Effect of centrifugation treatment before vitrification on the viability of porcine mature oocytes and zygotes produced in vitro
Effect of centrifugation treatment before vitrification on the viability of porcine mature oocytes and zygotes produced in vitro
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DOI：
10.1262/jrd.19150
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发表时间：
2008-06-01
影响因子：
1.8
通讯作者：
Kikuchi, Kazuhiro
中科院分区：
文献类型：
--
作者：
Somfai, Tamas;Kashiwazaki, Naomi;Kikuchi, Kazuhiro
本研究的目的是探讨离心预处理对猪体外成熟（IVM）卵母细胞的活力和核状态以及玻璃化冷冻（固体表面玻璃化; SSV）后体外受精（IVF）卵母细胞（受精卵）发育能力的影响。将IVM后具有第一极体的成熟卵母细胞和IVF后10小时具有第二极体的受精卵在37 ℃以10，000 × g离心20分钟，然后进行SSV。通过形态学观察和荧光素二醋酸盐染色评价其存活能力。玻璃化冷冻后6 h评价卵母细胞的核状态。玻璃化冷冻后的受精卵体外培养6天后评价其发育至囊胚期的能力。虽然离心并不直接损伤卵母细胞，但它大大降低了SSV后的活卵率。玻璃化诱导的孤雌激活率在离心和非离心卵母细胞中相似（分别为42.4%和47.4%）。冷冻对原核卵母细胞的存活率无显著影响。无论离心预处理与否，玻璃化冷冻的受精卵发育到囊胚期的速度均显著低于对照组。玻璃化冷冻后，对照组和离心组受精卵的卵裂率和囊胚率无差异。无论玻璃化冷冻与否，对照和离心受精卵之间的囊胚总细胞数也没有差异。这些结果表明，在IVM猪卵母细胞，离心预处理是非常有害的cryotolerance，然而，在受精卵，它只有轻微的影响，并没有改变存活的受精卵的发育能力。
The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 x g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes.