Whole blood optimization and genetic association of ex vivo TNF-α responsiveness to killed E. coli in Danish Holstein cows

Abstract Whole blood stimulation assay (WBA) has been widely used to study production of the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α) and is considered a relatively good predictor for the in vivo release of TNF-α during endotoximia in dairy cattle. However, it requires a standardized and optimized dose of lipopolysaccharides (LPS) to simulate the in vivo cytokine response during WBA when using killed bacteria. Moreover, if an association between genome (e. g., Quantitative trait loci) and ex vivo TNF-α responsiveness can be established; it helps to reduce mastitis in dairy cattle using selective breeding. Three experiment scenarios for pathogen dose, stimulation time and estimation of genetic variables effect in ex vivo TNF-α production were carried out to investigate differences of TNF-α responsiveness in early lactation in 4, 38 and 40 Danish Holstein-Friesian dairy cows respectively. Statistical analysis done using ANOVA, t.test and lm functions in R software. Results showed that, ex vivo TNF-α response to E. coli is dose and time dependent and a dose of 2.5×10 6 CFU/mL for 3.5 h is optimal for observing TNF-a response after ex vivo stimulation. Relatively consistent inter-individual cow differences in the TNF-α response ex vivo were observed from week 3 to week 8. No statistically significant association were found between the ex vivo TNF-α responsiveness and the two E. coli associated mastitis QTLs which may be due to the low number of animals (n=) in the study.