Deep-freezing of bovine embryos derived from in vitro fertilization (IVF)

The authors examine the effect of a direct rehydration after freezing in ethylene glycol (EG) on the survival rate of embryos derived from in vitro maturation (IVM), fertilization (IVF) and culture (IVC) and the effect of bovine cumulus cells monolayer on the viability of embryos during in vitro culture. Bovine oocytes were matured, fertilized and cultured in vitro according to the method previously described by HERRLER, LUCAS-HAHN and NIEMANN (1992). At days 6, 7, 8, 9, and 10 after in vitro insemination (Day o), good qual ity embryos were directly equilibrated at room temperature (20 to 25 degrees C) with 1.8 M EG for 15 min. in a modified phosphate buffered saline (mPBS) supplemented with 10% fetal calf serum (FCS). After equilibration, embryos were cooled at 0.3 degrees C/min. to -33 degrees C at which point they were plunged into liquid nitrogen. Embryos were thawed by placing the straws in a 25 degrees C water bath. The embryos were then transferred into culture medium for rehydration. For assessment of viability, embryos were cultured (36 to 48 hours) on feeder layers of bovine cumulus cells in TCM-199. Only a few, day 6 morulae survived the freezing and thawing procedure (2/33, 6.0%). High survival rates were found in day 7 and 8 blastocysts (14/19, 73.6%; 15/21, 71.4%, respectively). The survival rate of day 9 to 10 blastocysts was lower (8/20, 40%). Thus, day 7 and 8 IVMFC blastocysts seemed to be suitable for the present freezing and direct rehydration procedure (p<0.01, Table). In conclusion, the data have indicated that the age and time of blastocyst formation are important factors for the successful freezing of bovine IVMFC embryos. The reduced survival rates for day 9/10 blastocysts have shown to be accompanied by a lower cell numer (JIAING et al., 1992). The latter is not favourable in the context of freezing. The most appropriate age and developmental stage for freezing of bovine embryos were the day 7 and 8 blastocysts.