LAUSR

&NA; Preselection of sex before conception has been one of the objectives pursued by scientists and breeders for many years. The donkey milk industry has shown a greater commercial interest, since jennies' demand is increasing nowadays. Therefore, to get a female animal, the oocyte must be fertilized with previously sexed semen. The current technique used for sperm sexing separates spermatozoa containing the X or Y chromosome based on each cell DNA content. However, this technique exposes spermatozoa to high‐pressure speed flow, DNA fluorescent stain, and UV light, factors that may affect sperm quality. The aim of this study was to test a new technique to isolate spermatozoa carrying the X chromosome by means of magnetic nanoparticles (MNPs). Results show that the MNP technique is highly effective in select X spermatozoa, without affecting several physiological sperm parameters. In conclusion, the MNP technique provides an X sperm population with similar sperm physiological state than the control sample. The new sexing technology presented here can be mainly applied to improve the donkey's milk industry. HighlightsMagnetic nanoparticle technique for sperm sexing may be applied in animal‐assisted reproduction.Magnetic nanoparticles are effective for sex‐sorting donkey spermatozoa.Magnetic nanoparticle technique may be applied for jennies' production by donkey‐assisted reproduction.