Effects of Slow Freezing Procedure on Late Blastocyst Gene Expression and Survival Rate in Rabbit1

Abstract

Studies of embryo cryopreservation efficiency have focused mainly on technical and embryo factors. To determine how a slow freezing process affects embryo and fetal development, we studied in vivo development ability after the freezing procedure by assessing blastocyst development at Day 6, implantation, and birth rates. A transcriptional microarray study was also performed to compare gene expression of 6-day-old rabbit embryos previously frozen and transferred into recipient rabbit females to their in vivo counterparts. Our goal was to study which alteration caused by the freezing procedure still remained in late blastocyst stage just at the time when the implantation process began. A microarray specifically designed to study rabbit gene expression profiling was used in this study. Lower implantation and birth rates were obtained in frozen embryos than in the control group (29.9% and 25.7% vs 88.5% and 70.8% for frozen and control embryos, respectively). Likewise, differences were also observed in gene expression profiles. Compared to 6-day-old in vivo-derived embryos, viable frozen embryos presented 70 differentially expressed genes, 24 upregulated and 46 downregulated. In conclusion, our findings showed that the slow freezing process affected late blastocyst development, implantation, and birth rates and that the gene expression alterations identified at late blastocyst stage could be useful in understanding the differences in developmental potential observed and the deficiencies that might hinder implantation and fetal development.

Embryo vitrification in rabbits: Consequences for progeny growth

“…To this end, rabbit embryo banking has allowed the preservation and spread of genetically superior animals over different countries [34] and the possibility of evaluating the genetic gain in maternal lines [4][5][6][7] without cumulative genetic drift variance [35]. Nevertheless, it is known that these techniques induce changes in environ-mental and maternal side effects modifying the embryo gene expression and methylation pattern [19,20,36], the transcriptomic and proteomic placental profile [21], and their viability [29,37], but little is known regarding the long-term effects on the derived progeny. Some authors postulate that these procedures do not induce major anomalies but can lead to morphologic and behavioral features in adult mice derived from frozen embryos [16].…”

“…Over the past decade, research works have questioned whether embryo cryopreservation and transfer procedures are neutral to survivors [16][17][18], as preimplantation em-bryos are removed from their natural environment and subjected to manipulation (cryopreservation and transfer procedures) which in fact affect their RNA expression [19,20], placental transcriptome, and proteome [21].…”

A Chemically Defined Medium for Rabbit Embryo Cryopreservation

“…These effects would consequently not have an influence on the in vitro embryo survival rates in contrast to the in vivo embryo survival rates. On the other hand, slow-freezing protocols alter the gene expression of rabbit embryos [48]. It is possible that the genetic alterations induced by the FCS-based solutions could lead to more in vivo embryo death than those induced by the CRYO3-based solutions.…”

Endoplasmic Reticulum Stress Signaling in Mammalian Oocytes and Embryos: Life in Balance

“…Heat stress induces changes in gene expression in surviving embryos (Hickman et al, 2013). Cold stress associated with cryopreservation, particularly slow freezing can also affect embryo development and gene regulation (Larman et al, 2011; Saenz-de-Juano et al, 2012). Prolonged exposure to room temperature can inhibit cleavage and alter the Golgi complex in early embryos (Hegele-Hartung et al, 1991), and can degrade meiotic spindle structure in ovulated oocytes (Van der Elst et al, 1988).…”