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.

不同育种者对牲畜的偏好性别有所不同。例如，奶农更喜欢雌性犊牛来生产牛奶，而肉类生产者通常更喜欢雄性牛。在某些研究领域，包括生殖生物学和代谢研究中，对实验动物进行性交也是有益的。大多数性别鉴定方法都是使用细胞分选仪将X精子和Y精子分开。在这里，我们描述了一种系统，其中用TLR7 / 8配体（R848）进行处理可将X精子与Y精子分开。由于此协议不需要任何特殊设备或专业技能，因此可以轻松地应用于进行体外受精（IVF）的实验室。用0.03 µM R848在1 mL改良人输卵管液（mHTF）培养基（小鼠精子）或3 mL mHTF培养基（公牛精子）中处理精子60分钟，然后分别收集上层（小鼠精子中400 µL或公牛精子中1 mL）和沉淀物。通过离心洗涤每个样品后，将精子悬浮在无配体的IVF培养基中，然后用于IVF。在小鼠和牛中，上层精子产生的胚胎中有90％以上都是XY，在这两个物种中，沉淀精子产生的胚胎中> 80％是XX。X精子和Y精子的IVF分离可以在2小时内完成。