A method for the collection of early-stage sea turtle embryos

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.