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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY ABSTRACT OF DOCTORAL DISSERTATION AQUACULTURE AND FISHERIES CODE: 62 62. 03. 01 TIEN HAI LY STUDY OF BIOLOGICAL CHARACTERISTICS AND SEED PRODUCTION TECHNIQUES OF CHANNA LUCIUS CUVIER 1831 SCIENTIFIC SUPERVISORS: 1.Dr. BUI MINH TAM 2.Assoc. Prof. Dr. TRAN THI THANH HIEN Can Tho, 2016 The work was completed at the College of Aquaculture and Fisheries, Can Tho University Scientific supervisors: Supervisor 1: Dr. Bui Minh Tam Supervisor 2: Assoc.Prof. Dr. Tran Thi Thanh Hien The dissertation was defended before the committee marking the university-level dissertation Meeting at………………………………………………………………… Time……………………………Date…………………………………….. Reader 1: …………………………………………………………… Reader 2:…………………………………………………………… Reader 3:…………………………………………………………… The dissertation available at: 1. The LRC, CTU 2. The National Library 1 GENERAL DESCRIPTION OF THE DISSERTATION 1.1. Introduction Channa lucius Cuvier 1831 which belongs to snakehead is found in freshwaters like rivers, lakes, canals, rice fields and forests of nature conservation in the Mekong River Delta (MRD). Channa lucius meat that is tasty and appetizing people has respiratory organs with natural air, so it is easy to feed and the fish can live well in water from 5.5 to 6.0 low pH (Rainboth, 1996; Lee and Ng, 1994). Many people feeding fish in the MRD think that the fish has very promising development of commercial products, especially cultured in ponds, pens and cages. Currently, the main sources of Channa lucius fries are exploited naturally in small quantities and with insustainable quality. Meanwhile, the studies of this species is not much and the information is only limited in the initial basic research on biological characteristics and natural reproductive techniques. Therefore, the study of growing Channa lucius into a new cultured species which will have a great effect in the diversification of cultured species, thus contributing to reducing risks in farming and food supply needs for society, is a matter of urgency. As a result, the topic "Study of biological characteristics and seed production techniques of channa lucius Cuvier 1831" is performed. 1.2. Goal It is to provide scientific arguments about the biological characteristics, and reproductive and rearing techniques of Channa lucius. The success of the project will contribute greatly to the development process of Channa lucius hatchery to supply artificial fish for aquaculture systems and renewable fish resources in the wild in the Mekong Delta as well as in the whole country. 1.3 Research content - Research on the biological characteristics of Channa lucius. - Research on feeding Channa lucius to grow out in the pond. - Research on stimulating Channa lucius to reproduce artificially. - Research on the development characteristics of the digestive tract and the optional index of feed of fry. - Research on timing to substitute fresh food by processing food (PF) in the fry stage. - Assessing the possibility of using efficient industrial pellets in the phase rearing fish fry to breeding. 1.4 The scientific and practical significance of the dissertation The dissertation is a systematic and comprehensive research on Channa lucius. The results of the dissertation is the important scientific database and good reference source for teaching and further studies. The results of this study will contribute to enriching the basic research on biological characteristics, traits of developed gastrointestinal tract and the food choices of Channa lucius in Vietnam. The results of the growout techniques for brood, stimulating fish reproduction and techniques rearing Channa lucius from fry stage to fingerling are the original science-based support for the practical local apps in highly effective productivity. 1.5 The breakthrough of the dissertation The thesis is the first comprehensive study on the biological characteristics of Channa lucius as follows: identifying ecological features, growth characteristics, nutrition and reproductive biology. Channa lucius are mature in the pond by growout with cheap fish (75.0%) and industrial pellets (72.7%). It is able to identify the measures to stimulate effective reproductive Channa lucius from a combination of physiological and ecological factors. At 2,000 UI HCG. kg-1 male and 500UI HCG 2 mg combination with cerebral lobes. kg-1 females at pH 5.5 to 6.0, 28-29oC 2 temperature, dissolved oxygen 5-6 mg/l and the substrate for fish nesting slender vegetables, after injecting females 37-40 hours, the percentage of spawning with farrowing rate is 83.3%, the recovery rate is 95.3% and hatchability is 82.6% The development of the digestive tubeof Channa lucius is complete when the fish is 20 days old; the thesis has identified food choice index from fry stage to 30 days old; it is determined that the time of the 16th day is suitable for the replacement of live food by processing food (20% degree of substitution of FP/day) to ensure high fish survival rate and good growth. In particular, the thesis has been successful in using industrial pellets to run Channa lucius from fry to breed. These results contribute to solving the problems of fresh food in the nursery, help reduce costs, improve production efficiency and expand scale of commercial fish farming in the Mekong Delta. The success of the thesis is a huge motivation and a basis to accelerate breeding career of initiative development of Channa lucius, fish supply seeds for regeneration of Channa lucius natural resources and aquaculture in order to create a lot of fresh fish products to meet the needs of society. * The layout of the thesis The thesis consists of 139 pages (excluding appendices) which include the following sections: Chapter 1: Introduction of 4 pages; Chapter 2: Overview of 26 pages; Chapter 3: Materials and Research Methods of 25 pages; Chapter 4: Results and discussions of 68 pages; Chapter 5: Conclusions and recommendations of 2 pages; (5) References of 14 pages; The thesis has 36 tables and 51 figures. RESEARCH METHODS 3.1 Time, place and subject of research The study was conducted in 2010-2014. The study of biological characteristics was done with 968 samples in U Minh District-Ca Mau Province and Long My District-Hau Giang Province. The studies were conducted in freshwater fish farms, the CAF Laboratory, the College of Agriculture and Applied Biology, Can Tho University. The research is on Channa lucius (Channa lucius Cuvier, 1831). 3.2 Research Methods 3.2.1 Research chart: The research on biological characteristics was conducted first, then the next research contents were conducted as subsequent growout, spawning and fish hatchery. 3.2.2 Research Methods of biological characteristics 3.2.2.1 Methods of analysing morphological characteristics Body shape, head shape, location and size of the mouth of Channa lucius specimens were studied by the method of Pravdin (1973); Rainboth (1996). The quotas counted as scales and fin rays followed Holden and Raitt (1974). The morphological indicators were measured according to Lowe-McConnell (1971), Grant and Spain (1977) (quoting Pham Thanh Liem and Tran Dac Dinh, 2004). The research on digestive systems of fish focused on organs like the mouth, teeth, comb gills, esophagus, stomach, intestine, cecum (Lagler et al., 1977) and Bond (1996). 3.2.2.2 Methods of analysing the growth characteristics The correlation equation between length and weight is determined by the formula of the King (2007) W = aLb Where: W: whole body volume (g); L: fish body length (cm); a: constant of conditions; b: growth Coefficient. 3.2.2.3 Methods of analysing nutritional attributes 3 a. Relative Lengh of the Gut RLG (RLG - Relative Lengh of the Gut) The Relative Lengh of the Gut between intestinal length and body length is calculated according to the formula of Al-Hussainy (1949). RLG = Li/ L Where: Li: length of the fish intestine; L: length of the fish body. b. Determination of Channa lucius nutrition spectre. Nutrition spectre of mature Channa lucius is determined by the volume method of Biswas (1993). Determination of the dry weight and of each food sample by analytical method AOAC (2000) 3.2.2.4 Methods of analysing reproductive biological characteristics * Determining the condition factor (Condition factor-CF) Determination of condition factor by sex and formula-based King (2007). CF = W/Lb Where: W: Weight of fish body (g); L: fish body length (cm). b: the growth factor is determined from the equation W = aLb (a is condition factor). * Gonado Somatic Index (GSI) Gonado Somatic Index is defined by gender and by the formula of Biswas (1993). GSI (%) = 100 * Wg/ W Where: Wg: gonads weight (g);W: Body Weight (g). Determining the stage of gonad development is based on hierarchy of sexual mature of fish by Nikolsky (1963). Templates of histological staining gonads is done with haematoxylin and eosin according to standard histological methods of Drury and Wallinton (1967) and Kiernan (1990) * Fecundity Fecundity is determined on the amount of female fish eggs with the gonads in stage IV and oocyte number of Banegal formula (1967). - Absolute fecundity (Absolute fecundity-Fa ) Fa (egg/ female) = (n * Wg )/ Wm Where: Wg: ovary weight (g);Wm : Sample egg weight retrieved to count (g); n: Sample egg number retrieved to count. - Relative fecundity (Relative fecundity-Fr ) Fr (egg/kg of female) = Fa /W Where: Fr: Relative fecundity; Fa: Absolute fecundity;W: Weight of fish body (g). * The average length of first maturity The average length of first maturity (Lm) calculated by the formula King (2007). P=1/(1+e -r*(Ltb-Lm)) Where: P: Percentage of maturity (mature fish when gonad reached stage III according to maturity ladder Nikolsky (1963); r: correlation coefficient; Ltb: The medium length of fish body; Lm: The average length of first maturity. 3.2.3 Hatchery Method 3.2.3.1 Survey on aquatic environment where Channa lucius reproduce naturally The work conducted a survey on environmental factors on Long My II channel (KC.II), 2 channels of grade III (KC.III) and three interior canals (K.ND) in regions of 2 communes Luong Tam and Luong Nghia, Long My district, Hau Giang province. The environmental survey period 4 lasted from January to June, 2011, monthly water was measured twice: in phase 1 it was measured on days 14, 15, 16 (al) and in phase 2 it was measured on days 24, 25, 26 (al), each channel surveyed 2 points (the beginning and the end of the sources) and at each measurement point 3 indicators are pH, temperature, dissolved oxygen. Determination of pH and temperature indicators was measured by the ECO pH (HI 9813-5) and Oxygen indicators were measured by HANNA (HI 9142). 3.2.3.2 Research methods of broodstock growout *Testing Systems Broodstock Channa lucius growout experiments were conducted for 4 months (12/2011 to 3/2012). The experiment was arranged in a grid system (2x4x1,5 m) with a mesh size of 0.5 cm and put in the earth pond with an area of 500 m2, 1.2-1.5 m deep, the water in the pond is exchanged regularly according to tide and a minimum water level of 1 m was maintained * Experiment Fish: Healthy fish with uniform size of about 90-115 g / head. * Dietary treatments: Trash feed was ground and mixed with 1% wool to increase adhesion and industrial pellets are moistened with water before feeding. Feed was put on the floor and placed in the grids in feeding period. * Arranging experiment The experiment was completely random, including two treatments (trea). Each treatment was repeated 3 times, and each iteration has 30 pairs of Breeders: Treatment 1 (trea 1) used 100 % trash fish feed; Treatment 2 (trea 2) used 100% industrial feeding pellets. * Feeding Positive growout period was in 2 months and feeding in all treatments was as follows: 6%/day (pellets); 10%/day (trash); growout period maturing in 2 months left feeding 3%/day (pellets), 5%/day (trash fish meat). * Analysing indicators Before the experiment layout, we randomly collected 20 fish breeders in the fish's anatomical experiments to determine the initial biological indicators. During growout periodically on the last day of each month we collected 20 broodstock (randomly by gender) in each treatment to analyze some indicators of reproductive biology (maturation rate, GSI, CF, fecundity) and measured environmental factors (pH, temperature, dissolved oxygen). 3.2.3.3 Methods of spawning a) The experiment probe We chose healthy broodstock, with ripe gonads and 90-450g weight. The fish were injected with reproductive stimulants right of chest fin position. Then, the fish were put in the grids (0,5m3) in an area of 2m3 cement tanks, each tank had 2 grids. Experiment 1: Stimulating Channa lucius to reproduce with HCG Table 3.3: Dosage of HCG injections for broodstock. Treatment HCG injection on females (UI)/kg HCG injection on males (UI )/kg 1 2 3 Females (number) Dosage Males (number) Dosage 3 3 3 500 1,000 1,500 3 3 3 1,000 2,000 3,000 5 The experiment consists of 3 treatments with completely random layout, and each treatment has 3 pairs of broodstock. Females and males are injected at the same time and with the same HCG dosage presented in Table 3.3. Experiment 2: Stimulating Channa lucius to reproduce by LH-RHa + DOM The experiment consists of 3 treatments with completely random layout, and each treatment has 3 pairs of broodstock. In the laboratory experiments, the male fish are injected with LH-RHa + DOM before the females, in the control treatment the males and and females are injected with only physiological saline water(0.9%). The injectable time and dosage with LH-RHa + DOM is presented in Table 3.4. Table 3.4: The injectable time and dosage with LH-RHa+DOM for broodstock. Treatment Injection with LH-Rha on the males Injection with LH-Rha+DOM on the (µg)/ kg females (µg+mg)/ kg number Start 24h 48h Number (unit) Start 24h 48h (unit) Controlled 3 0 0 0 3 0 0 0 1 3 0 80 120 3 0 0 100+4 2 3 80 120 150 3 0 0 100+4 Experiment 3: Stimulating channa lucius to reproduce with HCG + pituirary extracts (PE) The experiment consists of 4 treatments with completely random layout, and each treatment has 3 pairs of broodstock, the males are injected before females are; the controlled group has injections as similar as in experiment 2. The time and dose of injections of HCG + PE are shown in Table 3.5. Table 3.5: The injectable time and dosage with HCG + PE for broodstock Treatment Injection with HCG on the males Injection with HCG + PE on the (UI/ kg cá) females (UI+mg)/kg number Start 24h 48h number start 24h 48h (unit) (unit) Controlled 3 0 0 0 3 0 0 0 1 3 0 2,000 0 3 0 0 500+1 2 3 1,000 2,000 0 3 0 0 500+1 3 3 1,000 1,000 2,000 3 0 0 500+1 Experiment 4: Stimulating Channa lucius to reproduce with HCG + PE and reducing water pH (5.5 to 6.0) Table 3.6: The injectable time and dosage of HCG and PE on the broodstock in combination with reducing water pH (5.5 to 6.0). Treatment Injection with HCG on the males Injection with HCG + PE on the (UI/ kg fish) females (UI+mg)/kg number start 24h 48h number start 24h 48h (unit) (unit) Controlled 3 0 0 0 3 0 0 0 1 3 0 2,000 0 3 0 0 500+2 2 3 1,000 2,000 0 3 0 0 500+2 3 3 1,000 1,000 2,000 3 0 0 500+2 The experiment consists of 4 treatments with completely random layout, and each treatment has 3 pairs of broodstock. HCG Injections on the males and HCG + PE on the females. The males 6 are injected before the females are. In the controlled treatment both the males and females only receives 0.9% physiological saline water. The time, dosage and interval between 2 injections of hormone are shown in Table 3.6. b. Main experiment: stimulating Channa lucius to reproduce This experiment was designed based on the results of the exploratory study on stimulating fish spawning in the experiments 1, 2, 3, 4 and selecting the best exploration results such as selecting concentrations of HCG + PE and methods of injection in treatments 1 and 2 of experiments 4 to arrange this experiment. The experiment was conducted with completely random layout consisting of 3 treatments, and each treatment had 12 pairs of brood stock and each pair was located in the same grids set in a cement tank of 2 m3. The time, dosage and the gap between the male and female injections are presented in Table 3.7 Table 3.7: The injectable time and dosage with HCG and PE lobe and the injection for broodstock are combined with reducing water pH (5.5 to 6.0) in the main experiments. Treatment number Rate HCG on the males HCG + PE on the females (unit) male/female (UI/kg) (UI+mg)/kg start 24h materia start 48h materi l al ĐC 24 1/1 0 0 0 0 0 0 1 24 1/1 0 2,000 2,000 0 500+2 500+2 2 24 1/1 1,000 2,000 3,000 0 500+2 500+2 Note: In the controlled treatment both the male and female are injected with only physiological saline water 0,9%. The water used in the experiment was over filtered and adjusted pH dropping from 7.0-8.0 to 5.5-6.0 with phosphoric acid (H3PO4), 28-29 oC temperature, dissolved oxygen 5-6 mg/l and the substrate for fish nesting with slender vegetables. c. The criteria for assessing reproductive outcomes Effective time (hours), spawning rate (%), fertility rate (%), hatching rate (%) 3.2.4 Methods of development characteristics of gastrointestinal tract and selecting indicators of fish feed for fry Channa lucius * Testing Systems: The fish, after eating the yolk, was transferred to spawning in small ponds with the size 2x3x0,5m, the pond bottom with 25 cm thick layer of mud, the stocking density of 2/liter; the water in the pond is aired slightly to ensure dissolved oxygen for fish to grow. * Sources of food: Before stocking fish in the pond, the water is breeding natural food by mixing concentrated feed (42.2% protein) with water at a dose of 10 g/m3 and successive fertilizers for 2 days. During the nursery, we hang fabric bags containing fish fry 5 g/m3 to maintain the natural food in the duration of the experiment. * Collecting and analyzing morphological samples of digestive tract - Collecting samples: Fish samples are collected in the old days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 and 10 specimens are collected each day to observe life and photograph the development stage of gastrointestinal tract, intestinal length measurement, body length, yolk size and the open mouth of the fish. - Method of analysis: Individuals from the age of 1-15 days are observed shapes of the digestive tube on a microscope with eyepiece micrometer to capture and measure the length. When the fish are 16-30 days old, the digestive tube indicators are measured by the naked eye on a ruler with an accuracy of 1 mm. * Method of determining the width of the fry mouth 7 Shirota (1970) describe fish mouth widths by the following formula MH (90o) = AB x 2 Where: - AB is the length of the upper jaw; MH is the width of the mouth (mm) * Method of determining RLG RLG is the ratio between the length of the intestines on the body length which is calculated according to the formula of Al - Hussainy (1949). * Method of histological analysis of gastrointestinal tract Fish samples are collected in the old days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 and 30 specimens are collected each day to observe life. The tissue template of the fish digestive tract is coloured with hematoxylin-eosin (H & E) by histological method described by Drury & Wallington (1967) and Kiernan (1990). We conduct the specimen observation of the gastrointestinal tract tissue on a microscope (4X, 10X, 40X) to photograph to identify the structural variation of the digestive organs such as oral cavity, esophagus, stomach, intestines. * Determination of food choice index (E) - Sampling: Samples of plants, aquatic animals and fish are collected on days 2, 3, 4, 5, 6, 8, 10, 15, 20, 25, 30 after the layout. Every day 20 units / samples are received preserved in commercial formalin solution 10%. - Analysis of water samples: The qualitative analysis of phytoplankton species and animals is under the document classification of Shirota (1966), Dang Ngoc Thanh et al (1980), Boltovskoy (1999). The quantitative analysis follows method of Boyd and Tucker (1992). - Analysis of food in the digestive tract of fish: the nutrition spectro of Channa lucius fry is determined by the number method of Biswas (1993). - The electivity fish food index (E): Ivlev (1961) expressed food electivity index E (electivity Index) by formula ri – pi E = (ri + pi) Where: ri: is the percentage of food found in the gut out of foods in the intestines of fish; pi: means the percentage corresponding foods found in the environment in general foods in the environment. 3.2.5 Method of channa lucius neursing 3.2.5.1 Determining the time of processed food (PF) of channa lucius in stage 4-30 days old * The laboratory system: an experimental system consists of 12 plastic tanks with a volume of 60 liters per tank. The experiment was completely random layout with 4 treatments (Trea) converting various PF including: (Trea 1) converter of PF from the 16th day after the run; (Trea 2) converter of PF from the 13th day after the run; (Trea 3) converter of PF from the 10th day after the run and (Trea 4) converter of PF from day 7 after the run. Each treatment was repeated 3 times. * Experiment Fish: Fish used to arrange experiments are from artificial breeding source and we selected 4-day-old larvae which had spent yolk and fish with an average initial length of 0.87±0.01 cm (0.002g/unit). We randomly assigned 100 fish in each plastic tank containing 50 liters (density of 2 fish/liter) and run for 30 days. * Experiment Food: Live foods used in the experiment is moina, earthworms; Processed 8 food (PF) were processed between concentrated food (42.2% protein, 3.20% fat, 5.40% ash, 24.8% NFE) mixed with minced trash fish meat (81,65% protein, 2.68% fat, 5.47% ash) with the blending ratio of 1/1. - During the experimental feeding on demand, every day feeding 2 times at 7 and 16 o’clock in the foods moina, earthworms and PF. The food intake from day 1-3 was added with moina at 24 larvae/ml/day; 4-15 day with cut-chunk worms around 2-4g/tank/day; PF amount from 7-15 days is around 2-4g/tank/day and PF amount from days 16-30 with 4-6 g/tank/day. - Until the replacing day, treatments have not replaced PF and the fish are fed with worms. By the time of days 7, 10, 13, 16 after the run, the worms are replaced gradually by PF presented in Table 3.9 Table 3.9: Methods of PF gradually replacing in all treatments Day Methods of replacing PF 1 80% worms + 20% PF 2 60% worms + 40% PF 3 40% worms + 60% PF 4 20% worms + 80% PF 5 0% worms +100% PF 3.2.5.2 30-60 day old Channa lucius Nursery with industrial pellets with different stocking density on the tank * Testing Systems: Experiments run from fry to fingerling on plastic tank of 60 liters (50 liters), run time is 30 days. The experiment had 4 treatments run in the stocking density of 1 head/liter; 1.5 units/liter, 2 fish/liter and 2.5 fish/liter and were arranged completely randomly and each treatment was repeated 3 times. * Experiment Fish: Fish derived from semi-artificial reproduction and used concentrate feed well. Originally, fish size is 2.40 to 2.46 cm/head (wieght from 0.135 to 0.153 g/head) and the difference in the length and weight of fish initially in the treatments are not significant (P> 0.05). * Treatment Dietary: Concentrate dietary with 42.2% protein, 3.20% fat, 5.40% ash, 24.8% NFE. The amount of feed used in the experiments is 7-10% body weight and feeding 2 times a day at 7 o’clock and 16 o’clock. 3.2.5.3 The track indicators - Environmental indicators: temperature, oxygen and pH are measured 2 times a day at 7 o’clock and 14 o’clock. Temperature (°C) and pH are measured by the ECO pH; dissolved oxygen is measured by HANNA 98172. Nitrite index is determined by Griess llosvay method, colorimetric spectrophotometer is at 540 nm wavelength and it is measured 1 time/1 week at 7 pm. - Ending the experiment, we caught 30 fish in the tank randomly to determine volume, measure the length, calculate the growth rate according to DWG day (g/day), specific growth rate - SGR (%/day), coefficient of variation in the volume of fish (CV) and compute survival rate (Survival Rate-SR), 3.3 Data processing Data was analyzed with average value (Mean), standard deviation (Standard deviation) using Excel version 6.0 program. We analyse Factor ANOVA compare compare the difference between the means of the treatments by DUNCAN test in SPSS 16.0 software. 9