Improving the Efficiency of Porcine Embryo Vitrification and its Impact on the Epigenome

Abstract

The cryopreservation of embryos is an essential assisted reproductive tool for the treatment of human infertility, agriculture and biomedical research. With sheep and cattle embryo cryopreservation commercially available, a reliable method is not yet offered for the pig. Recent studies have revealed that the in vitro environment of assisted reproductive technologies may affect epigenetics. To date few studies have examined the effect cryopreservation has on the expression of developmentally important genes, none of which have been conducted in porcine embryos. The aim of this study was to establish a reliable and epigenetically safe vitrification protocol for the cryopreservation of in vitro produced porcine blastocysts. A number of vitrification devices, cryoprotectants, cryoprotectant combinations and protocols were directly compared. In the final experiment, the effect these different vitrification methods had on the expression of developmentally important genes, IGF2 and IGF2R was examined. Results showed all devices and cryoprotectant combinations assessed were equally effective at supporting cryosurvival, with an increase in post warm survival seen when vitrification was carried out in 22°C media. Contrary to findings in other species, collapsing the blastocoele cavity prior to vitrification decreased cryotolerance. Although IGF2 expression was reduced following vitrification, IGF2R was maintained in some of the protocols examined. These results provide evidence to support the hypothesis that vitrification has a negative impact on the expression of developmentally important genes. This has major implications not only for the production of animals in advanced breeding programs but also for the vast number of children born each year from frozen embryo origins. Although embryo cryopreservation has proved an extremely useful tool, the effect that this procedure has on the genome deserves much greater attention with the health of future offspring in question.