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Acute puerperal metritis (APM) is one of the most common diseases during the puerperal period. Systemic administration of ceftiofur for 5 consecutive days has been shown to be effective for treatment of APM. The objective of this study was to determine concentrations of ceftiofur derivatives in serum, endometrial tissue, and lochia of cows with fever postpartum or APM 4 to 6d after treatment with a single subcutaneous dose of 6.6 mg of ceftiofur crystalline free acid (CCFA)/kg of estimated BW at the base of the ear. In the first experiment, samples from CCFA-treated cows with fever postpartum or APM (n=42) were taken on d 4, 5, or 6 after treatment. Concentrations of ceftiofur derivatives were quantified using an HPLC assay. Concentrations of active ceftiofur metabolite desfuroylceftiofuracetamide (DCA) were greatest at d 4 after treatment with CCFA in all samples, but they were considerably lower than the concentrations of DCA in healthy postpartum cows treated with the same dose of CCFA. The concentrations of DCA in serum, endometrial tissue, and lochia were affected by odor of vaginal discharge before treatment with CCFA. Mean concentrations of DCA could be detected above the reported minimal drug concentrations (minimum inhibitory concentrations, MIC) required to inhibit relevant pathogens such as Escherichia coli and Arcanobacterium pyogenes in serum on all days and in endometrial tissue and lochia only on d 4 in CCFA-treated cows with fetid vaginal discharge before treatment. In the second experiment, samples from CCFA-treated cows with APM (n=8) were taken on d 0 (before treatment) and d 4, 5, and 6 after treatment. Mean concentrations of DCA in serum and lochia were similar on d 4 to 6 in both laboratories. Furthermore, determined concentrations of DCA from both laboratories were correlated for serum and lochia. Mean concentrations of DCA could be detected above the reported MIC in serum and lochia only on d 4. Our 2 experiments demonstrated that in postpartum cows with fever postpartum or APM concentrations above the MIC for relevant bacteria (>0.5 μg/mL or >0.5 μg/g) of DCA could be sustained only for 4 (serum: 15/17; endometrial tissue: 2/17; lochia: 1/16) to 5d (serum: 10/13; endometrial tissue: 1/13; lochia: 2/12) after a single treatment with CCFA only in a certain proportion of cows. Overall, our data provide first pharmacological evidence that a single subcutaneous administration of 6.6g of CCFA/kg of BW might not be sufficient to efficaciously treat APM in postpartum dairy cows.