EFFECTS OF EGTA AND SLOW FREEZING OF BOVINE OOCYTES ON POSTTHAW DEVELOPMENT IN-VITRO

Experiments were conducted to assess the morphological viability and i n vitro developmental potential of bovine oocytes after exposure to Et hylene Glycol-bis(-aminoethyl Ether) N,N,N,N-Tetra-acetic Acid (EGTA) prior to slow freezing. Different concentrations of EGTA (0, 1, 5 and 10 mM) and exposure intervals (5, 10 and 15 min) were tested on immatu re (GV) and in vitro matured (IVM) oocytes equilibrated in 1.5 mM prop ylene glycol (PG) without (experiment 1) or with slow freezing (experi ment 2). In addition, PG and ethylene glycol (EG) were compared for cr yoprotective efficacy. In vitro maturation (IVM), in vitro fertilizati on (IVF) and embryo culture (IVC) were performed in defined conditions . Pretreatment of both types of oocytes with 1 mM EGTA for 5 min witho ut freezing yielded morphological and functional results comparable to those obtained for controls while results from higher concentrations of EGTA were lower (P < 0.05). Higher rates of freeze-thaw survival an d embryonic development were obtained after pretreating GV oocytes wit h 1 or 5 mM EGTA for 5 min. Similarly, better results were obtained wh en 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 oocyte s exhibited higher survival rates (P < 0.05) than those frozen with EG . However, no significant differences were observed in the in vitro de velopment of surviving GV or IVM oocytes frozen with either PG or EG. Results suggest that a prefreeze treatment with 1 mM EGTA for 5 min ca n enhance oocyte viability. Conditions described enabled blastocyst de velopment of 2.9% of GV oocytes and 8.0% of IVM oocytes after cryopres ervation and IVF. Mol. Reprod. Dev. 52:86-98, 1999. (C) 1999 Wiley-Lis s, Inc.