This study aims to investigate factors that affect the efficiency of blastocyst development and enhanced green fluorescence protein (EGFP) expression in porcine embryos following intracytoplasmic sperm injection (ICSI)-mediated DNA transfer. Frozen-thawed dead spermatozoa were exposed to different concentrations (0.01 microg/mL, 0.05 microg/mL or 0.1 microg/mL) of EGFP DNA solution, and then microinjected into in vitro matured oocytes. The optimal concentration for EGFP expression of resultant embryos was 0.05 microg/mL. When oocytes were microinjected on a warm stage at 30 degrees C, the percentage of EGFP-expressing embryos was higher than that at 38.5 degrees C (40.1% vs. 20.9%, P<0.01). The efficiency of EGFP expression in embryos following ICSI using linear EGFP DNA-exposed spermatozoa was higher than using circular DNA (40.8% vs. 28.2%, P<0.05). ICSI oocytes treated with 6-DMAP after electro-activation had a higher percentage of embryos expressing EGFP than those not treated (52.5% vs. 26.3%, P<0.01). However, neither incubation temperatures of spermatozoa and DNA (4 degrees C, 24 degrees C or 39 degrees C) nor BSA addition to the incubation medium affected the efficiency of producing EGFP-expressing embryos. Furthermore, treatment with DNase I after preincubation of sperm and DNA prevented the embryos from expressing EGFP. The EGFP expression of ICSI oocytes was affected neither by intracytoplasmic injection using sperm heads or whole spermatozoa, nor by washing of the sperm after preincubation. The above-mentioned factors did not affect embryonic developmental competence, apart from 6-DMAP treatment after electro-activation. In conclusion, most exogenous DNA molecules were tightly bound on the membranes of sperm head after incubation of DNA and sperm, and the temperature during ICSI, 6-DMAP treatment, exogenous DNA concentrations and constructs could significantly affect EGFP expression in porcine embryos following ICSI-mediated DNA transfer.