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Documente Cultură
& HOW TO
REDUCE IT
Introduction
Normal Fertilization : Monospermic
In vivo condition : regulate by oviduct,
oocyte
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
Polyspermic
Polyspermic
Polyspermic
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
systems
Sheep and goats : almost 20%
Coy & Aviles 2010
Polyspermic
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
male
Variation between Breed
Difficulty of cryopreservation of boar
semen
Quality of the matured oocytes in the
pig
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.
Polyspermic
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
Polyspermic
In pigs, decreasing the sperm
concentration during IVF induces a
parallel reduction in penetration
frequency, decreasing polyspermy
but not eliminating it
Method to Reduce
Polyspermic
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
Encapsulation
Boar Sperm Encapsulation Reduces In Vitro Polyspermy,
Faustini et al 2010
Adenosin Pretreatment +
Co-incubation in Caffeine
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
Encapsulation
Faustini et al 2010
Encapsulation
On average, the POLY rate lower in
encapsulated spermatozoa than that of
diluted semen after 24 and 48 h of
storage
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
Encapsulation
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.
Conclusion
Polyspermic high in in-vitro
No oviduct role to prevent
polyspermic
Effort to prevent polyspermic mostly
to mimic in-vivo condition