Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts.
Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts.
复制标题
升温后恢复培养过程中敲除血清替代对玻璃化孤雌猪囊胚发育和质量的影响。
DOI：
10.1089/bio.2018.0132
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Wu Guo Quan
中科院分区：
文献类型：
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作者：
Xiang De Cai;Jia Bao Yu;Quan Guo Bo;Zhang Bin;Shao Qing Yong;Zhao Zhi Yong;Hong Qiong Hua;Wu Guo Quan
复苏培养作为冷冻保存过程中的一个环节，直接关系到胚胎的存活和质量。通常，回收培养基包括不确定的血清或血清组分，这可能导致结果不稳定和其他问题。本研究的目的是评估敲除血清替代物（KSR）作为恢复培养期间血清的替代物对玻璃化冷冻猪孤雌激活囊胚发育和质量的影响。胎牛血清（FBS）用作阳性对照。将第5天的扩张囊胚通过Cryotop方法玻璃化冷冻，并在加温后用10%（v/v）KSR或10%（v/v）FBS恢复48小时。添加KSR或FBS可显著提高玻璃化冷冻囊胚的存活率和孵化率。玻璃化冷冻囊胚在KSR或FBS中复苏后，细胞膜损伤和凋亡率显著降低，细胞总数增加。在恢复培养过程中添加KSR或FBS显著降低了活性氧水平，并提高了玻璃化囊胚中线粒体活性和腺苷三磷酸含量。玻璃化不影响PCNA、CDX 2和CPT 1的基因表达，但显著增加POU 5 F1和uPA的mRNA水平。KSR可显著上调PCNA和CPT 1的mRNA水平，下调POU 5 F1的mRNA水平。玻璃化冷冻囊胚中PCNA、CDX 2、CPT 1和uPA的表达水平在复苏液中添加FBS后显著上调。此外，BAX：BCL 2L 1比率在新鲜和玻璃化冷冻囊胚之间是相似的，并且KSR或FBS补充对该值没有影响。总之，我们的数据表明，在恢复培养过程中添加KSR可以改善玻璃化冷冻猪孤雌胚泡的发育和质量。这些结果为KSR作为玻璃化囊胚复苏培养的血清补充剂替代FBS提供了有益的参考。
The postwarming recovery culture, as one of the steps in cryopreservation process, is directly correlated with the survival and quality of embryos. Generally, recovery medium includes undefined serum or serum components that may cause the instability of results and other problems. The objective of this study was to evaluate the effect of knockout serum replacement (KSR) as a substitute for serum during recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Fetal bovine serum (FBS) was used as a positive control. The expanded blastocysts on day 5 were vitrified by the Cryotop method, and recovered with 10% (v/v) KSR or 10% (v/v) FBS for 48 hours after warming. Survival and hatching rates of vitrified blastocysts were significantly increased by KSR or FBS supplementation. The vitrified blastocysts recovered in KSR or FBS exhibited significantly decreased percentages of membrane damage and apoptosis, and increased total cells. Addition of KSR or FBS during recovery culture significantly reduced reactive oxygen species levels, and improved mitochondrial activity and adenosine triphosphates content in the vitrified blastocysts. Vitrification did not affect the gene expression of PCNA, CDX2, and CPT1, but significantly increased mRNA levels of POU5F1 and uPA. KSR added to the recovery medium significantly upregulated mRNA levels of PCNA and CPT1, and downregulated POU5F1 mRNA levels. The expression levels of PCNA, CDX2, CPT1, and uPA in vitrified blastocysts were significantly upregulated by addition of FBS to recovery medium. Moreover, the BAX: BCL2L1 ratio was similar between fresh and vitrified blastocysts, and KSR or FBS supplementation had no effect on the value. In conclusion, our data showed that KSR supplementation during recovery culture can improve the development and quality of vitrified parthenogenetic porcine blastocysts. These findings provide a useful reference that KSR could be used to replace FBS as a defined serum supplement for recovery culture of vitrified blastocysts.