Re-suspension of frozen-thawed ram sperm with ovine and bovine whole seminal plasma (SP) is beneficial for its post-thawing viability. However, neither the influence of SP incubation duration nor the re-suspension with equine SP has been tested. In the first experiment, frozen-thawed ram sperm were incubated with SP from bovine (BSP), equine (ESP) or ovine (OSP) or without SP (-SP) for short (5 min) or long (6 h) periods. Viability parameters such as sperm progressive motility, percentage of live cells and membrane functionality were assessed every 2 h for 6 h. All SP treatments showed higher spermatozoa viability than the -SP treatment in most evaluations. Incubation time did not affect sperm viability for BSP treatment, however, ESP and OSP induced a transitory benefic effect in the short incubation period and detrimental effect in the longer period. In the second experiment, frozen-thawed sperm, with or without short incubation with SP, were selected by swim-up, and their DNA fragmentation rate was assessed using comet assay immediately after swim-up completion and after 5 h of incubation. BSP, ESP and OSP protein profiles were determined by SDS-PAGE. Only ESP was associated with sperm DNA stabilization capacity and SP from rams and bulls showed protein profiles different from that of stallions. These experiments indicate that equine or ovine, but not bovine whole SP supplementation to post-thawing incubation medium of frozen-thawed ram sperm affects its viability in a time dependent manner. The beneficial effect of ESP on stabilizing DNA integrity, even after sperm washing with swim-up method and incubation for 5 h, can be determined by SPP or by antioxidant components from SP.