The kodkod (Leopardus guigna) is a small felid endemic of Chile and is considered a vulnerable species. Domestic cat oocytes have been successfully used as recipient cytoplast to reprogram somatic cells from different felids by interspecific somatic cell nuclear transfer (iSCNT). The developmental competence of felid embryos generated by iSCNT can be improved by the aggregation method using a zona-free culture system. The objective of this research was to evaluate the developmental competence of kodkod embryos generated by iSCNT using domestic cat oocytes and the aggregation method. For this purpose, five experimental group were done: (1) cat embryos generated by IVF, (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by iSCNT (K1x) and (5) aggregated kodkod embryos generated by iSCNT (K2x). Cleavage, morulae and blastocyst rates were estimated. The blastocyst diameter was evaluated. The gene expression level of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6) was analyzed in blastocysts. Morulae rate was higher in the IVF group and when cloned embryos were cultured in aggregates (IVF: 68.2%, Ca2x: 58.0% and K2x: 62.4%) compared to individually cultured kodkod embryos (K1x: 37.0%) (P < 0.05). Embryo aggregation increased blastocysts formation in the Ca2x group (30.9%) to a similar rate compared to the IVF group (44.5%) (P > 0.05). No blastocysts were generated in the K1x group, whereas blastocysts formation was obtained in K2x group (5.9%). The diameter of blastocysts from the K2x group (172.8 μm) was significantly lower than blastocysts from the Ca2x group (P < 0.05). The relative expression of OCT4 was lower in blastocysts from Ca1x than in blastocysts from IVF (P < 0.05). Furthermore, CDX2 expression was lower in blastocysts from Ca2x than in blastocysts from Ca1x and IVF groups (P < 0.05). In kodkod embryos, only one blastocyst from the K2x group expressed OCT4. No expression of SOX2, NANOG, CDX2 and GATA6 was detected in kodkod blastocysts. In conclusion, after iSCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. The aggregation method increases the morulae rate of kodkod cloned embryos and allows blastocysts formation. However, kodkod blastocysts have a poor morphological quality and a lacking expression of pluripotency and differentiation markers, probably caused by an incomplete nuclear reprogramming.

中文翻译：

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胚胎聚集允许在种间 SCNT 后产生 kodkod（Leopardus guigna）囊胚

kodkod (Leopardus guigna) 是智利特有的一种小型猫科动物，被认为是一种脆弱的物种。家猫卵母细胞已成功用作受体细胞质，通过种间体细胞核转移 (iSCNT) 重新编程来自不同猫科动物的体细胞。使用无带培养系统的聚集方法可以提高 iSCNT 产生的猫科动物胚胎的发育能力。本研究的目的是评估使用家猫卵母细胞和聚集方法由 iSCNT 产生的 kodkod 胚胎的发育能力。为此，做了五个实验组：（1）IVF产生的猫胚胎，（2）SCNT（Ca1x）产生的猫胚胎，（3）SCNT（Ca2x）产生的聚合猫胚胎，(4) iSCNT (K1x) 产生的 kodkod 胚胎和 (5) iSCNT (K2x) 产生的聚合 kodkod 胚胎。估计卵裂、桑椹胚和胚泡率。评估囊胚直径。在胚泡中分析了多能性（OCT4、SOX2 和 NANOG）和分化标志物（CDX2 和 GATA6）的基因表达水平。与单独培养的 kodkod 胚胎 (K1x: 37.0%) 相比，IVF 组和克隆胚胎在聚集体中培养 (IVF: 68.2%, Ca2x: 58.0% 和 K2x: 62.4%) 的桑椹菌率更高 (P < 0.05)。与 IVF 组 (44.5%) 相比，胚胎聚集使 Ca2x 组 (30.9%) 的囊胚形成增加到相似的比率 (P > 0.05)。K1x 组未产生囊胚，而 K2x 组有囊胚形成（5.9%）。来自 K2x 组的囊胚直径 (172. 8 μm）显着低于 Ca2x 组的囊胚（P < 0.05）。OCT4 在 Ca1x 囊胚中的相对表达低于 IVF 囊胚（P < 0.05）。此外，CDX2 在来自 Ca2x 的囊胚中的表达低于来自 Ca1x 和 IVF 组的囊胚（P < 0.05）。在 kodkod 胚胎中，只有一个来自 K2x 组的囊胚表达 OCT4。在 kodkod 囊胚中未检测到 SOX2、NANOG、CDX2 和 GATA6 的表达。总之，在 iSCNT 之后，家猫卵母细胞支持 kodkod 胚胎的发育，直到桑椹胚阶段。聚合方法增加了 kodkod 克隆胚胎的桑椹胚率，并允许囊胚形成。然而，kodkod囊胚的形态质量较差，缺乏多能性和分化标志物的表达，