August 22, 2017 M. D. Lindemann

August 22, 2017 M. D. Lindemann University of Kentucky Phone: 859-257-7524 merlin.lindemann@uky.edu

Arabinoxylans: A major NSP in wheat The main component of wheat cell wall (aleurone layer and pericarp) Increase endogenous loss/reduce digestibility Increase viscosity and reduce passage rate of digesta Proliferate gut microbes Xylanase: a glycosidase enzyme Hydrolyzes arabinoxylan structure Improved fiber digestibility in corn-sbm (Passos et al., 2015), wheat-based diet (Atakora et al., 2011) and in the diet containing corn DDGS (Jang et al., 2013). 2

Cereal grains and oil seed meals: Indigestible portion: arabinoxylans and phytate Arabinoxylans, % DM Phytate P, % as-fed Corn 3.8 0.21 SBM 4.5* 0.38 Corn germ meal (CGM) 18.5 0.58 Wheat middlings 18.8 0.61 Corn DDGS (>10%) 12.3 0.26 (Arabinose + xylose) Jaworski et al., 2015; *Bach Knudsen, 1997 NRC, 2012; Rojas et al., 2013 Usage of by-product the content of arabinoxylans and phytate 3

Phytate (phytic acid): A storage form of P in cereal grains and oil seeds The main accumulation site is the aleurone layer Phytic acid bound to Ca, Mg (Chelate), fat, protein, and starch Reduce the availability of P and the other nutrients Phytase: Sequential removal of phosphate groups and release P from phytate. 4

Phytic acid and arabinoxylans are both found in the aleurone layer of wheat. Xylanase increases the permeability of the aleurone layer (the site of phytic acid storage). Then, the accessibility and action of phytase to the phytic acid in the cell wall can be improved. However, the results are inconsistent and research on the inclusion of xylanase and phytase to corn-based diets containing multiple by-products is limited. 5

When pigs are fed high fiber diets containing corn DDGS, wheat middlings, and corn germ meal (CGM), Objective 1. The effect of xylanase supplementation on ATTD and energy release for growing pigs fed diets containing phytase. Objective 2. Effect of combined xylanase and phytase on growth performance, ATTD, and carcass characteristics in growing pigs. 6

25 pigs (mean initial BW: 76.5 kg) 5 treatments in 5 replicates: PC: A corn-sbm basal diet with 15% each of corn DDGS, CGM, and wheat middlings NC: low ME diet by 103 kcal/kg from the PC diet NX1: NC + 8,000 BXU of xylanase/kg diet NX2: NC + 16,000 BXU of xylanase/kg diet, and NX3: NC + 24,000 BXU of xylanase/kg diet All diets contained 250 FTU/kg of phytase (Quantum Blue; AB vista Feed Ingredients, Marlborough, UK). Xylanase used: Econase XT; AB vista Feed Ingredients, Marlborough, UK 7

10-12 d of adaptation on the farm (ad libitum) Daily feed allowance: 2.7% of BW After a 7-d adaption period to the metabolic crates, feces and urine were collected for 5 consecutive days (total collection method). 8

Item Positive control Negative control Ingredient Corn 26.52 26.52 SBM (dehulled; 48% crude protein) 19.10 19.10 Corn germ meal 15.00 15.00 Corn DDGS 15.00 15.00 Wheat middlings 15.00 15.00 Corn starch 2.00 4.50 Grease 4.50 2.00 Others 1 2.88 2.88 Total 100.00 100.00 Calculated composition, % ME, kcal/kg 3,360 3,256 Crude protein 21.40 21.40 Lysine 0.982 0.982 Methionine 0.376 0.376 Calcium 0.66 0.66 STTD P 0.29 0.29 Phytate P, analyzed 0.34 0.34 Available P, analyzed 2 0.28 0.28 1 Others included L-lysineHCl, L-threonine, limestone, salt, vitamin and trace mineral premix, Santoquin, and AB-20 (clay). 2 Available P was calculated by subtracting phytate P from total P. 9

Apparent Total Tract Digestibility of Dry matter (DM), gross energy (GE), Ether extract (EE), crude protein (N), Neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose Ca, and P Retention GE, N, Ca, and P 10

Treatment: PC NC NX1 NX2 NX3 Items Xylanase, BXU/kg: 0 0 8,000 16,000 24,000 SEM ATTD, % DM 81.15 81.49 81.47 81.37 82.40 0.41 GE 81.76 81.26 81.18 81.16 82.08 0.38 N 84.68 83.88 83.62 84.10 84.66 0.53 EE 92.71 a 86.38 b 86.78 b 88.06 b 87.33 b 1.08 Ca 43.10 b 57.76 a 60.28 a 56.70 a 61.59 a 2.95 P 48.84 50.34 51.07 48.90 49.65 2.05 DE in diet, kcal/kg 3,567.3 a 3,383.0 b 3,379.8 b 3,378.9 b 3,417.2 b 15.85 ME in diet, kcal/kg 3,437.7 a 3,225.8 b 3,230.8 b 3,214.8 b 3,256.8 b 17.41 a,b P < 0.05. PC treatment had the greatest ATTD of EE, DE and ME but the lowest ATTD of Ca among dietary treatments (P < 0.05). No significant xylanase effects in ATTD of DM, GE, N, EE, Ca and P. 11

72.0 * 70.0 68.0 68.3 % 66.0 64.0 62.0 60.0 58.0 57.7 58.7 57.4 58.0 57.6 61.9 60.8 60.5 60.3 64.0 64.6 62.2 62.6 61.8 56.0 54.0 52.0 50.0 ADF NDF Hemicellulose PC NC NX1 NX2 NX3 Xylanase, BXU/kg: 0 0 8,000 16,000 24,000 ATTD of hemicellulose was improved by increasing xylanase level (*linear, P < 0.05). ATTD of NDF in NX3 treatment has a numerical improvement (linear, P=0.15). 12

Treatment: PC NC NX1 NX2 NX3 Items Xylanase, BXU/kg: 0 0 8,000 16,000 24,000 SEM Retention, g/d GE, kcal/d 7,158 a 6,624 b 6,651 b 6,602 b 6,738 b 97.1 N 29.57 26.18 26.87 22.41 26.64 2.26 Ca 5.39 b 7.65 a 7.90 a 7.37 a 8.08 a 0.49 P 5.69 5.60 5.52 5.30 5.39 0.33 Retention, % of intake GE 78.79 c 77.48 d 77.60 d 77.22 d 78.23 cd 0.41 N 40.41 36.80 37.36 32.10 36.84 2.72 Ca 38.34 b 54.88 a 56.94 a 52.83 a 57.85 a 2.93 P 44.12 43.74 43.13 41.46 41.53 2.20 a,b P < 0.05. c,d P < 0.10. GE retention (% of intake) in NX3 treatment slightly increased (P<0.10). 13

Xylanase supplementation to the high fiber diet also containing phytase can improve some aspects of fiber digestibility and thereby, energy utilization. The energy uplift under the conditions of this study was about 33 kcal/kg diet. 14

45 individually-fed pigs (mean initial BW: 26.4 kg) 9 treatments in 5 replicates (1 + 2 x 4 factorial arrangement) High and low-energy control diets (PC and NC) same with Exp. 1 Factor 1: Phytase level (0, 500, 1,000, and 2,000 FTU/kg) Factor 2: Xylanase supplementation (0 vs. 24,000 BXU/kg) This level was decided based on the result of Exp. 1 All nutrients including P met or exceeded NRC (2012) nutrient requirement estimates. Treatment: PC NC NP500 NP1000 NP2000 NX NXP500 NXP1000 NXP2000 Phytase, FTU/kg: 0 0 500 1,000 2,000 0 500 1,000 2,000 Xylanase, BXU/kg: 0 0 0 0 0 24,000 24,000 24,000 24,000 15

Item, % Ingredient Positive control Phase 1 Phase 2 Phase 3 Phase 4 Negative control Positive control Negative control Positive control Negative control Positive control Negative control Corn 27.672 27.672 33.800 33.800 38.474 38.474 43.106 43.106 SBM (dehulled, 48% CP) 21.000 21.000 15.000 15.000 10.500 10.500 6.000 6.000 Corn germ meal 13.000 13.000 13.000 13.000 13.000 13.000 13.000 13.000 Corn DDGS 15.000 15.000 15.000 15.000 15.000 15.000 15.000 15.000 Wheat middlings 15.000 15.000 15.000 15.000 15.000 15.000 15.000 15.000 Corn starch 1.500 4.000 1.500 4.000 1.500 4.000 1.500 4.000 Grease 4.000 1.500 4.000 1.500 4.000 1.500 4.000 1.500 Others 1 2.828 2.828 2.700 2.700 2.526 2.526 2.394 2.394 Total 100.000 100.000 100.000 100.000 100.000 100.000 100.000 100.000 Calculated composition, % ME, kcal/kg 3,344 3,241 3,355 3,251 3,365 3,262 3,374 3,271 Crude protein 21.90 21.90 19.56 19.56 17.78 17.78 16.01 16.01 Lysine 0.980 0.980 0.850 0.850 0.730 0.730 0.610 0.610 Calcium 0.67 0.67 0.59 0.59 0.52 0.52 0.46 0.46 Total P 0.60 0.60 0.57 0.57 0.55 0.55 0.53 0.53 STTD P 0.29 0.29 0.28 0.28 0.27 0.27 0.26 0.26 Phytate P, analyzed 0.40 0.40 0.38 0.38 0.38 0.38 0.40 0.40 Available P, analyzed 2 0.22 0.22 0.21 0.21 0.19 0.19 0.18 0.18 1 Others included L-lysine HCl, limestone, salt, vitamin and trace mineral premix, Santoquin, and AB-20 (clay). 2 Available P was calculated by subtracting phytate P from total P anlayzed. 16

Growth performance from 26 to 120 kg BW ADG, ADFI, and G:F ratio Carcass characteristics at 120 kg Backfat thickness, Longissimus muscle (LM) area, carcass lean percent, lean daily gain, lean gain to feed ratio Apparent Total Tract Digestibility in Phase 4 DM, GE, EE, N, NDF, ADF, hemicellulose, Ca, and P 17

P<0.05 ADG, kg/d 0.92 0.88 0.84 0.80 0.76 PC NC NP500 NP1000 NP2000 NX NXP500 NXP1000 NXP2000 0.38 P<0.05 G:F ratio 0.36 0.34 0.32 0.30 PC NC NP500 NP1000 NP2000 NX NXP500 NXP1000 NXP2000 18

0.920 0.910 ADG, kg/d +1.0% 3.50 3.00 2.50 ADFI, kg/d -1.4% 0.900 2.00 1.50 0.890 1.00 0.50 0.880 Overall 0.00 Overall PC 0 24,000 BXU/kg PC 0 24,000 BXU/kg 0.39 0.38 0.37 0.36 0.35 0.34 G:F ratio +2.3% No sig. xylanase effect. Only numerical improvement in ADG and G:F ratio. 0.33 Overall PC 0 24,000 BXU/kg 19

0.940 ADG, kg/d * Linear (P < 0.05) Quadratic (P < 0.05) 0.900 0.860 0.820 0.780 0.390 0.380 0.370 0.360 0.350 0.340 0.330 0.320 0.310 +7.5% Overall PC 0 500 FTU/kg 1,000 FTU/kg 2,000 FTU/kg G:F ratio +2.9% Overall PC 0 500 FTU/kg 1,000 FTU/kg 2,000 FTU/kg * +8.8% +5.5% +5.0% +5.9% ADFI, kg/d 2.700 2.600 2.500 +4.4% +3.7% 2.400 2.300-0.9% 2.200 Overall PC 0 500 FTU/kg 1,000 FTU/kg 2,000 FTU/kg 20

0.37 G:F ratio within phytase treatment 0.36 0.35 +5.0% +4.6% -0.3% 0.34 0.33 0.32 0 500 1,000 2,000 Phytase, FTU/kg 0 24,000 BXU/kg No statistical interaction observed. Up to 1,000 FTU phytase/kg, xylanase numerically increased G:F ratio. 21

Xylanase, BXU/kg P-values 2 Item, % 0 24,000 SEM Xyl DM 78.58 78.54 0.24 0.91 GE 78.01 77.87 0.28 0.73 N 76.24 76.21 0.46 0.96 EE 71.61 70.60 1.48 0.46 ADF 51.93 50.70 0.91 0.35 NDF 53.12 52.67 0.56 0.58 Hemicellulose 53.96 54.09 0.87 0.92 Ca 45.64 47.83 1.52 0.32 P 47.84 46.94 1.05 0.55 No xylanase effect of in ATTD. 22

55 Phosphorus *, * Linear (P < 0.05) Quadratic (P < 0.05) 50 % 45 40 35 30 PC 0 500 FTU/kg 1,000 FTU/kg 2,000 FTU/kg 23

80 * Linear (P < 0.05) Quadratic (P < 0.05) % 75 70 65 60 Dry matter +1.5 ~ 2.0% +8.0 ~ 15.8% Ether extract PC 0 500 FTU/kg 1,000 FTU/kg 2,000 FTU/kg PC treatment had greater ATTD of EE but lower ATTD of P than NC treatments (P < 0.05). % 60 55 50 45 40 35 +5.8 ~ 8.4% *, +6.9 ~ 11.3% +11.3 ~ 24.0% No phytase effect of in ATTD of GE, N, ADF, and Ca. 30 Neutral detergent fiber Hemicellulose Phosphorus PC 0 500 FTU/kg 1,000 FTU/kg 2,000 FTU/kg 24

Xylanase, BXU/kg P-values Item 0 24,000 SEM Xyl Scan weight*, kg 120.32 121.36 1.30 0.57 Backfat, mm 15.35 15.35 0.52 0.99 LM depth, mm 62.29 60.46 1.17 0.28 LM area, % 41.47 40.37 0.70 0.28 Carcass lean, % 56.60 56.39 0.36 0.68 Lean gain, g/d 358.48 354.22 3.19 0.35 Lean gain/feed 0.139 0.140 0.003 0.76 PC treatment was slightly greater in lean gain to feed ratio than NC treatment (P=0.06). No xylanase effect in carcass measurements. 25

58.00 57.00 56.00 55.00 54.00 Carcass lean, % PC 0 500 FTU/kg * 1,000 FTU/kg 2,000 FTU/kg 365 360 355 350 340 Lean gain, g/d PC 0 500 FTU/kg *Linear (P < 0.05) **Linear (P < 0.10) +1.4% +2.9% +2.8% 345 +1.7% +3.2% +4.0% * 1,000 FTU/kg 2,000 FTU/kg 0.155 0.145 0.135 0.125 Lean gain/feed * 17.00 16.00 15.00 14.00 Backfat thickness, mm +2.2% +6.0% +8.2% 13.00-6.1% -13.4% -10.8% 12.00 PC 0 500 1,000 2,000 PC 0 500 1,000 2,000 FTU/kg FTU/kg FTU/kg FTU/kg FTU/kg FTU/kg ** 26

Xylanase supplementation to the high fiber diet had no effect on growth, carcass characteristics, and ATTD. Increasing phytase level (super-dosing) enhanced growth rate, feed efficiency, carcass leanness, and ATTD; the energy uplift was about 44 kcal/kg diet. There was no statistically significant interaction observed between xylanase and phytase but potentially in G:F ratio. 27

With a similar diet composition between 2 trials Exp. 1: xylanase improved fiber digestibility. Exp. 2: Xylanase had no effect on the ATTD of fiber. Possible reasons Different amount of substrates for xylanase A difference in feeding regime between the restricted (Exp. 1) and ad libitum (Exp. 2) Duration of feeding the treatment diets A relatively greater BW (about 118 kg in Exp. 2) than 76.5 kg (Exp. 1) when fecal collection was performed 28

Hanczakowska et al. 2012. Efficiency and dose response of xylanase in diets for fattening pigs. Ann Anim Sci 12:539 548. O Shea et al. 2014. The effect of protease and xylanase enzymes on growth performance, nutrient digestibility, and manure odour in grower finisher pigs. Anim Fd Sci Tech 189:88-97. Passos et al. 2015. Effect of dietary supplementation of xylanase on apparent ileal digestibility of nutrients, viscosity of digesta, and intestinal morphology of growing pigs fed corn and soybean meal based diet. Anim Nutr 1:19-23. Ndou et al. 2015. Comparative efficacy of xylanases on growth performance and digestibility in growing pigs fed wheat and wheat bran-or corn and corn DDGS-based diets supplemented with phytase. Anim Fd Sci Tech 209:230-239. Yang et al. 2017. Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets. AJAS 30:57-63. 29

Xylanase can improve fiber utilization in diets containing multiple by-products. Super-dosing phytase can be an useful strategy to enhance nutrient utilization resulting in improved growth performance, and carcass leanness. 1,000 FTU phytase/kg diet Further investigation may be needed to clearly confirm the xylanase effect and the potential interaction between phytase and xylanase. with a large number of pigs and different physiological statuses 30

Thank you. 31