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 degrees C and frozen in 0.25 ml French straws. Frozen straws were then thawed at 37 degrees C for 20s 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. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.