M. Vailati-Riboni1, B. Qadir2, and J. J. Loor1 Abstract #17767 Methionine supplementation modulates the inflammatory response of dairy cow blood neutrophils in response to lipopolysaccharide M. Vailati-Riboni1, B. Qadir2, and J. J. Loor1 1Department of Animal Sciences, University of Illinois, Urbana; 2Veterinary division, Kurdistan region Government, Sulaymaniyah INTRODUCTION RESULTS Methionine (Met) is one of the two most-limiting amino acids for milk production in dairy cow diets. Methionine and some of its metabolites display an immunonutrition role, supporting and boosting the immune system (Grimble and Grimble, 1998; Li et al., 2007). The accepted “optimal” ratio when formulating lactating cow diets is Lys:Met of 3:1. However, blood from cows fed corn silage-based diets without supplemental rumen-protected Met averages ~3.6:1. Gene P-value L:M LPS L:M * LPS Cytokines IL1B 0.41 <0.01 0.83 TNF 0.13 0.44 IL10 0.60 0.08 IL6 0.85 Recognition CXCR1 0.52 0.27 HLA-A 0.37 SELL 0.50 0.30 TLR2 0.19 TLR4 0.71 0.14 0.63 Nuclear receptors   IRAK1 0.45 0.24 NFKB1 0.10 NR3C1 0.29 0.02 0.16 Killing mechanism LYZ 0.70 0.05 MPO 0.43 0.82 SOD1 0.48 Glutathione pathway GPX1 0.07 0.28 GSR DNMT3A 0.04 0.34 MAT1A 0.15 SAHH 0.69 0.53 MATERIALS AND METHODS Blood neutrophils were isolated from 5 mid-lactating pluriparous Holstein cows (238 ± 20 DIM, 33.8 ± 3.9 kg/d average milk production). Neutrophils were pooled together and diluted to 6 x 106 cells/mL. Treatments were allocated in a 3 x 2 factorial arrangement, including: Three Lys:Met ratios (L:M, 3.6:1, 2.9:1, 2.4:1) Two levels of lipopolysaccharide (LPS, 0 and 50 μg /mL) Samples were incubated in triplicate for 2 h, at 37°C and 5% atmospheric CO2. RNA was extracted and use used to measure expression of genes related to immune function and oxidative stress. Data were log2 normalized and subjected to ANOVA using the Proc MIXED procedure of SAS. The model included L:M, LPS, and their interaction as fixed effect. Significance was determined at P < 0.05, and tendency at P < 0.10

Abstract #17767 Methionine supplementation modulates the inflammatory response of dairy cow blood neutrophils in response to lipopolysaccharide M. Vailati-Riboni*, B. Qadir*, and J. J. Loor* *Department of Animal Sciences, University of Illinois, Urbana Figure 1. Effect of LPS stimulation on neutrophil gene expression RESULTS: The LPS effect Stimulation with LPS increased expression of pro- (IL1B, IL6, TNF), and anti-inflammatory (IL10, IL6) cytokines, as well as inflammation-related nuclear factors and receptors (NFKB1, NR3C1). In contrast, LPS stimulation decreased neutrophil expression of pathogen recognition (CXCR1) and killing mechanism-related genes (LYZ). Surprisingly, stimulation of neutrophils with LPS seemed to increase their consumption of Met (greater MAT1A) and decrease their antioxidant capacity (lower GPX1 and GSR). Met GSSG MAT1A GPX1 GSR Hcy GSH Cys

Abstract #17767 Methionine supplementation modulates the inflammatory response of dairy cow blood neutrophils in response to lipopolysaccharide M. Vailati-Riboni*, B. Qadir*, and J. J. Loor* *Department of Animal Sciences, University of Illinois, Urbana Figure 2. Effect of Met supplementation on neutrophils gene expression Figure 3. Effect of Met and LPS stimulation on neutrophil gene expression RESULTS: The MET effect CONCLUSIONS Greater supplementation of methionine, compared with control (3.6:1), decreased expression of inflammatory-related nuclear receptors (IRAK1, NFKB1), while it increased the expression of IL6 (both pro- and anti-inflammatory cytokine). Overall, additional Met decreased the need to regenerate reduced glutathione. When non stimulated, neutrophils cultured with greater amounts of Met had greater expression of the antiinflammatory citokyne IL10 and lysozyme (LYZ). LPS stimulation modulated the immune response as well as oxidative status of circulating bovine neutrophils. Increasing methionine availability enhanced neutrophil defense mechanisms, inflammatory and oxidative status.

Abstract #17767 Methionine supplementation modulates the inflammatory response of dairy cow blood neutrophils in response to lipopolysaccharide M. Vailati-Riboni*, B. Qadir*, and J. J. Loor* *Department of Animal Sciences, University of Illinois, Urbana REFERENCES Grimble, R. F., and G. K. Grimble. 1998. Immunonutrition: role of sulfur amino acids, related amino acids, and polyamines. Nutrition, 14:605-610. Li, P., Y. L. Yin, D. Li, S. W. Kim, and G. Wu. 2007. Amino acids and immune function. British Journal of Nutrition, 98:237-252.