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Successful vitrification of whole juvenile testis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens)

  • Shogo Higaki (a1), Natsue Kuwata (a1), Kotaro Tanaka (a1), Ikuo Tooyama (a2), Yasuhiro Fujioka (a3), Noriyoshi Sakai (a4) and Tatsuyuki Takada (a5)...


Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.


Corresponding author

All correspondence to: Tatsuyuki Takada. Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu, Shiga 525–8577, Japan. Tel: +81 77 561 2569. Fax: +81 77 561 2569. E-mail:


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