Effects of EGTA and slow freezing of bovine oocytes on post-thaw development in vitro.

摘要

The morphological viability and in vitro developmental potential of cattle oocytes after exposure to ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetra-acetic acid (EGTA) prior to slow freezing were assessed. Concentrations of EGTA (0, 1, 5 and 10mM) and exposure intervals (5, 10 and 15 min) were tested on immature (germinal vesicle; GV) and in vitro-matured (IVM) oocytes equilibrated in 1.5 mM propylene glycol (PG) without (experiment 1) or with (experiment 2) slow freezing. Ethylene glycol (EG) and PG were also tested for cryoprotective efficacy. In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC) were performed in defined conditions. Pretreatment of both types of oocytes with 1 mM EGTA for 5 min without freezingyielded morphological and functional results similar to those obtained for controls but lower (P<0.05) for higher concentrations of EGTA. Higher rates of freeze-thaw survival and embryonic development were obtained after pretreating GV oocytes with 1 or5 mM EGTA for 5 min or when IVM oocytes were pretreated with 1 mM EGTA for either 5 or 10 min. When pretreated with 1 mM EGTA for 5 min and frozen with PG, IVM oocytes exhibited higher survival rates (P<0.05) than those frozen with EG. However, the in vitro development of surviving GV or IVM oocytes frozen with either PG or EG was similar. It is suggested that a prefreeze treatment with 1 mM EGTA for 5 min can increase oocyte viability. This can be used to optimise cryopreservation methods for unfertilized cattle oocytes.