Abstract
Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.
Highlights
Cryopreservation [1,2,3] of porcine embryos is the best tool for exchange and conservation of genetics and has important applications for agriculture and biomedical research [4, 5]
Considering the stage-specific gene expression profiles between morulae and blastocysts, which is proper of preimplantation embryonic development [28], and that the morula is a suitable stage for vitrification and embryo transfer [5], studies focusing on the embryonic developmental stage of morulae are needed
Vitrification and warming procedures significantly modified the transcriptome profile of porcine morulae
Summary
Cryopreservation [1,2,3] of porcine embryos is the best tool for exchange and conservation of genetics and has important applications for agriculture and biomedical research [4, 5]. The altered genes in this study [27] were mainly involved cell cycle pathways, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK All of these studies are useful, information on the effects of vitrification on the porcine embryo transcriptome was limited to blastocyst stage embryos. Better insight into the effects of vitrification on embryonic gene expression may help identify molecular lesions that may be associated with reduced embryo developmental capacity and more frequent pregnancy loss after transfer of vitrified embryos. This information would allow a better understanding of the adaptation process of embryos after vitrification and warming. The aim of this study was to use microarrays to investigate the effects of vitrification on the gene expression profile of porcine in vivo derived morulae
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