A major obstacle for successful in vitro production of porcine embryosis the polyspermic fertilization. One possibility to reduce polyspermicpenetration is decreasing the number of spermatozoa added to thefertilization medium. Unfortunately, the lower rate of polyspermy isaccompanied by a reduced penetration rate. A short gamete coincubationperiod of 10 min has been described to obtain fertilization ratessimilar to 6 h of coincubation and may improve IVF efficiency(number of monospermic fertilized oocytes/total number inseminated)depending on sperm-oocyte ratio (Gil, 2007, Theriogenology, 67(3),620–626). Here we demonstrate that the optimal coincubation periodin our IVF conditions is between 10 min and 6 h. In vitro maturedoocytes (n = 600) were inseminated with frozen-thawed epididymalsemen with 600 spermatozoa per oocyte and coincubated for 2, 4 and6 h. At 2 and 4 h post insemination (hpi), oocytes were vortexed andtransferred to fertilization medium without spermatozoa. At 6 hpi,presumed zygotes of all groups were washed three times in culturemedium and cultured. At 22 hpi, zygotes were fixed overnight andstained with Hoechst 33,342 for the assessment of fertilization andpolyspermy. The IVF efficiency was higher for the 4 h group(40 ± 5%) than the 2 and 6 h group (19 ± 8% and 17 ± 5%).Between 4 and 6 h of gamete coincubation, the increase in the numberof polyspermic oocytes was relatively higher than the increase inpenetration rate (+39% vs. +15%), resulting in a decline in efficiency.(This study was supported by Research Foundation-Flanders).
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