Viability Of Frozen-Thawed Bovine Ivm/Ivf Embryos In Relation To Aging Using Various Cryoprotectants

Abstract

Bovine IVF embryos developed on Days 7, 8 and 9 were equilibrated with 1. 6 M propylene glycol (PG) , 1. 8 M ethylene glycol (EG) , 1. 1 M diethylene glycol (DEG) or 1 . 3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphate buffered saline (mPBS) supplemented with 10% superovulated cow serum. The embryos were loaded into 0. 25-ml plastic straws and were placed directly into a O"C alcohol bath chamber and held for 2 min. They were cooled from O"C to -5.5"C at 1"C /min and then seeded, followed by a 10-min holding period at - 5. 5"C . The straws were then cooled to -30"C at 0. 3"C /min before plunging into liquid nitrogen. Embryos were thawed and placed directly into the culture medium and washed 3 times. The survival rates of the Day-9 embryos based on reappearance of blastocoele, expansion, and hatching after 48 h of post-thaw culture were significantly lower (P<0.01) than those of the Day-7 and 8 embryos, in all of the cryoprotectants tested. On the other hand, while the reappearance of blastocoele and expansion of blastocysts after 48 h of post-thaw culture were not significantly different among each cryoprotectant, the percentage of hatching blastocysts were significantly different between DEG and EME (P<0.05) 1 between DEG and EG (P<0.01) and between PG and EG (P<0.05). These findings demonstrate that the age of the embryo (Day 7 and 8) is very important for the successful freezing of IVF bovine embryos. Also, as to the hatching rates, EME and EG are superior as cryoprotectants than the other 2 cryoprotectants tested.