Lab Activity III

FERTILIZATION AND EMBRYONAL DEVELOPMENT

OF NILEM FISH

Day : Thursday
Date : 27 September 2018

Name : Isnaeni Rachmawati

Student ID : B1B017036
Group :2
Subgroup : VIII
Assistant : Monica Widianti

LABORATORY OF ANIMAL STRUCTURE AND DEVELOPMENT

FACULTY OF BIOLOGY
JENDERAL SOEDIRMAN UNIVERSITY
PURWOKERTO

2018
 I. INTRODUCTION

A. Aims

The aims of this practical class are to be able doing fertilization in fish, recognize
fertilized egg cells of fish and identify factors that affecting the success of fertilization.

B. Benefits

The benefits of this practical class are students will be able to do fertilization in
fish alone, and know the process of embryonal development before becoming a
complete individual.
 II. MATERIALS AND WORK PROCEDURE

A. Materials

The tools that used in this practical class are well plate, transferring pipette, 1
mL syringe, plastic bowl, plastic plate, light microscope, timer, cavity slide, aerator,
and filter.
The materials that used in this practical class are milt of Nilem Fish
(Osteochillus hasselti), physiological NaCl solution, fresh egg cells and milt, also tap
water/ well water.

B. Work Procedures
Time Space
The work procedures that used in this time space method are:
1. All the tools and materials needed are prepared.
2. Milt that has been taken undergo dilution 10x.
3. Mature female Nilem fish is stripped and egg cells are taken out, put in plastic plate.
4. 1 mL milt from 10x dilution is added into the plate of egg cells, agitated for (1/ 3/
5 minutes) while water is also being added. Then both of them are filtered, leave
for 20 minutes.
5. Ten egg cells are randomly picked and put upon the cavity slide for observation
under the microscope.
6. Repetition of observation under microscope in 20, 30, and 40 minutes are done.
Dilution
The work procedures that used in this dilution method are:
1. All the tools and materials needed are prepared.
2. Milt that has been taken undergo dilution10x.
3. Mature female Nilem fish is stripped and egg cells are taken out, put in a plastic
plate.
4. Milt that will be added into plastic plate containing egg cells, are first diluted
according to the needs (10x, 100x, or 1000x).
5. Milt and egg cells are agitated for 3 minutes.
6. Ten egg cells are randomly picked and put upon the cavity slide for observation
under the microscope.
7. Repetition of observation under microscope in 20, 30 and 40 minutes are done.
 III. RESULT AND DISCUSSION

A. Result
Tabel 1. Presentation of Fertilized Egg in Different Time Space
Presentation of Fertilized Egg (%)
Total Average
Time Space Repetition
Repetition II Repetition III (%) (%)
1
Control 76% 46,64% 122,64 40,88
1 minute 52,55% 46,66% 50% 149,21 49,73
3 minutes 50% 95,6% 63% 208,60 69,53
5 minutes 74% 6% 0,23% 80,32 26,74

Tabel 2. Presentation of Fertilized Egg in Different Level of Milt Dilution

Level of Presentation of Fertilized Egg (%) Total Average
Milt Dilution Repetition I Repetition II Repetition III (%) (%)
Control 76% 46,64% 122,64 40,88
10x 53% 30% 83 27,66
100x 80,10% 23,3% 83% 186,40 62,13
1000x 81,65% 81,65 27,21

Calculation Data:
1. Fertilization Rate (FR) = ∑ Fertilized egg x 100%
∑ Total egg cell
20
= 30 x 100%

= 67%

2. Hatching Rate (HR) = ∑ Hatched eggs x 100%

∑ Total fertilized eggs
90
= x 100%
0,67 𝑥 288
90
= x 100%
192,96

= 46, 64%
Tabel 3. Presentation of Egg Cells in Every Stage of Development during
Observation within Time Space Treatment
Treatment Time of Stage of % of Eggs in Every Total Average
Observation Development Development Stage (%) (%)
Rep. Rep. Rep
I II III
Unfertilized 70 100 170 56,67
20’ Hylock 30 30 10

Hylock 50 40 90 30
2-cell 50 60 110 36,67
30’

Control

2-cell 70 50 120 40
Hylock 30 10 40 13,33
4-cell 40 40 13,33
40’

50’

Unfertilized 10 100 90 200 66,67

Hylock 10 10 3,33

20’

1 min
space
Hylock 20 40 60 120 40
Unfertilized 60 60 30 150 50
30’ 2-cell 10 10 20 6.67
Damaged 10 10 3.33
 40’ 2-cell 50 30 80 26,67
Hylock 40 50 40 130 43,33
Unfertilized 60 30 90 30

Tabel 3. (continue).
% Eggs in every
Treatment Time of Stage of Total Average
stage of
Observation Development (%) (%)
development
Rep. Rep. Rep.
I II III

50’

Hylock 100 50 150 50

Not forming 70 50 120 40
Damaged 30 30 10
20’

Hylock 40 80 10 130 43,33

2-cell 20 20 6,67
30’ Not forming 70 70 23,33
Damaged 60 60 20
Jeda 3 1-cell 20 20 6.67
menit
Hylock 60 60 120 40
Not forming 40 40 13,33
Damaged 100 100 33,33
40’
1-cell 10 10 3,33
2-cell 10 10 3,33
4-cell 20 20 6,67

50’

Jeda 5 Hylock 20 30 50 16,67

20’
menit Unfertilized 80 70 150 50
 Hylock 10 10 20 6,67
30’ 2-cell 50 30 80 26,67
Unfertilized 30 60 90 30
4-cell 10 10 3,33

Tabel 3. (continue).
Time of Stage of % Egg in Every Total Average
Treatment
Observation Development Stage Development (%) (%)
Rep. Rep. Rep.
I II III
Hylock 10 20 30 10
2-cell 80 80 26,67
40’
4-cell 90 90 30

50’

Tabel 4. Presentation of Eggs in Every Development Stage during Observation in

Dilution Level
Time of Stage of % Eggs in every Total Average
Treatment
Observation Development development stage (%) (%)
Rep. Rep. Rep.
I II III
Unfertilized 70 100 170 56,67
20’ Hylock 30 30 10

Control

Hylock 50 40 90 30
2-cell 50 60 110 36,67
30’
 2-cell 70 50 120 40
Hylock 30 10 40 13,33
4-cell 40 40 13,33
40’

50’

Tabel 4. (continue).
% Eggs in every Averag
Treatment Time of Stage of Total
stage of e
Observation Development (%)
development (%)
Rep. Rep. Rep.
I II III
Hylock 10 10 20 6,67
20’ Not forming 90 90 180 60

2-cell 70 70 23,33
Hylock 10 20 30 10
30’ Not forming 20 80 100 33,33
Dilution
Level 10x

4-cell 10 10 3,33
2-cell 50 50 16,67
Hylock 10 60 70 23,33
40’
Not forming 30 40 70 23,33

50’
 Hylock 50 50 100 33,33
Fertilized
Unfertilized 50 10 50 110 36,67
20’

4x division 30 30 10
Dilution Hylock 30 10 20 60 20
Level 30’ Fertilized 50 50 16,67
100x 2-cell 40 10 50 16,67
Unfertilized 30 80 110 36,67

2x division 40 20 80 140 46,67

Hylock 10 20 20 50 16,67
Unfertilized 50 50 16,67
40’
Deformed 10 10 3,33
4-cell 50 50 16,67

Tabel 4. (continue).
Time of Stage of % Egg in every stage of Total Average
Treatment
Observation Development development (%) (%)
Rep.
Rep. I Rep. II
III

50’

Hylock 10 10 3,33
Unfertilized 100 90 190 63,33

20’
Dilution
Level
1000x
Hylock 50 50 100 33,33
Unfertilized 10 50 150 50
30’ 2-cell 40 40 13,33
 Hylock 50 50 16,67
Unfertilized 60 40 100 33,33
Damaged 10 10 3,33
40’
4-cell 40 40 13,33

50’

Details
RI RII RIII RIV RV RVI RVII RVIII
K1 1’ K1 5’ K1 1’ K1 3’ K1 3’ K1 1’ K1 K1 1’
Control
K2 3’ K2 1’ K2 3’ K2 5’ K2 5’ K2 3’ K2 1’ K2
Control
K3 K3 K3 5’ K3 K3 K3 5’ K3 5’ K3 3’
10X Control 10X 10X
K4 K4 10X K4 K4 K4 K4 K4 K4
100X 1000X 100X 100X 10X 100X 100X
K5 K5 K5 K5 K5 K5
1000X 100X Kontrol 1000X 1000X 100X

ACC ACC ACC ACC ACC ACC ACC ACC

Pertukaran data: RI sampai RIV; RV sampai RVIII;

Figure 3.1 Unfertilized eggs Figure 3.2. Hylock formation

Figure 3.3. Two cells stage Figure 3.4. Four cells stage

Figure 3.5. Hatched larva

B. Discussion
Nilem fish (Osteochillus vittatus) is a native Indonesian fish and has high
economic value. Naturally, the gonad development of this fish group is strongly
depending on its habitat leads to a very limited frequency of spawning to twice per
annum. Spawning frequency of the nilem, however, might happen monthly though it
will reach maximum spawning frequency on a particular month on the year
(Setyaningrum et al., 2017).
Fertilization is a fusion between two gametes; male and female gametes which
ends with the fusion of nucleus consisting genetic materials, so zygote is formed.
Fertilization can be divided into two, internal and external fertilization. External
fertilization is a fertilization that happens outside the female body and commonly
occurs in aquatic animals and some land animals. In external fertilization, spermatozoa
is only motile after getting in contact with water to reach the place where egg cells
located (Soeminto, 2004).
Success or failure of the fertilization process is determined by several factors.
Internal factors include heredity and sex chromosome that carry genetic traits from
nature are difficult to be contributed. External factors influence embryo growth are
temperature, salinity, food, pollution which indirectly reduce the water quality
(Prabowo et al., 2016).
Other external factor such as condition of water flow during spawning also
contribute into success of fertilization. Water that flows profusely can cause
spermatozoa that released from male fish to drift and lessen the chance for ovum to
meet spermatozoa, therefore fertilization cannot occur. Water quality also play role in
fertilization process, water that has many toxic materials can cause death to ovum and
sperm. Acid water can free CO2 from bicarbonate. Humidity cause egg cells cannot be
fertilized which leads into damage to them. The damage can be torn up of periviteline
space and the chorion. Eggs that are not fertilized will die and have white feculent
color because of its brightness have gone (Harinadi, 2010).
Another factor that affecting the entire process of fertilization is the water
temperature. Temperature affects the speed of development process or developmental
fractions. Some types of fish develop under temperature that is not optimal like the
ones develop in laboratory condition. Temperature that toolow or too high will disturb
the development. Extreme temperature or sudden changes may cause death to the egg
cell also (Andriyanto, 2014).
 Internal factors that affecting fertilization process, such as physiological
condition of the fish that has genital disease. The higher the dilution level of milt, then
the longevity of motility becomes shorter, and so the opposite. This shows that the less
the motility, the harder it will be for observation of spermatozoa. Because higher
dilution level gives different osmolarity level (Wijayanti, 1997).
Fish of different developmental stages need different handling. Early
embryonic stages, from cleavage to late gastrula, are very susceptible to damage. Fish
larvae, particularly in the stage of yolk absorption and fin formation, are fragile.
Therefore they needed a fast penetrating fixative solution to prevent them from damage
(Wijayanti, 2017). The fish survival during post larvae is strongly influenced by the
availability of food because fish larvae will die in a short time if it does not manage to
get food; larvae will experience nutritional deficiencies leading to running out of
energy (Rukmini, 2016).
In this lab activity of fertilization and embryonal development, there are two
types of testing; based on the time space contact between egg cells and spermatozoa
(20, 30, and 40 minutes) and based on dilution level. Both of the testing are supported
with control variable. Our group got the control variable in third repetition.
Observation began with taking 10 random egg cells then added with 1 mL milt of 10x
dilution and agitated slowly to activate the motility of spermatozoa. When agitating is
done, the mixture of egg cells and spermatozoa is moved into another container with
aerator to keep the water rich in oxygen. After 20 minutes pass, take 10 egg cells
randomly and observe under microscope with low magnification (40x). The result is
all of the egg cells are still not fertilized yet. Observation after 30 minutes result in 4
cells already fertilized identified with the existence of hylock, and 6 cells are in 2-cell
division stage. After 50 minutes, observation is redone again under the microscope and
it can be seen that only one hylock left, 5 cells already in 2-cell stage, and 4 cells are
in the 4-cell stage. Later on, the data is calibrated and obtain presentation of fertilized
cell rate (FR) is 67%.
Average result from repetition 1, II, and III in time space of 1 minute is 49,73%,
in 3 minutes is 69,53% and in 5 minutes is only 26,74%. From these results show that
the longer time contact between sperm and egg cells, the higher the presentation of
fertilized egg cells. 4-cell stage division happens 50 minutes to one hour after
fertilization then continued to division into 8-cell, 16 cell, and 32 cell (Shukla, 2010).
 Meanwhile in the dilution level, 10x dilution has average presentation of
fertilized egg cells 27,66% consists of 53% in repetition I, and 30% in repetition II.
100x dilution resulted in FR 80,10% in repetition I, 23,3% in repetition II, and 83% in
repetition III so the average presentation is 62,13%. As for the dilution 1000x, data
obtained has total of fertilization rate 81,65% with average rate is only 27,21%.
Meanwhile for the control variable is the same as in the time space testing, which is
average 40,88%.
Aside from the result of dilution 100x, it can be said that this practical activity
is compatible with reference which says that in dilution testing, the higher the dilution
level then the motility of sperm will be shorter. So there will be less spermatozoa that
able to fertilize the eggs. It can be seen from 10x dilution to 1000x dilution there is
0,45% difference. Incompatible data obtained from the practical activity and the
references might be caused by several reasons such as agitation process is too fast, the
fish has been stripped too many times and inaccuracy of counting the fertilized eggs
(Arsianingtyas, 2009).
The cause of the low level of hatching is also suspected to be related to the
spawning time of fish. If the spawning season happens in rainy season, can lead to the
poor quality of sperm and result in the failure of spermatozoa to merge into the egg
cell nucleus, hence the eggs do not divide at blastocyte stage after fertilization, and the
embryos die before hatching (Rukmini, 2016).
According to Health (1995), the cause of insufficient result of fertilization and
development in Nilem fish can be because of the fish is in stress condition, it is not yet
sexually matured, the hormone injection is not very careful causing wounds to the fish,
and the sperm is weak, therefore cannot survive before penetrating the egg.
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion, it can be concluded that fertilization in fish
is categorized as external type of fertilization. It can be done by doing test based on
time space of contact and serial dilution. The longer the time is given, the more egg
cells are fertilized by spermatozoa. Fertilized egg cells are identified by the lighter
color of egg surface, and the presence of hylock. Several factors affecting the success
of fertilization are such as water flow, water quality, and humidity.

B. Suggestion

In this practical lab activity, it would be better if students are let to take the milt
and egg cells from fish directly and not just watching. So the students will understand
the procedure of stripping better.
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