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Objective:To determine the possibility of presence of bacterial biofilm in the uterus of repeat breeder cows and to evaluate the effect of mucolytic agent in cleanup of uterus from biofilm. Methods: Twenty repeat breeder cows were selected from a large commercial dairy farm near Shiraz, Fars province, south Iran. Uterine secretion samples were collected before and after uterine lavage with dimethyl sulfoxide (DMSO) 10% solution and periodic acid Schiff (PAS) staining was used to detect bacterial biofilm in uterine samples. After sampling, all cows were treated with two doses of PGF2 and intrauterine infusion of Cefquinome sulphate. Artificial insemination (AI) was performed after that.Results: Bacterial biofilms were found in 12 out of 20 animals (60%) in the first sampling with sterile saline lavage (before DMSO) and in 7 cows (35%) after DMSO lavage. Fourteen cows (70%) became pregnant after AI. This evidence showed the presence of bacterial biofilm in the uterus of dairy cows for the first time. Although non-significant, decrease in biofilm detection after DMSO lavage may suggest the potential ability of mucolytic agent for cleaning the uterus from bacterial biofilm. Also, high pregnancy rate after antibiotic treatment in the present study might be attributed to improved effect of antibiotic following lavage of uterine by DMSO.Conclusions:These findings should be investigated in future researches with more sample size.