Gut Physiology of Pigs Fed Carbohydrate Degrading Enzymes National Pork Board Webinar Series Summer 2017 P. E. Urriola*, Z. Zeng, M. Ferrandis-Vila, C. Chen, M. Saqui- Salces, and G. C. Shurson Department of Animal Science
Executive summary a. Goal of NPB: increase nutrient extraction in low energy feedstuffs b. Dietary fiber is a complex mix of multiple components that differ among cereal grains c. Zeng al, 2017 Current methods poorly measure the impact of fiber on animal performance d. Enzymes may increase nutrient extraction, but there are multiple products and multiple cereal co-prducts e. Principal findings on the project: a. In vitro (very small differences among 11 sources of enzymes): a. Wheat middlings enzymes in vitro AID of GE, but fermentability b. Corn DDGS with carbohydrases: negligible effects b. In vivo: a. Increase growth and feed intake of pigs fed wheat middlings; no corn DDGS b. Still observed differences in digesta viscosity and cytokine profile c. Extra-caloric effects of enzymes or oligosaccharides d. Future research: chemometric and microbiome (under analysis) http://research.pork.org/results/researchdetail.aspx?id=1998
Challenges of feeding grain by-products High in fiber content - NDF > 18.7% (Sauvant et al., 2004) Variability of NE and nutrient content among sources Reduce crude protein and ether extract digestibility (Dégen et al., 2007) Increase size and weight of the gastrointestinal tract (Kass et al., 1988) Reduce carcass yield
Alternative feed ingredients have high content of poorly digestible nutrients Water Protein Fat Sugars Starch Fiber Other Corn DDGS 0% 20% 40% 60% 80% 100%
Analytical vs. physiological characteristics of dietary fiber NRC (2007)
What are enzymes, how do they work? Enzymes modify activation energy to accelerate chemical reactions Key-lock specific activity of enzymes
Xylanases are a group of enzymes that degrade hemicellulose GH = glycoside hydrolase McKee et al. (2012)
Three step in vitro digestibility of DM and fiber Enzymatic hydrolysis - gastric I Enzymatic hydrolysis small intestine Serum Bottles Gas Measurement II Filtration and drying Filtration Large intestine fermentation III Filtration and drying Water Bath Huang et al. (2017); NPB Project # 13-014
Gas increases with in vitro fermentation of fiber SBH A DDGS C WS A/2 G = A / (1+ (B C /t C )) B Modified Van Soest (2015); Groot et al., (1996)
Sources of insoluble fiber with variable fermentable fiber Sources of fiber: Wheat Straw Corn dried distillers grains with solubles Soybean hulls NDF 85.0% 44.0% 67.0% TDF SDF 0.5% 0.7% 8.4% IDF 71.0% 42.2% 75.5% Fermentability Non-Fermentable Moderately fermentable Fermentable NDF Neutral detergent fiber TDF Total dietary fiber SDF Soluble dietary fiber IDF Insoluble dietary fiber Huang et al. (2016) ASAS Midwest Meeting
ATTD of TDF, % In vitro fermentation predicts in vivo apparent total tract digestibility of total dietary fiber 90 80 70 60 50 40 30 20 10 0 Wheat straw y = 0.1643x + 2.8959 R² = 0.819 Corn DDGS 0 100 200 300 400 500 Asymptotic gas production (A), ml/g DM Soybean hulls Huang et al. (2017); NPB Project # 13-014
Three step in vitro digestibility adapted to test 2 Ingredients + 11 Enzyme products Enzymatic hydrolysis - gastric I Enzymatic hydrolysis small intestine enzymes II Filtration and drying Large intestine fermentation A / 2 A B C SB H DDG S WS G = A / (1+ (B C /t C )) III Filtration and drying Huang et al. (2017); NPB Project # 13-014
Gas production increases with in vitro fermentation of fiber SBH A DDGS C WS A/2 G = A / (1+ (B C /t C )) B Modified Van Soest (2015); Groot et al., (1996)
Pentose release, % Enzymes break fiber into smaller fractions, but not monosaccharides % 30 20 10 Arabinose Xylose % 30 20 10 Acid hydrolyzed filtrate Arabinose Xylose Enzyme: P =0.01 Ingredient: P < 0.01 Enzyme ingredient = 0.24 Enzyme: P < 0.01 Ingredient: P < 0.01 Enzyme ingredient < 0.01 0 Control Wheat middlings E2 E5 Control E2 E5 Corn DDGS 0 Control E2 E5 Control E2 E5 Wheat middlings Corn DDGS
Gas production, ml/g DM Decrease in total gas production in wheat middling with carbohydrase enzymes 400 300 200 100 Con E1 E2 E3 E4 E5 0 E6 E7 E8 E9 E10 E11 0 10 20 30 40 50 60 70 80 Fermentation hours
Gas production ml/g Negligible impact of enzymes on in vitro fermentation of fiber in corn DDGS 400 350 300 250 200 150 100 50 0 0 10 20 30 40 50 60 70 80 Fermentation time, h T0.5 16 h Con E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11
Fiber degradation Fiber in cereal co-products is heterogenous, enzymes act on readily degradable fiber Un-digestible Hind gut fermentation AID 100% 80% 60% 40% Readily degradable fiber: susceptible to acid and mastication (mainly hemicellulose) 8-12 h Fermentable fiber: breakdown by microbial activity 24-36 h 20% 0% corn DDGS Corn basal diet Wheat basal diet Wheat grain Wheat millrun diet Recalcitrant fiber: resistant to attacks from exogenous enzyme or bacteria (mainly cellulose and entrapped hemicellulose Urriola et al., 2012; Shrestha, 2012, and Ndou et al., 2015
Materials and methods Animals: pigs (n = 54, 25kg BW ) were individually fed experimental diets for 28-d Feed and water: ad libitum and free access Factorial design : 2 3 arrangement Basal diets: corn-soybean (CSB), CSB + 40% corn DDGS, CSB + 30% wheat middlings (WM) Carbohydrases: with or without 100 mg/kg cocktail (1,500 U/g xylanase, 1,100 U/g beta-glucanase, 110 U/g mannanase, 35 U/g galactosidase)
Growth performance Diet = 0.02 Enzyme =0.051 Diet Enzyme = 0.42 Diet < 0.01 Enzyme =0.08 Diet Enzyme = 0.43 a b ab a b b ab Means within a row without same superscript are different (P < 0.05)
Digestibility (AID and ATTD) of DM Diet < 0.01 Enzyme =0.44 Diet Enzyme = 0.04 Diet < 0.01 Enzyme =0.53 Diet Enzyme = 0.50 AID of DM, % 100 90 80 70 60 AID of DM Control Enzyme ATTD of DM, % 100 90 80 70 60 ATTD of DM Control Enzyme a c b CSB DDGS WM CSB DDGS WM abc Means within a row without same superscript are different (P < 0.05)
Digestibility (AID and ATTD) of GE Diet < 0.01 Enzyme =0.44 Diet Enzyme = 0.04 Diet < 0.01 Enzyme =0.26 Diet Enzyme = 0.24 AID of GE, % 100 90 80 70 60 AID of GE Control Enzyme ATTD of GE, % 100 Control Enzyme 90 80 70 60 a ATTD of GE c b CSB DDGS WM CSB DDGS WM abc Means within a row without same superscript are different (P < 0.05)
Peak shear stress, Pa Peak shear stress (whole digesta) Diet = 0.111; Enzyme =0.01 Diet Enzyme = 0.22 Digesta liquid < 0.01 Diet = 0.04; Enzyme =0.72 Diet Enzyme = 0.86 Digesta liquid < 0.01 Diet < 0.01; Enzyme =0.89 Diet Enzyme = 0.89 Digesta liquid = 0.09 b b a a a b ab Means within a row without same superscript are different (P < 0.05)
IL 1 (p g /m L ) IL 1 (p g /m L ) Feeding enzymes increases expression of interleukin-1 β in ileum 2 5 0 0 Ileum 2 5 0 0 Colon 2 0 0 0 a 2 0 0 0 1 5 0 0 b b 1 5 0 0 1 0 0 0 c c c 1 0 0 0 5 0 0 5 0 0 0 C O N D D G S W M C O N + E n z D D G S + E n z W M + E n z 0 C O N D D G S W M C O N + E n z D D G S + E n z W M + E n z
Fiber diets promote anti-inflammatory, while enzymes promote pro-inflammatory response Ileal tissue Diet Th1 Th2 Th17 Treg CSB DDGS = = = = WM = = = Colonic tissue Diet Th1 Th2 Th17 Treg CSB DDGS = = = WM = = = CSB+E = = = DDGS+E = = = WM+E = = CSB+E = = = DDGS+E = = WM+E = =
Enzymes decrease MUC 2 expression and may decrease fermentable losses of Thr Ferrandis-Villa et al. (2017) and NPB report # 014-045
Overview of LC-MS based chemometrics Basal diet Enzyme Marker analysis Digesta samples Jejunum Ileum Cecum Sample preparation Extraction Derivatization Clustering analysis LC-MS analysis LC column MS system Structural analysis Multivariate data analysis Scores plot Modeling Loadings plot Marker identification Quantitation Bioinformatics Pathway analysis
Chemometric (LC/MS) composition of ileal digesta differs by diet, but not by enzyme addition 60 Corn-SBM diet 40 20 DDGS diet t[2] 0-20 -40 CN_CSB CN_DDGS CN_WM EN_CSB EN_DDGS EN_WM -60 Wheat middlings diet -80-60 -40-20 0 20 40 60 80 t[1] SIMCA-P+ 12-2017-06-21 15:24:56 (UTC-6)
Chemometric (LC/MS) composition of cecal digesta differs by diet, but not by enzyme addition 60 40 Corn-SBM diet t[2] 20 0-20 CN_CSB CN_DDGS CN_WM EN_CSB EN_DDGS EN_WM -40-60 DDGS diet Wheat middlings diet -80-60 -40-20 0 20 40 60 80 t[1] SIMCA-P+ 12-2017-06-21 15:30:51 (UTC-6)
IL 1 (p g /m L ) Carbohydrases modify fiber in wheat middlings and corn DDGS, but do no increase digestibility Decrease fermentation Change in cytokines 2 5 0 0 2 0 0 0 a Increase in viscosity 1 5 0 0 1 0 0 0 c c c b b 5 0 0 0 C O N D D G S W M C O N + E n z D D G S + E n z W M + E n z Increase in AID, but not ATTD of DM Decrease in mucin Microbes
Fiber degradation, % Increase degradation of recalcitrant fiber and implications of different portions on gut physiology 100% Recalcitrant or indigestible fiber Next NPB project - composition and pretreatment to increase degradation Slow fermentable fiber Rapidly fermentable fiber T1 Fermentation time T2
Conclusions In vitro experiments demonstrate that carbohydrase enzymes Little effect on digestibility of corn fiber and marginal effects on wheat fiber Marginal differences among products In vivo experiments demonstrate that carbohydrase enzymes degrade wheat fiber and to less extend corn fiber Products of fiber degradation (oligosaccharides) impact Viscosity Immunity Data supports hypothesis of extra-caloric effects, but can you measure it?
Funding and collaborations Funding: National Pork Board (NPB) Collaborations: Minnesota Discovery, Research, and InnoVation Economy (MnDRIVE) Danisco Animal Nutrition (DuPont) Archer Daniels Midland (ADM) USDA ARS
We are looking forward to seeing you in September September 16-19, 2017 @ St Paul RiverCentre Pedro E. Urriola 1988 Fitch Ave St Paul MN 55108 Urrio001@umn.edu (612)624-1244 http://animalsystemsbiology.cfans.umn.edu/