118 VITRIFICATION OF PRONUCLEAR-STAGE MOUSE EMBRYOS ON ALUMINUM FOIL FLOATING ON LIQUID NITROGEN

Abstract

The cryopreservation of pronuclear-stage embryos has a special importance in transgenic technology, cloning, and human-assisted reproductive technology. The objective of this study was to investigate the efficiency of a vitrification method modified in our lab for pronuclear-stage embryos. In experiment I, groups of 10 pronuclear-stage mouse embryos were cultured in 20 μL drops of three different culture media (G1.3/G2.3, CZB and M16) covered with mineral oil (Sigma M-8410, St. Louis, MO, USA). Twenty-four hours later, embryos cultured in G1.3 were transferred into G2.3 medium. In experiment II, 25–30 pronuclear-stage embryos were transferred into a 50-μL drop of equilibration medium containing 4% ethylene glycol (EG, Sigma E-9129) in TCM-199 (Sigma M-2520) supplemented with 10% FCS at 37°C for 12–15 min; then they were rinsed three times in 30-μL drops of vitrification medium containing 35% EG, 5% polyvinylpyrrolidone (PVP, Sigma P-0930) and 0.4 M trehalose (Sigma T-0167) in TCM-199 supplemented with 10% FCS at 37°C for 20–30 s. Embryos rinsed in vitrification solution were aspirated into a micropipette as a 1–2-μL drop containing 25–30 embryos and dropped onto aluminum foil floating on liquid nitrogen (LN₂). Vitrified droplets were stored in cryovials in LN₂. Warming was performed by moving the vitrified droplets into 0.3 M trehalose in TCM-199 supplemented with 10% FCS at 37°C. Embryos having normal morphological appearance under stereomicroscope examination were cultured in G1.3/G2.3 medium. Differences in the two experiments were analyzed by one-way ANOVA. In experiment I, development rates to the blastocyst stage were 26%, 10% and 4% for G1.3/G2.3, CZB and M16 media, respectively. The highest development rate in experiment I was obtained in G1.3/G2.3 culture media (P < 0.05). Therefore, G1.3/G2.3 media were used for culturing of vitrified-warmed and control embryos. In experiment II, the rate of embryos having normal morphology was 98.5%. There were no significant differences between the development rates of vitrified (13.1%) and control (18.7%) embryos to the blastocyst stage (P > 0.05). Although the vitrification method resulted in a high survival rate based on the morphological appearance, developmental rates of vitrified and control embryos were found to be lower than expected and reported previously by other researchers. We believe that the low developmental rates in this study were due to our culture conditions but not our vitrification method. Therefore, it could be concluded that this vitrification method is an efficient one for pronuclear-stage embryo cryopreservation and better development rates could be obtained by improving the culture conditions. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F. Ergin is a young volunteer researcher.