Abstract：
Nobiletin (NOB) is a bioflavonoid compound isolated from citrus fruit peels. The present study aimed to elucidate whether NOB facilitates the porcine sperm cryosurvival and embryo development after in vitro fertilization (IVF). To this end, spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 50, 100, 150, and 200 μM Nobiletin. The kinematic patterns of frozen-thawed (FT) sperm were assessed after 30 and 90 min incubation using a Sperm Class Analyzer (SCA). Viability, acrosome integrity, and mitochondrial membrane potential (MMP) were measured by fluorescence microscopy 30 min after thawing using SYBR-14/PI, PSA/FITC, and R123/PI, respectively. Lipid peroxidation was determined using MDA assay after incubation for 90 min. The addition of 100 μM and 150 μM NOB to the extender significantly improved sperm progressive motility, and acrosome integrity compared to the control group (P < 0.05). The proportion of viable spermatozoa was significantly higher in the 150 μM NOB group. MDA levels were less in 50 μM and 150 μM NOB treated groups compared to the control. In addition, IVF with FT sperm was used to assess the embryo developmental competence. Treatment with 150 μM NOB before cryopreservation increased the cleavage and blastocyst formation rates compared to the control group. Furthermore, the relative expression of POU5F1 and AMPK, genes related to pluripotency and cell differentiation were significantly upregulated in embryos resulting from NOB-treated sperm compared to the control group. These results suggest that Nobiletin is a functionally novel phytochemical to mitigate oxidative stress during the freezing-thawing of porcine spermatozoa as reflected by improved FT sperm quality and IVF outcome.
摘要：
Nobiletin（NOB）是从柑橘果皮中分离出的生物类黄酮化合物。本研究旨在阐明NOB是否促进体外受精（IVF）后猪精子冷冻存活和胚胎发育。为此,将精子稀释并冷冻保存在补充有0（对照）的冷冻补充剂中，50、100、150和200μM诺比林。使用精子类分析仪（SCA）在孵育30和90分钟后评估冻融（FT）精子的运动学模式。活力,顶体完整性,和线粒体膜电位（MMP）通过荧光显微镜在解冻后30分钟使用SYBR-14/PI测量，PSA/FITC,和R123/PI，分别。孵育90分钟后使用MDA测定法测定脂质过氧化。添加100μM和150μMNOB到延长剂显著提高了精子的进行性运动性,顶体完整性与对照组比较(P<0.05)。150μMNOB组中有活力的精子比例明显更高。与对照组相比，50μM和150μMNOB治疗组的MDA水平较低。此外,使用具有FT精子的IVF来评估胚胎发育能力。与对照组相比，在冷冻保存前用150μMNOB处理增加了卵裂和胚泡形成率。此外，POU5F1和AMPK的相对表达,与对照组相比，NOB处理的精子产生的胚胎中与多能性和细胞分化相关的基因显着上调。这些结果表明，Nobiletin是一种功能新颖的植物化学物质，可以减轻猪精子冻融过程中的氧化应激，这反映在FT精子质量和IVF结局的改善上。
