RNA-seq of in vivo, IVF, and vitrified porcine embryos

Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for research purposes. To date, vitrification is the only efficient method for pig embryo cryopreservation. Despite a high number of embryos survive in vitro after vitrification/warming (VIT) procedures, the in vivo embryo survival rates after embryo transfer is variable and with non-repeatable results among laboratories. So far, most studies have focused on technical factors (cryoprotectant (CPA); devices), while the VIT effects on the pig embryonic transcriptome and the underlying molecular basis remain unknown. Therefore, our objectives were: 1) To determining the effects of VIT on the pig embryonic transcriptome when compared to in vitro culture (IVC) during 24h and fresh embryos (control, CO) by RNA-sequencing, to unreveal the real effect of VIT excluding IVC; 2) To test a new vitrification protocol (sVIT) with reduced exposure to CPAs compared to objective 1 (VIT) on different in vivo and in vitro parameters as well as embryonic gene expression. RNA-sequencing data revealed three clear different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles of VIT versus CO, IVC versus CO and VIT versus IVC identified 1901, 1519 and 321 differentially expressed genes (FDR<0.001), respectively. When the sVIT protocol was examined, no differences were found between sVIT and VIT in terms of in vitro embryo survival, pregnancy and farrowing rates. Gene expression analysis of sVIT and VIT embryos based on a panel of genes derived from objective 1 did not reveal differences between VIT protocols. Only a decrease in the number of piglets born from sVIT compared to VIT was observed that requires further consideration. These results show that both VIT and IVC have an important impact on the embryo, being part of these alterations shared by VIT and IVC procedures while others associated only to VIT. These findings contribute to a better understanding of the impact of both procedures on pig embryos that can affect their development and fate. Besides, the genes identified here could be used as biomarkers of embryo quality after VIT and IVC procedures.Keywords: embryo, vitrification, in vitro culture, gene expression, porcine, embryo transfer, RNA-sequencing.