322 SPERM PRESERVATION BY FREEZE-DRYING IN ENDANGERED ANIMALS

Abstract

Sperm preservation is a useful tool for conservation of endangered animals. Freeze-drying sperm have been studied as new preservation method in various mammals as samples can be preserved in a refrigerator at 4°C or ambient temperature. Sperm preservation by freeze-drying is the ultimate method by which sperm can be stored that neither required specialised cryoprotectants nor constant supply of liquid nitrogen. We established the freeze-drying method that mouse and rat sperm could be preserved long-term at 4°C after freeze-drying using a simple solution containing 10 mM Tris and 1 mM EDTA (TE buffer; 2012 PLoS ONE 7, e35043; 2012 Cryobiology 64, 211–214). Using this method, the fertility of the chimpanzee, giraffe, and jaguar sperm after freeze-drying were estimated. Ejaculated chimpanzee and giraffe and cauda epididymal jaguar sperm were freeze-dried using TE buffer. Sperm were rehydrated with sterile distilled water after storage at 4°C for 1 month. Sperm with normal shape were injected into mouse oocytes in CZB medium with HEPES, and oocytes were then cultured in vitro for 6 to 8 h in the same media. In all animals, pronuclei and sperm tail were observed into oocytes without artificial activation after injection of freeze-dried sperm. When chimpanzee, giraffe, and jaguar sperm were injected into oocytes, 86% (12/14), 100% (12/12), and 96% (22/23) of oocytes formed 2 distinct pronuclei. This study demonstrated that the sperm of various animals could be decondensed into the mouse oocytes after freeze-drying using the same protocol. A further advantage is that freeze-dried sperm can be transported oversea at ambient temperature. Freeze-drying preservation without using liquid nitrogen can be protected strongly valuable gametes of endangered animals even in the event of unexpected accidents and disaster such as earthquakes and typhoons. Freeze-drying of sperm has been applied as a “freeze-drying zoo” for conservation of endangered animals (http://www.anim.med.kyoto-u.ac.jp/reproduction/home.aspx).