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.

Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas)

“…The characteristic white spot on the upper surface of the egg is indicative of the breaking of arrest, since it occurs once the vitelline membrane attaches to the shell membrane at the top of the egg, which in turn only occurs once the embryo develops past the gastrula stage and neurulation begins (Thompson, 1985). All five eggs incubated in normoxia (36 h N) developed a “white spot” (see Figure 1 in Garriz et al, 2020). In contrast, none of the five eggs incubated in hypoxia (36 h H) developed a white spot, indicating lack of development of the embryo past the gastrula stage.…”

“…The eggs were cleaned of adhering sand and debris and placed on a petri dish, stabilized with an aluminium foil ring. The method of isolation, which differs slightly for eggs before and after development of the white spot, has been described in detail elsewhere (Garriz et al, 2020). Once isolated, embryos were immediately frozen in liquid nitrogen before being stored at −80°C.…”

“…The full list of genes and their level of expression is available as Supporting Information data (Supplement S1) (Thompson, 1985). All five eggs incubated in normoxia (36 h N) developed a "white spot" (see Figure 1 in Garriz et al, 2020). In contrast, none of the five eggs incubated in hypoxia (36 h H) developed a white spot, indicating lack of development of the embryo past the gastrula stage.…”

Effects of postovipositional hypoxia and hyperoxia on leatherback turtle reproductive success and hatchling performance

“…Eggs that failed to hatch or were determined to have died (no signs of embryonic development, fungal growth, or abnormal coloration) were removed from the incubators. Dead eggs were opened (Gárriz et al, 2020) and the embryonic stage of death was assessed using Miller's (1985) 31‐stage and Leslie et al's (1996) four‐stage developmental chronologies. Hatching success (%) was calculated as the number of eggs to hatch of the total number of eggs for each treatment.…”