34 EVALUATION OF DIFFERENT SEQUENTIAL CULTURE SYSTEMS ON THE DEVELOPMENT AND QUALITY OF BOVINE EMBRYOS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER

Abstract

Different culture systems have been developed that support development of bovine embryos up to the blastocyst stage. However, the use of sequential culture systems has been studied less. The objective of the present study was therefore to examine the effect of 3 sequential culture systems, which involve the use of different culture media for early cleavage and later stage embryos, on the development and quality of bovine embryos generated by somatic cell nuclear transfer (SCNT). These systems were mainly based on different combinations of KSOM culture medium regularly used in our laboratory. Skin fibroblasts, at passage 5 to 6, were microsurgically placed into the perivitelline space evacuated during enucleation. Fusion was carried out by a single DC pulse of 1.7 kV cm^–1 with an Electrocell Manipulator 830 (BTX Inc., San Diego, CA, USA) and activation by treatment of NT units in Ionomicin (5 μM, 4 min) and DMAP (1.9 mM) for 4 h. Embryo culture was carried out in 50-μL drops under mineral oil at 38.5°C and 5% CO₂, 5% O₂, and 90% N₂, in a humidified atmosphere, according to the following sequential culture systems without co-culture: 1) KSOM + 0.4% BSA (FAF, A8806, Sigma, St. Louis, MO, USA) for 3 days and then KSOM + 5% FBS (characterized, Hyclone, Logan, UT, USA) to Day 7; 2) KSOM + 0.1% BSA for 3 days and then SOF + 0.8% BSA to Day 7 and 3) KSOM + 0.1% BSA for 3 days and then KSOM + 0.8% BSA to Day 7. Cleavage rate was evaluated on Day 3 and the number of blastocysts was recorded on Day 7. A total of 730 NT embryos randomly distributed were analysed for embryo development in 9 replicates and 5 blastocysts of each group were stained with bisbenzimide (Hoechst 33242) to assess the quality. ANOVA was used to test for statistically significant differences (P < 0.05) using Satgraphics Plus 2 Software. In cases where statistically significant differences were observed, a multiple comparison test was run using Tukey’s test. Sequential culture systems had no effect on cleavage rate (73, 76, and 73%, respectively). However, there was a significant difference (P < 0.01) in the rate of blastocysts. The sequential culture system consisting of KSOM + KSOM 5% FBS yielded a higher rate of blastocysts than other treatments (28, 18, and 16%, respectively). Despite this difference in embryo development, the quality of embryos as assessed by the total number of cells was not different (135 ± 6.5, 129 ± 7.5, and 128 ± 8.5, respectively). In conclusion, a sequential culture system consisting of KSOM + 0.4% BSA for 3 days and then KSOM + 5% FBS to Day 7 generated a higher number of cloned blastocyst than other treatments evaluated, although the quality of the embryos did not differ between treatments. Future studies are under way to establish the gene expression profile of NT embryos generated under these culture systems. The final aim is to evaluate the possibility of modulating the gene expression profile through changes in the culture medium composition.