Abstract: 【Objective】To clarify the feasibility of transfection of modified genome into spermatozoa by transmembrane peptide and the effect of transmembrane peptide on spermatozoa and fertilized eggs,so as to provide technical support for the safe and effective mass production of pig transgenic embryos.【Method】With buffalo Sohlh2 gene as the target gene,pig spermatozoa were transfected by cell membrane penetrating peptide(C105y,MPG and TAT)and lentivirus.Morphological characteristics of transfected spermatozoa were observed by laser confocal scanning microscope. Sperm viability,mean curvilinear velocity(VCL),mean linear velocity(VSL)and forward tropism(STR)were detected by sperm image analyzer(CASA),Sperm acrosome staining kit(psa-fitc)was used to detect pig sperm acrosome reaction,and in vitro fertilization was used to further verify the effect of different vector transfection on the production of Sohlh transgenic pig embryos.【Result】The acrosome structure of transfected pig sperm was intact,and the cell membrane was not broken. The positive rates of pig sperm transfection with different cell penetrating peptides(C105y,MPG and TAT)were 56.74%,50.44% and 43.58% respectively,which were lower than that of lentivirus(61.48%),but the difference was not significant(P>0.05,the same below). After transfection with membrane penetrating peptide C105y for 24 hours,the pig sperm viability(70.09%)and the average linear velocity(35.36 μm/s),average curve velocity(42.20 μm/s),forward directivity(1.03 μm/s),compared with the control group,the difference was not significant. After transfection with membrane penetrating peptides MPG,TAT and lentivirus,the indexes of pig sperm showed a downward trend to some extent. There was no significant difference in the incidence of spontaneous acrosome reaction in pig spermatozoa transfected with cell penetrating peptide compared with the control group,but there was a significant upward trend in lentivirus transfection(P<0.05,the same below). In the aspect of inducing acrosome reaction rate,the rate of inducing acrosome reaction in pig sperm transfected with lentivirus was significantly lower than that in the control group,while the rate of inducing acrosome reaction in pig sperm transfected with different cell penetrating peptides had no significant effect.The cleavage rates of the fertilized eggs obtained from porcine sperm transfected with lentivirus and transmembrane peptide C105y after in vitro fertilization were 64.82% and 65.91% after 24 hours of culture,which were not significantly different from those of the control group(64.52%). There was no significant difference in the blastocyst rate(15.8%)between the transfected group and the control group,but the blastocyst rate(9.11%)in the lentivirus transfected group was significantly lower than that in the control group. In terms of the positive rate of transgenic embryos,the membrane penetrating peptide C105y transfection group was significantly lower than that of the lentivirus transfection group(8.54%vs 10.38%).【 Conclusion 】Membrane penetrating peptide C105y has little effect on the fertilization ability of pig sperm and the development of transgenic pig embryos,and its toxic effect is not obvious. It is a safe vector for efficient transduction of foreign genes.