Latrodectus mactans aracnotoxin (Atx) induces changes in sperm function that could be used as a co-adjuvant in male contraceptive barrier methods. This effect includes the suppression of intracellular reactive oxygen species (ROS), an event necessary for capacitation, chemotaxis and acrosome reaction (AR). The sperm that are not trapped by the barrier method can reach the oviduct before fertilisation and be exposed to the secretions of the oviducts. This study evaluated the effect of bovine tubal explants (TU) and conditioned media (CM) from the ampullar and isthmal regions on spermatozoa exposed to Atx. Thawed bovine sperm were incubated with Atx, TU and CM from the ampullar and isthmal regions for 4h and then DNA integrity, intracellular ROS and lysophosphatidylcholine-induced AR were determined. Spermatozoa exposed to Atx and co-incubated with TU and CM for 4h produced an increase in sperm DNA damage, a decrease in ROS production and a decrease in %AR, compared with the control. A similar result was obtained from the co-incubation of spermatozoa with Atx. In conclusion, the effect of Atx is not modified by tubal cells or their secretions and this opens the door to future studies to evaluate the application of synthetic peptides obtained from Atx as a co-adjuvant of contraceptive barrier methods.