家兔超数排卵和体外受精技术的研究
摘要
本文主要以41只雌性新西兰白兔为实验素材,采用FSH+hCG激素组合进行超排处理,比较了FSH不同总剂量,季节,以及递减法与恒量法肌注对超排效果的影响,并分析了不同获能液,培养液,培养液中L-谷氨酰胺添加量及卵龄对兔卵母细胞体外受精和受精卵体外发育的影响。同时研究了卵龄对显微授精的影响,比较了显微授精与常规体外受精效果,结果如下:
1.不同FSH总剂量超排结果显示,60单位与100单位试验组平均排卵点(分别为36.13±8.13个/只和39.00±9.17个/只),平均回收卵子数(分别为28.88±6.71枚/只和30.67±7.71枚/只)均显著高于40单位试验组(分别为25.83±4.07个/只和19.50±4.42枚/只,P﹤0.05)。但三者卵子平均回收率(分别为75.48%,79.93%和78.63%)之间差异均不显著(P﹥0.05)。
2.春季无论平均排卵点(39.00±9.17个/只),平均回收卵子数(30.67±7.71枚/只)还是卵子平均回收率(78.63%)均显著高于秋季(21.58±7.10个/只,15.17±4.97枚/只,70.27%,P﹤0.05)。
3.用恒量法和递减法给母兔注射相同总剂量的FSH,两者之间超排结果差异不显著(P﹥0.05)。
4. DM-His法的8-16细胞百分率(13.3%)显著高于DM法与His法(分别为3.4%和3.3%,P﹤0.05)。DM法,His法及DM-His法三种获能方法总的卵裂率(分别为51.7%,38.3%和41.3%)之间差异不显著(P﹥0.05),但以DM法效果好些。
5.卵龄14-15h的卵母细胞体外受精后卵裂率(41.3%)和8-16细胞百分率(13.3%)均显著高于卵龄16-17h卵母细胞(分别为25.0%和2.9%,P﹤0.05)。
6.在培养液TCM-199 + 10%FBS中添加L-谷氨酰胺对卵母细胞体外受精及受精卵体外发育具有促进作用,但随着添加量的增加作用减弱,以0.5mmol/L添加量效果最好。
7.培养液DMEM + 10%FBS实验组卵裂率(55.4%),桑椹胚百分率(21.7%),囊胚百分率(6.5%)均显著高于TCM-199 + 10%FBS组(分别为41.3%,6.7%和0)以及RM-199组(分别为37.9%,5.7%和0,P﹤0.05);培养液RM-199组8-16细胞百分率(25.3%)显著高于TCM-199 + 10%FBS组(13.3%,P﹤0.05)。
8.卵龄19-20h卵母细胞显微注射后,注射卵形态正常率(30.4%)显著低于13-14h和16-17h卵母细胞(分别为61.9%和64.6%,P﹤0.05),19-20h卵母细胞显微注射后卵裂率(42.9%)显著高于13-14h卵母细胞(19.2%,P﹤0.05)。
9.常规体外受精卵裂率(41.3%)显著好于显微授精(22.9%)和孤雌激活(3.8%)结果(P﹤0.05),显微授精卵裂率显著优于孤雌激活卵裂率(P﹤0.05)。
In this paper,41 female New Zealand Rabbits were superovulated with FSH + hCG by muscle injected. Compared the influence about different FSH dose, season and the methods of constant and degressive on the results of superovulated. Analyzed the effects of different capacitation liquid, medium, the quantity of L-glutamine and the age of oocytes on the in vitro fertilization and zygote development. And studied the effects of the oocytes age on microinsemination, compared the results of microinsemination and normal in vitro fertilization. The results were as follows.
1. Rabbits were superovulated with different dose of FSH, the results showed that, the average number of ovulation point and the average number of oocytes recovered from experiment group of 60 unit (36.13±8.13 and 28.88±6.71, respectively) and 100 unit (39.00±9.17 and 30.67±7.71, respectively) were more than which recovered from 40 unit (25.83±4.07 and 19.50±4.42, respectively, P<0.05). The average recovered rate (75.48%, 79.93% and 78.63%, respectively) between each other showed no significant difference (P>0.05).
2. The average number of ovulation point (39.00±9.17), the average number of oocytes (30.67±7.71) and the average recovered rate (78.63%) recovered from spring were higher than that from autumn (21.58±7.10, 15.17±4.97 and 70.27%, respectively, P<0.05).
3. Injected the same dose of FSH with the methods of constant and degressive, there was no significant difference between two groups (P>0.05).
4. The percentage of 8-16 cells of DM-His method (13.3%) was higher than that of DM and His methods (3.4% and 3.3%, respectively, P<0.05). The cleavage rate of the methods of DM, His and DM-His were 51.7%, 38.3% and 41.3%, respectively, there was no significant difference between each other (P>0.05), but the effect of DM method was a little better.
5. The cleavage rate and the percentage of 8-16 cells from the age of 14-15h oocytes (41.3% and 13.3%, respectively) were higher than that of 16-17h oocytes (25.0% and 2.9%, respectively, P<0.05).
6. Added L-glutamine in TCM-199 + 10% FBS could promote embryonic development, but as the quantity increased, the function was downing. The best adding quantity was 0.5 mmol/L.
7. The cleavage rate, the percentage of morulae and blastulae from DMEM + 10% FBS medium (55.4%, 21.7% and 6.5%, respectively) were higher than TCM-199 + 10% FBS (41.3%, 6.7% and 0, respectively) and RM-199 medium (37.9%, 5.7% and 0,respectively, P<0.05); the percentage of 8-16 cells from RM-199 medium (25.3%) was higher than TCM-199 + 10% FBS medium (13.3%, P<0.05).
8. After microinsemination with sperms, the morphologically normal rates of 19-20h oocytes (30.4%) was lower than that of 13-14h and 16-17h oocytes (61.9% and 64.6%, respectively, P<0.05), The cleavage rate from the age of 19-20h oocytes (42.9%)was higher than that of 13-14h oocytes (19.2%, P<0.05).
9. The cleavage rate of normal IVF (41.3%) was higher than that of ICSI (22.9%) and Sham-operation (3.8%, P<0.05). It showed a significant difference between the cleavage rate of ICSI and Sham-operation (P<0.05).
1.不同FSH总剂量超排结果显示,60单位与100单位试验组平均排卵点(分别为36.13±8.13个/只和39.00±9.17个/只),平均回收卵子数(分别为28.88±6.71枚/只和30.67±7.71枚/只)均显著高于40单位试验组(分别为25.83±4.07个/只和19.50±4.42枚/只,P﹤0.05)。但三者卵子平均回收率(分别为75.48%,79.93%和78.63%)之间差异均不显著(P﹥0.05)。
2.春季无论平均排卵点(39.00±9.17个/只),平均回收卵子数(30.67±7.71枚/只)还是卵子平均回收率(78.63%)均显著高于秋季(21.58±7.10个/只,15.17±4.97枚/只,70.27%,P﹤0.05)。
3.用恒量法和递减法给母兔注射相同总剂量的FSH,两者之间超排结果差异不显著(P﹥0.05)。
4. DM-His法的8-16细胞百分率(13.3%)显著高于DM法与His法(分别为3.4%和3.3%,P﹤0.05)。DM法,His法及DM-His法三种获能方法总的卵裂率(分别为51.7%,38.3%和41.3%)之间差异不显著(P﹥0.05),但以DM法效果好些。
5.卵龄14-15h的卵母细胞体外受精后卵裂率(41.3%)和8-16细胞百分率(13.3%)均显著高于卵龄16-17h卵母细胞(分别为25.0%和2.9%,P﹤0.05)。
6.在培养液TCM-199 + 10%FBS中添加L-谷氨酰胺对卵母细胞体外受精及受精卵体外发育具有促进作用,但随着添加量的增加作用减弱,以0.5mmol/L添加量效果最好。
7.培养液DMEM + 10%FBS实验组卵裂率(55.4%),桑椹胚百分率(21.7%),囊胚百分率(6.5%)均显著高于TCM-199 + 10%FBS组(分别为41.3%,6.7%和0)以及RM-199组(分别为37.9%,5.7%和0,P﹤0.05);培养液RM-199组8-16细胞百分率(25.3%)显著高于TCM-199 + 10%FBS组(13.3%,P﹤0.05)。
8.卵龄19-20h卵母细胞显微注射后,注射卵形态正常率(30.4%)显著低于13-14h和16-17h卵母细胞(分别为61.9%和64.6%,P﹤0.05),19-20h卵母细胞显微注射后卵裂率(42.9%)显著高于13-14h卵母细胞(19.2%,P﹤0.05)。
9.常规体外受精卵裂率(41.3%)显著好于显微授精(22.9%)和孤雌激活(3.8%)结果(P﹤0.05),显微授精卵裂率显著优于孤雌激活卵裂率(P﹤0.05)。
In this paper,41 female New Zealand Rabbits were superovulated with FSH + hCG by muscle injected. Compared the influence about different FSH dose, season and the methods of constant and degressive on the results of superovulated. Analyzed the effects of different capacitation liquid, medium, the quantity of L-glutamine and the age of oocytes on the in vitro fertilization and zygote development. And studied the effects of the oocytes age on microinsemination, compared the results of microinsemination and normal in vitro fertilization. The results were as follows.
1. Rabbits were superovulated with different dose of FSH, the results showed that, the average number of ovulation point and the average number of oocytes recovered from experiment group of 60 unit (36.13±8.13 and 28.88±6.71, respectively) and 100 unit (39.00±9.17 and 30.67±7.71, respectively) were more than which recovered from 40 unit (25.83±4.07 and 19.50±4.42, respectively, P<0.05). The average recovered rate (75.48%, 79.93% and 78.63%, respectively) between each other showed no significant difference (P>0.05).
2. The average number of ovulation point (39.00±9.17), the average number of oocytes (30.67±7.71) and the average recovered rate (78.63%) recovered from spring were higher than that from autumn (21.58±7.10, 15.17±4.97 and 70.27%, respectively, P<0.05).
3. Injected the same dose of FSH with the methods of constant and degressive, there was no significant difference between two groups (P>0.05).
4. The percentage of 8-16 cells of DM-His method (13.3%) was higher than that of DM and His methods (3.4% and 3.3%, respectively, P<0.05). The cleavage rate of the methods of DM, His and DM-His were 51.7%, 38.3% and 41.3%, respectively, there was no significant difference between each other (P>0.05), but the effect of DM method was a little better.
5. The cleavage rate and the percentage of 8-16 cells from the age of 14-15h oocytes (41.3% and 13.3%, respectively) were higher than that of 16-17h oocytes (25.0% and 2.9%, respectively, P<0.05).
6. Added L-glutamine in TCM-199 + 10% FBS could promote embryonic development, but as the quantity increased, the function was downing. The best adding quantity was 0.5 mmol/L.
7. The cleavage rate, the percentage of morulae and blastulae from DMEM + 10% FBS medium (55.4%, 21.7% and 6.5%, respectively) were higher than TCM-199 + 10% FBS (41.3%, 6.7% and 0, respectively) and RM-199 medium (37.9%, 5.7% and 0,respectively, P<0.05); the percentage of 8-16 cells from RM-199 medium (25.3%) was higher than TCM-199 + 10% FBS medium (13.3%, P<0.05).
8. After microinsemination with sperms, the morphologically normal rates of 19-20h oocytes (30.4%) was lower than that of 13-14h and 16-17h oocytes (61.9% and 64.6%, respectively, P<0.05), The cleavage rate from the age of 19-20h oocytes (42.9%)was higher than that of 13-14h oocytes (19.2%, P<0.05).
9. The cleavage rate of normal IVF (41.3%) was higher than that of ICSI (22.9%) and Sham-operation (3.8%, P<0.05). It showed a significant difference between the cleavage rate of ICSI and Sham-operation (P<0.05).
引文
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