Kinetics of whole blood oxidative responses to in vivo and ex vivo lipopolysaccharide challenge in dairy cows

Abstract

Immune adaptation in dairy cows depends on the ability to manage inflammatory challenges without compromising productivity. The objective of this study was to evaluate how a systemic lipopolysaccharide (LPS) challenge alters the timing and magnitude of whole blood oxidative responses and their ex vivo reaction to LPS stimulation. Eight multiparous Holstein cows (646 ± 85 kgBW, 3.0 ± 1.4 BCS, 17 ± 9 days postpartum) received an i.v. LPS challenge (E. coli O55:B5; 62.5 ng/kgBW; LPS). Blood samples were collected at 0, 3, 6, and 24 h post-infusion. Cows were fed a TMR (18% CP and 36% NDF at DM) at 100% of nutrient requirements and milked twice daily. Infusion with LPS reduced DMI by 36% and milk yield by 13% while increasing rectal temperature (maximum mean 40.3°C at 3 h), heart rate (103 beats/min), and respiratory rate (70 breaths/min). Reactive oxygen species (ROS) production was assessed in whole blood using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and fluorescence was recorded kinetically and summarized as area under the curve. Incubation conditions used to separate basal fluorescence, probe background, and ex vivo LPS-stimulated oxidative responses were: whole blood alone, with LPS, with DCFH-DA, and with both DCFH-DA and LPS. Fluorescence values were background-corrected, and fold change relative to baseline was calculated. Mixed models (GLIMMIX, SAS) were used, with cow as a random effect and time as a fixed effect. Basal fluorescence increased over time, reaching its maximum at 24 h following the in vivo LPS challenge (P < 0.05), whereas the combined in vivo and ex vivo LPS stimulation resulted in an earlier peak, observed at 6 h (P < 0.0001). Fold change did not differ among time points (P = 0.10), indicating that systemic inflammation primarily determined baseline oxidative activity. Ex vivo stimulation accelerated the timing of responses without increasing overall oxidative output. These results indicate prior systemic inflammation affects kinetics but not magnitude of oxidative responses in whole blood, consistent with immune adaptation.