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
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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.
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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.
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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
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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
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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
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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
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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.
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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
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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).
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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
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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
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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
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