Efforts to improve nutrient digestibility by the pigs can have effects on profitability of the pork industry (National Pork Board, 2012). Studies indicate that non-starch polysaccharides (NSP) in corn and soybean meal negatively affect nutrient digestibility (Moeser et al., 2002; van Kempen et al., 2006). Whole corn grain contains 27e32 g of xylose/kg (Knudsen, 1997) as arabinoxylans in pericarp and aleurone (Landis et al., 2001). Soybean meal contains 18e19 g of xylose/kg (Knudsen, 1997; Irish and Balnave, 1993) as xyloglucan in the structural polysaccharides (Karr-Lilienthal et al., 2005).
Feed enzymes supplementation to corn (Cozannet et al., 2012; Li et al., 2010), soybean meal (Cozannet et al., 2012), and complete feed (Ji et al., 2008; Jo et al., 2012; Kim et al., 2003; Pettey et al., 2002) fed to pigs were previously reported. Endo-1,4-b-xylanase (xylanase) catalyzes endohydrolysis of 1,4-b-D-xylosidic linkages in xylans (International Union of Biochemistry and Molecular Biology, 1992) releasing oligosaccharides from corn and wheat fiber (Katapodis and Christakopoulos, 2008; Katapodis et al., 2003). Xylanase has been evaluated to improve nutrient digestibility in pigs (Moehn et al., 2007; Nortey et al., 2007; Woyengo et al., 2008). The mechanism proposed to explain the effect of fiber degrading enzymes involves degradation of polysaccharides in the cell wall (Adeola and Cowieson, 2011; Masey et al., 2012; Meng et al., 2005; Tervila-Wilo et al., 1996) and reduction of digesta viscosity (Garcia et al., 2008; Mathlouthi et al., 2002). However, viscosity might not be the most important factor affecting nutrient digestibility in pigs (Bartelt et al., 2002). Type of fiber and intestinal fermentation should be considered (Hooda et al., 2010; Jensen, 1996).
The hypothesis of this study is that supplementation of xylanase in corn-soybean meal based diets reduces digesta viscosity and thus enhances digestibility of nutrients. The objective of this study is to measure viscosity of jejunum digesta, intestinal morphology, and ileal digestibility of dry matter (DM), energy, acid detergent fiber (ADF), neutral detergent fiber (NDF), and crude ash of a corn-soybean meal based diet supplemented with xylanase fed to pigs.
2. Materials and method
The experimental protocol was approved by North Carolina State University Animal Care and Use Committee.
2.1. Experimental diets and pigs
The experiment was conducted at the Swine Educational Unit at the North Carolina State University (Raleigh, NC). Pigs were used to evaluate digestibility of DM, energy, protein, ADF, NDF, and crude ash of a diet (Table 1) supplemented with feed enzyme. Corn was ground to 400 mm. Xylanase (Carboflex, Lohmann Animal Nutrition GmbH, Cuxhaven, Germany) was supplemented at 0 (C), 100 (T1), and 200 mg/kg of diet (T3) to provide 0, 700, and 1400 LXU of xylanase/kg of diet respectively. LXU is the amount of enzyme which releases 1 mmol of reducing sugars equivalents (as xylose or glucose) from birch xylan or barley glucan per minute at pH 5.5 and 50 C (EURL, 2013).
Thirty six barrows (17.6 ± 3.3 kg) were placed in metabolic cages (0.6 m wide, 1.8 m long) equipped with stainless-steel feeder attached to the front of the pen, nipple water drinker next to the feeder, and slatted flooring. There were 12 cages available for the study and 3 groups of 12 pigs were allotted in the metabolism room. Pigs received one of the 3 treatment diets based on a randomized complete block design with initial body weight as block. The experimental period consisted of 10 days. Ileum content of ADF and NDF, ileal digestibility of ADF and NDF, villus height/crypt depth in jejunum and digesta viscosity were measured.
2.2. Experimental procedures, chemical analyses, and digesta viscosity
Pigs received experimental diets twice daily (0700 and 1700 h) at a fixed amount based on BW of pigs (0.09 x BW0.75 kg). Dietary treatments were fed to pigs for 10 days. Chromium oxide was added to experimental diets (0.3%) from day 6 as an indigestible external marker for calculation of ileal digestibility. Pigs were euthanized via captive-bolt stunning and exsanguination at day 10 for sample collection 8 h after the last meal. Immediately after the euthanasia, an ileal portion (a portion of 20 cm prior to ileo-cecal connection) of small intestine was used to obtain digesta in ileum. Digesta from ileum was stored in sterile container and kept frozen at -20 C. Jejunum tissue sample (3 cm) was collected and stored in formaline for further histological analysis. Intestine (20 cm) from distal portion of jejunum was also used to obtain digesta to measure viscosity. Jejunal contents were emptied into 50 mL tubes, samples were kept on ice and viscosity was measured immediately after the collection.
Frozen Ileal digesta were freeze-dried (24D x 48, Virtis, Gardiner, NY) for storage and chemical analysis. Diets and freeze dried digesta were analyzed for moisture (Method 934.01, AOAC, 2006), ADF (Method 973.18, AOAC, 2006), NDF (Van Soest et al., 1991), ash (Method 942.05, AOAC, 2006), chromium (Williams et al., 1962), and energy using a calorimeter (6200, Parr Instrument Company, Moline, IL). Apparent ileal digestibility (AID, %) of ADF and NDF were calculated using the chromium concentration in the diets and digesta by using AID - 100 = [(ND/NF) x (CrF/ CrD) x 100], where ND is the nutrient concentration present in the ileal digesta, NF is the nutrient concentration in the diet, CrF is the chromium concentration in the feed, and CrD is the chromium concentration in the ileal digesta.
Viscosity was done using a viscometer (Brookfield Digital Viscometer, Model DV-II Version 2.0, Brookfield Engineering Laboratories Inc., Stoughton, MA). The tubes were centrifuged at 3000 rpm for 5 min and then 2 mL of the supernatant was centrifuged at 12,500 rpm for 5 min. Viscometer was set at 25 C, 0.5 mL of digesta supernatant was placed in the viscometer. Viscosity measurement was the average between 45.0 s-1 and 22.5 s-1 shear rates.
Jejunum morphology were analyzed according to Fan et al. (2001) to obtain villus height, crypt depth and the relation villus height to crypt depth. Jejunum samples (2 sections per pig) were fixed in formaline and sent to North Carolina State University histology laboratory for hematoxylin and eosin staining and sectioning according to standard histological technique. The sections were dehydrated and embedded in paraffin. Staining was done using hematoxylin and eosin dyes (Junqueira and Carneiro, 2005).
Villus height, crypt depth, and relation villus height and crypt depth were measured in the microscope (Micromaster, Fisher Scientific International Inc., Pittsburgh, PA). For each section, 15 measurements of adjacent villus height and crypt depth were obtained. The measurements were done with ImageJ software (NIH, 2013) and transferred to Microsoft Excel software. The relation villus height to crypt depth of each measurement was calculated. The averages of the 30 measurements per pig were calculated and reported as one number per pig.
2.4. Statistical analysis
Data were analyzed using polynomial contrasts in the Mixed procedure of SAS version 9.3 (SAS Inc., Cary, NC, USA). The experiment was a randomized complete block design using initial BW and group of pigs allotted in the metabolism as blocking factor. The experimental unit was the individual pig. Initial BW and group of pigs were considered random effect. Statistical differences were considered significant with P < 0.05. Probabilities less than 0.10 and equal or greater than 0.05 were considered as a tendency.
The average BW of pigs utilized on this study was 17.6 kg and average daily feed intake (ADFI) was 757 g/d (Table 2). The weight gain of the pigs during the metabolism study was 352 g/d and the feed conversation ratio (G:F) was 0.47 in average. Increasing the level of xylanase in the diet (0-1400 LXU/kg) did not affect (P > 0.10) growth performance of pigs individually house in the metabolism cages. Pigs received a limited amount of feed based on their BW and this study was not designed to measure growth performance.
Increasing xylanase in the diet from 0 to 1400 LXU/kg did not affect histological measurements (Table 3) including villus height, crypt depth, and relation villus height to crypt depth. Increasing xylanase resulted in a quadratic change (P - 0.023) in viscosity of jejunal digesta from 2.94 to 2.52 centipoises (cP) when xylanase increased from 0 to 700 LXU/kg and from 2.52 to 3.20 cP when xylanase increased from 700 to 1400 LXU/kg respectively.
Increasing xylanase (0-1,400 LXU/kg) yielded greater AID of DM (linear increase from 55.43 to 64.58%, P - 0.006), organic matter (OM) (linear increase from 59.19 to 67.70%, P - 0.006), and energy (linear increase from 58.78 to 68.04%, P - 0.003). Similarly, AID of crude ash increased by 16% (quadratic increase from 18.71 to 34.34%, P - 0.045) and AID of NDF by 12% (linear increase from 27.91 to 40.32%, P - 0.042). However AID of ADF was not affected by supplementation of xylanase (P > 0.10).
The digesta viscosity obtained on this study ranged from 2.52 to 3.20 cP. The viscosity can be affected by the type of ingredient in the diet (Willamil et al., 2012). Digesta viscosity in the ileum was reported to be 2.8 cP for a corn-soybean meal based diet (Willamil et al., 2012), 1.7 cP in corn-soybean meal-DDGS based diet (Agyekum et al., 2012), 4.6 cP in a wheat based diet (Mavromichalis et al., 2000), and 7.0 cP in a rye-wheat based diet (Bartelt et al., 2002). Corn was the major ingredient in the diet of the present study, and it has lower content of soluble NSP than wheat, rye, barley, and oats (Knudsen, 1997) yielding low viscous solutions (Mathlouthi et al., 2002).
This study indicated that by increasing the use of xylanase yields a quadratic change in the viscosity of the digesta in pigs fed corn-soybean meal based diets. Corn grain NSP contains arabinoxylans (Landis et al., 2001), and contains 30 g total xylose/kg of corn (Knudsen, 1997). Soybean meal contains 18-19 g xylose/kg of soybean meal (Irish and Balnave, 1993; Knudsen, 1997) as xyloglucan (Karr-Lilienthal et al., 2005), therefore the main substrate for xylanase in a corn-soybean meal-based diet will be the arabinoxylans in the corn. The effect of xylanase on corn fiber was previously demonstrated by in vitro studies (Grabber et al., 1998; Hu et al., 2008; Saha, 2001). The limitations regarding the xylanase activity on corn fiber (Rose and Inglett, 2011) involve the arabinose sidechains in the xylan backbone of the arabinoxylan (Doner et al., 2001; Rose et al., 2010). However, arabinofuranosyl groups attached to xylan can be partially released under acidic pH conditions in the stomach (Zhang et al., 2003). In addition, the corn fiber utilization on xylanase production increases the number of side activity enzymes (b xylosidase and a-L-arabinofuranosidase) that enhance the release of arabinose and xylose from arabinoxylans (Saha, 2001). Arabinoxylans can form viscous solutions (Izydorczyk and Biliaderis, 1982, 1992) and increase viscosity of digesta (Choct and Annison, 1992). Xylanase can break arabinoxylans (Grabber et al., 1998; Pedersen et al., 2012) and reduce viscosity of in vitro solutions (Mathlouthi et al., 2002) and also digesta viscosity (Adeola and Bedford, 2004; Yin et al., 2001).
Increasing supplementation of xylanase yields a quadratic response on digesta viscosity. Corn contains a greater proportion of xylose in the insoluble NSPs (Knudsen, 1997) and some xylanases have affinity to insoluble xylan (Connerton et al., 1999; Sun et al., 1998). There is evidence that xylanases can degrade insoluble NSP into soluble NSP increasing digesta viscosity (Choct et al., 2004). Therefore, one can speculate that at greater dosages of xylanase (as treatment T2 in the study reported herein), the insoluble NSP become more soluble, and thus increase digesta viscosity. Therefore, the NDF result of this study supports the degradation of corn NSP. However, this needs further investigation with limited biological and practical meaning at this moment.
This study observed that by increasing the dietary supplementation level of xylanase, there will be a linear increase in the ileal digestibility of DM, OM, energy, and NDF (Table 4). The mode of action of xylanase on enhancing nutrient digestibility may involve the degradation of the cell wall NSPs, thus enabling endogenous digestive enzymes to access nutrients trapped (Adeola and Cowieson, 2011; Masey et al., 2012; Tervila-Wilo et al., 1996). The greater NDF digestibility can be explained by the method utilized to analyze NDF. Xylanase release oligosaccharides (xylobiose to xylopentose) from arabinoxylans (He et al., 2010; Rajagopalan et al., 2013). The filter bags utilized in the NDF analysis procedure have pore sizes of 25 mm (F57, ANKOM, Macedon, NY) and the smaller particles of xylotriose to xylopentose released by xylanase might not be retained by the filter bags. There are nutritional benefits of NSP degradation (Choct and Annison, 1992). The use of xylanase in corn-soybean meal based diets improved ileal digestibility of energy by 2% (Nian et al., 2011) and also an enzyme blend containing xylanase, protease, and amylase improved protein digestibility (Zanella et al., 1999). Improvement in NDF, DM, gross energy (GE), and starch digestibility were observed utilizing in vitro and in vivo digestibility methods in pigs when an enzyme blend composed of xylanase, protease, and amylase was added to the diet (Li et al., 2010). The present study indicated that as dietary level of xylanase increased, digestibility of DM, OM, energy, NDF, and crude ash increased by 9.2, 8.5, 9.3, 12.4, and 10.7%, respectively.
Dietary level of NSP can affect intestinal morphology (Montagne et al., 2003). Diets with high content of NSP from wheat and barley affected villus height and the relation villus height to crypt depth in the ileum of pigs compared to diet formulated with corn and soybean meal (Willamil et al., 2012). The use of feed enzyme can also mitigate the negative effect of NSP from wheat and barley on intestinal morphology, however, it does not affect intestinal morphology in corn-soybean meal based diet (Willamil et al., 2012). Similarly, there was no significant effect of dietary xylanase supplementation of corn-soybean meal diet on intestinal morphology measured in the study reported herein.
The ileal nutrient digestibility of a corn-soybean meal based diet improved when dietary xylanase supplementation level increased from 0 to 1400 LXU/kg. There was a quadratic change in viscosity of jejunum digesta, but no effect on intestinal morphology. The results confirm our hypothesis that xylanase can be supplemented to swine diets in order to improve nutrient digestibility.
This article was originally published in Animal Nutrition 1 (2015) 19e23. http://dx.doi.org/10.1016/j.aninu.2015.02.006. This is an Open Access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).