Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

DOI Web Site Web Site PubMed 被引用文献14件 参考文献47件
  • HASEGAWA Ayumi
    RIKEN BioResource Center, Ibaraki 305-0074, Japan
  • YONEZAWA Kazuya
    RIKEN BioResource Center, Ibaraki 305-0074, Japan Department of Life Science, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan
  • OHTA Akihiko
    Department of Life Science, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan
  • MOCHIDA Keiji
    RIKEN BioResource Center, Ibaraki 305-0074, Japan
  • OGURA Atsuo
    RIKEN BioResource Center, Ibaraki 305-0074, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki 305-8572, Japan The Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan

この論文をさがす

抄録

The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 μl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.

収録刊行物

被引用文献 (14)*注記

もっと見る

参考文献 (47)*注記

もっと見る

キーワード

詳細情報 詳細情報について

問題の指摘

ページトップへ