【作者】 卢春霞；

【导师】 石国庆；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 本研究着重探讨和研究了绵羊胞质内显微受精(ICSI)各技术环节当中一些因素对绵羊ICSI整个过程的影响,初步建立了一套比较完善的操作体系。研究结果如下:实验一,对精子DTT处理后进行ICSI,结果如下:(1)采用DTT处理与不处理的精子比较,卵裂率(79.6%、81.6%;P>0.05)没有明显差异,囊胚发育率(31.2%、20.9%;P<0.05)差异显著。实验二,分别采用活精子和死精子进行ICSI,卵裂率(80.0%、83.1%)和囊胚发育率(27.6%、20.2%)均没有显著影响(P>0.05)。实验三,为了研究激活方法对绵羊胞质内显微受精的影响,分别对卵子采用:(1)假注射,不激活;(2)注射精子,不激活;(3)假注射,Ionomysin +6-DMAP激活;(4)注射精子, Ethanol+6-DMAP激活;(5)注射精子,Ionomysin+6-DMAP激活。结果:第1、2组无论注射精子与否,都不激活,卵裂率(7.5%、5.3%)和囊胚发育率(0.94%、1.8%)都很低,差异均不显著。第3组与第4、5组比较,卵裂率(65.7%、71.6%、75.6%;P>0.05)没有明显差异,但囊胚发育率(5.7%、15.6%、26.0%;P<0.01)却明显低于4、5组。采用Ethanol+6-DMAP和Ionomysin+6-DMAP两种方法处理时,卵裂率差异不显著(71.6%、75.6%;P>0.05),但囊胚发育率前者要显著低于后者(15.6%、26.0%;P<0.05)。实验四,本实验对显微注射时对第一极体位置进行分析,发现第一极体位于6点和12点位置,卵裂率(82.4%%、85.8%;P>0.05)和囊胚发育率(28.8%、26.9%;P>0.05)没有显著影响。实验五,精子操作液中PVP浓度分别为10%、8%和5%时,卵裂率(80.2、83.6%、72.0%;P<0.05)和囊胚发育率(27.8%、30.5%、16.0%;P<0.05)均差异显著。实验六,在培养液中添加VEGF和不添加,卵裂率(78.7%%、83.9%;P>0.05)和囊胚发育率(27.7%、23.6%;P>0.05)没有显著影响。实验七,操作液中添加CB和不添加CB,卵裂率(77.5%、74.7%;P>0.05)没有显著影响,但添加CB的囊胚发育率(29.2%、14.3%;P<0.05)却显著高于对照组。实验八,对显微注射时的温度进行分析,恒温和室温条件下,卵裂率(80.1%、90.6%;P<0.05),差异显著,囊胚发育率(31.3%、28.2%;P>0.05)没有显著影响。实验九,显微注射时间在4h以前注射卵裂率(84.7%、86.0%、80.6%;P>0.05)没有显著影响。0~1h注射的囊胚发育率要显著高于1~2h注射的,并极显著高于3-4h的(33.6%、18.3%、13.0%;P<0.05-0.01)。1-2h与3~4h注射的囊胚发育率(18.3%、13.0%;P>0.05没有明显差异。更多还原

【Abstract】 The aim of this study was to establish an steady and efficient ICSI system for sheep oocytes maturation in vitro in our lab. Experiments were carried out to analyze the diffetent factors which been reported influence the results of ICSI and to optimize the technology of ICSI in sheep and establish a preliminary IVF system. Results:1.The sperm were treatment by DTT and untreated.after ICSI, The Cleavage rate (79.6%、81.6%;P>0.05)were not significantly different between the two group;whereas,The blastocys rate(31.2%、20.9%;P<0.05)was higher for treatment than untreated. 2.ICSI with live and dead status of the sperm, There was no significant difference of Cleavage rate((80.0%、83.1%)and blastocys rate(27.6%、20.2%) between the two group( P>0.05).3. After ICSI ,the oocytes were activated by different method:(1) Sham-injected, no activated. (2) Sperm-Injected, no activated. (3) Sham-injected, oocytes were activated with Ionomysin+6-DMAP.(4)Sperm-injected, oocytes were activated with Ethanol+6-DMAP.(5) Sperm-injected, oocytes were activated with Ionomysin+6-DMAP. Results : In Sham-injected and Sperm-injected oocytes,but no activated, both the cleavage rate(7.5%、5.3%)and blastocyst rate(0.94%、1.8%)were very low. The cleavage rate(65.7%、71.6%、75.6%;P>0.05)of 3、4、5 groud was no significant difference,but blastocyst rate(5.7%、15.6%、26.0%;P<0.01) of the third groud was lower than others. There was no significant difference of cleavage rate(71.6%、75.6%;P>0.05)of the sperm-injected groud treated with Ethanol+6-DMAP and Ionomysin+6-DMAP,while blastocyst rate (15.6%、26.0%;P<0.05)was lower than latter. 4. This study was to evaluate the Effects of first polar body position on fertilization of sheep oocytes by intracytoplasmic sperm injection .There was no significant difference of cleavage rate (82.4%%、85.8%;P>0.05)and blastocyst rate (28.8%、26.9%;P>0.05 ) to first polar body positioned at 12 o’clock or 6 o’clock. 5. The different concentration(10%、8%、5%) PVP were added to manipulation medium ,The results showed that cleavage rate (80.2%、83.6%、72.0%; P<0.05)and blastocyst rate (27.8%、30.5%、16.0%;P<0.05) was higher for 10% and 8%PVP than 5%PVP . (6) Added VEGF in culture medium , There was no significant difference of cleavage rate (78.7%、83.9%;P>0.05)and blastocyst rate (27.6%、23.6%;P>0.05 ) in the same condition with or without VEGF. (7) There was no significant difference of cleavage rate (77.5%、74.7%;P>0.05)in the same condition with or without CB,but blastocyst rate (30.5%、16.0%;P<0.05) was higher for with than without CB. (8) Injection eggs in room temperature and 37.0℃, respectively. the cleavage rate(90.6%、80.1%;P<0.05)was significantly higher for room temperature than that of latter, but the blastocyst rate(31.3%、28.2%;P>0.05) was not significantly different between the two group. (9) Oocytes were randomly injection for 0-1h、1-2h and 3-4h , cleavage rate were 46.07%、43.33%、30.56% (P>0.05),respectively.The blastocyst rate was higher for oocytes injection for 0~1 h than 1~2 h、3~4 h(33.6%、18.3%、13.0%;P<0.05-0.01), There was no significant difference of blastocyst rate between oocytes injection for 1-2h and 3-4h (P>0.05)更多还原