Nghiên cứu đặc điểm sinh thái phân bố, sinh trưởng, sinh sản và thử nghiệm nuôi sinh khối trùn chi (limnodrilus hoffmeisteri claparede, 1862)TTLA tieng anh

MINISTRY OF EDUCATION AND TRAINING
NHA TRANG UNIVERSITY

TRUONG THI BICH HONG

“STUDY ON CHARACTERISTICS OF ECOLOGICAL
DISTRIBUTI, GROWTH, REPRODUCTION OF
OLIGOCHAETE (Limnodrilus hoffmeisteri Claparede, 1862)
AND TRIAL OF THEIR BIOMASS CULTURE”

Major:
Aquaculture
Major code: 9620301

SUMMARY OF DOCTORAL THESIS

Supervisors:
1. Associate Prof. PhD Nguyen Dinh Mao
2. PhD Dinh The Nhan

KHANH HOA -2017


Thesis was comlpeted in Nha Trang University

1. Assoc. Prof. Dr. Nguyen Đinh Mao

Supervisor:

2. Dr. Đinh The Nhan

Reviewer 1:

Prof. Dr. Đo Cong Thung

Reviewer 2:

Dr. Tran Suong Ngoc

Reviewer 3:

Prof. Dr. Đoan Nhu Hai

Thesis will be defended at the Thesis university committee level at Nha
Trang University. Date:…………………….h,……………./…………….2018

The thesis can find out at:
- National Library
- Library of Nha Trang University
1


INTRODUCTION
Oligochaete (Limnodrilus hoffmeisteri) is essential food for most freshwater fish
species. In the past few years, the demand for oligochaete biomass has been increasing
for the production of native fish and ornamental fish, especially for the production of
cold water fish such as sturgeon and rainbow trout. However, information related to
culture of L. hoffmeisteri in Vietnam is not known. So that, worms have not yet been
cultured to provide freshwater fish hatcheries and ornamental fish.
With high application potential in aquaculture, the study of distribution
ecological characteristics, reproduction, growth as well as culture of oligochaete is

very necessary. According to above reasons, the thesis: “Study on characteristics of
ecological distributi, growth, reproduction of oligochaete (Limnodrilus hoffmeisteri
Claparede, 1862) and trial of their biomass culture” was conducted.
1. Objectives of thesis
Oligochaete (L. hoffmeisteri) biomass culture used to feed the ornamental fish and
native freshwater fish.
Detail objectives
1. Database on distribution, growth and reproduction of oligochaete (L. hoffmeisteri)
2. Determine the suitable substrate structure, substrate thickness, feed type, ration and
inoculation density when culture of oligochaete (L. hoffmeisteri)
3. Trial of oligochaete biomass culture.
2. The main contents
1.
Study on characteristics of ecological distributi of oligochaete in freshwater
ecosystems.
2.
Study on reproduction of oligochaete in laboratory
3.
Study on growth of oligochaete in laboratory
4.
Effect of substrata structure, substrata thickness, feed type, ration and
inoculation density on biomass and density of oligochaete (L. hoffmeisteri)
5.
Trial of oligochaete biomass culture
6.
Application biomass oligochaete to culture zebrafish (Danio rerio) and siamese
fighting (Betta splendens)
3. Scientific and practical significance
Scientific significance: The study provided a database on the distribution,
growth and reproduction of L. hoffmeisteri, assessing the potential of natural food

sources in related waters.
Practical significance: The results of study provided data of scientific value in
research and application, contributing to the improvement of the culture process of
oligochaete (L. hoffmeisteri). Oligochaete biomass contributes actively to live food
sources in freshwater fish hatcheries and ornamental fish farms.
4. New results of thesis:
The first works studied the distribution, growth, reproduction of oligochaete (L.
hoffmeisteri).
The study provided the scientific data to culture oligochaete (L. hoffmeisteri).
Reduction of the Feed conversion ratio when oligochaete (L. hoffmesteri) were fed a
mixture of soybean meal, corn meal and rice bran with a ratio of 1: 1: 1.
2


CHAPTER 2. MATERIALS AND METHODS
2.1 Time, locality and object of Research
2.1.1 Research time: 10/2012 - 05/2017
2.1.2 Research locality: Khanh Hoa province
2.1.3 Research object : Oligochaete (Limnodrilus hoffmeisteri Claparede, 1862)

Figure 2. 1.

Limnodrilus hoffmeisteri

2.2 Research block diagramamatic

Study on growth of oligochaete in laboratory

Study on reproduction of oligochaete in
laboratory

Study on ecological distributi of oligochaete in
freshwater ecosystegms at Nha Trang and suburb

Study on characteristics of ecological distributi, growth, reproduction of oligochaete
(Limnodrilus hoffmeisteri Claparede, 1862) and trial of their biomass culture

Study on Limnodrilus hoffmeisteri biomass culture
Effect of substrata structure on survival and growth of
Limnodrilus hoffmeisteri
Effect of substrata thickness on biomass and density of
Limnodrilus hoffmeisteri
Effect of feed type on biomass, density and quality of
Limnodrilus hoffmeisteri
Effect of ration on biomass, density and quality of
Limnodrilus hoffmeisteri
Effect of inoculation density on biomass and density of
Limnodrilus hoffmeisteri

Trial of oligochaete (L. hoffmeisteri) biomass culture

Application biomass oligochaete to culture zebrafish (Danio rerio) and siamese fighting
(Betta splendens)

Figure 2.2: Research block diagramamatic
3


2.3 Methods
2.3.1 Study the distribution of L. hoffmeisteri in different ecosystems

2.3.1.1 Sewage discharged
Drainage ditches in the urban area, in the countryside and drainage ditches
affected by industrial waste water. Each drainage ditches was collected organism samples
(oligochaete), water sample and bottom sample at 3 points: head, middle and end.
2.3.1.2 Static water with a water depth of more than 1 m
Natural ponds, ponds for aquaculture. Each pond was collected 5 samples: 4
points in 4 corners and one in the middle.
2.3.1.3 Lowland areas with a water depth of less than 1 m
Lowland areas are: ricefields, spinachfields, papyrusfields. Each of the lowland
areas was collected 5 samples: 4 points in the coastal area between the two fields; 1
point in the middle of the field.
2.3.2 Study on reproduction and biological behavior of L. hoffmeisteri in laboratory
• Reproductive season of L. hoffmeisteri
Oligochaete were collected randomly at domestic sewage ditches twice per
month for one year from December 2013 to November 2014 with 30 individuals at a
time. The worm was placed on a binocular optics microscope equipped with the
Olympus BX41 Olympus C-7070 magnification camera to determine the maturity of
the worm, according to Kennedy (1966).
*Age of maturity and cocoon production (E1)
Experimental conditions: Oligochaeta were cultured in a 250ml plastic
beakers containing 50mg of mud under a drip water system. Every day, we provided
1g of rice bran for oligochaete.
Experimental design: Five pair of oligochaete they had just hatched a day
were inoculated into plastic beakers (repeat 30 times). When the oligochaete were 4
weeks. We collected a test to keep a pair in a beakers. When the worms were 30 days
old. We collected oligochaete samples to check maturity. If oligochaete are mature,
then we check the bottom evryday to determine the reproductive age.
Mud used to made substrata: Mud were collected from sewage discharged in
Vinh Phuong – Nha Trang where oligochaete lived with high density. Mud washed
through 500 µm mesh sieve to remove extraneous materials and any organisms. A large

plastic container was used to allow for the retention of sediment fines. The fine mud
was put into plastic bag (each bag holds 2 kg of mud). Place the mud bag in autoclave
at 125 ° C for 120 minutes.
 Reproductive viability and number of embryos in cocoon of L. hoffmeisteri (E2).
Experimental conditions: Oligochaete were cultured in a 250ml plastic
container containing 50mg of mud under a drip water system for 2 months from 1
April to 1 June 2015. Every day, we provided 5g of rice bran for oligochaete.
Experimental design: L.hoffmeisteri worms were inoculated at the density of 5
mature individual in plastic box was prepared (repeat 10 times). Periodically, check the
number of cocoons and embryos in a cocoon once every 2 days for a period of 2 months.
Determine the number of cocoons by the method of Marchese and Brinkhurst (1966).
• Embryo development time and hatching rate (E 3)
Experimental conditions: Cocoons are incubated in a 100ml plastic beakers
containing 25mg of mud, under a drip water system.
Experimental design: 30 new cocoons were inoculated into 01 plastic beakers
with 100ml. we counted the number of embryos of a cocoon and calculated the total
number of embryos of 30 cocoons before putting the cocoons into incubate. The
4


experiment was repeated 10 times. We analyzed cocoon under a microscope everydays
for observation embryo development.
• Research on regeneration of worm (E 4)
Experimental conditions: Randomly placed in 15 plastic beakers of 250 ml,
each containing 50 mg of mud and 30 parts of oligochaete. The test beakers were
placed under the drip system. Every day, feed 1g of rice bran powder to the
oligochaete into a plastic beakers.
Experimental design: The worm was cut into 3 parts by a razor refills. We Put
3 body parts into 3 different treatments: first treatment; Treatment 2: Body; Treatment
3: The tail. In about one-third of the body, the location of the genital belt belongs to

the head. Each treatment was repeated 5 times. Periodically check the wound healing
and worm regeneration for 3 days. The experiment ends when the body parts
regenerate into a new body or complete death.
2.3.3 Study on growth characteristics of oligochaete (E5)
Experimental conditions: worms were cultured in a 250 cm2 plastic tray, 1cm
thick bottom mud. Stocking density of worms was 2 individual/cm2. The tray was placed in
the trough, under dripping water. Worms were fed in the way that they felt satisfied their
needs.
Experimental design: The stocking of 500 oligochaete hatched 1 -2 days. Once
a week, collected sample, measure the length and weight of 30 individual to assess the
growth of the oligochaete. The research period was 15 weeks.
2.3.4 Oligochaete (L. hoffmeisteri) culture study
2.3.4.1 Effect of substrata structure on survival and growth of L. hoffmeisteri (E6)
Experimental conditions: The experiment was conducted in a laboratory,
under the overflow system consisting of 25 plastic trays, each tray 150 cm2 (15x10),
the height of the tray 6.5 cm. The plastic trays were stacked between 1 and 5 and
placed under the tap. Stocking density of oligochaete was 5 individual/cm2. Stocking
oligochaete is a week old. Oligochaete were fed rice bran. Oligochaete were fed in the
way that they felt satisfied their needs. Experimental time was 5 weeks.
Experimental design: The experiment was conducted with 5 different
treatments on the ratio of sand and mud. Tr 1: 75% mud 25% fine sand; Tr2: 50% mud
50% fine sand; Tr 3: 25% mud and 75% fine sand; Tr 4: 100% fine sand; Tr 5: 100%
sludge. The sand used for substrata structure is freshwater sand. The dry mud and fine
sand were mixed according to the ratio of the treatments. Each treatment was repeated
5 times. Weekly, sampling was used to calculate the density, weight and body length
of 30 individual per treatment.
Sample collection: samples were taken at 5 points (4 points in 4 corners and 1
point in the middle) in the tray. We used plastic frames 4 cm2 (2x2cm) and spoons to
collect samples. First, we pressed the frame down to the bottom and then use plastic
scoop to collect all the mud and the oligochaete inside the frame. A quantity of mud

and the oligochaete washed through 2a = 0,5mm sieve with 600 cm2 tray to remove the
bottom. Oligochaete were retained to calculate the necessary indicators
2.3.4.2 Effect of substrata thickness on biomass and density (E7)
Experimental conditions: Experimental conditions was the same E6. However,
the hole position in the tray wall was different between treatment groups, ensuring the
water level from the substrate surface to the hole position was 2 cm. Substrata
structure was 75% mud 25% fine sand. This was selected from the treatment of E6.

5


Experimental design: Substrate thickness was arranged with four levels. Four
substrate thickness levels were tested: 1 cm, 2 cm, 3 cm and 4 cm. Each treatment was
repeated 5 times for 5 weeks. Weekly, sampling was used to calculate the density and
biomass of oligochaete. Sampling method was similar to E6
2.3.4.3 Effect of feed type on biomass, density and biomass quality of L. hoffmeisteri (E8)
Experimental conditions: The oligochaete were cultured in indoor cemented
culverts 4.000 cm2 (160 x 25 cm) system to protect from rain and sunlight. Flowing water.
Initial stocking density was 5 individual/cm2. Substrata structure was 75% mud 25% fine
sand. Thickness was 4cm. They were selected from the treatment of E6 and E7.
Oligochaete were fed once a day, the amount of food provided in the way that they felt
satisfied their needs.
Experimental design:The experiment was conducted with 4 treatments: Tr1:
soybean meal; Tr 2: corn meal; Tr 3: rice bran; Tr 4: mixed feed: soybean meal, corn
meal with rice bran at a ratio of 1: 1: 1. Weekly, sampling was used to calculate the
density and biomass of oligochaete. Each treatment was repeated 6 times. Sampling
method was similar to E6
2.3.4.4 Effect of ration on density, biomass and biomass quality of oligochaete (E 9)
Experimental conditions: Experimental conditions was the same E8. Substrata
structure 75% mud 25% fine sand with 4cm thickness. Feed type was mixed feed:

soybean meal, corn meal with rice bran at a ratio of 1: 1: 1. They were selected from
the treatment of E6, E7 and E8.
Experimental design: The experiment was conducted with 4 different
treatments: Tr1 0.00 % (control treatments, no feeding); Tr 2 fed 5%; Tr 3 fed 10%; Tr
4 fed 15% body mass/day. Each treatment was repeated 6 times, for 5 weeks. Weekly,
sampling was used to calculate the density and biomass of oligochaete. Sampling
method was similar to E6
2.3.4.5 Effect of inoculation density on biomass and density of oligochaete (E10)
Experimental conditions: Experimental conditions was the same E8. Substrata
structure 75% mud 25% fine sand with 4cm thickness. Feed type was mixed feed with
15% body mass/day. They were selected from the treatment of E6, E7, E8 and E9.
Experimental design: inoculation density was designed with four levels. Four
initial stocking biomass levels were tested including 1mg/cm2, 10mg/cm2, 20mg/cm2
and 30mg/cm2. Each treatment was repeated 6 times, for 5 weeks. Weekly, sampling
was used to calculate the density and biomass of oligochaete. Sampling method was
similar to E6
2.3.4.6 Experimentation of oligochaete biomass culture (E 11)
Experimental conditions: Oligochaete were cultured in indoor cemented
culverts. The area of the culverts 2.4 m2. Conditions on substrata structure was 75%
mud 25% fine sand with 4cm thickness. Feed type was mixed feed with 15% body
mass/day. Inoculation density was 10mg/cm2. They were selected from the results from
E 6 to E10. The culture time was 5 weeks.
Experimental design: Oligochaete (L. hoffmeisteri) cultured biomass in two
cement slurry systems. Identification the biomass, biochemical and amino acid of
oligochaete.
2.3.5 Application biomass oligochaete to culture zebrafish (Danio rerio) and
siamese fighting (Betta splendens)
2.3.5.1 Effects of culture oligochaete on gonadosomatic index and fecundity of
zebrafish (Danio rerio, F.Hamilton, 1822) (TN12)
Experimental conditions: The experiment was arranged randomly in 8 tanks.

Continuous air 24/24. Fish were fed twice a day in the way that they felt satisfied their
6


needs. Male and female were cultured in a glass tank 5 days prior to the experiment.
They adapted to the food and laboratory conditions.
Experimental design: The experiment was conducted with four different
treatments: Tr1: 100% live oligochaete; Tr 2: 100% frozen oligochaete; Tr3: 50% live
oligochaete + 50% industrial feed; Tr4: 100% industrial feed (control).
Male and female were cultured in different tanks with density of 30 fish / tank.
We evaluated effects of food on the sexual maturity of zebrafish at the end of the
experiment (30 days). Each indicator (gonadosomatic index and fecundity) of the
experiment was repeated 5 times.
2.3.5.2 Effects of culture oligochaete on growth and survival of siamese fighting
(Betta splendens, Regan, 1910) juveniles (E13)
Experimental conditions: The experiment was arranged randomly in 12 tanks
of size 25x25x40cm3. Fish were fed twice a day in the way that they felt satisfied their
needs. Daily substituting 20% of water after second feeding (15h). Fish were cultured
in a glass tank 5 days prior to the experiment. They adapted to the food and laboratory
conditions.
Experimental design: The experiment was conducted with four different
treatments: Tr1: 100% live oligochaete; Tr 2: 100% frozen oligochaete; Tr3: 100%
industrial feed (Kaokui); Tr4: 50% live oligochaete + 50% industrial feed (Kaokui).
Each treatment was repeated 3 times. We weighed weigh and measured the length of
fish every two weeks. The survival rate of fish was determined by recording the
number of dead fish daily in each experiment.
2.4. Data analysis
Inputting all raw data into the Excel software:
Determining the density, cocoon size, embryo hatching rate, characteristic
growth rate of length and weigh, absolute growth, absolute fecundity, relative

fecundity, fertilization rate, survival rate, biological diversity by formula.
Use SPSS 18.0 to compare mean values. Analysis of variance (ANOVA) was
used to compare these production metrics. Comparison of mean difference after post
hoc analysis with Duncan's reliability of 95% (P <0.05).

7


CHAPTER 3: RESULTS AND DISCUSSION
3.1 MORPHOLOGICAL CHARACTERISTICS AND DISTRIBUTION OF L.
hoffmeisteri IN FRESHWATER ECOSYSTEMS
3.1.1 Morphological characteristics
Mature L. hoffmeisteri in this study was characteristics by light red, a total
length between 14-40 mm, average was 21.3 ± 5.7 mm. The body of oligochaete had
many segments which the total count ranging from 47-85 (60.7 ± 9.6) segments. The
correlation equation between the length and segments of the worm body was Y =
1.41x + 30.595 R2 = 0.7796 (Figure 3.1)

number segments

100

y = 1.41x + 30.595
R² = 0.7796
n = 180

90
80
70
60

50
40
30
20
10
0
0

5

10

15

20

25

30

35

40

45

Length (mm)

Figure 3.1: Correlation between body length and segments
Dorsal pectinate and anterior ventral were S-shape. Anterior ventral bundles

in front of genital segments were composed of from 4 to 9 setae and reduced to 1 to
2 setae in the last segments (Figure 3.2: a, b). Penis-sheaths was about 400-500 μm
long, about 40-60 μm wide with wall thin (Figure 3.2c). Sperm bundle was club shaped (Figure 3.3 d).

a
b

0,05mm

d

c

Figure 3.2: Characteristics of L. hoffmeisteri: Anterior ventral bundles in front of genital
segments(a), Anterior ventral bundles in the bodily endings(b), Penis-sheaths(c), Sperm bundle(d)

8


3.1.2 Distribution of oligochaete in different ecosystems
3.1.2.1 In sewage discharged
a. Oligochaeta community
A total 6 species were found in sewage discharged. They belonged to order
Haplotaxida. Of which four species belonged to the Tubicidae family. Dominant species
in worm population were L. hoffmeisteri (Table 3.1).
Table 3.1: Oligochaeta community in sewage discharged
IS
DS
Genus
Family

Species
BB S ĐL VP
NP
VN
VT NH VH
- +
Naididae
Aulophorus
A. furcatus
+
+
+
Chaetogaster C. limnsei
- +
+
+
Tubificidae Branchiura
B. sowerbyi
++++
+++
++++
Limnodrilus L. hoffmeisteri
+
+
+
Tubifex
Tubifex sp.
+
+
Aulodrilus

A. prothecatus
Note:IS Industry sewage, BB Dong A Packaging Factory, S fiber factory and tobacco factory, ĐL F17
seafood processing factory, DS domestic sewage; VP V Vinh Phuong, NP V.Ninh Phung, VN V-Vinh
Ngọc, C- Vĩnh Thai, NH W-Ngoc Hiep, VH W-Vinh Hai, - not distributed; + rare; ++ regular, +++
abundant, ++++ very abundant

b. Density and percentage of L. hoffmeisteri / total number of oligochaete in
sewage discharged
Table 3.2: Distribution of oligochaete in sewage discharged
L. hoffmeisteri
% L.
Factors
Frequency
Total oligochaete
2
(individual/m )
hoffmeisteri
Sewage ditches
Appear
(individual /m2)
/total
Dong A Packaging
Not
0.0
0.0
0.0
Factory
Industry F17
seafood
Not

0.0
0.0
0.0
sewage processing factory
Fiber factory and
Not
0.0
0.0
0.0
tobacco factory
100,826±86,874 131,311± 104,233 76.78±8.34
Vinh Phuong village ++++
Ninh Phung village
854±404
++
1,689±634
50.56±6.41
domestic Vinh Ngoc village
32,292±10,033
+++
46,422±10,744
69.56±9.33
sewage Vinh Thai commune
Not
0.0
0.0
0.0
Ngoc Hiep ward
Not
0.0

0.0
0.0
Vinh Hai ward
Not
0.0
0.0
0.0
Average ± standard deviation
The locations where the worm was distributed, L. hoffmeisteri were always
dominant. The highest density of L. hoffmeisteri was 100,826 ± 86,874 individuals/m2
in sewage ditches of Vinh Phuong Village.
3.1.2.2 In ponds
a. Oligochaeta community in ponds
The results showed that Oligochaeta were not distributed in the catfish ponds.
There were from 3 to 7 oligochaeta species in the studied ponds (Table 3.3).

9


Table 3.3: Oligochaeta community in ponds
Genus
TN TrP NG
Species
Aulophorus
A. furcatus
+
A. hymanae
+
Chaetogaster Ch. limnsei
+

Dero
D. digitata
+
Tubificidae Branchiura
B. sowerbyi
Limnodrilus
L. hoffmeisteri
+
+
Tubifex
Tubifex sp.
+
+
Aulodrilus
A. prothecatus
+
+
Family
Naididae

TrD
+
+
+
+
-

CG
+
+

+
+
+
+

RĐ
+
+
+

Note: + distributed, - not distributed, TN Natural pond, TrP Shutchi catfish pond, NG
Polyculture pond , CG Tilapia nursing pond, RĐ Common climbing perch pond

b. Density and percentage of L.hoffmeisteri species / total oligochaeta in ponds
The presence of L. hoffmeisteri in natural ponds and aquaculture ponds
confirmed the distribution of L. hoffmeisteri in ponds. Percentage of L. hoffmeisteri
/total oligochaeta in the ponds ranged from 28.44 ± 4.25% to 42.06 ± 8.36%. Percentage
of L.hoffmeisteri species was lowest 28,44 ± 4,25% in polyculture ponds, highest in
tilapia nursing ponds by processed food, taking 42,06 ± 8,36 % (Figure 3.3).
500

60
50

400
350

40

300

250

30

200
20

150
100

% L.hoffmeisteri

Density (in/m2)

450

10

50
0

0

Natura

Catfish
ponds

Polyculture
ponds

Density (individual/cm2)

Shutchi
Tilapia
Common
catfish
ponds
climbing
ponds
perch ponds
Percentage L.hoffmeisteri/Olygochaeta

Figure 3.3: Density and percentage of L.hoffmeisteri species / total oligochaeta in ponds
3.1.2.3 Ricefields, spinachfields, papyrusfields
a. Oligochaeta community in fields
The results showed that a total of 12 oligochaeta species were found in the
studied fields. Three species of B. sowerbyi, L. hoffmeisteri, Tubifex.sp were found at
all the studied fields (Table 3.4). Table 3.4: Oligochaeta community in fields
Genus
RL
RC RR
Family
Species
Aelosomatidae
Aeolosoma
A. bengalense
+
A. travancorense
+

Aeolosoma sp.
+
+
Naididae
Aulophorus
A. furcatus
+
+
A. hymanae
+
Aulophorus sp.
+
+
Dero
D. digitata
+
+
D. indica
+
+
Tubificidae
Branchiura
B. sowerbyi
+
+
+
Limnodrilus
L. hoffmeisteri
+
+

+
Tubifex
Tubifex sp.
+
+
+
Aulodrilus
A. prothecatus
+
Note: RL Ricefields, RC Papyrusfields, RR Spinachfields, + distributed, - not distributed

10


50

90000

45

80000

40

70000

35

60000

30

50000

25

40000

20

30000

15

20000

10

10000

5

0

0

Density(ind/m2)

100000

Ricefields

Spinachfields

Density

% L.hoffmeisteri/giun ít tơ

b. Density and percentage of L.hoffmeisteri species / total oligochaeta in fields
The difference about density of oligochaete at the studied location was very large.
Density of oligochaete in the middle of the field was low (14,600 individuals/m2), but in
the coastal area where divided between fields was very high (99,133 ind/m2) (Figure 3.4).

Papyrusfields

Percentage L.hoffmeisteri/Olygochaeta

Figure 3.4: Density and percentage of L.hoffmeisteri species / total oligochaeta in fieds
3.2 REPRODUCTION AND REGENERATION OF L. hoffmeisteri
3.2.1 Reproduction of L .hoffmeisteri
3.2.1.1 Reproduction season
L. hoffmeisteri populations had mature throughout the year. But the reproductive
season of them was twice a year: First April and May, second August and September
90

40.00

80

35.00

70

30.00

60
50

Juveniles

40

Immature

Percent (%)

Percentage (%)

100

Mature

30

25.00
20.00

Percentage

15.00

20

10.00

10

5.00

0

The average embryos of cocoons X=3,40 ± 1,66

0.00

12

1

2

3

4

5

6

7

8

9

10 11

1

Month

2

3

4

5

6

7

8

Embryos of a cocoons

Figure 3. 5: Maturation ratio of
L. hoffmeisteri from 12/ 2013 to 11/2014

Figure 3.6: The number of embryos of
a cocoon

3.2.1.2. Life cycle of L. hoffmeisteri

Cocoon

mature
immature

11

The
young
break
out of
the
cocoon

Figure 3.7: Life cycle of L. hoffmeisteri


3.2.1.3 Age of maturity and cocoon (E1)
The mean maturation age of oligochaeta was only 35.33 ± 2.32 days. Oligochaeta
laid cocoon earliest when they were 31 days old and latest when they were 39 days old
3.2.1.4 Reproduction and number of embryos in cocoons (E2)
The average of cocoons per adult per day was 0,66 ± 0,25. The number of
embryos per cocoon varied between 1 and 8, with an average of 3,40 ± 1,66 (Figure 3.6)
The results of cocoon per adult per day and embryo per cocoon in the study
were higher than those of Lobo and Galves on reproductive of L.hoffmeisteri. The L.

hoffmeisteri when they cultured on fine sandy substrates (0.555-0.250) mm laid 0.37 ±
0.75 cocoons per day. Cocoon each had 3.12 ± 1.03 embryos. The L. hoffmeisteri
when they cultured on medium sandy substrates (0,25-1,00 mm) laid 0.23 ± 0.24
cocoons per day. Cocoon each had 3.06 ± 1.21 embryos (Lobo and Galves, 2011a).
Table 3.5: Reproduction results of L.hoffmeisteri
Tray
1
2
3
4
5
6
7
8
9
10
Total or
Average

Total
cocoons
248
137
143
151
342
149
108
183
233

286

Total
embryos
803
418
526
555
1182
489
342
674
805
942

embryos
of a
cocoons
3.25±1,58
3.05±1,28
3.68±1,82
3.68±1,73
3.46±1,74
3.28±1,56
3.17±1,61
3.68±1,82
3.45±1,78
3.29±1,63

1980

6736

3,40±1,66

The
young
610
318
410
412
892
370
265
517
615
710
5119

Hatching
rate
75.97
76.08
77.95
74.23
75.47
75.66
77.49
76.71
76.40

75.37

Cocoons
per day
0.83
0.46
0.48
0.50
1.14
0.50
0.36
0.61
0.78
0.95

76,13±1,07

0,66±0,25

Embryos of a cocoons, Hatching rate and cocoons per day were Average ± standard deviation

The average length of cocoon was 549.17 ± 187.39μm. The average width of the
cocoon was 337.39 ± 97.53μm. The length, width and number of embryos of the cocoons
were correlated with line equation y =100,15x + 213,26 and y = 53,621x + 157,73
respectively (Fig. 3.8 and Fig. 3.9).
y = 100.15x + 213.26
R ² = 0.7659
n = 240

1000

700

Width of cocons (µm)

Length of cocons (µm)

1200

800
600
400
200
0
0

1

2

3

4

5

6

7

8

y = 53.621x + 157.73
R ² = 0.8106
n= 240

600
500
400
300
200
100
0

9

0

Embryos of a cocoons

1

2

3

4

5

6

7

8

9

Embryos of a cocoons

Figure 3.8: Correlation between length Figure 3.9: Correlation between width
and number of embryos in cocoon
and number of embryos in cocoon
3.2.1.5 Embryo development time and hatching rate (E3)
The results showed that, The embryos of L.hoffmeisteri were protected in the
cocoon. The new hatchling ate albumen that was stored in the cocoon.
The period between cocoon laying and hatching varied from 6 to 12 days
(averaged 8.13 ± 1.64 days). This results was similar to the interval found by (Thai
Tran Bai, 2005), the development of oligochaeta is direct without any larval stage. The
12


youngsters get out of the cocoon after 8-10 days, depending on the species and
temperature.
Table 3.6: Embryo development time
Number
Period of
Characteristics
time (hours)
the zygote divides by holoblastic and spiral cleavage

1
0 -24
resulting in the formation of the blastula to gastrula
The gastrula open exterior through a wide aperture the
2
24 – 48
blastopore.
Body shape was formed. Initially, body was short arc3
48 -72
shaped, then developed long to form a circle in the
embryonic sac.
The oligochaete started to move. But this time, they only
4
72-94
moved in circles in the embryonic sac.
Youngsters broke the membrane of the embryo to go out. They
5
94-144
ate the albumen in the cocoon, they were still protected by the
cocoon. They were constantly moving in the cocoon. The
oligochaete can be wrapped together.
The Youngsters broke the cocoon to go out
6
144-288
3.2.2 Regeneration of L. hoffmeisteri (E4)
After 9 days, The head part regenerated the body and tail to form a completely
new body. the regeneration rate was 61.08 ± 11.48%. In contrast, the body and tail part
could not regenerate (Figure 3.10).

Regeneration percentage (%)

80

c

b

b

70
60

a

50
40

a

Head
Body
Tail

b

30

a

20

10
0

After 3 days

After 6 days

After 9 days

Times

Figure 3.10: Regeneration of L. hoffmeisteri
The results of this study was contrary to the previous view found. Oligochaete
can regenerate. When the worm is cut in two part, they will form two new oligochaete.
The results of this study can be confirmed. The regeneration capacity of L. hoffmeisteri
was limited, only the head part could regenerate into a complete body. The body and tail
part could recover wound but could not regenerate the head to form a new individual.
3.3 GROWTH OF OLIGOCHAETE (L. hoffmeisteri) (E5)
3.3.1 Weight growth

The results showed that the body weight of L. hoffmeisteri increased
continuously during the study period. After lay, the body weight was still increasing but
their growth rate was slow down (Figure 3.11). The curves of L. hoffmeisteri growth
show almost a constant weight gain during the 25 weeks of observation (Lobob and
Alves, 2011 a).

13


7

Weight (mg/individual)

6
5
4
3
2
1
0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Weeks

Figure 3.11: Growth of the weight of the worm (L. hoffmeisteri)
3.3.2 Length growth
The body length of oligochaete grew continuously from hatching to the 15th
week Figure 3.12. The length of mature was than 19.23 ± 3.13 mm. The end of the
experiment, the average length of L. hoffmeisteri was 30.27 ± 3.56 mm. Population
structure of the oligochaete was estimate by grouping their sizes into 4 length classes
varying from 0.3-0.5, 1.0-1.5, 2.0-2.5 and 3.0-3.5 cm. The body length of adult was
3.0-3.5 cm (Warucha và Saran, 2008).
40.00
35.00

Length (mm)

30.00
25.00
20.00

15.00
10.00
5.00

15

14

13

12

11

9

10

8

7

6

5

4

3

2

1

H
at
ch
e

d

0.00

Weeks

Figure 3.12: Growth of the length of the worm (L. hoffmeisteri)
3.3.3 Relationship between length and weight
The relationship equation
7
between length and weight was
W = 0.1254L
6
R² = 0.9512
W = 0.1254L1.1086 with
n = 450
2
5
relationship coefficient R =
4
0.9512 Figure 3.13. The value

3
of the exponent b = 1,1086
2
(b<3)
indicated
that
L.
hoffmeisteri was heterogeneous
1
growth. The
relationship
0
0
5
10
15
20
25
30
35
40
equation of Limnodrilus sp W =
0.74
Length (mm)
0.0779L
(b<3). Limnodrilus
sp
belongs
of
the

Figure 3. 13: Relationship between length and weight of
L. hoffmeisteri
heterogeneous growth.species
(Maria Miserendion, 2001).
Weight (mg)

1.1086

14


3.4 OLIGOCHAETE (L. hoffmeisteri) CULTURE STUDY
3.4.1 Effect of substrata structure on survival and growth of L. hoffmeisteri (E6)
3.4.1.1 Effect of substrata structure on survival of oligochaete
. The results showed that the different substrata structure significantly affected
the survival rate of the L. hoffmeisteri . Table 3.7
Table 3.7: Survival rate (%) of oligochaete were cultured in different substrata
Nghiệm thức
Day of
100%
75% mud
50% mud
25% mud
100%
culture
Mud
+25% sand
+50% sand
+ 75% sand
sand

7
93,4±3,7d 86,8±5,5c
81,6±4,6bc
80,2±4,1b
74,0±3,74a
c
c
b
a
14
91,0±3,4
85,7±3,6
79,5±3,6
69,8±4,7
67,2±4,96a
c
c
b
a
21
88,9±3,8
84,1±3.5
75,8±4,7
66,0±5,6
61,8±3,2a
c
c
b
a
28

86,8±2,6
81,6±4,8
74,2±4,9
65,2±4,5
60,5±3,5a
Average ± standard deviation,Different caps on the same row showed a statistically significant (P <0.05).

3.4.1.2 Effect of substrata structure on growth of worm
a. Length growth
Length growth of L. hoffmeisteri was difference between weeks. Growth in all
treatments was highest in the first week and then tended to decrease. Table 3.8
Table 3.8: Length growth of oligochaete were cultured in different substrata
Treatments
Day of
Length
100%
75% mud
50% mud
25% mud
100% sand
culture
growth
mud
+25% sand +50% sand + 75% sand
7
ADGL 1,24±0,25b 1,20±0,23b 1,08±0,39b 1,06±0,42b
0,81±0,28a
SGRL 9,04±2,45c 8,90±2,16c 8,40±1,95bc 7,51±1,61b
6,23±1,27a
ab

a
b
ab
14
ADGL 0,51±0,26
0,40±0,19
0,52±0,23
0,50±0,21
0,67±0,27c
ab
a
bc
b
SGRL 2,49±1,49
1,96±0,73
3,09±1,94
2,86± 1,89
3,92±1,96c
a
b
a
a
21
ADGL 0,26±0,15
0,5±0,21
0,19±0,09
0,25±0,11
0,31±0,13a
ab
c

a
ab
SGRL 1,07±0,48
1,91±1,35
0,94±0,54
1,11±0,51
1,48±0,71b
28
ADGL 0,21±0,10a 0,40±0,23b 0,67±0,36c 0,2±0,11a
0,17±0,07a
ab
b
c
ab
SGRL 0,84±0,27
1,16± 0,92 2,54±1,24
0,81±0,33
0,72±0,17a
Average ± standard deviation,Different caps on the same row showed a statistically significant (P <0.05).

b. Weight growth
The oligochaete were cultured in mud substrata grew higher than the worm
were cultured in different substrata Table 3.9. The L. hoffmeisteri individuals
maintained in medium sand grew less than L. hoffmeisteri individuals maintained in
fine sand (Loboa and Alves, 2011a).
Table 3.9: Weight growth of oligochaete were cultured in different substrata
Day of Weight
Treatments
culture growth
100%

75% mud
50% mud
25% mud
100% sand
mud
+25% sand +50% sand + 75% sand
7
ADGw 0,18 ±0,02c 0,16±0,03b
0,15± 0,02ab 0,14 ±0,01a
0,13±0,01a
b
ab
a
a
SGRw 19,66±3,84 18,33±3,63
17,73±3,12
17,36±2,74
17,09±3,05a
c
b
ab
a
14
ADGw 0,44±0,16
0,33±0,19
0,27±0,14
0,25±0,09
0,23±0,09a
c
b

ab
ab
SGRw 14,23±2,45 11,85±3,99
11,19±3,33
10,59±2,99
10,03±3,19a
a
b
bc
bc
21
ADGw 0,06±0,02
0,09±0,02
0,10±0,05
0,11±0,05
0,13±0,05c
SGRw 1,52±1,46a
3,09±2,62b
3,29±2,46b
3,71±2,42b
4,37±2,33b
a
a
a
a
28
ADGw 0,05±0,02
0,06±0,01
0,07±0,03
0,07±0,03

0,05±0.02a
a
ab
b
ab
SGRw 1,12±0,77
1,63±1,35
1,83±1,25
1,78±1,26
1,59±1,32ab
c
b
a
ab
Average ADGw 0.19±0,03
0.16± 0,04
0,14± 0,03
0,15±0,03
0,13±0,03 a
b
ab
a
a
SGRw 9,13± 0,99
8,73± 0,76
8,51± 0,89
8,36±0,97
8,27±0,67a
Average ± standard deviation,Different caps on the same row showed a statistically significant (P<0.05).

15


3.4.2 Effect of substrata thickness on biomass and density (E7)
3.4.2.1 Effect of substrata thickness on biomass of oligochaete
The biomass of worm populations during the experiment period is shown in the
column graph (Figure 3.14). At the first week of culture, the worm biomass in all
treatments was significantly different but did not comply with the law. From the second
week, The worm biomass maintained in thicker substrata grew more than the worm
biomass maintained in thinner substrata.
180
160

180

c

160

120 140

b

d

Sinh khối (mg/cm2)

2
Biomass
)

(mg/cm2)
mật độ (mg/cm

140

d

b

a

100 120

a
c

80 100

b

60 80

a a

c
b

b

40 60 ab a c bc

a

a

20 40
0 20
0

1

2
1

1cm nền đáy
4cm nền đáy

3

4

Weeks
Tuần
nuôi
Tuần
nuôi
3
4
2 cm nền đáy
3 cm nền đáy

2

3 cm nền đáy

2 cm nền đáy

5
5

4 cm nền đáy
1 cm nền đáy

F 3.14: Biomass of the oligochaete populations in the different substrata thicknesses
Results of research was suitable with biological behaviors of L. hoffmeisteri.
The oligochaete were vertically distributed throughout the whole sediment. They
generally penetrated as deep as 4-5 cm in the sediment but mostly concentrated in the
first centimeter (Wanchana , 2009). The juveniles (<0.1 mg), were restricted to a
layer between the surface and 2 cm depth, the mature (>5.0 mg) oligochaete
between 2 and 4 cm depth, and the immature (from 0.1 to 5.0 mg) ones from the
surface to 4 cm depth (Marian and Pandian, 1984).
3.4.2.2 Effect of substrata thickness on density of oligochaete
Substrata thickness in the study affected the density of worm population.
Comparing the four different substrata thickness, The density of the worm population
were cultured in 4cm thickness substrata increased faster the density of the worm
population were cultured in different substrata thickness (Figure 3.15). This can be
explained by biological behavior of oligochaete, which ofter lay cocoons on the
surface of the substrate or the base of the aquatic plant (Smith, 2001). However
oligochaete were very sensitive to light. When the light intensity was high ( 70
lux) over 90% of the oligochaete remained between the surface and a depth of
4 cm from the surface. Normally, over 90% of the worms remained at the

surface between 20.00 h and midnight, when light intensity is lowest (Marian và
Pandian, 1984). Thus, the high substrate thickness increased the space for oligochaete.

16


F3.15: Density of the oligochaete populations in the different substrata thicknesses
3.4.3 Effect of feed type on density, biomass and biomass quality of oligochaete
3.4.3.1 Effect of feed type on biomass of oligochaete (L. hoffmeisteri)
At 4th weekend, Treatments, oligochaete were fed soybean meal had the
highest biomass (137,35 ± 5,79 mg/cm2), followed by the mixture feed (130,83 ±
11,03 mg/cm2), not significant differences (P>0.05). The lowest biomass (94,51 ±
6,98 mg/cm2) were recorded when feeding L. hoffmeisteri with corn meal,
significant differences with other difference treatments (P<0.05) (Figure 3.16).
160

c

c
140

b

Biomass (mg/cm2)

Sinh khối (mg/cm2)

120

c

a

bc

100

Soybean
Bột
đậu
Corn
Bột
ngô
Cám
Ricegạo
Hỗn
hợp
Feed
mixture

b
c

a

80

b

b

60

a
40
20

b
a

a

a

0

1

2

Weeks

3

4

Tuần nuôi

F3.16: The biomass of oligochaete populations were cultured by feed different
Consideration about the biomass showed that, the feed within the study had

clearly affected to increase the biomass of L.hoffmeisteri oligochaete population. This
may be explained by the nutrient quality of feed type (Table 3.10).
Soybean was suitable feeds for worm. The highest significant (P<0,05) of
oligochaete was found in the culture media containing a mixture of 30% soybean meal
and the lowest of oligochaete was found in the culture media containing a mixture of
10% soybean meal (Hossain et al., 2011).

17


Table 3.10: The biochemical ingredients of feed type
Ingredient
(%)
Feed types
(DW) Soybean Corn meal Rice bran
Fed mixture
Protein
%
37,85
14,78
12,66
21,76
Lipid
%
16,25
6,05
24,04
15,45
Tro
%

21,00
12,20
9,89
14,36
Xơ
%
5,46
5,37
13,18
8,10
Cacbonhydrat %
15,40
44,30
18,30
25,20
(Fed mixture included soybean, corn meal, rice bran with the ratio of 1: 1: 1)
3.4.3.2 Effect of feed type on density of oligochaete (L. hoffmeisteri)
At 4th weekend, Treatments, oligochaete were fed soybean meal had the highest
density (49,08 ± 5,36 individuals/cm2), followed by the mixture feed (45,03 ± 4,76
individuals/cm2.The lowest density (33,16 ± 5,03 individuals/cm2) were recorded when
feeding L. hoffmeisteri with corn meal, significant differences (P<0.05) Figure 3.17.
60

c

2
Density
(con/cm2) )
Mật độ(individual/cm

50

bc

b

40

ab
a
c

30

a
a

b
20

b

a

a

Bột
đậu
Soybean
Corn

Bột
ngô
Ricegạo
Cám

10

Feed mixture

a

Hỗn hợp

0
Thả nuôi
Inoculation

tuần 1

tuần 2

tuần 3

tuần 4

Weeks
Tuần
nuôi

Figure 3.17: Density of oligochaete populations were cultured by feed different

In week, different character means that there was significant differences (P<0.05).
3.4.3.3 Effect of feed type on biomass quality of oligochaete (L.hoffmeisteri)
Results of biochemical ingredients analysis showed that protein and
carbohydrate were different between treatments. In contrast, other ingredients such as
lipids, ash, fiber were quite similar (Table 3.11).
Table 3.11: The biochemical ingredients of fresh oligochaete were cultured by
feed types (DW)
Ingredient
Feed types
(%)
Soybean meal Corn meal
Rice bran
Fed mixture
Crude
protein
55,79±1,68b
53,24 ± 4,14b 45,74 ± 4,42a 53,58 ± 2.29b
Crude pipid
15,41±1,07a
15,32±1,07a
16,83 ± 1,13a
15,95±2,00a
a
ab
b
Ash
12,53 ± 1,92
14,51±1,98
17,45±3,18
14,13 ± 0,33ab

Crude fiber
1.48 ± 0.54a
1,13±0,26a
1,06±0,16a
1,22±0,36a
Cacbonhydrat
14,79±1,53a
15,79±1,70a
18,91±0,62b
15,12±1,54a
Average ± standard deviation (SD. Different caps on the same row showed a statistically difference (P <0.05).

18


Table 3.12. The biochemical ingredients of fresh natural worms (L. hoffmeisteri)
Ingredient
% (DW)
43,15 ± 3,09
Crude protein
Crude pipid
18,35 ± 1,02
Ash
16,44 ± 0,81
Crude fiber
5,23 ± 0,70
Cacbonhydrat
16,84 ± 3,00
Average ± standard deviation
Results of biochemical analysis showed that the crude protein of L. hoffmeisteri in

the treatments was >45.74 ± 4.42% higher than the crude protein of L. hoffmeisteri in
natural at Nha Trang (43.15 ± 3.09%) (Table 3. 12).
Table 3.13: Fatty acid ingredients of fresh L. hoffmeisteri were cultured by feed types
Ingredient (%)
Feed types
Soybean meal Corn meal
Rice bran
Fed mixture*
% total fatty acid
16,34±1,45a
17,42±1,46a
16,78±1,39a
16,00±1,62a
SFA
22,94±1,41a
21,64±2,31a
21,89±2,06a
23,12±0,49a
MUFA
b
b
b
20,96±0,44
21,12±1,47
18,09±1,87a
PUFA (excep HUFA) 21,31± 1,16
39,50±2,58a
39,99±2,99a
40,20±2,60a
42,78±2,09a

HUFA
mg/g
12,79±2,24a
12,13±1,15a
13,42±1,21a
12,73±1,86a
FA
a
a
a
2,07±0,0,28
2,10±0,11 3
2,23±0,31
2,06±0,50a
SFA
2,68 ±0,18a
2,61±0,13a
2,94±0,38a
2,95±0,47a
MUFA
2,72±0,43ab
2,55±0,28ab
2,83±0,27b
2,29±0,27a
PUFA
4,19±0,73a
4,87±0,83ab
5,39±0,64ab
5,44±0,77b
HUFA

Average ± standard deviation. Different caps on the same row showed a statistically difference (P <0.05).

In terms of percentages of total fatty acid in this study, the percentage of fatty acids
was quite similar. Only the difference of PUFA was statistically significant (P <0.05).
In terms of content (mg/g), the difference between PUFA and HUFA in all
treatments was statistically significant (P <0.05). But this difference did not follow the
rules (Table 3.13).
3.4.4 Effect of ration on density, biomass and biomass quality of oligochaete (E9)
3.4.4.1 Effect of ration on biomass of oligochaete
During the experiment, the biomass of L.hoffmeisteri in the negative control
had increased very slowly. Conversely, the biomass of L.hoffmeisteri when this species
fed at the 5%, 10% and 15% ration of this food had increased rapidly. 5th weekend,
The treatment that L.hoffmeisteri were fed at ration of 15% had highest biomass
(153.03 ± 10.56 mg/cm2), followed by the ration of 10% (127.32 ± 8.59 mg/cm2 ).
Conversely, lowest biomass (10,24 ± 1,19 mg/cm2) were recorded when feeding L.
hoffmeisteri with ration of 0%, statistically significant (P <0.05) (Figure 3.18).
Ration had clearly effect on the biomass of the worm population. Same as
previous research. The total mass recovered was lowest at the 0% ration. The second
lowest mass was recovered at the 2.5% ration, and the total mass recovered was greatest
10.0% rations (Oplinger et al., 2011).

19


d

180

d

(mg/cm2)
Biomass
Sinh khối (mg/cm2)

160
140
120

d

100

c

80
60

b

40
20

c

c

b

b

0%

b

b

c

5%

b

c

10%
15%

b
a

a

a

a

a

0
W1

W2

W3

W4

W5

Weeks
Tuần
nuôi

Figure 3.18: Biomass of L. hoffmeisteri populations at different feeding ratio
3.4.4.2 Effect of ration on density of oligochaete
Density of L. hoffmeisteri in the negative control had increased slightly in the 3rd
week after that reduced in the 5th week. Conversely, L.hoffmeisteri density of the other
treatments had increased continuously throughout the entire experimental period. 5th
wekend, the treatment that oligochaete were fed at ration of 15% had highest density
(63,7±4,9 individuals/cm2), followed by the ration of 10% (53,1±4,0 individuals/cm2).
Conversely, lowest density (4,6 ± 0,5 individuals/cm2) was recorded when oligochaete
were fed at ration of 0%, statistically difference (P <0.05) (Figure 3.19)

2
Density
)
độ (con/cm2)
Mật(individual/cm

d

d

70
60

d

c

c

50

c

K0%

c

40
30

b

K5%
K10%

bc

b

b

20

b

K15%

b

10

a

a

a

a

a

0
Thả nuôi

Inoculation

T1

T2

T3

T4

T5

Tuần nuôi

Culture week
Figure 3.19: Density of L. hoffmeisteri populations at different feeding ratio
3.4.4.3 Effect of ration on biomass quality of oligochaete (L. hoffmeisteri)
Ration had effect on the crude protein, crude pipid and ash of the oligochaete
(P<0,05). Conversely, the difference of crude fiber and carbohydrate was not
statistically significant (P> 0.05), Table 3. 14.

20


T 3. 14: The biochemical ingredients of oligochaete at different feeding ratio (DW)
Ingredient
Rate (%)
0
5
10
15
a
ab

b
Crude protein 47,51± 1,81
48,93 ± 2,30
51,98 ± 1,57
52,51 ± 1,15b
b
b
a
Crude pipid
17,65 ± 0,94
18,08 ± 0,92
15,50 ± 1,47
16,98 ± 0,69ab
Ash
16,98 ± 0,69c 15,20 ± 0,57b 14,03 ± 0,13b 13,52 ± 0,10a
Crude fiber
1,26 ± 0,16a
1,23 ± 0,03a
1,34 ± 0,15a
1,33 ± 0,12a
Cacbonhydrat
16,68±2,10a
16,58 ± 0,89a 17,24±1,67a
15,70 ± 1,66a
Average ± standard deviation (SD). Different caps on the same row showed a statistically difference (P <0.05).

Results showed, percentage of MUFA, PUFA and HUFA was quite similar
between the treatments. Only, the difference of saturated fatty acids (SFA) between
treatments was statistically significant (P<0,05) (Table 3. 15).
Table 3.15 : The fatty acids of oligochaete at different feeding ratio (DW)

Ingredient
Rate (%)
0
5
10
15
% total fatty acid
16,44 ± 1,35ab
17,70 ± 1,63b 17,37 ± 1,38 ab 15,34 ± 0,71a
SFA
22,64 ± 1,49a
22,68 ± 2,42a
22,29 ± 2,83a
22,72 ± 0,86a
MUFA
21,64 ± 2,43a
21,99 ± 2,43a
20,87 ± 1,13a
19,81 ± 4,23a
PUFA
38,93 ± 2,81a
38,38 ± 4,01a
39,47 ± 4,02a
42,13 ± 3,36a
HUFA
mg/g
13,67 ± 1,76a
14,85 ± 1,75a
12,45±1,90a
13,44±1,54a

FA
2,24 ± 0,25ab
2,62 ± 0,22b
2,17 ± 0.43ab
2,07 ± 0.32a
SFA
3,09 ± 0.43a
3,34 ± 0.25a
2,76 ± 0.41a
3.06 ± 0.45a
MUFA
3,01 ± 0,49a
3.16 ± 0.43a
2.59 ± 0.29a
2,62 ± 0.36a
PUFA
5,33 ± 0.86a
5.74 ± 1.21a
4.93 ± 1,08a
5,69 ± 1,01a
HUFA
Average ± standard deviation (SD). Different caps on the same row showed a statistically difference (P <0.05).

3.4.5 Effect of density on biomass and density of oligochaete (L. hoffmeisteri) (E10)
3.4.5.1 Effect of ration on biomass of oligochaete
Initial stocking biomass had affected the increase in biomass of L. hoffmeisteri.
From week 1 to week 5, worm biomass increased and reached the highest in the
treatments that initial stocking density was 30mg/cm2 (Hình 3.20). However, the biomass
growth rate of this treatment decreased at the end of the experiment. 5th wekend, the
treatments that initial stocking density was 10 mg/cm2 had highest growth rate (0.07 ±

0.02 %/day), statistically difference (P <0.05) (Table 3.16).
Table 3. 16: Growth rate of oligochaete biomass (%/day) by initial stocking density
Initial stocking density
Cultured
2
weeks
1mg/cm
10mg/cm2
20mg/cm2
30mg/cm2
1
0,15± 0,02b
0,08±0,03a
0,07±0,02a
0,08±0,01a
2
0,04 ± 0,02a
0,09±0,04b
0,06±0,03ab
007±0,02ab
3
0,04 ± 0,03a
0,05±0.01ab
0,06±0,01b
0,03±0,01a
4
0,05 ± 0,04a
0,08±0,03b
0,08±0,02a
0,05±0,01a

5
0,03 ± 0,02a
0,03±0,02a
0,03±0,01a
0,07±0,02b
Average ± standard deviation. Different caps on the same row showed a statistically difference (P <0.05).

21


The results of this thesis was similar to those of previous studies. The highest
biomass growth rate was 0.0080 mg/day/beakers in the treatments that initial stocking
density was the lowest (2,674 individual/ m2). In contrast, the lowest biomass growth
rate was 0.0072 %/day in the treatments that initial stocking density was the highest
(133,720 individual /m2 (Oplinger et al., 2011).
250

d
2
(mg/cm
Biomass
)
(mg/cm2)
Sinh khối

200

d

c

c

150

d

b

c

100

c

50

b
a

d

1mg/cm2
10mg/cm2
20mg/cm2
30mg/cm2

b

d

c
b

b

a

a

a

a

0
T. 1

T. 2

T.3

T.4

T.5

Weeks
Thời gian
nuôi (tuần)

Figure 3.20 : Biomass of oligochaete populations at different initial stocking density
3.4.5.2 Effect of initial stocking density on density of oligochaete

The results showed that initial stocking density had a large influence on
production. Oligochaete population densities were always highest in treatment that
initial stocking density with 30mg/cm2 and the lowest in the treatment that initial
stocking density with 1mg/cm2, statistically significant (P <0.05) (Figure 3.21).
100

2
được (con/cm2)
Mật độ đạt
(individual/cm
Density
)

90
80
70

1mg/cm2
10mg/cm2
20mg/cm2
30mg/cm2

60
50
40
30
20
10
0
1

2

3

4

5

weeks
ThờiCulture
gian nuôi
(tuần)

Figure 3.21: Density of oligochaete populations at different initial stocking density
22


The growth rate of the density was also tended to decrease at the end of the
experiment. 5th wekend, the growth rate of the densities was low in all treatments. The
highest growth rate was 0.07 ± 0.02 %/day in the treatments that initial stocking density
was 10 mg/cm2, statistically difference with other treatments (P <0.05) (Table 3.17).
Table 3. 17: Growth rate of oligochaete density (%/day) by initial stocking density
Cultured
Initial stocking density
weeks
1mg/cm2
10mg/cm2
20mg/cm2
30mg/cm2

1
0,07± 0,02ab
0,08±0,02b
0,04±0,02a
0,04±0,01a
a
a
a
2
0,07 ± 0,02
0,09±0,04
0,06±0,03
0,08±0,02a
3
0,04 ± 0,03a
0,05±0.02a
0,06±0,01a
0,03±0,01a
4
0,05 ± 0,03a
0,08±0,02a
0,08±0,02a
0,05±0,01a
a
b
a
Tuần 5
0,02 ± 0,01
0,07±0,01
0,03±0,02

0,03±0,01a
Average ± standard deviation. Different caps on the same row showed a statistically difference (P <0.05).

The results showed that initial stocking density had affected on density. Density
of oligochaeta had a large influence on production. However, after reaching peak
density, there is strong intra-stage competition between adults and juveniles (Bonacina
et al, 1989). Reproduction reduced after highest density (Elwell et al. 2006).
3.4.6 Culture of L. hoffmeisteri with the results of the study (E11)
The results of previous experiments had determined: The suitable substrate
structure was containing a mixture of mud (75%) and fine sand (25%). The substrata
was 4cm. The feed type was containing a mixture of soybean meal, corn meal and rice
bran at a ratio of 1: 1: 1 with ration of 15%. The initial stocking density was 100 g/m2.
3.4.6.1 Productivity of oligochaete (L.hoffmeisteri)
Average biomass was 1.58 ± 0.12 kg/m2 (Table 3.18). This result shows that
biomass productivity in experimental culture was relatively high with previous studies.
Soybean meal and mustard oil cake had a large affect oligochaete population biomass.
The most suitable substrate for culture oligochaete was containing 30% soybean meal
and 20% mustard oil cake with other media. The average biomass of oligochaete at 40
day was 118.13 ± 7.35 mg/cm2 in treatments that culture media having 30% soybean
meal and 20% mustard oil cake (Hossain et al. , 2011).
Table 3. 18 Oligochaete biomass productivity in experiment 14
Indicator
culture culvert
Average
1
2
Total biomass (kg/2,4 m2/5 week)
3,57
3,98
3,78 ± 0,29

Productivity (kg/m2/5 week)
1,49
1,66
1,58 ± 0,12
3.4.6.2 Biomass quality of oligochaete (L. hoffmeisteri)
Although the protein content of L.hoffmeisteri (54,90 ± 1,71%)(Table 3.19)
was lower than protein content of Olygochaeta (59.13%) (Supenya Chittapun et al),
But higher than the protein content of L.hoffmeisteri that were collected naturally
(Table 3.12).

23


Table 3. 19: The biochemical
ingredients of oligochaete in
experiment 14
Ingredients (%) Culture worms
DW
54,90 ± 1,71
Crude protein
Crude pipid
15,79 ± 0,44
Ash
10,71 ± 0,31
Crude fiber
1,55 ± 0,73
Cacbonhydrat
17,04 ±acid
0,65 (Valine,
The essential amino

Giá trị Isoleucine,
trung bình ±Threonine,
độ lệch chuẩn
(SD).
Leucine,
Methionine,

Table 3.20 Amino acid composition of
worms in experiment 14
Amino acid
Percentage (%)
Alanine
2,88 ± 0,12
Glycine
2,48 ± 0,21
Valinea
2,56 ± 0,06
Leucinea
4,38 ± 0,11
Isoleucinea
2,43 ± 0,11
a
Threonine
2,33 ± 0,05
Serine
2,03 ± 0,04
Proline
2,66 ± 0,15
Asparagine
5,06 ± 0,07

Methioninea
0,67 ± 0,07
Hydroxypronine
0,05 ± 0,02
Glutamine
7,09 ± 0,10
Phenylalaninea
3,06 ± 0,08
Lysinea
4,20 ± 0,02
a
Histidine
1,49 ± 0,05
Cystine
0,23 ± 0,06
Tyrosina
1,81 ± 0,02
Tổng
45,41 ± 0,74

Phenylalanine, Lysine, Histidine, Cysteine,
Tyrosine) reached for 51% of the total free
amino acids present in the biomass
oligochaete. This result was suitable with
some analyzes of free amino acid of
freshwater oligochaete. Essential amino acids Average ± SD; a essential amino acids
of 5 species of freshwater worm (L.
hoffmeisteri, T.tubifex, Potamothrix vejdovskyi, Srylodrilus heringianus và P.moldaviensis)
ranged from 38.4% to 54.8% of total free amino acids (Graney et al., 1986).
3.4.6.3 Feed conversion ratio of oligochaete

Experimental results with positive results. After a day of inoculation , oligochaete
are distributed throughout the culvert. At the end of the first week, biomass reached
17.40 ± 0.05 mg/cm2. This biomass increased of 1.74 times compared to the initial
biomass. At the end of the 5-week, the average biomass was 146.95 ± 2.99 mg /cm2.
Feed conversion ratio of complete feed (corn meal, corn meal and rice bran at a ratio of
1: 1: 1) was low (1.46 ± 0.11). Feed conversion ratio of L. hoffmeisteri in the thesis was
much lower than that of some previous studies using organic fertilization or combination
of organic matter and fine sand to culture oligochaete. Feed conversion ratio of
oligochaete when oligochaete were cultured on a substratum containing a mixture of
fresh cow dung (75%) and fine sand (25%) ranged from 18-25 (Marian and Pandian,
1984; Marian et al., 1989). Feed conversion ratio of oligochaete when oligochaete were
cultured on pig manure was 11.11, on cow dung was 20 (Dinh The Nhan, 1999). Feed
conversion ratio of oligochaete when oligochaete were cultured on a substratum
containing a mixture rice bran (35%), mustard oil cake (20%), cow dung (25%) and fine
sand (20%) wwas 3.5 (Mollal and Ahamed, 1993). Feed conversion ratio of oligochaete
when oligochaete were cultured on a substratum containing a mixture rice bran, soybean
meal, mustard oil cake, cow manure and fine sand with 20%, 30%, 20%, 20% and 10%
percentages respectively was 2.65 (Hassain et al., 2011).
3.5 Application biomass oligochaete to culture zebrafish (Danio rerio) and siamese
fighting (Betta splendens).
Experimental result of worm culture was very good. Protein, lipid, ash and fiber
content in feeds that were used for zebrafish (Danio rerio) and siamese fighting (Betta
splendens) in Table 3.21.

24