Temporal impact of metritis and systemic antibiotics on immune cell function inferred from the blood leukocyte transcriptome

Approximately one-third of dairy cows develop metritis within 2 weeks postpartum, a uterine infection associated with reduced fertility and increased risk of reproductive culling. Antibiotics improve clinical cure and milk production following metritis but do not consistently increase subsequent fertility postpartum compared with untreated control. To better understand why antibiotic treatment improves clinical recovery but not fertility, we tracked changes over time in blood leukocyte gene expression to examine circulating immune cell function during the resolution of metritis in antibiotic-treated and untreated cows. Twenty-three primiparous Holstein cows diagnosed with metritis (n = 11) or classified as healthy controls (n = 12) between 7 and 10 d postpartum were enrolled. Cows were assigned to receive systemic ceftiofur (1.25 g/d for 3 d) or remain untreated in a 2 × 2 factorial design. Blood samples were collected at diagnosis (wk 1), and at 1 and 3 wk after treatment (wk 2 and wk 4) postpartum. The blood leukocyte RNA was isolated and sequenced and differentially expressed genes were analyzed for functional enrichment and immune cell type. Healthy cows exhibited enrichment of B cell activation and receptor signaling pathways, whereas metritis cows showed upregulation of inflammatory and innate immune responses, including monocyte and neutrophil markers. In untreated cows (healthy versus metritis), transcriptomic differences in blood leukocytes were resolved by wk 4 postpartum. In contrast, antibiotic-treated metritis cows displayed a distinct immune trajectory. At wk 2 postpartum (one wk after antibiotic treatment), cytotoxic T cell and natural killer cell gene expression signatures increased, differing clearly from untreated metritis cows. By wk 4, a second wave of inflammatory cell-associated differentially expressed genes emerged exclusively in antibiotic-treated metritis cows, suggesting prolonged immune cell activation. These findings demonstrate that antibiotic treatment reshapes systemic immune dynamics during recovery from metritis. The altered sequence (e.g., early cytotoxic activation followed by extended inflammation) may explain why antibiotic-induced clinical cure in metritis cows does not consistently translate into improved fertility.