145COMBINED GLUCOSE AND FRUCTOSE SUPPLEMENTATION IN PROTEIN-FREE KSOM IMPROVES PREIMPLANTATION DEVELOPMENT OF BOVINE TRANSGENIC CLONED EMBRYOS

Abstract

The present study investigated the effect of fructose supplementation in protein-free potassium simplex optimization medium (KSOM) on the preimplantation development of bovine transgenic cloned embryos. An expression plasmid containing bovine mutant PrP gene and enhanced green fluorescent protein (eGFP) as a marker gene was constructed and transfected into bovine fetal fibroblasts using FuGene6 (Roche, Indianapolis, IN, USA) as a lipid carrier. Transfected cells were cultured for 5 to 6 days to achieve chromosomal integration of the gene and then used for nuclear transfer. The somatic cell nuclear transfer was carried out by transferring a GFP-expressing donor cell into the perivitelline space of an enucleated oocyte. After electrical fusion and chemical activation, 525 (11 replicates) fused embryos were cultured in KSOM supplemented with 0.01% (w/v) PVA for 192 h at 39°C under 5% CO2, 5% O2 and 90% N2 gas atmosphere. The embryos were randomly allocated for 4 culture groups; KSOM supplemented with 1) 0.2mM glucose, 2) 1.5mM fructose, 3) 0.2mM glucose+1.5mM fructose and 4) vehicle (without glucose and fructose). We used 0.2mM glucose as per formulation of KSOM (Biggers JD et al. 2000 Biol. Reprod. 63, 281–293) and 1.5mM fructose due to its benefical effect on embryo development (Kwun J et al. 2003 Mol. Reprod. Dev. 65, 167–174). As a control experiment, 1043 (17 replicates) in vitro fertilized (IVF) embryos were cultured in the same culture system. The data were analyzed by PROC-GLM using SAS program. In IVF embryos, no significant differences in rates of cleavage (71.7 to 75.5%), morulae (34.1 to 37.1%) and blastocysts formation (21.0 to 24.5%) among the culture groups were observed. In contrast, significantly (P<0.05) higher rate in blastocysts formation (19.2%) was obtained when transgenic cloned embryos were cultured in KSOM supplemented with 0.2mM glucose+1.5mM fructose than with 0.2mM glucose (10.0%). However, the differences in blastocysts formation rates among other culture groups (13.2% for 1.5mM fructose and 11.9% for vehicle) were not significant. Moreover, the rates of cleavage (76.2 to 79.1%), morulae (19.3 to 23.8%) and GFP expression in blastocysts (68.0 to 78.6%) did not differ significantly among the culture groups for transgenic cloned embryos. This study demonstrated that the requirement of energy substrates in culture medium for bovine transgenic cloned and IVF embryos might be different. Now, we are conducting experiments to confirm whether the beneficial effect on transgenic cloned embryo development is due to combined effect of glucose and fructose or due to total concentration of available energy. This study was supported by Biogreen 21-1000520030100000.