A thesis submitted in partial fulfillment of the requirements for the degree of bachelor of aquaculture

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CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES THE DIGESTIVE TRACT DEVELOPMENT OF SNAKEHEAD FISH Channa striata LARVAE By TRUONG THI TU NGA A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2012 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES THE DIGESTIVE TRACT DEVELOPMENT OF SNAKEHEAD FISH Channa striata LARVAE By TRUONG THI TU NGA A thesis submitted in partial fulfillment of the requirements for The degree of Bachelor of Aquaculture Supervisor Dr. PHAM THANH LIEM Can Tho, December 2013 4 Acknowledgements First of all, I wish to express my deep appreciation and sincere gratitude to my supervisor Dr. Pham Thanh Liem for his constant guidance, advice and encouragement in my thesis research and writing. I am gratefully indebted to Mrs. Dang Thuy Mai Thy for her careful guidance in Histological laboratory. I also greatly indebted to Mr. Nguyen Hong Quyet Thang and Nguyen Ngoc Quy for their assistance in the wet laboratory. Many thanks to my classmate in Advanced Aquaculture Class course 35 for their support during my work in the college of Aquaculture and fisheries. Finally, I really want to thank my academic adviser, Dr. Duong Thuy Yen, who was guiding and encouraging me over the last four years, and my family for their great lifetime support which makes everything possible for me. i Abstract The purpose of this study was to observe changes of digestive tract as nutritional characteristics of snakehead fish Channa striata larvae in order to understand feeding behaviour of this species. Development of digestive tract was examined by morphological and histological characteristics. The fish was sampled at day 1, 3, 5, 7, 9, 12, 15, 18, 21, 25, 30 after hatching. For morphological observation, specimen of 10 fishes was sampled to observe the digestive tract under microscope. At the same time, 40 fishes was collected and fixed in buffer formalin 10% or Bouin’s solution to observe the digestive tract by histological method. The morphological result showed that at hatching, development of snakehead larvae exclusively depended on nutrients stored in the yolk. At this time, the digestive tract just was a straight line tube. At 3 day after hatching (DAH) when the fish larvae started exogenous feeding, the digestive tract still was undifferentiated but the zone from which the stomach will be differentiated could be identified. The digestive tract was divided into buccopharynx, esophagus, stomach and intestine distinctly on 5 DAH. The intestine started to coil on day 7. The results of digestive tract development were observed by histological method indicated that the first signs of lipid absorption could be identified as lipid vacuoles in intestine on 7 DAH. The intestine epithelium accumulated of lipid in posterior region from 15 DAH. The gastric gland appeared on day 12, revealing that the stomach became functional. At this time, the digestive tract of this fish was completed in both morphology and histology. ii Table of contents Page Acknowledgements ............................................................................................i Abstract ..............................................................................................................ii Table of contents ................................................................................................iii List of tables.......................................................................................................iv List of figures .....................................................................................................v CHAPTER I: INTRODUCTION ....................................................................1 1.1 General introduction .....................................................................................1 1.2 Research Objectives .....................................................................................2 1.3 Research Contents ........................................................................................2 Chapter 2: LITERATURE REVIEW .............................................................3 2.1 Biology of snakehead ..................................................................................3 2.2 Status of snakehead culture in Mekong delta ................................................4 2.3 Histology overview ......................................................................................5 2.4 Histological studies on fish ...........................................................................6 2.5 General morphology and histology of digestive tract ....................................8 Chapter 3: RESEARCH METHODLODY .....................................................12 3.1 Time and place .............................................................................................12 3.2 Materials and methods ..................................................................................12 3.3 Morphological characteristics analysis .........................................................13 3.4 Histological method .....................................................................................13 3.5 Result recording ...........................................................................................15 Chapter 4: RESULTS AND DISCCUSSION .................................................16 4.1 Morphology development .............................................................................16 4.2 Histological development .............................................................................18 Chapter 5: CONCLUSION AND RECOMMENDATION ............................30 5.1. Conclusion ..................................................................................................30 5.2. Recommendation .........................................................................................30 REFERENCES .................................................................................................31 iii List of tables Table 3.1: Chemicals and duration of tissue processing Table 3.2: Chemicals and duration of staining step Table 4.1: Mean total lengths, yolk sac lengths, gut lengths, mouth size and relative gut of lengths (RGL) of Channa striata larvae iv List of figures Figure 2.1: Snakehead fish Channa striata Figure 4.1: 1 DAH larva Figure 4.2: 3 DAH larva Figure 4.3: 5 DAH larva Figure 4.4: 7 DAH larva Figure 4.5: 9 DAH larva Figure 4.6: 12 DAH larva Figure 4.7: 15 DAH larva Figure 4.8: 18 DAH larva Figure 4.9: 21 DAH larva Figure 4.9: 21 DAH larva Figure 4.11: 30 DAH larva Figure 4.12: Longitudinal section of 1 day old snakehead Channa striata larva Figure 4.13: Longitudinal section of buccopharynx of 7 days old larva Figure 4.14: longitudinal section of anterior buccopharynx Figure 4.15: longitudinal section of the esophagus of 7 days old larva Figure 4.16: longitudinal section of the stomach of 3 days old larva Figure 4.17: longitudinal section of 3-day old larva Figure 4.18: Longitudinal section of the stomach of 12 days old larva Figure 4.19: Longitudinal section of the somach og 18 days old larva Figure 4.20: longitudinal section of the stomach Figure 4.21: cross section of stomach Figure 4.22: longitudinal section of the intestine of 7 days old larva Figure 4.23: cross section of the posterior intestine of 7 days old larva Figure 4.24: cross section of intestine of 18 days old larva v vi 0 Chapter 1: Introduction 1.1 General Introduction In recent years, aquaculture has been developed continuously and plays an important role in Vietnamese economy. The culture area increased from 445,300 ha to 570,300 ha and production also increased from 365,141 tones to 518,743 tones in 2002-2004. The culture area continued to increase from 658,500 ha to 685,800 ha with increasing production of 773,294 tones to 983,384 tones in 2004 – 2005 and total production reached 5,157,600 tones in 2010. This development leads to the increase of both quantity and quality fingerlings. Thus, the main cultured species such as tra catfish, tilapia, red tilapia, climbing perch…especially snake head fish need to be studied on early life stage that can understand larval characteristics as feeding behaviors and food type of fish larvae, which can increase fingerling production. According to Truong Thu Khoa and Tran Thi Thu Huong (1993) there are four snakehead species belonging to Channidea family including Channa striata, Channa micropeltes, Channa lucius and Channa gachua naturally distributed in the south of Vietnam. However, Channa striata is an important species and cultured widely in Mekong delta with total production of 1,094,879 tones in 2008. The fish is an air-breathing species; therefore, they can survive in harsh environment with low oxygen, and high ammonia contents (Marimuthu and Haniffa, 2007). Hence it’s often cultured in grow out ponds at high densities. In addition, the fish is well known for its taste, high nutritive value, recuperative and medicinal qualities (Khanna, 1978) so it’s one of high value freshwater species. Because of prominent characteristics mentioned above, this species was chosen to culture in intensive system since1990 up to now this system has been developed rapidly, and snakehead culture becomes a major job for many farmers in the Mekong delta. It is cultured in various culture models such as earthen ponds, canvas tanks, concrete tanks, hapas… However, when culturing this species; farmers sometime get trouble because of high mortality of larvae and juvenile fish. The main reason comes from cannibalistic behavior observed 2–3 days after hatching, which occurs when food supply is inadequate. So food supply during larval stage is an important factor to improve fish survival rates. Therefore, some characteristics of morphological and histological changes of digestive tract of fish need to be studied carefully to provide 1 the information that can help improve fingerling quality and commercial production. Therefore, the thesis “Study on the digestive tract development of snakehead fish (Channa striata) larvae” were conducted to observe changes of digestive tract of snakehead fish from newly hatch up to completed development. 1.2 Research Objectives Research carried out to examine the morphological and histological change in digestive tract in order to understand feeding behaviour of this species 1.3 Research Contents - Observation and description (by drawing) on morphological changes of the digestive tract of snakehead larvae. - Observations on histological changes in the digestive tract from newly hatch up to 30 days old. 2 Chapter 2: LITERATURE REVIEW 2.1 Biology of snakehead According to Truong Thu Khoa and Tran Thi Thu Huong, 1993, the taxonomy of Channa striata is as following: Class: Actinopterygii Order: Perciformes Family: Channidae Genus: Channa Species: Channa striata Figure 2.1: Snakehead fish Channa striata Snakehead fish Channa striata, Bloch 1793 is air breather species and popular presence in India and some South East Asia countries as Vietnam, Laos, Thailand, Cambodia, Myanmar. Areas with an abundance of wild resources are found in swamp, canal, lagoon, rice field. The fish can live in bad water quality, brackish water and tolerate to water temperature above 300C (Ngo Trong Lu and Thai Ba Ho, 2001). Most of Channa striata distributes in fresh water but it can be found in brackish water at the salinity of 5-7ppt (Pham Van Khanh, 2000). External morphology: snakehead has sub-cylindrical body; depress head; round caudal fin. The dorsal surface and sides is dark and mottled with a combination of black and ochre, and white on the belly; a large head reminiscent of a snake's head; deeply-gaping, fully toothed mouth; very large scales. Internal morphology: the fish has short esophagus and thick wall, inside of esophagus have many folds. Stomach has sacciform and Y shape. Observing 3 digestive tract, it contain 63.01% of fish, 35.94% of shrimp, 1.03% of frog, 0.02% of insect and organic matter (Duong Nhut Long, 2003). According to Duong Nhut Long (2003) snakehead larvae use yolk for nutrition source during first 3 days after hatching, after 5-7 days, it can be feed by Moina, Daphnia, tubifex, or chironomid. The juvenile eat superworm and muckworm. When becoming adult fish, the diets are shrimp, fish on small size in nature or pellet feed in commercial pond. This species can breed all year round but spawning season occurs mainly from May to July. It usually spawns 1-2 hours after showery, in the morning or in quiet places with more phytoplankton. The fertilized eggs hatch after 3days at temperature of 25-30oC. The larvae metamorphosed to juvenile within 20 days after hatching (Marimuthu and Haniffa, 2007). 2.2 Status of snakehead culture in Mekong delta In recent years, when tra catfish culture has problems due to market price of fish is not stable and diseases, many farmers change to culture snakehead that can supply for domestic consumption because it is high price (more profit for farmers), delicious (high quality of meet), easy to culture or high tolerance to adverse environment, the fish can be cultured at high density,… (Trieu Thi Y Vanne, 2011) Snakehead was wildly culture in Đong Thap, Hau Giang, Can Tho, Soc Trang, especially in An Giang province with culture area was 67 ha (occupy 26.2% of aquaculture aera) and reached 22.273 tons/year of snakehead production (An Giang statistics, 1/11/2010). The fish is culture effectively with various models as earthen pond, cage, pen, hapas, canvas tank, concrete tank… Culturing snakehead in earthen pond was one of common models has been developed early. In common pond, the cultural area is 100-1000m2 with stocking density of 20-30 fingerlings/m2. Trash fish, frog, small prawn or commercial feed are used to feed snakehead. After 6-7 months the fish can attain 0.8-1.0 kg (Duong Nhut Long, 2003). The fish can be cultured in grow out ponds at high density of 40-80 fish/m2 with annual yield ranging 7-156 tonnes/ha (Wee, 1982). 4 Besides that, culturing in hapas also is effective model (typically in Ben Tre province). Hapas is rectangle shape (5x3x2m) and the distance from pond bottom to haps bottom is upper 0.5cm. Advantages of this model are high stocking density (50 fishes/m2) and production; safety in flooding season; famer can put many hapas in a pond but still remain a part of pond to culture other species that can use waste feed from hapas, which reduce pollution and get more profit; fishes cannot touch or engulf inside the mud pond bottom so they have less rub and grow faster. The other hand, if farmers have poor management about feeding (quality, quantity…) and water exchange, they will lose money by disease. ( Duong Nhut Long, 2012) However, nowadays culturists tends is culture snakehead in canvas tank with using commercial feed. This is a model that can help farmers get high profit but not require large culture area, easy to invest and reduce environment pollution (Trieu Thi Vanne, 2011). Snakehead fish culture is more developed in many provinces of the Mekong delta, which have creative methods. For instances, in Tra Vinh province just apply a new culture method that is using well water to culture snakehead in earthen pond. Now, having more than one hundreds farmers are applying this method successfully and brings out more net income (Le Hoang Vu, 2013) 2.3 Overview of histological history Histology was developed early; the first histologist is Malpighi (1628-1694) – founder of histological anatomy (study on hedgehog). He own many discoveries about taste buds, capillaries, chick embryology, insect don’t use lungs to breath…describe bile, spleen, skin and other organs so some structure bring his name. In 1665, Hook (1635-1703), an English microscopist and physic, when examining a piece of cork with rudimentary microscopic, saw an abundance of empty small compartments - the cell was discovered. In that very years Hook discovered the cell, he and Malpighi were the first to observe the true unit that form the tissues of animals but now properly speaking cells. After that, leeuwenhoek (1700) who first described the nucleus when examining the red blood cells of salmon. The first description of nuclear envelope was accomplished by Purkinje (1787-1869) in 1830, he also introduced the term protoplasma (1840). However, who verified the constancy of organelle and who introduced the term of nucleus under microscopic was Brown (1773-1858) in 1831, after examination of epidermal cells of some orchids and some Asclepiadacea. Virchow (1821-1902), a great German pathologist, contributed to establish cell theory, he demonstrated that the pathological injuries also has a cellular 5 structure; thus he is considered to be pioneer of the cellular pathology. Since this time, histology also was applied for studies on abnormal structures. Bichat (1771-1802), a French pathologist is known as father of histology, he was first person to look beyond the recognizable organ system and suggest that each part of the body was composed of various tissues. In addition, he suggested that disease acts upon these tissue is ways that could be seen and studied. Reichenbach (1830) discovered paraffin wax that is used as embedding medium for histological analysis of natural tissues. Paraffin wax is mostly found as a white, odorless, tasteless, waxy solid with a typical melting point between about 46 and 680C (115 and 1540F). It is insoluble in water, but soluble in ether, benzene, and certain esters. Paraffin is unaffected by most common chemical reagents but burn readily. Furthermore, Wilhelm (1865) - a Swiss anatomist and professor invented the microtome. By treating animal flesh with acids and salts to harden it and the slicing very thinly with the microtome, scientists were able to further researches the organization and function of tissues and cells in a microscope. In the area of aquaculture, histology also has significant contributions. For instances, histological studies on the reproductive phenomenon of fish have been very useful in devising fry production methods, and histopathology is of extreme importance in the diagnosis, etiology, and prevention of diseases. In addition, histological investigations may also help elucidate the effect of waste water on fish. Understanding these points, Hibiya (1982) described normal and pathological organ systems of some species such as rainbow trout, carp, eel… he have emphasized both the morphological and physiological aspects of tissue of fish. 2.4 Histological studies on fish Olurin et al. (2006) based on histology to explain histopathological responds of gills and liver tissues of Clarias gariepinus fingerlings to the herbicide, glyphosate. The gills showed marked alterations in the epithelia in response to glyphosate treatment. There was fusion in adjacent secondary lamellae resulting in hyperplasia, with profound oedematous changes, characterised by epithelial 6 detachment. In the liver, the enlargement of the hepatocytes was related to the concentration and duration of exposure to glyphosate. There were also large vacuoles in the hepatocytes, with pyknotic nuclei, and cytolysis that increased with concentration. Focal necrosis was also observed in the hepatocytes. They concluded that glyphosate has a deleterious effect on the organs of C. gariepinus Camargo and Martinez (2007) observed histological changes in gills, kidney and liver to evaluate the health of the Neotropical fish species Prochilodus lineatus. This research showed that the fish caged in the urban stream the most common lesions were epithelial lifting, hyperplasia and hypertrophy of the respiratory epithelium lamellar fusion, and aneurysms in the gills; enlargement of the glomerulus, reduction of Bowman’s space, occlusion of the tubular lumen, cloudy swelling and hyaline droplet degeneration in the kidneys; hepatocytes with hypertrophy, cytoplasmic and nuclear degeneration, melanomacrophage aggregates, bile stagnation and one case of focal necrosis in the liver. The lesions were comparatively most severe in the liver. Tagrid (2010) had a histological study of the gill sections of Cyprinus carpio it found that marked histological lesions, include hyperplasia and hypertrophy of the respiratory epithelium, bloody congestion with hemorrhage and abundance of mucous substance, this at high temperature, while at low temperature also showed hyperplasia, shrinkage of blood vessels, fusion of secondary lamellae, cellular atrophy, damage and lamellar disorganization. Lesions were comparatively most severity at low temperature. Sayrafi et al. (2011) had a histological study of hepatopancreas in hi Fin pangasius (Pangasius sanitwongsei). The results showed that the structure of hepatopancreas in this species was similar to the other fishes. However, there were also considerable structural differences. 2.5 General morphology and histology of digestive tract Digestion of fish is the process of converting food into smaller compounds that can be used by the body. Food is eaten through the mouth of the fish using the jaws. Most fish have teeth and an immoveable tongue. The food then passes through the pharynx (throat) into the esophagus and the stomach. Partial digestion 7 takes place in stomach using gastric juices (including acids and enzymes), and then the food proceeds to the intestine for more digestion and absorption into the blood (Huck, 2002). The gut length change depends on feeding habit (carnivorous or herbivorous) of fish; the gut length of herbivorous fish is higher than the gut length of carnivorous fish. However, the digestive tract is not developed as adult fish, Govoni et al. (1986) reported that at first feeding, the larval alimentary canal is functional, but is structurally and functionally less complex than that of adults. The larval alimentary canal remains unchanged histologically during the larval period before transformation. During transformation, major changes that result in the development of the adult alimentary canal occur. The ontogeny of the alimentary canal differs in different taxa, and experimental evidence suggests that functional differences exist as well. Assimilation efficiency may be lower in larvae than it is in adult fishes, due to a lack of a morphological and functional stomach in larvae. When the larvae open the mouth and start to exogenous feeding, the digestive tract becomes functional with differentiation of bucoopharynx, esophagus, future stomach and convoluted gut. According to Boulhic and Gabaudan (1992), after hatching, the digestive tract of Dover sole was a simple undifferentiated tube, close anteriorly. The first signs of intestinal absorption appear quickly after first feeding and can be identified as vacuoles in the midgut and eosinophilic granules in the hindgut. This is followed by the formation of muscle layers, tooth development and swim bladder inflation The mouth exhibits a variety of fascinating adaptations for capturing, holding and sorting food but at hatching, mouth is undifferentiated until it open. In a study of mouth development of Mystus nemurus larvae, Hag et al. (2012) showed that the larval mouth opened at the end of the 1 day-post-hatch (dph) and the commencement of external feeding began on 4 dph following the jaw movement. Khalil et al. (2011) stated that The buccopharyngeal epithelium of Oreochromis niloticus is composed of a few numbers of squamous cells covered differentiated taste buds; scattered in the anterior and posterior region of the buccal cavity. Mucus-secreting cells are arranged in one layer in the buccopharyngeal epithelium. The histology of buccopharynx of Schizothorax plagiostomus showed goblet cells interdispersed within the epithelium and appeared to increase substantially in numbers posteriorly toward the pharynx and as development proceeded, 8 stratification of the oral mucosa becomes more pronounced in 6.0 cm. long larvae (Bahuguna and Gargya, 2009). Esophagus is usually lined with a squamous epithelium, it surface shows concentric microridges, just as epithelial cells in the skin of teleosts. In the epithelium, mucous cell are abundant and taste bud may present (citied by Stroband, 1979). According to Arellano et al. (2001) the oesophagus and oesogaster of Solea senegalensis were made up of four distinct layers: mucosa, submucosa, muscular and serous. Two morphological types of epithelial cells were distinguishable in the oesophageal mucosa: the more numerous type cells possessed an electron-dense cytoplasm, whereas the cyto-plasm was electron-clear in the other cells. Mucus-secreting cells were the dominant feature of the epithelium throughout the oesophagus. These gob-let cells were filled with numerous mucous droplets of low electron-density. The oesophagus was devoid of taste buds. The function of fish’s stomach is to break down food so the stomach develops when fish larvae start to feed outside. The stomach usually shows two distinct sections: a corpus part with a lining of mucous-producing cells with underlying gastric glands and a pyloric part without gastric gland and it was made up of four distinct layers: mucosa, lamina propria-submucosa-, muscularis and serosa. Surface epithelial, glandular and rodlet cells were present in the mucosa (histological study of Senegal sole-Solea senagalensis). Cells of the columnar epithelium contained a basa1 nucleus. The lysosomes were small, round and dense. The gastric glands were numerous in the pyloric and fundic regions but absent in the cardiac stomach. These glands were formed by two cell-types: light and dark cells. The light cells were characterised by numerous mitochondria, while dark cells had slightly fewer mitochondria and a tubulo-vesicular system. Rodlet cells similar to those observed in other teleostean fish were present among the epithelial cells (Arellano et.al, 2001). Groman, (1982) stated that cardiac stomach had a thicker tunica mucosa than the other parts of digestive tract and added that the cardiac stomach in formed from epithelium, serous cardiac glands, lamina propria, granulosa layer and comptactum and tunica muscularis. According to Infante and Cahu (2001) In red drum the stomach is well differentiate as early as day 7 post-hatching. Besides that, some species don’t have stomach. Day et.al. (2011) stated that lacking of a stomach is 9
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