昆白系小鼠体外受精
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摘要
体外受精在医学、生物学等相关领域已得到广泛的应用,但是体外受精及受精卵体外培养体系还有待于进一步完善。昆白小鼠是我国自行繁殖的近交系小鼠,由于其遗传背景清楚、生殖周期短、环境适应能力强等优点,是研究哺乳动物体外受精的良好的实验动物。为了建立更为简单、高效的体外受精及胚胎培养体系,提高体外受精率和体外发育的胚胎质量。本研究以昆白小鼠为研究对象,从胚胎培养液的筛选、肝素对精子活力的影响、不同获能液对受精率的影响、不同培养液对胚胎发育的影响、不同培养体系对胚胎发育的影响等,5个方面对昆白小鼠体外受精进行了研究。结果表明:
1.添加10%NBS的KSOM和G1/G2培养液均能较好支持小鼠2-细胞胚胎发育到囊胚(89.52%和87.08%)。同时,添加10%FBS组的囊胚率也显著高于相应的添加BSA组(P<0.01)。
2.筛选小鼠精子获能液中的肝素的最佳添加量。未添加肝素组受精率(41.75%)高于添加1μg/mL、3μg/mL及5μg/mL组(31.00%、18.80%、22.43%),差异显著。
3.原装进口精子获能液HTF(MR-070)受精率(85.71%)显著高于自配的三种体外受精HTF、T6、Whittingham的体外受精率(38.58%、10.22%、24.64%)。受精卵培养24h观察,HTF、T6、Whittingham中碎裂率(23.62%、20.44%、32.61%),显著高于原装HTF(3.57%)。
4. G1/G2中培养的体外受精胚胎囊胚率达到77.50%,明显高于原装KSOM和自配KSOM组(22.78%、0)。
5.与非共培养体系相比,颗粒细胞共培养,能够促进昆白小鼠胚胎克服2-细胞阻滞,显著提高小鼠体外受精胚胎的4-细胞(37.97%、59.72%)、桑椹胚(27.85%、48.61%)及囊胚率(22.70%、45.83%)。
In vitro fertilization and the culture of fertilized eggs have been widely used in the field of medicine and biology, but still need further optimizing. Kunming mouse(KM), bred in China by near inbred, has been an ideal laboratory animal model because of the well-known inheriting background, short reproduction cycle and the strong environmental adapt ability. The purpose of this study is to establish optimal, effective system for in vitro fertilization and embryo culture and improve the quality of embryo. In these experiments, we studied the in vitro fertilization of KM mouse from five aspects of screening the optimum embryos culture medium, effecting of heparin on sperm, the effect of different capacitation solution on rate of vitro fertilization, the effect of different culture media on the development of Kunming mouse embryos, the effect of different culture systems on the development of Kunming mouse embryos. And the result showed that:
1. G1/G2 and KSOM medium containing 10%NBS could support 2-cell embryos to development blastocyst (87.08% and 89.52%).The development rates of blastocyst in KSOM containing 10%FBS was significantly higher than that in the media containing BSA, the difference were extreme(P<0.01).
2. To screen the optimum heparin concentrations in the capacitation solution HTF of mouse. The rate of fertilization in vitro in the media without heparin (41.75%)was significantly higher(P<0.01) than those with heparin at 1μg/mL (31.00%)、3μg/mL(18.80%)、5μg/mL(22.43%).
3. The rate of vitro fertilization of HTF (MR-070,85.71%) which was foreign imported reagent was significant higher than those of HTF、T6 and Whittingham (38.58%、10.22% and 24.64%) self-prepared. The fracture rate of fertilized eggs in HTF was lower (3.57%) than those three other self-made media (23.62%、20.44%、32.61%).
4. The rate of blastocyst in G1/G2 was 77.50%, which was higher than imported KSOM(22.78%)、and self-made KSOM (0).
5. Granulosa cell co-cultured with mouse embryos could support IVF embryos to overcome 2-cell block, and the rates of 4-cell(37.97% vs 59.72%)、morula(27.85% vs 48.61%)、blastocyst (22.70%vs45.83%)could be all improved significantly than non-culture system.
1.添加10%NBS的KSOM和G1/G2培养液均能较好支持小鼠2-细胞胚胎发育到囊胚(89.52%和87.08%)。同时,添加10%FBS组的囊胚率也显著高于相应的添加BSA组(P<0.01)。
2.筛选小鼠精子获能液中的肝素的最佳添加量。未添加肝素组受精率(41.75%)高于添加1μg/mL、3μg/mL及5μg/mL组(31.00%、18.80%、22.43%),差异显著。
3.原装进口精子获能液HTF(MR-070)受精率(85.71%)显著高于自配的三种体外受精HTF、T6、Whittingham的体外受精率(38.58%、10.22%、24.64%)。受精卵培养24h观察,HTF、T6、Whittingham中碎裂率(23.62%、20.44%、32.61%),显著高于原装HTF(3.57%)。
4. G1/G2中培养的体外受精胚胎囊胚率达到77.50%,明显高于原装KSOM和自配KSOM组(22.78%、0)。
5.与非共培养体系相比,颗粒细胞共培养,能够促进昆白小鼠胚胎克服2-细胞阻滞,显著提高小鼠体外受精胚胎的4-细胞(37.97%、59.72%)、桑椹胚(27.85%、48.61%)及囊胚率(22.70%、45.83%)。
In vitro fertilization and the culture of fertilized eggs have been widely used in the field of medicine and biology, but still need further optimizing. Kunming mouse(KM), bred in China by near inbred, has been an ideal laboratory animal model because of the well-known inheriting background, short reproduction cycle and the strong environmental adapt ability. The purpose of this study is to establish optimal, effective system for in vitro fertilization and embryo culture and improve the quality of embryo. In these experiments, we studied the in vitro fertilization of KM mouse from five aspects of screening the optimum embryos culture medium, effecting of heparin on sperm, the effect of different capacitation solution on rate of vitro fertilization, the effect of different culture media on the development of Kunming mouse embryos, the effect of different culture systems on the development of Kunming mouse embryos. And the result showed that:
1. G1/G2 and KSOM medium containing 10%NBS could support 2-cell embryos to development blastocyst (87.08% and 89.52%).The development rates of blastocyst in KSOM containing 10%FBS was significantly higher than that in the media containing BSA, the difference were extreme(P<0.01).
2. To screen the optimum heparin concentrations in the capacitation solution HTF of mouse. The rate of fertilization in vitro in the media without heparin (41.75%)was significantly higher(P<0.01) than those with heparin at 1μg/mL (31.00%)、3μg/mL(18.80%)、5μg/mL(22.43%).
3. The rate of vitro fertilization of HTF (MR-070,85.71%) which was foreign imported reagent was significant higher than those of HTF、T6 and Whittingham (38.58%、10.22% and 24.64%) self-prepared. The fracture rate of fertilized eggs in HTF was lower (3.57%) than those three other self-made media (23.62%、20.44%、32.61%).
4. The rate of blastocyst in G1/G2 was 77.50%, which was higher than imported KSOM(22.78%)、and self-made KSOM (0).
5. Granulosa cell co-cultured with mouse embryos could support IVF embryos to overcome 2-cell block, and the rates of 4-cell(37.97% vs 59.72%)、morula(27.85% vs 48.61%)、blastocyst (22.70%vs45.83%)could be all improved significantly than non-culture system.
引文
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