Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillán, Chile.
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillán, Chile.
Theriogenology. 2020 Dec;158:148-157. doi: 10.1016/j.theriogenology.2020.09.006. Epub 2020 Sep 12.
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.
山猫（Leopardus guigna）是一种小型猫科动物，仅分布于智利，被认为是易危物种。已成功使用家猫卵母细胞作为受体胞质体，通过种间体细胞核移植（iSCNT）重编程来自不同猫科动物的体细胞核。通过无透明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产生的山猫 iSCNT 胚胎的发育能力。为此，进行了五个实验组：（1）通过 IVF 产生的猫胚胎，（2）通过 SCNT 产生的猫胚胎（Ca1x），（3）通过 SCNT 聚集产生的猫胚胎（Ca2x），（4）通过 iSCNT 产生的山猫胚胎（K1x）和（5）通过 iSCNT 聚集产生的山猫胚胎（K2x）。评估了卵裂、桑葚胚和囊胚的比率。评估了囊胚的直径。分析了囊胚中多能性（OCT4、SOX2 和 NANOG）和分化标记物（CDX2 和 GATA6）的基因表达水平。与单独培养的山猫胚胎（K1x：37.0%）相比，IVF 组和克隆胚胎在聚集培养时（IVF：68.2%，Ca2x：58.0%和 K2x：62.4%）桑葚胚的比率更高（P<0.05）。胚胎聚集增加了 Ca2x 组囊胚的形成（30.9%），达到与 IVF 组相似的比率（44.5%）（P>0.05）。K1x 组未产生囊胚，而 K2x 组则产生了囊胚（5.9%）。与 Ca2x 组（172.8μm）相比，K2x 组囊胚的直径明显较小（P<0.05）。Ca1x 组囊胚中 OCT4 的相对表达低于 IVF 组（P<0.05）。此外，Ca2x 组囊胚中 CDX2 的表达低于 Ca1x 和 IVF 组（P<0.05）。在山猫胚胎中，只有一个来自 K2x 组的囊胚表达了 OCT4。在山猫囊胚中未检测到 SOX2、NANOG、CDX2 和 GATA6 的表达。总之，iSCNT 后，家猫卵母细胞支持山猫胚胎发育至桑葚胚阶段。聚集方法增加了山猫克隆胚胎的桑葚胚比率，并允许囊胚的形成。然而，山猫囊胚的形态质量较差，多能性和分化标记物的表达缺失，可能是由于核重编程不完全所致。
