The present study investigated the effect of a novel density gradient centrifugation (DGC) treatment using recombinant trypsin on the in vivo fertilising capability of bovine spermatozoa compared with a standard method. In Trial 1, semen collected from Boran and Ankole (Bos indicus) bulls was treated either with a silane-coated silica particle colloid formulated for humans with a recombinant trypsin or processed using a standard method (dilution in an egg yolk-based diluent). Semen processed by the two methods was used to artificially inseminate (AI) superovulated cattle. Day 7 embryos were flushed and assessed for fertilisation rates and embryo quality. Trial 2 used a trypsinised silane-coated silica particle colloid formulated specifically for bovine semen. Trial 1 resulted in significantly higher fertilisation rates using the trypsinised human DGC treatment than cows inseminated using the standard method (75.2% v. 67%, respectively; P < 0.01), but the numbers of transferable-quality Day 7 embryos did not differ between the two groups (P > 0.05). Results for Trial 2 indicated that cows inseminated with the trypsinised bovine DGC treatment had significantly increased fertilisation rates compared with the standard method (88.4% v. 63.1%, respectively; P < 0.01) and had significantly higher numbers of transferable-quality embryos (70.3% v. 51.8%, respectively; P < 0.01). In summary, bovine sperm treatment before AI by DGC and recombinant trypsin increases fertilisation rates and can result in more transferable-quality embryos compared with standard methods.