comparing different methods to determine digestibility of snakehead (channa striata) juvenile

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

COMPARING DIFFERENT METHODS TO DETERMINE
DIGESTIBILITY OF SNAKEHEAD (Channa Striata) JUVENILE

BY
NGUYEN THI THU NGAN

A thesis submitted in partial fulfillment of the requirements for the degree of
Bachelor of Science in Aquaculture

Can Tho, January 16, 2013



CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

COMPARING DIFFERENT METHODS TO DETERMINE DIGESTIBILITY
OF SNAKEHEAD (Channa Striata) JUVENILE

BY
NGUYEN THI THU NGAN

A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor
of Science in Aquaculture

Supervisor
Ass. Prof. Dr. TRAN THI THANH HIEN

Can Tho, January 16, 2013
i


Acknowledgements
I wish to express my deep appreciation and sincere gratitude to my advisor Ass.Prof. Dr
Tran Thi Thanh Hien for her constant guidance and also good advises in during the time
I did these experiments and wrote thesis.
The second thing I want to send nice messages to Mss. Ngo Minh Dung and Mss. Tran
Le Cam Tu who transfer to me all of cultural professional knowledge when I cultured
snakehead in wet lab 3 or analyzed in the nutritional laboratory.
Thanks to students of advanced aquaculture course 34, Aquaculture A1 course 35, and
Fisheries management course 35 who was enthusiastic to help me finish this thesis.
Finally, thanks my family and also relatives make all of good things from finance to
spirit help me strong during time I did it.

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Abstract
In this study, a series of experiment was done to compare different fecal-collection
methods and digestibility of some ingredients of snakehead. A reference diet was fed 2
hours interval on 24 hours to find down what time was appropriate for collecting feces
in the next experiment and defined dry matter and protein digestibility of snakehead by
settling technique. No significant effect between groups of digestibility through 24 hours
and time space from eight and ten hours was appropriate for applying three different
methods (settling, dissection, and stripping). Stripping was ineffective in this case.
Dissection method was also assessed. Dry matter and protein digestibility of this method
were lower than those on settling method. Therefore, settling was chosen as an
appropriate method to determine dry matter and protein digestibility of test ingredient

mixing with a control diet following 30:70 on ratio. Ingredients included fishmeal,
soybean meal, meat bone meal and blood meal. The results showed that the diet which
was made from fish meal was the best protein digestibility percentage (93.63±0.62), and
the lowest one was meat bone meal (88.93 ± 0.58%). Soybean meal and blood meal
were similar, 91.27 ± 0.58%, and 91.23±0.93%, respectively. In digestibility of
ingredients, fish meal was also the best one with 85.75± 1.90% on value, following by
soybean meal (69.73 ± 2.3%), and lowest one being meat bone meal (52.28 ± 1.73%). In
conclusion, some ingredients with animal protein sources were digested better than
plant origin, except meat bone meal.

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Table of Contents
CHAPTER I INTRODUCTION ......................................................................................... 1
1.1 Background .................................................................................................................... 1
1.2 Research objectives ....................................................................................................... 1
1.3 Research contents .......................................................................................................... 1
CHAPTER II LITERTURE REVIEW ................................................................................ 2
2.1 Characteristics of snakehead ......................................................................................... 2
2.1.1 Classification .............................................................................................................. 2
2.1.2 Biology characteristics ............................................................................................... 3
2.1.3 Nutrition characteristics .............................................................................................. 3
2.2 Study on on fecal collection and digestibility of some ingredients for fish .................. 3
CHAPTER III RESEARCH METHODOLOGY ................................................................ 7
3.1 Experimental site and period ......................................................................................... 7
3.2 Experimental fish ........................................................................................................... 7
3.3 Experimental facilities ................................................................................................... 7
3.3.1 Facilities...................................................................................................................... 7
3.3.2 Experimental feed ....................................................................................................... 7

3.4 Digestible series of experiments .................................................................................... 8
3.4.1 Experiment 1: Detection appropriate time for collecting feces. ................................. 8
3.4.2 Experiment 2: Determination of appropriate fecal collection method for
digestibility of snakehead .................................................................................................... 8
3.4.2 Experiment 3: Determination of digestibility of feed ingredients for snakehead. ... 12
3.5 Sampling and data analysis methods ........................................................................... 14
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CHAPTER IV RESULTS AND DISCUSSION ............................................................... 16
4.1 Environmental conditions ............................................................................................ 16
4.2 Comparison of fecal quantities and ADC settlement periods. .................................... 17
4.3 Comparing three fecal- collection methods ................................................................. 18
4.4 Diets digestibility......................................................................................................... 19
4.5 Ingredients digestibility ............................................................................................... 19
CHAPTER V CONCLUSION AND RECOMMENDATION ......................................... 22
5.1 Conclusion ................................................................................................................... 22
5.2 Recommendation ......................................................................................................... 22
Reference list ..................................................................................................................... 22
Appendix ........................................................................................................................... 25

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List of tables
Table 3.1: Chemical composition of experimental ingredients ...................................... 8
Table 3.2: Table for predicted fecal-collection time ..................................................... 11
Table 3.3 Pellet – feed from mixing reference diet and ingredients ............................. 13
Table 4.1: Environmental condition of experiment ...................................................... 16
Table 4.2: The ADCs for three methods ....................................................................... 18

Table 4.3: Diet digestibility of reference and treatment ............................................... 19
Table 4.4: Digestibility of some ingredients ................................................................. 20

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List of figures
Figure 3.1: Settling system .............................................................................................. 9
Figure 3.2 Stripping method ......................................................................................... 10
Figure 3.3 The structure of an intestine ........................................................................ 10
Figure 3.4 Pellet feed after mixing ................................................................................ 12
Figure 4.1: Dry weight and ADCs of snakehead on two hours interval ....................... 17

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List of abbreviations
ADCs

Apparent digestibility coefficients

FM

Fishmeal

SBM

Soybean meal

BM

Blood meal

MBM

Meat bone meal

RD

Reference diet

AIA

Acid insoluble ash

viii


CHAPTER I
INTRODUCTION
1.1 Background
In recent year, aquaculture is becoming majority in the Mekong Delta where national
economy can be improved and developed through domestic consumption and export to
many countries in the world. The area for culturing aquaculture estimates 737,600 ha
with production 1,869,484 tones, which accounted for over 73% the production of the
country (General Statistic Office, 2010). The main cultured species are Tra catfish,
shrimp, snakehead, and red tilapia. Snakehead (Channa striata) is one of the new species
which are cultured recently, but it is becoming potential aquaculture, which is production
get 40,000 tons in 2011 and some households can get 14tons per year. The average feed
cost which typically comprises over 80% of the total variable production cost in the

country (Tran Thi Thanh Hien and Nguyen Anh Tuan 2009). However, scientifically,
there has not been any study on digestibility of this kind of species. Practically, research
on the digestibility is also essential to cost effective conduction of aquacultural diets and
it contributed to establish feed formula for aquatic animals (Glencross et al. 2007).
However, researches showed that the digestibility of feed ingredients was influenced by
fecal- collection methods (Weatherup and Mc Cracken 1998; Vandenberg and de la Noue
2001; Glencross et al 2005). Therefore, the study aimed to detect the appropriate fecal
collection methods and determine the protein digestibility of different feed ingredients on
snakehead (Channa striata) juvenile.
1.2 Research objectives
Detecting the appropriate fecal collection to determine the digestibility of some
ingredients of snakehead juvenile in order to determine the protein digestibility of this
species.
1.3 Research contents
Comparing different fecal collected methods to determine digestibility of snakehead
(stripping, dissection and settlement)
Comparing digestibility of the different rich protein ingredients of snakehead
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CHAPTER II
LITERTURE REVIEW
2.1 Characteristics of snakehead
2.1.1 Classification
Channidea is the family of snakehead, including 2 genuses: one genus is Channa, lived in
Asia and other is Parachana, lived mostly in Africa. There are 30 species of snakehead
today in the world, 5 species distributed in Vietnam and some Asia countries. According
to Rainboth (1996), snakehead belongs to:
Order: Perciformers
Family: Channaidae (Ophiocephalidae)

Genus: Channa (Ophiocephalus)
Species: Channa striata

Some sneakhead species lived in Mekong Delta:
- Snakehead murrel (Channa striata)
- Dwarf snakehead (Channa gachua)
- Giant sneakhead (Channa micropeltes)
- Splendid snakehead (Channa lucius )
2


2.1.2 Biology characteristics
According to Duong Nhut Long (2003), snakehead is abundant in rice fields, rivers, canal
and lagoon. It can adapt with the environment which is low oxygen, high turbidity and
high temperature (over 300C) in freshwater and brackish water (8-12‰).
Snakehead is likely to live in where there have much ceratophyllum demersum, aquatic
plants, and a mass of duck weed. There are good places for snakehead to hide for prey.
These elongated and predatory fish are distinguished by a long dorsal fin, large mouth
and shiny teeth. The fish have short gullet, thick wall, and wrinkle inside gullet. The big
stomach is likely Y shape. Observing digestive duct, it contains 63.01% of fish, 35.94%
of prawn, 1.03% of frog, and 0.02% of insects and organic matter.
2.1.3 Nutrition characteristics
According to Duong Nhut Long (2003), snakehead use yolk for nutrition source during
first 3 days after hatching and yeast, egg custard and power feed were the next chosen
feed for dry duration. After 5-7 days, snakehead can choose Moina, Daphnia, tubifex, or
chironomid. The juvenile eats likely with superworm and muckworm. When becoming
adult fish, they are good prey, mainly diets are shrimp, fish on small size in nature or
pellet feed in commercial pond.
2.2 Study on fecal collection and digestibility of some ingredients for fish
A research on effects of protein and energy ratio on fingerling snakehead (Channa

striata) conducted by K. Samantary and S.S Mohanty (1997). Fingerlings on 12 ± 2g
was used with labolatory – made brown fish meal being a major source of protein in all
the test diet which was fed 3% on body weight per day. There are four protein levels and
three energy levels at each protein level were used. The result showed that 40% crude
protein diets with 90.9 mg protein per kcal of digestible energy were the best result for
highest growth and 45% crude protein diets with 93.8 mg per kcal on P/E ratio were
lower result.

3


Indirect technique through inert marker is used popularly in aquaculture and terrestrial
animal. According to Tran Thi Thanh Hien (2004), the characteristic of marker is: (1)
the speed of movement like as nutrient, (2) indigestibility and solubility in water, (3) no
affecting digestibility and absorption of nutrients. Because the inert markers do not
affect digestibility and absorption of nutrients, the rate of marker in feed and feces is the
digestibility of feed. Some markers are used for research, namely, Cr2O3, HROM, HRA,
Cs137, Cr51. One of them, Cr2O3 is used mostly popular with the rate between feed and
marker 0.5 – 1%
The research of Sales and Britz (2001) compared the use of 3 markers including:
Chromic oxide, acid-insoluble ash (AIA) and crude fiber in South Africa abalone
(Haliotis midae L.) feed. Results showed that AIA was the only marker that yielded
consistence realistic apparent digestibility coefficients (ADCs), the amount of Cr2O3 and
crude fiber in feces was equal or lower than in feed, causing negative result. Therefore,
Cr2O3 was used popularly marker in the research about digestibility of aquatic animal
but not in mollusk as abalone.
Beside, fecal-collection was divided into 2 groups: (1) direct technique: using gentle
pressure contact to abdomen of fish to expel feces ( Nose, 1960), using tool to suck
feces out of intestine or cutting final part of intestine to take feces (Windell, 1978).
These above methods had disadvantages including: the feces had mixed with

unabsorbed feed, digestible enzyme, urine, and death sock fish. (2) fecal-collection in
culture system to determine the digestibility of feed was more accuracy and reduced
cons from direct technique, researchers applied fecal collection by culturing fish in
water. Two mail methods are settling and continuous methods (Guillaume et al. 1999).
Stripping method was applied popularly in many kinds of fish: white salmon fish
(Oncohynchus tshawytscha) grouper, Morone saxatilis x Monrone chrysops (Sullivan and
Reigh 1995) and Sciaenops ocellatus L., (Gaylor and Gatlin 1996). The advantage of this
method was that it could be repetition in experiment. However, Hajen et al. (1993)
showed that this repletion could make fish shock and reduce apparent digestibility
4


coefficient (ADC). Beside, expelling feed suddenly in anaesthetized situation, it could
encourage fish spawn unexpected (cited by Weatherup and Mc Cracken, 1998).
The research of Amirkolaie et al. (2005) compared between 2 indirect techniques was
that fecal collection way of Choubert and settling method on tilapia. The system of
Choubert with mesh size was 1 mm and continuously collected, discharge water from the
tank would lead to metal layer to separate feces and water. The feces were hold on metal
tray. The settling tank is commercial tans Aqua Optima with 170ml of volume and 44cm
of height with diameter 24.5 cm. There was the tube, connecting with fecal containers,
which kept cool by ice for prevent of bacterial infection in collecting time. As a result,
water in the tanks had just moved a little which gave correct result and collected feces
was higher than continuous method.
Hien et al. (2009) research on Tra catfish, determine the digestibility of some ingredients
was significant for research nutrition of fish. The results of research could help to find
down the material, which fish could digest well to improve the efficient of culturing of
fish. Therefore, there were many kinds of research about digestibility, especially, the
digestibility of material in different fish.
According to Tran Thi Thanh Hien (2004), the amount of fiber in feed could highly affect
to the digestibility of feed of aquatic animal. Fiber could increase the speed of feed

through digestive system so it could increase amount of intake feed and made slow
growth of aquatic animal. It is relevant with a research of Umesh et al. (1994) about
digestibility of protein and dry matter from Spirulina platensis algae by common carp
(Cyprinus carpio). The experiment was conducted with one control and nine treatments
with adding Spirulina platensis algae with increase amount from 10-90% in formulated
feed. The diets with adding algae were increase in protein and reduction on fiber value. A
result was that the dry matter digestibility was lower in control than in treatment. They
checked the result among group of treatments that the digestibility increased when the
amount of algae in treatments went up. It meant that digestibility was inversely
proportional with the amount of fiber in feed. The protein digestibility also increased in
5


the quantity of algae, accepting two treatments adding 60% and 70% digestibility slightly
went down. However, the protein digestibility increase in remain treatments and highest
in adding 50% of algae (96.55%), fiber content in that treatment was 7.08% on diet.
Therefore, the highest protein digestibility was done by common carp when fiber content
7% on diet.
The research of ingredient digestibility showed that the amount of ingredients mixed with
feed was usually 30% of control and 70% of treatments. However, some results
convinced that the quantity of mixed material did not affect ingredient digestibility. Kim
et al. (2006) studied about the digestibility of haddock (Melanogrammus auglefinus L.),
when changing the amount of tested material to herring fish meal in experimental diet
with quantity was about 10%, 20%, 30%, 40% and 50%. A result showed that protein and
energy digestibility among treatments was highly significant difference with 10-15%
amount of herring meal. Protein and energy digestibility with the average amount of
herring fishmeal among treatments were 94.0% and 95.5%.

6

CHAPTER III
RESEARCH METHODOLOGY
3.1 Experimental site and period
The study was conducted from June/1/2012 to December/15/2012. The experimental
diets formulated in the laboratory in Department of nutrition and products processing,
college of Aquaculture and Fisheries, Can Tho University.
3.2 Experimental fish
All of snakeheads (Channa striata) used in three experiment was 120g/individual in size.
The snakehead fishes were bought from farmers in Can Tho city.
The experimental fish, which was healthy, without graze fin, no infection with disease.
Those fish fed different diets and tamed with the experimental condition.
3.3 Experimental facilities
3.3.1 Facilities
Experiment conducted with six settling tanks (170L). These tanks designed as rounded
tanks with conical bottom, which had a valve in each to avoid the feces following the
flow going out and connected with composite fecal settling bottle to collect the feces for
experiment. In addition, these tanks used flowing out water system to be clean water and
slightly aeration to ensure snakehead having good condition to live.
3.3.2 Experimental feed
The formulated feed (45% crude protein) used in this experiment which was sinking in
the tanks; it is based on a protein and energy ratio of snakehead of Mohanty and
Samantaray (1997). The main ingredients contained fishmeal, soya meal, wheat flour,
vitamin, mineral, oil, adhesive substance. The feed should be stored in refrigerator.

7


3.4 Research methodology
3.4.1 Experiment 1: Detection appropriate time for collecting feces.

a. Experimental feed
The fish fed by formulated diet containing 45% crude protein, which mixed with chromic
oxide (Cr2O3) marker with 1% in weight.
Table 3.1: Chemical composition of experimental ingredients
Chemical composition
Treatment

Moisture

Protein

Lipid

Mineral

Fiber

(%)

(%)

(%)

(%)

(%)

Fishmeal

14.37±073

63.02±0.47

4.55±0.61

1.39±0.08

0.01±0.002

Soya bean meal

7.49±0.08

47.20±0.66

2.9±0.25

0.1±0.0034 0.04±0.005

Meat bone meal

16.36±0.14

52.99±1.11 10.00±0.46

2.16±0.02

0.03±0.002

Blood meal

6.29±0.29

91.51±1.41

0.39±0.02

0.03±0.01

0.36±0.17

c. Experimental design
Fish fed seven day prior to make acclimation with cultural condition. All feces were
collected on eighth day, feces analyzed, dry matter and protein digestibility for control
diet( 45% crude protein) was calculated and compared with feces collected every 2 hours
after feeding (2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours).
During acclimation period, fish fed once a day at 8a.m by hand. The quantity of feed
depended on demand of fish. After feeding 1 hour, tanks were cleaned by taking uneaten
feed out.
3.4.2 Experiment 2: Determination of appropriate fecal collection method for
digestibility of snakehead
a. Experimental fish and feed
The fish which was used in experiment 2 was also used in experiment 1
Experimental fish: reference diet mixed with chromic oxide 1% in weight.
8


b. Experimental design
The experiment was conducted randomly three treatments for three fecal collection
methods: stripping, dissection, and settlement (three replications per treatment). Six

conical tanks used for this experiment which three used for settlement and three for
striping method.
- Treatment 1: settling method, the snakehead stocked 45 individuals in three composite
settling tanks (170L), appropriately 88inds/m3.
- Treatment 2: striping method, the snakehead stocked 45 individuals in three composite
tanks (170 L), appropriately 88ind/m3
- Treatment 3: dissection method, killing all of fish of treatment 1 and treatment 2.

Figure 3.1: Settling system

9


Figure 3.2 Stripping method

Figure 3.3 The structure of an intestine

10


After finished seven day of fish feeding, fecal-collecting was recommenced in eight day
with covering of three techniques.
Table 3.2: Table for predicted fecal-collection time
Day

Settling technique

1st - 7th
8th - 10th
11th

feeding
collecting feces+ feeding

Dissection technique

Stripping technique
feeding
stripping + feeding

dissection (all of fish)

After 7 prior days, stripping and settlement was applied on eighth day.
Settling technique: after 8 hours of fish feeding, fecal samples were collected by using
accumulating method naturally and the samples were collected continuously next three
days. The sample containers were cleaned before starting collecting new samples. The
time for settling feces naturally was about 10 hours; the settling containers should keep
cool in ice to prevent decomposition of fecal sample. After the fecal sample had already
settled, valve opened for discharging to containers, 250 mL of volume. Those containers
stored in the refrigerator a day to completely deposit of feces. Then, water and feces
separated again, feces transferred to tumblers and dried it for analysis.
 Stripping technique: after 8 hours, the sample collected by using gentle pressure
applying to abdomen near the final intestine of the fish to expel feces onto fingertips.
All of fish should be done as quickly as possible and fecal sample was dried for
analysis.
 Dissection technique: after 8 hours, the fish from settling tanks used for this method.
After taking sample from settling method, the fish continuously fed a day, all of fish
within each tank killed. The distal intestine dissected to expel feces with gentle pressure
into sample container. The fecal samples dried from analysis.

11


3.4.2 Experiment 3: Determination of digestibility of feed ingredients for
snakehead.
a. Experimental diet:
 Control diet: base feed (45% protein) likely experiment 1.

 Treatment diet: feed determined digestibility of ingredients, containing 70% control +
30% required raw material. The test ingredients, containing include: fish meal, soya

meal, meat bone meal and blood meal.
All of used feed mixed with chromic oxide (Cr2O3) marker with 1% on concentrated.

Figure 3.4 Pellet feed after mixing

12


Table 3.3 Pellet – feed from mixing reference diet and ingredients

Treatment

Amount (%) Amount (%) Amount (%) Amount (%) Amount (%)

Fishmeal

36.27

25.39

25.39

25.39

25.39

Soybean meal

34.98

24.48

24.48

24.48

24.48

Wheat flour

18.92

13.24

13.24

13.24

13.24

Vitamin

1.98

1.39

1.39

1.39

1.39

Oil

5.86

4.10

4.10

4.10

4.10

CMC

0.99

0.69

0.69

0.69

0.69

Cr2O3

1.00

0.70

0.70

0.70

0.70

Fishmeal

-

30

-

-

-

Soybean meal

-

-

30

-

-

-

-

-

30

-

-

-

-

-

30

100

100

100

100

100

Meat

bone

meal
Blood meal
Total

b. Experimental design
 The experiment contained a control and four treatments with four different ingredients:
fishmeal, soybean meal, meat bone meal, blood meal. It was designed randomly with
three repetitions.
 Stocking density, feeding, management, sample-collection methods were similarity
with comparing three above fecal-methods experiment.

13

3.5 Sampling and data analysis methods
a. Sampling analysis
The environmental factors in these experiments contained: using thermometer for
measuring temperature (2 times/ day at 9 am and 2 pm), pH tool for measuring pH (2
times/day),
The criteria for chemical components of feed and fish on these experiments were
analyzed by usual methods according AOAC (2000)
Moisture: using oven (1050C)
Protein: determined by Kjedahl method
Lipid: determined by Soxlhet method
Ash: determined by burning at 550OC
Crude dry matter: determined after analyzed sample in diluted acid and base minus the
ash in sample
Carbohydrate (NFE): 100% - (% moisture + %protein + %lipid +% fiber + % ash)
Chromic oxide (Cr2O3): determined be method of Furukawa and Tsukahara(1996)
b. Data collection and computation
 Feed digestibility (ADC – apparent digestibility coefficient – ADC)
ADC = 100 – (100 x

)

There are:
% A: % marker in feed (dry weight)
% B: % marker in feces (dry weight)
 Nutritional digestibility (protein, energy) in feed
x

)

There are:
%A: % marker in feed (dry weight)
%B: % marker in feces (dry weight)
14


% A’: % nutrition in feed (dry weight)
%B’: % nutrition in feces (dry weight)
 Digestibility in ingredients
ADCingredients = (ADCT – 0.7 x ADCR)/ 0.3
ADCR: % control diet digestibility (R diet)
ADCT: % control diet digestibility (T diet)
 Nutrition apparent digestibility of ingredients

ADC Nu-treat: apparent digestibility coefficient of nutritional treatment
ADC Nu-ref: apparent digestibility coefficient of nutritional reference
N diet-ref: Nutrition of referent diet (%)
N ingredient: Nutrition of ingredient (%)
c. Data analysis
The criteria of these experiments (digestibility, chemical component of feed and feces)
were analyzed mean, standard deviation by Excel 2010 and SPSS 16.0 and comparing
difference between means among treatments basing on one way ANOVA with DUNCAN
test, significantly, p < 0.05.

15