| Intracytoplasmic sperm injection (ICSI) can raise the animal gamete utilizing efficienty, gain certain sex individuals.and solve productivity difference animal fertilization question, as an auxiliary technicalaspect it is important in transfers the genetic engineering. During ICSI,the majority of the laboratories use a solution of PVP (polyvinylpyrrolidone-a macromolecule) as a medium for spermatozoon injection, in order to slows down the movement of spermatozoa, being lubricated duing ICSI, reduce the conglutination between sperm and injection pipette. PVP is injected intracytoplasmically and the presence of PVP in the oocyte stabilizing the plasma membra and makes the sperm nucleus inaccessible for agents involved in sperm nucleus decondensation and oocyte activation. Spermatozoon injection can be performed successfully in the absence of PVP, but the absence of PVP makes the ICSI procedure more laborious.This study investigated several problems existent in ICSI process,such as slow down the movement of the spermatozoon, conglutination,the flexibility of membrane,PVP surrounding the spermatozoon,the difficulty of sperm decondensation,and oocytes activated insufficiency, this research studied the use of HA instead of PV, reduced the influence of PVP, improve the efficiency of ICSI in porcine.The purpose of this study is to diminish the effect of PVP in the operation medium, and ultimately improve the developmental potential of ICSI embryos. The study included: investigated the time of sperm decondensation and the pronuclear formation, and its morphological changes by adding PVP into the operation medium, after ICSI, investigated the effect of the electrical pulse and chemical activation methods on embryos, and the effect of CB in operation medium. Based on those experiments mentioned above, this experiment study the effect of the concentration of PVP and HA in operation medium. Finally, compared the cleavage rate and the proportion of embryos with more than 8-cell of the oocytes between the two groups. The results as following:Experiment 1: The time of sperm decondensation and the pronuclear formation after ICSI were investigated in this study. After ICSI, the oocytes were stained with Hoechst33342 to assess the sperm decondensation and the pronuclear formation. At 7h after ICSI, only 5.2% of the sperms was decondensed while the male or female pronuclear hadn't been found, at 10h and 13h after ICSI, 12.5% and 29.0% of sperm decondensed respectively, and 6.5% sperm formed male pronucleus at 13h., at 16h after ICSI, 16.7 % of sperm decondensed while 28.8% sperm formed male pronucleus, At 19h after ICSI, 12.5 % of sperm decondensed while 41.7% sperm formed male pronucleus. The results indicated that sperm decondensation mostly in 13-16h after ICSI, the rate of the female pronucleus increased along with the time. According to the result, the investigation time of sperm decondensation and the pronuclear formation at 16h after ICSI in later experiment is appropriate.Experiment 2: Because of insufficient in oocytes activation, sperm injected oocytes were activated by chemical substance after electrical pulse (a single dc-pulse of 0.4KV/cm for 90us), control group oocytes were cultured in embryo culture medium, other groups incubated in 2mmol/L 6-DMAP or 10ug/ml CHX for 6h after electrical activation. The results were that cleavage rates of control group lower than other two groups, which significantly lower than 6-DMAP group(41.0% vs 44.3%) (p＜0.01),lower than CHX group(41.0% vs 43.5%) (p＜0.05). The proportion of embryos with more than 8-cell of the oocytes in control group were lower than other two groups, which significantly lower than 6-DMAP group(14.1% vs 19.0%) (p＜0.0l),lower than CHX group(14.1% vs 17.6%) (p＜0.05), 6-DMAP group and CHX group had similar cleavage rates and the proportion of embryos with more than 8-cell (p＞0.05). These results indicated that the IVM oocytes just activated by electrical pulse is not sufficient, activated by 6-DMAP after electrical activation was appropriately, this method could decrease the effect of MPF, accelerate the sperm decondensation and the pronuclear formation, improve the effect of embryo, oocytes were activated by 6-DMAP 6h after electrical activation were used in later experiment.Experiment 3: Effect of CB on the oocytes in vitro development after ICSI. The cleavage rate of the oocytes treated with cytochalasin B (CB) was higher than that of the untreated ones(46.7% vs 43.7%) (p＜0.01), the proportion of embryos with more than 8-cell of the oocytes treated with CB group was higher than untreated ones(18.7% vs 18.3%), but the difference was not significant(p＞0.05). It demonstrated that adding CB in operation medium could ameliorate the effect of cleavage.Experiment 4: The effect of different concentration of PVP on porcine embryo development following ICSI. The decondensation of the sperm nucleus of oocytes in 5% and 10% groups were similar(16.5% vs 16.8%) (p＞0.05), the pronuclear formation rate of oocytes treated by 5%PVP was higher than that treated by 10%PVP(28.7% vs 23.7%) (p＜0.05), the cleavage rate of oocytes treated by 5%PVP was higher than that treated by 10%PVP(46.7% vs 42.2%) (p＜0.05), the proportion of embryos with more than 8-cell of the oocytes similar in these two groups (18.7% vs 17.6%) (p＞0.05). The result indicated that reduce the concentration of PVP could avail the pronuclear formation, increase the cleavage rate of oocytes.Experiment 5: The effect of different concentration of HA on porcine embryo development following ICSI. The decondensation of the sperm nucleus (16.0% vs 15.7%, 16.2%) and the pronuclear formation rate of oocytes (30.2% vs 30.4%, 29.3%) in 0.5mg/ml,1.5mg/ml and 2.5mg/ml groups were similar (p＞0.05). The cleavage rate of the oocytes in 0.5mg/ml HA were lower than other groups(46.0% vs50.1%, 49.6%) (p＜0.01) , the cleavage rate and the proportion of embryos with more than 8-cell of the oocytes in 1.5mg/ml HA and 2.5mg/ml HA were similar (50.1% vs 49.6%) (22.4% vs 21.2%) (p＞0.05). Those results indicated that use 1.5mg/ml HA could improve development quality of embryo after ICSI.HA was found to be as effective as PVP in facilitating the injection of spermatozoa during the ICSI procedure. There was no significant difference in the number of decondensation sperm nucleus between 1.5mg/ml HA and 5%PVP groups (p＞0.05). The pronuclear formation rate,the cleavage rate and the proportion of embryos with more than 8-cell of the oocytes in 1.5mg/ml HA were higher than 5%PVP (30.4% vs 28.7%) (50.1% vs 46.7%) (22.4% vs 18.75%) (p＜0.05).Therefore, the use of 1.5mg/ml HA was investigated as a replacement for 5 %PVP during ICSI.In conclusions, during ICSI, add 5μg/ml cytochalasin B (CB) in the injection medium,and use 1.5mg/ml HA instead of 5% PVP, porcine oocytes incubated in 6-DAMP for 6h after electrical activation was preferably. Compared with conventional method, the sperm decondensation, pronuclear formation, the cleavage rate and the proportion of embryos with more than 8-cell of the oocytes in HA group is better than PVP group, facilitated interaction effect between sperm and oocytes, the efficiency of ICSI in porcine improved obviously.
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