Abstract
Identification of polyspermic embryos by non-invasive selection markers could be a possible way to select only diploid embryos for embryo transfer. Although previous studies suggested altered cleavage patterns in polyspermic porcine embryos, convincing evidence has not been provided to support this theory. Therefore, we compared the early developmental kinetics of monospermic and polyspermic porcine embryos by time lapse cinematography. Follicular oocytes were in vitro matured, fertilized (IVF) and cultured according to Kikuchi et al. (2002 Biol Reprod). Ten h after IVF, zygotes were centrifuged at 10000 × g and those with 2 pronuclei (monospermy, 2PN zygotes ; n=32) or more than 2 pronuclei (polyspermy, PPN zygotes; n=32) were selected under an inverted microscope. PPN, 2PN and non-centrifuged zygotes (n=64) were cultured separately in the well of well dishes. The timing and pattern of the first embryonic cleavage of each embryo were recorded by a Primo Vision Timelapse System for 48 h. Four replications were performed. Data were analyzed by paired t-test and ANOVA. There was no difference in the pattern of first cleavage between 2PN and PPN zygotes regarding the frequencies of fragmentation, protrusions or direct cleavage resulted in the first cleaved embryos with 3 or 4 blastomeres. However, PPN zygotes cleaved significantly earlier (P<0.05) compared to 2PN zygotes (29.1±1.2 and 31.0±1.1 h post IVF, respectively). Centrifugation treatment had no effect on the timing and pattern of first cleavage. In conclusion, the timing of the first cleavage but not its pattern can be considered as a potent marker for polyspermy in pigs. This project was supported by JSPS-HAS/JHRCP and 8526-5/2014/TUDPOL.
