Simple method for emulsification of lipophilic nutrients that affect preimplantation development of bovine embryos in vitro
Simple method for emulsification of lipophilic nutrients that affect preimplantation development of bovine embryos in vitro
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乳化影响牛胚胎植入前体外发育的亲脂性营养物质的简单方法
DOI：
10.1071/rdv26n1ab86
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Ikeda S
中科院分区：
文献类型：
--
作者：
Asada;M.;Goto;Y.;Yahata;K.;Yokoyama;N.;Kawai;S.;Inoue;N.;Kaneko;O.;and Kawazu;S.-I;Ikeda S
为了研究生物活性亲脂性营养素对体外哺乳动物植入前胚胎的影响，通常使用两亲性载体将亲脂性物质溶解到培养基中。然而，这些营养物质的容易乳化将有利于培养基的制备。我们在这里报告了一种影响牛植入前胚胎体外发育的亲脂性营养物质乳化的简单方法。我们研究了乳化油酸 (OA) 或抗氧化维生素混合物——维生素 E (VE) 和 β-胡萝卜素 (BC) 的作用。聚甘油-10 月桂酸酯 (P10L) 用作乳化剂，并以 5.05%（wt/wt）的浓度溶解在玻璃瓶中的无菌水中。将百分之一(wt/wt)的OA或VE(α-生育酚)和BC的混合物(VE : BC = 1000 : 1 wt/wt)添加到小瓶中并通过使用磁力搅拌器混合。溶液首先呈现白色浑浊后变得透明且稳定，表明乳化稳定。将乳化的OA和VE+BC分别指定为emOA和emVEBC。从屠宰场牛卵巢获得的卵母细胞在补充有 10% (vol/vol) 胎牛血清和 0.2 IU mL–1 FSH 的改良合成输卵管液 (mSOF) 中体外成熟 (IVM) 22 小时。 IVM 后，使用 Percoll 梯度选择的单头公牛精子在基于 mSOF 的培养基中对卵母细胞进行 IVF 20 小时。 IVF 后，将推定受精卵从卵丘细胞中分离出来并在 mSOF 中培养。第3天（IVF = 第0天），已发育至8细胞阶段或以上（≥8细胞）的胚胎随后在补充有0.05%（体积/体积）的emOA或emVEBC的培养基中培养。在第 8 天评估 ≥8 细胞胚胎的囊胚发育。在无添加对照和 emVEBC 的情况下，还在第 10 天评估孵化率。所有培养物均在 38.5°C、5% CO2、5% O2 和 90% N2 下进行，并复制 4 次，每次重复每组约 18 个胚胎。开发数据采用一般线性模型进行统计分析。 emOA组的囊胚率（36.4%）显着（P<0.05）低于无添加对照组（54.1%）。 emVEBC组的囊胚率（53.9%）与对照组相似；然而，emVEBC 组的孵化率 (22.6%) 显着高于对照组 (9.2%)。这些数据表明，用 P10L 乳化亲脂性营养物质是一种简单的方法，可以将其添加到培养基中，以研究其对体外植入前发育的有利（例如抗氧化维生素）或抑制（例如 OA）作用。
In order to investigate the effects of bioactive lipophilic nutrients on mammalian pre-implantation embryos in vitro, amphipathic vehicles are commonly used to dissolve the lipophilic substances into culture media. However, easy emulsification of these nutrients would facilitate medium preparation. We report here a simple method for emulsification of lipophilic nutrients that affect bovine pre-implantation embryonic development in vitro. We investigated the effects of emulsified oleic acid (OA) or a mixture of antioxidative vitamins – vitamin E (VE) and β-carotene (BC). Polyglyceryl-10 laurate (P10L) was used as an emulsifier and was dissolved in sterile water at 5.05% (wt/wt) in glass vials. One percent (wt/wt) of OA or a mixture of VE (α-tocopherol) and BC (VE : BC = 1000 : 1 wt/wt) was added into the vial and mixed by using a magnetic stirrer. After first exhibiting white turbidity, the solution became transparent and stabilised, indicating stable emulsification. The emulsified OA and VE+BC were designated as emOA and emVEBC, respectively. Cumulus-enclosed oocytes obtained from abattoir bovine ovaries were in vitro-matured (IVM) for 22 h in modified synthetic oviduct fluid (mSOF) supplemented with 10% (vol/vol) fetal calf serum and 0.2 IU mL–1 FSH. After IVM, the oocytes were subjected to IVF with Percoll gradient-selected sperm from a single bull in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from the cumulus cells and cultured in mSOF. On Day 3 (IVF = Day 0), embryos that had developed to the 8-cell stage or more (≥8-cell) were subsequently cultured in medium supplemented with 0.05% (vol/vol) of emOA or emVEBC. Blastocyst development from ≥8-cell embryos was assessed on Day 8. In the case of no-additive control and emVEBC, the hatching rate was also assessed on Day 10. All the cultures were performed at 38.5°C under 5% CO2, 5% O2, and 90% N2 and replicated 4 times with ~18 embryos per group per replicate. The development data were statistically analysed by the general linear model. The blastocyst rate in the emOA group (36.4%) was significantly (P < 0.05) lower than that in the no-additive control (54.1%). The blastocyst rate in the emVEBC group (53.9%) was similar to that in the control; however, the hatching rate was significantly higher in the emVEBC group (22.6%) than in the control (9.2%). These data suggest that emulsification of lipophilic nutrients with P10L is an easy method to allow their addition into culture media for investigating their favourable (e.g. antioxidative vitamins) or inhibitory (e.g. OA) effects on pre-implantation development in vitro.