Effects of Differential Supplementation of Calcium Salts of Fatty Acids (CSFAs) in Lactating Dairy Cows on Plasma Acute Phase Proteins and Leukocyte Responses: Phagocytic and Oxidative Burst, CD62L/CD18 Expression and Cytokine Production.

Abstract

The objective was to determine immune responses to different CSFAs (Virtus Nutrition) supplemented during -30 to 30 days postpartum (dpp; CON [Palm oil, 47% C16:0] vs LN [Safflower oil, 64% C18:2n-6]) and from 30 to 80 dpp (CON [C16:0] vs FO [Fish oil, 11% of C20:5n-3 + C22:6n-3]). Supplementation was at 1.5% of CSFAs in the diet. Plasma samples were collected daily from 0 to 10 dpp for measurements of PGF2α; metabolite (PGFM) and thrice weekly until 30dpp for haptoglobin (HP) and fibrinogen (FB). Vaginoscopy was performed at 8dpp to evaluate cervical discharge. At -35, 0, 4, and 7 dpp, blood samples were collected to evaluate phagocytic and oxidative burst of neutrophils in whole blood (100μl) using a dual color flow cytometry method and expression of the adhesion molecules CD62L and CD18 using monoclonal antibodies against bovine CD62L (L-selectin) and canine CD18 (β2-integrin). Mouse control antibody was used to correct for non-specific binding. All antibodies were conjugated with a goat anti-mouse IgG1:RPE. Flow cytometry was used to acquire and analyze the neutrophil and mononuclear cells populations for CD62L and CD18 mean fluorescence intensity (MFI). At 30 and 80 dpp, neutrophils were isolated, suspended in RPMI, and added (5x105 in 100μl) to 96 well plates in duplicate for non-or LPS (20μl of 1mg/ml) stimulation, volume was completed to 200μl with RPMI for incubation (18h at 37° 5% CO2), and subsequent measurement of TNF-α in supernatant. Frequency of cervical discharge classified as clear or flecks (66/84) and mucopurulent or purulent (18/84) did not differ between CON and LN diets. Plasma concentration of HP and FB were higher (P<0.05) for LN (0.034 OD and 248.8 mg/dl, n=17) compared with CON (0.02 OD and 205.3 mg/dl, n=15). PGFM was not affected by diets (206 pg/ml, n=32). Percent neutrophils that phagocytized with oxidative burst did not differ between diets for samples challenged with E. coli (49%, n=47) or S. aureus (33%, n=39), but increased (P<0.01) from 0 (38%, 23%) to 4 (53%, 38%) and 7 (57%, 34%) dpp, respectively. Neutrophil MFI of CD62L was higher in LN at 4 (P<0.05) and 7 (P<0.08) dpp and increased (P<0.05) from 0 to 4 and 7 dpp in cows across diets (n=45). The MFI of CD62L in mononuclear cells and CD18 in neutrophil and mononuclear cells were not affected by diet or dpp. Number of mononuclear cells positive for CD62L was higher in LN at 4 (P<0.01) and 7 (P<0.01) dpp and increased more in the LN (diet*dpp; P<0.05). Number of mononuclear cells positive for CD18 was not affected by diet, but decreased (P<0.01) from 0 to 4 and 7dpp. Concentration of TNF-α from neutrophil cultures for non-stimulated and LPS at 30dpp was higher (P<0.05) for LN (52.7 and 107 pg/ml, n=17) compared with CON (30 and 63.2 pg/ml, n=16); however, the incremental increase did not differ. Concentration of TNF-α from neutrophil cultures at 80dpp for non-stimulated samples was not affected by diets, but LPS stimulation and mass increase were lower (P<0.01) for FO (42.5 and 4.6 pg/ml, n=14) compared with CON (82.7 and 47.5 pg/ml, n=14), respectively. In summary, feeding CSFAs enriched with 18:2n-6 during the peri-partum elevated both neutrophil CD62L expression and TNF-α production, and increased acute phase proteins in plasma. At 80dpp FO suppressed production of TNF-α. In conclusion, differential immune responses to n-6 or n-3 FAs in dairy cows may serve as a mean to enhance or suppress inflammatory responses according to stage of lactation.