Preservation of Spermatogonia cell in Shrimp

Abstract

Prawns and shrimp are the most interesting group of resources, they are also one of the most widely traded fishery products on the international market and they are one of the few that can be considered a "commodity," generating significant economic benefits, especially for many developing countries. In Thailand, shrimp culture (such as giant freshwater prawn, banana shrimp and black tiger shrimp) is economically important of market. Nowadays, the number of shrimps in natural is decreased. Therefore, the conservation of shrimp species is important to prevention. Cryopreservation has been used to preserve germ cell of many endangered species. However, this method has not been established for cryopreservation of spermatogonia cell in shrimp. Therefore, in this study aimed to identify the spermatogonia cells developmental stage for cryopreservation and optimize the equilibration time (15, 30 and 60 min) for slow freezing method, type and concentration of cryoprotectants (dimethyl sulfoxide (DMSO), glycerol (GLY), and magnesium chloride (MgCl_2) at 5%, 10%, and 15%), and thawing temperature (10 and 27 °C) for preserve spermatogonia cell of giant freshwater prawn, banana shrimp and black tiger shrimp by compare between slow freezing and vitrification methods for a long-term preservation of spermatogonia cells. The viability and recovery percentage of fresh and frozen spermatogonia cell of shrimp were assessed by staining spermatogonia cell with trypan blue. The result was obtained in equilibration time tested of giant freshwater prawn spermatogonia cells were incubated in cryomedium containing cryoprotectants (10% DMSO, 10% GLY and 10% MgCl_2) and extender for 15, 30 and 60 min, there was not significantly difference. In next experiment, among three cryoprotectants tested, the best result of viability and recovery percentage was obtained with 10% DMSO for spermatogonia cell of giant freshwater prawn and banana shrimp and 10% GLY for spermatogonia cell of black tiger shrimp. The best thawing was found at 10 ᵒC for giant freshwater prawn, banana shrimp and black tiger shrimp. For long-term cryopreservation, the recovery of spermatogonia cells preserved in liquid nitrogen at 6 months with vitrification method were significantly higher than of preserved with slow freezing method in all shrimps and the total cells were observed after preserved not apoptosis during cryopreservation for both methods. This study provide evidence that spermatogonia cell in giant freshwater prawn, banana shrimp and black tiger shrimp can be preserved long-term in liquid nitrogen. Importantly, our study demonstrate that cryopreservation can be successfully performed without requiring no special or expensive equipment (freezing container and -80 ᵒC freezer) by using vitrification method.