| Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization technique valuable for studying fertilization, treating human infertility and producing transgenic animals. Although live ICSI-derived offspring have been generated in many species, it was not until 2000 that live ICSI-derived piglets were born. In comparison to ICSI in other species, ICSI in swine has not been well established.The objective of this study was to investigate the effects of various methods of oocyte activation, sperm pretreatment and cysteine treatment on development of porcine embryos derived from intracytoplasmic sperm injection (ICSI) of in vitro-matured oocytes. Experiment 1: Based on motile sperm, Calcium ionophore A23187, Calcium ionophore A23187+6-DMAP (a combination of Calcium ionophore A23187, with 6-DMAP 4 h later) and 1 DC (twice directed current electrical pulse, 1.20 kv/cm for 30μs) three activation methods were applied for porcine embryos activation, the control group were not applied any additional activation. Experiment 2: Motile sperm was designed as control group. Sperm rendered immotile by one-time freezing and thawing without cryoprotectant in liquid nitrogen and pre-incubated with 0.1% Triton X-100 were designed as treatment group. Experiment 3: Presumed zygotes after ICSI were cultured in PZM-3 with 1.71 mmol/L cysteine for 0 h (control), 4 h, 12h and 168 h and then were cultured in normal PZM-3.The results showed that 1) The oocyte activation and fertilization rates significantly increased in the three activation groups (86.57%,96.88%,94.12% vs 71.88%;32.84%,43.75%,36.76% vs 21.88%)(p<0.05). The fertilization rates of A23187+6-DMAP group were significantly higher than A23187 and DC groups (43.75% vs 32.84%,36.76%)(p<0.05). The rates of secondary polar body extruded of A23187+6-DMAP and A23187 groups were significantly higher than control and DC groups (74.19%,74.14% vs 58.90%,54.69%)(p<0.05). The cleavage and blastocyst rates of A23187+6-DMAP and DC groups were significantly higher than control group (69.57%,70.15% vs 53.33%;20.29%,25.37% vs 6.67%)(p<0.05). 2) The fertilization rates of 0.1%TritonX-100 incubated sperm and frozen/thawed sperm groups were significantly higher than control group (48.15%,56.36% vs 39.34%)(p>0.05). The male pronucleus rates of frozen/thawed sperm group were significantly higher than control group (74.55% vs 52.46%)(p<0.05). There were no significantly difference in the cleavage and blastocyst rates among the three groups (P>0.05). 3) Fertilization and male pronucleus rates significantly increased when presumed zygotes after ICSI were cultured in PZM-3 with 1.71 mmol/L cysteine for 4 h and 12 h compared to 0 h group (58.73%,53.70% vs 37.29%;71.43%,64.81% vs 47.46%)(p<0.05). The blastocyst rates of 4 h group were significantly higher than 0 h, 12h and 168 h groups (24.78% vs 13.56%,18.52%,12.17%)(p<0.05).These results indicated that additional activation of the oocytes is necessary for the porcine ICSI, and A23187+6-DMAP is a reasonable choice; Pretreatment of sperm by TritonX-100 or freezing and thawing without cryoprotectant in liquid nitrogen can promote fertilization of the presumed zygotes after ICSI; Culture of presumed zygotes after ICSI in PZM-3 with 1.71 mmol/L cysteine for 4h can promote fertilization, male pronucleus and blastocyst formation.
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