Development of Germ Cell Isolation and Optimal Cryopreservation Method for Lissachatina fulica (L. fulica)

Abstract

This study aims to develop an optimized method for cryopreserving the germ cells of Lissachatina fulica (L. fulica) using vitrification, as an alternative approach for conserving endangered snail species. First, we isolated several key reproductive organs, including the sperm oviduct, albumen gland, hermaphrodite gland (ovotestis), and hermaphrodite duct from L. fulica. When the ovotestis was finely chopped, numerous sperm with long tails and distinct heads were observed. The staining of sperm nuclei was confirmed using Hoechst 33342 dye. Since the hermaphrodite gland, referred to as the ovotestis, contains both male and female germ cells, we performed tissue staining on the ovotestis using hematoxylin and eosin (H&E) dye. H&E staining of the ovotestis revealed numerous oval-shaped acini containing sperm and early germ cells. Spermatocytes and spermatids were observed within distinct boundaries, with mature sperm appearing following spermatogenesis. To preserve the species of the L. fulica, we introduced vitrification technology to cryopreserve its reproductive organs. The non-vitrification group showed an average cell viability of 96.6%, while the vitrification group had 86.8% after thawing. This study presents a reliable cryopreservation protocol for L. fulica, with potential applications for other endangered snails, supporting conservation efforts to preserve genetic resources and biodiversity.