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

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Comparative efficacy of xylanases on growth performance and digestibility in growing pigs fed wheat and wheat branor corn and corn DDGS-based diets supplemented with phytase S.P. Ndou a , E. Kiarie a,b,∗ , A.K. Agyekum a , J.M. Heo c , L.F. Romero b , S. Arent d , R. Lorentsen d , C.M. Nyachoti a a b c d

Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 DuPont Industrial Biosciences – Danisco Animal Nutrition, Marlborough, Wiltshire SN8 1XN, United Kingdom Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, South Korea DuPont Nutrition Biosciences – Enzyme Research & Development, Brabrand, Arhus DK-8220, Denmark

a r t i c l e

i n f o

Article history: Received 15 May 2015 Received in revised form 27 August 2015 Accepted 28 August 2015 Available online xxx Keywords: Digestibility Diet type Xylanase Pigs Growth performance Phytase

a b s t r a c t Two experiments were conducted to determine the effects of supplemental xylanases on growth performance, coefficients of apparent ileal (CAID) and total tract (CATTD) digestibility in growing pigs fed wheat- or corn -based diets. Two basal diets were formulated based on either corn plus 40% corn distillers dried grains with solubles or wheat plus 25% wheat co-products and fed without or with five xylanases from different microbial origins. The xylanases were identified as A, B, C, D and E and fed at 75 mg of xylanase protein/kg of feed. All diets contained added phytase. In Experiment 1, the diets were offered for 42 d to 96 individually penned gilts (29.0 ± 0.2 kg) to give 8 pigs per diet. An interaction between diet type and xylanase was observed for ADG (P < 0.05) such that pigs fed wheat diets with xylanases A and C and corn diets with xylanases A and D had greater (P = 0.044) ADG than pigs fed xylanase-free diets or wheat diets with xylanase D or corn diets with xylanases B and C. A main effect of xylanase was observed for G:F in which case xylanase A improved G:F by 10% (P = 0.010) compared to the control. In Experiment 2, TiO2 (3 g/kg) was added in all diets and pigs were allotted diets in a Youden Square design, to give 6 replicates per diet. Interaction (P < 0.05) between diet type and xylanase were observed on CAID of gross energy (GE), insoluble arabinose and xylose (iAX), total arabinose and xylose (tAX), insoluble NSP (iNSP), and total NSP (tNSP). In this context, pigs fed wheat-based diets with xylanase C and D and those fed corn-based diets with xylanases B and E had greater (P < 0.05) CAID of GE compared to those fed xylanase-free diets. The CAID of tAX and tNSP in pigs fed wheat-based diets with xylanase C and corn-based diets with xylanases B and E were higher (P < 0.001) than in pigs fed the other diets. There was no treatment effects on CATTD of GE, DM and crude protein, however, xylanases independently improved (P = 0.040)

Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; CAID, coefficient of apparent ileal digestibility; CATTD, coefficient of apparent total tract digestibility; BW, body weight; cDDGS, corn-distillers’ dried grains with solubles; DM, dry matter; EE, ether extract; G:F, gain:feed ratio; GE, gross energy; iAX, insoluble arabinose + xylose; iNSP, insoluble non-starch polysaccharide; N, nitrogen; NSP, non-starch polysaccharides; P, phosphorus; sAX, soluble arabinose + xylose; tAX, total of arabinose and xylose; sNSP, soluble non-starch polysaccharide; tNSP, total non-starch polysaccharide; WB, wheat bran. ∗ Corresponding author at: DuPont Industrial Biosciences – Danisco Animal Nutrition, Marlborough, Wiltshire SN8 1XN, United Kingdom. E-mail address: [email protected] (E. Kiarie). http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011 0377-8401/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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CATTD of fat compared to the control. Different xylanases responded differently in improving dietary components digestibility and growth performance depending on the diet type. However, the comparisons made in the present study are only valid for the test conditions as some of the xylanases may have been dosed at levels below the biological optimum and others well above. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cereal co-products can be incorporated in swine diets as replacement of substantial amounts of conventional feed ingredients to offset feed cost. Wheat bran (WB) and corn-distillers’ dried grains with soluble (cDDGS) are common agro-industrial co-products that can be used as alternative low-cost feed resources. However, dietary inclusions of these co-products increase non-starch polysaccharides (NSP) that escape enzymatic digestion or mask other nutrients from hydrolysis by endogenous enzymes in the pig gut (Zijlstra et al., 2010; Kiarie et al., 2014). Xylans are the predominant component of NSP in both cereal co-products from biofuel and milling industries (Zijlstra and Beltranena, 2013; Woyengo et al., 2014). Wheat co-products have high phytate content (Lenis and Jongbloed, 1999; Cowieson et al., 2004). Phytate binds nutrients and prompts nutrient excretion, thereby reducing nutrient digestibility and growth performance (Bedford, 2000; Cowieson et al., 2004). Because of the heterogeneity and complex nature of xylans; complete hydrolysis requires a large variety of cooperatively acting enzymes (Wong et al., 1988; Coughlan and Hazlewood, 1993; Harris and Ramalingam, 2010). Endo-1,4-␤-d-xylanase is a crucial component that cleaves the backbone of xylan, ␤-d-xylosidases cleave xylose monomers from the non-reducing end of xylo-oligosaccharides and xylobiose while removal of the side groups is catalyzed by numerous accessory or debranching enzymes depending on the nature of the xylan. With regard to feed application, only a partial hydrolysis of xylan is needed to counteract the anti-nutritive effects of dietary fiber (Bedford and Schulze, 1998). However, many factors related to the enzyme source and biochemical characteristics, diet and animal have been suggested to (singly or interactively) influence variable responses seen with application of xylanases in practical animal feeding (Bedford and Schulze, 1998; Partridge, 2001; Ravindran, 2013). With respect to enzyme, it is known some xylanases require lengths of un-substituted xylan in order to bind and hydrolyse the backbone, while others do not (Wong et al., 1988; Coughlan and Hazlewood, 1993; Harris and Ramalingam, 2010). Furthermore, some micro-organisms produce multiple xylanases as a strategy for complete xylan hydrolysis (Sunna and Antranikian, 1997; Wong et al., 1988). Thus, xylanases from different microbial sources are likely to facilitate unique hydrolytic effects on digesta components during transit in the gut. These differences are rarely evaluated to optimize application of xylanases in animal nutrition. Historically, xylanase and phytase had been widely used to reduce anti-nutritional effects of NSP and phytate, respectively (Rosenfelder et al., 2013; Kiarie et al., 2014). For example, if xylanase and phytase are included in the same diet, the former hydrolyze the NSP, and thus providing better access for the latter to reduce the interaction of phytate with protein and starch molecules as well as reducing binding of elemental phosphorus (Zijlstra et al., 2010). Previous studies have demonstrated synergetic effects of phytase and xylanase on nutrient digestibility in pigs (Kim et al., 2008; Kiarie et al., 2010) and broilers (Liu et al., 2011; Kiarie et al., 2014), but others have pinpointed that beneficial effects originates mainly from phytase alone (Olukosi et al., 2007). Therefore, inconsistences in these findings warrant further investigations using xylanases of different microbial sources. Furthermore, although phytase is a common additive in majority of monogastric diets, too much emphasis had been placed on interpretation of xylanases evaluation without phytase in the background. Understanding efficacy of xylanases from different microbial origin could assist nutritionists to identify appropriate enzyme activities that optimize hydrolysis for a particular cereal co-product during feed formulation. It was hypothesized that xylanases of different microbial origin will differentially influence growth performance and nutrient digestibility in growing pigs fed different diet types with added phytase. Thus, the objective of the present study was to evaluate the effect of five xylanases from different microbial sources on growth performance, coefficient apparent ileal digestibility (CAID) and apparent total tract digestibility (CATTD) in growing pigs fed wheat-wheat bran-based or corn-corn distillers dried grains with soluble (DDGS)-based diets with added phytase. 2. Materials and methods The experimental procedures for this study were approved and performed according to the ethical guidelines specified by the Animal Care Committee of University of Manitoba (Reference Number: F09-008/1/2) and standard guidelines of the Canadian Council on Animal Care (CCAC, 2009). 2.1. Experiment 1: growth performance 2.1.1. Diets Two basal diets, based on either wheat plus 25% wheat co-products or corn plus 40% corn DDGS were formulated to meet or exceed the NRC (1998) nutrient requirements for growing pigs (20–50 kg BW). These two diets were fed without or with Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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Table 1 Ingredient and calculated nutrient composition of experimental diets (as-fed basis). Item

Wheat-based diet

Corn-based diet

Ingredient, g/kg Corn Corn DDGS Wheat HRW Wheat middlings Wheat bran Soybean meal Beef tallow Limestone Monocalcium phosphate Salt Vitamin-Mineral Premixa l-Lysine-HCl dl-Methionine Threonine Tryptophan TiO2

– – 565.7 199.4 50.6 135 9.9 13.8 0.8 4.7 5.0 7.3 1.7 3.0 0.1 3.0

414.4 400 – – – 150 – 13.0 – 3.0 5.0 7.5 1.0 2.5 0.6 3.0

Calculated provisions Dry matter, g/kg Crude protein, g/kg Net energy, MJ/kg SID lysine, g/kg SID methionine, g/kg SID methionine + cysteine, g/kg SID threonine, g/kg SID tryptophan, g/kg Neutral detergent fiber, g/kg Calcium, g/kg Total phosphorus, g/kg Available phosphorus, g/kg

890.9 214 10.1 11.6 3.5 6.7 7.6 1.9 206 7.6 6.1 2.3

882.3 191 9.95 11.6 3.5 7.0 7.4 1.9 189 6.5 5.7 2.4

a Provided the following nutrients (per kg of air-dry diet): Vitamins: A, 2000 IU, D3 200 IU, E, 40 mg, K, 2 mg, B1 , 1.5 mg, B2 , 7 mg, B6 , 2.5 mg, B12 , 25 ␮g, calcium pantothenate, 14 mg, folic acid, 1 mg, niacin, 21 mg, biotin, 70 ␮g. Minerals: Cu, 10 mg (as copper sulphate), iodine, 0.4 mg (as potassium iodine), iron, 120 mg (as ferrous sulphate), Mn, 10 mg (as manganous oxide), Se, 0.3 mg (as sodium selenite), Zn, 110 mg (as zinc oxide).

either xylanase A, B, C, D or E, to produce 12 experimental diets. Xylanases were of different microbial origins and were mono-component preparation of endo-1,4-␤-d-xylanase. The molecular weights of the xylanases were 33, 21, 23, 21 and 23 for xylanase A, B, C, D and E, respectively. Xylanase A was from Fusarium verticilloide, B from Aspergillus Clavatas, C from Bacillus substilis and xylanase D was from Trichoderma reesei. Xylanases A, B and D were produced from modified strain of Trichoderma reesei and xylanase C was produced from Bacillus substilis. Xylanase E was a commercial product and the source organism is not known but was indicated to be produced by modified strain of Trichoderma reesei. In a preliminary assay, xylanases showed more than 80% recovery in pepsin tests at pH 2 and 3. In brief, pepsin was measured by incubating the enzyme in 100 mM glycine buffer, pH 3.5 containing 0.2 g/l pepsin at 40 ◦ C for 2 h with shaking. Subsequently, the residual xylanase activity was determined using Megazyme xylanase assay kit (Megazyme International Ireland Ltd., Bray, Ireland). Xylanases were dosed at 75 mg of xylanase protein/kg of feed. The enzymes along with the enzyme assay procedures were supplied by Danisco Animal Nutrition (Danisco UK Ltd., Marlborough, Wiltshire, UK). Diets were fed as mash and all diets contained supplemental microbial phytase with an activity of 500 FTU/kg (Axtra® PHY). The 500 FTU phytase/kg supplied 0.093% Ca and 0.12% digestible or available P. The ingredient compositions and calculated nutrient compositions of the control diets are presented in Table 1.

2.1.2. Pigs, management and performance measurements Ninety-six Genesus gilts [(Yorkshire − Landrace) × Duroc] with an initial average body weight (BW) of 29.0 (s.d. = 0.23) kg were obtained from Glenlea Swine Research unit, University of Manitoba. Upon arrival, pigs were individually penned and based on BW assigned to 12 experimental diets in a randomized complete block design to give 8 pens per treatment using the Experimental Animal Allotment Program of Kim and Lindemann (2007). Each pen measured 1.5 m × 1.2 m and was equipped with a stainless steel self-feeder and a low-pressure nipple drinker that allowed unlimited access to feed and water throughout the experiment. The experiment lasted for 42 days. Weight of each pig on Day 0 and 42, and feed intake during the experimental period were measured to calculate average daily feed intake (ADFI), average daily gain (ADG) and gain: feed ratio (G:F). Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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2.2. Experiment 2: ileal and total tract digestibility 2.2.1. Diets The experiment used 12 dietary treatments used in Experiment 1, except that titanium dioxide (TiO2 ; 3 g/kg) was added to all diets as an indigestible marker at the expense of either corn or wheat (Table 1). 2.2.2. Pigs, management and sample collections Twelve Genesis ([Yorkshire − Landrace dam] × Duroc sire) barrows with an initial average body weight (BW) of 23.0 ± 0.41 kg (mean ± standard deviation) were obtained from Glenlea Swine Research Unit, University of Manitoba. Pigs were housed in individual pens (1.5 m × 1.2 m) within a temperature-controlled room of 21 ± 2 ◦ C (mean ± standard deviation). Each pen was fitted with a stainless steel self-feeder and a low-pressure nipple drinker that allowed unlimited access to water. Lighting was provided for 16 h daily from 7:00 AM to 11:00 PM. Upon arrival in the experimental unit, pigs were allowed a 5 day acclimation period, and subsequently fed with a commercial growing pig diet ad libitum. Thereafter, the 12 barrows were surgically fitted with simple T-cannula at the distal ileum as described by Nyachoti et al. (2002). A post-surgical recovery period of 14 days was allowed. After the recovery period, the ileal cannulated barrows were randomly allocated to the experimental diets in a Youden Square design with 12 treatments, 6 columns (experimental periods) and 12 rows (pigs), to give 6 replicates per treatment. Pigs were fed twice daily (0830 and 1530 h) at 2.6 times maintenance energy requirement (NRC, 1998). Each experimental period lasted for 14 days; adaptation periods of 10 days, followed by grab fecal collection for 2 days and ileal digesta collection for 2 days. Digesta and fecal samples were stored immediately at −20 ◦ C for further analysis. 2.3. Laboratory analysis Digesta samples were thawed and pooled for each pig by period, homogenized in a blender (Waring Commercial, Torrington, CT, USA), sub-sampled and freeze dried. Fecal samples were dried in an oven at 60 ◦ C for 4 days, pooled per pig and period and subsampled. Both digesta and fecal samples were then finely ground in a Thomas Wiley mill model 4 (Labwrench, Midland, ON, Canada) and mixed prior to chemical analysis. The diets, ileal digesta, and fecal samples were analyzed for dry matter (DM), gross energy (GE), ether extract (EE), nitrogen (N), minerals (Ca, P and Ti) and NSP contents. Ileal digesta was further analyzed for starch. Dry matter content was determined according to AOAC (1990); method 925.09. Nitrogen content was determined using a combustion analyzer (Model CNC-2000; LECO Corporation, St. Joseph, MI, USA). Gross energy was determined using an automated adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL, USA) with benzoic acid as the reference material. Samples for Ca and P analyses were prepared according to AOAC (1990); method 990.08 and read on a Varian Inductively Coupled Plasma Mass Spectrometer (Varian Inc., Palo Alto, CA, USA). Titanium dioxide was determined according to Lomer et al. (2000) using a Varian Inductively Coupled Plasma Mass Spectrometer. Ether extract was determined using hexane as the solvent according to AOAC (1990; method 920.39). Total starch was determined by a modified version of Englyst (Englyst et al., 2000), which involved initial heat dispersion together with heat stable amylase followed by treatment with alkali to disperse any retrograded type III resistant starch. A pH 4.5 buffered aliquot was treated with amyloglucosidase to release glucose which was quantified by high performance anion exchange chromatography with pulsed amperometric detection. The mono sugars composition of the NSP were determined by the method of Englyst (Englyst et al., 1994), whereby starch was completely dispersed and then hydrolysed enzymatically. The NSP was isolated by precipitation in 80% ethanol then hydrolysed by sulphuric acid and the released component sugars measured by gas chromatography as their alditol acetate derivatives. The basal diets were further subjected to multi-mycotoxins analysis according to AOAC 2008.02 (modified) in a commercial laboratory (Midwest Laboratories Inc., Omaha, NE). Briefly, ground samples were extracted using a polar extraction solvent and filtrated through immunoaffinity columns. The extract was analyzed using liquid chromatography with tandem mass spectrometry and different mycotoxins were determined by retention time, molecular weight, and molecular fragmentation ions. The samples were analyzed for major mycotoxins including aflatoxin, deoxynivalenol, fumonisin, ochratoxin, zearalenone and T-2 toxin. The detection limits were: aflatoxin = 1 ppb, deoxynivalenol = 0.1 ppm, fumonisin = 0.1 ppm, Ochratoxin = 1 ppb, zearalenone = 50 ppb and T-2 = 0.1 ppm. The in-feed phytase activity was analyzed according to the method of Engelen et al. (2001). One FTU of enzyme activity is defined as the amount of enzyme that liberates 1 ␮mol of inorganic P per min at 37 ◦ C and pH 5.5 (Engelen et al., 2001). 2.4. Calculations and statistical analysis The CAID and CATTD values of DM, GE, N, Fat, Starch, Ca and P and NSP were calculated as follows:



CAID/CATTD =



(NT/Ti) diet − (NT/Ti) ileal digesta or feces , (NT/Ti) diet

where (NT/Ti) diet = ratio of component and titanium in diet, (NT/Ti) ileal digesta or feces = ratio of component and titanium in ileal digesta or feces. The component can be DM, GE, N, fat, starch, Ca, P, or NSP. Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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Table 2 Analyzed composition (g/kg, as-fed basis) of experimental diets. Item

Wheat-based

Corn-based

Dry matter Gross energy, MJ/kg Crude protein Fat Starch Calcium Phosphorus

867 17.4 228 48.8 466 7.7 6.0

882 16.5 201 53.9 331 6.2 5.9

Non-starch polysaccharides (NSP) Soluble arabinose + xylose Insoluble arabinose + xylose Total arabinose + xylose Soluble NSP Insoluble NSP Total NSP

7.70 60.3 68.0 20.7 104 125

3.03 65.3 68.3 19.1 125 144

All statistical analyses were performed using SAS (SAS Software Release 9.2, SAS Institute, Inc., Cary, NC, USA). Data generated for the growth performance were analyzed as a 2 × 6 factorial arrangement of treatments with eight randomized replicates. The main effects of cereal type, xylanase and their interaction on ADFI, ADG and G:F were determined by GLM procedure. Data from Experiment 2 (digestibility trial) were analyzed using the mixed procedure for repeated measures. The model included period, cereal type, xylanase and their interactions as fixed effects, while the pig nested within each square was a random factor. The experimental period was the repeated term in the model. The individual animal was used as the experimental unit for all analyses done. Treatment mean values were separated using Tukey’s test. Differences observed among treatment means were considered significant at P < 0.05 and trends (0.05 > P < 0.10) were discussed. 3. Results Aflatoxin, zearalenone and T-2 toxins were not detected. Fumonisin was not detected in wheat diets but was detected in corn diets at 0.20 ppm. In wheat diets, deoxynivalenol and ochratoxin were 0.3 ppm and 1.7 ppb, respectively; and the corresponding values in corn diets were 0.5 ppm and 2.0 ppb, respectively. Analyzed chemical composition of the basal diets is shown in Table 2. Wheat diets had higher crude protein (228 vs. 201 g/kg) and starch (466 vs. 331 g/kg). Although the concentration of total arabinoxylans (arabinose + xylose) was comparable between wheat and corn diets, the wheat diets had 2.5 times more concentration of soluble arabinoxylans relative to the corn diets. In contrast, the corn diets had greater concentration of total insoluble NSP (125 vs. 104 g/kg) than wheat diets and as a result corn diets had higher concentration of total NSP (144 vs. 125 g/kg). Xylanase activities in the feed were not determined because the tested xylanases were prototypes and their assays in feed matrix have not been developed and optimized. However, to ensure proper inclusion in the diets, the formulation of the product was such that each xylanase was mixed with precise amount of phytase and shipped to the research site as a single product. Therefore, phytase was assayed in the feed to confirm inclusion of the product and to assess uniformity of feed mixing. The assayed phytase activity (FTU/kg of feed) in wheat based diets was 701, 721, 729, 761, 741 and 703 for control, A, B, C, D and E respectively and the corresponding values for corn diets were 442, 498, 567, 504, 557, and 521, respectively. 3.1. Experiment 1: growth performance There was a significant interaction between cereal type and xylanase on ADG but not (P > 0.05) on final BW, ADFI and G:F (Table 3). Pigs fed wheat diets with xylanase A and C and corn diets with xylanase A and D had greater (P = 0.044) growth rates than pigs fed xylanase-free diets, wheat diets with xylanase D and corn diets with xylanase B and C. Average daily feed intake, final BW, and G:F ratio were not affected (P > 0.05) by diet type. However, the main effect of xylanase was observed for G:F in which case pigs fed diets with xylanase A had 10% better G:F (P = 0.01) compared to pigs fed diets without xylanase. 3.2. Experiment 2: ileal and total tract digestibility 3.2.1. Coefficients of apparent ileal digestibility There was an interaction (P < 0.05) of diet type and xylanase on CIAD of GE, but not (P > 0.05) on CAID of DM, N, Ca, P, fat and starch (Table 4). In this context, pigs fed wheat diets with xylanase C and D and those fed corn diets with xylanase B, and E, had greater (P < 0.05) CAID of GE compared to those fed xylanase-free diets. Diet type effects were observed in which pigs fed corn-based diets had greater CAID of P (P < 0.001) and fat (P < 0.003) than in pigs offered wheat-based diets. There were interactions (P < 0.05) between diet type and xylanase on CAID of insoluble arabinose and xylose (iAX), total arabinose and xylose (tAX), insoluble NSP (iNSP) and total NSP (tNSP) (Table 4). Pigs fed xylanase C had higher (P = 0.002) CIAD of iAX that pigs fed any other treatment. The CAID of tAX in wheat diets was greater (P = 0.011) for xylanase C relative to Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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Table 3 Influence of xylanase on the performance characteristics of growing pigs fed wheat and WB- and corn or cDDGS-based diets (Experiment 1). Item Wheat Wheat Wheat Wheat Wheat Wheat Corn Corn Corn Corn Corn Corn Diet

Xylanase

Control A B C D E Control A B C D E SEM Wheat Corn SEM Control A B C D E SEM

Initial BW (kg)

Final BW (kg)

ADFI (g)

ADG (g)

G:F (g/kg)

23.0 23.0 23.0 22.9 22.9 22.9 22.9 22.9 23.0 22.9 22.9 22.9 0.46 22.9 22.4 0.65 23.0 22.9 23.1 22.9 22.9 22.9 0.85

60.8 63.5 61.6 63.6 61.2 61.9 59.7 62.3 60.8 60.4 62.7 61.7 6.23 61.7 61.4 1.23 61.2 62.5 61.8 60.9 61.7 60.5 2.13

2120 2010 2056 2136 2103 2016 2073 2069 2010 2011 2060 2081 52.9 2060 2054 20.62 2098 2038 2035 2040 2081 2055 35.96

902b 965a 929ab 968a 913b 928ab 877b 939a 901b 892b 945a 924ab 20.8 919 912 8.78 891y 952x 916xy 909xy 929xy 926xy 14.63

427 480 452 462 434 459 424 455 448 445 459 444 1.10 450 447 5.50 425y 469x 449xy 449xy 446xy 451xy 0.80

P-value Diet Xylanase Diet × xylanase

0.788 0.568 0.458

0.894 0.256 0.456

0.786 0.868 0.887

0.614 0.038 0.044

0.786 0.006 0.056

abxy Means in the same column with different superscripts differ (P < 0.05); SEM, standard error of means; WB, wheat bran; cDDGS, corn distillers’ dried grains with solubles; BW, body weight; ADFI, average daily feed intake; ADG, average daily feed intake; G:F, gain:feed ratio; A, Xylanase A; B, Xylanase B; C, Xylanase C; D, Xylanase D; E, Xylanase E.

Table 4 Effect of xylanase on the coefficients of ileal digestibility of dietary components in pigs fed wheat and WB- and corn and cDDGS-based diets. Item Wheat Wheat Wheat Wheat Wheat Wheat Corn Corn Corn Corn Corn Corn Diet

Xylanase

P-value Diet Xylanase Diet × xylanase

Control A B C D E Control A B C D E SEM Wheat Corn SEM Control A B C D E SEM

DM

GE

N

Ca

0.642 0.623 0.666 0.722 0.682 0.666 0.674 0.658 0.669 0.658 0.661 0.683 0.017 0.667 0.667 0.070 0.658 0.641 0.667 0.690 0.671 0.674 0.121

0.626b 0.606b 0.649ab 0.707a 0.669a 0.648ab 0.641b 0.653ab 0.671a 0.646ab 0.662ab 0.681a 0.019 0.651 0.664 0.008 0.648 0.630 0.660 0.677 0.665 0.665 0.013

0.789 0.775 0.812 0.841 0.821 0.801 0.799 0.785 0.797 0.796 0.796 0.791 0.013 0.806 0.795 0.005 0.794 0.780 0.805 0.818 0.808 0.799 0.009

0.616 0.605 0.643 0.675 0.629 0.619 0.639 0.621 0.682 0.621 0.674 0.675 0.039 0.631 0.652 0.012 0.628 0.613 0.663 0.648 0.652 0.647 0.021

0.989 0.147 0.051

0.192 0.225 0.044

0.130 0.073 0.219

0.234 0.607 0.505

P 0.450 0.402 0.448 0.497 0.428 0.434 0.589 0.545 0.596 0.558 0.582 0.585 0.024 0.443g 0.576f 0.010 0.520 0.477 0.52. 0.527 0.505 0.509 0.017 <0.001 0.284 0.368

Fat

Starch

sAX

iAX

0.590 0.636 0.654 0.752 0.619 0.654 0.704 0.704 0.688 0.711 0.699 0.707 0.026 0.651b 0.702a 0.011 0.647 0.670 0.671 0.731 0.659 0.680 0.002

0.943 0.935 0.941 0.943 0.942 0.953 0.965 0.946 0.966 0.965 0.945 0.960 0.013 0.943 0.966 0.011 0.935 0.946 0.954 0.945 0.943 0.945 0.001

−0.778 −0.691 −0.108 −0.876 −0.876 −0.932 −0.221 0.001 0.210 −0.262 −0.029 0.003 0.143 −0.87g −0.081f 0.559 −0.500 −0.345 −0.531 −0.569 −0.452 −46.4 0.097

0.240b 0.301bc 0.368b 0.501a 0.365b 0.335bc 0.335bc 0.361bc 0.310bc 0.293bc 0.345bc 0.381b 0.037 0.352 0.337 0.015 0.318 0.301 0.339 0.397 0.355 0.358 0.031

0.003 0.352 0.075

0.546 0.254 0.898

<0.001 0.668 0.343

0.513 0.167 0.002

tAX 0.063b 0.219ab 0.222ab 0.346a 0.226ab 0.193b 0.154b 0.262ab 0.353a 0.248ab 0.317a 0.351a 0.035 0.212g 0.281f 0.015 0.109y 0.241x 0.287x 0.297x 0.271x 0.272x 0.003 <0.001 <0.001 0.011

sNSP −0.026 −0.035 −0.009 0.011 −0.076 −0.07 0.171 0.464 0.382 0.258 0.392 0.291 0.071 −0.04g 0.326f 0.029 0.073 0.230 0.142 0.135 0.158 0.110 0.005 <0.001 0.374 0.203

iNSP 0.260b 0.318b 0.361ab 0.463a 0.389ab 0.353ab 0.248b 0.358ab 0.373ab 0.289b 0.360ab 0.382ab 0.025 0.357 0.335 0.010 0.254y 0.338x 0.367x 0.376x 0.374x 0.367x 0.002 0.131 <0.001 <0.001

tNSP 0.181e 0.271cd 0.291bcd 0.375a 0.321abc 0.277cd 0.234de 0.364ab 0.403a 0.283cd 0.349abc 0.391a 0.026 0.286g 0.338f 0.011 0.207y 0.318x 0.347x 0.329x 0.335x 0.334x 0.002 <0.001 <0.001 0.002

a–y Means in the same column with different superscripts differ (P < 0.05); SEM, standard error of means; sAX, soluble arabinose + xylose; iAX, insoluble arabinose + xylose; tAX, total of arabinose and xylose; sNSP, soluble non-starch polysaccharide; iNSP, insoluble non-starch polysaccharide; tNSP, total non-starch polysaccharide; A, Xylanase A; B, Xylanase B; C, Xylanase C; D, Xylanase D; E, Xylanase E.

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Table 5 Effect of xylanases on the coefficients of total tract digestibility of dietary components in pigs fed wheat and WB- and corn and cDDGS-based diets. Item Wheat Wheat Wheat Wheat Wheat Wheat Corn Corn Corn Corn Corn Corn Diet

Xylanase

Control A B C D E Control A B C D E SEM Wheat Corn SEM Control A B C D E SEM

P-value Diet Xylanase Diets × xylanase

DM

GE

N

Ca

0.827 0.826 0.833 0.849 0.827 0.832 0.836 0.838 0.829 0.832 0.842 0.840 0.007 0.832 0.836 0.003 0.831 0.832 0.831 0.840 0.835 0.836 0.005

0.808 0.814 0.820 0.842 0.811 0.818 0.818 0.820 0.815 0.821 0.824 0.825 0.009 0.819 0.821 0.004 0.813 0.817 0.818 0.831 0.818 0.821 0.006

0.877 0.893 0.887 0.907 0.887 0.892 0.891 0.891 0.881 0.892 0.891 0.883 0.008 0.890 0.888 0.003 0.884 0.892 0.884 0.899 0.889 0.887 0.006

0.696 0.639 0.682 0.693 0.659 0.687 0.673 0.638 0.656 0.558 0.662 0.633 0.043 0.676 0.637 0.017 0.685 0.639 0.669 0.626 0.661 0.660 0.030

0.340 0.745 0.197

0.225 0.192 0.544

0.130 0.073 0.219

0.114 0.777 0.607

P

Fat 0.540 0.485 0.503 0.542 0.479 0.528 0.572 0.601 0.601 0.636 0.604 0.611 0.030 0.513b 0.590a 0.013 0.556 0.550 0.552 0.539 0.541 0.569 0.012

<0.001 0.927 0.167

0.710 0.764 0.772 0.819 0.793 0.788 0.823 0.819 0.808 0.807 0.809 0.813 0.021 0.774b 0.813a 0.009 0.767z 0.792y 0.790y 0.813x 0.801y 0.801y 0.002 0.001 0.040 0.087

sAX 0.734 0.854 0.785 0.767 0.852 0.843 0.686 0.592 0.715 0.704 0.795 0.712 0.065 0.806a 0.701b 0.027 0.710 0.723 0.750 0.736 0.823 0.777 0.046 <0.007 0.551 0.560

iAX

tAX

sNSP

0.530 0.567 0.563 0.581 0.542 0.530 0.539 0.567 0.537 0.545 0.573 0.567 0.024 0.548 0.554 0.009 0.535 0.554 0.549 0.563 0.557 0.548 0.015

0.557 0.576 0.573 0.602 0.564 0.565 0.551 0569 0.551 0.558 0.591 0.578 0.018 0.573 0.566 0.007 0.554 0.573 0.562 0.580 0.577 0.572 0.012

0.836 0.876 0.814 0.847 0.806 0.881 0.845 0.806 0.854 0.847 0.886 0.863 0.032 0.843 0.850 0.013 0.840 0.841 0.834 0.847 0.846 0.872 0.023

0.601 0.839 0.393

0.533 0.682 0.405

0.705 0.887 0.297

iNSP 0.523 0.552 0.546 0.576 0.540 0.525 0.558 0.619 0.587 0.594 0.619 0.613 0.019 0.544b 0.598a 0.008 0.541 0.586 0.566 0.585 0.580 0.569 0.013 <0.001 0.175 0.416

tNSP 0.584 0.602 0.599 0.629 0.592 0.594 0.605 0.654 0.636 0.641 0.668 0.659 0.015 0.600b 0.644a 0.003 0.594 0.628 0.617 0.635 0.630 0.627 0.010 <0.001 0.097 0.238

abxyz Means in the same column with different superscripts differ (P < 0.05); SEM, standard error of means; sAX, soluble arabinose + xylose; iAX, insoluble arabinose + xylose; tAX, total of arabinose and xylose; sNSP, soluble non-starch polysaccharide; iNSP, insoluble non-starch polysaccharide; tNSP, total non-starch polysaccharide; A, Xylanase A; B, Xylanase B; C, Xylanase C; D, Xylanase D; E, Xylanase E.

xylanase E and control whilst in corn diets xylanases B, D and E had higher (P = 0.011) CIAD of tAX than control. All xylanases improved (P < 0.01) CIAD of tNSP across diet types relative to respective controls; except C in corn and the magnitude of responses among xylanases was dependent on the diet types. In wheat diets xylanase C (0.375) had higher CIAD of tNSP compared to xylanases A (0.271), B (0.291) and E (0.277) whilst in corn diets, xyalanase B (0.403) and E (0.391) showed greater CIAD of tNSP compared to xylanase C (0.283). Pigs fed diets corn-enriched diets had higher CIAD of soluble arabinose and xylose (sAX) and soluble NSP (sNSP) than pigs fed wheat-based diets. 3.2.2. Coefficients of apparent total tract digestibility There were no interactions (P > 0.05) between diet type and supplemental xylanase on CATTD of components evaluated (Table 5). Main effects of the diets were such that pigs fed corn-based diets had higher (P < 0.001) CATTD of P, fat, iNSP and tNSP than pigs fed wheat-based diets. In contrast, pigs offered wheat diets had higher (P < 0.001) CATTD of soluble arabinoxylans than pigs offered corn diets. Although pigs fed diets with xylanases had higher (P = 0.040) CATTD of fat compared to control fed pigs, xylanase C fed pigs had higher CATTD of fat relative to pigs receiving xylanases A, B, D, and E. 4. Discussion Pigs fed wheat diets with xylanase A and C and those fed corn diets with xylanase A and D had similar and the highest growth rates. Substrate-directed response could explain the observations that high growth rates occurred in pigs supplemented with different xylanase products in both corn- and wheat-based diets (Sunna and Antranikian, 1997; Bedford and Schulze, 1998; Collins et al., 2005). These findings further suggest the efficacy of xylanase depend not only on the nature of dietary substrates (Wong et al., 1988; Collins et al., 2005), but also on microbial origin of the xylanase. These results are in agreement with the observations of Courtin and Delcour (2001) that xylanases of different microbial origin could have variable effects on hydrolysis of dietary fibers. The higher growth rates and G:F observed in pigs fed diets supplemented with xylanase A than in pigs fed xylanase-free diets suggested that this product has a great catalytic versatility perhaps linked to physicochemical properties related to higher molecular weight (Wong et al., 1988; Coughlan and Hazlewood, 1993; Harris and Ramalingam, 2010). However, better growth performance effects due to xylanase A over the control were not accompanied with improved nutrients and fiber digestibility. Perhaps suggesting the improved performance was as a result of other mechanisms not measured in the present study. Earlier studies have reported that NSP-degrading enzymes hydrolyze the cells walls, increase permeability of cell walls or cut long chain polysaccharides (Bedford and Schulze, 1998; Partridge, 2001). As observed in the current study, pigs offered wheat-based diets supplemented with xylanase C and D and those on corn-based diets supplemented with xylanase B Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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and E had greater CAID of GE compared to those fed xylanase-free diets. These findings are in agreement with Lv et al. (2013) who reported that carbohydrase supplementation improves digestibility of GE, but the variation in our results might be due to uniqueness of xylanolytic activity of different xylanases. This variation can also be ascribed to differences in dietary composition and NSP contents across diets (Adeola and Cowieson, 2011). The paradox that ileal digestibility of GE was influenced by supplemental xylanase but digestibilities of energy yielding substrates, such as starch, fat and nitrogen were the same across all treatments is difficult to explain. Nevertheless, it supports the assertion that there could be other dietary components (e.g. NSP), that were degraded during digesta transit and indirectly contributed to ileal digestibility of GE. The lack of significant differences of xylanase types on ileal digestibility of fat observed in this study is in accordance with several previous studies (Bedford, 2000; Esmaeilipour et al., 2012). However, a trend was observed for the interaction between xylanase and diet type on CIAD of fat, in which case xylanases tended to improve CIAD of fat in wheat-based diets and but not in corn-based diets. A proportion of fat in wheat-based diets was added, whereas the fat in corn-based diets was intact. Thus, the greater fat digestibility in wheat-based diets indicates that extracted fat has physicochemical properties that make it easier for pigs to digest free fat than intact fat (Kil et al., 2014). Nevertheless, our results, along with those of Bedford (2000) and Esmaeilipour et al. (2012), further suggests that due to differences in their catalytic activities, xylanases could reduce entrapment of energy substrates and increase their digestibility. Moreover, the commonality of the xylanase on the fat digestibility observed more so in wheat-based diets were not by coincidence but likely due to increased gut viscosity ochestrated by the higher concentration of soluble arabinoxylans in those diets. The interaction between cereal and xylanase type was evident on iAX, tAX, iNSP and tNSP. Coefficients of ileal digestibility of tAX and tNSP in pigs fed corn-based diets with xylanase B and E were similar but numerically superior to CAID in pigs fed wheat diets with xylanase C. This observation can be ascribed to the fact that corn-based diets had a great composition of both insoluble and total NSP contents, available for hydrolysis. Conversely, pigs fed wheat diets with xylanase C had highest CIAD of iAX and iNSP compared with pigs fed other diets including corn-based diets with xylanase C. The mechanism which led to the unexpected poor digestibility of iAX, iNSP and tNSP observed in pigs fed corn diets with xylanase C is unclear, but it suggests that there could be interactive factors other than the physicochemical nature of the cereal. These observations augments the postulation that, even though xylanases could be from be the same microbe, substratespecificity plays the central role in enhancing xylanolytic activities, and consequently influence its requirement for accessory activities if maximum depolymerisation is to be achieved (Bedford and Schulze, 1998). This is supported by variations in digestibility of tAX and iNSP in both wheat- and corn-based diets in the present study, when xylanase E was used in the two rations. The CAID of P, fat, sAX, tAX, sNSP and tNSP, as well as CATTD of P, fat, iNSP and tNSP were greater in pigs fed corn-based diets than in pigs offered wheat-based diets. These findings can be ascribed to the common paradigm that increased intestinal digesta viscosity due to soluble NSP is the key mechanism that deteriorated feed utilization in pigs fed wheat-based diets (Bedford and Schulze, 1998; Barrera et al., 2004; Kiarie et al., 2013). It is worth noting that although fiber degrading enzymes target well defined targets the interpretation of animal responses across feedstuffs probably suffers from the fact that the knowledge on fiber in swine nutrition is based on chemical entities defined by analytical methods and ability to adequately relate analytical measures to fiber utilization in swine is uncertain. For example, it has been reported that fiber from wheat co-products depressed growth performance and carcass characteristics more than fiber from the corn DDGS whether fed singly or in combination on the basis of equal neutral detergent fiber (Asmus et al., 2012). The diversity and concentration of chemical characteristics that exists among plant-based fibrous feed ingredients, as well as interactions among constituents within feed ingredients and diets, suggests that improvements in nutrient digestibility and pig performance depends on a better understanding of these characteristics and how they affect pig nutrition and metabolism. The lower digestibility of P in pigs fed wheat-based diets than in pigs fed corn-based diets can furthermore be attributed to that the former have high phytate contents that bind elemental P thereby reducing its digestibility and absorption (Kim et al., 2005; Rosenfelder et al., 2013). Observation that CATTD for sAX were higher in pigs fed wheat-based diets compared to those fed corn-based diets could be explained by functional differences of fiber on changes of the microbial ecology of the gut. This is supported by Rodriguez et al. (2012) who observed that differences in microbial community between broiler chickens fed wheat- and barley-based diets versus those fed corn diets. It was unlikely that the level of mycotoxins affected the differences in the performance of pigs fed different diet types. This is because the assayed mycotoxins in the diets were below the FDA maximum limits in complete diets: aflatoxin (20 ppb), deoxynivalenol (1 ppm), fumonisin (10 ppm), ochratoxin (<2 ppb) and zearalenone (<1500 ppb) and t-2 toxin (<1 ppm). In spite of the observations that xylanase A did well across diets for performance, it is interesting to note that it struggled in ileal digestibility of iAX in all diets and iNSP in wheat-based diets. The findings that xylanase A supplementation did not affect feed intake but improved G:F is likely to be a result of improved nutrient utilization, as noticed with increased digestibilities of tNSP especially in wheat-based diets. In contrast, xylanase C improved both digestibility and performance in wheat-based diets better than in corn-based diet. Pigs fed diets with xylanase E had superior growth rates but digestibility of tAX, iNSP and tNSP was lower in wheat-based diets than corn-based diets. The variations in the effects of different xylanase on both performance and NSP digestibility could have been due to a change in the activity of the bacteria in the digestive tract of pigs when fed different diets with enzyme supplementation. This postulation is supported by the study of Garry et al. (2007) showing that enzyme supplementation to different cereal based pig diets resulted in changes in the activity of bacteria in the caecum and large intestine. Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011

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5. Conclusion It should be noted that many factors related to the biochemical properties of the enzyme have been suggested to influence responses seen in application of xylanases in animal feeds (e.g. Ravindran, 2013). In this respect although xylanases were fed at equal protein basis, the comparisons made in the present study are only valid for the employed dosages and as a result some of the xylanases tested may have been dosed at levels below the biological optimum and others well above. Nonetheless, supplementation with xylanase C in wheat-based diets seemed to outperform other xylanases in terms of digestibilities of NSP, particularly in the ileum. However, other xylanases were efficacious across both corn- and wheatbased diets, particularly xylanase A and D, effectively improved growth rates and CAID of NSPs. Therefore, the successful use of xylanase in improving dietary component utilization and growth performance is dependent on its microbial origin and dietary substrate. 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Please cite this article in press as: Ndou, S.P., et al., 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. Feed Sci. Tech. (2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.08.011