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R. Taufiq Purna Nugraha

Program Studi Biologi
Sekolah Pasca Sarjana
Institut Pertanian Bogor

Normal Fertilization : Monospermic
In vivo condition : regulate by oviduct,
Polyspermic : multiple sperm fertilize 1 egg
Diploid : two copy of each parent
chromosome, Polyspermic : three or more
copy : Inviable zygot
When more than one sperm manages to
enter the ovum (dispermy= 2; triploidy=
3), the fetus nearly always aborts


a secretion reaction, the "cortical reaction" modifies the

extracellular coat of the egg


High incidence of polyspermic in-vitro
ZP and membrane blocks to polyspermy
is not fast enough to prevent entry of the
excess spermatozoa in vitro compared to
the in vivo situation
Human : incidence of polyspermy during
IVF ranges from less than 3% to over 30%
Cattle range from 5% to 45%for in vitro
Sheep and goats : almost 20%
Coy & Aviles 2010

Pig : high incidence of polyspermic
during IVF, 14% to 93% (with
percentages of penetration ranging
from 19 to 95%, respectively)
higher than that of in vivo matured
and fertilized oocytes : 5%

Polyspermic in Pig
IVF : high concentrations of spermatozoa
are usually introduced to microdrops in
which oocytes have been placed
Excess spermatozoa and suboptimal IVF
conditions are the major reasons for
polyspermy in pig oocytes
Dead Sperm also introduced :
detrimental factor ?
Reducing sperm number decreased
polyspermic penetration, also reduced
sperm penetration rates
Li et al 2003

Polyspermic in Pig
Quality of Semen
High variation between individual
Variation between Breed
Difficulty of cryopreservation of boar
Quality of the matured oocytes in the

Suzuki et al 20

Polyspermic in Pig
Monospermic zygote,
paternal and maternal microtubule domains
unified during PN apposition. Interaction with
microfilaments may be important for merging
of two microtubule domains.

Multiple microtubule domains in the
polyspermic zygote became merged, but some
delay or interruption was noted in the oocyte
showing a higher degree of polyspermy
(usually over trispermy).

Effort to Reduce
In pigs, decreasing the sperm
concentration during IVF induces a
parallel reduction in penetration
frequency, decreasing polyspermy
but not eliminating it

Method to Reduce
IVF in the straw
Reduced Polyspermic Penetration in Porcine Oocytes
Inseminated in a New In Vitro Fertilization (IVF) System:
Straw IVF, Li et al 2003

Pretreatment with adenosine & co-incubation with

Reduction of the incidence of polyspermic penetration into
porcine oocytes by pretreatment of fresh spermatozoa with
adenosine and a transient co-incubation of the gametes
with caffeine, Funahashi & Romar 2004

Modified Swim up method

A modified swim-up method reduces polyspermy during in
vitro fertilization of porcine oocytes, Park et al 2008

Boar Sperm Encapsulation Reduces In Vitro Polyspermy,
Faustini et al 2010

IVF in the StrawLi et al 2003

Try to mimic oviduct condition

Sterile 0.25-ml embryo cryopreservation straws
Plugs in the straws were cut off with scissors.
One end of the straw was connected to a 1-ml syringe
through a hosepipe
Loaded in the following sequences.
straw with air columns :
1 cm medium with spermatozoa, 5 cm insemination medium, 1 cm
medium with 60 oocytes, 1 cm air, and 1 cm paraffin oil were loaded.
Then the hosepipe was connected to the other end of the straw, 1 cm air
and 1 cm paraffin oil were loaded. In the straw

without air columns :

1 cm medium with spermatozoa, 7 cm insemination medium, 1 cm
medium with 60 oocytes, and 1 cm paraffin oil were loaded.
Then the hosepipe was connected to the other end of the straw, and 1 cm
paraffin oil was loaded. After being loaded, the straws

Put on the adhesive tape holders horizontally, incubated for 6

h at 398C in an atmosphere of 5% CO2 in air.

IVF in the Straw

IVF in the Straw

IVF in the Straw

IVF in the Straw

Air columns in the straws had no obvious
effect on fertilization parameters,
especially on the rates of normal fertilized
oocytes when oocytes were inseminated
in the straws.
The rate of cleavage (2- to 4-cell embryos)
was significantly higher in the straws with
air columns than in straws without air c
Air columns for air equilibration when the
columns incubated in CO2

IVF in the Straw

In microdrop sperm concentration
higher (3.5x) but penetration lower
than straw method
Dead sperm also introduced
detrimental to oocytes, thus affecting
subsequent embryo development

Adenosin Pretreatment +
Co-incubation in Caffeine

Funahashi & Romar 20

Caffeine inhibits cyclic nucleotide

phosphodiesterase, resulting in an
increase in intracellular cyclic adenosine
30,50-monophosphate and induction of
capacitation and spontaneous acrosome
reactions of boar spermatozoa
longer duration of co-culture of gametes
in the presence of caffeine increase the
chance for more spermatozoa bind to the
zona pellucida
Adenosine and fertilization-promoting
peptide (FPP, which is a tripeptide, pGluGlu-ProNH2) induce capacitation of both
fresh and frozenthawed boar
spermatozoa in vitro but prevent a
spontaneous acrosome reaction via the
adenylyl cyclase/cAMP pathway, even in
the presence of caffeine
Supplementation with adenosine and FPP
during co-culture of oocytes with frozen
thawed spermatozoa increased the
proportion of monospermic penetration

Adenosin Pretreatment +
Co-incubation in Caffeine
5 mmol caffeine/l supplemented during periods of co-culture
and additional culture periods until 8 h after insemination
Shortened co-incubation period of gametes from 30 to 5 min
increased the monospermy rate
Spermatozoa binding to the zona surface lower in oocytes cocultured for 5 min than 8 h
Limited exposure of gametes to 5 mmol caffeine/l only during
a transient co-culture period for 5 or 30 min significantly
reduced sperm cells that penetrated into the oocyte.
5 min Transient exposure of spermatozoa to caffeine increased
the percentage of capacitated cells but not acrosome-reacted
cells, compared with a whole exposure treatment.

Adenosin Pretreatment +
Co-incubation in Caffeine
Preincubation of spermatozoa with 10mmol
adenosine/l for 90 min increased the
incidence of capacitated cells and
monospermy rate
Preincubation of fresh spermatozoa with
adenosine before the transient co-incubation
IVF can also improve the monospermy rate
Asynchrony in the morphology of sperm
nuclei in polyspermic oocytes was reduced
by the pretreatment with adenosine and a
brief exposure to caffeine.

Adenosin Pretreatment +
Co-incubation in Caffeine

Modified Swim Up
Park et al 2003 Method
Semipermiable 70
m pore sized cell
strainer separate the
sperm pellet placed
at the bottom of a
tube from the mature
oocytes placed within
the upper region.
To ensure that only
motile sperm gained
access to the oocytes

The sperm pellet, diluted to a concentration

of 1.0109 cells/ml in IVF medium, was
added to the bottom of each test tube.
Immediately following insemination, a 70m
pore sized cell strainer gently inserted into
the upper region of the test tube
Oocytes were then carefully distributed
evenly across the surface of the cell
strainer, and the tube was incubated at 39
C for 6 h in an atmosphere containing 5%
CO2 and 5% O2 at 100% humidity.

swim-up method reduce the incidence of

polyspermy and hence yield more high
quality embryos with a higher incidence of
chromosome normality
The cell strainer, through the limitation of
sperm-binding sites on the zona pellucida
may also further prevent polyspermic

Faustini et al 2010

Boar sperm encapsulation is an

emerging technique to solve several
problems linked to spermatozoa
preservation and artificial insemination
Able to fertilize in vitro and in vivo
Maintains high percentages of nonreacted acrosomes at 18oC over 72 h
of storage

4.4 x 108 cells ml;

motility 95%; average
velocity 65 m s and
secondary anomalies
A saturated BaCl2 solution
added to semen to reach a
Ba2+ ion concentration of
25 mmol l

medium (Brackett &

medium with 12% foetal
calf serum and 7 mg ml
caffeine) to a
concentration of 1 . 106
50 matured denuded oocytes
sperm ml
transferred in 500 l of spermcontaining IVF medium. After 1 h
of co-incubation,
transferred in fresh IVF medium.
Oocytes collected 18 h after
exposure to spermatozoa and
fixed in acetic acid : ethanol (1 : 3
v v) for 24 h, stained with 1%
(w v) lacmoid solution

On average, the POLY rate lower in
encapsulated spermatozoa than that of
diluted semen after 24 and 48 h of
MONO rate was higher for all time of
storage if encapsulated
POLY rate diminished from 24 to 48 h
and increased from 48 to 72 h of storage
time for both treatments : ageing
IRR = 0.766

Encapsulated spermatozoa have
lesser damage of sperm membranes
and preserved specific ligand or
ligands for the oolemma receptor.
morphological and functional
alterations during semen processing
influences the penetration ratio and
the incidence of polyspermy.

Polyspermic high in in-vitro
No oviduct role to prevent
Effort to prevent polyspermic mostly
to mimic in-vivo condition

IVF in the Straw