OmniGen-AF® supplementation improves leukocyte responses and hematology in multiparous peripartum cows C.R. Nightingale1, M.D. Sellers1, A.R. Pepper-Yowell1, J.D. Chapman2 and M.A. Ballou1 1Texas Tech University, Lubbock 2Phibro Animal Health Corporation, Quincy, IL 1/12/2019 www.theOmiGenDifference.com

Objective To evaluate changes in indicators of immunological stress both pre- and postpartum in healthy multiparous dairy cows supplemented with OmniGen-AF from dry-off through 28 DIM 1/12/2019 www.theOmiGenDifference.com

Experimental Design Location: Texas Tech U. & a commercial Holstein dairy Design: Control (CT) or basal diets (no OmniGen-AF) vs. basal diets + OmniGen- AF (OG, 56g/h/d) Diets: Diets fed as TMR’s (far-off, close-up negative DCAD, lactation) OmniGen-AF pre-blended with wheat middlings to provide 56g/h/d in a 0.25 lb./d feeding rate OmniGen-AF blend was added on-farm to respective TMR’s after control cows were fed Diets fed beginning at dry-off through 28 DIM 1/12/2019 www.theOmiGenDifference.com

Experimental Design Metrics: Neutrophil response Inflammatory Markers L-selectin (CD62L) expression, oxidative burst capacity Neutrophil to Lymphocyte ratio Inflammatory Markers Haptoglobin Tumor Necrosis Factor-Alpha Blood metabolites NEFA, BHBA, blood urea nitrogen (BUN) Milk & milk components: tested at 30 d intervals Milk yield, 2x milking Milk fat & milk protein (% and lb.) Somatic cell count 1/12/2019 www.theOmiGenDifference.com

Experimental Design Metrics: Neutrophil response Inflammatory Markers L-selectin (CD62L) expression, oxidative burst capacity Neutrophil to Lymphocyte ratio Inflammatory Markers Haptoglobin Tumor Necrosis Factor-Alpha Blood metabolites NEFA, BHBA, blood urea nitrogen (BUN) Milk & milk components: tested at 30 day intervals Milk yield, 2x milking Milk fat & milk protein (% and kg.) Somatic cell count 1/12/2019 www.theOmiGenDifference.com

Experimental Design Cow selection: Data collection: Cows enrolled at dry-off using Dairycomp305 records based on parity (avg. 2.55), expected calving date (-60 ± 3 d) and previous production and health history Only clinically healthy cows that successfully completed the entire trial period were used in the final statistical analyses (CT=24, OG=23) Data collection: Blood samples collected: at dry-off, mid-dry, calving and 14 and 28 DIM Statistics: Linear mixed model with repeat measures using baseline (-60 d) measures as a covariate 1/12/2019 www.theOmiGenDifference.com

Figure 1. Neutrophil surface expression of L-selectin ** Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. L-selectin measures as surface bound protein. Used Ab to measure l-selectin (GMFI = geometric fluoresence intensity). Graph illustrates the CD62L (L-selectin) profiles from OG and control cows from -30 to +28 days post-partum. OG cows had significantly greater levels pre-partum and at calving. These data mirror those reported by Burton et al. for the control cows and reflect the ‘rescue’ effect observed by Mullarky et al. FYI: GMFI = geometric fluorescense intensity **P<0.001 1/12/2019 www.theOmiGenDifference.com

Figure 2. Neutrophil oxidative burst capacity * Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. Graph illustrates oxidative bursts of neutrophils. Oxidative bursts are the release of reactive oxygen species (ROS), chemical compounds used to kill pathogens. Although effective in destroying harmful pathogen, excessive production of ROS can damage healthy tissue (ie: mammary gland). These data suggest that OG is reducing or attenuating this release of ROS thereby potentially avoiding tissue damage while still effectively able to ‘kill’ pathogens. To test for oxidative burst capacity, neutrophils were harvested from OG and control cows and incubated with E. coli until 50% of neutrophils produced an oxidative burst. 1/12/2019 www.theOmiGenDifference.com

Figure 3. Plasma haptoglobin concentrations ** Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. Haptoglobin (Hp) is an acute phase protein produced in response to any inflammation process (infection, stress, burns, injury, etc.) or an immunological challenge (calving). Hp is a measure of systemic inflammation. In this study Nightingale et al., observed Hp response to be less in the OG fed cows verses the controls at d 14 post-partum, suggesting that OG may be attenuating the inflammation response. 1/12/2019 www.theOmiGenDifference.com

Figure 4. Tumor necrosis factor-alpha #P<0.10, **P<0.048 # ** Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. Tumor Necrosis Factor alpha, involved in systemic inflammation, is a member of a group of cytokines that stimulate the acute phase reaction. Produced chiefly by macrophages and other immune cells, CD4+lymphocytes, NK cells and neurons. TNF-alpha primary role is regulation of immune cells. TNF an endogenous pyrogen (fever inducer), apoptoic cell death, cachexia (loss of body mass) and inflammation. 1/12/2019 www.theOmiGenDifference.com

Milk and Milk Components Control OmniGen-AF Milk, lb. First Test 104.3 103.0 Avg. All Tests 98.3 98.8 Fat, % 3.82 3.74 3.37 3.38 Fat, lb. 3.98 3.30 3.32 Protein, % 2.95 2.94 2.92 Protein, lb. 3.06 2.88 No differences were detected between the control or OmniGen-AF fed cows for milk or milk components. The ‘average all tests’ for milk and components is calculated from 5 consecutive test-days (30 through 150 DIM). 1/12/2019 www.theOmiGenDifference.com

Milk and Milk Components Control OmniGen-AF Milk, kg. First Test 47.41 46.82 Avg. All Tests 44.68 44.90 Fat, % 3.82 3.74 3.37 3.38 Fat, kg. 1.81 1.50 1.51 Protein, % 2.95 2.94 2.92 Protein, kg. 1.39 1.31 No differences were detected between the control or OmniGen-AF fed cows for milk or milk components. The ‘average all tests’ for milk and components is calculated from 5 consecutive test-days (30 through 150 DIM). 1/12/2019 www.theOmiGenDifference.com

Milk Yield, lb. www.theOmiGenDifference.com Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. 1/12/2019 www.theOmiGenDifference.com

Milk Yield, kg. www.theOmiGenDifference.com Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. 1/12/2019 www.theOmiGenDifference.com

Somatic Cell Count * * Test-day 120 , SCC’s differed P = 0.024 Reference: J. Dairy Sci., 2013. vol. 96, E suppl 1 (abstract & poster) - OmniGen-AF supplementation improves leukocyte responses and hematology of multiparous peripartum cows. No differences in SCC were detected across all test-days (CT=131,000 cell/ml vs. OG=54,000 cell/ml, P=0.065), however SCC’s at test-day 120 were different (P=0.024). * Test-day 120 , SCC’s differed P = 0.024 1/12/2019 www.theOmiGenDifference.com

Summary Cows fed OmniGen-AF (OG) from dry off through 28 day in milk were observed to have a more functional innate immune response during the transition period as measured by: A greater surface expression of neutrophil L-selectin (P < 0.001) A lower neutrophil to lymphocyte ratio (P<0.001) An elevated plasma concentrations of tumor necrosis factor-alpha (P < 0.10) Haptoglobin concentrations were lower (P < 0.002) at 14 DIM in OG cows, suggesting an attenuation of the inflammation response associated with parturition Milk or milk components were not different. Overall SCC’s tended (P < 0.065) to be lower for OG cows and were different (P < 0.024) at 120 DIM 1/12/2019 www.theOmiGenDifference.com