The Effect of a Modified GH11 Xylanase on Live Performance, Gut Health, and Clostridium perfringens Excretion of Broilers Fed Corn-Soy Diets

Xylanase enzymes and other feed additives are being used more commonly in poultry feed to reduce feed cost, improve performance, and maintain gut health. Five corn-soy-based dietary treatments were designed to compare the effect of different inclusion levels of high-efficiency GH11 xylanase on live performance, gut lesions, and Clostridium perfringens excretion in littler samples of broiler chickens. Diets were the standard diet (positive control; PC); a diet of reduced energy by 130 kcal/kg diet (negative control; NC); NC with xylanase at 10 XU/g of feed (NC + 10); NC with xylanase at 12.5 XU/g of feed (NC + 12.5); NC with xylanase at 15 XU/g of feed (NC + 15). Data were analyzed with one-way ANOVA. At 42 d, birds fed NC + 12.5 and NC + 15 were heavier (P < 0.05) than NC and comparable improvement to birds fed PC. Significant Improvement in FCR (P = 0.0001) was observed from 1 to 42 d for NC + 12.5 and NC + 15 compared with NC. Supplementation of xylanase reduced (P < 0.005) 21 d intestinal lesion score at 21 d with further improvement (P < 0.0001) at 42 d. NC + 15 reduced lesion scores by 24% compared with NC. Xylanase supplementations reduced litter C. perfringens cell forming unit per gram (CFU/g) compared with NC with the highest reduction of NC + 15 treatment by ~27%. In conclusion, xylanase can be included in reduced-energy diets up to 15 XU/g of feed to improve live performance, energy digestibility, and reduce intestinal lesion scores in broilers.


INTRODUCTION
Providing adequate quantities of animal protein to meet the demands of a continuously growing population requires that livestock producers must increase the production efficiency of farm animals. This can be achieved through improved genetics, management, and nutrition. Furthermore, feed ingredient prices continue to increase, resulting in minimizing the profit for the producers and forcing them to seek cheaper alternative ingredients, typically of lower quality and higher fiber content (1).
Feed composition plays an important role in efficiently providing birds with the required nutrients for maintenance and growth. It is well-known that various feed ingredients have antinutritional factors that would either negatively affect digestion, or trap nutrients within their cell wall structure, making them unavailable for the bird to digest and utilize (2, 3) simply because the bird lacks the endogenous enzymes needed to hydrolyze these structures (4,5). In cereal grains, such as wheat and barley, and in legumes, such as soybeans, the presence of non-starch polysaccharides (NSP) and lignin fiber reduces the extent of digestion and absorption of nutrients by broilers due to increased digesta viscosity (6), as well as encapsulating nutrients within the cell wall structure. Furthermore, birds, especially at a young age, lack the enzymes required to hydrolyze these fibers; arabinoxylan, β-glucan, cellulose, and the non-carbohydrate component lignin are the predominant polymers in cereals. Several commercially available exogenous enzymes have been proven to increase the digestibility of poorly digested cereals to a much greater extent than well-digested cereals (7)(8)(9)(10); however, to achieve the maximum improvement in NSP hydrolysis, multiple enzymes may be needed depending on the ingredients used in a diet to hydrolyze the various fibers resulting in an improved energy recovery. Another approach is to feasibly maximize hydrolysis through targeting the most abundant fraction of the NSP in a specific diet by selecting the most effective enzyme for that fiber fraction. Arabinoxylans are the predominant NSP in corn compromising ∼5% of the DM (11,12) and ∼50% of the total carbohydrate fraction (13). This fraction can be hydrolyzed by a glycoside hydrolase (GH); the forms most commonly found commercially belong either to the GH10 or GH11 families (14). The GH11 xylanases usually have a larger active site, and they are more substrate specific (14), and degradation produces shortchained oligosaccharide subunits that act as prebiotics for the beneficial bacteria in the gut (15). It has been shown that adding xylanase to poultry diets improves nutrient utilization by increasing its digestibility as well as alleviating the negative effect of many anti-nutritional compounds that the animal cannot digest (16,17). Furthermore, xylanase has been shown to have a protective effect on the intestinal mucosa barrier to alleviate the negative effect of Clostridium perfringens infection in 21 d old broilers (18). However, few studies have focused on the effect of GH11 xylanases on performance and gut health of broilers.
Therefore, the objective of this study was to evaluate the efficacy of different inclusion rates of high efficiency GH11 endo-1,4-β-xylanase, on live performance, gut lesions, and Clostridium perfringens excretion of broiler chickens fed standard corn-soy diets reared under typical broiler production conditions.

METHODS AND MATERIALS
Practices conducted during this trial were in compliance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (19).

Experimental Design
A total of 2,080 Ross 708 mixed sex 1-day-old broilers were obtained from a commercial hatchery and placed in floor pens to evaluate the effect of different xylanase inclusion rates on live performance, severity of gut lesions, and Clostridium perfringens excretion of broiler chickens fed standard cornsoy diets. Birds were reared under typical broiler production conditions without any imposed microbial challenge except for what was naturally occurring in the used litter. The experiment was conducted in a completely randomized design and consisted of 5 experimental treatments with 8 replicate floor pens per treatment, each containing 52 chicks and were reared to day 42 of age.
Chicks were reared on used litter topped with fresh pinewood shavings in floor pens (3.05 × 1.52 m) with a minimum of 0.08 m 2 per bird, provided age-appropriate environmental conditions, and given ad libitum access to feed and water. The lighting program included continuous light for the first week (>3 foot candle), then dimmed to 1 foot candle for the remainder of the trial.

Experimental Diets
Diets were formulated to either meet or exceed the NRC (20) and Ross Broiler Management Handbook (21) requirements for broilers. Three phases were provided: starter (0-21 d), grower (22-35 d), and finisher (36-42 d). The 5 dietary treatments consisted of (1) Positive Control (PC), a standard diet formulated at normal dietary energy level; (2) Negative Control (NC), a standard diet formulated at 130 kcal/kg lower than PC; (3) NC with xylanase at 10 XU/g of feed (NC + 10); (4) NC with xylanase at 12.5 XU/g of feed (NC + 12.5); and (5) NC with xylanase at 15 XU/g of feed (NC + 15). Energy reduction in NC was achieved by adding soy hulls. The xylanase is a GH11 endo-β-1,4-xylanase; a glycosidase that hydrolyzes 1→4-β-Dxylosidic linkages in xylan, it is produced by fermentation of a Komagataella phaffii microorganism expressing a gene coding for the xylanase enzyme. One unit of endo-1,4β-Xylanase activity (XU) is defined as the amount of enzyme needed for the release of one nanomole of reduced sugars (xylose equivalents) per second from 0.5% xylan at 50 • C in 50 mM trisodium citrate buffer pH 6.0. All diets were corn-soybean meal-based ( Table 1). Inclusion levels were consistent at all phases of diet to achieve the activity unit of xylanase in each treatment.

Live Performance
Live performance measurements were taken at placement and at 21, 35, and 42 days of age. Mortality was recorded daily. Body weight (BW), body weight gain (BWG), feed consumption (FC), feed conversion ratio (FCR) adjusted for mortality, BW coefficient of variation (CV; flock uniformity), and percent mortality were determined.

Apparent Metabolizable Energy
At both 19 and 40 d of age, digestibility of apparent metabolizable energy was estimated in four randomly selected birds per pen (two males and two females). Birds were moved to raised-wire cages, and both feed consumption and feces were collected for 3 days. Feces were pooled, processed, and analyzed for dry matter, gross energy, and nitrogen. Feed was also analyzed for dry matter, gross energy, and nitrogen. The following calculations were used to determine apparent metabolizable energy (AME) and nitrogen corrected (AMEn): Lesions were scored using a scale from 0 (no lesions found) to 4 (diffused necrosis typical of field cases).

Clostridia Perfringens Excretion
Litter samples were analyzed for Clostridium perfringens (C. perfringens) as an indicator of environmental pathogen load and excretion of C. perfringens. Cell forming units of C. perfringens (CFUs) per gram of litter was determined prior to placement, at 21 and at 42 days of age following procedure described in FDA BAM, Ch 16 in quadruplicates of 25 grams of litter after homogenizing in 225 ml of peptone diluent (0.1% peptone) and diluting 10-fold, then plating on Tryptose-sulfite-cycloserine agar (TSA) plates. Plates were incubated under anaerobic conditions at ∼35 • C for ∼24 h. Colonies were counted using dilution plates with ∼20-200 CFUs. Data was expressed as log 10 per gram.

Statistical Methods
Data were analyzed with one-way ANOVA in a completely randomized design (CRD) with five dietary treatments and eight

Live Performance
Live performance results are shown in  (26) showed that the addition of a xylanase and arabinofuranosidase combination improved duodenum villi length; an indicator of enhanced growth performance of the broilers. The coefficient of variation (CV) calculation of individual BW data was analyzed at d 0, 21, 35, and 42 of age as an indicator of flock uniformity. All CVs for BW were < 10% at 35 and 42 d of age indicating that birds' growth was uniform. Mortality was not affected by treatments.
Overall mortality in the entire trial was < 3%. As the xylanase activity increased, performance was improved. Digesta viscosity was not measured in this trial; however, this improvement could probably be due to the action of xylanase on degrading the nonstarch polysaccharides (NSPs), thus reducing digesta viscosity as well as minimizing the caging effect NSPs have on other nutrients blocking endogenous enzymes from working on them (6,27).   (28) also demonstrated that adding an enzyme cocktail that included xylanase at 300 U/kg of feed to broiler diets improved AME. By xylanase enzyme action on the NSPs, additional nutrients, mainly energy, become available for the bird to utilize, leading to improved digestibility.  (29) that xylanase at 15 XU/g of feed reduced lesion score at 21 and 42 days of age. There was no imposed C. perfringens challenge implemented in this trial; however, birds were raised on used litter, which could potentially contain Clostridia and other pathogens. Xylanase helped in ameliorating the negative effect of naturally occurring Necrotic Enteritis probably by mainly reducing the amount of nutrients available to pathogens in the hindgut and environment as well as providing prebiotics of the short units of xylooligosaccharides (XOS) required to support the growth of beneficial bacteria, which ultimately aid in creating a healthy microbiota in the gut.

Clostridia perfringens Excretion Over Time
The abundance of C. perfringens present in litter is shown in Figure 1 and expressed as log 10 (x) per gram of litter. Litter samples from each pen were enumerated for its C. perfringens counts prior to placement, and at 21 and 42 d. Counts remained relatively constant for PC and NC throughout the trial; however, adding xylanase to NC reduced (P < 0.05) C. perfringens counts by ∼28% compared with control diets. This could be caused by a reduction of nutrients in the hind gut that could be utilized as source of food for pathogen such as C. perfringens leading to subsequently improving the intestinal integrity and reducing the lesion score and thus reduce the load in the litter at 21 and 42 days of age as xylanase dose increased. From these findings it can be concluded that xylanase can improve the nutritional value of corn-SBM based diets, and improve performance, intestinal gut health, and reduce pathogen load in the litter. The concentration of substrate and different feed ingredients in the diet may aid in determining the proper xylanase dose to use. In this study, a supplement of 15 XU/g provided the greatest improvement of body weight gain and lesion scores numerically but was statistically comparable to a dose of 12.5 XU/g. However, energy digestibility improved as the xylanase dose increased. Close attention should be paid to using the right dose of xylanase to optimize economic outcomes, availability of ingredients, and their prices.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee.