Xiang De Cai, Jia Bao Yu, Quan Guo Bo, Zhang Bin, Shao Qing Yong, Zhao Zhi Yong, Hong Qiong Hua, Wu Guo Quan
1Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Yunnan Animal Science and Veterinary Institute, Kunming, P.R. China.
2College of Veterinary Medicine, Yunnan Agricultural University, Kunming, P.R. China.
Biopreserv Biobank. 2019 Aug;17(4):342-351. doi: 10.1089/bio.2018.0132. Epub 2019 Apr 19.
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.
解冻后复苏培养作为冷冻保存过程中的一个步骤，与胚胎的存活和质量直接相关。一般来说，复苏培养基包含未定义的血清或血清成分，这可能会导致结果不稳定及其他问题。本研究的目的是评估在复苏培养过程中使用敲除血清替代品（KSR）替代血清对玻璃化孤雌生殖猪囊胚发育和质量的影响。胎牛血清（FBS）用作阳性对照。第5天的扩张囊胚通过Cryotop法进行玻璃化处理，并在解冻后用体积分数为10%的KSR或体积分数为10%的FBS复苏48小时。添加KSR或FBS可显著提高玻璃化囊胚的存活率和孵化率。在KSR或FBS中复苏的玻璃化囊胚表现出膜损伤和凋亡百分比显著降低，总细胞数增加。在复苏培养过程中添加KSR或FBS可显著降低玻璃化囊胚中的活性氧水平，提高线粒体活性和三磷酸腺苷含量。玻璃化处理不影响增殖细胞核抗原（PCNA）、尾型同源盒转录因子2（CDX2）和肉碱/有机阳离子转运体1（CPT1）的基因表达，但显著增加了八聚体结合转录因子4（POU5F1）和尿激酶型纤溶酶原激活剂（uPA）的mRNA水平。添加到复苏培养基中的KSR显著上调了PCNA和CPT1的mRNA水平，并下调了POU5F1的mRNA水平。向复苏培养基中添加FBS可显著上调玻璃化囊胚中PCNA、CDX2、CPT1和uPA的表达水平。此外，新鲜囊胚和玻璃化囊胚之间的BAX:BCL2L1比值相似，添加KSR或FBS对该值无影响。总之，我们的数据表明，在复苏培养过程中添加KSR可改善玻璃化孤雌生殖猪囊胚的发育和质量。这些发现为KSR可用于替代FBS作为玻璃化囊胚复苏培养的明确血清补充剂提供了有用的参考。
