Direct-fed Microbials, Alternative to Antibiotic Growth Promoters

Combination of Xylanase and Bacillus Direct-fed Microbials, as an Alternative to Antibiotic Growth Promoters, Improves Live Performance and Gut Health in Subclinical Challenged Broilers

Published on: 5/2/2019
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Objective: This study evaluated the effect of xylanase, Bacillus direct-fed microbials (DFM) and their combination on performance under a mild, subclinical challenge with two Eimeria species and Clostridium perfringens in broilers raised to 42 days. Materials and Methods: A total of 6 dietary treatments were used throughout the trial. Diets were supplemented with one of the following; no xylanase or Bacillus (control), xylanase only, Bacillus L. only, Bacillus A. only, xylanase plus Bacillus L. or xylanase plus Bacillus A. Data were analyzed as randomized complete block design. Results: When compared to control at 42 days, the xylanase, Bacillus L. and Bacillus A. improved (p<0.05) BW gain by 93, 94 and 53 g, respectively and FCR by 4, 4 and 6 points, respectively. When compared to control at 42 days, the combination of xylanase and Bacillus L. or Bacillus A. improved (p<0.05) BW gain by 142 or 147 g, respectively and FCR by 9 or 11 points, respectively. The combination of xylanase and Bacillus L. or Bacillus A. reduced (p<0.05) BW coefficient of variation from 15.09% (control) to 8.27 or 8.22%, respectively at 42 days. The combination of xylanase and Bacillus L. or Bacillus A. reduced (p<0.05) gross lesion scores in small intestine and C. perfringens count at 42 days compared to control. Conclusion: Results suggest that xylanase and Bacillus alone may improve broiler performance and reduce the severity of intestinal lesions due to Eimeria and C. perfringens challenges and that the effect of xylanase and Bacillus DFM are additive.

Key words: Broiler, xylanase, direct-fed microbial, Clostridium perfringens, Eimeria


Sub-therapeutic levels of antibiotics have been used in feed as growth promoters (AGP) in poultry and other species in an attempt to control enteric diseases and improve feed conversion since the 1940s. However, the indiscriminate use of AGPs in production animals has resulted in the emergence of antibiotic-resistant pathogens, which has serious human health implications. With the recent FDA guidance to voluntarily reduce antibiotics for the use of growth promotion, the demand for alternative solutions has risen. This has led to increased interest in the use of feed additives in feed formulation to promote growth and limit pathogen presence.

One potential AGP alternative is the use of exogenous enzymes, which are commonly used to improve the nutritive value of feedstuffs. Among these are carbohydrases, their mode of action is predominantly through degradation of non-starch polysaccharides (NSP), which are the major dietary fiber component within the cell wall of cereal grains. Specifically, xylanases can improve the nutritive value by either reducing digesta viscosity, releasing encapsulated nutrients by disruption of the cell wall structure5 or through a prebiotic effect in which xylo-oligosaccharides (XOS) are produced by xylanase. The XOS can be utilized as a substrate for beneficial bacteria thus help in improving intestinal health, consequently, improving poultry performance.

Another option for AGP alternatives are direct-fed microbials (DFM) or probiotics. The DFM help positively shift the balance of the microbial population in the gastro-intestinal tract (GIT), improve digestion and absorption of nutrients and to inhibit the growth of pathogenic microorganisms. A wide range of microorganisms have been used as DFM in commercial poultry production, with Bacillus and Lactobacillus constituting the majority of species used. It has been shown that Bacillus subtilis DFM can reduce clinical signs of experimental avian coccidiosis with concomitant increase in protective immunity and improve growth performance of chickens.

The DFM are typically naturally occurring microorganisms supplemented as either dried or soluble fermentation products. The interaction between exogenous enzymes and DFMs has been reported in the literature. It is well known that feed enzymes, such as carbohydrases, can improve the utilization of carbohydrates and provide “prebiotics” to beneficial bacteria, whereas proteases can reduce the indigestible protein and reduce substrate availability for pathogenic bacteria. The DFMs can improve the microbial balance, intestinal health and provide an environment that may stimulate the activity of enzymes.

The objective of this study was to investigate the impact of combining Bacillus DFMs and xylanase enzyme on improving the live performance and gut health of broilers challenged with Clostridium perfringens and multiple Eimeria spp.



Experimental design: A total of 2,496 Ross 708 mixed sex 1-day old broilers (males and females) were obtained from a commercial hatchery and placed in floor pens to evaluate the effect of xylanase, DFMs and the combinations of xylanase and DFMs on live performance, coccidiosis observations (severity of intestinal lesion scores), Salmonella incidence, C. perfringens, Escherichia coli and aerobic plate count (APC) intestinal load. The experiment was conducted in a completely randomized block design and consisted of 6 experimental treatments with 8 replicates per treatment, each containing 52 chicks, for a 42 days assay period.

Chicks were reared on used litter in 5×10 ft. floor pens with a minimum of 0.87 ft2 per bird, provided age appropriate environmental conditions and given access of feed and water ad libitum. The lighting program included continuous light for the first week (>3 fc), then dimmed to 1 fc for the remainder of the trial. Litter was dosed with C. perfringens, E. maxima and E. acervulina at day 1, 7 and 10 post-hatch (Table 1). Mortality was recorded as it occurred and a record of mortality weight was kept. On day 21, 35 and 42, at each dietary change, birds and remaining feed were weighed for determination of average body weight (BW), feed consumption for the calculation of mortality-adjusted feed conversion ratio (FCR) and BW coefficient of variation (flock uniformity). Intestinal lesions, as indication of coccidiosis, were scored on day 21 and 42 of age (2-males and 2-females per pen at each age). Clostridium perfringens count per gram of intestinal content, E. coli count and Salmonella incidence were detected at 21 and 42 days of age.

Animal care practices conformed to the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.


direct-fed microbials



Experimental diets: Diets were formulated to either meet or exceed the NRC nutrient requirements for broilers and provided in three phases: Starter (day 0-21), grower (day 22-35) and finisher (day 36-42). The 6 dietary treatments consisted of an unsupplemented negative control (no DFM and no xylanase, NC), NC+Bacillus L., NC+Bacillus A., NC+xylanase, NC+Bacillus L.+xylanase and NC+Bacillus A.+xylanase. The xylanase is an endo-$-1, 4-xylanase derived from Orpinomyces species and produced by Pichia pastoris strain added at an inclusion level of 15 XU/g of feed. Two DFMs, Bacillus L. and Bacillus A., were used in this study at an inclusion level of 1×106 CFU/g of feed. All diets were corn-soybean meal-based (Table 2). Inclusion levels were consistent at all phases.

Statistical methods: A one-way ANOVA using completely randomized design with 8 replicate pens per dietary treatment was employed. The GLM of SAS was used to analyze the data and differences among means were partitioned by LSMEANS. The experimental unit for live performance parameters was the pen, while for microbial and lesion score data the experimental unit was an individual bird.



Live performance: Throughout the entire study (1-42 days of age) inclusion of either Bacillus L. and Bacillus A. alone, xylanase alone or a combination of Bacillus L. plus xylanase or Bacillus A. plus xylanase, significantly (p<0.05) improved 21 and 42 days broiler performance as compared to the negative control (Table 3).

The BW and FCR were improved (p<0.05) by the combination of either Bacillus L. or Bacillus A. with xylanase (Table 3). The combination of either Bacillus A. or Bacillus L. with xylanase improved FCR at 42 days compared to single product supplementation. Zhang and Kim reported that when multi-strains of Bacillus are fed to broilers to 35 days improved body weight gain (BWG), FCR, but not feed intake is observed, as found in this study. Treatment of pen litter with C. perfringens, E. maxima and E. acervulina resulted in a mortality of 4.79% at 42 days. Supplementation of Bacillus A., Bacillus L. or xylanase alone resulted in reduce (p<0.5) mortality. This is in agreement with Lee et al., who reported that DFMs have shown promise as an alternative to in-feed antibiotics in reducing enteric disease and eliminating subsequent contamination of poultry products. The combination of enzyme and either DFM reduced mortality (p<0.05) compared to negative control, suggesting an additional benefit when xylanase is utilized in combination with DFMs.

Intestinal pathogen load: Results for lesion score, E. coli, APC and C. perfringens count and Salmonella incidence (%), measured at 21 and 42 days of age are presented in Table 4. Supplementing diets with the combination of Bacillus L. or Bacillus A with xylanase were found to improve (p<0.05) lesion scores at 14, 21 and 42 days and reduce (p<0.05) intestinal bacteria counts of APC and E. coli counts and Salmonella incidence (Table 4). This is likely due to the DFM functioning to maintain the presence of beneficial microorganisms and inhibit growth of pathogenic bacteria. Single product supplementation significantly reduced (p<0.05) the levels of C. perfringens in small intestines (1.22 CFU/g xylanase, 0.80 CFU/g Bacillus A. and 0.82 CFU/g Bacillus L. log reduction). All combinational supplementation outperformed single product supplementation. A log reduction of 1.49 CFU/g in C. perfringens with the combination of xylanase and Bacillus A. and 1.55 with the combination of xylanase and Bacillus L. was observed. Similar results are observed when measuring the Salmonella incidence in intestinal contents across treatments. Single product supplementation significantly reduced (p<0.05) Salmonella incidence (30% xylanase, 39% Bacillus A., 33% Bacillus L.). The combinational supplementation outperformed single product supplementation in reducing Salmonella incidence, with a 50 and 48.8% decrease for the combination of xylanase with Bacillus A. and Bacillus L., respectively. The combination of DFM and xylanase likely have a combinatorial effect on improving the survival of beneficial microorganisms and their colonization in the intestinal tract.





It can be concluded that supplementing xylanase to broiler feed may improve live performance and gut health. Furthermore, supplementing Bacillus DFMs can also improve live performance and reduce pathogen load in the digestive tract when supplemented to broilers up to 42 days. When xylanase and Bacillus DFM supplemented simultaneously, FCR and lesion score were improved further (additive effect).



Current study shows that xylanase and Bacillus DFM can be used in combination to improve live performance and reduce pathogen load in an antibiotic-free environment. These findings are significant given the increased interest in AGP-free poultry production.


This article was originally published in International Journal of Poultry Science, 17: 362-366. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.

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