【作者】 苗德强；

【导师】 谭景和； 姜运良；

【作者基本信息】 山东农业大学 ， 基础兽医， 2005， 硕士

【摘要】 各种实验中所使用的卵母细胞多数来自屠宰场宰杀后的动物。死后动物机体会发生一系列生理生化变化,这些变化对卵母细胞质量的影响有待研究。本实验研究了处死小鼠后短时间滞留对卵巢卵母细胞、排卵后卵母细胞和着床前胚胎的影响,为更好地利用卵母细胞和胚胎,以及为合理地进行组织保存提供科学依据。实验结果如下:1.随滞留时间延长排卵后卵母细胞死亡率和自发激活率升高,同样滞留时间,随卵龄增加排卵后卵母细胞死亡率和自发激活率升高。2.输卵管在M2中放置、腹腔全开口体内滞留以及腹腔半开口体内滞留三组相比,只有半开口体内滞留长达15min时死亡率（27.8%）较高,其余处理死亡率较低且差异均不显著;自发激活率三组依次升高,其中输卵管在M2中放置不引起卵母细胞死亡和自发激活。3.不同品系小鼠卵母细胞对输卵管在体内滞留产生的反应不同,滞留10min C57BL/6系小鼠卵母细胞死亡率为56.5%,显著高于昆明鼠（47.6%）;自发激活率分别为13.3%和46.0%,C57BL/6系小鼠显著低于昆明鼠。4.hCG后24h体内滞留5min卵母细胞在CZB中培养自发激活率为81.1%,显著低于SrCl₂孤雌激活率（96.4%）;自发激活的卵母细胞有较高的卵裂率（93.2%）和4-cell比率（87.3%）,但囊胚率（18.7%）较低,同卵龄的卵母细胞经SrCl₂孤雌激活囊胚发育率为22.9%,差异不显著。HCG后18h卵母细胞经SrCl₂孤雌激活卵母细胞囊胚发育率为64.6%,显著高于24h卵龄卵母细胞自发激活和诱导孤雌激活囊胚发育率。5.滞留20min不引起原核胚、2-cell胚胎和桑椹胚以及囊胚的死亡。滞留10min获取的原核胚体外培养囊胚率为81.2%显著低于对照组（93.2%）,随滞留时间延长,滞留20min囊胚率为67.5%,显著下降。6.滞留30min从卵巢获取的卵丘卵母细胞复合体体外成熟率为97.2%,与对照组（98.5%）差异不显著,孤雌激活后囊胚发育率分别为15.7%和16.7%,差异也不显著。结论:1.短时间滞留不会导致卵巢卵母细胞功能退化,但却能严重影响排卵后卵母细胞,导致其死亡和自发激活,且随着排卵后卵母细胞在体内的老化这种影响更明显。2.短时间滞留不会引起着床前胚胎死亡,但却使原核胚发育到囊胚能力显著下降。3.输卵管在死后小鼠体内滞留,对不同品系小鼠卵母细胞影响存在差异。4.腹腔开口会降低滞留对排卵后卵母细胞的影响。5.hCG后24h的卵母细胞虽然经短时间滞留自发激活后能发育到囊胚,但卵母细胞老化导致其囊胚率较低。更多还原

【Abstract】 Most oocytes used in all kinds of experiments were from animals killed in abattoirs. The body will take place physiological and biochemical changes,the impact on oocytes quality is in need of study. we studied the effect of the length of time the ovaries, oviducts and uterus remained in the mice after cervical dislocation on ovary oocytes, ovulation oocytes, zygotes and other stage preimplantation embryos. This study provided scientific proof to make better use of oocytes and embryos, as well as to effectively conserve organs. We obtained the results as follows: 1.With the time the oviducts remained in the body after the mice were killed extending, ovulation oocytes death rate and spontaneous activation rised. At the same time, with the increase of the oocytes age oocytes death rate and spontaneous activation rose too. 2.Among three groups, one was that oviducts were layed in M2, another one was that oviducts were obtained after delay while abdomen was opened wholly, the third one was that oviducts were obtained after delay while abdomen was opened half. Except the third group delayed for 15minute with the death rate of 27.8%, others had low death rate and the difference is not significant. Among three groups spontaneous activation rose in turn. In the first group no oocyte died or took place spontaneous activation. 3. Different strains have different reaction to the delay of oviducts in body after animal death. After 10min delay C57BL/6 mouse oocytes have death rate of 56.5%,significantely higher than Kunming mouse （47.6%）;Spontaneous activation rates were respectively 13.3% and 46.0%, C57BL/6 were obviously lower than Kunming mouse.4. The oviducts were obtained after being delayed 5min 24h after the mice were injected with hCG. The oocytes were cultured in CZB. About 81.1% occurred spontaneous activation, evidently lower than parthenogenetic rate （96.4%） with SrCl₂. Spontaneous activable oocytes had high cleavage rate（93.2%） and 4-cell rate（87.3%）. However, spontaneous activable oocytes had blastula development rate（18.7%） as low as parthenogenetic oocytes by SrCl₂（22.9%）. 18 hours after the mice wereinjected hCG, oocytes were parthenogenetic activated about 64.6%, significantely higher than spontaneous and induced activation rate of 24h aged oocyte.5.20min delay didn’t bring the pronucleus, 2-cell embryos, morula and blastula into death. After lOmin delay only 81.2% pronucleus developed to blastula, significantly lower than control （93.2%）. 20min delay reduced the blastula development rate to 67.5% more lower than lOmin delay.6.After 30min delay cumulus-oocytes complexions were cultured in vitro, the mature rate was 97.2%, not different significantly compared to the control（98.5%）.The treated group activated oocytes had 15.7% blastula development rate, not different significantly compared to the control （16.7%）. Conclusion: 1. Short time delay don’t result in ovary oocyte degeneration, but can interfere severely in ovulation oocytes, leading to death and spontaneous activation .With the aging of the oocytes in vivo, the effect gets more obvious. 2.Short time delay don’t result in preimplantation embryos death, but impacted blastula rate of pronucleus embryos. 3.The postmortem delay of the oviducts in the mouse body made different effect on different strains mice. 4.0penning the abdomen reduced the effect of the delay on the ovulation oocytes. 5.Oocytes obtained after the oviducts were delayed in dead animal body for a short while 24h after the mice were injected hCG took place spontaneous activation and developed to blastula, but the blastula rate was very low because of the aging of the oocytes.更多还原