MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY

INSTITUTE OF SCIENCE AND TECHNOLOGY ------

TA THI BINH

STUDY ON BIOLOGICAL CHARACTERISTICS AND DEVELOPMENT OF SEED PRODUCTION TECHNOLOGY FOR THE BLOTCHED SANKEHEAD maculata (Lacepède, 1801) Major: Zoology Code: 9 42 01 03

The abstract of PhD Thesis in Biology Sciences

HANOI– 2020

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THIS THESIS IS COMPLETED AT INSTITUTE OF SCIENCE AND TECHNOLOGY, VIETNAM ACADEMY

Supervisors: 1. Assoc.Dr. Nguyen Huu Duc 2. Dr. Do Van Tu

Examiner 1: … Examiner 2: … Examiner 3: …. The thesis will be defended infront of thesis defense comitee at Institute of Science and Technology-Vietnam Academy at/in …2020.

This thesis can be found at: - Institute of Science and Technology Library - Vietnam National Library

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INTRODUCTION Among freshwater fish species, Channidae (belonging to ordo) are of interest to both international and vietnames researchers due to their large size, delicious meat and high vitality. Many species have been well studied for their biological characteristics, seed production and important aquaculture development in Asian countries such as Vietnam, Thailand, Cambodia, Philippines, India and Malaysia (Muntaziana et al., 2013). In Vietnam, Channidae has only one Channa genus which comprises 12 species distributed across the country with many different names based on local languages. In particular, the the striped (C. striata) and the giant snakehead (C. micropeltes) have been extensively studied and developed for aquaculture because of their large size and high economic value (Nguyen Huan and Duong Nhut Long, 2008). Recently, much attention has been paid on biological characteristics and seed production of the forest snakehead (C. lucius) (Tien Hai Ly, 2016), the dwarf snakehead (C. gachua) (Ho My Hanh, 2017). While the blotched snakehead(C. maculata) is a potential species for aquaculture, however, its information of biological characteristics is limmited. There have been a few studies on and some biological characteristics of the blotched snakehead as well as preliminary results on artificial seed production (Nguyen Thai Tu, 1983; Mai Dinh Yen, 1978; Nguyen Van Hao, 2005; Nguyen Dinh Vinh et al., 2015; Ta Thi Binh et al., 2015). The study of biological characteristics, a basis for development of artificial breeding techniques and introduction of C. malacuta to aquaculture, is essential to contribute to the diversification of cultured species, maintaining and developing resources, protecting gene pool and preserving biodiversity. According to Pravdin (1973), understanding biological characteristics of a species is an extemely important step to successfully domesticate and introduce the species to aquaculture. Therefore, the project "study on biological characteristics and development of seed production technology for blotched snakehead Channa maculata (Lacepède, 1801)" was carried out to address the above important need. Research objectives: To determine the nutritional, reproductive characteristics of wild blotched snakehead as a scientific basis for developing its breeding and nursery techniques. To establish a number of specifications in the production of blotched snakehead in artificial conditions. Research contents: Study some biological characteristics of the blotched snakehead in the nature : - Nutritional biology - Reproductive biology Development of techniques to produce blotched snakehead seed in artificial conditions: - Broodstock rearing - Spawning stimulation techniques - Nursing of larvae to fry - Nursing of fry to fingerlings

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CHAPTER 1. LITERATURE REVIEW 1.1. CLASSIFICATION According to Eschmeyer (2018), the blotched snakehead C. maculata (Lacepède, 1801) is classified as below: Kingdom Animalia. Phylum: Chordata. Class: Actinopteri. Ordo: Anabantiformes. Sub-ordo: Channoidei. Family: Channidae. Genus: Channa. Species: Channa maculata (Lacepède, 1801) 1.2. Status of research on the blotched snakeheadin the world and in Vietnam 1.2.1. Status of research on the blotched snakeheadin the world The blotched snakehead was discovered before 1900 in Oahu, Hawaii and is widely distributed in Oahu (Yamamoto and Tagawa, 2000). Studies of the distribution characteristics of the blotched snakehead have revealved that this species is distributed in tropical and subtropical countries such as China, Taiwan, the Philippines and North Vietnam. In addition, they have been introduced to Japan, Hawai and Madagascar (Water and James, 2004). In China, Fang Fang et al. (2002) reported that the blotched snakehead was the second most important cultured species and was mainly cultured in Guangdong province. At present, this species is considered to be one of the food sources in Taiwan and Nara, Hyogo, Hiroshima (Japan) and Philippines (Okada, 1960; Liang et al., 1962; Hay and Hodgkiss, 1981; Uyeno and Akai, 1984 cited from Water and James, 2004). Some other studies focused on nutrition, disease and comercial production techniques. For instance, according to Yamamoto and Tagawa (2000), the blotched snakeheadis a predatory species. Chen (2012) isolated the bacteria Aeromonas schubertii in the diseased C. maculata. Ju and Woof (1987) studied the metabolism of C. maculata in the hypoxic conditions. Zhao et al. (2016) evaluated the effect of dietary lipid concentration on the growth, oxidation of liver and serum metabolites of fingerlings which are hybrids of C. argus and C. maculata. Chen (1976) briefly described the comercial production techniques of C. maculata species in Taiwan. 1.2.2. Research status of the blotched snakeheadin Vietnam Currently, there have been few studies on taxonomy, some biological characteristics and comercial seed production. Mai Dinh Yen (1978) reported 4 snakehead species, C. orientalis, C. striata, C. maculata and C. asiatica in Northern Vietnam. Nguyen Van Hao (2005), Mai Dinh Yen (1978) describeed their morphology. While Nguyen Thai Tu (1983); Mai Dinh Yen (1978) and Nguyen Van Hao (2005) described the environment, habits, nutrition, growth and reproduction of of C. maculata. Nguyen Dinh Vinh and Ta Thi Binh (2015) first time investigated some reproductive characteristics of C. maculata in the North Central region of Vietnam. Ta Thi Binh et al. (2015), initially conducted experiments on reproduction of C. maculata under artificial conditions.

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In general, the results of research on C. maculata have been very limited, therefore, it is necessary to continue basic research on nutrition, reproductive, and especially research should focus on stimulating spawning and developing rearing techniques of fry and fingerlings in order to establishing and stabilizing the seed production technology of C. maculata in the future. Chapter 2. MATERIALS AND METHODOLOGY 2.1. Study time and location The work described in this thesis was conducted between January 2016 and June 2019. Blotched snakehead were collected in districs Ha Trung, Vinh Loc, Hau Loc, Nga son, Hoang Hoa (Thanh Hoa province). Experiments were conducted at aquaculture lab, School of Agriculture and Resources, Vinh University.

Figure 2.2. Location of blotched snakehead sampling 2.2. Materials 2.2.1. Sample 2.2.1.1. Sample used for investigating biological characteristics A totoal of 820 blotched snakehead were collected from fishmen in districs Ha Trung, Vinh Loc, Hau Loc, Nga son, Hoang Hoa (Thanh Hoa province) form January 2016 to June 2018. 2.2.1.2. Sample used for studying seed production - Brood fish: 96 pairs of brood fish (weight >500g/fish) provided by fishmen in Thanh Hoa province, were acclimatised at Hung Nguyen Centre for Freshwater Aquaculture, Vinh University. - Fish larvae: from artificial seed production - Feed: Trash fish, pellets, homemade food.

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2.3. Methods 2.3.1. Project outline

Research contents

Study on biological characteristics Development of seed production techniques

- Broodstock rearing techniques - Nutritional biology - Spawning stimulation techniques - Reproductive biology - Nursing techniques

Broodstock rearing Induced spawning methods Nursing techniques

Determin Determin Determin Determin Nursing of larvae Nursing of fry to Induced Induced suitable suitable time for suitble by by to fry fingerlings foods diets injecting time for pituitary hormone male fish injection

female fish

Determin Determine Determin suitable suitable suitable Determine foods time for stockist suitable changing density diets foods

CONCLUSION AND RECOMENDATION

Figure 2.2. Project outline

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2.3.2. Study methods of biological characteristics 2.3.3.1. Nutritional biology The ratio of intestinal length and trunk length (RLG - Relative Lengh of the Gut) was calculated using the formula of Al-Hussainy (1949). Food components were classified using the methods of Dang Ngoc Thanh et al. (1980), Mai Dinh Yen et al. (1979) and Shirota (1966). The nutritional spectrum of adult fish was determined using the method of Biswas (1993). Dry weight of samples of each food type was determined based on AOAC analysis method (2000). 2.3.3.2. Reproductive biology Gonadal development stages of blotched snakehead samples were determined based on the observation of the shape, size and color of the gonads on a 6-step scale of Xakun and Buskaia (1968). The gonadal histology was performed using the method of Drury and Wallington (1967). Samples were embedded in paraffin, cut into slices then stained with Haematoxyline and Eosin. The Gonadal Somatic Index was determined by the formula of Biswas (1993). Fercunlity was determined based on the egg mass of a female with gonads in stage IV and the number of oocytes was calculated according to Banegal's formula (1967). 2.3.3. Seed production technology 2.3.3.1. Broodstock rearing a/ Effects of food types on fecundity and quality of eggs and larvae The brood fish were fed with trash fish at 5-7% body weight for 1 month prior being put into the experiment system. Brood fish with an average weight of 0.85kg (ranging 0.65 to 0.97 kg) are cultured in 3 cages (4 mx 3 mx 2m) put in a pond of 1000 m2 with a stocking density of 24 fish/ cage (equivalent to 2 fish/ m2, 1 fish/ m3), the male to female ratio was 1: 1.5. There are 3 broodstock rearing periods corresponding to 3 treatments: – Treatment 1 fed 100% trash fish – Treatment 2 fed 50% trash fish: 50% comercial feed pellets. – Treatment 3 fed 100% comercial feed pellets. b/ Effects of food components on fecundity and quality of eggs and larvae Culture conditions, number of fish per treatment, stocking density, methods for spawning and assessment criteria are similar to those in the experiment 1, excepting weight of brood fish is smaller, 0.65 kg (from 0.55 to 0.71). The experiment was repeated 3 times (30-35 days each). Brood fish were fed trash fish with the following diet: - Treatment 1: fed with a diet of 5% body weight - Treatment 2: fed with a diet of 7% of body weight - Treatment 3: fed with a diet of 9% body weight - Treatment 4: fed with a diet of 11% body weight 2.3.3.2. Spawning stimulating methods a. Experiment 1. Determine a suitalbe time for hormonal injection The experiment consists of 4 treatments, each treatment was replicated 3 times and designed in a completely random manner. + Treatment 1: male fish were injected at the same time of giving the decision dose to females (0h). + Treatment 2: male fish were injected 8 hours before the time of of giving the decision dose to females 5

+ Treatment 3: male fish were injected 16h before the time of of giving the decision dose to females + Treatment 4: male fish were injected 24 hours before the time of of giving the decision dose to females b. Experiment 2. Determine a suitale time for injecting decision dose of female fish The experiment consists of 4 treatments, each treatment was replicated 3 times and designed in a completely random manner. + Treatment 1: Final dose from premilary dose 6h + Treatment 2: Final dose from premilary dose 12h + Treatment 3: Final dose from premilary dose 18h + Treatment 4: Final dose from premilary dose 24h c. Experiment 3. Spawning stimulation by pituitary The experiment consists of 5 treatments, each treatment was replicated 3 times and designed in a completely random manner. Each treatment has 3 pairs of brood fish. + Treatment 1: 9 mg/kg female + Treatment 2: 10 mg/kg female + Treatment 3: 11 mg/kg female + Treatment 4: 12 mg/kg female + Treatment 5: 13 mg/kg female d. Experiment 4: Effects of hormone types on quality of eggs and larvae The experiment consists of 4 treatments and designed in a completely random manner. Each treatment has 3 pairs of brood fish. + Treatment 1: 3500 IU HCG/kg female. + Treatment 2: 60µg LRHa + 15mg DOM/kg female). + Treatment 3: 12 µg Pituitary /kg female. + Treatment 4: Injection of saline solution at a dose of 0,5 mL/kg 2.3.3.3. Methods of nursing a. Effects of food types on growth, survival rate and coefficient of variance of larvae to fry stage The experiment consists of 4 treatments and designed in a completely random manner. Each treatment was replicated 3 times. Treatment 1: Moina + commercial food Treatment 2: Moina + Filariae Treatment 3: Moina + marine shrimp b) Effect of feeding conversion regime on growth, survival and coefficient of variance of fry stage The experiment was designed completely randomly with 6 treatments of feeding practices from trash feed to comercial feed at different time points, each treatment was replicated 3 times. Treatment 1: Changing to processed food from day 7; Treatment 2: Changing to processed food from day 9; Treatment 3: Changing to processed food from day 11; Treatment 4: Changing to processed food from day 13; Treatment 5: Changing to processed food from day 15;

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Treatment 6: Changing to processed food from day 17. c. Effect of stoking density on growth, survival rate and coefficient of variance of fry to fingerlings stage. The experiment was designed completely randomly and each treatment was replicated 3 times, including 4 nursery treatments at the density of 1 fish/L; 1.5 fish /L, 2 fish /L and 2.5 fish/L. d. Effect of diets on growth, survival and coefficient of variance of fry to fingerlings stage. The experiment was designed completely randomly with 5 treatments corresponding to the diets of 3, 6, 9, 12 and 15% body weight/day. Each treatment was replicated 3 times. 2.4. Data analysis All collected data were analyzed and graphed on excell software v2010. Analysis of variance was performed on SPSS 16.0. One-way- ANOVA and Ducan test were then used to verify the statistically significant difference (P <0.05) among the treatments in each experiment.

Chapter 3. RESULTS AND DISCUSSION

3.1. Biological characteristics 3.1.1. Nutritional biology 3.1.1.1. Morphology of digestion system a/ Mouth, teeth and gills The blotched snakehead C. malacuta has a large mouth, front facing and a protruding lower jaw with a row of sharp teeth. There are many small and pointed teeth in the mouth, growing in rows on the jaws and vomer. The upper jaw is longer than the lower jaw. There are several sharp teeth on two jaws. Tongue is long and sharp. Upper lip is thick. The jaw anterior teeth and vomer are are continuous arc shaped. The large mouth and well- developed teeth indicate that this is a predatory species (Figure 3.1). Gill rakers are arranged in two rows on gill arch and with hard spines. The first gill arch has an average of 22.33 ± 1.01 rakers (ranges from 19-24 gill rakers) (Figure 3.2).

Figure 3.1. Mouth and teech Figure 3.2. Gill rakers b/ Esophagus The esophagus of the blotched snakehead is short, but its wall is thick. There are many white folds located behind the oral sinus inside the esophagus, which indicates that the esophagus has a high elasticity and can store a lot of food and catch large preys (Figure 3.3).

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a b Figure 3.3. Esophagus Figure 3.4. Transversal slices f a/ Outside of the esophagus; b/ Inside of the esophagus esophagus (a: Sphincter layer, b: Vertical muscle layer, c: Mucosa, d: adipose tissue)

c/ Stomash The stomach of the blotched snakehead is U-shaped, short, large in size and with thick walls. The inner surface of stomach has many folds making it easy to be expanded and to have a large contraction force to accommodate large-sized preys (Figure 3.5). Cross section of the stomach shows that its wall has 3 layers: outer membrane, mucous membrane, smooth muscle layer (Figure 3.6).

a b

Figure 3.5. The blotched snakehead stomash a/ Outside of the stomash; b/ Inside of the stomash

a. Outer membrane b. Sphincter layer c. Vertical layer of muscle d. The submucosa e. Lining f. Crease

Figure 3.6. Stomash structure e/ Intestine The intestine is a continuation after the stomach. The intestine receives digestive enzymes from the pancreas and bile fluid from the liver to digest food and absorb nutrients 8

through the intestinal wall into the bloodstream to serves organs, organizations, tissues in the body. Therefore, the intestine is considered an important digestive organ. The blotched snakeheadinstestine is straight, short, but its wall is thick (Figure 3.7).

Stomash Manh tràng

Instestine

Figure 3.7. Digestive track

Similar to other parts of the digestive tract, the wall of the intestine is composed of the mucosa, submucosa, muscle and outer membrane (Figure 3.8, Figure 3.9)

Figure 3.8. Transverse slice of intestine Hình 3.9. Intestine structure a. Wall; b. Below submucosa; a. submucosa; b. below submucosa c. submucosa c. Smooth muscle layer; d. Outer membrane; e. Folds

Morphological characteristics and structure of digestive tract such as the position of the mouth, teeth, gill rakers, esophagus, size, structure of the stomach and intestines indicate that the blotched snakehead is a preadtory species. 3.1.1.2. The relative length of gut (RLG) The result of determining RLG is presented in Table 3.1. Table 3.1. RLG in different sizes. Body length Mean Min Max Sample size

(mm) (Li/Lt) (Li/Lt) (Li/Lt) (n=344) <50 0.51±0.02 0.47 0.55 34 50 – 99 0.52±0.07 0.38 0.61 22 100 – 149 0.56±0.03 0.49 0.61 23 150 – 199 0.57±0.06 0.40 0.71 59 200 – 249 0.59±0.06 0.46 0.74 79 250 – 299 0.60±0.05 0.45 0.79 63 ≥ 300 0.60±0.02 0.57 0.65 64 Mean 0.58 ± 0.06 0.38 0.79 9

3.1.1.3. Natural food components

W <100 g Spills Others Reptile 5% 1% 100 8%

83.33 81.08 78.38 Worm (%) 80 63.33 61.67 56.08 11% 60

35.83 40 15.5419.17 20 14.19 10.14 Crustatio fish 0.00 0.00 0.00 ns 57% Occurence frequency Occurence 0 18%

Food types

Figure 3.11. Occurence frequency of food for fish Figure 3.13. Food spectrum of blotched smaller than 100 g (n=120) and bigger than 100 g snakehead (n=148). 3.1.2. Reproductive biology 3.1.2.1. Female fonadal development stages

Figure 3.18. The histolorical image of Figure 3.20. The histolorical gonadal development at stage I (40X) image of gonadal development at stage II (40X)

Figure 3.22. The histolorical image of female Figure 3.24. The histolorical image of female gonadal development at stage III (10X) gonadal development at stage IV(4X)

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Figure 3.25. The image of ovary at stage V Figure 3.26. The histological image of ovary cells at stage V (4X) b/ Male gonadal development satges

Figure 3.18. The histological image of male Figure 3.19. The histological image of male gonadal development at stage III (40X) gonadal development at stage VI (40X)

3.1.2.2. Gonado somatic index Table 3.3. Gonado somatic indexof C. maculate during study time Month Female Male /Year W(g) Wg(g) GSI (%) W(g) Wg(g) GSI (%) 1/2017 83483±105.77 3.41±1.02 1.20±0.28 446.08±29.19 1.60±0.16 0.45±0.04 2/2017 398.69±144.16 4.01±1.34 1.30±0.47 361±116.18 1.58±0.23 0.56±0.09 3/2017 441.76±184.21 5.17±1.47 1.45±0.46 394.23±125.22 1.69±0.44 0.69±0.11 4/2017 436±132.57 5.44±0.45 2.02±0.56 457±164.88 2.84±0.26 1.10±0.42 5/2017 496.94±113.07 8.57±0.44 2.71±0.56 423.67±79.91 3.52±0.30 1.32±0.16 6/2017 441.76±130.27 6.95±0.35 2.54±0.68 399±104.09 3.12±0.31 1.24±0.27 7/2017 422.71±128.23 5.88±0.33 2.31±0.23 432.38±85.72 3.29±0.32 1.20±0.13 8/2017 436±102.61 4.53±0.32 1.44±0.25 412.33±68.44 2.59±0.32 0.85±0.09 9/2017 410±91.19 4.66±0.29 1.35±0.24 431.5±105.61 2.40±0.31 0.65±0.09 10/2017 450.94±89.07 3.02±0.45 0.90±0.07 422.79±106.96 1.67±0.37 0.53±0.06 11/2017 352.73±93.02 2.86±0.47 0.77±0.12 448±101.12 1.20±0.33 0.36±0.04 12/2017 386.14±86.82 1.63±0.37 0.57±0.06 392.5±106.97 0.69±0.41 0.22±0.09

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3.1.2.3. Condition factor (CF)

Figure 3.32. The condition factor of C. maculata following investigated months 3.1.2.4. Spawning season

Figure 3.23. Fluctuation of gonado somatic index and fatness

Figure 3.34. The maturation period of C. malacuta in the months 12

Thus, within the scope of this study, we found that the spawning season of C. malacuta occurs between March to October, mainly focuses from May to June. Therefore, it is can be predicted the main spawning sason of C. malacuta is from April to June. 3.1.2.5. Fecundity Table 3.4. Fecundity of C. malacuta in diffennt groups Absolute Body weight Samples Individual weight Relative fecundity fecundity (g) Size (n) (g/fish) (eggs/g fish) (egges/fish) < 300 15 262.33 ± 36.0 3.985 ± 777 15.262 ± 2.2249 301 - 400 15 360.60± 29.63 5.288 ± 813 14.697 ± 2.132 401-500 15 455.67 ± 27.50 5.918 ± 1.004 12.973 ± 1.947 501- 600 15 563.07 ± 27.03 6.631 ± 1.004 11.759 ± 1.528 > 600 15 746.00 ± 79.59 7.283 ± 654 9.799± 701 3.1.2.6. Egg diameter Table 3.5. Egg diameter in different female groups Body weight Individual weight Samples size (n) Egg diameter (mm) (g) (g/fish) < 300 12 252.50 ± 35.33 1.17 ± 0.01 301 – 400 10 347.20± 22.44 1.19 ± 0.01 401-500 15 454.80 ± 26.78 1.23 ± 0.02 501- 600 14 555.07 ± 22.46 1.24 ± 0.01 > 600 7 755.20 ± 80.48 1.25 ± 0.01 3.2. Development of seed production techniques for C. malacuta 3.2.1. Broodstock rearing 3.2.1.1. Effects of food types on fecundity, quality of eggs and fish arvae Table 3.7. The maturation rate, fecundity, quality of eggs and larvae of C. malacuta fed by different foods. Treatment 2 (Trash fish Treatment 3 Treatment 1 Reproductive performance food+ (Comcercial (Trash fish food) comcercial feed feed pellets) pellets) Maturation rate (%) 82.26 ± 5.23a 81.76 ± 6.35a 65.76 ± 3.97b Fecundity (eggs/kg female) 45.346 ± 5.009b 46.776 ± 5.526b 32.645 ± 3.821a Egg size (mm) 1.21 ± 0.006 1.23 ± 0.015 1.22± 0.010 Oil droplet size (mm) 0.27 ± 0.00 0.28 ± 0.006 0.27 ± 0.006 Yolk-sac size (mm) 1.13 ± 0.015b 1.16 ± 0.015b 1.08 ± 0.020a Fertilization rate (%) 80.43 ± 2.32b 82.54 ± 3.21b 72.51 ± 3.12a Hatching rate %) 81.87± 1.49b 83.54 ± 1.46b 78.30 ± 1.18a Larvae size (mm) 2.57± 0.025b 2.61 ± 0.030b 2.42 ± 0.030a Abnormality rate (%) 2.54± 0.04a 2.73 ± 0.12a 4.34 ± 0.66b Survival rate (%) 62.50± 3.69a 65.7 ± 5.45a 66.61 ± 2.70a Note: different letters in the same row represents a statistically significant difference (P <0.05). 13

The results show that feeding 50% of trash fish combined with 50% of comercial feed pellets not only improves the reproductive performance of the brood fish, but also significantly improves the quality of eggs and larvae. 3.2.1.2. Effect of diets on fecundity, quality of eggs and fish larvae Table 3.8. Weight, maturation rate, fecundity, quality of eggs and fish larvae of brood fish fed with different diets

Reproductive performance Diets 5%BW 7%BW 9%BW 11%BW Weight before experiment (g) 643 ± 37 653 ± 61 658 ± 15 643 ± 47 Weight after experiment (g) 857 ± 19a 952 ± 54b 954 ± 46b 973 ± 80b Weight gained (g) 214 ± 19a 299 ± 54b 313 ± 46b 330 ± 80b Maturation rate (%) 81,23 ± 3,05b 82.48 ± 1.47b 89.56 ± 2.07c 71.43±4.49a Fecundity (eggs/kg female) 45.321 ± 3.582a 47.886 ± 8.175a 65.325 ± 6.842b 51.637± 5.442a Egg size (mm) 1.19 ± 0.015a 1.21 ± 0.017b 1.22± 0.058b 1.23±0.058b Oil droplet size (mm) 0.27 ± 0.058 0.28 ± 0.058 0.27 ± 0.058 0.28± 0.00 Fertilization rate (%) 75.54± 4.99 81.56 ± 5.17 79.24 ± 3.66 77.35± 2.33 Hatching rate (%) 81.61± 3.17 85.52 ± 1.54 85.31 ± 2.38 87.25± 3.26 Larvae size (mm) 2.51± 0.026a 2.61 ± 0.026b 2.62 ± 0.030b 2.62± 0.030b Abnormality rate (%) 3.71± 0.25 3.42 ± 0.16 3.88 ± 0.12 3.87± 0.41 Note: different letters in the same row represent a statistically significant difference (P <0.05). The results showed that the brood fish fed a diet of 9% BW not only improved their maturity, fecundity, and egg quality but also improved their growth rate. 3.2.2. Spawning stimulation 3.2.2.1. Determine a suitable time for hormones injection of male fish

Figure 3.42. Male gametogensis rate

Thus, injecting male fish 24h before the decision dose of the female is also a solution to overcome the asynchronous maturation of C. malacuta. 3.2.2.2. Determine a suitalbe time for injecting decision dose of female fish The results of determining a suitalbe time for injecting decision dose of female fish is showed in Figure 3.44 and Figure 3.46. 14

Figure 3.44. Spwaning rate

Figure 3.46. Fertilization and hatching rate of C. malacuta at different premilary and decision doses

The experimental results recomend that injections time of preliminarily doses is 18h from the decision dose of the female. 3.2.2.3. Spawning stimulatation of C. malacuta using pituitary Table 3.9. Spawning rate, fecundity and responding time of brood fish stimulated by pituitary Responding time Spawning rate Fecundity Treatment (h) (%) (Eggs/kg female) Saline solution - - - Pituitary 09 mg/kg - - - Pituitary 10 mg/kg - - - Pituitary 11 mg/kg 37.53 11.11 ± 9.24 a 20.476 ± 0000a Pituitary 12 mg/kg 35.42 100.00 ± 0.00b 27.580 ± 834b Pituitary 13 mg/kg 36.43 100.00 ± 0.00 b 22.633 ± 2.055a Note: Different letters in the same column represent a statistically significant difference (P<0,05) 15

Table 3.10. Hatching time, quality of eggs and larvae from brood fish stimulated by pituitary at different dosages. Treatments Reproductive performance Pituitary 11 Pituitary 12 Pituitary mg/kg mg/kg 13mg/kg

Hatching time 45h 30 min 40 h15 min 43h 30 min

Fertilization rate (%) 81.67 ± 1.47a 91.47 ± 1.88c 86.58 ± 0.83b Floating rate of eggs (%) 66.70 ± 4.98a 82.21 ± 1.58b 76.21 ± 1.93b Hatching rate (%) 75.55 ± 2.10a 85.01 ± 1.62b 81.87 ± 1.41b Egg size (mm) 1.21 ± 0.000a 1.23 ± 0.006b 1.22 ± 0.006b Oil droplet (mm) 0.27 ± 0.006a 0.28 ± 0.006a 0.28 ± 0.006a Larvae size (mm) 2.40 ± 0.02a 2.67 ± 0.02c 2.57 ± 0.04b Yolk-sac size (mm) 1.09 ± 0.02a 1.16 ± 0.02b 1.14 ± 0.02b Abnormality rate (%) 4.55 ± 0.88ab 3,.80 ± 0.66a 5.74 ± 0.70b Survival rate of 3-day old fish (%) 59.31 ± 2.87a 67.99 ± 4.93b 61.67 ± 2.40ab Note: different letters in the same row represent a statistically significant difference (P <0.05). Thus, through reproductive performance such as spawning rate, responding time of hormones, actual fecundity, egg size, oil droplet, floating egg rate, fertilization rate, hatching rate, larva size, yolk-sac size, abnormality rate and survival rate of 3-day-old fish, it is recomended to use pituitary at a dose of 12mg/kg to stimulate the reproduction of C. malacuta. 3.2.2.4. Effects of hormone types on quality of eggs and larvae Table 3.11. Maturation rate, gametogenesis recurrence and fecundity of brood fish injected with different hormones. Spawn Fecundity Maturation rate Gametogenesis Treatment ig rate (eggs/kg (%) time (day) (%) female) Saline solution 56.85 ± 7.44a 38.00 ± 3.61b - - HCG 81.47 ± 6.96b 30.67 ± 2.52a 100 24.221 ± 3.315 LHRHa + DOM 77.36 ± 5.64b 37.67 ± 2.88b 100 27.404 ± 2.017 Pituitary 75.46 ± 9.58b 33.67 ± 1.53ab 100 25.779 ± 959

Note: Different letters in the same column represent a statistically significant difference (P<0,05).

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Table 3.12. The effects of hormones types on responding time and reproductive performance of C. malacuta. Hormone types Reproductive performance HCG LHRHa + DOM Pituitary Responding time (h) 34.36 ± 1.49a 40.79 ± 1.76b 35.90 ± 0.65a Fertilization rate (%) 82.31 ± 3.36a 87.91 ± 1.38b 83.66 ± 2.59a Floating egg rate (%) 88.13 ± 2.14 91.31 ± 2.40 90.80 ± 2.47 Hatching time (h) 41.77 ± 3.66 44.61 ± 1.09 42.23 ± 1.34 Hatching rate (%) 79.80 ± 2.47a 85.85 ± 3.45b 79.40 ± 3.64a Egg size (mm) 1.23 ± 0.015 1.23 ± 0.006 1.22 ± 0.006 Oil droplet (mm) 0.28 ± 0.006 0.27 ± 0.010 0.28 ± 0.006 Larvae size 2.65 ± 0.015 2.67 ± 0.020 2.65 ± 0.025 Yolk-sac size (mm) 1.16 ± 0.015 1.16 ± 0.020 1.17 ± 0.015 Larvae abnormal rate (%) 3.20 ± 0.28ab 3.79 ± 0.60b 3.03 ± 0.24a Survival rate of 3-day old fish (%) 69.31 ± 2.84 68.95 ± 4.16 73.25 ± 2.14

Note: Different letters in the same row represent a statistically significant difference (P<0,05). Based on the results obtained, injection of LHRHa + DOM showed the highest fercundity (27,404 eggs/kg), fertilization rate (87.91%) and hatching rate (85.85%). Meanwhile, no significant difference was observed in other criteria such as the rate of floating eggs, deformities, survival rate of 3 days old larvae, egg size, oil droplets, yolk-sac and fry in 3 treatments LHRHa + DOM, HCG, pituitary. Therefore, it can be concluded that LHRHa + DOM is the most suitable hormone to stimulate the reproduction of the blotched snakeheadC. malacuta. 3.2.3. Nursing of C. malacuta at the larvae to fry stage 3.2.3.1. Effects of different food types on growth, survival and coefficient of variance of C. malacuta at the larvae to fry stage a/ Survival rate

Figure 3.47. Survival rate of C. malacuta at the larvae to fry stage fed by different foods 17

c/ Growth The nursing experiment was conducted for 28 days (4 weeks). The growth results of C. malacuta at the larvae to fry stage are presented in Table 3.14 Table 3.14. The growth of C. malacuta at the larvae to fry stage fed with different foods Moina – thread Moina – Pellets Moina -Shrimp Worm a a a W0 (g) 0.0025±0.0003 0.0025±0.0003 0.0025±0.0003 Release a a a L0(cm) 0.89±0.03 0.89±0.03 0.89±0.03 a b ab Wfl (g) 0.38±0.02 0.45±0.03 0.42±0.03 Harvest a b b Lfl(cm) 3.10±0.10 3.47±0.15 3.37±0.06 DWG(g/day) 0.013±0.00a 0.016±0,001b 0.015±0.001ab Daily growth rate DLG(cm/day) 0.08±0.000a 0.093±0.005b 0.090±0.001b a b ab Specific growth SGRW(%/day) 17.94±0.18 18.51±0.20 18.29±0.26 a b b rate SGRL(%/day) 4.46±0.12 4.85±0.16 4.68±0.21 Note: Different letters in the same row represent a statistically significant difference (P<0,05). The results showed that fry fed by moina - threadworms and moina - shrimp developed better and more uniformly than larvae fed by other foods. 3.2.3.2. Effects of procecced food regime on growth, survival and coefficient of variance of C. malacuta at the larvae to fry stage. a/ Survival rate

Figure 3.53. Survival rate of larvae following feeding practice of processed food b / Growth The growth of length of the larvae to fry satge following feeding practices of processed food is shown in Table 3.16+3.17. Table 3.16+3.17. The growth rate of length of larvae following feeding practice of processed food during nursing time. Treatment Fish at the end of experiment Daily growth rate Specific growth rate

DWG DLG SGR SGRL W (g) TL (cm) W fl fl (g/day) (cm/day) (%/day) (%/day) Feeding at day 7 0.21 ± 0.020a 2.20 ± 0.10a 0007 ± 0.001a 0.043 ± 0.003a 14.62 ±0.32a 2.96 ±0.15a Feeding at day 9 0.24 ± 0.015a 2.40 ± 0.10ab 0.008 ± 0.0006a 0.050 ± 0.003ab 15.02 ±0.22a 3.25 ±0.14ab Feeding at day 11 0.22 ± 0.006a 2.53 ± 0.06b 0.007 ± 0.0006a 0.054 ± 0.002b 14.83 ± 0.09a 3.43 ± 0.10b Feeding at day 13 0.32 ± 0.02b 3.10 ± 0.27c 0.010 ± 0.0006b 0.073 ± 0.009c 15.99 ± 0.22b 4.10 ± 0.29c Feeding at day 15 0.34 ± 0.017b 3.33 ± 0.21c 0.011 ± 0.0006b 0.081 ± 0.007c 16.23 ± 0.17b 4.35 ± 0.21cd Feeding at day 17 0.41 ± 0.037c 3.64 ± 0.06d 0.014 ± 0.001c 0.091 ± 0.002d 16.82 ± 0.30c 4.64 ± 0.10d

Note: Different letters in the same column represent a statistically significant difference (P<0,05). TLfl (cm): fish length at the end

of the experiment; Wfl (g): fish weight at the end of the experiment; DLG (cm/day): daily length growth; DWG (g/day): daily weight growth; SGR(%/day): specific growth rate. 18

c/ Coefficient of variance and differientation of growth

Figure 3.61. Coeeficient of variance and growth differientation of C. malacuta at the larvae to fry stage during nursing time Based on results of survival, growth and differientation during experimental time, it is suggested that processed food can be used for nursing C. malacuta at the larval to fry stage. This is the basis to open up the prospect of replacing trash foods with processed food in rearing C. malacuta. The appropriate time to start feeding processed foods is 15 days old with the method of gradually replacing trash foods by processed foods at the rate of 20% per day. 3.2.4. Nursing of C. malacuta at the fry to fingerling stage 3.2.4.1. Effects of nursing density on growth, survival rate and coeficiente of variance of C. malacuta at the fry to fingerling stage a/ Survival

Figure 3.64: Survival rate of C. malacuta at the fry to fingerling stage at different nursing densities b/ Growth In addition to the increase in body weight, body length was also observed increase and presented in Table 3.19+3.20.

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Bảng 3.19+3.20 . Growth rate of C. malacuta at the fry to fingerling stage at different nursing densities Treatment Fish at the end of Daily growth rate Specific growth rate experiment DWG DLG SGR SGR W (g) TL (cm) W L fl fl (g/day) (cm/day) (%/day) (%/day) 1.0 fry/L 0.87 ± 0.020c 5.25 ± 0.026c 0.022 ± 0.001a 0.099 ± 0.001c 4.45 ± 0.08c 2.71 ± 0.018c 1.5 fry/L 0.86 ± 0.015c 5.16 ± 0.036c 0.021 ± 0.001a 0.096 ± 0.002c 4.40 ± 0.14c 2.65 ± 0.025c 2.0 fry/L 0.80 ± 0.021bc 4.83 ± 0.032b 0.019 ± 0.001ab 0.085 ± 0.001b 4.14 ± 0.06bc 2.41 ± 0.024b 2.5 fry/L 0.75 ± 0.02ab 4.47 ± 0.09a 0.018 ± 0.001b 0.072 ± 0.003a 3.92 ± 0.09ba 2.13 ± 0.072a 3.0 fry/L 0.71 ± 0.040a 4.30 ± 0.7a 0.017 ± 0.001b 0.065 ± 0.006a 3.74 ± 0.24a 1.99 ± 0.14a

Note: Different letters in the same column represent statistically significant difference (P<0,05). TLfl (cm): fish length at the end of

the experiment; Wfl (g): fish weight at the end of the experiment; DLG (cm/day): daily length growth; DWG (g/day): daily weight growth; SGR(%/day): specific growth rate. c/ Coefficient of variance

Figure 3.67. Weight coefficient of variance of C. malacuta at the fry to fingerling stage at different nursing densities Nursing of the fry to fingerling satge at the density of 1-1.5 fish/L gave the best results because of the low coefficient of variance (1.72-1.90), good growth in weight and length and a high survival rate (91.92-93.76%). 3.2.4.2. Effect of diets on growth, survival and coefficient of variance of C. malacuta at the fry to fingerling stage. a / Survival rate

Hình 3.68. Survival rate of C. malacuta at the fry to fingerling stage fed by different diets.

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b/ Growth Table 3.22+3.23. Growth of C. malacuta at the fry to fingerling stage fed by different diets Diets Fish at the end of experiment Daily weight growth Specific growth rate DWG DLG SGR SGR W (g) TL (cm) W L fl fl (g/day) (cm/day (%/day) (%/day) 3%BW 0.52 ± 0.031a 5.25 ± 0.026c 0.009 ± 0.001a 0.76 ± 0.11a 2.33 ± 0.20a 0.89 ± 0.13a 6%BW 0.67 ± 0.036b 5.16 ± 0.036c 0.014 ± 0.001b 0.91 ± 0.08b 3.15 ± 0.18b 1.70 ± 0.04b 9%BW 0.82 ± 0.030c 4.83 ± 0.032b 0.019 ± 0.001c 0.99 ± 0.09c 3.82 ± 0.12c 2.49 ± 0.05c 12%BW 0.85 ± 0.020c 4.47 ± 0.09a 0.020 ± 0.001c 0.94 ± 0.14c 3.95 ± 0.08c 2.53 ± 0.04c 15%BW 0.87 ± 0.020c 4.30 ± 0.17a 0.020 ± 0.001c 1.19 ± 0.08c 4.03 ± 0.08c 2.58 ± 0.04c Note: Different letters in the same column represent statistically significant difference (P<0,05) c/ Coefficient of variance and feed conversion ratio

Figure 3.75. Weight coefficient of variance of C. malacuta at the fry to fingerling stage during experiment

Firue 3.76. Feed conversion ratio of C. malacuta at the fry to fingerling stage during experiment

The results of this experiment showed that C. malacuta at the fry to fingerling satge fed on a diet of 9% BW (similar to 12, and 15% BW) grew faster than ones fed on 3-6% BW. However, due to the fact that FCR of 9% BW diet is lower than that of 12 and 15% BW diet, 9% BW is therefore recommended for nursing of C. malacuta at the stage of fry to fingerlings.

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GENERAL CONCLUSION

1. The blotched snakehead C. malacuta belongs to the group of predatory with nutritional characteristics can be described as follows: lower large mouth; the upper jaw is longer than the lower jaw; the protruding lower jaw has a row of sharp teeth. The stomach is U-shaped with a thick wall; the inner surface of the stomach has many folds making it easy to be expanded and generated a largely contraction force. C. malacuta interstine is short with a relatively thick wall; relative gut length/body length rate is 0.58 (ranges from 0.38 -0.79). The origin food accounts for 93.54%, while other foods only account for 6.46%. 2. The spawning season of C. malacuta mainly occurs between April and June. The maturity sizes of males and females are between 20-25 cm. C. malacuta eggs are in the form of floating eggs, the absolute and relative fecundity ranges from 3,985-7,283 eggs/fish and 9,799-15,261 eggs/kg of female fish, respectively. 3. The combination of 50% trash fish and 50% comercial feed pellets in broodstock rearing was the most suitalbe feeding practice as it improved the fecundity, quality of eggs and larvae. The diet of 9% of BW improved the reproductive performance of C. malacuta brood fish. 4. In terms of spawning stimulation, C. malacuta males were injected 24 hours prior decision dose on females, the injection time for the female between the decision dose and the preliminary dose was 18h. Using the pituitary at a dose of 12mg/kg fish resulted in the best spawning results, however, C. malacuta did not breed when injected pituitary at a dose of 9-11 mg kg fish. Injection of LHRHa + DOM showed the best spawning results with high fecundity (27.404 eggs/kg), fertilization rate (87.91%) and hatching rate (85.85%). 5. The combination of moina-threadworms and moina –shrimp in nursing of the larvae to fry stage significantly improved survival rate, shock resistance, growth rate and uniform size compared to other foods. The suitalbe time to start changing trash feed to processed feeds is 15 days of age after hatching. 6. In nursing of the fry to fingerling stage, comercial feed pellets should be used with a diet of 9% BW and the optimal stocking density is 1.0-1.5 fish/ L. This stocking density ensures a good quality of fingerling because of its low coefficient of variance (1.72- 1.90) and high survival rate (91.92-93.76%)

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NEW CONTRIBUTION OF THE THESIS The thesis has academic, theoretical and practical contributions as follow: 1. The thesis has discovered the nutritional and reproductive biology of C. maculata in natural environments. The C. maculata has a lower large mouth; the upper jaw is longer than the lower jaw; the protruding lower jaw has a row of sharp teeth. The stomach is U- shaped with a thick wall; the inner surface of the stomach has many folds making it easy to be expanded and generated a large force of contraction. C. maculata interstine is short with a relatively thick wall; relative gut length/body length rate is 0.58 (ranges from 0.38 -0.79). The animal origin food accounts for 93.54%, while other foods only account for 6.46%. The spawning season of C. maculata occurs mainly from April to June. The maturity sizes of males and females are between 20-25 cm. C. maculata eggs are in the form of floating eggs, the absolute fecundity ranges from 3,985-7,283 eggs/fish and the relative fecundity ranges from 9,799-15,261 eggs/kg of female fish. 2. The thesis has established some important technical aspects in seed production of the blotched snakehead C. maculata. Particularly, in broodstock rearing, a combination of 50% trash fish and 50% comcercial feed pellets with a feeding regime of 9% BW are the most suitable feeding practices for C. maculata broodfish to improve their fecundity and quality of eggs and larvae. Males are injected 24 hours before the deciding dose of females; the injection time between the decision dose and the preliminary dose of females is 18h. Using pituitary at a dose of 12mg/kg results in the best success in stimulating C. maculata spawning. The hormones RHLHa + DOM contributes significantly to improving fecundity, quality of eggs and larvae and shorten the recurrence time of brood fish. The thesis determined suitable foods and a feeding change time for the larvae to fry stage. Feeding moina - threadworms and moina – shrimp significantly improve survival rate, shock resistance, growth rate and minimize the coefficient of variance. The suitalbe time to start changing trash foods to processed foods is 15 days of age after hatching. 3. Based on the scientific experiments on artificial reproduction of C. maculata, the thesis has proposed technology solutions to enable this species breeding in artificial conditions, thereby proactively supplying its fingerlings, promoting diversification of aquaculture species.

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LIST OF PUBLICATIONS 1. Ta Thi Binh, Nguyen Dinh Vinh, Nguyen Huu Duc and Do Van Tu. The effects of food and diet of C. maculata (Lacepède, 1801) broodfish on fecundity, quality of egg and fry. Journal of Aquatic Science and Technology - Nha Trang University, No. 2, 2018, pages 9- 15. 2. Ta Thi Binh, Nguyen Dinh Vinh, Nguyen Huu Duc and Do Van Tu. The effects of feeding conversion regime on growth, survival rate of C. malacuta at 4 to 30 days old. Journal of Agriculture and Rural Development - No. 10, May 2, 2018, pages 118-125. 3. Ta Thi Binh, Nguyen Huu Duc, Do Van Tu and Nguyen Dinh Vinh. Premilary results on reproductive stimulating C. malacuta in artificial conditions. Scientific report - National conference on biotechnology 2018. Natural Science and Technology Publishing House, October 2018, pages 1696-1704. 4. Ta Thi Binh, Nguyen Huu Duc, Do Van Tu, Nguyen Dinh Vinh and Nguyen Cong Thanh. Study on nutritional characteristics of wild C. malacuta (Lacepède, 1801). Scientific report on Fishery in Vietnam - The first National Conference on Fishery Science 2019. Natural Science and Technology Publishing House, June 2019, pages 11-21. 5. Ta Thi Binh, Nguyen Huu Duc, Do Van Tu, Nguyen Dinh Vinh and Nguyen Cong Thanh. Seasonal spawning cycle in nature of blotched snakehead Channa maculata (Lacepède, 1801). Journal of Biology. No 2se1&2se2, September 2019, pages 255-264.

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