Supplementary information Running tittle: Dietary

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Supplementary information

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Running tittle: Dietary fiber alters intestinal milieu

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Dietary supplementation with flaxseed meal and oat hulls modulates intestinal

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histomorphometric characteristics, digesta- and mucosa-associated microbiota in pigs

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S.P. Ndoua, H.M. Tuna, E. Kiariea,b, M.C. Walshd, E. Khafipoura,c,* and C.M. Nyachotia,*

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a

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b

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c

Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada. R3T 2N2

Department of Animal Biosciences, University of Guelph, Guelph, ON, Canada, N1G 2W1

Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada. R3T

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2N2 d

DuPont Industrial Biosciences-Danisco Animal Nutrition, Marlborough, Wiltshire, United

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Kingdom SN8 1XN

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*Co-corresponding author:

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Dr. Martin Nyachoti, Tel: +1-204-474-7323; Fax: +1-204-474-7628; EMail: [email protected]

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P = 0.612

P = 0.712

P = 0.575 345

P = 0.522

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Supplementary Figure S1: Alpha-diversity indices of microbiota in ileal digesta of pigs fed with control (CON), flaxseed meal (FM) and oat hulls (OH) diets

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P = 0.019

P = 0.828

P = 0.064

P = 0.365

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Supplementary Figure S2: Alpha-diversity indices of ileal mucosa-associated microbiota of pigs fed with control (CON), flaxseed meal (FM) and oat hulls (OH) diets

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P < 0.001

P = 0.050

P = 0.313

P < 0.001

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Supplementary Figure S3: Alpha-diversity indices of microbiota in cecal digesta of pigs fed with control (CON), flaxseed meal (FM) and oat hulls (OH) diets

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P < 0.001

P = 0.303

P = 0.015

P = 0.144

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Supplementary Figure S4: Alpha-diversity indices of cecal mucosa-associated microbiota of pigs fed with control (CON), flaxseed meal (FM) and oat hulls (OH) diets.

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Table S1. Ingredient composition and calculated provisions of the control, flaxseed meal and oat

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hulls diets1

CON

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Diet1 FM

OH

Ingredients Corn 64.53 57.40 52.99 Oat hulls 10.00 Flaxseed meal 12.00 Soybean meal, 44 % CP 31.00 25.49 31.500 Vegetable oil 1.354 2.044 2.390 Limestone 0.683 0.667 0.640 Monocalcium phosphate 0.752 0.637 0.750 Salt 0.35 0.35 0.35 2 Vitamin-mineral premix 1.00 1.00 1.00 0.086 0.010 L-Lysine HCl -Methionine 0.031 0.010 0.060 DL Threonine 0.012 0.010 Titanium dioxide 0.30 0.30 0.30 3 Calculated dietary provisions ME kcal/kg 3,330 3,334 3,326 CP, % 18.10 18.70 18.01 SID Lys, % 0.99 0.99 0.99 SID Met, % 0.30 0.31 0.30 SID Met + Cys, % 0.58 0.61 0.55 SID Thr, % 0.62 0.60 0.60 SID Trp, % 0.21 0.17 0.18 1 CON = control diet; FM = flaxseed meal-containing diet; OH = oat hulls-containing diet. 2 Provided the following nutrients per kg of air-dry diet: 8,250 IU retinol (Vitamin A); 200 IU cholecalciferol (Vitamin D3); 40 UI α-tocopherol (Vitamin E); 4 mg Vitamin K; 1.5 mg Vitamin B1; 7 mg Vitamin B2; 2.5 mg Vitamin B6; 25 µg Vitamin B12; 14 mg calcium pantothenate; 2 mg folic acid; 21 mg niacin (Vitamin B3); and 200 µg biotin (Vitamin B7). Minerals: 15 mg Cu (as copper sulphate); 0.4 mg iodine (as potassium iodine); 120 mg iron (as ferrous sulphate); 20 mg Mn (as manganese oxide); 0.3 mg Se (as sodium selenite); 110 mg Zn (as zinc oxide). 3 The nutrient compositions were calculated based on ingredient composition values from NRC (2012) and our laboratory.

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Table S2. Analyzed composition of the experimental diets Item Diets1 CON FM OH Chemical composition Crude protein (N × 6.25), % 19.14 19.33 19.24 Gross energy, kcal/kg 3879 3886 3977 Fat content, % 4.58 5.32 5.94 Calcium 0.61 0.62 0.60 Phosphorus 0.58 0.63 0.58 Neutral detergent fiber, % 9.37 17.9 18.6 Acid detergent fiber, % 3.6 4.8 7.2 2 NSP constituent sugars, % Total NSP 9.48 (0.80) 13.3 (3.50) 15.0 (1.79) Arabinose 1.80 (0.16) 2.20 (0.36) 1.84 (0.06) Xylose 1.83 (0.06) 2.76 (0.79) 4.81 (0.38) Mannose 0.26 (0.00) 0.23 (0.06) 0.22 (0.01) Galactose 1.50 (0.15) 1.75 (0.50) 1.30 (0.06) Glucose 2.78 (0.43) 4.26 (1.29) 5.60 (0.94) Uronic acids 1.32 (0.12) 2.10 (0.50) 1.20 (0.35) Physical properties Bulk density, g/ml 1.56 1.43 1.36 Swelling capacity, ml/g 2.67 4.51 2.85 Water holding capacity, ml/g 3.31 5.98 4.52 3 Particle size distribution, % >1700 µm 19.8 20.6 18.2 > 850 µm 19.3 16.6 22.7 < 600 µm 61.0 62.8 59.1 1 CON = control diet; FM = flaxseed meal-containing diet; OH = oat hulls-containing diet. NSP = non-starch polysaccharides. 2 Values in parentheses are for the soluble proportions of the NSP. 3 100 g of sample size for each diet.

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Table S3. Intake of dietary components and growth performance of growing pigs fed the control, flaxseed meal and oat hulls diets1 2 Diet 3 Item SEM3 P-value CON FM OH Intake of dietary components Gross energy, MJ/d 30.02b 26.80c 32.21a 0.724