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

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.