离心参数对猪卵母细胞早期去脂效果的影响  

The Influence of Centrifugal Parameters on the Performance of Early-delipated Pig Oocytes

作　　者：刘凤娟 刘晋冀[1] 李小兵[1] 李思[1] 潘伟荣[1] 成文敏[1] 贾宝瑜 赵红业 卿玉波[1] 魏红江[1] LIU Fengjuan;LIU Jinji;LI Xiaobing;LI Si;PAN Weirong;CHENG Wenmin;JIA Baoyu;ZHAO Hongye;QING Yubo;WEI Hongjiang(College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China;College of Plant protection, Yunnan Agricultural University, Kunming 650201, China)

机构地区：[1]云南农业大学动物科学技术学院,云南昆明650201 [2]云南农业大学植物保护学院,云南昆明650201

出　　处：《云南农业大学学报（自然科学版）》2018年第2期240-248,共9页Journal of Yunnan Agricultural University:Natural Science

基　　金：云南省应用基础计划研究重大项目(2014FC006)

摘　　要：【目的】卵母细胞脂肪含量较高是影响猪卵母细胞和胚胎冷冻效果不佳的主要原因,离心去脂可以提高冷冻成功率。本研究选用不同离心参数对猪卵母细胞进行离心去脂,通过研究其对卵母细胞早期去脂效果及其去脂后克隆胚胎的体内外发育和冷冻的影响,筛选最适离心参数。【方法】设定不同离心参数对猪卵母细胞进行早期去脂,利用体细胞克隆技术构建重构胚,通过离心后卵母细胞脂肪层的分离、重构胚的卵裂率和囊胚率、冷冻—解冻后的存活率以及代孕母猪妊娠等分析重构胚玻璃化冷冻前后体内外的发育效果。【结果】离心转数为10 000 r/min、离心时间为20 min时,卵母细胞的早期去脂效果最好。早期去脂重构胚的卵裂率和囊胚率分别是65.38%和13.46%;冷冻—解冻的2~4细胞、5~8细胞、9~16细胞的重构胚的存活率和囊胚率分别为49.09%、52.75%、78.57%和12.72%、2.20%、21.42%,而且冷冻胚胎移植到代孕母猪体内后成功妊娠。【结论】本研究证明了猪卵母细胞早期去脂可直接用于胚胎冷冻,最适离心参数为10 000 r/min×20 min。[Purpose]High fat content in oocytes is the main factor that affects porcine oocyte and embryo freezing effect, the method of centrifuging delipation can improve the freezing success rate. In this study, we removed the fat from the porcine oocytes with different centrifugal parameters, and screened out an optimal centrifugal parameter through investigating the effect of early delipation on oocytes, in vitro and in vivo embryo development and freezing.[Method]We set various centrifugal parameters to perform the early delipation of porcine oocytes, established the reconstructed embryos using somatic cell nuclear transfer, and analyzed in vitro and in vivo developmental effect of the reconstructed embryos before and after vitrification by separating the fat layer of porcine oocytes, calculating the cleavage rate and blastocyst rate of reconstructed embryos, the survival rate of frozenthawed embryos and the pregnancy rate of surrogate sows.[Result]When the centrifugal speed ×the centrifugal time was 10 000 r/min × 20 min, the early-delipated effect was the best. Under such centrifugal parameter, the cleavage rate and blastocyst rate of early-delipated reconstructed embryos were 65.38% and 13.46%, respectively; the survival rate and blastocyst rate of frozen-thawed 2-4 cells, 5-8 cells and 9-16 cells were 49.09% and 12.72%, 52.75% and 2.20%, and 78.57% and 21.42%,respectively; the surrogate sows transferred the frozen embryos were successfully pregnant.[Conclusion]This study suggests that the porcine oocytes after early-delipation can be directly used for cryopreservation of embryos and the optimum centrifugation parameter is 10 000 r/min×20 min.

关 键 词：离心参数 早期去脂 重构胚 玻璃化冷冻 

分 类 号：Q813.7[生物学—生物工程]