Influence of Plasmid DNA Concentrations on IVP Ovine Embryo Development and EGFP Expression

Genetic engineering applications that are employed to improve targeted livestock traits and to generate large animal models for biomedical

research have drawn considerable interest recently. In this study, effects of intracytoplasmic injection of different pEGFP-N1 concentrations on fluorescing EGFP+ embryo yield and embryonic development were investigated. Ovaries obtained from slaughterhouse were transported to the laboratory in PBS at 30-35°C and oocytes were isolated by slicing and matured for 24h. IVF was carried out using fresh semen which was prepared with Percoll density gradient technique. Sperm concentration was adjusted to 800000 spz/mL and oocytes were co-incubated for 20-22 h. Following denudation putative zygotes were divided into four; control and 3 study groups (5, 10 or 20 ng/μL of pEGFN) for microinjections. Zygotes were subjected to ICI, cultured and observed for development through morula to hatched blastocyst. On 7th day postfertilization embryos were assessed by fluorescence microscopy to determine EGFP expression. The cleavage rate in control, 5, 10 and 20 ng/μL groups were 82.1%, 79.4%, 75.4% and 35.0% respectively which revealed that 20 ng/μL group exhibited significantly lower cleavage rate than other groups (p<0.01). Development rates to the morula through hatched blastocyst stages in all injected zygotes were 46/67 (68.7%), 68/107 (63.5%), 73/114 (64.1%), 14/40 (35.0%) respectively. EGFP expression rates in all development stages of 5, 10 or 20 ng/μL groups were 35/107 (32.7%), 50/114(43.8%), 5/40 (12.5%) respectively. Results of EGFP expression in 10 ng/μL group was significantly higher than 20 ng/μL although there is no significancy between 5 and 10 ng/μL group (p<0.01) Developmental competence of putative zygotes was found higher in 5 and 10 ng/μL compared to 20 ng/μL group as there is a probability of higher plasmid concentration may compromise the development of zygotes. In scope of cleavage rates showed that implemented microinjection procedure’s effect on zygotes are not significantly harmful when compared to culture control. Cytoplasmic microinjection of 10 ng/μL pEGFP-N1 into in vitro produced sheep zygotes reproducibly results in higher EGFP expressing embryos then other groups and does not impede embryonic development.