FORMAT OF ANIMAL DEVELOPMENT PRACTICAL REPORT:

Practical report typed using Times New Roman font, font size 12, spacing 1.5 with
margin 4 cm left and top, 2.5 cm right and bottom on Quarto Paper (A4) 70 grams.
- Report consist of :
Cover Page
I. INTRODUCTION
a. Background
b. The Aims
II. MATERIAL AND METHODS
a. Material
b. Methods
III. RESULT AND DISCUSSION
a. Result
b. Discussion
IV. CONCLUSION AND RECOMMENDATION
REFERENCES
- Jurnal berbahasa Indonesia atau Inggris (5)
- text book berbahasa Indonesia atau bahasa Inggris (5)
- melampirkan 1 jurnal berbahasa Inggris. Jurnal yang dilampirkan harus
dimasukkan ke dalam pembahasan dan sudah ditandai.
- Gambar atau data hasil praktikum wajib dilampirkan dalam laporan
 FERTILIZATION AND DEVELOPMENT NEMBRYO OF NILEM FISH
(Osteochillus hassselti)

By:
Name : Safira Dwi Oktaviani
Student ID : B1B015002
Entourage : VII
Group :1
Assistant : Sarah Nurul

PRACTICAL REPORT OF ANIMAL DEVELOPMENT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2016
 I. INTRODUCTION
A. Background

Every living organism is created with a limited life time la. So that they can
maintain the existence of its kind, they have to produce the successor generation, or
the term is generally capable of reproducing. Reproduction is the ability of living
organisms to produce offspring (Effendy, 1997).
In order to produce offspring living things must reproduce the circumstances
that fertile or infertile, there will be a process of fertilization. According Soeminto
(2000), fertilization is a process that starts with the convergence of sex cells of male
and female sex cells. Penetration of sperm into the egg topped with the merger of the
two pronuklei. Because after fertilization, the egg will become more active to
cleavage segmentation and evolving, whereas when the fertilized ovum is not going
to die. Meanwhile, according to Hoysak and Liley (2001) fertilization is the process
of joining of the sperm nucleus with the cytoplasm of the egg cell nuclei to form a
zygote, which this association is a chain of early and very important in the process of
fertilization.
Practicum this time we used Osteochillus hasselti or nilem fish as animals
that will be tested in the lab this fertilization. Nilem fish are fish that are in the
fertilisasinya or external environment, because milt and egg is released in waters
with a sequence of first egg is released and then fertilized by male.Milt can fertilize
the egg because the egg has milt and chemical signals and receiving signals to each
other via protein receptors that are owned respectively (Soeminto, 2004).
Fertilization and embryonic development of fish is done with a concentration level of
dilution varying aims to determine the number of ovum and sperm concentration that
is most appropriate in order to achieve fertilization. Pause a different time aims to
determine the right time for the ovum and fertilization of eggs can perform
(wijayanty 2010).
The use nilem fish because these animals easily available and relatively cheap
price.The fish Nilem (Osteochilus hasselti) has type telolechital egg weight, yolk
means are unevenly distributed and can be considered almost fill entire sphere of
eggs. Bioplasma only as a thin layer on the polar animal in which there is a core of
eggs. Type of fish Nilem cleavage are meroblastik (Moeller, 2004).

B. Purpose
 The purpose fertilization and embryonic development of fish nilem is
fertilization in fish, recognize the fertilized egg cell, identify the factors that affect
fertilization, and identify the stages of embryonic development of fish.
 II. MATERIAL AND METHODS

A. Material

The tools used in this lab is well plate, large-scale transfer pipette, 1 ml volume
syringe injeeksi, plastic cups, plastic bowls. Cavity slides, light microscopy,
timekeeper, haemocytometer, timekeeper, cotton fabrics, filter, aquarium aeration
system.
The materials used in this lab is nilem male fish milt, fish eggs nilem female
cells, physiological saline or Ringer, hormones for ovulation induction and spermiasi
(keenjar pituitary gonadotropins GnRH analog or analog), and distilled water

B. Methods

The method used in this lab are:

B.1 Induction to get fresh gametes

1. Fish nilem (Osteochillus hasselti) mature gonads selected, a mature female fish
gonad is characterized by an enlarged abdomen, feels soft when touched, and the
area around the porous urogenital slightly reddish .; Gonads mature males, which
is characterized by ease milt out at the time of stripping.
2. The fish will be weighed in order to hormone doses of the hormone can be
determined .the next step is done when the egg is really hard to get out when the
stripping process.
3. Hormones will be used for the induction prepared with the following provisions.
When the inducer in the form of extracts pituitary hormone dosages are given
based on the ratio of the weight of fish donor and recipient fish is 3: 2 means that
if a recipient fish weight of 1 kg, the donor fish weight was 1.5 kg. If the
hormone GnRH inducers such ovaprim example, the dose given was 0.5 ml / kg
body weight.
4. Preparations hormone that has been prepared is injected into the muscle tissue
and maintained until no spilling out.
5. Male parent put together with females that had induced put into the aquarium
with clean water and aeration is complete. The upper part of the aquarium is
covered with gauze or strimin so that the fish do not jump out of the aquarium.
6. The behavior of fish observed during 6-8 hours after inducing, when iakn been
doing spinning motion, following each other from each other yamg these things
indicate that the fish are ready to spawn.
7. After the lifting of the observed fish in the aquarium. The abdominal wall is
finely sorted starting from the front fin abdomen toward the genital pore, thus
exiting thick white liquid like milk. The liquid is milt and egg, if it has come out
the stripping process can be carried out using a 1 ml syringe.
8. Milt and egg is removed from the syringe accommodated in the container is clean
and dry, and miltp and cell egg ready for fertilization.

B.2 fertilization with various ratios of sperm: egg

1. The whole equipment to be used is prepared
2. Gond mature male fish from the aquarium removed and cleaned abdominal
especially around the genital pore using tissue paper.
3. The abdominal wall gently massaged starting from the front fin abdomen toward
the genital pore, thus exiting thick white liquid like milk. The liquid is milt
(sperm in the seminal plasma).
4. Milt obtained accommodated in 1 ml syringe without a needle that also functions
as a measure of volume of milt. Volume milt obtained measured.
5. Milt was diluted using well plate. In the first well put one part of miltyang
volume of 0.05 ml physiological saline plus 9 parts volume of 0.45 ml
homogenized with pipette several times to obtain a 10x dilution. Take 1 part milt
of the first dilution was added to the second well plate, is added 9 parts of
physiological saline solution volume of 0.45 ml and homogenized until milt
obtained by dilution 100x. The same way done for dilution 1000X
6. Fish mature female gonads removed from the aquarium, the abdominal wall is
cleaned, especially around the genital pore used tissue paper.
7. The abdominal wall sevara finely sorted, swab of the front fin abdomen toward
the genital pore, thus exiting the egg with a dark brown color yellowish,
clustered, contains no water and fresh-looking. The egg cell housed in a small
plate that is dry and clean.
8. One small spoon of egg cells is taken and diluted 10x, stirring gently so the egg
and milt homogenous, water dripped little by little to activate spermatozoa while
still agitated gently for 3 minutes.
9. After 3 minutes of mixing egg and milt, slowly add the medium conception that
fit into each cup until its volume reached 100mL. Allow the egg cell that has
been mixed with milt in each medium for 5 minutes, later the egg wash with
plain water.
10. Egg cell that has been washed entered into a basin of water wells or tap water
with a water volume of about 1.5 ml.
11. 10 egg cells are taken from each fertilization using a transfer pipette and place it
above the cavity slide and observed under a microscope using 4x magnification
objective.
12. The proportion of eggs fertilized and unfertilized calculated. The fertilized egg
yolk color characterized by a sharp, yolk high integrity and the animalis pole is
formed buds fertilization. Unfertilized egg yolk color characterized by a dull, the
integrity of the yolk decreased and there are no buds fertilization.
13. Stage 8-12 performed equally on milt by diluting 10, 100, 1000, data are
recorded into a table,
14. Counte the consentration at each dilution using a haemocytometer whilst
awaiting the observations of embryos.

C.3 fertilization with different contact time of spermatozoa with egg

1. The whole equipment to be used is prepared.
2. Milt diluted 10x prepared in 0.9% NaCl solution.
3. Fish mature female gonads removed from the aquarium, the abdominal wall,
especially around the genital pore cleaned using tissue paper.
4. The abdominal wall gently massaged starting from the front fin abdomen toward
the genital pore, thus exiting the egg with a dark brown color yellowish. Note: at
the time of egg dtripping good will come out easily are not clustered, contains no
water and fresh-looking. The egg cell is accommodated on a small plate that is
dry and clean.
5. One small spoon of egg cells is taken and added to 1 ml milt diluted 10x,
agitation slowly so the egg and miltbercampur average, while water dripped little
by little to activate spermatozoa (up to volume 50 ml).
6. After 1 minute mixing or agatasi ovum and spermatozoa poured into a fine sieve
to remove spermatozoa after one minute of mixing egg and sperm rinsed by
dipping a sieve into a clean water as much as 3x.
7. The egg that had been washed incorporated into a basin of water wells or tap
water with a volume of about 1.5 liters.
8. 10 egg cells are taken from each fertilized using a transfer pipette and place it
above the cavity slide and observed under a microscope with a magnification of
4x every 20 minutes, 30 minutes and 50 minutes.
9. The proportion of eggs fertilized and unfertilized calculated. The fertilized egg
yolk color characterized by a sharp, yolk high integrity and the animalis pole is
formed buds fertilization. Unfertilized egg yolk color characterized by a dull, the
integrity of the yolk decreased and there are no buds fertilization.
10. Perform 5-9 phase contact time 3 and 5 minutes, and then filled into the ta
 B. Discussion

Fertilization is the fusion of two gametes that is the male gamete and
female gamete, which ended with the merger of the nuclei of two gametes to form a
zygote (Farooq et al, 2011). Consolidation is usually involves the incorporation of
the cytoplasm and nucleus material unification, by meiosis, the zygote formed a
fundamental characteristic of most eukaryotic mitotic cycle, and basically gametes
fuse is haploid. Fertilization in animals there are two kinds of external fertilization
and internal fertilization. External fertilization typical of aquatic animals, which is
the process of fertilization where the gametes are expelled from the body before
fertilization. Typical internal fertilization for adaptation to life on land, the sperm is
inserted into the female reproductive area and was followed by fertilization. After
fertilization, the egg fertilization form membranes (membrane feripitelina) to further
thwart sperm entry. Sometimes the sperm is needed only for mengaktivkan egg
(Linder, 1992).
Fertilization or also called fertilization is the process of joining of the sperm
nucleus with the cytoplasm of the egg cell nuclei to form a zygote. Basically
fertilization is the union of the male gamete or cell fusion and cell female gametes to
form a single cell (zygote). In the process of fertilization, the sperm into the egg
through the hole in the chorion micropyle contained. Each spermatozoa have the
same opportunity to fertilize an egg. However, because the space where the
fertilization of the egg by the sperm that is meeting on ovipar very big fish, then the
spermatozoa opportunity to meet with the egg is actually very small. Effendie (1997)
states to overcome these so that fertilization is successful, spermatozoa released the
numbers are very large compared to total eggs will be fertilized. In optimum
conditions the new fish spermatozoa expelled from the body has the power to move
around in the water for 1-2 minutes.
Telolecital nilem fish eggs are heavy with meroblastik division. Fish
embryo development itself starts from cleavage segmentation, morulasi, b; astulasi,
gastrulation until finally the process of organ formation perfect body (Linder, 1992).
Type of cell division is meroblastik nilem fish. Meroblastik division produces cells
that will become the embryo's body is located in the middle (Nurman, 1998).
 The process that occurs after fertilization is a division segmentation,
morulasi, blastulasi, and gastrulation. Phase segmentation division, the cells are still
very small and the maximum number is 32 cells. Morula form of late and early
blastula so similar that it is hard to distinguish (Effendy, 1997).
Developments in the fish embryo Nilem (Osteochillus hassselti) females
began after the egg is fertilized by a spermatozoon nucleus are all haploid, diploid
zygote into the core. The zygote is what has the ability to perform segmentation
division through mitosis process fast. The zygote-segment segmented into smaller
parts (cleavage), originated from a single cell then divides into two cells, 4-cell, 8-
cell, 16 cells, up to 32 cells called morula stage (Linder, 1992).
Zygote cell division in fish generally are the type meroblastik (partial)
although there are also holoblastik (total). In type meroblastik which divide only the
cell nucleus and cytoplasm course, being in the yolk also participated holoblastik
dividing. Both types of cell division is determined by the amount of egg yolk and
spread (Harinadi, 2010). In fish, reptiles and birds groups main cells is also called
disc sprouts (germinal disc) consisting of embryonic tissue (blastodisc) will be the
body of the embryo and tissue periblast that serves as distributor of food derived
from egg yolk (Dudek 2011 ). After the cleavage stage, a subsequent stage is the
formation of blastula. Blastulasi called blastula formation process in which a group
of cells child of cleavage-shaped objects that are relatively rounded adjoining empty
cavity called suloblastula (coeloblastula) while the hollow massif called
steroblastula. Suloblastula contained in Amphioxus and toads, steroblastula found in
fish and amphibians are no legs (gymmophonia) (Soeminto et al, 2010). After the
blastula stage, a subsequent stage is gratulasi process. Gastrulation is closely related
to the formation of the nervous system (neurolasi) so it is a critical period. In this
process the displacement area of ectoderm, mesoderm, and the notochord entoderm
towards a definitive (Wijayanti, 1997). Next is the stage of organogenesis.
Organogenesis, which is the process of formation of organs creature is growing.
System of organs derived from three layers, ie, ectoderm, and mesoderm entoderm.
From the ectoderm will form the organs arrangement (system) nerves and skin
epidermis. From entoderm will form along the digestive tract glands, digestive and
respiratory apparatus. While the mesoderm will appear skeleton, muscles, blood
circulation tools, tool excretory, reproductive organs and skin korium (Soeminto et
al, 2010).
 The development can be influenced by several factors that can determine
whether or not been able to a growing embryo. These factors affect the speed of
development and determine the shape and arrangement. Among these factors is the
water temperature. Temperature affects the speed of the whole process of
development or fractions developments. The speed can be expressed as the inverse
developmental periods in the day. The greater the fraction of the increasingly rapid
development. For example if the fish had during the development period of 88 days
then the speed is 1/88 (Robert, 2004) .According to the opinion parker and Begon
(1993) No Affect Four factors can influence the likelihood of fertilization success in
fish: (i) the proximity of lazy to eggs during ejaculation, (ii) the timing of sperm
release and coordination with female egg release, (iii) the number of sperm released
and (iv) sperm traits such as swimming speed (Taborsky, 1998). The first two are
intrinsic to sexual behavior in animals whereas the others depend upon the
individual. Occurs Sperm competition between the sperm of different lazy and Also
Among the sperm of a single male.
Based on the observations of our group fertilization describes the various
treatments that percentage fertilized 0% caused due to poor quality of the ovum.
While the average percentage of dilution of 10x, 100x, and 1000x the result
fertilization sperm and ovum shows quantity well. This assumption is based on the
literature stating that if fertilization has a percentage of >50%, it can be said that
sperm quality is quite excellent (Setu, 1962). Physical injury or stress affects the
percentage of fertilization, the female fish can produce a failure oocit stress in ovary
(Basaran, 2008). The quality of spermatozoa will be even better when the percentage
of conception which indicates that the egg and sperm used good quality can reach up
to 99% (Shukra, 2005). Further data, when picking up random or random data by
viewing the data obtained per treatment data tend varied and have the decrease the
accordance with the repetitions 20 minutes as the initial stage of development after
fertilization there is a difference observed physical properties of the cell, the cell the
start bright and there are such fine lines round the egg is fertilized it. Average cell
that successfully fertilized in this stage is 36.75% which includes all phases of
development of cells ranging from not have cleavage, hylock, two cells to sixteen
this sel.Jumlah terbeilang small and far enough away from the existing literature that
assumes that it should fertilization can be more than 90% (Watung, 2004). For
dilution 100 times showed that the percentage increase each repetition implies that
the fertilized egg cell accretion due to a repetition of the second and the third is an
advanced stage of a more modest stage (Effendie, 2002). Many factors affect this one
sperm quality and also an error of observation phase of development of the zygote
..TO rest there are no issues regarding the division and growth of these cells. Each
repetition over 20 minutes (35 and 50 minutes) is the development of phase
fertilization and most definitely fertilized, if there are unfertilized may imply that these
cells undergo abnormal or damage (Healt, 1980).
Observation of fertilization and embryonic development in nilem fish done with
different treatments including dilution rate and lag time different observations. This is done
to determine the appropriate concentration of sperm that can fertilize the egg to achieve
fertilization. Dilution rate milt conducted also vary aims to determine how much time is
required by the spermatozoon penetrates the ovum to the wall. The time difference during
a meeting between the egg and sperm have in order to determine the rate at fertilization
occurs, how long it takes the spermatozoon penetrates to the wall ovum and to determine
the stage of development that occurs in every time, sperm easily hung by the atmosphere,
the temperature of the medium which is too high. Conversely, changes in pH will damage
the growth of the ability to fertilize. Water quality greatly affects cell division (hatching
eggs), especially that the water temperature media (Health, 1995).
Based on lab results on average 10x dilution fertilized egg at 20minute was 20%
and 80% were not fertilized. 35 minutes into the fertilized formed hylock as much as 30%,
10% has entered the second stage, and 60% are not fertilized. At minute 50 percentage
fertilized formed hylock much as 10%, 70% already entere stage 4 with a cell number 8, and
20% in stage 2 with the cell number 2. On 100x dilution in the 20th minute a fertilized egg
as much as 60% formed hylock , and the remaining unfertilized. At 35minute was 45%
fertilized with leftover fertilize and die or lethal, and at the 50 minute that was not fertilized
by about 15% and the rest is not fertilized, dead or lethal. For dilution 1000X in the 20th
minute fertilized as much as 30% and the remaining unfertilized. At minute 50 minutes
35And the fertilized egg reaches 100%. In the treatment of the lag time of 1 minute
percentage of a fertilized egg in the 20th minute was 35% and 65% were not fertilized. 35
minutes into the eggs were not fertilized only 0% and at minute 50 percentage fertilized egg
reaches 80%. For the treatment of lag time of 3 minutes in the 20th minute a fertilized egg
as much as 45%, to 35 minutes a fertilized egg until 80% and on 50 minutes a fertilized egg
as much as 90%. Treatment lag time 5 minute percentage of the fertilized egg in the 20th
minute, namely 20%, min to 35 eggs was fertilize reached 55% and at 50 minutes into the
fertilized eggs as much as 100%.
 Observations from group 1 group VII has been subject to treatment with a lag
time of one minute by the repeated 3 times. At 20 minutes 3 eggs fertilized at an early
stage, namely stage hylock emergence. In the 35th minute 10 pieces egg was fertilized with
8 pieces forming hylock, and 2 pieces has entered the stage 2 by forming two cells. At
minute 50, 9 eggs was fertilize with 8 already formed hylock stage, and 2egg was 4-cell
stage. This is consistent with the statement Djuhanda (1981), originated from a single
zygote cell then divides into two cells, 4-cell, 8-cell, 16 cells, up to 32 cells called morula
stage.
The percentage of fertilized eggs in the control treatment intervals 20, 35, and 50
minutes is calculated from when mixing eggs and milt are 10%, 70% and 100%, the result of
the constant rise. According Wijayanti and Simanjutak (2005), the time will affect
terbuahinya an egg. The increasing time the lower the result of fertilization of the egg itself.
Similarly, the high dilution rate, the greater the dilution that will result in fertilization of the
egg slightly and lower.
Based on observational data treatment dilution rate obtained negative correlation
because the percentage of fertilized eggs in the group with no dilution rate of 100% is
fertilized. According to Gilbert (1991), the higher the dilution rate, then the long motility
spermaozoa getting shorter, and vice versa. This shows that the shorter the sperm motility
means the fewer the number of spermatozoa are alive and able to fertilize an egg.
According Wijayanti and Simanjuntak (2005), the percentage of fertilization which indicates
that the egg and sperm used good quality can reach up to 99%. This generated a large
percentage of the eggs and milt are easily stripped out at the time, the eggs are not
clustered in a relatively uniform diameter, yolk color sharp, do not have the space perivitelin
and milt are produced in large quantities with normal consistency.
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the results and the previous discussion it can be concluded as follows:
1. Volume Milt = 0.4 mL.
2. Color striping sperm results in fish nilem ( Osteochilus hasselti ) is milky
white, it indicates that the fish sperm nilem used in the lab are healthy.
3. Sperm examination results showed fishy smell, no foul, mean sperm
produced normal, prostate active and no infection.
4. Nilem fish sperm were observed showed pH 7, indicates that the sperm in
normal circumstances.
5. Calculation of fish sperm motility obtained nilem motile sperm count is 80%,
while the number of non-motile spermatozoa is 20%.
6. The result of the calculation of the average sperm count was 12,9 x
10 9 spermatozoa / ml Milt .
7. The milt in healthy condition

A. Suggestion

Sperm fish for observation should not be taken from some fish and Pruit united in
one injection. We recommend that every fish taken sperm with each one spruit
injection, so that we can know where the fish are most fertile
 REFERENCES
Djuhanda, T. 1981. Comparative Embryology. Armico. duo
Dudek, Ronald W. 2011. Embryology 5 th Edition. Lippincott William & Wilkins
Effendie, M. I. 2002. Fisheries Biology. Bogor ,Reader Nusantara
Effendiei.1997. Biology Perikanan.Yayasan Nusatama.j Library journal, the effect of
different concentrations of materials in fish sperm dilution "skim yolk" to
lauju penetsan fertilization rate and survival rate of carp (Cyprinus
carpoio), volume V (1): 5 Yogyakarta
Effendy, M.I. 1997. Fisheries Biology. Bogor. Nusatama Foundation.
Faruk. A, Erdinc. S, Zafer. D. Embronic larval Development. Journal of
Acquaculture. Department of Fisheries. Turkey.Bozova. Sanliurva..
Soeminto, Wijayanti, G.E., Atang and A.N. Habibah. 2010.Power On Growing
Power. The proportion of male and female fish Nilem Ginogenesis Up to
Cook Sex (laboratory scale). Purwkerto. Research Report. Faculty of
Biology UNSOED.
Gilbert, S. F. 1991. Developmental Biology. Sinauer Associates Inc., Sunderland.
Harinadi. 2010. Embryology and Development.Jakarta. Erland.
Health, A. G. 1980. Water Pollution and Fish Physiology 2. New York. Lewis
Publishers
Linder, M.C. 1992. Biochemistry of nutrition and metabolism (translation).
University of Indonesia, Jakarta. 781 things
Moeller, R. B. 2004. Biology of Fish. California Animal Health and Food Safety
Laboratory. Californis System University of California.
Squest M, Billard. R, Cosson J., G. Dorange, Chauvaud l., Mugnier C., & L. Fauvel,
1994. Sperm features in turbot (Scophtalmus maximus): A comparison eith
of freswater and marine fish species. Aquatic living Resaurces. 7: 283-294.
Parker, G. A .; Begon, M. E., 1993: Sperm competition games - sperm size and
number gametic under control. relationshop journal between morphology,
motility and fertilization capacity in rainbow trout spermatozoa, volume 24:
4
Setu, R. 1962. Experimental Embryologi. Burgess Publishing Company, Minnesota
Soeminto, Wijayanti, GE, Atang and AN Habibah. Power On Growing
Power 2010. The proportion of male and female fish Nilem Ginogenesis Up
 to Cook Sex (laboratory scale) . Research Report .Faculty of Biology
UNSOED Purwkerto.
Soeminto. 2004. Vertebrate Embryology. Unsoed, Purwokerto
Shukra, Y. 2000. Embryonic Science Insight: Seeds of the Future. Directorate
General of Higher Education Ministry of National Education, Bogor
Taborsky, 1998
Wijayanti, Grantiana.E. 2005. Embryonic Development and Viability and fish larvae
Nilem (Osteochillus hasselti C.V.) Post Chorion Puncture. J. Biol. Indon.
Vol. III, No. 10: 411-419
Wijayanti, G.E. 1997. Fertilization Fish Eggs and Sperm Nilem (Osteochillus hasselti
C.V.) Post Striping in Natural Media. Faculty of Biology Unsoed,
Purwokerto