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奶牛X性控冻精的卵胞质内单精子注射研究
Studies on Intracytoplasmic Sperm Injection of X-Sorted Frozen-Thawed Sperm in Dairy Cow
【作者】 徐红强；
【导师】 马保华；
【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2009， 硕士
【摘要】 本试验比较了奶牛X性控冻精和常规冻精的胞质内单精子注射(ICSI)效果,同时研究了奶牛X性控冻精的运动状态、聚乙烯吡咯烷酮(PVP)的浓度和卵母细胞的第一极体位置对奶牛X性控冻精ICSI的影响。1.分别用奶牛X性控冻精和常规精子进行ICSI,比较了两种精子的ICSI卵的卵裂率和囊胚发育率。结果表明,显微注射常规精子和性控冻精ICSI卵的卵裂率(88.3 %,84.1 %)、正常卵裂率(75.5 %,72.2 %)、囊胚发育率(29.3 %,27.2 %)差异不显著(P > 0.05)。2.性控冻精解冻后,运动精子和不运动精子经过同样浓度的PVP处理,分别进行ICSI操作。结果表明,注射运动精子的ICSI卵的卵裂率和囊胚发育率(86.4 %,28.1 %)高于不运动精子组(82.3 %,25.7 %),但无统计学差异(P > 0.05)。3.分别用5 %、8 %和10 %PVP的精子操作液处理奶牛X性控冻精,选择运动精子进行ICSI操作,ICSI卵的卵裂率(87.3 %,82.3 %,81.7 %)差异不显著;正常卵裂率(78.4 %,73.8 %,69.1 %)和囊胚发育率(29.7 %,24.8 %,20.4 %)随着PVP浓度的升高而下降,PVP浓度在5 %和10 %时,正常卵裂率分别是78.4 %和69.1 %(P < 0.05),囊胚发育率分别为29.7 %和20.4 %(P < 0.05)。4.注射时卵母细胞极体位置于相当于时钟6点的位置与12点的位置相比,ICSI卵的卵裂率(84.6 %,83.3 %)差异不显著(P > 0.05);而正常卵裂率(76.4 %,66.7 %)和囊胚发育率(28.6 %,19.0 %)明显提高(P < 0.05)。结论表明:用奶牛X性控冻精进行ICSI是可行的;在进行奶牛X性控冻精ICSI操作时,选用运动精子,并用含5 %PVP的精子操作液进行处理,而且注射在卵母细胞极体置于6点的位置,有利于奶牛X性控冻精ICSI卵体外发育。
【Abstract】 This study compared x-sorted frozen-thawed sperm with conventional sperm in intracytoplasmic sperm injection(ICSI), and investigated the effect of the state of x-sorted frozen-thawed sperm, the polyvinyl pyrrolidone(PVP)concentration in the spermic manipulation medium, and the first polar body(PB)on the in vitro development of the ICSI zygotes of x-sorted frozen-thawed sperm in dairy cow.In experiment 1, two different sperm types ( conventional sperm and x-sorted frozen-thawed sperm)in ICSI were compared. The result indicated that there were no significant differences in the ICSI zygotes cleavage rate(88.3 %, 84.1 %), regular cleavage rate(75.5 %, 72.2 %), blastocyst rate(29.3 %, 27.2 %)between the two types of the sperm(P > 0.05).In experiment 2, two different sperm kinestates(motile sperm and immotile sperm)were used in ICSI, the higher cleavage and blastocyst rate of ICSI zygotes were obtained from the motile sperm(86.4 %, 28.1 %)than those from the immotile sperm(82.3 %, 25.7 %), but there was no significant different(P > 0.05).In experiment 3, three different concentrations(5 %, 8 % and 10 %)of PVP in the sperm injection vehicle solution were compared. There was no effect of the three PVP concentrations on cleavage rate after sperm injection, but regular cleavage rate and blastocyst rate were descended with increasing concentration of PVP, regular cleavage rat(e78.4 %, 69.1 %)and blastocyst rate(29.7 %, 20.4 %)were significantly higher with 5 % than 10 % PVP(P < 0.05).In experiment 4, two positions(6 and 12 o’clock)of the first polar body(PB)located in oocyte at ICSI were examined. There was no significant difference in the cleavage rate(P > 0.05)between the two positions of the PB , but regular cleavage rate(76.4 %, 66.7 %)and blastocyst rate(28.6 %, 19.0 %)were significantly improved at 6 o’clock of the polar body(P < 0.05).The results showed that a preliminary procedure for ICSI in dairy cow was established. At the same time, they indicated that the motile sperm be used in ICSI and the sperm was treated in the manipulation medium with 5 % PVP, and the oocytes should be injected with the polar body at 6 o’clock, can improve the in vitro development of the ICSI zygotes in dairy cow.