MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY TRUONG THI BICH HONG “STUDY ON CHARACTERISTICS OF ECOLOGICAL DISTRIBUTI, GROWTH, REPRODUCTION OF OLIGOCHAETE (Limnodrilus hoffmeisteri Claparede, 1862) AND TRIAL OF THEIR BIOMASS CULTURE” Major: Aquaculture Major code: 9620301 SUMMARY OF DOCTORAL THESIS Supervisors: Associate Prof PhD Nguyen Dinh Mao PhD Dinh The Nhan KHANH HOA -2017 Thesis was comlpeted in Nha Trang University Assoc Prof Dr Nguyen Đinh Mao Supervisor: Dr Đinh The Nhan Reviewer 1: Prof Dr Đo Cong Thung Reviewer 2: Dr Tran Suong Ngoc Reviewer 3: Prof Dr Đoan Nhu Hai Thesis will be defended at the Thesis university committee level at Nha Trang University Date:…………………….h,……………./…………….2018 The thesis can find out at: - National Library - Library of Nha Trang University INTRODUCTION Oligochaete (Limnodrilus hoffmeisteri) is essential food for most freshwater fish species In the past few years, the demand for oligochaete biomass has been increasing for the production of native fish and ornamental fish, especially for the production of cold water fish such as sturgeon and rainbow trout However, information related to culture of L hoffmeisteri in Vietnam is not known So that, worms have not yet been cultured to provide freshwater fish hatcheries and ornamental fish With high application potential in aquaculture, the study of distribution ecological characteristics, reproduction, growth as well as culture of oligochaete is very necessary According to above reasons, the thesis: “Study on characteristics of ecological distributi, growth, reproduction of oligochaete (Limnodrilus hoffmeisteri Claparede, 1862) and trial of their biomass culture” was conducted Objectives of thesis Oligochaete (L hoffmeisteri) biomass culture used to feed the ornamental fish and native freshwater fish Detail objectives Database on distribution, growth and reproduction of oligochaete (L hoffmeisteri) Determine the suitable substrate structure, substrate thickness, feed type, ration and inoculation density when culture of oligochaete (L hoffmeisteri) Trial of oligochaete biomass culture The main contents Study on characteristics of ecological distributi of oligochaete in freshwater ecosystems Study on reproduction of oligochaete in laboratory Study on growth of oligochaete in laboratory Effect of substrata structure, substrata thickness, feed type, ration and inoculation density on biomass and density of oligochaete (L hoffmeisteri) Trial of oligochaete biomass culture Application biomass oligochaete to culture zebrafish (Danio rerio) and siamese fighting (Betta splendens) Scientific and practical significance Scientific significance: The study provided a database on the distribution, growth and reproduction of L hoffmeisteri, assessing the potential of natural food sources in related waters Practical significance: The results of study provided data of scientific value in research and application, contributing to the improvement of the culture process of oligochaete (L hoffmeisteri) Oligochaete biomass contributes actively to live food sources in freshwater fish hatcheries and ornamental fish farms New results of thesis: The first works studied the distribution, growth, reproduction of oligochaete (L hoffmeisteri) The study provided the scientific data to culture oligochaete (L hoffmeisteri) Reduction of the Feed conversion ratio when oligochaete (L hoffmesteri) were fed a mixture of soybean meal, corn meal and rice bran with a ratio of 1: 1: CHAPTER MATERIALS AND METHODS 2.1 Time, locality and object of Research 2.1.1 Research time: 10/2012 - 05/2017 2.1.2 Research locality: Khanh Hoa province 2.1.3 Research object : Oligochaete (Limnodrilus hoffmeisteri Claparede, 1862) Figure Limnodrilus hoffmeisteri 2.2 Research block diagramamatic Study on growth of oligochaete in laboratory Study on reproduction of oligochaete in laboratory Study on ecological distributi of oligochaete in freshwater ecosystegms at Nha Trang and suburb Study on characteristics of ecological distributi, growth, reproduction of oligochaete (Limnodrilus hoffmeisteri Claparede, 1862) and trial of their biomass culture Study on Limnodrilus hoffmeisteri biomass culture Effect of substrata structure on survival and growth of Limnodrilus hoffmeisteri Effect of substrata thickness on biomass and density of Limnodrilus hoffmeisteri Effect of feed type on biomass, density and quality of Limnodrilus hoffmeisteri Effect of ration on biomass, density and quality of Limnodrilus hoffmeisteri Effect of inoculation density on biomass and density of Limnodrilus hoffmeisteri Trial of oligochaete (L hoffmeisteri) biomass culture Application biomass oligochaete to culture zebrafish (Danio rerio) and siamese fighting (Betta splendens) Figure 2.2: Research block diagramamatic 2.3 Methods 2.3.1 Study the distribution of L hoffmeisteri in different ecosystems 2.3.1.1 Sewage discharged Drainage ditches in the urban area, in the countryside and drainage ditches affected by industrial waste water Each drainage ditches was collected organism samples (oligochaete), water sample and bottom sample at points: head, middle and end 2.3.1.2 Static water with a water depth of more than m Natural ponds, ponds for aquaculture Each pond was collected samples: points in corners and one in the middle 2.3.1.3 Lowland areas with a water depth of less than m Lowland areas are: ricefields, spinachfields, papyrusfields Each of the lowland areas was collected samples: points in the coastal area between the two fields; point in the middle of the field 2.3.2 Study on reproduction and biological behavior of L hoffmeisteri in laboratory • Reproductive season of L hoffmeisteri Oligochaete were collected randomly at domestic sewage ditches twice per month for one year from December 2013 to November 2014 with 30 individuals at a time The worm was placed on a binocular optics microscope equipped with the Olympus BX41 Olympus C-7070 magnification camera to determine the maturity of the worm, according to Kennedy (1966) *Age of maturity and cocoon production (E1) Experimental conditions: Oligochaeta were cultured in a 250ml plastic beakers containing 50mg of mud under a drip water system Every day, we provided 1g of rice bran for oligochaete Experimental design: Five pair of oligochaete they had just hatched a day were inoculated into plastic beakers (repeat 30 times) When the oligochaete were weeks We collected a test to keep a pair in a beakers When the worms were 30 days old We collected oligochaete samples to check maturity If oligochaete are mature, then we check the bottom evryday to determine the reproductive age Mud used to made substrata: Mud were collected from sewage discharged in Vinh Phuong – Nha Trang where oligochaete lived with high density Mud washed through 500 µm mesh sieve to remove extraneous materials and any organisms A large plastic container was used to allow for the retention of sediment fines The fine mud was put into plastic bag (each bag holds kg of mud) Place the mud bag in autoclave at 125 ° C for 120 minutes  Reproductive viability and number of embryos in cocoon of L hoffmeisteri (E2) Experimental conditions: Oligochaete were cultured in a 250ml plastic container containing 50mg of mud under a drip water system for months from April to June 2015 Every day, we provided 5g of rice bran for oligochaete Experimental design: L.hoffmeisteri worms were inoculated at the density of mature individual in plastic box was prepared (repeat 10 times) Periodically, check the number of cocoons and embryos in a cocoon once every days for a period of months Determine the number of cocoons by the method of Marchese and Brinkhurst (1966) • Embryo development time and hatching rate (E 3) Experimental conditions: Cocoons are incubated in a 100ml plastic beakers containing 25mg of mud, under a drip water system Experimental design: 30 new cocoons were inoculated into 01 plastic beakers with 100ml we counted the number of embryos of a cocoon and calculated the total number of embryos of 30 cocoons before putting the cocoons into incubate The experiment was repeated 10 times We analyzed cocoon under a microscope everydays for observation embryo development • Research on regeneration of worm (E 4) Experimental conditions: Randomly placed in 15 plastic beakers of 250 ml, each containing 50 mg of mud and 30 parts of oligochaete The test beakers were placed under the drip system Every day, feed 1g of rice bran powder to the oligochaete into a plastic beakers Experimental design: The worm was cut into parts by a razor refills We Put body parts into different treatments: first treatment; Treatment 2: Body; Treatment 3: The tail In about one-third of the body, the location of the genital belt belongs to the head Each treatment was repeated times Periodically check the wound healing and worm regeneration for days The experiment ends when the body parts regenerate into a new body or complete death 2.3.3 Study on growth characteristics of oligochaete (E5) Experimental conditions: worms were cultured in a 250 cm2 plastic tray, 1cm thick bottom mud Stocking density of worms was individual/cm2 The tray was placed in the trough, under dripping water Worms were fed in the way that they felt satisfied their needs Experimental design: The stocking of 500 oligochaete hatched -2 days Once a week, collected sample, measure the length and weight of 30 individual to assess the growth of the oligochaete The research period was 15 weeks 2.3.4 Oligochaete (L hoffmeisteri) culture study 2.3.4.1 Effect of substrata structure on survival and growth of L hoffmeisteri (E6) Experimental conditions: The experiment was conducted in a laboratory, under the overflow system consisting of 25 plastic trays, each tray 150 cm2 (15x10), the height of the tray 6.5 cm The plastic trays were stacked between and and placed under the tap Stocking density of oligochaete was individual/cm2 Stocking oligochaete is a week old Oligochaete were fed rice bran Oligochaete were fed in the way that they felt satisfied their needs Experimental time was weeks Experimental design: The experiment was conducted with different treatments on the ratio of sand and mud Tr 1: 75% mud 25% fine sand; Tr2: 50% mud 50% fine sand; Tr 3: 25% mud and 75% fine sand; Tr 4: 100% fine sand; Tr 5: 100% sludge The sand used for substrata structure is freshwater sand The dry mud and fine sand were mixed according to the ratio of the treatments Each treatment was repeated times Weekly, sampling was used to calculate the density, weight and body length of 30 individual per treatment Sample collection: samples were taken at points (4 points in corners and point in the middle) in the tray We used plastic frames cm2 (2x2cm) and spoons to collect samples First, we pressed the frame down to the bottom and then use plastic scoop to collect all the mud and the oligochaete inside the frame A quantity of mud and the oligochaete washed through 2a = 0,5mm sieve with 600 cm2 tray to remove the bottom Oligochaete were retained to calculate the necessary indicators 2.3.4.2 Effect of substrata thickness on biomass and density (E7) Experimental conditions: Experimental conditions was the same E6 However, the hole position in the tray wall was different between treatment groups, ensuring the water level from the substrate surface to the hole position was cm Substrata structure was 75% mud 25% fine sand This was selected from the treatment of E6 Experimental design: Substrate thickness was arranged with four levels Four substrate thickness levels were tested: cm, cm, cm and cm Each treatment was repeated times for weeks Weekly, sampling was used to calculate the density and biomass of oligochaete Sampling method was similar to E6 2.3.4.3 Effect of feed type on biomass, density and biomass quality of L hoffmeisteri (E8) Experimental conditions: The oligochaete were cultured in indoor cemented culverts 4.000 cm2 (160 x 25 cm) system to protect from rain and sunlight Flowing water Initial stocking density was individual/cm2 Substrata structure was 75% mud 25% fine sand Thickness was 4cm They were selected from the treatment of E6 and E7 Oligochaete were fed once a day, the amount of food provided in the way that they felt satisfied their needs Experimental design:The experiment was conducted with treatments: Tr1: soybean meal; Tr 2: corn meal; Tr 3: rice bran; Tr 4: mixed feed: soybean meal, corn meal with rice bran at a ratio of 1: 1: Weekly, sampling was used to calculate the density and biomass of oligochaete Each treatment was repeated times Sampling method was similar to E6 2.3.4.4 Effect of ration on density, biomass and biomass quality of oligochaete (E 9) Experimental conditions: Experimental conditions was the same E8 Substrata structure 75% mud 25% fine sand with 4cm thickness Feed type was mixed feed: soybean meal, corn meal with rice bran at a ratio of 1: 1: They were selected from the treatment of E6, E7 and E8 Experimental design: The experiment was conducted with different treatments: Tr1 0.00 % (control treatments, no feeding); Tr fed 5%; Tr fed 10%; Tr fed 15% body mass/day Each treatment was repeated times, for weeks Weekly, sampling was used to calculate the density and biomass of oligochaete Sampling method was similar to E6 2.3.4.5 Effect of inoculation density on biomass and density of oligochaete (E10) Experimental conditions: Experimental conditions was the same E8 Substrata structure 75% mud 25% fine sand with 4cm thickness Feed type was mixed feed with 15% body mass/day They were selected from the treatment of E6, E7, E8 and E9 Experimental design: inoculation density was designed with four levels Four initial stocking biomass levels were tested including 1mg/cm2, 10mg/cm2, 20mg/cm2 and 30mg/cm2 Each treatment was repeated times, for weeks Weekly, sampling was used to calculate the density and biomass of oligochaete Sampling method was similar to E6 2.3.4.6 Experimentation of oligochaete biomass culture (E 11) Experimental conditions: Oligochaete were cultured in indoor cemented culverts The area of the culverts 2.4 m2 Conditions on substrata structure was 75% mud 25% fine sand with 4cm thickness Feed type was mixed feed with 15% body mass/day Inoculation density was 10mg/cm2 They were selected from the results from E to E10 The culture time was weeks Experimental design: Oligochaete (L hoffmeisteri) cultured biomass in two cement slurry systems Identification the biomass, biochemical and amino acid of oligochaete 2.3.5 Application biomass oligochaete to culture zebrafish (Danio rerio) and siamese fighting (Betta splendens) 2.3.5.1 Effects of culture oligochaete on gonadosomatic index and fecundity of zebrafish (Danio rerio, F.Hamilton, 1822) (TN12) Experimental conditions: The experiment was arranged randomly in tanks Continuous air 24/24 Fish were fed twice a day in the way that they felt satisfied their needs Male and female were cultured in a glass tank days prior to the experiment They adapted to the food and laboratory conditions Experimental design: The experiment was conducted with four different treatments: Tr1: 100% live oligochaete; Tr 2: 100% frozen oligochaete; Tr3: 50% live oligochaete + 50% industrial feed; Tr4: 100% industrial feed (control) Male and female were cultured in different tanks with density of 30 fish / tank We evaluated effects of food on the sexual maturity of zebrafish at the end of the experiment (30 days) Each indicator (gonadosomatic index and fecundity) of the experiment was repeated times 2.3.5.2 Effects of culture oligochaete on growth and survival of siamese fighting (Betta splendens, Regan, 1910) juveniles (E13) Experimental conditions: The experiment was arranged randomly in 12 tanks of size 25x25x40cm3 Fish were fed twice a day in the way that they felt satisfied their needs Daily substituting 20% of water after second feeding (15h) Fish were cultured in a glass tank days prior to the experiment They adapted to the food and laboratory conditions Experimental design: The experiment was conducted with four different treatments: Tr1: 100% live oligochaete; Tr 2: 100% frozen oligochaete; Tr3: 100% industrial feed (Kaokui); Tr4: 50% live oligochaete + 50% industrial feed (Kaokui) Each treatment was repeated times We weighed weigh and measured the length of fish every two weeks The survival rate of fish was determined by recording the number of dead fish daily in each experiment 2.4 Data analysis Inputting all raw data into the Excel software: Determining the density, cocoon size, embryo hatching rate, characteristic growth rate of length and weigh, absolute growth, absolute fecundity, relative fecundity, fertilization rate, survival rate, biological diversity by formula Use SPSS 18.0 to compare mean values Analysis of variance (ANOVA) was used to compare these production metrics Comparison of mean difference after post hoc analysis with Duncan's reliability of 95% (P 0.05) The lowest biomass (94,51 ± 6,98 mg/cm2) were recorded when feeding L hoffmeisteri with corn meal, significant differences with other difference treatments (P