Fertilization and Development of Frozen–Thawed Germinal Vesicle Bovine Oocytes by a One-Step Dilution Methodin Vitro

Abstract

The objective of this study was to evaluatein vitrofertilization and cleavage rates of frozen–thawed bovine oocytes at the germinal vesicle (GV) stage. In mouse oocytes, spindle microtubule reorganization after GV breakdown is particularly sensitive to cold and readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. The effects of various permeating cryoprotective agents [1.8Methylene glycol (EG), 1.3Methylene glycol monomethyl ether (EME), and 1.6M1,2-propanediol (PROH)] and different concentrations of trehalose (T) and polyvinylpyrrolidone (PVP) on post-thaw developmental capacity were examined. When bovine GV oocytes were frozen slowly in mixtures of 1.8MEG plus 5% PVP and 0.05MT, almost 80% developed to metaphase II; 22.2% degenerated afterin vitromaturation, and none of those that had been cryopreserved underwent parthenogenetic activation. The total fertilization rate was higher (P< 0.05) for oocytes frozen in a mixture of 1.8MEG plus 0.05MT or 0.1MT than in a mixture of 1.8MEG with or without 0.2MT; however, there was no difference in the number of normally fertilized or polyspermic oocytes that had been frozen in various cryoprotective solutions. No significant difference was observed in subsequent development using EG, EME, and PROH for GV oocytes. The addition of 0.05 or 0.1Mtrehalose to the freezing solution yielded significantly better cleavage and blastocyst rates than the solutions containing 0.2Mor no trehalose. For unfrozen controls, GV oocytes yielded significantly higher (P< 0.01) cleavage and blastocyst rates compared with frozen–thawed GV oocytes. It was found that 5% PVP had a beneficial effect compared with 10 or 20% concentrations for the development of blastocysts. Transfer of six blastocysts derived from frozen–thawed GV oocytes into three recipient heifers resulted in three pregnancies and the birth of one set of twins and one singleton calf.