Kinetic and Temporal Factors Influence Chilling Injury to Germinal Vesicle and Mature Bovine Oocytes

In this study we examined the effects of low, above freezing temperatures on the viability and functionality of bovine oocytes. Germinal vesicle (GV) stage andin vitromatured oocytes (MII) were exposed to various combinations of time (15 and 60 min) and temperature (4, 16, 23, and 39°C). After being treated, the ability of oocytes to undergo maturation and fertilizationin vitrowas examined, as well as their viability assayed by two fluorescent probes, fluorescein diacetate (FDA) and 5-carboxyfluorescein diacetate (cFDA). Cooling GV oocytes to 16°C for 15 min reduced the fertilization rate by more than 40%, compared with those left at 39°C. Surprisingly, cooling oocytes to 4°C reduced the fertilization rate by only 10% compared with control. Exposing GV oocytes to temperatures below 23°C reduced their viability. Similar to the reduction in fertilization, the viability of GV oocytes after exposure to 16°C was reduced by more than 50%, whereas exposure to 4°C reduced it by only 9%. Viability measurements using FDA and cFDA gave comparable results and showed a similar trend. The viability of MII oocytes and of GV oocytes pretreated with butylated hydroxytoluene, following exposure to low temperatures, was higher compared with that of GV controls. We interpret these results as indicating chilling effects on membrane integrity. Improving the chilling resistance of bovine oocytes may facilitate their short- and long-term preservation.