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A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos

Nature Protocols volume 15, pages 2645–2667 (2020)Cite this article

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Abstract

The preferred sex of livestock differs among breeders; for example, dairy farmers prefer female calves for the production of milk, whereas cattle meat producers often prefer males. Sexing of laboratory animals is also beneficial in some research fields, including reproductive biology and metabolic studies. Most sexing methods separate X sperm and Y sperm with a cell sorter. Here, we describe a system in which treatment with the TLR7/8 ligand (R848) separates X sperm from Y sperm. Because this protocol does not require any special equipment or professional skills, it can be easily applied in laboratories where in vitro fertilization (IVF) is performed. The sperm are treated with 0.03 µM R848 in 1 mL of modified human tubal fluid (mHTF) medium (mouse sperm) or 3 mL of mHTF medium (bull sperm) for 60 min, and then the upper layer (400 µL in mouse sperm or 1 mL in bull sperm) and the precipitate are separately collected. After each sample is washed by centrifugation, the sperm are suspended in ligand-free IVF medium and can then be used for IVF. More than 90% of the embryos made with upper-layer sperm are XY in both mice and cattle, and >80% of the embryos made with precipitated sperm are XX in both species. Separation of X sperm and Y sperm for IVF can be completed within 2 h.

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Fig. 1: Effects of R848 on sperm with different glucose concentrations.
Fig. 2: Effect of washing after sperm separation by R848 on the successful fertilization rate.
Fig. 3: The addition of creatine increases the swim-up ratio, fertilization ability and ratio of successful XY embryo production.
Fig. 4: The expression of TLR7 in murine sperm before/after treatment with R848.
Fig. 5: Expression of TLR7 in frozen bull sperm.
Fig. 6: Effect of R848 on frozen−thawed bull sperm.
Fig. 7: The effects of different R848 solvents, water and DMSO, on sperm motility.
Fig. 8: Overview of a novel frozen–thawed bull sperm separation method using R848.
Fig. 9: Overview of a novel mouse sperm separation method using R848.
Fig. 10: Method for collection of mouse sperm.
Fig. 11: Method for the collection of mouse COCs.

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Data availability

All raw data except those from the flow cytometry are available at Figshare (https://doi.org/10.6084/m9.figshare.12152712), and in the Supplementary Data. Other data are available on request from the authors.

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Acknowledgements

This work was supported in part by the Livestock Promotional Funds of the Japan Racing Association (JRA) and by the Hiroshima Cryopreservation Service Co. (M.S.).

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Authors and Affiliations

  1. The Research Center for Animal Science, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan

    Takashi Umehara, Natsumi Tsujita, Zhendong Zhu & Masayuki Shimada

  2. Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Bungo-Ono, Japan

    Moeka Ikedo

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  1. Takashi Umehara
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Contributions

T.U., N.T., Z.Z. and M.S. developed this protocol. Bovine IVF was performed by M.I. T.U. made the figures and wrote the manuscript. M.S. supervised all aspects of this study and wrote the manuscript

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Correspondence to Masayuki Shimada.

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Competing interests

Hiroshima University and Oita Prefecture have filed a provisional patent application on the mammalian sperm sexing technique using TLR7/8 ligands; the inventors on this application are T.U., M.I. and M.S.

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Peer review information Nature Protocols thanks Manuel Hidalgo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Umehara, T., Tsujita, N. & Shimada, M. et al. PLoS Biol. 17, e3000398 (2019): https://doi.org/10.1371/journal.pbio.3000398

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Umehara, T., Tsujita, N., Zhu, Z. et al. A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos. Nat Protoc 15, 2645–2667 (2020). https://doi.org/10.1038/s41596-020-0348-y

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