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: The LRC, CTU The National Library 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 mg combination with cerebral lobes kg-1 females at pH 5.5 to 6.0, 28-29oC 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 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 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 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), channels of grade III (KC.III) and three interior canals (K.ND) in regions of communes Luong Tam and Luong Nghia, Long My district, Hau Giang province The environmental survey period lasted from January to June, 2011, monthly water was measured twice: in phase it was measured on days 14, 15, 16 (al) and in phase it was measured on days 24, 25, 26 (al), each channel surveyed points (the beginning and the end of the sources) and at each measurement point 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 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 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 times, and each iteration has 30 pairs of Breeders: Treatment (trea 1) used 100 % trash fish feed; Treatment (trea 2) used 100% industrial feeding pellets * Feeding Positive growout period was in months and feeding in all treatments was as follows: 6%/day (pellets); 10%/day (trash); growout period maturing in 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 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 Females (number) Dosage Males (number) Dosage 3 500 1,000 1,500 3 1,000 2,000 3,000 The experiment consists of treatments with completely random layout, and each treatment has 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 treatments with completely random layout, and each treatment has 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 0 0 80 120 0 100+4 80 120 150 0 100+4 Experiment 3: Stimulating channa lucius to reproduce with HCG + pituirary extracts (PE) The experiment consists of treatments with completely random layout, and each treatment has pairs of broodstock, the males are injected before females are; the controlled group has injections as similar as in experiment 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 0 0 2,000 0 500+1 1,000 2,000 0 500+1 3 1,000 1,000 2,000 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 0 0 2,000 0 500+2 1,000 2,000 0 500+2 3 1,000 1,000 2,000 0 500+2 The experiment consists of treatments with completely random layout, and each treatment has pairs of broodstock HCG Injections on the males and HCG + PE on the females The males 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 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, and selecting the best exploration results such as selecting concentrations of HCG + PE and methods of injection in treatments and of experiments to arrange this experiment The experiment was conducted with completely random layout consisting of 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 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 24 1/1 2,000 2,000 500+2 500+2 24 1/1 1,000 2,000 3,000 500+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 days During the nursery, we hang fabric bags containing fish fry 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 mm * Method of determining the width of the fry mouth Shirota (1970) describe fish mouth widths by the following formula MH (90o) = AB x 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 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 after the run Each treatment was repeated 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 fish/liter) and run for 30 days * Experiment Food: Live foods used in the experiment is moina, earthworms; Processed 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 times at 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 80% worms + 20% PF 60% worms + 40% PF 40% worms + 60% PF 20% worms + 80% PF 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 treatments run in the stocking density of head/liter; 1.5 units/liter, fish/liter and 2.5 fish/liter and were arranged completely randomly and each treatment was repeated 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 times a day at o’clock and 16 o’clock 3.2.5.3 The track indicators - Environmental indicators: temperature, oxygen and pH are measured times a day at 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 time/1 week at 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 coefficient of 0.25±0.01% The features of tissue cells at this stage largely contain the oocyte cytoplasm of growth period and a number of cells in the nutritional growth period The cells are rounder than the ones in the synthetic core period and they are arrange closely together (Figure 4.11b) a b Figure 4.11: a) ovaries S II, b) Ovum oocytes S II (40X) - Stage III a b Figure 4.12: a) Ovaries S III; b) Ovum oocytes S III (10X) The Ovaries clearly increase in size, with straw yellow on the surface of the ovaries there are many tiny blood vessels distributed evenly across the surface Naked eye can see particles scattered eggs in the ovary (Figure 4.12a) Individuals in this stage have sexual maturity coefficient of 0.7±0.32% and 1.06 mm diameter eggs (0.98 to 1.11 mm) The features of ovarian cytology at this stage contain most of the cells in biomass growth period (Figure 4.12b) - Stage IV a b Figure 4.13: a) Ovaries S IV; b) Ovum oocytes S IV (4X) The ovaries constantly increase size and many large blood vessels are distributed evenly across the ovaries, the big egg particles, even and easily separate each egg beads (Figure 4.13a) Fish in 13 stage IV have coefficient sexual maturity reaching 1.27% and 2.88±1.18 mm diameter eggs (1.10 to 1.23 mm) Characteristics of ovarian cells at this time are mainly in the nutritional growth period and maturity Rounded oocytes, yolk particles stick around and catch dark pink eosin (Figure 4.13b) - Stage V: This is the reproductive stage, the fish egg cells reach the largest size, the particles now separate eggs and egg plates flowing into the environment outside the body when gently pressed into the belly of the fish This phase exists only in a very short time period - Stage VI: This is the completion of spawning stage when the fish ovaries shrivel, soft dough structure, ovarian membrane furrow, and eyes can see a few big and yellow oocytes that spawners residue (Figure 4.14a) In this period oocyte of nutrition growth is degraded and is reabsorbed, besides there are still some reserves in cell growth period cytoplasm (Figure 4.14b) a b Figure 4.14: a) Ovaries S VI, b) Ovum oocytes S VI (4X) 4.5.3 The development stages of male gonads - Stage I: The fish sperms are undeveloped At this stage gonads take the form of two small pieces, transparent white and underneath the organ In the chamber of stage I there are mainly gonialblasts - Stage II: Sperm chamber of this stage begins lobed Then the chamber is larger than the one of stage I with transparent white The fish have small gonads and the maturation coefficient reach 0.076±0.03% (Figure 4.15a) At this stage there is cell proliferation in the number of primary sperm cells The sperm cells form clumps and are surrounded by a membrane called the capsules (Figure 4.15b) b a Figure 4.15: a) Sperm chamber SI, b) Histological organization in sperm chamber SI (40X) - Stage III: Sperm chambers are opaque white with divided lobes On the surface of the chamber there are many tiny blood vessels Maturity coefficient of fish reach 0.198 ± 0.08% (Figure 4.16a) In 14 the gonad chamber of this stage the number of primary and secondary sperm cells is more than that at stagee II and also appear sperms catching green purple (Figure 4:16b) a b Figure 4.16: a) Sperm chamber S III, b) Histological organization in sperm chamber S III (40X) - Stage IV: Sperm chambers are slightly opaque white When the sperm chambers are cross-cut, there is liquid on the blade, but when we pat on the belly, the liquid will not run out At this stage the fish have matured coefficient reaching 0.533±0.17% (Figure 4.17a) Histological observation of chamber organization at stage IV shows that the chamber consists largely of sperms catching blue purple (Figure 4.17b) a b Figure 4.17: a) Sperm chamber S IV, b) Histological organization in sperm chamber S IV (40X) - Stage V : Sperm chambers become opaque white with glossy surface and more room than the one at stage IV Sperm chambers are reproductive status Sperm chambers develop to reach the maximum size and are in a state of reproductive ejaculation At this stage the fish have matured coefficient reaching 1.619 ± 0.80%, while we pat the belly of fish, liquid will run out - Stage VI: a b Figure 4.18: a) Sperm chamber S VI, b) Histological organization in sperm chamber S VI (40X) 15 After spawning, the mass and size of sperm chambers decrease significantly, the surface looks milky-colored, the surface is pale pink and sperm chambers become soft dough (Figure 4.18a) Inside the vas deferens are the empty capsules and a few leftover sperms (Figure 4.18b) 4.5.4 Maturity coefficient GSI (%) 4.00 GSI cáFemale 3.00 GSI cáfemale đực 2.00 1.00 0.00 10 11 12 -1.00 Time (month) -2.00 Figure 4.19: Variation of GSI coefficient of channa lucius Coefficient CF.10-2 Figure 4:19 shows GSI of male and female Channa lucius are constantly changing over time, the ratio of GSI female and male Channa lucius starts to increase from December last year and reaches the pinnacle of female and male GSI in June respectively 1.68% and 0.53% Lowest GSI coefficient for female Channa lucius was 0.69% in November but the male fish was 0.15% in September 4.5.5 Condition Factor CF 1.200 1.000 0.800 0.600 0.400 0.200 0.000 CF đực CFcá female CF CFcámale 10 11 12 Time (month) Figure 4.20: Condition factor (CF) of channa lucius Figure 4.20 shows survey results indicate the CF of female Channa lucius ranged 0.842.10-20.864.10-2 and male 0.848.10-2-0.874.10-2 The CF of female and male were highest in June, 0.864.10-2, 0.874.10-2 respectively 4.5.6 Seasonality and reproduction cycle Via survey results ovarian female ratio reached stage III (33.3%) and IV (58.3% ) in May Meanwhile, in September, the female ovary at stage IV (11.1%), at stage III only 11.1% and the female in stages I - II (70.2%) accounted for highest Based on the rate of maturation and GSI coefficient of female Channa lucius, we can identify spawning season thickness starting from and peaking in June of the year 16 4.5.7 The length of first maturity 1.0 0.9 0.8 0.7 P 0.6 0.5 0.4 P=1/(1 + e -0,30*(L-21,3958) ) 0.3 n = 391 0.2 R = 0,9565 0.1 0.0 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 L (cm) Figure 4.22: Corelation between maturity ratio and length of female channa lucius 1.0 0.9 0.8 0.7 P 0.6 0.5 0.4 P=1/(1+e -0,17*(L-21,3952) ) 0.3 n = 478 0.2 R = 0,9721 0.1 0.0 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 L (cm) Figure 4.23: Corelation between maturity ratio and length of male channa lucius Figure 4.22, 4.23 shows analytical results have determined that the first average length of maturity of female Channa lucius is 21.3958 cm thick (n = 391, R = 0.9565), and male fish is 21.3952 cm thick male (n = 478, R = 0.9721) 4.5.8 Fecundity A survey of wild fish (W from 94-295g) showed that average absolute fecundity is 2,065 ± 640 eggs/fish ranging from 905-3,519 eggs/fish and it is equivalent with the study on Channa lucius in Lake Singkarak Basin, east of Sumatera Indonesia It is 1,996-2,539 eggs/fish (Azrita and Syandri, 2013) Regression Correlation between absolute fecundity (F) and the fish weight (W) 17 according to the equation F = 102.47W0.5845 (R2 = 0.4631, n = 77) and this result is also consistent with the research on Channa lucius distributed in flooded areas Lindung Jambi-Indonesia (Azrita and Syandri, 2013) 4.5.9 Diameter of eggs Over 35 samples of eggs observed in stage III and 42 ovarian samples in stage IV showed that Channa lucius eggs are spherical, relatively round and the diameter of oocyte in stage III has average size of 1.06±0.03 (0.98 to 1.11 mm) and 1.18 ± 0.03 mm (1.10 to 1.23 mm) in stage IV It is smaller than the diameter of Channa lucius distributed in lake Singkarak Basin, east of Sumatera-Indonesia (Azrita and Syandri, 2013) 4.6 Growout of channa lucius 4.6.1 Environment in growout pond: pH (6.9-7.5), DO (4-6mg/l), temperature (28.1o 30.6 C 4.6.2 Rate of Channa lucius of sexual maturity in growout pond The result of sexual maturity of Channa lucius for four month’s growout is presented in Figure 4.25 75.0 72.7 Rate (%) 80 70 55.6 60 50.0 50 35.7 35,7 40 41.7 38.5 35.7 30.8 Trash fish 30 Industrial pellets 20 10 30/11/2011 31/12/2011 31/01/2012 28/02/2012 31/03/2012 Time (month) Figure 4.25: Rate of sex-matrue females in growout pond For treatments feeding industrial pellets reaching 72.7% and trash fish reaching 75.0% In each month’s growout the ratios of sexually mature females in treatment for industrial pellets are lower than the ones in treatment for trash fish This problem may be that industrial food is not the right food for the species, although the levels of protein in the feed is of 39.1%, 5.4% lipid, NFE 28.5% The study results showed that Channa lucius are totally capable of sexual maturity when growout is in ponds with trash fish or industrial pellets, which is similar to black snakehead fish, potted snakehead (Pham Van Khanh, 2003; Nguyen Huan and Duong Nhut Long, 2008) 4.6.3 Coefficient of sexually mature of female fish in growout ponds Coefficient of sexually mature in growout period of 120 days with trash fish feed and industrial pellets are shown in Table 4.7 Table 4.7: Coefficient of mature individuals over the months growout Treatment Coefficient of mature over the months growout (%) 30/11/2011 31/12/2011 31/01/2012 28/02/2012 31/03/2012 trea (trash fish) 1.34±0.42a 1.42±0.71b 1.64±1.47b 2.27±1.31a 3.61±1.10a trea (industrial pellets) 1.40±1.22a 2.40±2.01a 2.84±1.34a 3.01±1.37a 3.54±1.84a * Values shown are means and standard deviations The values in the same column with different letters are different with statistical significance (P 0.05) From Channa lucius growout results above it is able to confirm that Channa lucius can get normal sexual maturity in ponds with trash fish food or industrial pellet with protein of 39.1% 4.6.4 Indicators of female CF growout ponds Findings also noted CF index of Channa lucius presented in Table 4.8 Table 4.8: Evolution of the CF index of Channa lucius in growout ponds Time CF (with growth factor b=3.0376) Trash fish feed Industrial pellets a 30/11/2011 0.0090±0.0011 0.0090±0.0008a 31/12/2011 0.0091±0.0010a 0.0090±0.0011a 31/01/2012 0.0091±0.0009a 0.0091±0.0011a 29/02/2012 0.0092±0.0010a 0.0093±0.0017a 31/03/2012 0.0093±0.0013a 0.0095±0.0008a *Values in the table represent the mean and standard deviation The values in the same row with different letters have differences with statistical significance at (p 0.05) 4.6.5 Fecundity of Channa lucius in growout ponds Fecundity of Channa lucius in growout ponds with trash fish feed and industrial pellets are shown in Table 4.9 Table 4.9: Fecundity of Channa lucius in growout ponds Treatment Fecundity Absolute fecundity (egg/female) Relative fecundity (egg/kg female) a trea (trash fish) 5,764±1,580 41,951±7,820a trea (industrial pellets) 4,296±737a 42,106±7,201a * Values shown are means and standard deviations The values in the same column with different letters have differences with statistical significance at (p 0.05) Fecundity of Channa lucius in Table 4.9 higher than the fecundity of Channa lucius (13,105 eggs/kg fish) in the wild (Section 4.5.8) 4.7 Stimulating Channa lucius to reproduce 4.7.1 Experimental exploration in Channa lucius reproduction Experiment 1: Effect of HCG on the reproductive indicators of Channa lucius After the injection 31:30 minutes, the female fish begin to spawn in treatment 1, but in the other treatments the fish not spawn The offspring is presented in Table 4.10 19 For HCG injection treatments on male with dose of 1,000 IU/kg and 500 IU/kg spawning female with 66.7% rate, fecundity of 1,127 eggs kg-1 Eggs into the environment not concentrate and not fertilize Table 4:10: The fecundity indicators of Channa lucius with single dose of HCG Criteria Controlled trea trea trea HCG on male (UI/kg) 1.000 2,000 3,000 HCG on female (UI/kg) 500 1,000 2,000 Effective time (hour) 31:30±2:7 0 Rate of spawning fish (%) 66.7±57.7 0 Reality fecundity (egg/kg) 1,127±76 0 Rate of fertilizer (%) 0 0 Notes:ET: Effective time; F: Fecundity: trea: Treatment Experiment 2: The influence of LH-RHa + DOM on criteria of spawning Results of using LH-RHa + DOM injections for both males and females The reproductive indicators are presented in Table 4:11 Table 4:11: The criteria of exploration fecundity of fertility stimulant LH-RHa + DOM Criteria Controlled trea trea LH-RHa on male (µg/kg) 200 350 100+4 100+4 LH-RHa+DOM on female (µg+mg)/kg Effective time (hour) 40 Rate of spawning (%) 33.3 Real fecundity (egg/kg) 1,417 Rate of fertilizer (%) 0 Notes: trea:treatment Table 4:11 shows that fish not spawn in the controlled treatment (controlled) no LH-RHa injection and injection treatments LH-RHa 350μg/kg on males and 100μg injection 4mg LH-RHa + DOM/1 kg on females But 200μg injection treatments LH-RHa/kg on males and 100μg injection 4mg LH-RHa + DOM/1 kg on female, the rate of spawners was 33.3% However, the eggs into the environment are still not fertilized Experiment 3: Effect of HCG and Pituitary extracts on Channa lucius reproductive indicators The reproductive indicators in experiment HCG + PE are presented in Table 4:12 Table 4:12: reproductive criteria in the exploration HCG + PE Criteria Controlled trea trea trea HCG on male (UI/kg) 1,000 2,000 3,000 HGC+ PE on female (UI+mg)/kg 500+1 500+1 500+1 Effective time (hour) 30:0±2:48 33:0±2:36 32:0±2:39 Rate of spawning (%) 66.7±57.7 100 100 Real fecundity (egg/kg) 1,563±212 1,780±218 1,685±104 Rate of fertilizer (%) 0 0 Notes: ET: Effective time; RF: real fecundity;C: Controlled, trea:Treatment, PE: Pituitary extracts In this experiment the females in the injectable HCG treatments are spawning The fish spawn in trea 1, trea 2, trea 3, respectively 66.7%, 100% and 100% However, the reproductive activity of females appears otherwise than in the wild such as: male and female are not paired, no make nest before spawning and fish eggs after laying out water are still separate, not 20 concentrated in clusters with yellowish color Actual fecundity of fish in all the treatments are also very low (1,563 to 1,780 eggs/kg female) and eggs laid out in the treatments were not fertilized Experiment 4: Effects of HCG + PE and reducing water pH over the channa lucius reproductive indicators Results of stimulating Channa lucius spawning in cement tanks by combining HCG hormones and PE and stimulating ecology by lowering pH to 5.5 to 6.0 aquatic environment are shown in Table 4:13 Through Table 4:13 it is shown that the mother fish after injection of 33-40 hours at temperatures of 28,5oC spawn in the laboratory experiment Injectable treatments for female PE with 500UI+2mg/kg and males with HCG 2,000 IU/kg at a rate of 100% and 66.7% for spawners in 3000 injected experimental UI/kg It is more important that the fecundity of fish in two treatments was high (20004-25582 eggs/kg female) ratio from 92.0 to 95.0% of fertilized eggs and hatching rate reaches 82, to 83.0% However, experiment shows that when injected with HCG in males at high dose (4,000 IU/kg) or no HCG injections, the fish not spawn Lowering pH 5.5 to 6.0 and increase the dose to the PE mg/kg on females showed initial production results very positively Table 4:13: The Channa lucius reproductive criteria when using HCG hormones in combination with PE and decreasing pH (5.5 to 6.0) Criteria Controlled Treatment Treatment Treatment HCG on males (UI/kg) 2,000 3,000 4,000 HGC+ PE on females 500+2 500+2 500+2 (UI+mg)/kg Effective time (hour) 40:0±3:28 33:0±4:14 Rate of fish spawning (%) 100 66.7±57.7 a b Real fecundity (egg/kg) 25,582±4.329 20,004±625 Rate of fertilizer (%) 95.0±5.0a 92.0±4.0a a a Rate of hatching (%) 83.0±6.0 82.0±6.0 Notes: The values in the same column with different letters have differences with statistical significance (p[...]... different conditions of growout It is able to continue to study the influence of reproductive stimulants and ecological conditions on the quality of sperm of Channa lucius 26 LIST OF ARTICLES PUBLISHED WITH THE CONTENT OF THE THESIS 1 Tien Hai Ly1 and Bui Minh Tam2 (2013) Research on reproductive biology of Channa Lucius Cuvier 1831 Journal of Science and Technology, Ministry of Agriculture and Rural... massive and less growth variation than high-density growth CONCLUSION AND RECOMMENDATION 5.1 Conclusion Channa lucius are carnivores with characteristics of wide mouth, sharp teeth, thin gills, ushaped and thick-wall stomach, short and straight rectum (0.61), ingredients of animal origin account for 93.7%, others account for 6.30% Strong correlation between the length and weight of Channa lucius follows... size and transitions to sexual maturity, the growth in length slowdowns and the volume growth is fast 4.4 Characteristics of Channa lucius nutrition 4.4.1 Characteristics of mature Channa lucius feeding The results determined the rate of the gut length on the body length (RLG) of Channa lucius are shown in Table 4.3 Table 4.3 shows RLG