Cryopreservation of mouse 2-cell embryos and ova by vitrification: methodologic studies

Area of Science:

• Reproductive Biology
• Developmental Biology
• Cryobiology

Background:

• Cryopreservation is crucial for preserving reproductive cells.
• Vitrification offers a promising alternative to slow-freezing for cryopreservation.
• Optimizing vitrification protocols is essential for successful cryopreservation of mammalian oocytes and embryos.

Purpose of the Study:

• To evaluate the efficacy of vitrification for cryopreserving unfertilized mouse ova and 2-cell embryos.
• To determine the optimal conditions for vitrification of unfertilized mouse ova.
• To assess the impact of vitrification on subsequent in vitro fertilization and embryo development.

Main Methods:

• Vitrification of 2-cell mouse embryos and unfertilized mouse ova using a vitrification solution (VS1).
• Assessment of survival rates based on morphological integrity and development to the hatching blastocyst stage.
• Systematic variation of VS1 concentration and exposure time at 0°C to identify optimal conditions.
• In vitro fertilization of vitrified-unfertilized ova and subsequent embryo development assessment.

Main Results:

• Vitrification of 2-cell embryos resulted in 100% morphologic survival and 89% development to hatching blastocyst stage.
• For unfertilized ova, VS1 concentration and exposure time significantly affected survival rates (P < 0.01).
• Optimal conditions (100% or 90% VS1, 10 min exposure at 0°C) did not significantly differ from controls (52% survival).
• Lower concentrations (70-80%) or longer exposures (15-30 min) significantly reduced survival rates.
• Vitrification of unfertilized ova under optimal conditions followed by liquid nitrogen immersion resulted in 0% fertilization rates in vitro.

Conclusions:

• Vitrification is highly effective for cryopreserving 2-cell mouse embryos, yielding excellent developmental potential.
• Optimal vitrification conditions for unfertilized mouse ova at 0°C do not improve survival compared to controls.
• Cryopreservation of unfertilized mouse ova by vitrification, even under optimized conditions, significantly impairs their subsequent fertilization capacity.