Optimization of buffalo spermatozoa transfection with pbIFN-tau-EGFP gene by simple incubation technique

The present study was conducted to optimize and analyze the uptake of pbIFN-tau-EGFP by spermatozoa of Murrah buffaloes (Bubalus bubalis) by simple incubation technique. Fresh semen samples from two Murrah buffaloes were selected based on mass activity (+3 and above) from the herd. Three different variables i.e. bovine Interferon tau gene conjugated to enhanced green fluorescent gene (pbIFN-tau-EGFP) gene concentration, incubation time and number of spermatozoa, were considered for optimization of spermatozoa transfection by simple incubation technique. Before and after transfection, the spermatozoa samples were evaluated for sperm functional parameters, motility and viability. Spermatozoa incubated with different concentrations (10 and 20μg/ml) of gene (pbIFN tau-EGFP) and without gene which served as control were subjected to in vitro fertilization (IVF). A significant decrease (P<0.05) in percentage of motile spermatozoa of fresh semen sample was observed. For further 1 h of incubation the decrease observed was significant (P<0.05) when compared with control group. Similarly a significant decrease (P<0.05) in percentage of viable spermatozoa was observed after 1h with a further 1h of incubation. Positive results were obtained for uptake of the gene (amplified 585bp product) on PCR analyses of DNA extracted from sperm samples incubated with either concentration of the gene. A 40–47 percentage of cleaved embryos were obtained post IVF with spermatozoa of fresh semen samples. Either, concentration of the gene or spermatozoa used in the present study did not result in any significant difference in percentage of cleaved or morula stage embryos. The resulting embryos were also positive for uptake of pbIFN-tau-EGFP gene.