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Indonesian Animal Husbandry Journal, February 2011

ISSN 1907 - 1760 Vol13(1)

The Effect of Different Fertilization Times and Incubation Systems on

Fertilization Rate of Local Cattle In Wtro

The Effect of Fertilization Time and Different Incubation System on Fertilization Level of Indigenous
Cow by In-Vitro

F. L. Syaiful, R. Saladin, Jaswandi, and Z. Udin

Faculty of Animal Husbandry, Andalas

University Campus Unand Limau Manis Padang
25153 E-mail: ferrylismantonova@yahoo.com
(Received: 01 December 2010; Approved: 28 January 2011)

ABSTRACT
The purpose of this study was to determine the effect of fertility time and different incubation systems
on infertilization level by in-vitro. The mature of ovaries from indigenous cow and fresh cement from Holstein
Frisiancows(FH), 0.9% NaClphysiological PBS, NissuiJapan, I, TCM-199, HEPES30pM, 20 mL Heparin, 10%
goat serum, 250 g/ml FSH BO medium, gentamisain medium 50 mB -O, mineral oil, alcohol, aquabidest, and
I %o aceto orcein were materials and reagents. The Completely Randomized Design (CRD) in factorial pattern
was used. Results shown no significant ffict on the percentage of fertilized ooq) tests and level of development
of pronuclei (2PN and> 2PN) by different timing of fertilization and incubation system. The development of
pronucleus (IPN) showed significant (P <0.05) on I2 hours (37.60 %o), but no significant effect on different
incubation system. It concluded, the system of incubation and time of fertilization has no effect on oocyte
fertilization rate. Oocytes fertilization time can be performed at 6 hours I 2 hours, and I B hours, while the ,
extension of the period of fertilization until I 8 hours did not increase the I ev el offertilization.

Keywords : Incubation systems, fertilization time, FIV and pronucleus

PEI\DAIIULUAN In continue to be identified to increase the

vitro fertilization technology (FIV) is a technology
for the production of embryos in an artificial
environment outside the body in a cell culture system
(Hunter, 1995). The in vitro fertilization technique
(FIV) can use oocytes derived from living animals or
from slaughtered animal oocytes, so that this in vitro
fertilization technique (FIV) can be an alternative to
embryo production in the implementation of embryo
transfer (TE). Another benefit of in vitro fertilization
(FIV) technology is that it opens up greater
opportunities for developing gamete and embryo
manipulation techniques such as cloning production
( Gordon, I 994 ).
The application of this biotechnology requires
a large number of oocytes, then the oocytes obtained
are matured in vitro (in vitro maturation) for the
benefit of in vitro fertilization .
Successful in vitro fertilization requires adequate
success of in vitro fertilization @racket and
Zuelke, 1993).
According to Herdis (2000), embryos produced
from in vitro fertilization technology can be transferred
to recipient livestock to help accelerate the increase
in livestock population. With the in vitro fertilization
technique , the use of oocytes from slaughtered
animals is an economical way of producing embryos
because in this way the oocytes of slaughtered
animals can be used to make seeds , this will certainly
add value . In utilizing dead animal oocytes , not all
of the potential that exists can be exploited due to the
limited viability of oocytes, while ovarian storage
technology can maintain oocyte viability for quite a
long time or during transportation .
not yet available.

biological readiness of oocytes and sperm and
culture conditions that support the metabolic
effectiveness of male and female gametes .
Various aspects of culture conditions such as medium,
time of insemination and capacitation, culture system
To increase fertilization and the flexibility of in
vitro embryo production that can be done outside the
laboratory, several studies have tried to replace the
source or role of COrsoA in maintaining the pH of the
medium. PH

Local Cattle Fertilization (FLSyaiful, el a/) 27

Machine Translated by Google
the medium can be maintained by adding a buffer
such as Hepes. According to Jaswandi Q002), the
results of maturation and in vitro fertilization of sheep
oocytes with the addition of Hepes buffer in the
medium can provide optimal conditions . Thus the
use of straws needs to be linked to the addition of
Hepes buffer in the medium and the incubation time
so that the synergistic effect of its use can be
optimally achieved .
The gas composition of the air is one of
the important factors influencing the
development of embryonic oocytes in incubation.
The best levels of oocyte maturation and
fertilization and embryo growth were obtained
in CO gas , 2.5-5 yo and airborne Orlo/odi
(Pinyopummint and Bavister, 1995). Meanwhile,
according to Thompson (1996), the development
of a cow embryo is not absolutely dependent
on CO, 5% io. this can be seen in bovine
embryos which are able to develop to the
blastocyst stage if the medium is added with
ionic nvitter buffer in air without CO, 5 %.
Freshney (1987) suggested that cell culture in
the open requires incubation with CO.S% in air.
This situation can be used as a basis for
CO manipulation in the incubation system . The
in vitro fertilization process in the incubation
system without CO gas, in the field is felt to be
very efficient compared to the CO incubation
system .
For this reason , technology is needed that
allows the embryo production process to be
carried out during transportation or outside the
laboratory. The main obstacle in the production
of embryos outside the laboratory or during
transportation is the presence of CO and the
place of fertilization. Therefore it is necessary
to study the use of CO incubation systems , and
incubation systems without CO, and the
appropriate fertilization time for in vitro cow fertilizationmaturation.
Based on the problems above, the authors
raised a study on a study entitled "The Influence of
Different Fertilization Times and Incubation Systems
on In Wtro Fertilization Levels of Local Cattle ". This
study aims to determine the effect of wakfu on in
vitro fertilization rates and to obtain the best wakhr/
in vitro fertilization period .
28
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MATERIALS AND METHODS
This research was conducted at the
Reproductive Laboratory of the Faculty of
Animal Husbandry, Andalas University, Padang.
The material used in this study was the Ovary
as a source of oocytes obtained from local
adult cows . The semen used was fresh semen
of Frisian Holstein (FH) cattle. The materials
used were physiological NaCl 0.9o/o, Phosphate
Buffered Salline (PBS; Nissui Japan), Tissue
Culture Medium - 199 (TCM-199; Sigma, M
-5017), Hepes 30 pM (Sigma; H-1617), Heparin
20 pl, Goat serum 10%, FSH 250 ml,
Gentamicain (Sigma 6-1397) 50mg/ ml, Brackett
Oliphant medium (BO), Brackett Oliphant
modified medium (mB-O), Mineral oil, Alcohol,
Aquabidest, and Aceto Orcein 1% (Sigma O
7380). The tools used were flasks for
transporting the ovaries to the laboratory, razor
blades for slicing the ovaries, Pasteur pipettes ,
Eppendorf pipettes , incubators, stereo
microscopes, inverted microscopes ,
micropipette ( 50 1t"l and 250 ti), scales .
electronics, cover , straws 0.25 ml, pH meter,
glass petri dishes , glass objects , tissue,
aluminum foil and an artificial vagina to collect semen.
Experimental design
The design used for oocyte maturation during
the 24- hour incubation period used a completely
randomized design ( CRD ) and fertilization data
were analyzed using a 2 x 3 factorial complete
randomized design ( CRD) . Each treatment consisted
of three (3) replications, each of which The
experimental unit consisted of 10 oocytes. The
treatment is a. Factor I: The incubation :
system consists of two types , namely the CO
incubation system , 5%
and incubation system without COr 5%o.
Fertilization at 18 hours after
rates . b. Factor II : Time of fertilization
consists of three
treatments , namely: Pl = Time of fertilization at 6 hours aft
sorry.
P2 : Time of fertilization at 12 hours after
maturation.
P3 = Time of fertilization at 18 hours after
sorry"
Fertilization of Local Cattle (FLSyaiful, e/ aI)
Machine Translated by Google
Research procedure
The work procedure carried out in this study
was oocyte :
collection . For the collection of oocytes the
basic medium used is Phosphate Buffered Salline
(PBS). The media was sterilized in an autoclove
at 120'C for 30 minutes and after that it was stored
at room temperature . On the day of ovarian
retrieval , basic media was supplemented with 100
ru pennicillin/ml and 100 pg streptomycin/ ml. For
maturation , TCM-199 media was used plus 10%
goat serum . Media sterilization was carried out
by filtering using a 0.22 gm millipore filter .
The ovaries were obtained at the
slaughterhouse ( RPH) and then brought to the
laboratory in a flask containing 0.9 physiological
NaCl medium added o/o with 100 pglml steptomycin
and 100 IU/ ml penicillin and then stored in a
thermos at 350C, then the ovaries obtained were
washed until
clean.
Oocytes were obtained from bovine ovaries,
oocyte collection was carried out using the slicing
method . The ovaries were sliced in petridishes
containing medium Phosphate Buffer S al in e ( PBS )
supplemented with goat serum 10 %o and gentamicin
( Sigma , G - 1 3 97 ) 50 mg / ml . The number and
quality of oocytes obtained were observed under a
microscope. The oocytes used for maturation are
quality A and B oocytes.
Oocyte maturation . The oocytes used were quality
A and B oocytes. The maturation procedure was carried
out according to the procedure proposed by Jaswandi el
al., (2003). The quality A and B oocytes were then washed
3 times in Phosphate Buffered Saline (PBS) medium, then
the oocytes were matured in maturation medium . For
maturation in the CO.5 yo incubation system , oocytes were
matured in petri dishes on TCM - 199 medium supplemented
with 10 % goat serum , FSH mg /ml and 50 mglml
gentamicin . Ripening in the petri dish four microdrops
( droplets ) were made . Each milcrodrop (droplet) contains
100 ml of TCM-199 maturation medium , then 20 oocytes
are put into the microdrop and then mineral oil is added to
the petn cup .
Fertilization of Lokpl Cattle (FL Syaiful, et af
Vol 13(1)
Then each of the petri dishes was put into the CO
incubator , S%o,lab,s, in
oC
incubation at 3 8.5 for 24 hours.
Whereas for oocyte maturation in the
incubation system without CO, 5% was carried
out using a 0.25 ml straw on TCM 199 + Hepes
30 mM medium supplemented with 10% goat
serum , mgl ml FSH and 50 mgl ml gentamicin .
Then the oocytes are aspirated into the skaw by
attaching a syringe to the end of the straw that
has cotton. Oocytes are sucked into the straw as
much as 10-20 oosiV straws. Oocytes in the straw
are placed in the same way as frozen semen
placement as shown in Figure 1.
Furthermore, oocyte maturation was carried
out for 24 hours in both incubation systems ( CO
incubation system, 5yo and incubation system
without CO, 5%). after maturation / maturation of
the oocyte then the status of the oocyte is observed below
microscope. Oocytes that have undergone
metaphase-Il (M-II) will be used for fertilization.
Sperm capacity. The sperm used was fresh
sperm taken from a Frisian Holstein (FH) bull with
the help of an artificial vagina . Spefina washing
procedure on sperm capacitation was carried out
according to Jaswandi et al. (2003), namely by
adding 4 ml of capacitation medium , namely
Brackett Oliphant (BO) medium and Brackett
Oliphant (mB-O) modification medium into a
centrifuge tube containing 200 1tl of bovine
semen , then centrifuged for 10 minutes. In the
CO incubation system , 5
o/o
using
capacitation medium , namely BO medium , while
without CO, the grammatical medium for sperm
capacitation is mB-O medium.
Then centrifuged 500 G for 10 minutes .
The supernatant portion from the centrifugation was
discarded , and the precipitated sperm was diluted to
a concentration of 1x10' sperm/ ml using treatment
medium (BO and mB-O) supplemented with heparin
(sigma, H-3125) 20 goat serum I} oh and gentamicin
5 0 ml then incubated at 1yr,
temperature 38.5 oC for 30 minutes.
29
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000'0.,0000'06
Medium Air Medium and Oocytes
Figure 1. Oocyte in shaw
in vitro fertilization of oocytes. Oocytes
that had matured well in both incubation systems
( CO r5o/o incubation system and incubation
system without CO, 5%), each treated oocyte
was washed three times with fertilization medium
( BO medium and mB-O medium ) to remove
some of the oocytes. cumulus cells . Meanwhile,
for the treatment of the CO2 incubation system ,
50% used BO medium and the 50% CO2- free
incubation system used mB-O medium. The pH
of BO modified medium (mBO ) was adjusted
by adding 1N NaOH so that it reached pH + 1.4 .
In the incubation system with COr syo , 10-20
oocytes were put into 50 pl of Brackett Oliphant
@-O) medium in a petri dish and then added
with 50 pl semen with sperm that has undergone
capacitation so that the final concentration is lx10u sperm/ml.
Whereas in the incubation system without CO, 5%
oocytes were fertilized in sfaw. The sperm used was
diluted to a concentration of 1x10u spermlml with
fertilization medium. The oocytes and sperm are
then aspirated into the straw, each straw containing
10-20 oocytes. The position and placement of
oocytes in the straw is the same as in the maturation
process , as shown in Figure I.
While oocyte fertilization was carried out for
6, 12 and 18 hours which were incubated at 38.5
oC
in each different incubation system , namely the
CO incubation system, 5oA and the incubation
system without CO , 5o/o.
Evaluation of in vitro fertilization . Unh - rk
looked at the fertilization rate by observing the
development of the pronucleus (1 PN, 2 PN and >2
PIti) with the aceto orcein staining method. This
coloring procedure is based on Jaswandi (2003),
before being stained, the oocytes were cleaned
from cumulus cells by pipetting several times in
Phosphate Buffered Saline (PBS) medium , then
fixed in acetic acid solution and absolute ethanol
solution with a ratio (1:3)
30
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Air Medium Cotton
for 48 hours then stained with 1% aceto orcein
(Sigma, O-7380) for 10 minutes, then the oocyte
core status was observed under a microscope.
Oocytes that have one pronucleus (1 PN)
are assumed to consist only of female pronuclei,
whereas oocytes that have two pronuclei (2 PN)
are assumed to consist of male and female
pronuclei . Oocytes that have more than two
pronuclei (>2 PN) are assumed to consist of two
or more female pronuclei and one male
pronucleus , or vice versa. Oocytes are
categorized as fertilized if they have two or more
pronuclei (2 PN and >2 PN). The variables
observed were: the percentage of in vitro
fertilization and the level of pronucleus
development (PN). Fertilization results obtained
were analyzed using a 2 x 3 factorial Completely
Randomized Design (CRD) . If there is a
significant effect , then the analysis is continued
with Duncan's Multiple Range Test (Steel and
Tonie) , 1995).
RESULTS AND DISCUSSION
Percentage of in vitro fertilization
The results of this study show the average
percentage of in vitro fertilization in the two incubation
systems , namely the 5% CO2 incubation system
and 5% CO2 without in the three incubation periods
can be seen in Table 1.
The average percentage of oocyte fertilization
at 6 hours was 63.63 yo, 12 hours and 18 hours was
and the average o , 62.12 yo. of 56.31 oocytes Se
percentage
the Cor \ o/o incubation of fertilization
system was in p no
6I.460 while without COr 5olo it was 59.600 /0 as
shown in Table 1. But after statistical analysis, the
percentage of oocyte fertilization showed an
increase in fertilization time had no significant effect
(P>0.05) on the incubation system
different.
Fertilization of Local Cattle (FLSyaiful, er a/)
Machine Translated by Google
Table 1. Percentage of local bovine oocyte fertilization at different fertilization times and incubation systems
Incubation system Time
(%) fiam fertilization )
6 35
l With CO25
I2
18
2. No CO25 6 l2
18
The percentage of fertilization that did not
significantly affect the different incubation systems , this
was due to the addition of hepes into the mB-O medium
in the CO incubation system , 5% as a buffer in the
medium. Added by Shamsuddin et al (1993), the addition
of Hepes buffer in the medium and the incubation time
so that the synergistic effect of its use can be optimally
achieved . Reinforced by Jaswandi (2002), the level of
oocyte maturation in vitro using Hepes 10-30 mM in
medium is quite effective in conditions without CO. 5o/o
either using a petri dish or a straw. The results of
maturation and in vitro fertilization of sheep oocytes with
the addition of Hepes buffer in the medium can provide
optimal conditions.
From the results of the study, shown in Figure 2.
the fertilization rate of the three fertilization times of 6, 12
and 18 hours with different incubation systems was almost
the same. While the percentage of in vitro fertilization at
12 hours of fertilization decreased. This is in accordance
with Jaswandi's research (2002) that the oocyte fertilization
rate in sheep decreased during the 12- hour incubation
period . Added by Rehman et al (1994), that fertilization in
cows with an incubation time of less than 16 hours with a
fertility rate of 57.16yo .
Fertilization rates are almost the same between
incubation periods because the oocyte has a mechanism
that inhibits the entry of other sperm when penetration by
one sperm has occurred .
The extension of the incubation period to 18 hours had
no effect on the oocyte fertilization rate . According to
Hafez and Hafez (2000) that after fertilization, the oocyte
surface changes to prevent the fusion of other sperm .
Local Cattle Fertilization (FLSyaiful, el a/)
Vol13(1)
Number of Oocytes
Fertilization (%)
(fruit)
23 (65.71)
23 t3 (56.52)
37 23 (62.16
26 t6 (6t.54)
4T 23 (56.t0)
29 t8 (62.07)
From the results of this study , it was shown that
the incubation period of 6 hours had a
good effect on penetrating oocytes. Dode et al., (2002)
reported that oocyte penetration by qperma incubated
for 6 hours obtained a sperm penetration rate of 63.3
yo. While Jiang (1991) obtained levels of monospermia
and polyspermia in several incubation methods after
insemination of sperm in bovine oocytes matured in vitro
as follows : monospermia in the incubation period of 6
hours 45.3 % at 68.6 % and 20 hours 80.9 while ; 12
o
tinglot polyspermia is in the incubation
, period of 6 hours
is 0.0o/o; 12 hours 9.8 % while 20 hours 12.8o/o.
Long incubation of oocytes and sperm can reduce
the ability of oocytes to develop because too long sperm
can reduce the ability of oocytes to develop because
sperm has the potential to release hydrolytic enzymes
into the fertilization medium (Gordon, 1994) . The vito of
the two incubation systems at the 3 incubation periods
can be seen in Figure 3. It can be seen in Figure 3 that
the pronucleus development rate (IPN) in the different
incubation systems was obtained , namely for the CO
incubation system, 5% o y ain26,32 while in the
incubation system without CO, 5olo of 28.13 yo. The
results of statistical analysis showed that there was no
o/o
difference in LPN in different
incubation systems (P>
0.05). The rate of development was almost the same
between the two incubation systems because in this
study supplemented with Heparin with a dose of 20 1 @
nn into the fertilization medium.From the results of Lu's
study (1990), reported that the use of Heparin (10 pgnQ
obtained a development rate of 1 PN of 4.8% while at a
dose of 50 1 tg/nn (7.3%) and with a dose of 100 pgl ml
obtained IPN of 5.270.
31
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68
66
64
62
60

58
56
54
52
50

6 hours 12 hours l8 hours

Fertilization Time

Figure 2. Percentage of in vitro fertilization at different times of fertilization and incubation systems ( 0 with CO2 5%o, tr without CO2
5%)

60

50

IPN 2PN > 2PN

Prenucleus developmental level

Figure 3. Pronucleus development rate ( ( n lPN, 2PN, and >2PN) on different incubation systems
with 5 % CO2, r without 5% CO2)

The development rate of 2PN pronuclei in both both incubation systems can provide optimal
incubation systems , namely in the incubation system conditions at the level of 2PN development.
aA
using 5% CO2 was 52.08 while without 5o% CO2
o/o. After
it was 54.77 analyzed by the incubation The 5% CO2 incubation system uses BO
system which did not show a significant difference medium (without Hepes) while without aCA2 5o/o uses
(P> 0.05) to the development rate of 2 PN . mB-O medium (with Hepes).
This assumption agrees with Vsconfl et al.,
Almost the same research results were (1999) who found that fertilization success in
obtained from the two incubation systems used, medium containing Hepes and NaIICO3 was
namely the 5Yo CO2 incubation system and 5% almost the same as medium containing
CO2 without . This is due to the medium used in NaHCO3 without Hepes.

32 Fertilization of Local Cattle (FLSyaiful, et a/)

Machine Translated by Google
The results of a study by Jaswandi et al.
(2004) showed that the use of Hepas could replace
the role of 5% CO2 in embryo production . According
to Triwulanningsih (2002), oocytes cultured in
TCM-199 medium for 18 hours in a CO2 So/o
incubator at 39 0C can be fertilized and produce
more blastocysts than those cultured for 24 hours.
The results of this study indicate that the
oocyte development ability of the two incubation
systems in in vitro fertilization is almost the same.
The same trend may be caused by the level of
sperm penetration at fertilization.
The rate of fertilization is due to the mechanism
of the oocyte which prevents the entry of other sperm
fusions that have been penetrated by one sperm. Failure
this mechanism will result in the occurrence of
polyspermy which ends with the polyploid embryo
collapsing , this embryo will experience abnormal
development and die (Qlafez and Llafez, 2000).
The pronucleus development rate >2PN in
different incubation systems , namely in the 5o/o
CO2 incubation system was 8.87oA while in the
5o/o without CO2 incubation system . The rate
a 5% CO2 o of development >2PN at 7.51 with
incubation system was higher than an incubation
system without 5% COz . Although there were
differences in the rate of pronucleus development
>2 PN in the two incubation systems, statistical
analysis showed no significant difference (P>0.05).
In this study , the rate of development >2PN
was not much different from the results of a study
by Jaswandi (2002) who suggested that the
fertilization rate >2PN in sheep oocytes was relatively higher .
12 hours is 5I,70yo while 18 hours is
60-
50-
40-
30-
20-
10-
0t
1 PN
Developmental level of the pronucleus
Figure 4. Pronucleus development (lPN, 2PN, >zPN) at different fertilization
times (r 6h, t72h,r lSh)
Fertilization of Local Cattle (FLSyaifu1, er a/)
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high in the 5% CO2 incubation system was 9.68a/o
while in the incubation system without CO, 5o/o
was 7.19oh.
Pronucleus development rate (PN)
Seen in Figure 4 the level of
development of the pronucleus 1 PN to various
fertilization time obtained, namely 6 hours as
much as 22.03%o; 12 hours of 37.60yo and 18 hours
i.e. 24,23o/o.
During the 12- hour incubation period , the
development rate of 1 PN increased by 15.57o ,
then during
, the 18- hour incubation period there
was a drastic decrease of 25.48yo . The results
of the statistical analysis showed that the time of
fertilization greatly affected the flame (P<0.05)
on the development of the 1 PN pronucleus .
After the DMRT solubility test , the pronucleus
development rate was obtained . The highest PN
was the 12-hour fertilization time of 36.84
followed by the 18 - hour fertilization oh then
time of 24.47o/o. From the above it is assumed
that extending the fertilization time to 18 hours
does not increase 1PN pronucleus development
rate in bovine oocyte fertilization in vitro.
Reinforced by Jaswandi (2002), argued that
extending the fertilization period to 24 hours in
sheep did not increase the success of in vitro
oocyte fertilization .
Pronucleus development fertilization rate
2PN at various times of fertilization is obtained;
at 6 hours of fertilization as much as 57.42%o;
2pn >2PNs
JJ
Machine Translated by Google
of 48.47o/o. After statistical analysis showed that the
increase in fertilization time did not cause a significant
effect (P>0.05) on the development of the 2PN
pronucleus.
According to Jaswandi (2002) that the
fertilization rate which is almost the same up to the
24- hour incubation period is due to the oocyte having
a mechanism that inhibits the entry of the next sperm
if it has been penetrated by safusperm.
The pronucleus development rate >2 PN at various
times of fertilization was obtained, namely; 6 hours which is
6.2I o/o; 12 hours is 4.62 o/o and 18 hours is 13.66yo. After
statistical analysis, it was shown that there was no significant
difference (P>0.05) to the development of pronucleus >2PN
during fertilization . An increase in the incubation period can
lead to a decrease in the development rate of the pronucleus
>2PN. The results of this study indicate that a time period of
18 hours is effective for in vitro oocyte fertilization .
Confirmed by Chian et al. (1992), suggested
that sperm was still able to penetrate the oocyte
up to 24 hours after insemination but the Hunter, RHF 1995. Physiology and Reproductive Technology
increase in fertilization rate was not significant,
and was also followed by a fairly high increase
in the incidence of polyspermy . The increase in
fertilized oocytes after prolonged fertilization Jaswandi, Z. Udin and M. Mundana. 20A4.
time was also followed by an increase in oocytes
with more than two pronuclei (>2 PN). The less
favorable conditions and the prolongation of the
incubation period is that an increase in the
percentage of fertilization can be caused by oocytes undergoing
parthenogenesis.
CONCLUSION
Based on the results of this study, it can
be concluded that the incubation system and
fertilization time do not affect the oocyte
fertilization rate. Oocyte fertilization can be done
in the incubation period of 6 hours, 12 hours and
18 hours. Extension of the fertilization period to
18 hours does not increase the rate
fertilization.
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Brackett, BG, KA Zuelke, 1993. Analysis of
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34
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