【作者】 孙玉成；

【导师】 高建明； 王子荣；

【作者基本信息】 新疆农业大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 本研究主要探讨mCZB分别添加黄芩苷(Baicalin,Bai)、川芎嗪(Ligustrazine,Lig)、小檗碱(Berberine,BR)对小鼠2-细胞胚胎体外发育抗氧化作用的影响,并比较各组胚胎冷冻解冻效果及冷冻胚胎移植妊娠率和产仔率等,以进一步探讨利用三种中药有效成分改善小鼠早期胚胎体外培养微环境、提高体外培养胚胎质量和移植胚胎的发育潜力,为更好地改进哺乳动物胚胎培养液成分和培养条件提供可借鉴的资料和途径。试验一:以mCZB添加抗生素为对照组,分别添加4μg/mlBai、0.5μg/mlLig和0.1μg/mlBR为试验组,比较各组胚胎孵化率、微滴中一氧化氮(Nitric oxide,NO)和丙二醛(malondialdehyde,MDA)含量及孵化胚胎的细胞数量。结果:各试验组胚胎孵化率,Bai组(80.2%,81.3%)、Lig组(78.9%,77.6%)及BR组(76.7%,71.6%)极显著高于对照组(50.7%,47.8%)(P<0.01)。各组间NO含量,72 h较24 h均有减少,而120 h又均有增加且最高。各组间MDA含量,72 h和120 h与24 h比较呈现增加趋势,除120hBai组,对照组均高于试验组。孵化胚胎细胞计数结果显示,BR组、Lig组和Bai组(87.2±8.6,83.9±7.7,81.9±6.2)与对照组(77.4±5.6)差异极显著(P<0.01)。表明,三种中药有效成分均能显著促进小鼠早期胚胎体外发育及胚胎细胞增殖,并对NO和MDA含量有一定影响。试验二:以试验一为基础,2-细胞胚胎体外培养至桑椹、囊胚期进行常规冷冻,比较各组胚胎冷冻解冻效果及两种程序对胚胎冷冻解冻的影响。结果:冷冻解冻桑椹胚体外培养8～14 h囊胚发育率,程序一中Bai组(65.8%)显著高于对照组(48.6%)和BR组(46.7%)(P<0.05),Lig组(81.1%)极显著高于对照组(48.6%)和BR组(46.7%)(P<0.01);程序二中Bai组(80.6%)、Lig组(78.3%)极显著高于对照组(55.2%)和BR组(47.6%)(P<0.01)。冷冻解冻囊胚体外培养6～8 h囊胚发育率,程序一中各组间无显著差异(P>0.05);程序二中,中药有效成分各组间差异不显著(P>0.05),但均显著高于对照组(61.2%)(P<0.05)。两种冷冻程序冷冻效果差异不显著(P>0.05)。表明,添加Bai、Lig对提高胚胎体外发育质量效果明显,显著提高了冷冻胚胎发育率。试验三:以试验二为基础,将各组冷冻解冻胚胎培养发育至囊胚期进行子宫移植。结果:移植妊娠率以Bai组(56.3%)、Lig组(52.6%)和BR组(55.0%)均高于对照组(46.2%);产仔率以Bai组(63.3%)、Lig组(67.3%)和BR组(60.5%),均高于对照组(52.8%),但差异不显著(P>0.05)。综上结果表明:三种中药有效成份Bai、Lig、BR能显著提高小鼠2-细胞胚胎体外发育质量,并有利于提高冷冻解冻后移植胚胎的发育潜力。更多还原

【Abstract】 This study was mainly approached the antioxidation effect of adding Baicalin(Bai), Ligustrazine(Lig)and Berberine(BR)in mCZB respectively on 2-cell mouse embryos in vitro culture ,and comparedthe effect of froze-thaw, pregnancy rate and litter rate after embryos transfer etc. in order to furtherinvestigate the value of these three Chinese Medicine effective constituents on improving themicro-environment of preimplantation embryos in vitro culture on mouse and enhancing the embryoquality and the developmental potential of embryos after embryos transfer , which can provide valuabledata and access to enhance the mammalian embryo culture medium composition and culture condition.The group added antibiotics to mCZB was regarded as the control, while the others supplemented with0.1μg/ml BR, 0.5μg/ml Lig and 4μg/ml Bai respectively as experiment groups in the first experiment tocompare the rates of hatched embryos, the contents of nitric oxide(NO) and malondialdehyde(MDA) in themicrodrop, and the cell amount of hatched embryos. As a result, the rates of hatched embryos from thethree groups of Bai ( 80.2% , 81.3% ), Lig (78.9%,77.6% )and BR (76.7% ,71.6%) was significantly higher(P<0.01) than control group (50.7%、47.8%) . The content of NO decreased at 72h as compared with that of24h in every experiment group, but it increased to the highest point at 120h. As a whole, raise trend wasevident for the content of MDA in every group at 72h and 120h, and the content of MDA in the control washigher than that in experiment groups at 120h except for Bai group. According to the cell amount ofhatched embryos, the difference between experiment groups(BR(87.2±8.6), Lig(83.9±7.7) ,Bai(81.9±6.2))and control group(77.4±5.6)were extremely obvious (P<0.01). The result showed that these three kinds ofChinese Medicine effective constituents could efficiently promote early embryos development and increasethe cell amount of hatched embryo, and the contents of NO and MDA were affected as well in vitro cultureon mouse.Based on the first experiment, the second one in which each group were conserved throughconventional freezing process from 2-cell embryo in vitro culture to the stage of morula and blastocyst wasoriented to compare the effects of froze-thaw in each group and the difference between the two methods.The results of morula and blastocyst showed that in the first procedure the development rate of blastocystcultured for 8～14 hours after froze-thaw in the Bai(65.8%)was superior(P<0.05)to that of the control(48.6%)and the BR(46.7%), and the Lig(81.1%)was significantly higher (P<0.01) than that ofthe control and the BR(46.7%). In the second procedure, the Ba(i80.6%)was significantly higher (P<0.01)than the control(55.2%)and the BR(47.6%)in vitro culture. Through analysis of the development rate ofblastocyst cultured for 6～8 hours in the first procedure, there was no significant difference(P>0.05)ineach group. In the second procedure, there was no significant difference(P>0.05)in each group, but theeffect of Chinese Medicine was superior(P<0.05)to that of control group(61.2%)in vitro culture. Theresults indicated there was no significant difference(P>0.05)in the different procedures. Bai and Lig cansignificantly enhance the development quality of embryos and the development rate of freezing embryos invitro culture.Based on Experiment two, froze-thaw embryos were cultured to the stage of blastula and thentransferred to uterus in experiment three. As a result, the pregnancy rate from the three groups of Bai(56.3% ),Lig (52.6% )and BR (55.0%) was higher than that of control group (46.2%),but there was notobvious difference between experiment groups and control group;the birth rate from the three groups ofBai (63.3% ), Lig(67.3% )and BR (60.5%) was higher than control group (52.8%),but there was notsignificant difference either.These results indicated the three Chinese Medicine effective constituents(Bai, Lig , BR)can distinctlyimprove the quality of 2-cell mouse embryo in vitro culture and be propitious to the developmentalpotential of embryos following embryos transfer after froze-thaw embryos.更多还原