Ines Andretta
Catarina Stefanello
The authors declare no conflict of interest.
This study evaluated the effects of xylanase combined with increasing amylase levels in broiler diets containing protease and phytase on performance, intestinal histomorphology, cecal short-chain fatty acid profile, serum interleukin (IL) levels, and economic viability over a 28-d period. A completely randomized experimental design was adopted in a 2 × 3 + 1 factorial arrangement (100 or 200 U kg−1 amylase × 0, 1,000 and 2,000 U kg−1 xylanase + positive control (PC), without enzymes), with seven replicates of 20 birds. Diets with amylase and xylanase also included 2,500 U kg−1 protease, whereas all diets had 500 U kg−1 phytase. Enzyme matrix values accounted for 100 kcal of metabolizable energy and 6% of crude protein and digestible amino acids. At 14 d, broilers receiving xylanase levels presented better feed conversion ratio (FCR) compared with those fed diets without xylanase (P = 0.014). However, regardless of the enzyme supplementation level, FCR was worse compared with PC (P = 0.0001). Diets containing 100 U kg−1 amylase resulted in better histopathology scores (I See Inside®). No difference (P>0.05) was observed between PC and enzyme-supplemented groups for intestinal morphometric parameters, concentrations of acetic, butyric and isovaleric acids, or serum levels of IL-6, IL-10 and IL-16 at 28 d. Feed cost/t of broilers fed 100 U kg−1 of amylase or 1,000 U kg−1 of xylanase was 3.15% and 3.24% lower, respectively, than PC. Although the inclusion of amylase and xylanase in reduced-energy and protein diets containing protease and phytase did not improve the broiler performance and did not match the PC group, it maintained gut health and immunological parameters. The use of 100 U kg−1 of amylase and 1,000 U kg−1 of xylanase enabled an average 3.25% reduction in production cost.
Broiler diets are primarily composed of cereal grains such as corn, soybean meal, and wheat. However, these ingredients contain non-starch polysaccharides (NSPs), which act as antinutritional factors due to the absence of endogenous enzymes capable of degrading their complex carbohydrate structures. NSPs impair nutrient digestibility and gut health, especially when included at high levels or provided through less-refined byproducts such as wheat bran.
NSPs are classified as soluble and insoluble. Soluble NSPs – such as β-glucans and arabinoxylans – are prevalent in wheat grain and wheat bran. These polysaccharides increase digesta viscosity (
Given the NSP profile of these ingredients, exogenous enzymes are widely used to improve nutrient utilization. Xylanase can hydrolyze cell walls rich in soluble fibers, releasing starch, lipids, and proteins and thereby improving the accessibility of endogenous enzymes (
The inclusion of amylase also benefits broiler performance by acting on undigested starch. The digestibility of this component varies according to the grain type and the degree of processing. The rate of nutrient utilization is influenced by physiological limitations of broilers, such as age, the site of digestion, and the high feed intake of broilers, which provides a greater substrate for digestion (
In this context, we hypothesized that the inclusion of corn, soybeans, and wheat in broiler diets, despite their high digestibility, can negatively affect gut health and growth performance. Furthermore, we proposed that the dietary inclusion of amylase and xylanase, in combination with protease and phytase, could mitigate these effects, support broiler physiological responses, and enable the use of reduced dietary energy and protein levels. Therefore, the research question addressed in this study was whether broilers fed diets supplemented with exogenous enzymes formulated with reduced energy and protein levels would differ in performance, intestinal health, immune parameters, and economic viability when compared with broilers fed a nutritionally adequate control diet. The null hypothesis (H0) was that broilers fed enzyme-supplemented, reduced-nutrient diets would not differ from those fed the control diet in growth performance, intestinal histomorphology, cecal SCFA profile, serum interleukins levels, or production cost over a 28-d period.
The experiment was conducted at the Poultry Research Center of Universidade Estadual do Oeste do Paraná - Unioeste, Marechal Cândido Rondon, Paraná, Brazil. The trial was conducted in accordance with ethical principles in animal experimentation, with protocol approved by the Ethics Committee for the Use of Animals in Research of the University (protocol n° 23/2022).
The experiment was conducted in an experimental aviary, where birds were housed in 49 boxes, each measuring 1.96 m2 and equipped with a tubular feeder, a nipple drinker, and a concrete floor covered with pine shavings previously used for six flocks. The experimental period was divided into two phases, according to the feeding program adopted, the starter phase (1-14 d) and the grower phase (15-28 d). During the first three days, the temperature was maintained near 33 °C and was gradually decreased to 23 °C at the end of the experiment. When necessary, cooling and air renewal were carried out by exhaust fans and evaporative cooling pads. The lighting program provided 24 h (natural plus artificial) until day 3, 1 h of darkness between days 4 and 7, and 6 h of darkness from days 8 to 28, controlled by an automatic panel (model SMAAI 4).
A total of 980 one-day-old male Cobb 500® broiler chickens, with an initial average weight of 46 ± 0.40 g, were distributed in a completely randomized design in a 2 × 3 + 1 factorial scheme, with six combinations of two amylase levels and three xylanase levels and one positive control, totaling seven treatments, with seven replicates and 20 birds per experimental unit (EU). Diets were formulated based on the nutritional requirements of the lineage.
Birds received water and feed
PC - Positive control - feed formulated according to the nutritional recommendations; ENZ - PC reducing 100 kcal, 6% of crude protein and amino acids (lysine, met+cys, threonine and tryptophan) and adding enzymes. 1 Composition of starter vitamin premix (1 – 14 d), levels per kilogram of feed: vitamin A, 10,000 IU kg−1; vitamin D3, 2,500 IU kg−1; vitamin E, 20 IU kg−1; vitamin K3, 2 mg kg−1; vitamin B1, 2 mg kg−1; vitamin B2, 5 mg kg−1; vitamin B6, 3 mg kg−1; vitamin B12, 12 mg kg−1; niacin, 35 mg kg−1; pantothenic acid, 12 mg kg−1; folic acid, 1 mg kg−1; biotin, 50 mcg kg−1. Composition of grower vitamin premix (15 – 28 d), levels per kilogram of feed: vitamin A, 8,000 IU kg−1; vitamin D3, 2,000 IU kg−1; vitamin E, 16 IU kg−1; vitamin K3, 1.60 mg kg−1; vitamin B1, 1.60 mg kg−1; vitamin B2, 4 mg kg−1; vitamin B6, 2.40 mg kg−1; vitamin B12, 9.60 mg kg−1; niacin, 28 mg kg−1; pantothenic acid, 9.60 mg kg−1; folic acid, 0.80 mg kg−1; biotin, 40 mcg kg−1. 2 Composition of mineral premix (1 – 28 d), levels per kilogram of feed: manganese, 70 mg kg−1; zinc, 60 mg kg−1; iron, 50 mg kg−1; copper, 8 mg kg−1; iodine, 0.80 mg kg−1; selenium, 0.30 mg kg−1. 3 Bentonite. 4 Anticoccidial additive: Nicarbazin+Narasin (80%/80%), levels per kilogram of feed: 80 mg kg−1. 5 The inclusion of enzyme associations and doses on diets was done replacing the filler (kaolin), providing levels of amylase (100 and 200 U kg−1) and xylanase (1,000 and 2,000 U kg−1). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease.
Item
Initial
Grower
PC
ENZ
PC
ENZ
Ingredient (kg t−1)
Corn
368.55
437.93
370.46
435.95
Soybean meal (46%)
288.00
245.52
209.52
170.25
Wheat grain
150.00
150.00
175.00
175.00
Whole deactivated soybean
80.00
80.00
100.00
100.00
Wheat bran
50.00
50.00
75.00
75.00
Refined soybean oil
28.93
2.18
39.49
13.43
Dicalcium phosphate
10.32
10.32
7.71
7.71
Limestone (36%)
9.24
9.24
8.87
8.87
NaCl
4.287
4.292
3.835
3.839
L-Lysine HCl (78%)
2.746
2.949
3.141
3.325
DL-Methionine (98%)
3.294
2.975
2.977
2.680
L-Threonine (98%)
1.259
1.216
1.319
1.276
Choline chloride (60%)
0.500
0.500
0.350
0.350
Vitamin premix1
0.250
0.250
0.200
0.200
Mineral premix2
0.500
0.500
0.500
0.500
Mycotoxins adsorbent3
1.000
1.000
1.000
1.000
Anticoccidial additive4
0.500
0.500
-
-
Filler (kaolin)5
0.625
0.625
0.625
0.625
Calculated composition
Metabolizable energy (kcal kg−1)
3,000
2,900
3,100
3,000
Available protein (%)
22.00
20.66
20.00
18.80
Calcium (%)
0.743
0.734
0.645
0.672
Available phosphorus (%)
0.450
0.443
0.400
0.389
Sodium (%)
0.200
0.200
0.180
0.180
Chloride (%)
0.361
0.361
0.338
0.338
Digestible lysine (%)
1.200
1.127
1.100
1.034
Digestible methionine (%)
0.596
0.560
0.540
0.510
Digestible cystine (%)
0.292
0.274
0.274
0.255
Digestible met+cys (%)
0.888
0.834
0.814
0.765
Digestible threonine (%)
0.792
0.742
0.726
0.682
Digestible tryptophan (%)
0.249
0.230
0.224
0.205
Positive control (PC) - feed formulated according to the nutritional recommendations. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan).
Treatment (U kg−1)
Moisture (%)
Crude protein (%)
Ash (%)
Ether extract (%)
Crude fiber (%)
Starter phase
Positive control
13.50
21.93
4.44
6.58
3.22
100 U amylase + 0 U xylanase
13.14
20.74
4.46
4.28
3.16
100 U amylase + 1,000 U xylanase
12.95
20.23
4.45
4.78
3.17
100 U amylase + 2,000 U xylanase
12.94
21.24
4.48
4.62
3.15
200 U amylase + 0 U xylanase
12.52
20.79
4.06
4.63
2.84
200 U amylase + 1,000 U xylanase
12.50
20.66
4.39
4.83
3.01
200 U amylase + 2,000 U xylanase
12.13
20.67
4.47
4.68
3.02
Grower phase
Positive control
13.89
19.70
4.14
7.75
2.97
100 U amylase + 0 U xylanase
13.24
18.84
3.89
5.68
2.94
100 U amylase + 1,000 U xylanase
13.00
19.37
3.78
5.67
2.72
100 U amylase + 2,000 U xylanase
13.15
18.81
3.75
6.10
3.24
200 U amylase + 0 U xylanase
12.67
18.61
3.85
5.92
3.29
200 U amylase + 1,000 U xylanase
12.08
18.92
4.02
6.05
3.19
200 U amylase + 2,000 U xylanase
12.25
18.80
4.17
5.85
3.28
The experimental diets consisted of combinations of 100 or 200 U kg−1 of amylase with 0, 1,000, or 2,000 U kg−1 of xylanase, corresponding to 100 g and 200 g of amylase product and 0, 100 g and 200 g of xylanase product per tonne of feed, respectively, and one positive control (without enzyme supplementation). The inclusion of enzyme associations and doses in the diets was performed by replacing the inert material (kaolin). Diets supplemented with amylase and xylanase also contained 2,500 U kg−1 of protease (125 g of product per tonne of feed). The nutritional matrix was valued at 100 kcal of metabolizable energy and 6% of crude protein (CP) and digestible amino acids (lysine, methionine + cystine, threonine, and tryptophan), reflecting the expected nutrient release from protease and amylase. In contrast, xylanase was added on top of the matrix-reduced diets, without assigning a matrix value, to isolate its additive effects and potential interactions with amylase. All diets also contained 500 U kg−1 of phytase (100 g of product per tonne of feed), and its nutritional matrix value was considered equivalent to 0.12% calcium and available phosphorus. The recovery of protease and xylanase activities from the diets in the starter phase was analyzed by a commercial laboratory (Toledo, Paraná, Brazil) (
N/A - not analyzed. * Enzyme recoveries were performed before (mash feed) and after pellet processing (pelletized feed).
Treatment/Feed*
Protease
Xylanase
Mashed
Pelleted
Mashed
Pelleted
100 U amylase + 0 U xylanase
3180
3460
N/A
N/A
100 U amylase + 1,000 U xylanase
3780
3640
1321
402
100 U amylase + 2,000 U xylanase
3540
3770
2951
1614
200 U amylase + 0 U xylanase
3440
3580
N/A
N/A
200 U amylase + 1,000 U xylanase
3580
3060
1603
292
200 U amylase + 2,000 U xylanase
3440
3710
2391
623
The xylanase consisted of an endo-1,4-β-xylanase produced via fermentation of
The amylase consisted of an α-amylase produced via fermentation of
The phytase consisted of a 6-phytase produced via fermentation of
The protease was produced via fermentation of
At 28 d, one bird/EU was selected, weighed, and euthanized by cervical dislocation. Fragments of the jejunum were collected for intestinal morphometry analysis by light microscopy and I See Inside® (ISI) score index. The cecal content was stored in a sterile tube (50 mL) and immediately frozen at −20 °C for subsequent analysis of the SCFA profile. Blood samples were collected via brachial puncture into sterile vacuum tubes and immediately centrifuged, with the serum being separated and frozen to determine the serum cytokine concentrations.
To evaluate growth performance, birds and feed were weighed at the beginning of the experimental period and at 14 and 28 d to determine average feed intake (AFI), weight gain (WG), and feed conversion ratio (FCR). Mortality was recorded daily for subsequent performance correction, as recommended by
To analyze the morphometry of the intestinal mucosa, villus height, crypt depth, villus height to crypt depth ratio, and absorption area were determined. Two-centimeter cross-sectional histological fragments of the jejunum were collected immediately proximal to Meckel’s diverticulum, washed with saline solution, fixed in buffered formalin solution (10%) for 24 h, and then dehydrated in a series of increasing concentrations of alcohol, diaphanized in xylene, and embedded in paraffin (
The I See Inside methodology is an index based on a numerical alteration score applied to intestinal histological analysis, as described by
An impact factor (IF) is defined for each alteration observed in the analyses, according to the degree of reduction of the organ functionality. The IF ranges from 1 to 3, with 3 being the most impactful for the organ’s function. Furthermore, the extent of each lesion (intensity) or the observed frequency relative to the unaffected organs in each organ/tissue per animal was evaluated. The score ranged from 0 to 3: score 0 (absence of lesion or frequency), score 1 (alteration of up to 25% of the area or frequency observed), score 2 (alteration of 25 to 50% of the area or frequency observed) and score 3 (extent alteration of more than 50% of the area or frequency observed). To calculate the final ISI index value, the impact factor (IF) of each alteration was multiplied by its respective score number, and the results of all alterations were summed. The sum of all previously mentioned parameters resulted in the total ISI value for each bird (each bird was considered a replicate for statistical analysis).
The determination of SCFAs was performed according to
An aliquot of 1 μL from each sample was injected with a split ratio of 40:1, using helium as the carrier gas at a linear speed of 42 cm·s⁻1, with the separation of the analytes achieved in a chromatographic run of 11.5 min. The injector and detector temperatures were set at 250 °C and 300 °C, respectively, with an initial column temperature of 40 °C. The column temperature ramp began with a gradient from 40 °C to 120 °C at a rate of 40 °C min⁻1, followed by a gradient from 120 °C to 180 °C at 10 °C min⁻1, and finally from 180 °C to 240 °C at 120 °C min⁻1, maintaining the final temperature of 240 °C for an additional 3 min.
For the quantification of analytes, a calibration method was performed using dilutions of the WSFA-2 standard (Supelco©) and glacial acetic acid (Sigma-Aldrich©), analyzed under the conditions described above. The determination and integration of peaks were performed using the GCsolution software v. 2.42.00 (Shimadzu©). The results were expressed in mmol kg⁻1.
The serum concentration of cytokines (IL-6, IL-10, and IL-16) was measured using the Milliplex commercial kit specific for chicken (Catalog # GCYT1-16K; MilliporeSigma Co., Burlington, MA), following the manufacturer’s instructions. The thawed serum samples were diluted (1:4) with 25 μL of appropriate matrix solutions, standard solutions, and control solutions provided in the commercial kit. The samples were then incubated and agitated for 16–18 h at 2–8 °C. After reaching room temperature, 25 μL of the detection antibody preparation was added. The samples were incubated and agitated on a plate shaker for 1 h at room temperature. Subsequently, 150 μL of Sheath Fluid PLUS solution was added, and the samples were agitated for an additional 5 min. Cytokine quantification was performed using the Luminex xMAP® technology (Luminex Corp., Austin, TX), which employs magnetic microspheres conjugated with specific antibodies for each detectable target.
The economic analysis was conducted by considering the cost/t of feed (US$/t) for each feeding program by animal phase, average weight, and average feed consumption per phase according to
All statistical analyses were performed using SAS® Version OnDemand (2021) program (SAS Inst. Inc., Cary, NC, USA), considering a significant level of 5%. Data were initially tested for normality (Shapiro-Wilk test), homogeneity of variances (Levene’s test), and outlier removal using the PROC UNIVARIATE procedure. For normally distributed data, an analysis of variance (ANOVA) was conducted to evaluate the isolated and interactive effects of amylase and xylanase levels. Comparisons among amylase levels were performed using an F-test, while comparisons among xylanase levels were performed by Tukey’s test. The enzyme treatments were compared to the positive control by Dunnett’s test. All procedures were conducted using the PROC GLM procedure.
For ISI variables, which did not exhibit normal distribution, non-parametric tests were applied using the PROC NPAR1WAY procedure. The evaluation of the isolated effect of the enzyme combination and the comparison with the positive control were performed using the Kruskal-Wallis test. When significant effects were observed, xylanase levels were compared using Dunn’s test. To assess the interaction between treatments, the Friedman test was employed.
The following overall model was used:
in which Yij = value observed in the experimental unit that received the combination of factor A (i = 1, 2, …, I) with the factor B (j = 1, 2, …., J); µ = overall mean of the experiment; Ai = fixed effect of factor A; Bj = fixed effect of factor B; ABij = fixed effect of the interaction between factor i and factor j; and eij = random error in factor i and level j.
No interaction (P>0.05) was observed between amylase and xylanase supplementation in either of the evaluated phases for performance parameters. Considering the isolated effects, a difference was observed among the xylanase levels during the starter phase, in which birds receiving diets containing 1,000 U kg⁻1 of xylanase presented better feed conversion ratio (FCR; P = 0.014) compared to the group without xylanase supplementation (
WG - weight gain; AFI - average feed intake; FCR - feed conversion ratio; CV - coefficient of variation; SEM - standard error of the mean (n = 7 per treatment). PC - Positive control feed formulated according to the nutritional recommendations, without adding exogenous enzymes. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease. Means with different letters in the same column indicate differences (P<0.05) by Tukey’s test. * Mean different from the positive control by the Dunnett’s test.
Treatment
1-14 d
1-28 d
WG (g)
AFI (g)
FCR
WG (g)
AFI (g)
FCR
Positive control
451
551
1.216
1863
2547
1.376
Amylase (U kg−1)
100
449
559
1.246*
1823
2577
1.414*
200
454
566
1.246*
1822
2576
1.416*
Xylanase (U kg−1)
0
447
562
1.255a*
1814
2570
1.419*
1,000
458
568
1.241b*
1835
2588
1.411*
2,000
450
559
1.244ab*
1818
2571
1.415*
SEM
15.60
19.87
0.01
54.55
63.47
0.02
CV (%)
3.46
3.54
1.07
2.98
2.47
1.22
P-value
Amylase
0.357
0.250
0.395
0.879
0.915
0.710
Xylanase
0.157
0.535
0.014
0.558
0.704
0.448
Amylase
0.250
0.649
0.119
0.386
0.481
0.410
Dunnett amylase
0.613
0.188
<0.001
0.198
0.555
<0.001
Dunnett xylanase
0.331
0.362
<0.001
0.222
0.593
<0.001
There was no interaction between amylase and xylanase levels, nor isolated effect for the intestinal morphometric parameters (P>0.05), demonstrating that villus height, crypt depth, villus height to crypt depth ratio, and absorption surface area were similar among treatments (
VH - villus height; CD - crypt depth; VCR - villus/crypt ratio; AA - absorption surface area; CV - coefficient of variation; SEM - standard error of the mean (n = 7 per treatment). PC - Positive control feed formulated according to the nutritional recommendations, without adding exogenous enzymes. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease.
Treatment
VH (μm)
CD (μm)
VCR
AA (μm)
Positive control
729.59
83.44
9.35
15.39
Amylase (U kg−1)
100
722.27
75.01
10.33
13.95
200
694.38
74.09
10.24
13.53
Xylanase (U kg−1)
0
725.84
73.12
10.63
14.39
1,000
707.88
76.67
10.01
14.15
2,000
691.26
73.87
10.21
12.68
SEM
83.79
13.52
1.80
2.33
CV (%)
11.78
17.84
17.75
16.69
P-value
Amylase
0.288
0.825
0.880
0.557
Xylanase
0.557
0.762
0.631
0.110
Amylase
0.422
0.350
0.089
0.470
Dunnett amylase
0.466
0.277
0.531
0.200
Dunnett xylanase
0.674
0.388
0.483
0.059
Interactions were observed between amylase and xylanase levels for the parameters of lamina propria thickness (LPT) (P = 0.009), epithelial thickness (ET) (P = 0.020), enterocyte proliferation (EP) (P = 0.021), inflammatory infiltration in the epithelium (IIE) (P<0.001), inflammatory infiltration in the lamina propria (IILP) (P = 0.022), and total ISI score (P = 0.014;
LPT - lamina propria thickness; ET - epithelial thickness; EP - enterocytes proliferation; IIE - inflammatory cell infiltration in the epithelium; IILP - inflammatory cell infiltration in the lamina propria; GC - goblet cells; CONG - congestion; PO - presence of oocysts; SEM - standard error of the mean (n = 7 per treatment). PC - Positive control feed formulated according to the nutritional recommendations, without adding exogenous enzymes. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease. * Mean different from the positive control by the Dunn’s test.
Treatment
LPT
ET
EP
IIE
IILP
GC
CONG
PO
Total
Positive control
1.99
1.20
1.01
1.63
2.70
2.09
0.13
0.00
10.74
Amylase (U kg−1)
100
2.24*
1.36*
1.05
1.62
2.90
2.33*
0.05*
0.71*
12.26*
200
2.24*
1.41*
1.05
1.67
2.81
2.34*
0.02*
0.06
11.59*
Xylanase (U kg−1)
0
2.13
1.39*
1.02
1.60
2.70
2.35*
0.03*
0.31*
11.53*
1,000
2.29*
1.32*
1.05
1.58
2.95*
2.29*
0.05*
0.25*
11.77*
2,000
2.31*
1.44*
1.08**
1.74*
2.91*
2.38*
0.02*
0.60*
12.48*
SEM
0.02
0.01
0.01
0.01
0.03
0.02
0.01
0.04
0.09
CV (%)
34.23
35.18
23.04
29.93
39.14
34.18
68.57
74.11
25.78
P-value
Amylase
0.973
0.136
0.880
0.154
0.182
0.932
0.107
<0.001
0.062
Xylanase
0.009
0.020
0.021
<0.001
0.018
0.341
0.389
0.302
0.014
Amylase
0.009
0.020
0.021
<0.001
0.022
0.337
0.339
0.265
0.014
Dunnett amylase
0.002
<0.001
0.243
0.334
0.262
0.001
0.001
<0.001
<0.001
Dunnett xylanase
<0.001
<0.001
0.010
<0.001
0.032
0.001
0.004
<0.001
<0.001
LPT - lamina propria thickness; ET - epithelial thickness; EP - enterocytes proliferation; IIE - inflammatory cell infiltration in the epithelium; IILP - inflammatory cell infiltration in the lamina propria. Means with different lowercase letters in the same column or uppercase letters in the same row indicate differences (P<0.05) by the Friedman test.
LPT
ET
EP
IIE
IILP
Total
Amylase (U kg−1)
Xylanase (U kg−1)
100
200
P-value
100
200
P-value
100
200
P-value
100
200
P-value
100
200
P-value
100
200
P-value
0
2.10b
2.16
0.523
1.44a
1.34b
0.067
1.04
1.01b
0.281
1.56b
1.64
0.180
2.54bB
2.86A
0.031
11.59b
11.47
0.991
1,000
2.26ab
2.31
0.556
1.29b
1.36b
0.202
1.04
1.06a
0.436
1.51bB
1.65A
0.026
3.01a
2.89
0.229
11.98b
11.56
0.380
2,000
2.37a
2.26
0.266
1.34abB
1.53aA
0.002
1.08
1.08a
1.000
1.78a
1.71
0.189
3.13aA
2.69B
0.001
13.21aA
11.75B
0.021
P-value
0.019
0.186
0.021
0.002
0.255
0.034
<0.001
0.451
<0.001
0.284
0.009
0.587
Considering the isolated effects, the higher amylase dose decreased the presence of oocysts (PO) value (P<0.001). However, the inclusion of 100 U kg−1 amylase resulted in a higher PO value (P<0.001) than that observed in the PC group. Regardless of amylase level, higher values of lamina propria thickness (LPT) (P = 0.002), epithelial thickness (ET) (P<0.001), goblet cells (GC) (P = 0.001), and total ISI score (P<0.001) were observed in treatments containing this enzyme. The inclusion of 1,000 and 2,000 U kg−1 xylanase increased LPT (P<0.001) and inflammatory cell infiltration in the lamina propria (IILP) (P = 0.032). The highest xylanase dose increased enterocyte proliferation (EP) (P = 0.010) and inflammatory cell infiltration in the epithelium (IIE) (P<0.001). Regardless of xylanase level, higher values of ET (P<0.001), GC (P = 0.001), PO (P = 0.001), and total ISI score (P = 0.0001) were observed. Lower congestion values were observed regardless of amylase (P = 0.001) or xylanase level (P = 0.004) (
No interaction or isolated effects (P>0.05) were observed for the cecal SCFA profile (
CV - coefficient of variation; SEM - standard error of the mean (n = 7 per treatment). PC - Positive control feed formulated according to the nutritional recommendations, without adding exogenous enzymes. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease. Isobutyric, propanoic and valeric acids were not considered because they were below the limit of quantification.
Treatment
Acetic
Butyric
Isovaleric
Positive control
29.85
5.13
2.31
Amylase (U kg−1)
100
32.38
5.52
2.54
200
29.82
4.43
2.50
Xylanase (U kg−1)
0
29.57
4.86
2.59
1,000
30.30
5.21
2.59
2,000
33.43
4.83
2.43
SEM
8.03
1.80
0.57
CV (%)
25.97
35.63
22.92
P-value
Amylase
0.288
0.102
0.817
Xylanase
0.382
0.863
0.801
Amylase
0.230
0.219
0.358
Dunnett amylase
0.548
0.183
0.589
Dunnett xylanase
0.586
0.875
0.663
IL-6 - interleukin-6; IL-10 - interleukin-10; IL-16 - interleukin-16; CV - coefficient of variation; SEM - standard error of the mean (n = 7 per treatment). PC - Positive control feed formulated according to the nutritional recommendations, without adding exogenous enzymes. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease.
Treatment
IL-6
IL-10
IL-16
Positive control
54.32
21.14
150.55
Amylase (U kg−1)
100
41.81
20.62
146.93
200
41.95
19.95
148.65
Xylanase (U kg−1)
0
40.16
23.27
150.85
1,000
45.85
17.44
147.77
2,000
39.69
19.71
144.18
SEM
8.19
8.57
8.76
CV (%)
5.53
42.03
5.92
P-value
Amylase
0.967
0.851
0.601
Xylanase
0.468
0.220
0.111
Amylase
0.350
0.596
0.084
Dunnett amylase
0.617
0.943
0.641
Dunnett xylanase
0.288
0.345
0.227
No interaction (P>0.05) was observed between amylase and xylanase supplementation for the economic analysis (
CV - coefficient of variation; SEM - standard error of the mean (n = 7 per treatment). PC - Positive control feed formulated according to the nutritional recommendations, without adding exogenous enzymes. Treatments containing enzymes had a reduction of 100 kcal and 6% of crude protein and amino acids (lysine, methionine, met + cys, threonine and tryptophan). All diets contained 500 U kg−1 of phytase, and its nutrition valorization of calcium and phosphorus was 0.12%. Treatments containing amylase and xylanase contained 2,500 U kg−1 of protease. * Mean different from the positive control by the Dunnett’s test. The cost of feed per ton of chicken was calculated by the cost of feed based on the prices of ingredients for the month of April 2025.
Treatment
Feed cost (US$/t broiler)
Positive control
598.79
Amylase (U kg−1)
100
579.91*
200
584.25
Xylanase (U kg−1)
0
584.89
1,000
578.78*
2,000
581.77
SEM
15.72
CV (%)
2.70
P-value
Amylase
0.383
Xylanase
0.701
Amylase
0.521
Dunnett amylase
0.028
Dunnett xylanase
0.063
Broilers tend to increase feed intake to compensate for energy and nutrient deficits resulting from enzyme matrix valorization, which can reduce the feed efficiency (
One way to improve feed efficiency in poultry is to process feed, such as by pelleting, which can modify carbohydrate structures and improve nutrient digestibility. Pelleted diets increase feed intake in broilers because they facilitate feed consumption and reduce feed wastage (
Although thermal processing can improve feed utilization, this process can reduce enzyme activity due to the high temperatures used in the process, thereby reducing enzyme stability and efficiency (
The enzyme recovery analysis performed on the feed in our study demonstrated that thermal processing affected the enzymatic activity of xylanase. These results suggest that the inferior performance of broilers fed diets containing xylanase, when compared with the PC group, was affected by the low activity of the enzyme in the diets after pelleting. However, an improvement in FCR was observed during the initial phase in broilers supplemented with 1,000 U kg−1 of xylanase compared with those not receiving the enzyme. Therefore, although nutrient digestibility was not evaluated, the improved FCR observed indicates a potential response to xylanase supplementation, even under reduced enzymatic activity.
A limitation of our study is the absence of a negative control diet without enzyme supplementation formulated with the same nutrient levels as the enzyme-supplemented treatments. Therefore, the results observed in the enzyme-supplemented groups should be interpreted as the combined effect of nutrient reduction and enzyme inclusion, rather than as an isolated enzymatic response.
In addition, the PC diet differed in formulation from the enzyme-supplemented diets, particularly regarding oil inclusion. Dietary oil levels are known to affect pellet quality, palatability, digestion, absorption, and rate of passage through the gastrointestinal tract (
Exogenous enzymes such as xylanase, protease, and amylase exert complementary modes of action in broiler diets by improving nutrient availability and supporting gut health. Xylanase hydrolyzes arabinoxylans in cereal cell walls, reducing digesta viscosity and releasing encapsulated nutrients, which may enhance the access of endogenous enzymes to starch and protein fractions and improve feed utilization (
Villus height, crypt depth, and absorption area are key indicators of nutrient absorption capacity and intestinal health of broilers. A higher villus height to crypt depth ratio and greater absorptive surface indicate increased potential for nutrient absorption (
Bucław (2016) states that the need for greater villus height to increase absorption area requires faster epithelial growth, which requires more energy and nutrients for cell proliferation. The greater need for cell maintenance and renewal may occur when there are stressors, such as those coming from the diet, including the greater demand for nutrients for digestion, toxins, antinutritional factors and pathogens that cause lesions and desquamation in the intestinal epithelium (
A previous study reported that diets with reduced crude protein (CP) levels without protease supplementation impair broiler performance due to amino acid imbalances, particularly threonine deficiency (
Lower ISI scores are indicative of better intestinal health. Dietary stressors that alter the intestinal mucosa, as well as tissue renewal resulting from epithelial damage and desquamation, can increase nutrient requirements (
In general, 100 U kg−1 of amylase provided better health parameters when compared with treatments containing xylanase supplementation. However, this benefit was not reflected in a better ISI score or in the performance of the animals when compared to the PC group. Although improvements were found in some ISI score parameters when considering the interaction among amylase and xylanase, evaluating the parameters in isolation it was observed that, in general, the lowest scores were found in animals that received the diet without the enzyme supplementation, indicating that enzyme supplementation under reduced-nutrient conditions was not sufficient to fully match the performance and ISI scores of the positive control.
We observed no differences in SCFA concentrations among the treatments. Xylanase contributes to the release of nutrients through hydrolysis of the cereal cell wall, degrading the xylose units and releasing XOs. The formation of SCFAs depends on the amount of substrate contained in the ingredients, and the amount of NSPs varies among different cereals and may vary within them (
The experimental diets in our study contained a high proportion of ingredients with high levels of soluble NSPs. Therefore, it is possible to infer that the amount of substrate (arabinoxylans) present in the diets was similar and was not markedly affected by the action of the xylanase. The results found for SCFAs corroborate those obtained in morphometry. Xylooligosaccharides (XOs) act as a prebiotic substrate or as signaling molecules for specific groups of bacteria that produce SCFAs, such as butyric acid, which acts as the main source of energy for colonocytes and helps maintain the integrity of intestinal epithelial cells (
Isobutyric, propanoic, and valeric acids were below the quantification limit in our analysis, indicating that these substances are rapidly absorbed and used by the broiler. The digesta flow through the intestinal tract is dynamic, and reverse peristalsis in chickens can result in variability in the local concentrations of the different SCFAs, making their quantification difficult (
Both IL-6 and IL-16 are cytokines with pro-inflammatory characteristics (
The addition of enzymes to the broiler diets aims mainly to reduce feed costs through the contribution of nutrients that the enzyme can release, maintaining the nutritional requirements demanded by the animal in their respective production stages (
The inclusion of amylase and xylanase in reduced energy and protein diets was economically viable despite lower growth performance compared with the positive control. Although the weight of the chicken produced was lower, with a nutritional value of 100 kcal of energy and 6% of proteins and amino acids, the production cost of the chicken was lower.
This study presents a novel approach by evaluating the inclusion of xylanase in diets with reduced energy and protein levels combined with increasing amylase levels in diets already supplemented with protease and phytase under commercial-like pelleting conditions. While prior studies have examined individual enzymes or fixed combinations, few have addressed the layered effect of these four enzymes on both gut health and economic performance in nutrient-restricted conditions. These findings offer practical insights for optimizing enzyme strategies in modern broiler nutrition.
The inclusion of amylase and xylanase in diets with reduced energy and protein levels did not improve the growth performance of broilers at 28 d compared to broilers fed a diet with regular nutrient levels. Furthermore, the enzyme supplementation did not alter gut health or immunological parameters. The analysis of feed costs suggests that the use of 100 U kg−1 of amylase and 1,000 U kg−1 of xylanase enables an average reduction of 3.25% in the production cost/t of broilers produced.
The authors gratefully acknowledge the financial support provided by the Brazilian research agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance code 001) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), which partially funded this study.
The entire dataset supporting the results of this study was published in the article itself.