SummaryThe aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

中文翻译：

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卵母细胞选择、锌螯合剂激活和组蛋白去乙酰化酶抑制对克隆猪胚胎和化学激活卵母细胞发育的影响

摘要本研究的目的是评估替代方案对改善卵母细胞选择、胚胎激活和基因组重编程的影响体外通过体细胞核移植（SCNT）克隆的猪胚胎的发育。在实验 1 中，体外在显微操作之前通过暴露于高渗蔗糖溶液来选择成熟的卵母细胞。在实验 2 中，使用锌螯合剂作为佐剂（离子霉素 +ñ,ñ,ñ',ñ'-四(2-吡啶基甲基)乙二胺(TPEN) +ñ-6-二甲基氨基嘌呤 (6-DMAP)) 与标准方案 (离子霉素 + 6-DMAP) 进行比较，以激活猪卵母细胞或 SCNT 胚胎。在实验 3 中，假定的克隆受精卵在组蛋白去乙酰化酶抑制剂 (Scriptaid) 中化学活化后孵育 15 小时，评估第 7 天囊胚中的胚胎产量和总细胞数。在实验 1 中，蔗糖处理的卵母细胞的卵裂率往往高于对照组（分别为 123/199、61.8% 和 119/222、53.6%）；然而，各组之间的囊胚率相似。在实验 2 中，用 TPEN 处理的受精卵的卵裂率高于对照组，但组间的囊胚率没有差异。对于实验 3，接触 Scriptaid 并没有改善克隆后的胚胎发育。尽管如此，用 Scriptaid 处理的克隆受精卵中的细胞总数高于 SCNT 对照。总之，蔗糖的卵母细胞选择以及锌螯合剂和组蛋白去乙酰化酶抑制剂的处理并没有显着提高克隆和单性生殖的囊胚产量。然而，组蛋白脱乙酰酶抑制剂显着改善了胚泡质量。