Department of Agrarian Production, Technical University of Madrid (UPM), 28040, Madrid, Spain; Department of Biological Science, Technical University of Loja (UTPL), 1101608, Loja, Ecuador.
Department of Biochemistry and Molecular Biology, Veterinary Faculty, Complutense University of Madrid (UCM), 28040, Madrid, Spain.
Theriogenology. 2024 Jul 15;223:36-46. doi: 10.1016/j.theriogenology.2024.04.011. Epub 2024 Apr 22.
In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3',4' hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 μM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.
体外胚胎生产（IVP）对养猪业以及基础研究和生物医学应用具有重要意义。尽管世界各地的实验室都在努力改善不理想的培养条件，但猪的 IVP 仍然效率低下。在各种物种中，补充诺比汀（ Nob，5,6,7,8,3',4'六甲氧基黄酮）到体外培养（IVC）培养基中可提高体外胚胎的发育。然而，其对体外生产的猪胚胎质量和发育能力的影响尚未确定。本研究评估了不同浓度（2.5 和 5 μM）的 Nob 在体外生产的猪胚胎早期培养过程中对胚胎发育能力、线粒体活性、脂质含量、细胞内活性氧（ROS）和谷胱甘肽（GSH）含量、每个囊胚的总细胞数（TCN）以及与胚胎发育、质量和氧化应激相关的基因表达的影响。未添加 Nob 补充剂的培养基和添加 0.01%二甲基亚砜（ Nob 的载体 DMSO）的培养基中培养的胚胎分别构成对照和 DMSO 组。在 IVC 的第 2、6 和 7 天评估胚胎发育率。此外，选择代表性胚胎组评估线粒体活性、脂质、ROS 和 GSH 含量（IVC 的第 2 和 6 天）、TCN 评估和基因表达分析（IVC 的第 6 天）。在 IVC 的第 2 天，评估的所有参数均无显著差异。相比之下，在 Nob 2.5 存在下培养的胚胎在 IVC 的第 6 和第 7 天显示出更高的发育率。此外，与其他组相比，在 Nob 2.5 组中，第 6 天的胚胎显示出更高的线粒体活性，ROS 和 GSH 水平降低。与对照和 DMSO 组相比，Nob 2.5 和 Nob 5 胚胎的 TCN 更高。此外，Nob 2.5 和 Nob 5 上调了超氧化物歧化酶 1（SOD1）和葡萄糖-6-磷酸脱氢酶（G6PDH）基因的表达，这有助于在 IVC 期间对抗氧化应激。总之，在 IVC 的前 48 小时添加 Nob 提高了猪胚胎的发育率，并提高了其质量，包括上调了潜在地提高 IVP 系统整体效率的相关基因。
