J Vet Sci.  2014 Jun;15(2):217-224. 10.4142/jvs.2014.15.2.217.

Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers

• Concha C
• Carretta M
• Alarcon P
• Conejeros
• Gallardo D
• Hidalgo AI
• Tadich N
• Caceres DD
• Hidalgo MA
• Burgos RA

• KMID: 1784642
• DOI: http://doi.org/10.4142/jvs.2014.15.2.217

Abstract

Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response.

Keyword

cow; lactic acidosis; L-selectin; neutrophils; reactive oxygen species

MeSH Terms

Acidosis/chemically induced/immunology/*veterinary
Animals
Blood
Cattle
Cattle Diseases/chemically induced/*immunology
Female
Flow Cytometry/veterinary
*Immunity, Innate
L-Selectin/metabolism
Neutrophils/*drug effects
Oligosaccharides/*pharmacology/toxicity
Platelet Activating Factor/*pharmacology
Reactive Oxygen Species/metabolism
Rumen
L-Selectin
Oligosaccharides
Platelet Activating Factor
Reactive Oxygen Species

Figure

• 

  Fig. 1 Ruminal liquor pH and lactic acid concentrations in the rumen after oligofructose overload. (A) pH of ruminal samples obtained by rumenocentesis at 0, 24 and 48 h. (B) Lactic acid concentrations analyzed by HPLC in ruminal samples obtained at 0, 24 and 48 h. The bar graph shows the mean ± SEM of six different animals. *p < 0.05, **p < 0.01, ***p < 0.001 compared with 0 h.

• 

  Fig. 2 The plasma lactic acid concentration after oligofructose overload. Plasma samples obtained at -72, 0, 8, 16, 24, 32 and 48 h after oligofructose overload were analyzed by HPLC. The bar graph shows the mean ± SEM of six different animals. *p < 0.05, **p < 0.01 compared with 0 h.

• 

  Fig. 3 The effects of oligofructose overload on blood neutrophil reactive oxygen species (ROS) production in vitro. Blood neutrophils isolated from six animals with ruminal acidosis (oligofructose overload) or six animals fed with tap water were treated with vehicle (0.01% ethanol in HEPES buffer) or platelet activating factor (PAF; 100 nM), and ROS production was measured by a luminol chemiluminescence assay. The PAF-induced ROS production in neutrophils isolated from heifers treated with tap water (A, C, and E) or oligofructose (B, D, and F) 72 h before treatment or 24 h and 48 h after oligofructose overload is shown. The bar graph of basal ROS production (without PAF induction or negative) or PAF-induced ROS production in the neutrophils (positive) isolated from heifers at -72, 0, 8, 24, 32 and 48 h after being provided with tap water (G) or after oligofructose overload (H) are shown. The bar graph shows the mean ± SEM of six different animals. *p < 0.05, **p < 0.01. RLU: relative luminescence units.

• 

  Fig. 4 Changes in L-selectin expression in bovine neutrophils over the course of acute ruminal acidosis. Neutrophils isolated from animals treated with tap water or oligofructose were stimulated in vitro with vehicle (0.01% ethanol in HEPES buffer) or PAF (100 nM) for 30 min at 37℃. Afterwards, the cells were incubated with anti-L-selectin coupled with phycoerythrin, and fluorescence was analyzed by flow cytometry. In A, representative flow cytometry histograms of PAF-induced L-selectin changes in neutrophils isolated from heifers at 0, 8, 16 and 24 h after oligofructose or tap water treatment are depicted. The bar graph represents the mean of PAF-induced L-selectin changes represented as the fold change relative to the control ± SEM from six independent experiments with tap water (B) or oligofructose (C). *p < 0.05, **p < 0.01.

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