Human male fertility is in decline and one reason given for this is the increased incidence of sperm showing chromosomal or DNA damage. Previous work in laboratory and farm animals as well as humans has shown that elevated testicular temperatures increases the incidence of sperm with DNA damage. As part of a study into the effect of temperature on mutation rate in spermatozoa using rams as a model, measurements of sperm DNA damage were also recorded. Twelve mature Dorset x Romney rams, were trained to AV semen collection. Scrotal insulation harnesses were applied (plastic bag with cotton wool insulation inside a rubberised fabric protection cover) to two groups of 6 rams for either a 24 or 48 hour period at day 0. Semen was collected on days -4 and 0 as control samples for each ram before treatment and days 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 43, 45 and 49 post treatment. Semen was diluted in RSD-1 + TNE and snap frozen in liquid nitrogen. Samples were thawed and assessed by the Sperm Chromatin Structure Assay (SCSA) on the flow cytometer. This measures sperm susceptibility to 'in situ' acid denaturation by staining with acridine orange fluorochrome, which fluoresces differentially when binding to double stranded DNA (green) or single stranded DNA (red). The data indicate an increase in the percentage of sperm that have DNA damage, appearing about 21 days post treatment and reaching a maximum at 35 days. The proportion of sperm with moderate to high levels of DNA damage was significantly (P<0.001) greater in the 48 hour treatment group and was still observed at 49 days while most of the 24 hour treated animals had recovered by this stage. Of particular interest was the shift of the entire sperm population, in DNA fragmentation index (ratio of red fluorescence:total (red + green) fluorescence), indicating increased susceptibility of DNA denaturation in all cells. This was accompanied by an increase in DNA stainability (increase of green fluorescence) which is interpreted as being due to an increase in the number of immature sperm cells with less compacted chromatin which results from abnormal or unprocessed protamines. The sperm damage seen after a relatively mild heat treatment indicates possible mechanisms for the adverse effects of modern life styles on male fertility.