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To quantify the influence of intravenous regional limb perfusion (IVRLP) with amikacin on bacterial bioburden in limb wounds.In vivo, experimental.Four adult horses.Full thickness wounds created on each dorsal metacarpus were inoculated with Staphylococcus aureus. One forelimb was randomly assigned to cephalic vein IVRLP with amikacin on days 2, 3, and 4 post-wounding. Biopsies on day 2 prior to IVRLP, and on days 5 and 8 were cultured to quantify wound bioburden (CFU/g). Two horses repeated the study in a crossover design, receiving IVRLP on the opposite limb (n = 6 experiments). IVRLP was performed on both forelimbs simultaneously in 3 horses at the end of the study: limbs were perfused with a volume of new methylene blue equal to that of the amikacin, through the cephalic vein or palmar digital vein. After euthanasia, wounds were photographed to subjectively assess dye diffusion.The bioburden did not differ between control and IVRLP limbs overall or at any individual time point. No difference was detected between groups in terms of frequency of positive bacterial growth at any time. Methylene blue was visible in all excised tissues after IVRLP through the palmar digital vein, but was not visible in limbs perfused through the cephalic vein.IVRLP may not effectively concentrate amikacin within a wound bed and did not influence S. aureus bioburden in an experimentally created wound infection.