Effects of gas conditions, time of medium change, and ratio of medium to embryo on in vitro development of horse oocytes fertilized by intracytoplasmic sperm injection

Abstract

This study was conducted to evaluate the effects of two different gas conditions (5% CO 2 in air or 5% CO 2, 5% O 2, 90% N 2, mixed gas), time of medium change (Day 3 or 4) and ratio of medium to embryo (2, 5 or 10 μl per presumptive zygote) on the development of horse oocytes fertilized by intracytoplasmic sperm injection and cultured in G1.2/2.2 medium. Oocytes from slaughterhouse-derived ovaries were matured in vitro for 24 h and fertilized by injection of frozen–thawed sperm using micromanipulation with a Piezo drill. Presumptive zygotes were randomly assigned to 5% CO 2 in air or mixed gas and fixed after 96 h of culture. Cleavage rates between two gas conditions were similar (67 and 63%), but the mean nucleus number of embryos in the mixed gas treatment was significantly ( P<0.05) higher than that of embryos cultured in 5% CO 2 in air (15.2 versus 7.0, respectively). Further experiments were done with mixed gas incubation. Development of embryos was compared after change from G1.2 to G2.2 medium at Day 3 or 4. There was no significant difference in cleavage rate (56 and 65%, respectively) or development to the blastocyst stage after 7 days of culture (5% and 46%, respectively) between embryos changed on different days. To evaluate the effect of the ratio of medium to embryo, zygotes were cultured at a ratio of 2, 5 or 10 μl medium per zygote. There were no significant differences among ratio treatments in rates of cleavage or development to blastocyst.