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Artificial insemination programs for horses usually involve ex vivo handling and transporting of sperm. The present experiment was designed to: (i) assess the effect of transportation on sperm DNA integrity at different time post semen collection, and (ii) evaluate if sperm DNA quality deteriorates rapidly beyond 24 h of cooled storage. After collection, the ejaculates were extended using INRA 96 and semen was prepared for prompt analysis (A0) or 24 h/48 h cooled-shipping (B24 and C48 respectively). Each sample was assessed for sperm DNA fragmentation index (SDFI) at time 0 and after incubation for 2, 6 and 24 h at 37 °C. There was very little difference in SDFI between freshly extended (A0) and 24 h/48 h cooled-transported semen samples (B24/C48) at time 0. After 2 h of incubation at 37 °C, there was an increase in SDFI ranging from 2.7% to 7.5% per hour in freshly extended semen samples (A0: 5 …