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A whole blood stimulation assay was used to investigate the effects of parity, number of weeks after calving and Gram-positive and Gram-negative bacteria on the ex vivo TNF-α responsiveness of Danish Holstein-Friesian cows of first to third lactation (n = 28). Blood samples were collected in weeks 2, 3, 5 and 8 after parturition and stimulated with Escherichia coli LPS (10 μg/mL), Staphylococcus aureus peptidoglycan (PGN, 10 μg/mL) and dead Escherichia coli, Streptococcus uberis, Staphylococcus aureus, and Streptococcus dysgalactiae at a