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Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat

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Abstract

Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen–thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen–thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

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Fig. 1: Overview of procedures.
Fig. 2: Equipment and instruments required for sperm cryopreservation and embryo transfer.
Fig. 3: Preparation of the sperm suspension and freezing straw.
Fig. 4: Preparation and cryopreservation of the freezing straw containing rat sperm.
Fig. 5: Thawing and preincubation of the cryopreserved rat sperm.
Fig. 6: Preparation of oocytes and IVF procedure.
Fig. 7: Embryo transfer procedure.
Fig. 8: Motility analysis of frozen–thawed rat sperm.
Fig. 9: Fertilized oocytes derived from IVF using cryopreserved sperm from LE rats.
Fig. 10: Blastocysts and live pups (2 weeks old) derived from IVF using cryopreserved LE rat sperm.

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Data availability

The data supporting the study findings are available within the paper and its supplementary information files.

Change history

  • 21 October 2022

    In the version of this article initially published, the label “EGFP” now appearing on the second from bottom row of Table 4 was originally placed on the third from bottom row. The table has been amended in the HTML and PDF versions of the article.

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Acknowledgements

We thank K. Uchiyama (Livestock Improvement Association of Japan, Inc.), H. Yamashiro (Graduate School of Science and Technology, Niigata University) and A. Takizawa (Human & Molecular Genetic Centre, Medical College of Wisconsin) for invaluable advice and discussions. We are grateful to our staff: T. Kondo, Y. Haruguchi, K. Yamashita, E. Ishida, M. Sakaguchi and Y. Deshimaru, for their technical support and helpful discussions. We would like to thank Enago (www.enago.jp) for English language review. This work was supported by a grant from Kyudo Company.

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Authors

Contributions

N.N. designed the work, and N.N., N.M., S.N. and K.Y. contributed to the acquisition of the data. N.N., N.M., S.N. and T.T. wrote the initial draft of the manuscript. S.T. contributed to the illustrations of the procedures. E.N. contributed to the interpretation of the data. T.T. contributed to the analysis, interpretation of the data and revision of the manuscript. R.M. produced the video abstract. All authors approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Corresponding author

Correspondence to Naomi Nakagata.

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Competing interests

The authors declare competing financial interests. The present study was partially supported by a grant of Kyudo Co. Ltd. N.M. is a member of Kyudo Co. Ltd. K.Y. received a grant from Kyudo Co. Ltd. N.N. is a member of collaboration laboratory between Kyudo Co. Ltd and Kumamoto University. T.T., S.N., R.M., S.T. and E.N. declare no financial interests.

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Lab Animal thanks Elizabeth Bryda and Martha Koerner for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, and Supplementary Fig. 1.

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Supplementary Video 1

Animation abstract. The animation abstract introduces the optimized IVF protocol of cryopreserved rat sperm by combining preincubation of sperm with increased concentration of BSA and the removal of cumulus cells that surround the oocytes. This cryopreservation and IVF protocol has the potential to be implemented in several rat strains and will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

Supplementary Video 2

Motility of frozen–thawed rat sperm after 120min preincubation. Frozen–thawed rat sperm recovered motility during the preincubation. Sperm with good motility were observed at the edge of the drop.

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Takeo, T., Nakao, S., Mikoda, N. et al. Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat. Lab Anim 51, 256–274 (2022). https://doi.org/10.1038/s41684-022-01053-5

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