ウシ卵細胞質内精子注入法(ICSI)の技術改良

概要

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This study was conducted to investigate the effects of ooplasm clarification, sperm tail-cutting and reduction of the polyvinylpyrrolidone (PVP) concentration in the sperm injection vehicle solution on ICSI in cattle. In vitro matured oocytes, motile spermatozoa and a piezo system were used for ICSI. In experiment 1, oocytes were centrifuged at 6,000 × g for 7 min to polarize the ooplasm lipid before ICSI. Sperm tail-cutting was carried out and the effect on ICSI was tested in experiment 2. In experiment 3, the effect of the PVP concentration in the sperm injection vehicle solution on ICSI was investigated. After ICSI, oocytes were cultured in vitro in mTALP for 15 h before fixation and observation. It was found that ooplasm clarification and sperm tail-cutting before ICSI significantly enhanced the survival rate of oocytes (92.0% vs. 67.7% and 94.8% vs. 77.3%: p<0.001, respectively) and the pronuclear formation rate (87.6% vs. 57.8% and 86.6% vs. 67.7%: p <0.001, respectively) after ICSI. Reduction of the PVP concentration from 10% to 4% and 0% decreased the survival rate of oocytes (95.0% vs. 89.3% vs. 74.6%: p<0.05-0.001) but increased the pronuclear formation rate (74.5% vs. 84.9% vs. 88.6%: p<0.05-0.001). This study demonstrated that technical improvement by clarifying oocyte cytoplasm, cutting the sperm tail and reducing the PVP concentration can significantly benefit ICSI in cattle.
2000-12-01