小鼠不同杂交组合胚胎玻璃化冷冻的研究
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摘要
本文通过小鼠胚胎玻璃化冷冻的研究，确定对胚胎无毒性或毒性小的冷冻保护剂的种类、适宜浓度及适宜冷冻的胚胎发育阶段，以便达到提高胚胎冷冻存活率的目的。
1．小鼠不同杂交组合分为ICR♀×昆明♂、昆明♀×昆明♂、ICR♀×ICR♂、昆明♀×ICR♂组，受精率分别为76.0％、80.9％、72.4％及66.7％，平均排卵数为17.6、13.4、11.3及8.1枚。经统计分析，受精率在不同杂交组合之间差异不显著；超排效果ICR♀×昆明♂组显著高于ICR♀×ICR♂、昆明♀×ICR♂组(P＜0.05)，其它各组之间差异不显著。
2．用海藻糖和蔗糖处理的小鼠早期囊胚存活率分别为79.06％和77.07％；桑椹胚分别为46.31％和51.63％。经统计分析，小鼠早期囊胚存活率海藻糖组显著高于蔗糖组(P＜0.05)；桑椹胚存活率两组之间差异不显著。
3．小鼠桑椹胚和早期囊胚用0.5M海藻糖处理后存活率分别为61.84％和95.57％；用0.25M海藻糖处理后存活率分别为35.50％和67.22％。经统计分析，桑椹胚和早期囊胚在0.5M海藻糖溶液中的存活率均显著高于0.25M组(P＜0.05)。
4．0.5M海藻糖溶液做基础液，分别用0.0M(对照组)、2.0M、2.5M、3.0M、及3.5M甘油处理小鼠桑椹胚和早期囊胚，桑椹胚存活率分别为57.25％、56.43％、63.68％、60.48％及70.79％；早期囊胚存活率分别为96.98％、94.77％、98.15％、92.44％及96.10％。经统计分析，对照组与不同浓度甘油组之间小鼠桑椹胚和早期囊胚的存活率均差异不显著。
5、小鼠桑椹胚和早期囊胚用2.5M甘油处理存活率分别为73.7％和98.2％。经统计分析，早期囊胚的存活率显著高于桑椹胚(P＜0.05)。
6．不同冷冻方法对小鼠胚胎冷冻效果的影响实验中，缓慢冷冻法与玻璃化冷冻法之间解冻后回收正常胚数、培养12小时的囊胚存活率均差异不显著。
7．不同浓度乙二醇(EG)对小鼠胚胎冷冻效果的影响实验中，解冻回收正常胚、经培养12小时、24小时、36小时及48小时的囊胚存活率和囊胚孵化率，除对照组(不含EG)培养12小时的囊胚存活率显著高于10％EG组(P＜0.05)外，对照组与10％EG和20％EG组之间均差异不显著，但对照组的解冻回收正常胚、经
小鼠不同杂交组合胚胎玻璃化冷冻的研究一中文摘要
培养12小时、24小时、36小时及48小时的囊胚存活率和囊胚孵化率均显著高
于30%EG组(P<0 .05)。
8.小鼠不同杂交组合对胚胎玻璃化冷冻效果的影响实验中，昆明早x昆明
占、昆明早x工cR占、IcR早x昆明占、IcR早x工cR占组合间的解冻后正常胚胎数
差异不显著;培养12小时的囊胚发育率昆明早x工cR占组显著高于工cR早只昆明
古组(P<。.05)，其他组之间差异不显著;培养24小时、36小时的囊胚发育率
昆明早x昆明占、昆明早X工cR占、工cR早X工cR占组均显著高于工cR早x昆明占
组(P<0 .05);昆明早x昆明占、昆明早X工cR占、IcR早X IcR古组之间差异不显
著;培养48小时的囊胚发育率和囊胚孵化率四个杂交组合之间差异不显著。
These experiments were carried out to clarify the kinds, the best concentration of the cryoprotectant which has no or little toxicity to embryos as well as suitable stage of the embryos' development by studying on the toxicity experiments of the mouse embryos, so that achieve the aim to improve viability of the frozen embryos.
1. According to the crossbreed, the mice were divided into four groups-ICR Kunming , Kunming Kunming , ICR ICR and Kunming ICR . The oocytes of ovulation were 17.6, 13.4, 11.3 and 8.1 respectively. The effect of superovulation for the group of ICR Kunming was significantly higher than those of the groups of ICR ICR and Kunming ICR ( P<0.05), and the differences between other groups were not significant.
2. The survival rates of early blastocyst treated with trehalose and sucrose, were 79.1%, and.77.1%, respectively; and the survival rates of morula were 46.3% , and 51.6%, respectively. The survival rate of blastocyst treated with trehalose were significantly higher than that of those treated with sucrose (P<0.05); while there was no significant difference between two groups in the survival rate of morula.
3. The survival rates of mice early blastocyst and morula treated with 0.5M trehalose were 61.8% and 95.6% respectively; and treated with 0.25M trehalose were 35.5% and 67.2% respectively. The survival rates of both early blastocyst and morula treated with 0.5M trehalose were significantly higher than that of 0.25M trehalose treated ( P<0.05) .
4.The survival rates of mouse morula for 0.5M trehalose added with O.OM, 2.0M, 2.5M, 3.0M and 3.5M glycerol were 51.3%,56.4%,63.7%,60.5%, and 70.8% respectively; the survival rates of blastocyst treated using the
same method were 97.09, 94.8% , 98.2%, 92.4% , and 96.1% respectively. There was no significant difference between control and treatments for morula and early blastocyst treated with different glycerol concentration.
5. The survival rates of the early blastocyst and morula treated with 2.5M glycerol were 98.2% and 73.7% respectively. The survival rate of early blastocysts was significantly higher than that of the morula (P<0.05).
6. In the experiment of researching the frozen effects on mouse embryos treated with different freezing methods, there were no significant difference in the number of normal embryos retrieved after thawing and the survival rate of embryos cultured 12 hours.
7. In the experiment of freezing the mouse embryos treated with different concentration of ethylene glycol (EG), the number of normal embryos, the survival rates of blastocysts cultured 12 hours, 24 hours, 36 hours, and 48 hours and the hatching rates of the blastulas were compared. Except that the survivals rates of the contrast group and the cultured 12 hours group were significantly higher than those of the group with 10% EG(P<0.05), there were no significant difference between the contrast group and 10% group, 20% group in other aspects, but the contrast group was significantly higher than that of 30% group in every aspects.
8. In the experiment of researching vitrification effect on mouse embryos crossed differently, there were no significant difference for the number of normal embryos thawed among Kunming Kunming
, Kunming ICR , ICR Kunming and ICR ICR ; the blastocyst development rate of the embryo cultured 12 hours for the group of Kunming ICR was significantly higher than that of the group of ICR Kunming(p<0.05), and there were no significant difference between other groups; the blastocyst development rates of the embryos cultured for 24 hours and 36 hours
of the group of ICR Kunming were significantly higher than that of the other three groups(P<0.05); There were no significant difference among Kunming Kunming , Kunming ICR , ICR ICR ; the blastocyst development rate and hatching rate of the embryo cultured for 48 hours among four crossbred groups did not differ significantly.
1．小鼠不同杂交组合分为ICR♀×昆明♂、昆明♀×昆明♂、ICR♀×ICR♂、昆明♀×ICR♂组，受精率分别为76.0％、80.9％、72.4％及66.7％，平均排卵数为17.6、13.4、11.3及8.1枚。经统计分析，受精率在不同杂交组合之间差异不显著；超排效果ICR♀×昆明♂组显著高于ICR♀×ICR♂、昆明♀×ICR♂组(P＜0.05)，其它各组之间差异不显著。
2．用海藻糖和蔗糖处理的小鼠早期囊胚存活率分别为79.06％和77.07％；桑椹胚分别为46.31％和51.63％。经统计分析，小鼠早期囊胚存活率海藻糖组显著高于蔗糖组(P＜0.05)；桑椹胚存活率两组之间差异不显著。
3．小鼠桑椹胚和早期囊胚用0.5M海藻糖处理后存活率分别为61.84％和95.57％；用0.25M海藻糖处理后存活率分别为35.50％和67.22％。经统计分析，桑椹胚和早期囊胚在0.5M海藻糖溶液中的存活率均显著高于0.25M组(P＜0.05)。
4．0.5M海藻糖溶液做基础液，分别用0.0M(对照组)、2.0M、2.5M、3.0M、及3.5M甘油处理小鼠桑椹胚和早期囊胚，桑椹胚存活率分别为57.25％、56.43％、63.68％、60.48％及70.79％；早期囊胚存活率分别为96.98％、94.77％、98.15％、92.44％及96.10％。经统计分析，对照组与不同浓度甘油组之间小鼠桑椹胚和早期囊胚的存活率均差异不显著。
5、小鼠桑椹胚和早期囊胚用2.5M甘油处理存活率分别为73.7％和98.2％。经统计分析，早期囊胚的存活率显著高于桑椹胚(P＜0.05)。
6．不同冷冻方法对小鼠胚胎冷冻效果的影响实验中，缓慢冷冻法与玻璃化冷冻法之间解冻后回收正常胚数、培养12小时的囊胚存活率均差异不显著。
7．不同浓度乙二醇(EG)对小鼠胚胎冷冻效果的影响实验中，解冻回收正常胚、经培养12小时、24小时、36小时及48小时的囊胚存活率和囊胚孵化率，除对照组(不含EG)培养12小时的囊胚存活率显著高于10％EG组(P＜0.05)外，对照组与10％EG和20％EG组之间均差异不显著，但对照组的解冻回收正常胚、经
小鼠不同杂交组合胚胎玻璃化冷冻的研究一中文摘要
培养12小时、24小时、36小时及48小时的囊胚存活率和囊胚孵化率均显著高
于30%EG组(P<0 .05)。
8.小鼠不同杂交组合对胚胎玻璃化冷冻效果的影响实验中，昆明早x昆明
占、昆明早x工cR占、IcR早x昆明占、IcR早x工cR占组合间的解冻后正常胚胎数
差异不显著;培养12小时的囊胚发育率昆明早x工cR占组显著高于工cR早只昆明
古组(P<。.05)，其他组之间差异不显著;培养24小时、36小时的囊胚发育率
昆明早x昆明占、昆明早X工cR占、工cR早X工cR占组均显著高于工cR早x昆明占
组(P<0 .05);昆明早x昆明占、昆明早X工cR占、IcR早X IcR古组之间差异不显
著;培养48小时的囊胚发育率和囊胚孵化率四个杂交组合之间差异不显著。
These experiments were carried out to clarify the kinds, the best concentration of the cryoprotectant which has no or little toxicity to embryos as well as suitable stage of the embryos' development by studying on the toxicity experiments of the mouse embryos, so that achieve the aim to improve viability of the frozen embryos.
1. According to the crossbreed, the mice were divided into four groups-ICR Kunming , Kunming Kunming , ICR ICR and Kunming ICR . The oocytes of ovulation were 17.6, 13.4, 11.3 and 8.1 respectively. The effect of superovulation for the group of ICR Kunming was significantly higher than those of the groups of ICR ICR and Kunming ICR ( P<0.05), and the differences between other groups were not significant.
2. The survival rates of early blastocyst treated with trehalose and sucrose, were 79.1%, and.77.1%, respectively; and the survival rates of morula were 46.3% , and 51.6%, respectively. The survival rate of blastocyst treated with trehalose were significantly higher than that of those treated with sucrose (P<0.05); while there was no significant difference between two groups in the survival rate of morula.
3. The survival rates of mice early blastocyst and morula treated with 0.5M trehalose were 61.8% and 95.6% respectively; and treated with 0.25M trehalose were 35.5% and 67.2% respectively. The survival rates of both early blastocyst and morula treated with 0.5M trehalose were significantly higher than that of 0.25M trehalose treated ( P<0.05) .
4.The survival rates of mouse morula for 0.5M trehalose added with O.OM, 2.0M, 2.5M, 3.0M and 3.5M glycerol were 51.3%,56.4%,63.7%,60.5%, and 70.8% respectively; the survival rates of blastocyst treated using the
same method were 97.09, 94.8% , 98.2%, 92.4% , and 96.1% respectively. There was no significant difference between control and treatments for morula and early blastocyst treated with different glycerol concentration.
5. The survival rates of the early blastocyst and morula treated with 2.5M glycerol were 98.2% and 73.7% respectively. The survival rate of early blastocysts was significantly higher than that of the morula (P<0.05).
6. In the experiment of researching the frozen effects on mouse embryos treated with different freezing methods, there were no significant difference in the number of normal embryos retrieved after thawing and the survival rate of embryos cultured 12 hours.
7. In the experiment of freezing the mouse embryos treated with different concentration of ethylene glycol (EG), the number of normal embryos, the survival rates of blastocysts cultured 12 hours, 24 hours, 36 hours, and 48 hours and the hatching rates of the blastulas were compared. Except that the survivals rates of the contrast group and the cultured 12 hours group were significantly higher than those of the group with 10% EG(P<0.05), there were no significant difference between the contrast group and 10% group, 20% group in other aspects, but the contrast group was significantly higher than that of 30% group in every aspects.
8. In the experiment of researching vitrification effect on mouse embryos crossed differently, there were no significant difference for the number of normal embryos thawed among Kunming Kunming
, Kunming ICR , ICR Kunming and ICR ICR ; the blastocyst development rate of the embryo cultured 12 hours for the group of Kunming ICR was significantly higher than that of the group of ICR Kunming(p<0.05), and there were no significant difference between other groups; the blastocyst development rates of the embryos cultured for 24 hours and 36 hours
of the group of ICR Kunming were significantly higher than that of the other three groups(P<0.05); There were no significant difference among Kunming Kunming , Kunming ICR , ICR ICR ; the blastocyst development rate and hatching rate of the embryo cultured for 48 hours among four crossbred groups did not differ significantly.
引文
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