Dietary management of hypocalcemia through the use of a synthetic zeolite A

Dietary management of hypocalcemia through the use of a synthetic zeolite A A. L. Kerwin, C. M. Ryan, B. M. Leno, M. Jakobsen, P. Theilgaard, and T. R. Overton Cornell Nutrition Conference October 18, 2018 Charlie Sniffen Graduate Student Research Presentation Peripartum calcium function Skeletal muscle function Risk Down Cow / Clinical Milk Fever X X Curtis et al., 1983; Goff and Horst, 1997; Kimura et al., 2002; Kimura, et al., 2006; Goff, 2008; Chapinal et al., 2011; Ribeiro et al., 2011; Chapinal et al., 2012; Martinez et al., 2012 Peripartum calcium function Smooth muscle function and motility Risks DA DMI Ketosis Milk Produc on X Peripartum calcium function X Placental expulsion Risks Metritis Poorer Reproductive Performance Curtis et al., 1983; Goff and Horst, 1997; Kimura et al., 2002; Kimura, et al., 2006; Goff, 2008; Chapinal et al., 2011; Ribeiro et al., 2011; Chapinal et al., 2012; Martinez et al., 2012 Curtis et al., 1983; Goff and Horst, 1997; Kimura et al., 2002; Kimura, et al., 2006; Goff, 2008; Chapinal et al., 2011; Ribeiro et al., 2011; Chapinal et al., 2012; Martinez et al., 2012

Peripartum calcium function Peripartum calcium function Immune cell activation X Teat sphincter contraction Risks Mastitis Milk Production Risk Immune Function Oxida ve Stress?? Curtis et al., 1983; Goff and Horst, 1997; Kimura et al., 2002; Kimura, et al., 2006; Goff, 2008; Chapinal et al., 2011; Ribeiro et al., 2011; Chapinal et al., 2012; Martinez et al., 2012 Curtis et al., 1983; Goff and Horst, 1997; Kimura et al., 2002; Kimura, et al., 2006; Goff, 2008; Chapinal et al., 2011; Ribeiro et al., 2011; Chapinal et al., 2012; Martinez et al., 2012 Peripartum calcium function Smooth DA, Ketosis, muscle Decreased function and Milk Production motility Skeletal Down muscle Cow Clinical function Milk Fever Immune Decreased cell Immune activation Function X X X XX Metritis, Placental Poorer Reproductive expulsion Performance Teat Mastitis sphincter contraction Ca status during the transition period Plasma Total Calcium (mg/dl) 10.5 9.5 8.5 7.5 6.5 5.5 18 13 8 3 2 7 12 Day Relative to Calving Curtis et al., 1983; Goff and Horst, 1997; Kimura et al., 2002; Kimura, et al., 2006; Goff, 2008; Chapinal et al., 2011; Ribeiro et al., 2011; Chapinal et al., 2012; Martinez et al., 2012 Sweeney et al., 2015. J. Dairy Sci 98 (Suppl. 2):128.

Blood calcium mobilization PARATHYROID GLAND KIDNEY PTH Calcium excretion PTH secretion Activation of Vitamin D Mg Hypocalcemia prevalence Mg PTH BONE + + Low blood calcium P + Active Vitamin D INTESTINE Ca absorption Release of Ca Modified slide from B. M. Leno; Goff et al., 2008 Reinhardt et al., 2011 SCH Classification Chronic hypocalcemia (csch) Currently no standard; Literature cut-point = 1.85 to 2.15 mmol/l (7.4 to 8.6 mg/dl) for total Ca Literature sampling timing = 0 to 7 DIM Chamberlin et al. (2013) and Neves et al. (2017, 2018) found no association of SCH with RP or metritis when sample was collected within 24 h Sampling at 2 DIM for multiparous animals had increased risk of metritis and/or DA (Neves et al., 2018) Neves et al. (2017, 2018) found: Multiparous cows with lower Ca concentrations within 12 h of calving produced more milk Multiparous cows with higher Ca had an increased risk of being culled 20% 46% 34% 8.6 Parity csch Incidence 1 20% 2 32% 3 46% Ca Status % Pregnant OR P value at 1 st Service Eucalcemic 63 a Ref. SCH 44 a,b 0.46 0.2 csch 31 b 0.27 0.03 Caixeta et al., 2017

Hypocalcemia effect on pregnancy rate Preventative nutritional strategies SCH 8.6 mg/dl = no SCH = SCH = csch P > 0.2 SCH 8.56 mg/dl P = 0.06 1) Altering the prepartum DCAD Induces a metabolic acidosis Increases tissue sensitivity to PTH Decreases SCH, especially in older cows, however the best outcomes require urine ph monitoring, and non-commercial anion sources have palatability issues 2) Feeding a low calcium prepartum ration Induces PTH response Current US feedstuffs makes this difficult to achieve Actualize a low Ca approach by feeding a synthetic zeolite A Martinez et al., 2012; Caixeta et al., 2017 Synthetic Zeolite A Sodium aluminum silicate Capable of binding to dietary Ca, P, and Mg Shown to increase active form of vitamin D prepartum and improve calcium status Zeolite A feeding recommendations Alternative to DCAD Previous studies fed the compound for 2 weeks prior to expected calving This doesn t fit the North American prefresh grouping system Objective: To determine the effect of feeding a synthetic zeolite A to multiparous Holstein cows for 3 wk prior to expected calving on serum mineral status and prepartum and postpartum performance outcomes Thilsing Hansen et al., 2002; Thilsing et al., 2006; Pallesen et al., 2008; Grabherr et al., 2009

Zeolite A trial study design Covariate (Day 28 to 21 d) Control () 40% CS, 33% wheat straw, and 27% concentrate Prepartum (Day 21 to 0 d) (n = 29 cows) Experimental () + zeolite A (X Zelit) (n = 26 cows) Postpartum (Day 0 to 28) Fresh Data Collection Intake Rumination Milk Production (through 63 DIM) 4 3 2 1 0 1 2 3 4 Week Relative to calving Select blood samples: Minerals (Ca, P, Mg) Oxidant Status; Antioxidant potential (AOP) Reactive Oxygen and Nitrogen Species (RONS) Colostrum: Weight, volume IgG Blood BCS and Body Weight Milk Sampling Colostrum Additional measurements: Reproduction through 150 DIM Statistical analysis Prepartum and postpartum data were analyzed separately (SAS v. 9.4) Differences in SCH prevalence between treatments by day: Fisher s exact test (PROC FREQ) Data analyzed using ANOVA (PROC MIXED) w/repeated statement for time, if applicable Fixed effects: trt, time (when applicable), parity (2 nd vs. 3 rd ), all 2-way interactions, random effect of cow(trt), and covariate measure if applicable Tukey HSD adjustment for multiple comparisons Effect of treatment and Ca status (w/fixed effect of parity), on time to pregnancy within 150 DIM: Cox Proportional-Hazard models (PROC PHREG) As Fed Diets Prepartum Ingredient, % of diet DM Postpartum Corn silage 40.00 38.60 40.25 Alfalfa hay 3.58 Wheat straw 33.33 32.17 4.48 Alfalfa silage 10.74 Canola meal 8.33 8.03 5.37 Ground shelled corn 3.58 Steam flaked corn 6.27 Citrus pulp 3.33 3.24 2.02 Blood meal 1.67 1.62 Amino Plus 6.67 6.47 6.14 Smartamine M 0.03 0.05 LysAAmet 1.77 Soybean hulls 5.00 4.85 3.74 Dried molasses 4.08 X Zelit 3.40 Urea 0.41 Other 1.6 1.56 7.53

Chemical composition of diets Prepartum Postpartum Nutrient Fresh CP, % DM 13.6 ± 1.0 13.5 ±0.7 16.4 ± 0.4 NDF, % DM 46.4 ± 1.4 46.0 ± 1.7 30.6 ± 2.8 Starch, % DM 16.8 ± 1.7 16.3 ± 0.3 26.1 ± 1.6 Sugar, % DM 3.2 ± 0.8 3.3 ± 0.4 3.3 ± 0.5 Fat, % DM 2.24 ± 0.13 2.25 ± 0.30 2.64 ±0.24 Ash, % DM 6.12 ± 0.53 7.99 ± 0.36 9.14 ± 0.49 Ca, % DM 0.68 ± 0.05 0.65 ± 0.03 1.00 ± 0.07 P, % DM 0.39 ± 0.03 0.38 ± 0.02 0.38 ± 0.01 Mg, % DM 0.42 ± 0.05 0.42 ± 0.03 0.51 ± 0.04 K, % DM 1.14 ± 0.06 1.12 ± 0.04 1.91 ± 0.11 DCAD, meq/100g +11.0 ± 2.1 +26.9 ± 1.7 +40.8 ± 2.5 MP, g/kg of DM 87.24 85.41 123.04 Calcium Serum Ca (mg/dl) 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 Prepartum effects Trt x day P = 0.05 17 10 5 3 1 0 0.25 0.75 2 3 4 6 7 10 15 Day Relative to Calving Trt x day P = 0.0006 Serum P (mg/dl) Phosphorus 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Trt x day P = 0.04 17 10 5 3 1 0 0.25 0.75 2 3 4 6 7 10 15 Day Relative to Calving Trt x day P < 0.0001 Serum Mg (meq/l) Magnesium 2.2 2.0 1.8 1.6 1.4 1.2 1.0 Trt x day P = 0.007 Trt x day P < 0.0001 17 10 5 3 1 0 0.25 0.75 2 3 4 6 7 10 15 Day Relative to Calving

SCH prevalence Chronic SCH prevalence Percent of cows with serum Ca < 8.5 mg/dl 100 90 80 70 60 50 40 30 20 10 0 17 10 5 3 1 0.25 0.75 2 3 4 6 7 10 15 Day relative to calving Percent of cows per treatment 70% 60% 50% 40% 30% 20% 10% 0% No SCH SCH csch Calcium Status within 3 DIM Survival Probability (proportion nonpregnant) Time to pregnancy by treatment 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 70 days 89 days 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 DIM 43% increased risk HR (95% CI) = 1.76 (0.93 3.33; P = 0.08) Treatment ( vs. ) Pregnancy by 150 DIM 46% decreased risk HR (95% CI) = 0.54 (0.29 1.02; P = 0.06) Parity ( 3 lact vs. 2 nd lact)

Time to pregnancy by calcium status Survival Probability (proportion nonpregnant) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% No SCH SCH csch 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 DIM 82% decreased risk HR (95% CI) = 0.18 (0.06 0.57); P < 0.01 csch vs. No SCH Ca Status (P = 0.01) Pregnancy by 150 DIM P = 0.29 SCH vs. No SCH 41% decreased risk HR (95% CI) = 0.59 (0.31 1.11); P = 0.10 Parity ( 3 lact vs. 2 nd lact) Dry Matter Intake - kg Prepartum outcomes DMI (kg) 26 24 22 20 18 16 14 12 10 Trt x Week P = 0.04 Trt P = 0.51 Trt x Week P = 0.16 3 2 1 0 1 2 3 4 Treatment P value Variable SEM Trt Trt x Week EBAL (Mcal/d) 5.4 4.4 0.4 0.05 0.11 BW (kg) 808 790 4 0.0004 0.02 BW change (kg) 17 6 4 0.04 BCS 3.37 3.29 0.02 0.008 0.32

Postpartum outcomes Rumination Treatment P value Variable SEM Trt Trt x Week EBAL (Mcal/d) 11.8 11.9 0.7 0.91 0.66 BW (kg) 713 708 5 0.42 0.82 BW change (kg) 36 36 7 0.96 BCS 3.14 3.11 0.03 0.34 0.01 Rumination (min/d) 580 560 540 520 500 480 460 440 420 400 Trt P = 0.03 Trt x Week P = 0.77 Trt P = 0.61 Trt x Week P = 0.34 3 2 1 0 1 2 3 4 Milk Yield weeks 1-4 Energy Corrected Milk Yield weeks 1-4 Milk Production (kg) 55 53 51 49 47 45 43 41 39 Week 1 4 effects: Trt P = 0.58 Trt x week P = 0.99 1 2 3 4 ECM (kg) 58 56 54 52 50 48 46 Week 1 4 effects: Trt P = 0.50 Trt x week P = 0.57 1 2 3 4

Milk Yield (kg) Milk Yield weeks 1-9 60 55 50 45 40 35 Week 1 9 effects: Trt P = 0.37 Trt x week P = 0.84 1 2 3 4 5 6 7 8 9 Milk Composition weeks 1-4 Treatment P value Variable SEM Trt Trt x Week Fat (%) 4.17 4.32 0.08 0.17 0.05 Fat (kg/d) 1.98 2.03 0.04 0.35 0.26 Protein (%) 3.19 3.30 0.05 0.09 0.24 Protein (kg/d) 1.51 1.55 0.03 0.33 0.88 Lactose (%) 4.58 4.59 0.02 0.78 0.59 Lactose (kg/d) 2.22 2.20 0.04 0.67 0.54 TS (%) 13.03 13.32 0.11 0.07 0.18 TS (kg/d) 6.23 6.30 0.10 0.65 0.66 ECM (kg/d) 53.0 53.8 0.9 0.50 0.57 ECM/DMI 2.47 2.47 0.05 0.95 0.47 MUN (mg/dl) 12.46 10.90 0.43 0.01 0.87 SCS 1.07 1.08 0.25 0.98 0.72 Percent Fat Colostrum Percent Fat 5.5 5 4.5 4 3.5 3 1 2 3 4 Colostrum yield (kg) 9 8 7 6 5 4 3 2 1 0 P = 0.16 P = 0.29 10000 IgG (mg/dl) 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 IgG yield (g) 600 500 400 300 200 100 0 P = 0.35

Oxidant Status index (OSi; RONS/AOP) OSi (RFU/TE) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Trt P =0.54 Trt x day P = 0.81 Trt P = 0.66 Trt x day P = 0.76 7 3 0 3 7 14 Day relative to calving Summary Serum Ca concentrations were higher for cows fed compared to during periparturient period Serum P concentrations were lower for cows fed compared to during both prepartum and immediate postpartum periods Serum Mg concentrations were lower for cows fed compared to during the prepartum and immediate postpartum periods; however Mg concentrations remained within normal ranges There were no cases of csch in -fed cows compared to ~35% of -fed cows Cows fed the diet had a median time to pregnancy 19 days sooner than -fed cows Prepartum DMI tended to decrease more for -fed cows as parturition approached compared to ; however, there were no significant treatment differences postpartum Postpartum performance was similar between both treatment groups Acknowledgements Thank you! Funding provided by Protekta, Inc. Dr. Tom Overton Charlene Ryan Dr. Brittany Leno Lisa Furman CURC Staff Helen Korzec Sarah LaCount Dr. David Barbano (and his lab) Claira Seely Shana Coffey Reilly Pierce Brook Ryan Courtney Dearnley Conor McCabe Dr. Angel Abuelo (MSU) Dr. Sabine Mann Allison Kerwin abl37@cornell.edu

Study population at enrollment BW Treatment P value Variable Entering Parity 2 nd 13 11 0.85 3 rd + 16 15 Previous 305 d MEq (kg) 14870 ± 2081 15197 ± 1793 0.54 Days on prepartum 22 ±5 21 ± 5 0.47 treatment BW (kg) 777 ± 92 776 ±68 0.97 BCS 3.25 ± 0.24 3.29 ± 0.27 0.62 Body Weight (kg) 380 370 360 350 340 330 320 310 300 290 280 Trt x Week P = 0.02 Trt P = 0.42 Trt x Week P = 0.82 3 2 1 0 1 2 3 4 Week Relative to Calving BCS Dry Matter Intake - % of BW BCS 3.5 3.4 3.3 3.2 3.1 3 2.9 Trt P = 0.008 Trt x Week P = 0.31 Trt x Week P = 0.01 3 2 1 0 1 2 3 4 Week Relative to Calving DMI, % of BW 3.8 3.4 3 2.6 2.2 1.8 1.4 Trt P = 0.44 Trt x Week P = 0.10 Trt P = 0.36 Trt x Week P = 0.20 3 2 1 0 1 2 3 4

Health Events Disorder P value Clinical Mastitis 3 0 0.09 Metritis 0 0 RP 5 3 0.55 DA 0 3 0.06 Milk Fever 0 0 Injury 3 3 0.55 Hyperketonemia 10 8 0.77 Pneumonia 1 3 0.25 Reactive oxygen and nitrogen species (RONS) RONS (relative fluorescent units/μl) 80 75 70 65 60 55 50 45 40 Trt P =0.28 Trt x day P = 0.70 Trt P = 0.73 Trt x day P = 0.40 7 3 0 3 7 14 Day relative to calving Antioxidant potential (AOP) AOP (trolox equivalents/μl) 62 57 52 47 42 37 32 Trt P =0.36 Trt x day P = 0.47 Trt P = 0.89 Trt x day P =0.64 7 3 0 3 7 14 Day relative to calving Milk Fatty Acid weeks 1-4 Treatment P value Variable SEM Trt Trt x Week Fat (%) 4.17 4.32 0.08 0.17 0.05 Fat (kg/d) 1.98 2.03 0.04 0.35 0.26 DeNovo (%) 0.91 0.95 0.02 0.21 0.71 Mixed (%) 1.36 1.44 0.03 0.05 0.95 Preformed (%) 1.65 1.68 0.06 0.72 <0.01 DeNovo (% of TFA) 23.3 23.5 0.5 0.81 0.12 Mixed (% of TFA) 34.9 35.6 0.6 0.43 0.02 Preformed (% of TFA) 41.8 40.9 1.0 0.53 <0.01 DeNovo (g) 431 447 13 0.36 0.31 Mixed (g) 648 680 15 0.15 0.55 Preformed (g) 784 785 25 0.98 0.06 Predicted Blood NEFA (µeq/l) 511 518 34 0.87 0.04

Predicted blood NEFA Predicted NEFA (μeq/l) 800 700 600 500 400 300 200 1 2 3 4 Percent Fatty Acids FA % of milk 2.3 2.1 1.9 1.7 1.5 1.3 1.1 0.9 0.7 0.5 1 2 3 4 Preformed Preformed Mixed Mixed De Novo De Novo Proportion of TFA 50 45 Grams of Fatty Acids 950 850 % of TFA 40 35 30 25 Preformed Preformed Mixed Mixed De Novo De Novo Fatty Acids (g) 750 650 550 450 Preformed Preformed Mixed Mixed De Novo De Novo 20 1 2 3 4 350 1 2 3 4

Supplemental Ca treatment options IV Ca Intravenous Calcium Rapid supply of available calcium Appropriate for clinical milk fever Unnecessary for cows with no clinical signs Oral calcium boluses or drenches Will not provide Ca quickly enough for recumbent cow Slow release of different calcium salts or other compounds May be more appropriate for suspected subclinical cases Subcutaneous calcium Intermediate to the above two options Serum total Ca following postpartum prophylactic administration of Ca IV (500 ml 23% Ca borogluconate) or Ca Oral (2 Ca boluses 12 h apart). Blanc et al., 2014. J. Dairy Sci. 97:6901 6906 SQ Ca 7 farms, 984 cows w/o visible milk fever Administered Ca SQ immediately postcalving and at 12 to 24 h postcalving Ca gluconate in combination with Ca glucoheptonate (TheraCalcium) Total ~ 10 g of supplemental Ca Results Transient increase in blood Ca at 24 h; no difference at 48 h Treated cows slightly less likely to receive subsequent Ca treatments (5.0 vs. 8.4%) No effect on RP, metritis, ketosis, culling No effect on early lactation production, probability of pregnancy to first artificial insemination, or time to pregnancy. Oral Ca Bolus 2 herds, 927 multiparous cows 1 st bolus 0-2 h after calving, 2 nd bolus 8-35 h after calving (Bovikalc) Mean ica concentrations were not different Treated lame cows averaged 0.34 fewer health events (w/in 30 DIM) Treated cows w/higher previous ME305 milk production cows produced 2.9 kg more milk at first DHI test Miltenberg et al., 2016. J. Dairy Sci. 99:6550 6562 Oetzel and Miller., JDS, 2012

Oral Ca Bolus Supplementation Benefit Fewer health disorders Increased Milk Yield Reduced services per conception Multiparous cows w/ BCS >3.5 Cows w/ parity 3 Primiparous cows Questionable benefits to a calving >712 d old blanket treatment approach Primiparous cows w/ BCS >3.5 Multiparous cows w/ higher previous ME305 Multiparous Cows Detriment Reduced Milk Yield Increased services per conception Multiparous cows with lower previous ME305 Primiparous Cows Oetzel and Miller, 2012; Martinez et al., 2016; Leno et al., 2018 Improving Ca status via altering prepartum Dietary Cation Anion Difference (DCAD) Cations: Sodium (+1) Potassium (+1) Anions: Chloride (-1) Sulfate (-2) Impact on calcium metabolism: Improved sensitivity of PTH receptor to PTH stimulation Decreased urine ph increases urinary Ca excretion = increased calcium flux Systemic ph reduction may directly stimulate Ca resorption from bone More H + in blood to maintain electroneutrality Decreased blood ph Increased H + excretion Goff et al., 2008. Vet J. 176:50 57. Major strategies for application of DCAD for close-up dry cows Day -31 to Day -25 Day -24 to Day 0 Day 0 to Day 63 Focus on feeding low K (and Na) forages and feeds to close-up dry cows Calculated DCAD ~ +10 meq/100 g of DM Urine ph ~ 8.3 to 8.5 Feeding low K forages along with partial use of anionic supplement in close-up ration or one-group dry cow ration Calculated DCAD ~ 0 meq/100 g of DM Urine ph ~ 7.5 Feeding low K forages along with full use of anionic supplement in close-up ration or one-group dry cow ration Calculated DCAD ~ -10 to -15 meq/100 g of DM Urine ph ~ 5.5 to 6.0 need to monitor weekly and adjust DCAD supplementation if out of range Low K control +18.3 meq/100 g diet DM Low K control Low K control +18.3 meq/100 g diet DM +10 meq/100 n= 30 g diet DM Medium Medium DCAD +5.9 meq/100 g diet DM DCAD n = 30-3 meq/100 g diet DM Low DCAD -7.4-18 meq/100 meq/100 g diet diet DM DM n= 29 Lactating Ration Leno et al., 2017. J. Dairy Sci. 100:4604 4622.

As forumulated prepartum diets, lbs DM Ingredient (lb DM/d) Control MedDCAD LowDCAD BMR Corn Silage 12.77 12.77 12.77 Wheat Straw 8.00 8.00 8.00 Amino Plus 2.30 2.30 2.30 Citrus Pulp 0.95 0.95 0.95 Soybean Hulls 0.66 0.66 0.66 Canola Meal 0.63 0.63 0.63 Molasses 0.19 0.19 0.19 Calcium diphosphate 0.13 0.13 0.13 Ground corn grain 0.12 0.12 0.12 Salt 0.07 0.07 0.07 Vitamin Mix 0.04 0.04 0.04 Rumensin (mg) 318 318 318 Animate 0.56 1.14 Wheat Midds 0.92 0.74 0.55 Calcium carbonate 0.82 0.80 0.77 Corn Distillers Ethanol 0.63 0.37 0.11 Magnesium Oxide 0.16 0.12 0.07 Urea 0.12 0.06 Leno et al., 2017 100:4604 4622. Analyzed (mean +/- SD) composition of experimental diets MedDCAD LowDCAD Lactating DM (%) 46.3 ± 1.6 46.5 ± 1.3 46.4 ± 1.1 45.7 ±1.8 CP (% DM) 13.0 ± 0.3 13.2 ± 0.4 13.2 ± 0.5 15.7 ±0.2 ADF (% DM) 30.2 ± 0.7 30.5 ± 1.3 30.1 ± 1.3 20.6 ±0.8 NDF (% DM) 44.3 ± 1.2 44.0 ± 2.1 43.2 ± 1.8 31.1 ±1.0 Starch (% DM) 17.0 ± 0.5 16.0 ± 0.8 16.3 ± 0.9 26.0 ±0.7 NFC (% DM) 33.6 ± 0.9 34.3 ± 2.5 35.0 ± 1.9 45.8 ±1.2 Fat (% DM) 1.1 ±0.1 1.3 ±0.2 1.1 ±0.3 2.3 ±0.2 Ca (% DM) 1.54 ± 0.12 1.57 ± 0.14 1.57 ± 0.07 0.95 ±0.03 P(%DM) 0.44 ± 0.01 0.43 ± 0.01 0.41 ± 0.01 0.41 ±0.02 Mg (% DM) 0.47 ± 0.01 0.48 ± 0.03 0.50 ± 0.03 0.44 ±0.02 K(%DM) 1.28 ± 0.07 1.26 ± 0.06 1.24 ± 0.07 1.37 ±0.05 S(%DM) 0.20 ± 0.01 0.30 ± 0.02 0.41 ± 0.02 0.29 ±0.01 Na (% DM) 0.13 ± 0.01 0.13 ± 0.01 0.14 ± 0.01 0.44 ±0.02 Cl (% DM) 0.27 ± 0.03 0.47 ± 0.05 0.69 ± 0.04 0.40 ±0.02 DCAD (meq/100g DM) 18.3 ±0.8 5.9 ±3.4 7.4 ± 3.6 25.0 ±1.5 Predicted MP (g/kg DM) 93.8 93.23 92.26 116.56 Leno et al., 2017. J. Dairy Sci. 100:4604 4622. Urine ph Urine ph 9 8.5 8 7.5 7 6.5 6 5.5 5 4.5 4-23 -21-19 -17-15 -13-11 -9-7 -5-3 -1 Day Relative to Calving Control MedDCAD LowDCAD Quadratic P<.0001 Trt P<0.0001 Wk P=0.30 TrtxWk P=0.02 Postpartum Blood Ca Treatment by Parity Interaction Blood Calcium (mg/dl) 9.4 9.2 9 8.8 8.6 8.4 8.2 2nd Parity Group 3rd+ Control MedDCAD LowDCAD Trt x Parity P=0.06 Leno et al., 2017. J. Dairy Sci. 100:4604 4622. Leno et al., 2017. J. Dairy Sci. 100:4604 4622.

Dry Matter Intake Prepartum Diet P-values MED LOW SEM Linear Quad Trt Wk Prepartum DMI, kg/d 14.55 15.08 14.08 0.23 0.15 0.007 0.45 DMI, % of BW 1.87 1.89 1.80 0.03 0.16 0.22 0.38 Postpartum (wk 1 to 3) DMI, kg/d 20.99 21.74 22.30 0.50 0.07 0.88 0.24 DMI, % of BW 2.94 3.04 3.15 0.07 0.03 0.99 0.37 Leno et al., 2017. J. Dairy Sci. 100:4604 4622. Leno et al., 2017. J. Dairy Sci. 100:4604 4622. Milk Production: Weeks 1 to 3 Prepartum Diet P-values MED LOW SEM Linear Quad Trt Wk Milk yield, kg/d 40.54 42.13 43.79 1.05 0.03 0.97 0.35 Fat, % 4.38 4.36 4.24 0.08 0.21 0.63 0.10 True protein, % 3.54 3.49 3.27 0.07 0.005 0.33 0.36 Lactose, % 4.64 4.67 4.69 0.03 0.25 0.94 0.38 Total Solids, % 13.63 13.61 13.27 0.10 0.01 0.20 0.10 ECM, kg/d 46.12 48.04 49.50 1.35 0.08 0.89 0.39 MUN, mg/dl 10.32 9.72 9.44 0.30 0.04 0.67 0.17 SCS 2.62 3.26 2.73 0.25 0.75 0.06 0.27 What about Mg and P levels in close-up diets? Mg excellent consensus Magnesium is involved in Ca mobilization signaling Supplement ~ 0.45% Mg in close-up diet Magnesium oxide, magnesium sulfate, magnesium carbonate, commercial sources P good consensus 0.35 to 0.42% of diet No effect on P status as low as 0.25% of diet precalving High P (> 0.50%) may cause hypocalcemia? Leno et al., 2017. J. Dairy Sci. 100:4604 4622.

What about Ca levels in close-up diets? Lack of consensus Moore et al. (2000) determined better Ca status as Ca increased from 0.44 to 0.97 and 1.50% of the diet and DCAD decreased Recent data (Chan et al., 2006) suggest Ca at 0.99 or 1.50% in conjunction with anionic supplement maintained adequate serum Ca Lean et al. (2006) meta analysis Suggested less milk fever at either lower < 0.60% or higher Ca concentrations > 2.0% Current Overton recommendation 0.9 to 1.0% if no anionic supplement 1.4 to 1.5% if anionic supplement What about Vitamin D levels in close-up diets? Good consensus 25,000 to 30,000 IU of Vitamin D (cholecalciferol) Some recent research with calcidiol 25-hydroxyvitamin D3 Enhanced responses to negative DCAD diets in some studies (Martinez et al., 2015) but not others (Weiss et al., 2015)