Effect of two different dietary endo-1,4-β-xylanases on growth performance, intestinal histomorphology, caecal microbial population and short-chain fatty acid composition of broiler chickens

Abstract

Effects of two different dietary xylanases on broiler performance and gut integrity were determined. Totally, 7600-d-old Ross-308 chicks were used for 41 d. Chicks were randomly assigned to 4 dietary treatments with 10 replicates (95 males, 95 females per replicate); they were: positive control (PC) based on Ross 308 nutrient specification; negative control (NC) with less ME (80 kcal/kg), crude protein and amino acids compared to PC; NC + AO with addition of xylanase derived from Aspergillus oryzae (AO); NC + TB with addition of xylanase derived from Trichoderma bisset (TB). Birds fed on PC diet significantly (p < 0.05) had higher final body weight (BW) and body weight gain (BWG) compared to other treatments. The final BWG and feed conversion ratio (FCR) were not fully compensated with the addition of the enzymes compared to PC, and FCR was found significantly (p < 0.05) higher in birds fed on diets NC + AO and NC + TB. However, dietary xylanase significantly improved FCR, villus surface area and total goblet cell number in NC + AO and NC + TB birds compared to NC. Xylanase supplementation significantly increased the concentrations of branched-chain fatty acid (BCFA) and total caecal short-chain fatty acid (SCFA) (p < 0.05). Furthermore, both xylanases increased lactobacillus count (p < 0.05). Dietary treatments had no effect on carcase parameters and mortality rate. In conclusion, along with improving performance parameters, the inclusion of xylanase enzymes with nutrient matrix value in broilers’ diet may ameliorate intestinal morphology, stimulate caecal microflora and increase caecal SCFA concentrations, but nutrient contribution of both enzymes seems overestimated.

HIGHLIGHTS

• Dietary xylanase could not compensate the final BWG and FCR compared to positive control.

• Dietary xylanase increased villus surface area and total goblet cell number.

• Dietary xylanase increased the concentrations of branched-chain fatty acids and total caecal short-chain fatty acid.

Keywords:

• Broiler
• caecal microflora
• intestinal histomorphology
• short-chain fatty acid
• xylanase

Introduction

Plant cell walls contain different levels of non-starch polysaccharide (NSP). As digestive system of poultry has no proper enzyme to degrade the NSPs so, these compounds are serving as antinutritional factors in poultry nutrition. In order to overcome this problem, carbohydrase/NSPase enzymes are often added exogenously to poultry diets to improve nutrient utilisation and increase all over productivity (O’Neill et al. 2014; Choct 2015). The structure and composition of plant cell walls have been extensively researched and discussed in previous studies (Caprita et al. 2010; O’Neill et al. 2014; Choct 2015).

Arabinoxylans are a major part of the dietary NSP component in plant-based feed ingredients. Xylanase is one of the most important forms of NSPases. Xylanases are produced by fungi, bacteria, yeast, marine algae, protozoa, snails, crustaceans, insect, seeds, etc. but the principal commercial source is filamentous fungi (Polizeli et al. 2005). Difference between bacterial and fungal xylanases has been reviewed by Polizeli et al. (2005), Chakdar et al. (2016) and Basit et al. (2020). Briefly, the main advantage of fungal originated enzymes compared to bacterial ones is their higher activity levels. Furthermore, bacterial xylanases are more prone to losing their efficiency as they are not subjected to post-translation modifications such as glycosylation. However, enzyme manufacturers have claimed that bacterial xylanase may be more thermostable at a high temperature (Polizeli et al. 2005). Moers et al. (2003) also showed that three different kinds of commercial NSPase are available based on their origin and activity. Some of them mostly affect soluble NSP, some of them affect insoluble NSP; and others affect both soluble and insoluble NSP. Based on Moers et al. (2003) and Masey-O’Neill, Smith, et al. (2014) categorisation, plant cell wall has three different kinds of NSP, namely soluble, insoluble and fermentable. The first one increases viscosity by holding water and the second one has a cage effect to encapsulate nutrient like a physical trap. The cage effect properties of NSP depend on their ability to prevent the endogenous enzymes such as pancreatic proteases, amylases, etc., to penetrate the nutrients caught within the cell wall structures and finally, ability to produce short-chain fatty acids (SCFAs) by bacteria (prebiotic effect). So, xylanase supplementation of poultry diets works through first, decreasing the viscosity of the gastrointestinal tract (GIT), second breaking the ‘cage effect,’ and third abrading the cell walls of feedstuff by direct action of xylanase, which results in shorter lag time for bacterial attachment that eventually accelerates fermentation. Degradation of xylan in the distal sections of the intestine at the end of the mechanism releases xylo-oligosaccharides (XOS). These molecules act as prebiotics or signalling molecules for specific groups of beneficial bacteria. These bacteria are responsible for producing SCFA in gut (Choct et al. 2004; Bedford 2018; Gonzalez-Ortiz et al. 2019, 2021). One hypothesis about the positive effects of XOS on broiler performance is the direct stimulating effect of XOS on SCFA producing bacteria (Mroz et al. 2005; Van Hoeck et al. 2021; Mátis et al. 2022). Several researchers have reported better zootechnical performance of broilers because of higher butyric acid concentrations in the intestinal digesta by supplementation of dietary xylanase (Masey-O'Neill, Singh, et al. 2014; Lee et al. 2017; Gonzalez-Ortiz et al. 2019).

AfTable (2012) reported that dietary inclusion of NSPase improved ME by 3% and digestibility of protein by 5%. Such improvement leads to better BWG (0.8–10.4%; mean = 4.3%) and FCR (1.3–9.3%; mean = 3.7%). Based on a wide range of studies, possible physico-chemical mechanism behind such improvements are associated with direct and indirect effects of NSPases through their positive impact on gastrointestinal development, intestinal architecture (i.e. increasing villus length, villus length to crypt depth (CD) and villus surface area), influencing intestinal secretion and absorption (Cowieson et al. 2010; Saleh et al. 2019), changing gut microflora population and/or composition (Choct 2009; Mahmood and Guo 2020), improving nutrient digestibility (Saleh et al. 2019; Amer et al. 2020), up-regulating the expression of intestinal nutrient transporter genes (such as PEPT1, GLUT2, ACC and IL-2; Saleh et al. 2018), and increasing SCFA production, however, results about the physiological effects of dietary xylanase have been contradictory (Gonzalez-Ortiz et al. 2020; Singh, Mishra, et al. 2021). For example Gonzalez-Ortiz et al. (2020) did not find any significant effects of dietary xylanase on SCFA concentrations in turkeys, instead Singh, Mishra, et al. (2021) showed a significant increase in total SCFA by xylanase (p < 0.01) though, villus height (VH) CD ratio were not affected (p > 0.05).

Based on our knowledge in most of the studies about the effects of supplemental xylanase on broilers, enzyme was used as on top without applying any nutrient matrix value (energy and amino acid). The objective of the current experiment was to investigate the effect of supplementing corn-soybean meal-based diets, which were deficient in energy and amino acids, with two different commercial xylanases on growth performance, intestinal histomorphology, SCFA composition, and selected microbial population in caeca of broiler chickens. Moreover, two different NSPases were studied in the current study because different kinds of commercial NSPase are available based on their activity, methods of production and origin.

Material and methods

Birds and house arrangement

This study was conducted at the broiler research development centre of Beypilic, Bolu-Turkey. Totally, 7600-d-old Ross 308 broiler chicks (obtained from Bey Pilic Co. Hatchery, as hatched; average BW 43,91 g), were randomly distributed into four dietary treatments with 10 replicate pens (6.5 × 2 m), each containing 190 birds (95 male and 95 female) for 41 d. Birds were raised under standard and full automated controlled house (in terms of heating, cooling and ventilation systems) in floor pens covered with wood shavings as litter material. Birds had ad libitum access to water and feed during the entire period of experiment via nipple drinkers and feeders which were equipped in each pen. All birds were vaccinated once against ND and IBV on day 13 and once more against ND on day 27 of the trial after determination of serum antibody titre in each period. Ambient temperature was 33 °C from day 1–3 then reduced periodically to 22–23 °C based on Aviagen management guide (2018) with relative humidity of about 50 ± 5%%. From 0 to 7 d birds were exposed to 23-h light: 1-h darkness, after 7 d the lighting schedule was 18-h light: 6-h darkness for the whole period. All pens were checked on daily basis for general health and mortality. All experimental procedures were approved by the local Animal Ethics Committee of Bolu Province with the number of 2021-9-122.

Dietary treatments and experimental design

A diet was prepared to meet or exceed according to Ross 308 breeder guidelines containing 2995, 3015 and 3195 kcal of ME/kg in the starter, grower, and finisher phases, respectively. This diet was served as a positive control (PC). Another diet with 80 kcal ME/kg, %0.47 crude protein, %0.0175 dig. Lysine, %0.01 dig. Methionine, %0.02 dig. Met + Cys, %0.02 dig. Threonine, %0.02 dig. Isoleucine and %0.025 dig. Arginine reduced nutrients compared to PC was assigned as negative control (NC) (Table 1). These reduced nutrient amounts were provided by considering both manufacturers’ recommendation for nutrient levels of their xylanase as matrix values. The remaining two dietary treatments were prepared with the addition of commercial NSPase enzyme derived from Aspergillus oryzae (Ronozyme WX^®; AO, DSM) and Trichoderma Bisset (Hostazym X^®; TB, Huvepharma) to obtain NC + AO and NC + TB treatments. Hostazym X marketed as solid (Hostazym X Microgranulate). Based on manufacturers’ declaration thermostability (recovery rate after pelleting) of both enzymes is more than 80% under 80 °C heat and 70 s conditioner time. More information about stability, homogeneity and safety of Ronozyme WX and Hostazym X are presented in EFSA (2012) and EFSA (2013), respectively. All diets were supplemented with 2000 FTU phytase (Ronozyme Hiphos^®) and only Ca and avP matrix of phytase was applied in feed formulation. Withdrawal feeds (38-41 days) were the same as finisher diets in all treatments except for missing anticoccidial. Birds had free access to water and pellet feed. All raw materials used in the study were subjected to proximate analyses (AOAC 2006; data not shown) before formulation of experimental diets. Feeds were produced in Beypilic feed mill. Feeds were provided in two forms as crumble (starter, 0–10 d) and pellet (grower and finishers, 11–41 d). The mixing time was 3.5 min and conditioner temperature was 75 °C. Dietary oil was divided into two, half of it was added to the feed pre-pellet and the other half was added post-pellet. Analysed xylanase content for AO and TB was 152 FXU/kg (spectrophotometric method Xylanase assay in feed WX-201/02E performed by DSM BioPract GmbH Lab, 11. D-1248, Berlin, Germany) and 1690 EPU/kg (spepectrophotometric method Xylanase assay in feed QCD-FPC-0859-03 performed by Huvepharma Biovet lab, 4550, Peshtera, Bulgaria). These values are the average of final starter, grower, and finisher feeds.

Table 1. Ingredients and nutrient composition of the experimental diets through different periods, g/kg as-fed basis^a,b,c.

	Starter		Grower		Finisher
Ingredients	PC	NC	PC	NC	PC	NC
Corn	487.196	502.188	538.151	566.976	583.521	557.744
Soybean meal, 47 %	317.416	299.329	266.438	261.232	231.279	210.591
Full fat soybean, 34.6	71.500	60.000	60.000	50.000	50.000	52.403
Barley	50.000	58.710	55.000	55.000	50.000	100.000
Soy oil	17.823	7.000	26.316	11.806	35.226	24.456
Sunflower meal, 36.5%	25.000	40.000	25.000	25.000	25.000	30.000
Dicalcium phosphate	7.306	7.346	5.664	5.733	3.463	3.391
Limestone	7.522	7.521	6.832	6.849	6.009	6.105
DL-methionine	3.512	3.338	3.171	3.011	2.798	2.658
Common salt	2.300	2.313	2.300	2.300	2.200	2.000
L-Lysine HCL	2.184	2.524	2.294	2.390	1.936	2.114
Vitamin Premix^d	1.000	1.000	1.000	1.000	1.000	1.000
Mineral Premix^e	2.000	2.000	2.000	2.000	2.000	2.000
Sodium bicarbonate	1.669	2.128	2.310	2.350	2.414	2.408
L-threonine	1.379	1.367	1.241	1.037	0.883	0.852
Anticoccidial	0.500	0.500	0.500	0.500	0.500	0.500
Choline chloride, 75	0.492	0.535	0.583	0.615	0.571	0.581
Phytase	1.200	1.200	1.200	1.200	1.200	1.200
Xylanase	0.00	1.000	0.00	1.000	0.00	1.000
Total	1000.0	1000.0	1000.0	1000.0	1000.0	1000.0
Nutrients
Crude protein, %	23.26	22.79	20.874	20.481	19.077	18.72
AMEn, Kcal/Kg	2995.0	2915.0	3095.0	3015.0	3195.0	3115.0
Crude fat, %	5.652	4.405	6.350	4.797	7.111	6.071
Crude fibre, %	4.309	4.51	4.139	4.135	3.998	4.160
Crude ash, %	5.238	5.228	4.750	4.707	4.226	4.199
Dig. methionine, %	0.658	0.641	0.600	0.581	0.545	0.53
Dig. lysine, %	1.280	1.2625	1.150	1.1325	1.022	1.00
Dig. Met + Cys, %	0.950	0.93	0.870	0.85	0.800	0.78
Dig. arginine, %	1.42	1.392	1.258	1.229	1.137	1.10
Dig. threonine, %	0.860	0.84	0.770	0.738	0.680	0.66
Dig. leucine, %	1.624	1.588	1.483	1.469	1.385	1.343
Dig. isoleucine, %	0.867	0.843	0.770	0.754	0.700	0.68
Dig. valine, %	0.945	0.925	0.850	0.837	0.782	0.77
Dig. tryptophane, %	0.249	0.243	0.219	0.214	0.197	0.19
Ca, %	0.90	0.90	0.820	0.820	0.725	0.725
P available, %	0.45	0.45	0.410	0.410	0.363	0.363

^aNegative control diet supplemented with either Xylanase enzyme derived from TB or AO to obtain NC + AO and NC + TB treatments; PC: positive control; NC: negative control; NC + AO = negative control + Aspergillus oryzae; NC + TB = Trichoderma bisset; All diets supplemented with phytase 2000 FYT/kg of feed.

^bNegative control plus xylanase diet gives the applied matrix values for xylanase enzymes: 80 kcal ME per Kg, %0.47 crude protein, %0.0175 dig. Lysine, %0.01 dig. Methionine, %0.02 dig. Met + Cys, %0.02 dig. Threonine, %0.02 dig. Isoleucine and ve %0.025 dig. Arginine.

^cWithdrawal feeds (38–41 d) are the same as finisher diets except removal of anticoccidial.

^d,eSupplied the following per kg of complete feed: 4.13 mg retinyl acetate; 125 μg cholecalciferol; 100 mg dl-α-tocopheryl acetate; 3.5 mg menadione; 3.5 mg thiamine; 9 mg riboflavin; 20 mg calcium d-pantothenate; 65 mg niacin; 0.02 mg vitamin B12; 2.2 mg folic acid; 4.5 mg pyridoxine; 0.22 mg biotin; 120 mg manganese (Mn oxide);1.5 mg iodine (Ca iodate); 25 mg iron (Fe sulphate); 16 mg copper (Cu sulphate); 110 mg zinc (Zn oxide); 0.3 mg selenium (Na selenite).

Sampling and data collection

Hatchlings were weighed at the beginning, on days 10, 22 and 41 on pen basis. Feed intake and FCR were also calculated for the same periods. Dead birds were weighed and recorded for calculating adjusted FCR. At the end of the trial (d 41) 4 male birds per pen, close to the average pen weight, were leg-banded, exsanguinated by cutting the jugular vein, allowed to bleed for approximately 2 min, and then eviscerated. Whole dressed carcase (without abdominal fat and giblets), abdominal fat, breast meat (skin on and bone in), Maryland with backbone (skin on and bone in) and liver were weighed and yields expressed as a fraction of the individual live BW. The livers were also examined for the presence of lesions and haemorrhagic scoring. Liver lesions were graded on a scale from 0 to 5 based on method of Shini (2014). As contamination of raw materials with mycotoxins (specially aflatoxin) affect the liver lesion scores, so all raw materials used in this study analysed for mycotoxin levels, as well (data not shown).

Jejunal histomorphologic measurements

Jejunal samples (10 cm towards the proximal duodenum from Meckel’s diverticulum) were fixed in 10% formalin after being flushed with saline solution. Following histological procedures such as dehydration and clearation, samples were embedded in paraffin wax. Histological sections consisting of intestinal segments with a thickness of 4 μm laid on glass slide by using microtome, were examined under a light microscope (Olympus Optical) and photographed with a digital microscope camera, and evaluated using ImageJ software (Image J, U.S. National Institutes of Health, Bethesda, MD). The measured variables were VH, villus width (VW), CD, VH to CD ratio (VH:CD) and villus surface area. Surface area of the villus was measured using the following formula: (2π) × (VW/2) × (VH)/10⁶ (Solis de los Santos et al. 2005; Ceylan et al. 2023).

Caecal volatile fatty acids concentration

The SCFAs were analysed as free acids by gas chromatography, using pivalic acid as an internal standard (Apajalahti et al. 2019). Briefly, the process started with mixing 1 mL of H₂O with 1 g of caeca content, and then 1 mL of 20 mmol/L pivalic acid solutions was added as an internal standard. Being mixed, 1 mL of perchloric acid was added. The mixture was then shaken for 5 min to extract SCFA.

In order to precipitate the available perchloric acid, 50 mL of 4 mol KOH was mixed into 500 mL of supernatant after centrifugation. Being set aside for 5 min, saturated acid was added. Before the second centrifugation at 18,000 × g for 10 min, the mixture was left in incubator at 4 °C for 60 min. Samples were analysed by gas chromatography using a glass column packed with 80/120 Carbopack B-DA/4% Carbowax 20 mol stationary phase (Supelco, Bellefonte, PA), using helium as the carrier gas and a flame ionisation detector. The acids measured were acetic, propionic, butyric, iso-butyric, valeric and iso-valeric.

Caecal microflora population

The process to evaluate caecal microflora colonies started with homogenising a mixture of one gram of fresh sample with 9 mL of sterile physiological saline solution. After serially diluting the inoculants up to 10⁸, three of them,10⁶,10⁷ and 10⁸, were inoculated (100 μL of each dilution) to appropriate selective agar media to determine coliform, lactobacillus, total aerobic bacteria count. Mac-Conkey agar (Merck, Darmstadt, Germany), MRS agar (Merck, Darmstadt, Germany) and Plate Count Agar (Merck, Darmstadt, Germany) were used to enumerate coliform, lactobacillus and total aerobic bacteria, respectively. All dilutions were inoculated to selective agars in triplicate. Bacterial colonies were counted and averaged. Data were expressed as log₁₀ colony-forming units (cfu) per gram of ileal digesta (Sharifi et al. 2022).

Statistical analysis

A completely randomised experimental design involving 4 treatments and 10 replicate were subjected to statistical analysis of variance using the general ANOVA procedure of SAS release version 9.2 (SAS Institute, Inc., Cary, NC). When significant differences (p < 0.05) were found between groups, means were separated using the Tukey HSD test. The chi-square test assessed mortality results.

Results

Growth performance and carcass traits

The effects of experimental diets on zootechnical performance parameters are presented in Table 2. The BW weight on days 10, 22 and 41 were significantly the highest in PC compared to other treatments (p < 0.05). On the other hand, there was no significant difference between NC + AO and NC + TB treatments for BW during above mentioned periods. Whereas rather different results were found for the final BW as it was lower by 1.701% and 1.263% in NC + AO and NC + TB, respectively, compared to PC (p < 0.05; Table 2). Dietary treatments, regarding the whole period, had significant effect on final BWG (p < 0.05; Table 2). So that, birds fed on NC + AO and NC + TB significantly had the lowest BWG by 1.725% and 1.285%, respectively.

Table 2. Effects of different dietary xylanase on growth performance of broilers¹.

Parameters	PC	NC	NC + AO	NC + TB	Pooled SEM	p Value
BW, g
Initial weight, g	43.94	43.88	43.79	44.03	0.881	0.11
BW d.10	312.24 a	311.32 ab	307.77 b	308.75 ab	0.0001	0.7
BW d.22	1064.36 a	1043.00 b	1037.08 b	1036.00 b	0.0001	2.87
BW d.41	2822.07 a	2750.73 c	2774.05 bc	2786.45 b	0.002	8.92
BWG, g
0–10	268.29 a	267.44 ab	263.98 b	264.71 ab	0.0001	0.69
11–22	752.30 a	731.42 b	729.04 b	726.78 b	0.0001	2.48
23–41	1751	1700.82	1729.71	1738.11	0.102	7.6
0–41	2778.1 a	2706.7 c	2730.2 bc	2742.4 b	0.000	8.93
FI, g
0–10	312.17 b	319.80 a	313.21 b	316.20 ab	0.0001	0.71
11–22	863.38	864.27	855.3	854.6	0.572	2.67
23–41	3165.94	3145.66	3139.03	3175.76	0.326	8.1
0–41	4335.41	4328.69	4317.58	4354.8	0.112	10.26
FCR
0–10	1.164 b	1.196 a	1.186 a	1.193 a	0.0001	0.003
11–22	1.148	1.182	1.173	1.176	0.158	0.003
23–41	1.809 bc	1.851 a	1.815 bc	1.827 b	0.010	0.001
0–41	1.561 c	1.600 a	1.582 b	1.588 b	0.0001	0.003
Mortality, %
0–10	0.95	1	1.21	1.32	0.823	0.09
11–22	2.05	1.52	1.99	2.5	0.170	0.15
23–41	2.42	2	1.95	2.2	0.742	0.16
0–41	5.37	4.47	5.47	6.1	0.158	0.29

^a,cMeans within the same column without common superscripts are significantly different (p < 0.05).

¹PC = Positive Control; NC = negative control; NC + AO = negative control + Aspergillus oryzae; NC + TB = Trichoderma bisset.

BW: Body weight, BWG: Body weight gain, FI: Feed intake, FCR: Feed conversion ratio.

NC birds significantly had the highest FI during starter period (0–10 days; p < 0.05; Table 2). But, this finding did not apply to other treatments as no significant difference could be reported for the overll period. Birds fed on diet NC + AO and NC + TB had the highest final FCR by 1.345% and 1.729%, respectively, compared to PC (p < 0.05; Table 2). This difference comes from the lower BW and BWG in these treatments compared to PC. Dietary treatments had no significant effect on carcase parameters, liver weight and liver haemorrhage scores (p > 0.05; Table 3).

Table 3. Effects of different dietary xylanase on carcase parameters and liver score of broilers at 41 d.¹

Parameters^2	PC	NC	NC + AO	NC + TB	Pooled SEM	p Value
Carcase Yield,%	74.39	73.17	72.7	73.47	0.283	0.194
Breast meat yield,%	26.83	25.57	26.55	26.92	0.251	0.213
Maryland with backbone yield,%	20.3	20.42	19.42	19.99	0.182	0.220
Abdominal fat, %	1.57	1.56	1.66	1.53	0.046	0.756
Liver weight, %	2.01	1.94	1.95	2.08	0.031	0.326
Liver score	2.94	2.88	3	2.88	0.080	0.945

^a,bMeans within the same column without common superscripts are significantly different (p < 0.05).

¹PC = Positive Control; NC = negative control; NC + AO = negative control + Aspergillus oryzae; NC + TB = Trichoderma bisset.

²Skin-on and bone-in % of body weight (BW).

Jejunal histomorphologic measurements

Data on the effect of dietary treatments on jejunal histomorphometric analysis results are summarised in Table 4. Nutrient reduction between PC and NC diets significantly impaired VH, VH:CD, villus surface area and goblet cell number (p < 0.05). VH significantly was the highest in NC + AO (p < 0.05; Table 4). There was not difference between PC and NC + TB birds in terms of VH. Analyses showed significant reduction in CD by inclusion of xylanase in NC + AO and NC + TB by 21.59% and 11.0% compared to PC birds (p < 0.05; Table 4). Villus wide was not affected by dietary treatments (p > 0.05). Birds fed on diet NC + AO and NC + TB had the highest VH:CD ratio (10.53 and 9.21, respectively; p < 0.05).

Table 4. Effects of different dietary xylanase on histomorphological parameters of the jejunum at 41 d.¹

Parameters	PC	NC	NC + AO	NC + TB	Pooled SEM	p Value
Villus height, µm	1291.7 b	1201.5 c	1373.8 a	1326.7 b	13.171	0.001
Crypt depth, µm	176.07 a	189.29 a	138.04c	156.70 b	4.125	0.001
Villus wide, µm	186.63	172.18	180.38	184.77	3.943	0.527
Villus height to Crypt depth	7,66 c	6.68 d	10.53 a	9.21 b	0.284	0.001
Villus surface area, mm^2	0.758 a	0.649 b	0.778 a	0.769 a	0.018	0.029
Goblet cell number	189.04 a	149.26 b	214.18 a	191.65 a	6.685	0.008

^a-dMeans within the same column without common superscripts are significantly different (p < 0.05).

¹PC = Positive Control; NC = negative control; NC + AO = negative control + Aspergillus oryzae; NC + TB = Trichoderma bisset.

Based on the findings, feeding birds with NC diet significantly impaired villus surface area and goblet cell numbers while other birds statistically had the same villus surface area and goblet cell numbers (p < 0.05).

Caecal volatile fatty acids concentration

As shown in Table 5, the acetate (35.38 μmol/g of digesta), butyrate (11.25 μmol/g of digesta) and total SCFA (63.18 μmol/g of digesta) was the highest in NC + AO birds (p  < 0.05). On the other hand, birds fed on diet containing both kinds of xylanase significantly had the highest concentrations (μmol/g of digesta) of caecal propionate and branched chain fatty acids (BCFA) on day 41 d compared to PC and NC birds (p < 0.05). Interestingly, birds received dietary xylanase had greater acetate, butyrate, total BCFA and total SCFA in contrast to PC (p < 0.05).

Table 5. Effects of different dietary xylanase on short-chain fatty acid (SCFA) and branched-chain fatty acid (BCFA) concentrations (μmol/g of digesta) in the caecum at 41 d.¹

Parameters	PC	NC	NC + AO	NC + TB	Pooled SEM	p Value
Acetate	24.55 c	23.43 c	35.38 a	31.53 b	1.012	0.0001
Propionate	9.42 b	9.29 b	13.15 a	13.36 a	0.411	0.0001
Butyrate	8.21 c	7.83 c	11.25 a	9.71 b	0.300	0.0001
Isobutyrate	1.04	0.95	1.17	1.11	0.033	0.070
Valerate	1.05	1.01	1.15	1.22	0.031	0.098
Isovalerate	1.00 b	0.97 b	1.09 ab	1.15 a	0.025	0.047
BCFA^2	3.10 b	2.93 b	3.41 a	3.47 a	0.061	0.003
Total SCFA^3	45.28 c	43.49 c	63.18 a	58.06 b	1.587	0.000

^a-cMeans within the same column without common superscripts are significantly different (p < 0.05).

¹PC = positive control; NC = negative control; NC + AO = negative control + Aspergillus oryzae; NC + TB = Trichoderma bisset.

²BCFA = isobutyrate + valerate + isovalerate.

³Total SCFA = acetate + propionate + butyrate + isobutyrate + valerate + isovalerate.

Caecal microflora population

The data on the caecal selected bacterial counts of broilers fed on different treatments are summarised in Table 6. Although Coliform was not affected by dietary xylanase (p > 0.05), diet supplemented with different commercial xylanase led to higher total aerobe and lactobacillus population in the birds’ caecum (log10 cfu/g; p < 0.05). It is worth mentioning that the nutrient reduction between PC and NC treatments had no significant effects on Lactobacillus or Escherichia coli levels, but the total aerobe counts significantly (log10 cfu/g; p < 0.05) went down in NC compared to PC (8.21 vs. 8.60; Table 6).

Table 6. Effects of different dietary xylanase on the total aerobe bacteria, Lactobacillus and coliform populations in the caecum (log10 cfu/g) at 41 d.¹

Parameters	PC	NC	NC + AO	NC + TB	Pooled SEM	p Value
Total aerobe counts	8.60 bc	8.21 c	9.75 a	9.33 ab	0.203	0.010
Lactobacillus	8.48 b	8.29 b	9.61 a	9.52 a	0.170	0.007
Coliform	7.91	8.24	7.29	7.53	0.156	0.233

^a,bMeans within the same column without common superscripts are significantly different (p < 0.05).

¹PC = positive control; NC = negative control; NC + AO = negative control + Aspergillus oryzae; NC + TB = Trichoderma bisset.

Discussion

Due to price fluctuations in feed raw materials in poultry production industry, nutritionists always try different strategies to reduce feed costs. NSP degrading enzymes such as commercial xylanase is one of the most extensively used feed additives that can take part in bringing down the dietary AME without any negative effect on birds’ performance (Hew et al. 1998; Francesch et al. 2012). In this study, the hypothesis that inclusion of two different xylanases in a diet with low energy and amino acids would influence the performance and GIT integrity was put into practice. As it was discussed, lowering ME and AAs in NC birds by 80 kcal/kg and 3%, respectively, significantly reduced (p < 0.05) BWG and impaired FCR in comparison to the PC. Adding xylanase to NC diet alleviated the negative effects of nutrient reduction, leading to partially better performance in terms of BW, BWG and FCR. These results were parallel with what Gonzalez-Ortiz et al. (2021) had reported. In their study, NC birds (a 50 kcal/kg ME reduction and a 3% reduction in AA content) fed on diet containing combination of xylanase and XOS performed equal to those in PC. They concluded that dietary xylanase and XOS improved performance parameters such as, European production efficiency factor (EPEF) and FCR in broiler chickens particularly those with deficient ME and AA. Similarly, Singh, Mishra, et al. (2021) showed that dietary supplementation of xylanase linearly (p < 0.01) increased the average daily gain in finishers and total period, and final BWG (2940 and 2932 vs. 2760 g) of broilers. Luo et al. (2009) revealed that birds fed on diet containing 500, 1000 and 5000 U/kg xylanase had a lower FCR on day 42 by 9.97%, 6.67% and 11.27%, respectively, compared to the ones fed on unsupplemented diet. Observing the positive outcomes in terms of improving zootechnical performance and/or GIT integrity by dietary supplementation of xylanase in wheat-based diets (Bedford and Schulze 1998; Luo et al. 2009; Gonzalez-Ortiz et al. 2019), corn-based diets (Masey-O’Neill et al. 2012; Nguyen et al. 2021) and corn-wheat based diets (Gonzalez-Ortiz et al. 2021), it can be suggested that utility of this enzyme is possible in both cereals.

Reduction in hemicellulose integrity and digesta viscosity in GIT, and enhanced energy and nutrient utilisation can explain why applying xylanase in diet improves zootechnical performance (Choct et al. 2004; Choct 2006; Francesch et al. 2012; Kiarie et al. 2014). Improvement in digestibility of DM, CP, and energy has been approved by different studies (Hew et al. 1998; Kiarie et al. 2014; Inayah et al. 2022).

In this study, inclusion of dietary xylanases to NC group improved the performance compared to the NC group without additional enzyme. But this improvement was not as much as PC group. Similar results had been reported in previous studies (Kocher et al. 2003; Rabello et al. 2021; Simoes et al. 2023). For example, Simoes et al. (2023) reported lower performance results in the broiler chickens fed on corn-soy bean meal based diet plus exogenous NSPase (a basal diet with a reduction of 80 kcal/kg) compared to PC diet (a basal diet without enzyme and energy reduction).

On the other hand, addition of enzyme to NC resulted in improvement in SCFA and beneficial bacterial population in the intestine, even compared to PC diet that led to better intestinal histomorphology. All these ameliorated birds’ zootechnical performance in enzyme group compared to NC group without enzyme. However, the improvement with enzyme supplementation was not good enough to catch or compensate the growth of PC birds. This might be due to the higher amount of the energy and AA reduction in NC diet applied in the experiment by considering the matrix value of both enzymes, which may be overestimated for corn-soybean meal based diets. The underperformed efficacy of the enzymes may also be attributed to the NSP level and structure of corn-soybean meal diets. It has been shown that soluble and insoluble fraction of NSP can differ based on their plant source, chemical structure and chain length. For example, total NSP level in corn, wheat and barley is 9%, 11.3% and 18.6% of dry matter, respectively (Knudsen 2014). Gehring et al. (2013) concluded that responses to different exogenous xylanase supplementation are not constant and are influenced by the source of ingredients as well as the age of broilers. Kim et al. (2022) also showed that there is a marked difference in the amounts and types of NSP delivered to different GIT sections when birds are fed on wheat-compared to corn-based diets specially, in the gizzard and the lower GIT of birds.

Moreover, xylanase plays a part in destroying insoluble fibres and releasing trapped nutrients that later would be subjected to digestive enzymes in one hand and on the other hand, it increases the level of XOS in hind gut and boosts prebiotic effect (Masey-O’Neill, Singh, et al. 2014; Bedford 2018). In a recent study, Jha and Mishra (2021) discussed that fibre can stimulate the physiological process of GIT mechanically and enzymatically as a result, supplementing broilers’ diet with high amounts of fibre compounds can alter histologic parameters of gut. The result of such an improvement in nutrient digestibility surfaces when BW increases and FCR decreases, which in turn positively reflects birds’ zootechnical performance (Kiarie et al. 2014). However, the researchers do not appear to agree about the outcomes because the performance results can change due to the amount of insoluble and soluble NSP of digesta in different studies, and the type of exogenous xylanase included in the diet (Choct et al. 2004; Singh, Mishra, et al. 2021).

Shorter villi is a warning that an unfavourable condition has rosen up in intestine. In response to sloughing or inflammation from pathogenic bacteria and their toxins, crypts depth increases as an indicator of faster turnover to allow renewal of villi. Fast tissue turnover calls for more energy and protein requirements for maintenance of GIT which in turn lowers the birds’ performance (Awad et al. 2009; Choct 2009). In a research, VH and VH:CD ratio on day 35 were significantly lower in NC diet that contained 100 Kcal ME/kg less than PC (Van Hoeck et al. 2021). It has been shown that, feeding the birds with diets 5% below Aviagen recommendation for the whole period gives rise to significantly failure in VH (1238.82 vs. 1311.58 µm), VW (153.87 vs. 178.56 µm), and villus surface area (0.731 vs. 0.585 mm²) compared to PC birds (Ceylan et al. 2023). In this study xylanase supplementation significantly increased jejunal VH and reduced CD. According to Mathlouthi et al. (2002), supplementation of broiler diet with xylanase and β-glucanase enzymes increases the average VH. Meta-analysis output of 53 articles showed that the xylanase supplementation did not affect VH and VH:CD ratio of duodenum, jejunum, ileum, and the CD of duodenum and jejunum. However, xylanase supplementation linearly (p < 0.05) lowered the CD in ileum (Inayah et al. 2022). Previous studies documented a relation between enhancing VH and VH:CD ratio of different sections of the intestinal tract with FCR (Zou et al. 2013; Zhu et al. 2014). Some studies reported no effect of xylanase on intestinal morphology (Pekel et al. 2017).

Montagne et al. 2003 and Mateos et al. (2012) stated that fibre molecules remain in digestive villi of broiler chickens fed on a diet with high fibre. This phenomenon depends on factors such as, physico-chemical characteristics of the fibre, level of feed consumed, type of animal, the age and health status of the poultry. Supplementing the diet with xylanase facilitates digestion of indigestible fibre. Xylanase positively effects metabolic and digestive processes by letting more degraded nutrients being absorbed in the small intestine; in this way less nutrient will be delivered to caecum which reduces the workload of caecum (Inayah et al. 2022). Overall, this process has positive and favourable impact on the ratio of different SCFAs and microflora population of caeca.

In this study, supplementation of NC with enzyme, significantly increased caecal BCFA and SCFA concentration. This is inconsistent with what Rebolé et al. (2010) and Józefiak et al. (2007) presented. In line with our findings Van Hoeck et al. (2021) reported that the addition of xylanase significantly increased caecal butyrate, and total SCFA concentrations compared to birds fed on PC or NC diet. Furthermore, similar effects of xylanase on increasing acetate and total SCFA were shared by Dale et al. (2020) and Singh, Mandal, et al. (2021).

The importance and physiological role of SCFA and specially butyric acid in GIT as an energy source for enterocytes is well known. Some studies showed that SCFAs mainly acetate, propionate, and butyrate have an important role in cellular differentiation, total GIT integrity, balance of the caecal/gut microbiota and increasing beneficial microbial activity (Mroz et al. 2005; Gonzalez-Ortiz et al. 2019; Van Hoeck et al. 2021; Mátis et al. 2022).

Gut microbiota promotes enzyme secretion, contributes to the process of digestion and absorption. It also regulates energy metabolism, prevents mucosa infections and modulates the immune system (Jha and Mishra 2021). Regarding the indirect effect of dietary xylanase (prebiotic effect), it has been discussed that xylanase blocks the growth of pathogen in distal part of the GIT and protects it from infection by stimulating beneficial bacteria such as Lactobacillus which attaches to the gut mucosa. All of these in general, promote the growth of beneficial bacteria in gut and production of SCFA (Singh, Mishra, et al. 2021; Jha and Mishra 2021). Data from this study, accordingly, revealed that dietary xylanase, free from their commercial brand, could modulate caecal microflora by stimulating the growth of beneficial Lactobacilli. Mathlouthi et al. (2002), reported that xylanase and β-glucanase supplementation to a wheat- and barley-based diet reduced the counts of total facultative anaerobic bacteria and E. coli in the caeca, although their data did not show any significant effect on the number of Lactobacilli. Similarly, Van Hoeck et al. (2021) showed that dietary xylanase effectively reduced E. coli, and increased Lactobacillus spp. in the caeca of 35-day-old broiler chickens. On the other hand, Luo et al. (2009) presented totally different results as they could not find any positive effect of supplemented xylanase on intestinal microflora of broilers on day 42. The positive correlation of acetate production with Lactobacillus observed in this study was also reported previously (Liao et al. 2020).

Conclusion

It can be concluded that under the conditions of the current study, reducing the density of dietary energy and AAs up to 80 Kcal/kg and 3% respectively, impaired BWG and adjusted FCR for 2500 g BW by almost 1.5% and 0.035 points, respectively, compared to PC birds fed on diet based on Ross 308 nutrient recommendation, though, xylanase supplementation with the matrix values (without considering their commercial brand) had beneficial effects. It improved BWG, FCR jejunal histomorphology, SCFA composition and caecal microbial population compared to NC. However, the improvement with enzyme supplementation was not good enough to catch or compensate the growth of PC birds. In this case, in spite of beneficiary effects of both supplemented xylanase on improving growth performance by affecting gut structure, microbiota, and positive production of SCFA, nutritionists’ decisions for using and applying the nutrient matrix level for the enzyme will still depend on and be affected by the price of commercial enzyme and the cost of final diet.

Acknowledgment

The authors would like to thank Beypilic Co. for their valuable support and contribution during the research.

Disclosure statement

There is no conflict of interest for declaration.

Availability of data

The participants of this study did not give written consent for their data to be shared publicly, so due to the sensitive nature of the research supporting data is not available.