Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

Cristina Cuello; Cristina A Martinez; Josep M Cambra; Alejandro González-Plaza; Inmaculada Parrilla; Heriberto Rodriguez-Martinez; Maria A Gil; Emilio A Martinez

doi:10.3389/fvets.2021.771996

Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

Front Vet Sci. 2021 Nov 12:8:771996. doi: 10.3389/fvets.2021.771996. eCollection 2021.

Authors

Cristina Cuello¹, Cristina A Martinez², Josep M Cambra¹, Alejandro González-Plaza¹, Inmaculada Parrilla¹, Heriberto Rodriguez-Martinez², Maria A Gil¹, Emilio A Martinez¹

Affiliations

¹ Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum," Institute for Biomedical Research of Murcia (IMIB-Arrixaca), University of Murcia, Murcia, Spain.
² Department of Biomedical & Clinical Sciences (BKV), BKH/Obstetrics & Gynaecology, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden.

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.

Keywords: embryo; gene expression; morula; pig; transcriptome; vitrification.