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Reproductive Characteristics and Suitability of Sterile dead end Knockout Nibe Croaker as a Recipient for Intraperitoneal Germ Cell Transplantation

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Abstract

The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10 days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6 months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.

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Data Availability

The data supporting the findings of this study are available upon request.

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Funding

This work was partly supported by JSPS KAKENHI Grant Number 23H00344 and JST-Mirai Program Grant Number JPMJMI21C1.

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Authors and Affiliations

  1. Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo, 108-8477, Japan

    Ryosuke Yazawa, Kyoichiro Saitoh, Akihiro Yamauchi, Onur Eyüboğlu, Kana Ozawa & Goro Yoshizaki

  2. Institute for Reproductive Biotechnology for Aquatic Species, Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo, 108-8477, Japan

    Ryosuke Yazawa, Tetsuro Morita & Goro Yoshizaki

  3. Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto, 860-8555, Japan

    Wataru Kawamura

  4. Faculty of Biological Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa, 920-1192, Japan

    Yutaka Takeuchi

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  1. Ryosuke Yazawa
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Contributions

K. S., A. Y., O. E., K. O., W. K., and R. Y. conducted experiments and analyzed the data. R. Y., Y. T., T. M., and G. Y. designed the research. R. Y. and G. Y. conceived the study and wrote the manuscript. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report.

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Correspondence to Ryosuke Yazawa.

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Yazawa, R., Saitoh, K., Yamauchi, A. et al. Reproductive Characteristics and Suitability of Sterile dead end Knockout Nibe Croaker as a Recipient for Intraperitoneal Germ Cell Transplantation. Mar Biotechnol (2024). https://doi.org/10.1007/s10126-024-10323-x

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