Effect of cryopreservation on the efficiencies of goat sperm in picking up exogenous DNA and resulted transgenic embryos

The efficiency and the ability of fresh and frozen-thawed sperm in picking up exogenous DNA were investigated in this study. The fresh and frozen-thawed sperm was incubated with linearized end-labeled pEGFP-N1 plasmid DNA, and detected by in situ hybridization method. The in vitro producted embryos were screened by PCR assay. The ultrastructure of activated spermatozoa were observed by transmission electron microscope (TEM), and the integrity of sperm plasma membrane was evaluated with a combination of fluorescent probes-carboxifluorescein diacetate and propidium iodide (PI). The sperm genomic DNA damage was determined by single cell gel electricity assay (SCGE) methods. The results showed that the frozen-thawed treated goat sperm cells were more efficient and more reliable than untreated sperm in picking up exogenous DNA and subsequently internalizing the DNA into sperm nuclei（81.60%±16.59% vs 32.95%±2.93%，t=4.873，P=0.003；41.80%±6.26% vs 27.89%±8.64%，t=2.634，P=0.039）. The exogenous DNA retained its integrity in sperm as demonstrated with PCR and Southern Blotting assay methods. The rate of transgenesis embryos produced by exogenous DNA incubated sperm was enhanced significantly(45.45%±10.87% vs 24.44%±6.06%, t=1.750，P=0.013). The semen cryopreservation reduced the integrity of sperm plasma membrane (8.34%±4.21% vs 65.67%±6.46%，t=12.492，P=0.000) and enhanced the sperm genomic DNA strand breakage significantly. These results suggest that semen cryopreservation could enhance the sperm ability in picking up foreign DNA. The cryopreservation-induced changes in the sperm plasma membrane facilitate the internalization exogenous DNA [Acta Zoologica Sinica 54(6): 1089 – 1097, 2008].