The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10-50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.