Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes

Abstract

The purpose of this study was to evaluate effects of cooling and rewarming on the meiotic spindle apparatus of bovine oocytes. In experiment 1, in vitro-matured bovine oocytes were either maintained at 39°C or cooled abruptly to 4°C or ~25°C. Immunohistochemical and DNA staining for visualization of microtubules and chromosomes, respectively, revealed an anastral, barrel-shaped spindle in bovine oocytes. Exposure to 4°C for 10- 20 min caused complete disappearance of the spindle. Some chromosome dispersion occurred after 60 min at 4°C. After exposure to ~25°C for 30 min, 90% of oocytes appeared abnormal, having either an abnormal spindle or no spindle. In experiment 2, oocytes cooled to either ~25°C or 4°C for 30 min were rewarmed directly or in steps for 15 or 60 min. Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme. Step-wise rewarming was no more beneficial than direct rewarming. More of the oocytes rewarmed directly contained dispersed chromosomes as time at 39°C increased.