Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling

Abstract

The objective of the research described was to devise an efficient procedure to cryopreserve in vitro-matured bovine oocytes, using in vitro fertilization (IVF) and development of resultant zygotes into blastocysts as criteria of oocyte survival. Oocytes at metaphase II were found to be extremely sensitive to chilling. Cooling them to 0°C for as little as 5 sec significantly decreased their capability to cleave and develop further after IVF; after 80 sec at 0°C, only ∼10% of chilled oocytes developed into blastocysts. Oocytes were also adversely affected by brief exposures to 4 M and 5.5 M ethylene glycol (EG) solutions supplemented with sucrose; after being suspended in either of these EG solutions in plastic straws and plunged directly into liquid nitrogen (LN2), few of the oocytes were fertilized and developed. To "outrace" chilling injury, oocytes contained in < 1 μl of EG solution were placed onto electron microscope grids and plunged directly into N2 slush or LN2. After such ultra-rapidly cooled oocytes were warmed, 30% of them cleaved after IVF, and half of these developed into blastocysts - survival rates equivalent to those for oocytes that had been exposed to EG without any cooling. This method offers promise as a novel way to cryopreserve bovine oocytes.