Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes.

ABSTRACTAndrogenetic embryos are useful model for investigating the contribution of the paternal genometo embryonic development. Little work has been done with androgenetic embryo productionin domestic animals. The aim of this study was the production of diploid androgeneticsheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro;parthenogenetic and normally fertilized embryos were also produced as a control. Fifteenhours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored forthe number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid,and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportionof oocytes with polyspermic fertilization increased linearly with increasing spermconcentration. After IVF, there was no significant difference in early cleavage and morula formationrates between the groups, while there was a significant difference on blastocyst developmentbetween IVF, parthenogenetic, and androgenetic embryos, the last ones displayingpoor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploidandrogenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgeneticembryonic development, we produced diploid androgenetic embryos through pronucleartransfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploidembryos and transferred into the perivitelline space of other haploid embryos, and the zygoteswere reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfersignificantly increased blastocyst development (IVF, parthenogenetic, androgenetic:Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly,the Haploid Diploid group showed the highest development to blastocyst stage.The main findings of our study are: (1) sheep androgenetic embryos display poor developmentalability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryosproduced by pronuclear exchange developed in higher proportion to blastocyst stage,particularly in the Diploid–Haploid group. In conclusion, pronuclear transfer is an effectivemethod to produce sheep androgenetic blastocysts.[...]