ABSTRACT: Early-stage turtle embryos, immediately after oviposition, are very small (<5 mm diameter), hindering research on the initial period of embryonic development. For example, assessing whether turtle eggs had been fertilized and contained a viable embryo at oviposition, especially under field conditions, is complicated by the microscopic size of embryos that may have died at an early stage of development. Further, little is known about the molecular pathways that promote and regulate early developmental processes in turtles, such as pre-ovipositional embryonic arrest. To enable further investigation of the processes critical to early embryonic development in turtle species, a reliable method is required for extraction of early-stage embryos from the egg. Therefore, our aim was to develop a novel and reproducible method for extracting early-stage sea turtle embryos. Herein, we describe the technique for extracting Chelonia mydas embryos before and after white spot formation. Once the embryos were collected, the total RNA of 10 embryos was extracted to validate the method. The total RNA concentration was above 5 ng µl^-1 and the RNA integrity number varied between 7.0 and 10.0, which is considered acceptable for further RNA-sequencing analyses. This extraction technique could be employed when investigating fertilization rates of turtle nests and for further investigation of the molecular biology of embryonic development in turtles. Furthermore, the technique should be adaptable to other turtle species or any oviparous species with similar eggs.

中文翻译：

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一种收集早期海龟胚胎的方法

摘要：产卵后的早期乌龟胚胎非常小（直径小于5毫米），阻碍了胚胎发育初期的研究。例如，评估可能在发育初期已经死亡的胚胎的微观尺寸会使评估乌龟卵是否受精并在产卵时，尤其是在田间条件下是否含有存活的胚胎变得复杂。此外，关于促进和调节乌龟早期发育过程（例如卵前胚胎停滞）的分子途径知之甚少。为了进一步研究对龟物种早期胚胎发育至关重要的过程，需要一种可靠的方法从蛋中提取早期胚胎。因此，我们的目的是开发一种新颖且可重现的提取早期海龟胚胎的方法。在这里，我们描述提取技术白斑形成前和后的小叶Chelonia myda的胚胎。收集胚胎后，将提取10个胚胎的总RNA以验证该方法。总RNA浓度高于5 ng µl ^-1，RNA完整性数在7.0和10.0之间变化，这被认为可以用于进一步的RNA测序分析。在研究乌龟巢的受精率以及进一步研究乌龟胚胎发育的分子生物学时，可以使用这种提取技术。此外，该技术应适用于其他乌龟物种或具有相似卵的任何卵生物种。