The objective of the current study was to evaluate the effects of antioxidant ergothioneine added to cryopreservation extender on the DNA integrity of Merino ram sperm. Semen samples from 5 mature Merino rams (1 and 2. years of age) were used in the study. Semen samples, which were diluted with a Tris-based extender containing ergothioneine at different concentrations and no antioxidant (control), were cooled to 5. °C and frozen in 0.25. ml French straws. Frozen straws were then thawed at 37. °C for 20. s in a water bath for evaluation of sperm DNA damage using the Comet test. The addition of ergothioneine at concentrations of 1, 2 and 4. mM resulted in lower sperm with damaged DNA (5.4, 4.7 and 3.2%, respectively) than that of control (7.9%), thus reducing the DNA damage (P<0.01). Findings of this study showed that the increasing concentrations of ergothioneine in semen extenders, were of greater benefit to DNA integrity of frozen-thawed ram sperm. © 2012.