In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and immotile spermatozoa after their injection into ovulated mouse oocytes. In both groups, injected horse spermatozoa activated the mouse oocytes. This was followed by the extrusion of the second polar body (2 PB) and the formation of maternal pronuclei (Mo-fPN- mouse female pronucleus); in parallel, the horse sperm heads rapidly decondensed in the murine cytoplasm and formed paternal pronuclei (Ho-mPN- horse male pronucleus), which were larger than the female ones. With the exception of one stallion tested, DNA damage has been detected in almost all Ho-mPNs originating from immotile spermatozoa. DNA in motile (even sporadically) spermatozoa was mostly undamaged. Moreover, when the xenogeneic zygotes cleave to the two-cell stage, the incidence of micronuclei in blastomeres mirrors the extent of DNA damage in paternal pronuclei. In conclusion, and contrary to the mouse, where sperm DNA is very resistant to damage, we do not recommend the use of immotile horse spermatozoa for ICSI. On the other hand, even the sporadically motile mouse spermatozoa have no damaged DNA and can thus be used for intragenic ICSI.