Primary Studys On Intracytoplasmic Sperm Injection (ICSI) In Porcine

The objective of this study was taken to investigate the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). Meanwhile, the possibility of making transgenic porcine embryos by the ICSI was also explored.Effects of activation methods on the development of porcine embryos derived from ICSI were examined. Based on once frozen/thawed sperm, IDC (single directed current electrical pulse, 1.30 kv/cm for 80μs ), 1DC+6-DMAP and Ionomycin+6-DMAP three activation methods were applied for porcine embryos activation. Embryos with 2PN+2PB seemed as normal fertilization standard. The normal fertilization rates of 1DC+6-DMAP and Ionomycin+6-DMAP groups were significantly higher than that of IDC group(32.76%, 30.16% vs 16.67%, P<0.05), and the rates of cleavage and blastocyst formation were also significantly higher than that of IDC group(60.74%, 57.81% vs 33.62%, P<0.05; 16.82%, 13.28% vs 5.17%, P<0.05). However, there were no significantly difference between 1DC+6-DMAP and Ionomycin+6-DMAP groups(P>0.05). The rates of secondary polar body extracted among three activated groups were not significantly difference(66.67%, 68.97% vs 63.49%, P>0.05).Effects of sperm pretreatments on formation of male pronucleus and embryonic development of ICSI porcine embryos were investigated. Fresh sperm was designed as control group, 0.1%TritonX-100 incubated sperm and frozen/thawed sperm were designed as treatment group. The rates of male pronucleus and male pronucleus or decondensed sperm head formation of 0.1%TritonX-100 incubated sperms and frozen/thawed sperm were significantly higher than that of fresh sperm(38.46%,36.21% vs 20.83%; 50.0%, 48.26% vs 22.92%; 71.15%, 72.41% vs 47.92%, P<0.05), but there were no significantly difference between 0.1%TritonX-100 incubated sperm and frozen/thawed sperm(P>0.05). The rates of cleavage and blastocyst formation of 0.1%TritonX-100 incubated sperm and frozen/thawed sperm were also significantly higher than that of fresh sperm (66.13%, 65.18% vs 38.71%, P<0.05; 15.32%, 16.07% vs 9.68%, P<0.05).The possibility of producing transgenic porcine embryos by the ICSI was explored. When frozen/thawed sperm incubited with Linear pEGFP-N1 plasmid was applied in ICSI of IVM porcine oocytes, the cleavage rate was not significantly different (60.37% vs 65.18%, P>0.05) from the normal frozen/thawed sperm without incubated with exogenous DNA, but the blastocyst formation rate was significantly lower than sperm without incubated with exogenous DNA (8.49% vs 16.07%, P<0.05). The efficiency of GFP gene expressed in 2～4cells embryos was 32.61%.In conclusions, (1) Either ionomycin pluse 6-DMAP or direct current pluse 6-DMAP can be employed for activation of porcine embryos derived from ICSI; (2) Pretreatment of sperm with 0.1%TritonX-100 and liquid nitrogen freezing can promote the development of porcine embryos derived from ICSI; (3) Transgenic porcine embryos can be produced by ICSI, but the condition of sperm incubat with exogenous DNA should be optimizated.