混合保护液和冻前处理方法对小鼠胚胎一步冷冻效果的影响

Effects of Cryoprotectant Mixtures and prefreezing Treatment Methods on One-step Cryoprotective Effective-ness of Mouse Embryos

选用Ａ～Ｅ５种混合保护液：Ａ。二甲亚砜（ＤＭＳＯ）＋乙二醇（ＥＧ），Ｂ。甘油（ＧＬ）＋丙二醇（ＰＧ），ＯＧＬ＋ＥＧ，Ｄ。ＧＬ＋ＤＭＳＯ，Ｅ．ＰＧ＋ＥＦ，以２种冻前处理方法（Ⅰ法和Ⅱ法），对小鼠桑椹胚和囊胚进行一步冷冻，冷冻混合保护液（ＶＳ２）中各种保护剂浓度均为２５％。Ⅰ法的冻前平衡液（ＶＳ１Ⅰ）为ＶＳ２的对倍稀释液；Ⅱ法的冻前平衡液（ＶＳ１Ⅱ）中，前一种保护剂浓度为１０％，后一种保护剂浓度为２０％。胚胎于室温（２０℃）下在ＶＳ１Ⅰ中平衡５ｍｉｎ或ＶＳ１Ⅱ中平衡１０ｍｉｎ后，移入已在细管内经４℃预冷的ＶＳ２中５ｍｉｎ（Ⅰ法）或１０ｍｉｎ（Ⅱ法）。细管热封后直接投入液氮保存。经２０℃水浴解冻，０．５ｍｏｌ／Ｌ蔗糖ＰＢＳ脱除保护剂后进行荧光检查评价冻胚活力。结果，Ａ液和Ｅ液无论以Ⅰ法或Ⅱ法冻前处理，对桑椹胚和囊胚均表现出较好且稳定的冷冻保护效果。桑椹胚和囊胚的冻后存活率分别为ＡⅠ（即Ａ液以Ⅰ法，以此类推）９１．２％和８７．５％，ＡⅡ８６．７％和８９．２％，ＥⅠ７８．９％和７５．０／，ＥⅡ８３．３％和７９．３％。选择经ＡⅠ和ＥⅡ冷冻的桑椹胚和囊胚进行移植，其体内发育率分别为ＡⅠ１０．９％和９．７／；ＥⅡ８．３％和?

Five types of mixttire solutions were used for one-step freezing of mouseembrvo in two different wavs (method I and method Ⅱ).They each (VS2)were com-posed of two types of Cryoprotectants, namely, sohtion A : dimethyl sLilfoxide(DMSO)+ethylene glycol(EG),B:glycerol(GL)+propylene glycol(PG), C:GL+EG,D:GL+DMSO and E:PG+EG.The concentrations of all these ingredientswere 25%, The pre- freezing equilibrated soltition for method I(VS1I)was the 50%diluted solution of VS2 with 5%PBS and for method Ⅱ(VS1 Ⅱ),it was consisted of10%of the first cryoprotectant and 20% of the second in PBS. After having been ex-posed to VS1 Ⅰfor 5 minutes or 10 minutes to VS1 Ⅱ at room temperature(20℃),em-bryos were loaded into straws containing VS2 which were precooled at 4℃ for 5 minutes(method Ⅰ)or 10 minutes(method Ⅱ),then the straws were heat-sealed and droppedinto liqtid nitrogen directly.After thawing in water bath at 20℃ the cryoprotectantswere washed away with 0.5 μmol/L sucrose soltition,the embryos were examined underthe fltiorescence microscoue for their viabilities. The results indicated that the cryopro-tective effectiveness of morulae and blastocystS in solutiton A and E in both methods wasgood and stable. The survival rates of morulae aid blastocysts cryopreserved were 91.2% and 87.5% in A Ⅰ(i.e. solution A in methodⅠ); 86.7%and 89.2%in A Ⅱ;78.9%and 75.0%in E Ⅰ;83.3%and 79.3%in EⅡ,respectively.AⅠ and E6Ⅱwereselected for cryopreserving morulae and blastocysts which were transferred into recipi-ents。 The in vivo developmental rates of morulae and blastocysts cryopreserved in A Ⅰwere l0.9%and 9.7%and in E Ⅱ8.3%and 8.6%,respectively.