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Background Amniotic membrane extract enhances the rate of epithelialisation after corneal ulceration in several species but has not been studied in the equine cornea. Objectives To evaluate the effect of amniotic membrane extract on re-epithelialisation of equine corneal ulcers compared to ulcers treated with antibiotic, antifungal, and mydriatic medical therapy alone; to evaluate equine corneal healing after experimentally induced superficial ulceration. Study design Masked, randomised, controlled experimental trial. Methods Superficial, 8 mm corneal ulcers were created bilaterally in each horse. One eye was treated with amniotic membrane extract; the opposite was control. Both eyes were treated with medical therapy. Treatment eyes received amniotic membrane extract, and control eyes received the amniotic membrane extract vehicle. Ulcers were stained with fluorescein and photographed in 12-hour increments until completely healed. Ulcer surface area was determined by analysing photographs with ImageJ. A mixed linear model was used to compare ulcer surface area and hours until healing between treatment groups. A regression model was also used to calculate corneal re-epithelialisation rate over time. Results Regardless of therapy, healing occurred in two phases: an initial rapid phase of 0.88 mm2 /hr (95% CI: 0.81 - 0.94 mm2 /hr) for approximately 48-54 hours followed by a second, slow phase of 0.07 mm2 /hr (95% CI: 0.04 - 0.09 mm2 /hr). Most eyes healed within 135.5 ± 48.5 hours. Treatment (amniotic membrane extract vs. control) was not significantly associated with size of ulcers over time (P=0.984). Discomfort was minimal to absent in all horses. Main limitations Results achieved experimental studies may differ from outcomes in the clinical setting. Conclusions There was no significant difference in healing rate with addition of amniotic membrane extract to medical therapy for equine superficial corneal ulcers. A biphasic corneal healing process was observed, with an initial rapid phase followed by a slow phase. Further study will be needed to determine if amniotic membrane extract will be helpful for infected or malacic equine corneal ulcers.