Comparison of traditional and modified (VitMaster) methods of rabbit embryo vitrification

Abstract

In spite of their cryobiological efficacy, minimum-volume vitrification methods suffer from the risk of microbiological contamination and are technically and/or manually demanding. In this study, the effects of a traditional, slightly modified vitrification method and vitrification using supercooled liquid nitrogen (VitMaster) applied for rabbit morula-stage embryos were compared. Embryos were equilibrated in a solution containing 1,2-propanediol (2.72 M) and glycerol (1.36 M) for 7 min and vitrified in 0.25-ml insemination straws after 1-min exposure to a vitrification solution containing additionally 1.0 M sucrose. Cooling was performed in 'normal' or supercooled liquid nitrogen. Regardless of the cooling method applied, high in vitro survival and development rates of vitrified embryos were obtained. All embryos were intact after warming, and 61 out of 65 (93.8%) and 23 out of 24 (95.8%) embryos developed to the blastocyst stage after 48-h in vitro culture of embryos vitrified in 'normal' or supercooled liquid nitrogen, respectively. The results suggest higher developmental ability of embryos vitrified in supercooled liquid nitrogen (91.7% vs. 83.1% of embryos vitrified traditionally developed to more advanced, expanding and/or hatching blastocyst stages). In vivo survival rate, tested for the traditional vitrification system only, revealed that 36.8% of embryos developed to term. The results show promise for establishing a fully successful method for rabbit embryo vitrification.

Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

“…Survival of mouse oocytes after very slow cooling rate (!200 8C/min) with high warming rate (O2000 8C/min) was very high when compared with those cooled very rapidly and warmed slowly. However, we and others (see Table 1) have shown that this is not the case for chilling sensitive oocytes and embryos such as those of bovine, pig, rabbit, and human (Arav & Zeron 1997, Lee et al 2007, Papis et al 2009). …”

Μελέτη των παραγόντων που επηρεάζουν τη βιωσιμότητα και τη φυσιολογική ανάπτυξη των εμβρύων μετά από κρυοσυντήρηση με την τεχνική της ταχείας ψύξης (vitrification)

“…Σο φαινόμενο αυτό προκαλείται λόγω τθσ μεγάλθσ ποςότθτασ υγροφ και αζρα που βρίςκεται μζςα ςτθν παγιζτα κακϊσ και ςτθ μεγάλθ επιφάνειά τθσ.Θ χριςθ μια ςυςκευισ γνωςτισ ωσ VitMaster παρζχει τθ δυνατότθτα ελαχιςτοποίθςθσ του κερμομονωτικοφ μανδφα που δθμιουργείται γφρω από το δείγμα. Σο ςυγκεκριμζνο ςφςτθμα μειϊνει τθ κερμοκραςία του υγροφ αηϊτου ςτουσ -210°C και με αυτόν τον τρόπο εξαλείφεται ο ςχθματιςμόσ ατμοφ γφρω από τθν παγιζτα(Papis et al, 2009). Ωςτόςο ζνα ακόμθ πρόβλθμα είναι ότι οι κρυοπαγιζτεσ μπορεί να εκραγοφν και το δείγμα να χακεί κατά τθ διάρκεια του άμεςου εμβαπτιςμοφ ςτο υγρό άηωτο ι/και κατά τθν ανακζρμανςθ ςτο υδατόλουτρο.…”