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The CARD Method for Mouse Sperm Cryopreservation and In Vitro Fertilization Using Frozen-Thawed Sperm

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Microinjection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1874))

Abstract

In the last few years sperm cryopreservation was rapidly established as the technique to efficiently manage production, preservation, and transportation of genetically engineered mice. However, occasionally, the reduced fertility of the frozen-thawed mouse sperm can make it difficult to revitalize the mouse by in vitro fertilization (IVF). In particular, the frozen-thawed sperm of C57BL/6 mice, widely used as the background of choice for genetically engineered strains, show very low fertility after freezing and thawing. To overcome this problem, we have developed a new protocol for sperm cryopreservation and IVF with frozen-thawed C57BL/6 sperm as well as other mouse strains. This protocol has the following three modifications: (1) addition of l-glutamine to the sperm cryoprotectant, (2) addition of methyl-β-cyclodextrin to the sperm preincubation medium, and (3) addition of reduced glutathione to the fertilization medium. These modifications greatly enhanced the fertility of frozen-thawed C57BL/6 sperm, resulting in a stable fertilization rate >80% in IVF. Our results indicate that this robust protocol for sperm cryopreservation may improve the archiving and distributing system for genetically engineered mice.

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Acknowledgments

The authors thank our CARD research group members Kiyoko Fukumoto, Yukie Haruguchi, Tomoko Kondo, Yuko Nakamuta, Yumi Takeshita, Hiroko Matsunaga, Manami Nishimura, Tomoko Umeno, Eri Ishida, Fumi Takahashi, Mari Iwamoto, Eri Kohagura Tomoko Matsumoto and Shuuji Tsuchiyama, my students: Aki Tsutsumi, Taichi Omaru, Kazuhito Sakoh, Yuta Ishizuka, Satohiro Nakao, Hidetaka Yoshimoto, Yuki Sakai, and Yuka Horikoshi, Ayumi Mukunoki for their technical assistance and helpful discussions.

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Authors and Affiliations

  1. Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan

    Toru Takeo, Jorge Sztein & Naomi Nakagata

Authors
  1. Toru Takeo
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  2. Jorge Sztein
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  3. Naomi Nakagata
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Corresponding author

Correspondence to Toru Takeo .

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Editors and Affiliations

  1. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Chengyu Liu

  2. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Yubin Du

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Takeo, T., Sztein, J., Nakagata, N. (2019). The CARD Method for Mouse Sperm Cryopreservation and In Vitro Fertilization Using Frozen-Thawed Sperm. In: Liu, C., Du, Y. (eds) Microinjection. Methods in Molecular Biology, vol 1874. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8831-0_14

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  • DOI: https://doi.org/10.1007/978-1-4939-8831-0_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8830-3

  • Online ISBN: 978-1-4939-8831-0

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