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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY A SUMMARY OF DOCTORAL DISSERTATION Major: Aquaculture Major code: 9620301 NGUYEN THI KIM HA EFFECTS OF NITRITE, CO2 AND TEMPERATURE ON PHYSIOLOGICAL PARAMETERS AND GROWTH IN BASA CATFISH (Pangasius bocourti Sauvage, 1880) Can Tho, 2019 THE RESEARCH WAS PERFORMED AND COMPLETED AT CAN THO UNIVERSITY Supervisor: Assoc Prof Dr Do Thi Thanh Huong The dissertation will be defended at the Doctoral Dissertation Examination Committee at the University Level At: ………………………………………………………… Time and Date: …………………………………………… Reviewer 1: ……………………………………………… Reviewer 2: ……………………………………………… The dissertation can be found at: The Learning Resource Center, Can Tho University The National Library of Vietnam THE LIST OF PUBLISHED PAPERS Nguyen Thi Kim Ha, Nguyen Tran Phuong Thao, Nguyen Thanh Phuong, Mark Bayley and Do Thi Thanh Huong, 2017 Effects of nitrite on physiological parameters and growth in basa catfish (Pangasius bocourti) Can Tho University Journal of Science 52: 93-102 (in Vietnamese) Nguyen Thi Kim Ha, Nguyen Xuan Bieu, Nguyen Thanh Phuong and Do Thi Thanh Huong, 2018 Effect of CO2 on acid-base regulation and growth performance of basa catfish (Pangasius bocourti) Can Tho University Journal of Science 54 (2): 18-26 Chapter INTRODUCTION 1.1 Introduction Aquaculture has been playing an important and significant role in the Vietnam economy over the recent decades, which the total production reached 3,835 thousand tons in 2017 (General Statistics Office of Vietnam, 2017) Tra catfish and basa catfish are two of mainly farmed and exported freshwater species in Mekong Delta River (MDR) In Vietnam, the two species are largely farmed in freshwater regions of the river basin including An Giang, Dong Thap, Vinh Long and Can Tho provinces Basa catfish (Pangasius bocourti) has commercial value and been exported globally due to its white meat, good taste and fast growth Previous studies on basa catfish are mostly about nutrition demand (Phuong, 1998 Hung et al., 2004), culture and seed production techniques (Cacot et al., 2002 Cacot et al., 2003) However, there have not been studies about the tolerance and adaptation of basa catfish under impacts of environmental factors such as nitrite, temperature and CO2 Nitrite is a well – documented toxin for most of the aquatic animal (Lewis and Morris, 1986) Therefore, its presence in culture ponds is a concerned problem Nitrite enters into red blood cells, where it oxidises iron in hemoglobin (Hb) to methemoglobin (metHb) giving brown blood disease In addition, nitrite is also well-known as a disrupter of growth and physiological functions including iron regulatory, respiratory, endocrine and excretory processes (Kosaka and Tyuma, 1987; Siikavuopio and Saether, 2006; Jensen, 2009; Lefevre et al., 2011) Besides, CO2 concentrations in the air have been increasing, which is caused by economic development and industrialization (or greenhouse) In fish, the increase of CO2 in the water affects to its diffusion for blood to outside leading to unbalance of acid-base regulation and confusion of intracellular activities as ion regulation, respiratory increase,… (Ishimatsu et al., 2005; Brauner et al., 2004; Gilmour, 2001; Cameron and Iwama, 1987) Temperature is considered as one of important factors to affect to organism activities such as physiological process, growth, survival rate and so on…Some studies showed factors involved to nitrite toxicity, particularly the higher temperature increases the toxicity and converse (Jeberg and Jensen, 1994; Huey et al., 1984) In the current status, the temperature and CO2 related to climate changes and the intensive level in nursery ponds lead to more damages to fish of the environmental toxic gases as nitrite, especially in the case of combination of these factors together Base on the above reasons, it requires to carry out the research on the effects of nitrire, temperature and CO2 on fish at fingerling stage in order to provide information for the management of fish, in general and in basa catfish, in particular 1.2 The objective of study The objectives of this study were to understand the effects of environmental factors such as nitrite, temperature and the increase of CO2 concetration in the water on physiological parameters and growth performance of basa catfish The results of study will provide more information on physiological characteristics and adaptation of basa catfish on studied factors The results also help farmers to be aware of the effects of these factors on aquaculture species, especially basa catfish to minimize their inpact on fish health during nursery period 1.3 The main contents of the study a) Study on the effects of nitrite and temperature on physiological parameters and growth performance of basa catfish b) Study on effects of nitrite and CO2 on physiological parameters and growth performance of basa catfish c) Study on effects of nitrite on methemoglobin (metHb) reductase activity and oxygen consumption (MO2) of basa catfish d) Conducting a survey on some selected environmental parameters on basa catfish nursery ponds 1.4 Significant contributions and applications of the study The study provided evidences on the physiological changes, adaption and growth performance of basa catfish at fingerling stage exposed to the change of nitrite, CO2 and temperature The findings also contributed to the evaluation of the effects of environmental parameters related to climate change on aquaculture species, especially basa catfish Results showed that when temperature increased to 33oC, the predicted temperature for the 21th century, fish grew better than the lower temperature, 27oC Thus, the increase temperature related climate change will not affect to basa catfish growth However, when fish exposed to nitrite at concentration above or equal to 10% LC50-96 h or CO2 at concentration above mmHg, fish reduced growth rate and physiological parameters were negatively affected The study provided basic knowledge on elimination of nitrite and CO2 in aquatic pond which contributes to the sustainable development of this species In addition, the findings of this study also contributed to the reference’s sources for teaching and further follow-up studies on basa catfish or research on the effects of nitrite, CO2 and temperature on other species Chapter METHODOLOGY 2.1 Activity 1: Effects of nitrite and temperature on physiological parameters and growth performance of basa catfish This work was conducted with basa catfish of 10-20 g Experiment 1: Determination of acute nitrite toxicity (LC50-96 h) The experiment consisted of 11 nitrite concentrations such as 0; 0.54; 0.65; 0.76; 0.87; 0.98; 1.09; 1.20; 1.31; 1.42 and 1.53 mM at 27°C (the average level of water temperature in Vietnam) and 33°C (elevated temperature, the level is predicted to reach at the 21st century), with 12 fish per 100 L tank and four replicates The temperature of 27°C treatments was adjusted by coolers (Teco SeaChill TR10) The temperture of 33°C treatments was elevated from 27°C to 33°C at a rate of 2°C per day by heaters Nitrite was not added untill all treatments reaching the desirable temperatures Nitrite was added as NaNO2 (99.9% Merck) The required volume of a M NaNO2 solution was added to obtain the desired NO2concentrations Nitrite in the water was analyzed twice a day and subsequently extra nitrite was added for maintaining the chosen concentrations The numbers of dead fish were recorded at 1, 3, 6, 9, 12, 24, 48, 72 and 96 h LC50-96 h was computed by Probit using SPSS 16.0 Experiment 2: Effects of nitrite at different temperatures on haematological parameters in basa catfish The experiment was conducted with two temperatures (27°C and 33°C) and four nitrite concentrations (0 mM; 0.09 mM; 0.22 mM; 0.44 mM), which represent for 0%, 10%, 25%, 50% of LC50-96 h Fourty fish were cultured in 500 L tanks (containing 300 L water) with triplicates for a period of 14 days Temperatures and nitrite concentrations were maintained by the same way as the Experiment The blood samples of three fish per tank were collected at day 0, 1, 2, 3, 4, and 14 after nitrite exposure Fish were anaesthetized by benzocain (0.1 g/L) before blood sampling Blood was collected from the caudal artery of fish by a heparinized syringe of mL Then, blood samples were contained in eppendorf 1.5 mL and kept in the ice during sampling time A part of blood was used immediately for measurements of blood gas parameters including blood pH (pHe), pCO2 using the i-STAT analyzer with cartridges CG3+ [HCO3-] in the blood was calculated by the method of Henderson and Hasselbach (Boutilier et al., 1985) [HCO3-] = αCO2 pCO2 10pHe-pK This part of blood was also used to measure other haematological parameters such as red blood cells, Hb, Hct, metHb The remainder of the blood was centrifuged (6 at 6,000 g), and the plasma was stored at -80°C for subsequent analysis of plasma ions concentration ([NO2-], [NO3-], [HCO3-], [Na+], [K+], [Cl-]), glucose concentration and osmolality Experiment 3: Effects of nitrite at different temperatures on growth performance and digestive enzyme activities of basa catfish The experiment was carried out in 60 days with the same temperatures and nitrite concentrations as Experiment Each treatment included replicates (3 tanks, 45 fish/tank) During experimentation, fish were fed by commercial feed (Cargill 30% protein) with a feeding rate of 3% body mass After 30 minutes feeding, uneaten feed was removed for counting the total number of pellets to obtain the actual weight of feed comsumed Dead fish were recorded daily and counted number of fish in each tank at ending time of the experiment for determining survival rate Nitrite concentrations were maintained by the same way as the Experiment and Fish were collected at day 0, 30 and 60 for measuring growth performance parameters such as DWG, SGR and WG Five fish per tank were collected at the day 60 for measurement of digestive enzymes activities as chymotrypsine, trypsine, amylase in intestine and pepsine in stomach, and blood samples were used to analyze Immunoglobulin (Ig) and lysozyme activity 2.2 Activity 2: Effects of nitrite and CO2 on physiological parameters and growth of basa catfish This content was conducted with Pangasius bocourti (10-20 g) Experiment 1: Effects of CO2 on haematological parameters in basa catfish The experiment consisted of four treatments including control, mmHg (1% CO2), 14 mmHg (2% CO2) and 21 mmHg (3% CO2), with four replicates Fish were randomly distributed to 200-L tanks (45 fish per tank) Fish were fed twice a day with commercial feed (Cargill 30% protein) Water pCO2 was controlled with an Oxyguard Pacific system (Denmark) coupled with a G10ps CO2 probe and a K01svpld pH probe (Oxyguard International A/S, Farum, Denmark), which connected to a CO2 cylinder via a regulator to supply CO2 into water Blood samples were taken at 1, 6, 24, 48, 72, 96 h and 168 h (7 days) after CO2 exposure for the measurement of blood gas parameters including pHe, pCO2, [HCO3-] Haematological parameters such as red blood cells, Hb, Hct, metHb, glucose concentration, plasma osmolality and [Na+], [K+], [Cl-] were measured Experiment 2: Effects of CO2 on growth performance and survival rate of basa catfish The experiment included four treatments: control, mmHg (1% CO2), 14 mmHg (2% CO2) and 21 mmHg (3% CO2), with three replicates Fish were randomly exposed to 200-L tanks with the density of 30 individuals each tank The CO2 concentrations were adjusted similar to Experiment – Content The fish were fed twice a day by commercial feed (Cargill 30% protein and 3-5% lipid) with a diet of 3% total weight of fish Feed was weighted before feeding After 30 minutes feeding, uneaten feed was removed for counting the total number of pellets Thirty percent of the tank water was changed every week Growth samples were collected at day 0, 30, and 60 for measuring growth parameters: DWG, SGR and WG Dead fish were removed and recorded every day for determining FCR After experimentation, the remaining fish were counted to determine survival rate of fish Experiment 3: The combined effects of nitrite and CO2 on physiological parameters of basa catfish The experiment consisted of six treatments including control, 0.09 mM NO2-, 14 mmHg CO2, 0.09 mM NO2-+14 mmHg CO2, 0.22 mM NO2- and 0.22 mM NO2-+14 mmHg CO2 The CO2 concentrations were adjusted similarly as Experiment and Experiment of Activity Nitrite addition was similar with the experiments of Activity Blood samples were collected at 0, 1, 6, 24, 48, 72, 96 h and 168 h (3 fish per tank) for analysis of blood gas parameters (pCO2, pHe, [HCO3-]); haematological parameters (red blood cells, Hb, Hct, metHb); glucose concentration and plasma ions [NO2-], [NO3-] and [Cl-] Gill samples were collected at 1, 6, 24, 72, 96 h and days to check gill remodelling Gill samples were carefully immersed in PBS 0.1 M pH 7.4 After histological processing, the chosen tissues were embedded in methyl methacrylate Technovit®7100 solutions (Heraeus Kulzer, Germany), and then stained with Eosin and Haematoxylin Histological images were looked and captured under microscopes (20x) The characteristics of histological structure of healthy fish were based on the description of Wilson and Laurent (2002) and Evans et al (2005); of fish exposed to CO2 were based on the description of Good et al (2010) and Fivelstad et al (2003); and of fish exposed to nitrite were according to Gisbert et al (2004) 2.3 Activity 3: Effect of nitrite on metHb reductase enzyme activity and oxygen consumption of basa catfish Experiment 1: Effects of nitrite on metHb reductase enzyme activity of basa catfish at different temperatures Basa catfish (10-20 g) were randomly assigned into treatments including two nitrite concentrations (0 mM and 0.44 mM) at two temperature levels (27°C and 33°C) Each treatment was done in triplicate The density of fish was 40 fish/tank (200 L) The temperature and the nitrite exposure to tanks were carried out in similar ways as experiments in Activity After exposed to nitrite, blood (4 mL) was sampled from 8-10 fish/tank at day 0, and Blood samples were mixed together for the analysis of metHb reductase enzyme activity (Jensen and Rohde, 2010) Blood collected from fish exposed to nitrite were washed and diluted with Ringer solution Then, this suspension was equilibrated to 1% CO2 and 99% air and the metHb decay was followed, where after k value in exposed fish was calculated Experiment 2: Effect of nitrite on oxygen consumption (MO2) and oxygen threshold of basa catfish stored in plastic bottle (100 mL) under cool condition were transferred to the laboratory for analysis 2.5 Analytical methodology Physiological parameters analysis Red blood cells were stained with Natt-Herick method and counted in Neubauer chamber Hematocrit was centrifuged at 12,000 rpm in minutes Hemoglobin was determined by Drabkin reagent at the absorbance of 540 nm MetHb was determined with phosphate buffer 0.02 M pH 7.3 (Lefevre et al., 2011) Glucose concentration was analyzed according to Hugget and Nixon method (1957) Plasma concentration of Na+ and K+ was measured using a Flame photometer (Sherwood Model 420, Sherwood Scientific Ltd., Cambridge, UK) Plasma Cl- concentration was measured using a chloride titrator (Sherwood model 926S MK II Chloride analyzer) Plasma NO2- and NO3- was measured spectophotometrically using the Griess reaction (Miranda et al., 2001) Immunological parameters analysis The amount of Ig was analyzed by following the study of Siwicki and Anderson (1993) with some modifications of Milla et al (2010) The reference of Ellis (1990) modified by Milla et al (2010) was used to analyze lysozyme Digestive enzyme analysis Pepsine and chymotrypsine were analyzed using method described by Worthington (1982), whereas amylase was determined by the method described by Bernfeld (1951) 2.6 Statistics Data were calculated in average values, standard deviation (SD) and standard error (SE) by Microsoft excel 2007 Normally distributed data were tested with a one-way and two-way ANOVA A DUNCAN analysis, paired T-test and Independent T-test were used in the determination of oxygen consumption of basa catfish The analysis of variance (ANOVA) was performed using SPSS16.0 procedures (significance was defined at p < 0.05) Chapter RESULTS AND DISCUSSIONS 3.1 Effects of nitrite and temperature on physiological parameters and growth performance of basa catfish 3.1.1 LC50-96 hours of nitrite in basa catfish at different temperature The LC50-96 h of nitrite in basa catfish (10-20 g) at 27oC and 33oC were 0.88 mM (Figure A) and 0.60 mM (Figure B), respectively It indicated that the ability of basa catfish with nitrite reduced according to the increase of temperature Those values are lower than that of other fish which are air-water breath fishes in the Mekong Delta such as snakehead (Channa striata) with 4.7 mM (Lefevre et al., 2012); striped catfish (Pangasius hypophthalmus) with 1.65 mM (Lefevre et al., 2011); knife fish with 7.82 mM (Gam et al., 2017) B A Raw data Redicted mean Lower 95% Uppe 95% 1.0 1.0 Proportion mortality Proportion mortality 1.2 0.8 0.6 0.4 0.2 0.8 1.0 1.2 0.6 0.4 0.2 0.0 0.2 0.0 0.6 0.8 1.4 Raw data predicted mean Lower 95 % Upper 95 % 0.4 0.6 0.8 1.0 1.2 [NO2- ] (mM) [NO2- ] (mM) Figure 3.1: LC50-96 h of basa catfish at 27oC (A) and 33oC (B) 3.1.2 Effect of nitrite and temperature on physiological parameters of basa catfish At lower acute dose of exposure, after 24 hours, the number of red blood cell, Hb and Hct of fish reduced in comparison with control treatment; and the significant difference among nitrite exposure treatments and the control treatment was found in the treatments of 0.22 mM and 0.44 mM After days of exposure, these parameters were increased and showed no difference from control treatment (p>0.05) The change of proportion of plasma metHb, [NO2-] and [NO3-] resulted from the interaction effect of nitrite and 10 temperature in this study These parameters highly increased after 48 h of exposure then reduced, but they remained significantly higher than control (p
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