【作者】 杨兴东；

【导师】 刘长松；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2006， 硕士

【摘要】 本研究采用程序冷冻、直接冷冻、玻璃化冷冻和开放式拉管法(OPS)法对小鼠桑葚胚和早期囊胚进行了冷冻保存，探讨了不同冷冻方法对胚胎存活率的影响，并从冷冻保护剂、植冰温度等不同角度加以研究，以胚胎冷冻后的体外培养和移植来鉴定其冷冻效果。 结果表明：1．将6～8周龄的昆明种雌鼠15只随机分为三组，分别用PMSG和HCG以不同的处理剂量(5IU，10IU，15IU)进行超排处理，每只平均获得胚胎数分别为16.2、23.2和19.0枚，正常胚数分别为12.6、18.8和13.4枚，正常胚的比率分别为：77.78％、81.03％和70.53％，均达到了超排效果。结果表明10IU的处理剂量的超排效果较好(p＞0.05)。2．将超排获得的桑葚胚和早期囊胚随机分为(1)20％乙二醇(EG)+20％DMSO处理30秒，再经0.3M蔗糖处理1 min；(2)20％甘油(Gly)+20％1，2—丙二醇(1，2-Pro)处理30秒，再经0.3M蔗糖(Suc)处理1min；(3)经0.3M蔗糖处理(解冻液处理组)1 min；(4)不经任何冷冻液和解冻液处理(对照组)四组。分别24h培养表明：扩张囊胚率为91.23％、80.65％、89.58％和90.74％；48h后的孵化率为64.91％、51.61％、83.33％和85.19％。(1)和(2)组间差异不显著，二者与(3)和(4)相比，差异均显著(p＜0.01)。3．分别用F1、F2和F3三种冷冻保护剂，行常规程序冷冻方法冷冻，解冻后培养结果表明：扩张囊胚率分别为78.18％、73.58％和57.14％；孵化囊胚率为45.45％、41.51％和26.53％，F1和F2显著高于F3(p＜0.05)。4．将获得的桑葚胚和早期囊胚放入冷冻液F1中平衡5min，冷冻室内停留2min后植冰，再行程序冷冻。证明：-5.5℃、-6.0℃、-6.5℃植冰时，胚胎解冻后体外囊胚孵化率分别为40.38％、45.45％、12.50％，-5.5℃和-6.0℃组的显著高于-6.5℃组(p＜0.01)。5．分别用程序冷冻、常规玻璃化冷冻、OPS、直接冷冻法对小鼠桑葚胚进行冷冻保存。解冻后存活率分别为78.18％、70.37％、81.36％和44.92％；48h后的孵化率分别为45.45％、44.44％、50.85％和27.54％。前三种方法组间差异不显著，但直接冷冻法和其他各组相比差异极显著(p＜0.01)。6．将上述四种方法冷冻保存的83、68、75和71枚胚胎解冻后分别移植给8、6、6和6只受体，平均每只10～13枚，结果表明：妊娠率为12.5％(1/8)、16.67％(1/6)、16.67％(1/6)和16.67％(1/6)。更多还原

【Abstract】 The present research adopted four methods to cryopreserve the mouse morula and early blastocysts. They were programming freezing, quick cooling, vitrification and OPS. Probed into the effect of different Cryo-preservation on the development of mouse embryos, studied from the cryoptectant and seeding temperature etc. In vitro culture and the embryos surgical transfer were used to appraise the effect of mouse embryos cryopreservation.The results showed: 1. The experiments were divided into 3 groups according to three different dosages (5 IU, 10 IU andl5 IU). For the dosage of PMSG treatment, 10IU injection was better than both 5 IU and 10 IU groups(p<0.05).2. The super-ovulated embryos were exposed in four operation solutions as follows:(1) (20%Gly+20%1, 2-Pro) X 30sec.+0.3M Suc. X 1min; (2) (20%EG +20%DMS0) X 30sec. +0.3 M Suc. X 1min; (3) 0.3M Suc. X 1min; (4) Control (no treatment). The survival rates and hatching rates of groups (1) and (2) werelower than that of groups (3) and (4) (P<0.01). 3. Adopted the programming freezing with three cryoprotectant:F1,F2 and F3. percentage of hatching embryos of F1 and F2 were45. 45%, 41. 51%. they both had significantly higher developmental abilities than F3(26. 53%). 4. Mouse embryos were exposed to the cryoprotectant F1 and seeded at -5.5℃ and -6.0℃ followed lauching into liquid nitrogen at -35℃,after thawing and in vitro culture, percentage of hatching embryos were40.38%, 45. 45%. they both had higher developmental更多还原