.4.2 Effects of salinity on fish 37 2.4.3 Effects of nitrite on fish 43 2.5 Effects of plant extracts on fish 47 2.5.1 Effects of plant extracts on hematological parameters of fish 47 2.5.2 Effects of plant extracts on digestive enzymes activities and growth of fish 49 2.5.3 The effects of plant extracts on oxidative stress of fishes 60 2.5.4 Effects of plant extracts on striped catfish (P hypophthalmus) 62 a) Effects of Mimosa pudica on aquaculture species 67 b) Effects of Psidium guajava on aquaculture species 67 c) Effect of Phyllanthus amarus on fish 71 d) Effect of Euphorbia hirta on fish 77 e) Effect of Azadirachta indica on fish 79 CHAPTER 81 EFFECTS OF PLANT EXTRACTS ON SELECTED HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES, AND GROWTH PERFORMANCE v OF STRIPED CATFISH, Pangasianodon hypophthalmus (Sauvage, 1878) FINGERLINGS 81 Abstract 81 3.1 Introduction 81 3.2 Material and Method 83 3.2.1 Plant extract and feed preparation 83 3.2.2 Experimental fish acclimation, facilities, and feeding management 84 3.2.3 Haematological and biochemical parameters 85 3.2.4 Digestive enzyme activities 86 3.2.5 Growth performance and survival rate 86 3.2.6 Statistical analysis 87 3.3 Results 87 3.3.1 Effects of plant extracts on haematological parameters 87 3.3.2 Effects of plant extract on digestive enzyme activities 90 3.3.3 Effects of plant extracts on growth performance and the survival rate 91 3.4 Discussion 92 3.5 Conclusions 96 CHAPTER 97 EFFECTS OF Psidium guajava AND Phyllanthus amarus EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO HIGH-TEMPERATURE STRESS 97 4.1 Introduction 98 4.2 Materials and methods 100 4.2.1 Plant extracts and diet preparation .100 4.2.2 Experimental fish acclimation, facilities, and feeding trial 101 4.2.3 Haematological and biochemical parameters .102 4.2.4 Oxidative stress assays .103 4.2.5 Statistical analysis 104 4.3 Results 105 4.3.1 Effect of plant extract on P hypophthalmus haematological parameters 105 4.3.2 Effect of plant extract on digestive enzyme activities 109 4.3.3 Effect of plant extract on growth performance of P hypophthalmus 111 4.3.4 Effect of plant extract on P hypophthalmus oxidative stress 113 4.4 Discussion .115 4.5 Conclusions 122 Acknowledgments .122 CHAPTER .123 vi EFFECTS OF GUAVA (Psidium guajava L.) AND BHUMI AMLA (Phyllanthus amarus Chum et Thonn.) EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO SUBLETHAL SALINITIES .123 Abstract 123 5.1 Introduction 123 5.2 Material and Method .125 5.2.1 Plant extract and feed preparation 125 5.2.2 Experimental fish acclimation, facilities, and feeding management 126 5.2.3 Haematological and biochemical parameters .127 5.2.4 Digestive enzyme activities 127 5.2.5 Oxidative stress biomarkers .128 5.2.6 Growth performance and survival rate .129 5.2.7 Statistical analysis 129 5.3 Results 129 5.3.1 Effects of plant extracts on haematological parameters .129 5.3.2 Effects of plant extracts on digestive enzyme activities 133 5.3.3 Effects of plant extracts on oxidative stress .136 5.3.4 Effects of plant extracts on growth performance and the survival rate 140 5.4 Discussion 140 5.5 Conclusions 144 CHAPTER .126 EFFECTS OF Psidium guajava AND Phyllanthus amarus EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO NITRITE-INDUCED TOXICITY .126 Abstract 126 6.1 Introduction 126 6.2 Material and Method .128 6.2.1 Plant extract and feed preparation 128 6.2.2 Experimental fish acclimation, facilities, and feeding management 128 6.2.3 Haematological and biochemical parameters .130 6.2.4 Digestive enzyme activities 130 6.2.5 Oxidative stress biomarkers .131 6.2.6 Growth performance and survival rate .132 6.2.7 Statistical analysis 132 6.3 Results 132 6.3.1 Effects of plant extracts on haematological parameters .132 vii 6.3.2 Effects of plant extracts on digestive enzyme activities 136 6.3.3 Effects of plant extracts on oxidative stress .136 6.3.4 Effects of plant extracts on growth performance and the survival rate 142 6.4 Discussion 142 6.5 Conclusions 146 CHAPTER .147 GENERAL DISCUSSION 147 7.1 Effects of five selected extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings 147 7.2 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings exposed to hightemperature stress 150 7.3 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings exposed to sublethal salinities .152 7.4 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P hypophthalmus fingerlings exposed to nitriteinduced toxicity 153 CONCLUSIONS AND RECOMMENDATIONS 156 8.1 Conclusion .156 8.2 Recommendations for further studies 157 APPENDIX List of protocols of analysis used in the studies viii 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