The objective was to compare the effects of treating bovine semen with two trypsin products (the porcine pancreas extract and a recombinant) and a control (no trypsin) on in vitro embryo production. Our hypothesis was that the trypsin treatments would not cause any significant difference in fertilization and embryo development as compared to the control. Semen was washed through a gradient system containing a porcine-origin trypsin, a recombinant bovine-sequence trypsin, or the control (no trypsin). Oocytes (n=3036) were collected from abbatoir-derived ovaries, matured for 24h, and allocated into three groups: porcine trypsin (n=1040), recombinant trypsin (n=972), and control (n=1024). Ova were inseminated with 1 x 10(6) motile sperm/mL and cultured for 18-24h. Thereafter, presumptive zygotes were cultured for 7 days in 50 microL G1/G2 micro-droplets under mineral oil. Overall, sperm motility was lower before than after each treatment (mean of 51.4% versus 70.2%, respectively; P<0.001); however, motility was not significantly different among the three groups (porcine-origin trypsin=68.8%, recombinant trypsin=69.0%, and control=72.6%). Similarly, there was no significant difference among these groups for cleavage rates (70.1, 70.9, and 73.9%), or the number of morula/blastocyst stage embryos (53.4, 53.3, and 48.7%). In conclusion, treatment of bovine sperm with either porcine-origin trypsin or recombinant trypsin prior to insemination had no detrimental effects on in vitro embryo development.