Effect of Different Two-steps and Sequential Culture Systems on the Development, Quality and Gene Expression Profile of Bovine Embryos Produced in Vitro

In the present study, we examined the effect of different two-steps and sequential culture systems, which involve the use of different culture media for early cleavage and later stage embryos, on the development, quality and gene expression profile of bovine embryos generated by in vitro fertilization. Presumptive zygotes were randomly allocated to four different medium treatments without co-culture: 1) KSOM + 0.4% BSA for 3 days and then KSOM + 5% FBS to day 7; 2) KSOM + 0.1% BSA for 3 days and then SOF + 5% FBS to day 7; 3) KSOM + 0.1% BSA for 3 days and then SOF + 0.8% BSA to day 7 and 4) KSOM + 0.1% BSA for 3 days and then KSOM + 0.8% BSA to day 7. Culture medium had no effect on cleavage rate. However, a significant difference (P < 0.05) was observed with the two-steps culture systems yielding higher rate of blastocysts (37 and 32% for KSOM + KSOM 5% FBS and KSOM + KSOM 0.8% BSA, respectively), compared to sequential culture systems (26 and 28% for KSOM-SOF 5% FBS and KSOM-SOF 0.8% BSA, respectively). Embryos cultured in equential KSOM-SOF 5% FBS developed slowly, had lower hatching rate, lower total number of cells, and higher apoptosis rate compared to other treatments. Furthermore, gene expression analysis of 20 developmentally important genes showed alterations of DNMT1 in embryos cultured in sequential KSOM-SOF 5% FBS and DNMT1, SOD1 and GSS in embryos cultured in two steps KSOM + KSOM 0.8% BSA. In conclusion, the use of a two step culture system consisting of KSOM medium supplemented with 5% FBS after 3 days of culture, resulted in higher and better quality blastocysts. Future studies, including evaluation of pregnancy rates and development to term of embryos cultured in these systems will be necessary to better understand the differences observed in embryo culture efficiency.