Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea)

The number of sea turtles in the wild is decreasing because of human activities, such as fishery bycatch (Peckham et al. 2007), oil spills, and marine pollution (Witherington 2001). Furthermore, illegal hunting for their oil, meat, and shells are still continuing (Koch et al. 2013). Hence, protection measures are being implemented in various countries to conserve the endangered species of sea turtles. In the past few decades, a major concern regarding sea turtles in the wild has been hybridization. Interspecific hybridization has been reported in several areas of the world (Karl et al. 1995; Barber et al. 2003; Lara-Ruiz et al. 2006). The original sea turtle species, such as hawksbill, loggerhead, and olive ridley, had established as independent species for more than millions of years despite overlaps among their habitats (Bowen et al. 1993). However, recent reports on intensive hybridization among these species clearly indicate that these unique original species are at a high risk of extinction in the near future. Acquiring information about the basic genetic background of these species, such as karyotype, is necessary to understand the cause of this intensive hybridization. In this study, we established a primary culture from olive ridley sea turtles and determined the karyotype of the primary cells. Since 1976, the San Diego Zoo started the preservation of biological specimens derived from critically endangered species in order to facilitate the use of these specimens as research materials for the following generations; this project was named “Frozen Zoo.” The significance of this project was internationally recognized, and similar projects, such as the Frozen Ark project in the UK, are underway. The establishment of cell cultures from critically endangered animals might contribute to these cryopreservation projects by increasing the stability of cryopreserved materials and facilitating efficient expansion of cultured cells. Since cultured cells have intact genetic information of the critically endangered animals, the cells have a potential to be used for the genetic analysis of future generations. The olive ridley sea turtles were maintained at the Port of Nagoya Public Aquarium. Small (3×3 mm) dermal tissue biopsy specimens were obtained from the flipper-like fin of two olive ridley sea turtles. The tissue biopsy specimens were immediately immersed in the cell culture medium. The biopsy process was supervised by a veterinary doctor of the Port of Nagoya Public Aquarium. For the primary culture, a six-well cell culture dish was coated with type I collagen. The detailed method for collagen coating has been described in our previous study (Fukuda et al. 2012).