British Poultry Science

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Dietary microbial muramidase improves feed

efficiency, energy and nutrient availability, and
welfare of broilers fed commercial type diets
containing exogenous enzymes

V. Pirgozliev , A. Simic , S.P. Rose & E. Pérez Calvo

To cite this article: V. Pirgozliev , A. Simic , S.P. Rose & E. Pérez Calvo (2020): Dietary
microbial muramidase improves feed efficiency, energy and nutrient availability, and welfare of
broilers fed commercial type diets containing exogenous enzymes, British Poultry Science, DOI:
10.1080/00071668.2020.1817330

To link to this article: https://doi.org/10.1080/00071668.2020.1817330

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Publisher: Taylor & Francis & British Poultry Science Ltd

Journal: British Poultry Science

DOI: 10.1080/00071668.2020.1817330
Dietary microbial muramidase improves feed efficiency, energy and nutrient
availability, and welfare of broilers fed commercial type diets containing exogenous
enzymes

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V. PIRGOZLIEV 1, A. SIMIC1, S.P. ROSE1 AND E. PÉREZ CALVO2

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NIPH, Harper Adams University, Newport, Shropshire, UK
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DSM Nutritional Products, Animal Nutrition & Health R & D, Village-Neuf, F-68128

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Corresponding author: Dr V. Pirgozliev

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Email: vpirgozliev@harper-adams.ac.uk
The National Institute of Poultry Husbandry, Harper Adams University, Newport, UK

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Abstract
1. The aim of this study was to evaluate the effect of graded levels of the microbially-derived
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feed lysozyme, muramidase (MUR) on feed intake (FI), weight gain (WG), feed conversion

ratio (FCR), European Performance Index (EPI), dietary N-corrected apparent metabolisable
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energy (AMEn), footpad dermatitis score (FPD) and other welfare variables, when fed to
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broilers from 0 to 42d age.

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2. A four-phase dietary program and four experimental pelleted diets were used; a control

diet (following breeder recommendations without MUR supplementation), and three diets
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based on the control diet supplemented with 25,000, 35,000 and 45,000 LSU (F)/kg of MUR,
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respectively. In addition, all experimental diets contained exogenous xylanase, phytase and a

coccidiostat. Each diet was fed to birds in 24 pens (20 male Ross 308 chicks in each pen)

following randomisation. Dietary AMEn was determined at 21 d of age, and FPD was

evaluated at the end of the study. Data were analysed by ANOVA, using orthogonal

polynomials for assessing linear and quadratic responses to MUR activity.

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3. The inclusion of MUR did not change FI (P>0.05), but increased WG in a linear manner

(P<0.05) and reduced FCR in a quadratic manner, with optimum WG and FCR observed in

birds fed approximately 35000 LSU (F)/kg. In accordance with the improvement in FCR,

35000 LSU (F)/kg MUR supplementation produced the highest EPI (P<0.05). FPD score was

linearly decreased with increased addition of MUR (P<0.05). Dietary AMEn responded in a

quadratic fashion to the MUR inclusion, as the highest values were obtained with the highest

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inclusion rate (P<0.05).

4. In conclusion, the results showed that inclusion of MUR improved feed efficiency and the

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foot health of birds.

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Key words: Muramidase, feed efficiency, metabolisable energy, footpad dermatitis.
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Introduction

The use of feed additives to improve the efficiency of growth and/or egg production, prevent

disease and improve feed utilisation is a common strategy to improve efficiency in the

poultry industry (Pirgozliev et al., 2019). Exogenous enzymes are the most commonly used

feed additives. The enzymes widely used by the industry are non-starch polysaccharidases

that cleave the non-starch polysaccharides in viscous cereals and microbial phytases that

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target the phytate-complexes in plant ingredients (Pirgozliev et al., 2010; Adeola and

Cowieson, 2011; Ravindran, 2013). Recently a new category of feed enzymes, microbial

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muramidase (MUR) have become available, in which the substrate is not present in the feed

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but already present in the gastrointestinal tract. Muramidases (EC 3.2.1.17), also known as

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lysozyme or N-acetylmuramidase, are naturally found in a great variety of animal secretions,
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plants, or micro-organisms. Muramidases are glycosyl hydrolytic enzymes that cleave the β-

1, 4 glycosidic linkages between N-acetylmuramic acid and N-acetyl glucosamine in the

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carbohydrate backbone of bacterial cell wall components, called peptidoglycans (PGNs).

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Recent studies have demonstrated the efficacy of microbial MUR on feed efficiency and
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gastrointestinal tract functions, enhancing nutrient digestibility and absorption (Goodarzi

Booronjeri et al., 2019; Sais et al., 2019). Lichtenberg et al. (2017) suggested that catalysing
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the depolymerisation of PGNs from the bacterial cell debris present in the gut, as a result of

the continuous bacterial turnover, may best describe the mode of action of this enzyme.
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During this process, 50% of the pre-existing PGNs in a bacterial cell are released from the
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wall and recycled within one generation (Reith and Mayer, 2011), although the fate of the

remaining 50% is unclear. It can be speculated that accumulation of bacterial cell wall

fragments at the gut surface could impair nutrient digestion and absorption and, in that case,

the inclusion of microbial MUR in broiler diets could result in better nutrient availability and

higher growth performance (Goodarzi Boroojeni et al., 2019). Thus, the combined

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application of different categories of enzymes in commercial poultry diets may result in

additive or synergistic effects on nutrient utilisation and animal performance.

The present study investigated the impact of different inclusion levels of microbial MUR on

growth performance, including feed intake (FI), weigh gain (WG) and feed conversion ratio

(FCR), dietary N-corrected apparent metabolisable energy (AMEn), dry matter (DMR),

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organic matter (OMR), nitrogen (NR) and fat retention (FR) coefficients, sialic acid (SA) in

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excreta, foot bad dermatitis score (FPD), European poultry efficiency factor (EPEF) and

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some litter quality variables when fed to broilers from 0 to 42d age.

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Materials and methods
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The experiment was conducted at the National Institute of Poultry Husbandry (NIPH) and

approved by the Research Ethics Committee of Harper Adams University, Newport, UK.
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Animals and experimental design

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A total of 1960, male, Ross 308 broilers were obtained from a commercial hatchery (Cyril
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Bason Ltd, Craven Arms, UK). On the arrival, 1920 birds were divided into 96 floor pens
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with 20 birds in each (excluding ill and malformed birds). Each of the 96 pens had a solid

floor and measured 2.1 m2 and bedded with wood shavings.

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The room temperature was approximately 32ºC at day old and was gradually reduced to
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about 20ºC at 21 days of age. A standard lighting program for broilers was used, decreasing

the light:dark ratio from 23h:1h from one day old to 18h:6h at seven days old, which was

maintained until the end of the study. Access to feed and the water was ad libitum.

Four starter (day 1 to 10), grower (day 11 to 20), finisher-1 (day 21 to 35) and finisher-2

(day 35 to 42) wheat-soybean diets were produced (control; C) , three containing different

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levels of microbial MUR (BalanciusTM, DSM Nutritional Products Ltd, Kaiseraugst,

Switzerland); low (L, 417 g/t; 25,000 LSU(F)/kg, medium (M, 583 g/t; 35,000 LSU(F)/kg),

and high (H, 750 g/t; 45,000 LSU(F)/kg). Each single unit of LSU(F) is defined as the

amount of enzyme that increases the fluorescence of 12.5 μg/ml fluorescein-labelled

peptidoglycan per minute at pH 6.0 and 30 C by a value that corresponds to the fluorescence

of approximately 0.06 nmol fluorescein isothiocyanate isomer I.

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The tested MUR product was included in powder form with a minimum analysed MUR

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activity of 60,000 LSU(F)/g product. Diets were supplemented with exogenous xylanase

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(RONOZYME®WX, endo-1,4-beta-xylanase; DSM Nutritional Products Ltd, Kaiseraugst,

Switzerland), phytase (RONOZYME® HiPhos; DSM Nutritional Products Ltd, Kaiseraugst,

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Switzerland) and coccidiostat (CLINACOX®, Elanco Ltd., Guelph, CA). No antibiotic was
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included in feed during the experimental period. The diets were isocaloric and isonitrogenous

for each feeding phase, and met or exceeded breeder recommendations (Aviagen Ltd,
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Edinburgh, UK). The composition of the experimental diets is shown in Table 1.

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Table 1 here
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Mortality was recorded daily. A visual assessment for litter score of the entire pen was
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performed at 34 d old, using a five point scoring system, from 1 to 5, as previously described
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(Da Costa et al., 2014; Mirza et al., 2016). A lower score indicated better litter quality. The

litter pH was determined at 35 d of age using a pH probe with a stainless steel penetration

blade directly into the litter in four different sides in each pen. The pH probe was attached to

a Hanna HI 99163 meter (Hanna Instruments Ltd, Bedfordshire, UK). Litter dry matter was

determined at 35 d of age by taking five samples from the same locations of the floor in each

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pen, including the area near the drinker, and drying them in an oven (see method below). The

samples were then homogenised, milled and stored dry before further analysis.

Footpad and hock lesions were assessed and given a score at 35 d of age for both the left and

right leg of all birds, and classified according to a scale published by Hocking et al., (2008)

from 0 (no lesion) to 4 (very severe lesions). A mean value per pen for each of the

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measurements was used in statistical analysis.

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At 17 d of age, two randomly selected birds from each pen were transferred to one of 96

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raised-floor battery pens (60 × 60 cm floor area) in a controlled environment room. Each pen

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was equipped with a metal feeder, providing 40 cm feeding space, and two nipple drinkers

with spill cups. Treatments were randomly allocated to the pens. Feed and water were offered

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for ad libitum consumption. The selected birds were kept in the pens for 72 h, and total
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excreta were collected three times (every 24 h) from the trays beneath, and spilled feed and

feathers were removed before weighing. Feed intake was weighed for the same period. The
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N-corrected apparent metabolisable energy (AMEn) of diets was determined following the
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procedure of Hill and Anderson (1958).

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The coefficients of apparent retention of dietary dry matter (DMR) and N (NR) retention
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coefficients were determined as the difference between nutrient intake (feed intake multiplied

by the nutrient content in feed) and nutrient output (excreta voided for 72 h multiplied by the
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nutrient content in excreta) divided by the nutrient intake.

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The European Poultry Efficiency Factor, which standardises technical results by considering

FCR, mortality and daily weight gain, was determined for the broilers from 0 to 42 d age.

Chemical analysis

Dry matter in litter, feed and excreta was determined by drying samples in a forced draft oven

at 105°C to a constant weight (AOAC 2000; method 934.01). Crude protein (6.25 × N) in

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litter, feed and excreta samples was determined by the combustion method (AOAC 2000;

method 990.03) using a LECO FP-528 N (Leco Corp., St. Joseph, MI, USA). Oil (as ether

extract) was analysed using diethyl ether by the ether extraction method (AOAC 2000;

method 945.16) using a Soxtec system (Foss Ltd., Warrington, UK). The gross energy (GE)

values for feed and excreta samples were determined in a bomb calorimeter (model 6200;

Parr Instrument Co., Moline, IL, USA), with benzoic acid used as the standard.

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Endogenous mucin in the dry excreta was measured using the concentration of the SA as a

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marker, following the periodate-resorcinol method (Jourdian et al., 1971). In brief, the

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method involves conversion of free and glycosidically bound SA to chromogenic substances,

by treatment with periodic acid followed by resorcinol. The colour of the samples was

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stabilised by 2-methyl-propan-2-ol, and, after centrifugation, the absorbance of the
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supernatant was determined spectrophotometrically at 630 nm (Spectronic 301; Milton Roy

Company, Warminster, PA). This procedure detected total, free, and glycosidically bound N
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acetyl neuraminic (sialic) acid. The MUR activity in the feed samples was determined
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according to the method described by Lichtenberg et al. (2017).

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Statistical analysis
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Prior to statistical analyses, data were checked for normality and homogeneity, and

transformations were deemed not necessary. Statistical analyses were performed using
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GenStat (18th edition) statistical software package for Windows (IACR, Rothamstead,
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Hertfordshire, UK). The comparison between the experimental results was performed by

ANOVA, using orthogonal polynomials for testing linear and quadratic responses to MUR

inclusion. Differences were reported as significant at P<0.05, and trends towards significance

(P<0.1), were included in the report.

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Results

The birds remained healthy throughout the study period. No adverse effects due to feeding

the experimental diets were observed, and the overall mortality was low at 3.4% and not

treatment related. The determined chemical composition of the diets is presented in Table 1

and agreed with the calculated values.

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Table 1 here

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Results of analyses of MUR activity in the diets confirmed the correct addition of the product

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within the range of the expected values ± 20% (Table 2).
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Table 2 here
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During the first three weeks of the feeding trial there were no effects (P>0.05) of diet on any
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growth performance variables, although birds fed the control diet tended (P=0.053) to have

the lowest WG during the starter phase (1-10 d; Table 3).

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A change in performance was observed at 35 d of age when weight gain of the birds was
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improved in a significant linear fashion (P<0.05) with increasing MUR dosage. The high

dosage of MUR gave the lowest FCR, although the response was curvilinear (P<0.05), i.e.

low MUR dosage produced a higher FCR compared to medium and high dosages. Overall,

for the entire period from one to 42 d of age, weight gain increased in a dose dependent linear

manner (P<0.001). The significant quadratic response of FCR at 42 d to MUR

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supplementation (P=0.010) suggested that the optimum inclusion level at this age was at

35,000 LSU(F)/kg, where FCR was 2.6% lower than the control. In agreement with the FCR

at 42 d of age, the EPEF responded in the same way to MUR activity (P=0.016), being 6.7%

higher than the control when the diet was supplemented with 35,000 LSU (F)/kg. The

liveability of the birds was unaffected (P>0.05) by MUR dosage.

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The footpad dermatitis score, determined at 35 d of age, was reduced in a dose dependent

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linear manner (P<0.001; Table 3) in agreement with the improved WG and FCR for the same

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period.

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Table 3 here
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Dietary MUR significantly alter the litter dry matter, pH, N content or footpad dermatitis
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score (P>0.05; Table 4). Fat retention increased in a dose dependent linear manner (P<0.001;
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Table 4).
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Table 4 here
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There were no differences (P>0.05) in SA excretions. Exogenous MUR supplementation

significantly improved (P<0.05) dietary AMEn, and the coefficients of retention of dry

matter, organic matter and nitrogen (Table 5) in a quadratic manner.

Table 5 here

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Discussion

The positive responses in the growth performance variables and EPEF in this study are in

accordance with recently published studies. When feeding the same levels of the same MUR

product, Goodarzi Boroojeni et al. (2019) found a linear increase in WG and decrease in FCR

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at 35 d of age and the supplementation improved EPEF in similar way as in the present paper.

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Sais et al., (2019) reported reduced FCR in broilers fed MUR from day old to 36 d age. Most

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importantly, the improvement in FCR at 42 d of age in the current study agreed with the

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findings of Lichtenberg et al. (2017), who fed the same dosage of the same enzyme to

broilers. The latter authors found an even greater improvement in final weight of birds,

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although they were fed much higher MUR dosages (225,000 and 450,000 LSU (F)/kg),
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although no changes at FCR were noted.

Studies on the use of MUR from different origins, e.g. modified rice expressing lysozyme
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(Humphrey et al., 2002) or hen egg-white (HEW) lysozyme (Abdel-Latif et al., 2017), in
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broiler diets have been reported to improve feed efficiency. However, Gong et al. (2017)
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found no effect on growth performance, but saw changes in the microbiome when feeding a

HEW lysozyme preparation to broilers. Liu et al. (2010) and Zhang et al. (2010), reported
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improved growth when HEW lysozyme was fed to Clostridium perfringens challenged birds,
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but not in the unchallenged control group.

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The variation in growth responses between published reports may be attributable to

differences in dietary formulations, enzyme dose, application or the origin of the lysozyme or

the simultaneous use of other enzymes. Given the diversity in origin between different

lysozymes evaluated in vivo, it can be speculated that the mode of action can differ. In the

current study, the microbial-derived product used was encoded by the MUR gene from the

fungus Acremonium alcalophilum and was assessed to ensure it did not to possess any

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antibacterial activities at the intended doses (EFSA, 2018). Lichtenberg et al. (2017) showed

an increase in feed efficiency, without any significant differences in the caecal microbiome

for microbially-derived MUR supplemented broiler diets.

In the current study, significant growth performance in response to dietary MUR was only

observed in birds after 21 d of age. This suggested that the beneficial effect of MUR was

related to the changing importance of the caeca in birds as they aged, as at 7 d of age the

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caeca represents only 13% of the weight of the small intestine, whereas at 35 d it comprises

24% of the small intestine (Yang et al., 2020). Apajalathi et al. (2002) reported that the

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numbers of microbes reach 1011/g of caecal digesta and 109/g of ileal digesta during the first

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three days post hatch, and remain relatively stable for the following 34 d. As feed intake and

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the absolute size of the gastrointestinal tract (GIT) increases with the age of the birds, it is
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logical to assume that the content of digesta, i.e. the total number of microbes in the GIT,

increases proportionally. The life span of bacteria is relatively short (Fuller, 1978) and a
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continuous and natural bacterial turnover occurs, releasing bacterial cell debris into the GIT.
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Through this process, in one generation, up to half of the pre-existing PGNs from the
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bacterial cell wall is released and recovered (Reith and Mayer, 2011). However, it is still

unclear what happens with the remaining PGNs, and, as birds age, their GIT may accumulate
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bacterial cell debris, including PGNs. This might explain why the improvement of growth

performance was only seen in older birds in the present study.

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Metabolisable energy is a measurement of the available energy from dietary carbohydrates,

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fats and proteins, hence, it was expected that an improvement in nutrient retention

coefficients would improve dietary AMEn (Woods et al., 2020). The main ingredient in the

diets was wheat, which may cause an increase in digesta viscosity due to high non-starch

polysaccharide (NSP) content, that can reduce energy and nutrient availability (Pirgozliev et

al., 2015). Although viscosity was not measured in the reported study, the quadratic response

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between AMEn and the majority of the nutrients suggested that MUR may have an impact on

digesta viscosity. However, further research into any interaction between MUR and other

feed additives is warranted. Zanella et al. (1999) found that metabolisable energy and nutrient

digestibility differed when determined using ileal digesta or excreta. This may provide an

alternative explanation to the quadratic responses seen to MUR in the current study, where

AMEn was performed on excreta and was linear, whereas the Goodarzi Boroojeni et al.,

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(2019) study used digesta samples for evaluation.

In addition, increased digesta viscosity has been shown to reduce conjugated bile acids,

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affecting fat emulsification and digestibility (Langhout et al., 1997). In the present study, fat

retention increased with MUR in a dose dependent linear manner. Sais et al. (2019) showed

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that MUR inclusion increased ileal apparent digestibility of fat and increased fat-soluble
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vitamin A in plasma at 9 d of age. This suggested that MUR improves fat digestion and

absorption in young birds. Goodarzi Boroojeni et al. (2019) observed that supplementing
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MUR in a 30% wheat-based diet containing exogenous carbohydrase showed improvement in

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the apparent ileal digestibility of fat in a linear fashion after 35 d of supplementation.

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Goodarzi Boroojeni et al. (2019) suggested that MUR might catalyse the depolymerisation of

peptidoglycans from bacterial cell debris and reduce its accumulation in the gut, thus
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improving nutrient utilisation. During this process, negatively charged peptidoglycans

(Marquis and Bender, 1990) may lose their charge, reducing the number of interactions with
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fat micelles, thus benefiting fat absorption.

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Sialic acid has been used as a marker to measure the dynamics of mucin secretions in excreta

in enzyme fed birds. Early work with phytase (Cowieson et al., 2004; Pirgozliev et al., 2011)

showed a reduction in SA secretion due to supplementation, although feeding an enzyme

mixture to broilers (Abdulla et al., 2016, 2017) did not change the concentration of SA

secreted. In the current study, the SA data measured in excreta after 17 d of supplementation

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did not indicate differences due to MUR supplementation. Goodarzi Boroojeni et al. (2019)

did not observe any significant differences in goblet cell numbers at the jejunal and ileal level

after 35 d supplementation with microbial MUR in a diet containing other enzymes (phytase

and xylanase). However, Sais et al. (2019) detected an increase in goblet cell numbers after

36 d of microbial MUR supplementation in a diet without other feed enzymes. This can

probably be explained by direct or indirect changes promoted by MUR in the intestinal

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ecosystem or in the release of bioactive factors. The variability in response may be due to the

sampling region (small intestine or excreta), maturity of the birds, method of analyses or type

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of diet (with or without additives), and further research is needed to explore the mode of

action of this microbial MUR and its role in improving gastrointestinal function.

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Improvements in litter quality and footpad dermatitis contribute to welfare in poultry. The
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current study showed an improvement in FPD when animals were supplemented with

microbial MUR, but there was no impact on litter moisture and NH3 concentration. An
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increase in litter moisture and NH3 are the main predisposing factors for footpad dermatitis in
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broilers (Dawkins et al., 2004), although there was no obvious correlation between the
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improved FPD and the litter parameters. Mirza et al. (2016) reported that good litter scores

(based on physical appearance) were not related to litter NH3 or pH, showing that scoring per
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se is of limited value in terms of lowering FPD incidences in poultry production. This

suggests that dietary MUR may provide better nutrient availability and have a direct positive
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impact on the development of skin of the foot pad in poultry.

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It can be concluded that the exogenous microbial MUR (Balancius TM) used in this study was

effective in improving growth performance and welfare in broilers. This was attributed to

improved dietary nutrient and energy availability. There is a need to study potential

interactions of MUR in combination with other exogenous enzymes, plant extracts and feed

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additives. Strategies to incorporate MUR with other feed ingredients in poultry diets, in order

to improve production and welfare, may increase the profitability of broiler production.

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of Clostridial Necrotic Enteritis in Broiler Chickens.” Avian Diseases 54 (4): 1298-

1300. doi:10.1637/9370-041410-ResNote.1.

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Table 1. Composition and nutritive values of the experimental diets

Ingredients (g/kg) Starter Grower Finisher 1 Finisher 2
Wheat 586.9 680.7 700.9 724.9
Soybean meal (CP 480) 342.7 247.1 228.3 205.8
Soybean oil 36.3 41.9 42.4 43.2
Limestone 12.8 11.3 10.7 9.9
Monocalcium phosphate 9.2 7.5 6.6 5.6
Lysine HCL 2.7 3.3 3.1 2.9
Methionine DL 3.4 3.0 2.8 2.5
L-Threonine 1.3 1.5 1.4 1.3

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Salt 1.9 1.6 1.7 1.7

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Sodium bicarbonate 2.5 1.8 1.7 1.7
Xylanase1 0.0075 0.0075 0.0075 0.0075
Phytase2 0.0100 0.0100 0.0100 0.0100

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Premix (VitMin)3 0.2000 0.2000 0.2000 0.2000
Calculated values

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ME (MJ/kg) 12.70 13.20 13.29 13.40
Crude protein (g/kg) 235 198 190 181
Ether extract (g/kg) 51 57 57 58
Ash (g/kg)
Digestible Lys (g/kg)
53
12.9
45
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11.0
43
10.4
40
9.7
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Digestible Met+Cys (g/kg) 9.5 8.4 8.0 7.6
Ca (g/kg) 10.0 9.0 8.5 8.0
Available P (g/kg) 5.0 4.5 4.3 4.0
Determined values
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DM (g/kg) 904 902 898 898

GE (MJ/kg) 16.59 16.94 17.02 16.95
Crude protein (g/kg) 245 198 200 174
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Ether extract (g/kg) 50 58 56 56

Ash (g/kg) 54 53 47 43
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Xylanase (FXU/kg) 183 185 177 158

Phytase (FYT/kg) 2427 2720 2408 2537
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Ronozyme® WX2000: minimum 2 000 FXU/ g endo-1,4-beta-xylanase; 1 xylanase unit
(FXU) is defined as the amount of enzyme that releases 7.8 μmol of reducing sugar (xylose
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equivalents) from azo-wheat arabinoxylan per minute at pH 6.0 and 50 C

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Ronozyme ® HiPhos 20000GT: minimum 20 000 FYT/ g ; 1 phytase unit (FYT) is defined
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as the amount of enzyme that releases 1 µmol of inorganic phosphate from phytate per
minute under reaction conditions with a phytate concentration of 5.0 mM and pH 5.5 and
temperature 37°C.
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The vitamin and mineral premix contained vitamins and trace elements to meet breeder’s
recommendation (Aviagen Ltd., Edinburgh, UK). The premix provided is as follows (units/kg
diet): retinol 3600 μg, cholecalciferol 125 μg, α- tocopherol 34 mg, menadione 3 mg,
thiamine 2 mg, riboflavin 7 mg, pyridoxine 5 mg, cobalamin 15 μg, nicotinic acid 50 mg,
pantothenic acid 15 mg, folic acid 1 mg, biotin 200 μg, iron 80 mg, copper 10 mg, manganese
100 mg, cobalt 0.5 mg, zinc 80 mg, iodine 1 mg, selenium 0.2 mg and molybdenum 0.5 mg.

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Table 2. Analysed muramidase activity in samples of the experimental diets

Measured activity (LSU(F)/kg
Treatment Inclusion level
(LSU(F)/kg)* Starter Grower Finisher 1 Finisher 2
Control 0 - - - -
Low 25 000 26472 26469 30186 26500
Medium 35 000 31422 39569 38106 45180

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High 45 000 33932 49049 53036 51650

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* One unit of muramidase (LSU(F)) is the amount of enzyme that increases the fluorescence
of a 12.5 µg/ml fluorescein-labelled peptidoglycan suspension by a value that corresponds to

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the fluorescence of 0.077 mM fluorescein isothiocyanate (FITC), per minute at pH 7.5 and
30°C.

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Table 3. Effect of different inclusion levels of muramidase on growth performance of broiler
chickens
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Probability
Treatment SEM P L Q
groups1 Control Low Medium High
Starter period (1 to 10 d old)
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Feed intake 294 293 296 295 2.2 0.800 0.610 0.993
(g/b)
Weight gain 216 222 223 223 2.5 0.173 0.053 0.277
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(g/b)
Feed 0.0248 0.394 0.183 0.349
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conversion
ratio2 1.376 1.325 1.329 1.325
Grower period (10 to 21 d old)
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Feed intake 1142 1134 1136 1139 7.9 0.885 0.850 0.463
(g/b)
Weight gain 933 956 937 935 14.4 0.663 0.809 0.402
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(g/b)
Feed 1.218 1.181 1.224 1.233 0.0317 0.670 0.538 0.469
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conversion
ratio
Finisher period 1 (21 to 42 d old)
Feed intake 2961 2972 2951 2978 16.5 0.682 0.698 0.632
(g/b)
Weight gain 1957a 2008b 2003b 2014b 12.4 0.007 0.004 0.108
(g/b)
Feed 1.492a 1.454b 1.457b 1.451b 0.0067 <0.001 <0.001 0.019
conversion
ratio
Overall period (1 to 42 d old)

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Feed intake 4565 4536 4539 4562 26.3 0.809 0.970 0.331
(g/b)
Weight gain 2834a 2874ab 2910b 2896b 19.1 0.038 0.012 0.164
(g/b)
Feed 0.0069 <0.001 <0.001 0.010
conversion
ratio 1.579a 1.551b 1.538b 1.547b
Liveability 0.797 0.582 0.954 0.243
(%) 96.25 96.67 97.50 96.04
EPEF3 403.0a 417.9ab 430.9b 419.1ab 5.43 0.007 0.014 0.016

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1

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Control: control diet, low (25,000 LSU(F)/kg muramidase), medium (35,000 LSU(F)/kg
muramidase), high (45,000 LSU(F)/kg muramidase). 2 Gram feed intake per gram weight
gain. 3European poultry efficiency factor: averaged grams gained per day × survival rate (%)

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÷ feed conversion ratio × 10. Data are means of 24 replicate pens with 20 birds per pen. P

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value describes significance between treatments determined by ANOVA. Linear (L) and
quadratic (Q) effects of dietary treatment. Results are statistically significant when P<0.05.

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Table 4. Effect of dietary treatment on dry matter (DM), pH and N of litter, litter and footpad
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scores at 35 days of age
Probability
1
Treatment groups Control Low Medium High SEM P L Q
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Dry matter litter 0.0179 0.804 0.539 0.452

(g/kg) 0.667 0.652 0.643 0.654
pH litter 7.35 7.44 7.53 7.46 0.120 0.770 0.451 0.510
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N litter (g/kg) 40.8 41.8 40.5 40.9 0.81 0.693 0.808 0.680
Litter score 3.27 3.14 3.20 3.12 0.065 0.332 0.175 0.726
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Footpad score 26.0 24.0 13.0 15.8 4.45 0.123 0.039 0.590

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Control: control diet, low (25,000 LSU (F)/kg muramidase), medium (35,000 LSU (F)/kg
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muramidase), high (45,000 LSU (F)/kg muramidase). Data are means of 24 replicate pens
with 20 birds per pen. P value describes significance between treatments determined by
ANOVA. Linear (L) and quadratic (Q) effects of dietary treatment. Results are statistically
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significant when P<0.05.

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Table 5. Effect of dietary treatment on N-corrected apparent metabolisable energy (AMEn),

dry matter (DMR), organic matter (OMR), N (NR), fat (FR) retention coefficients and sialic
acid (SA) excretions.
Probabilit
y
Contro Mediu SEM P L Q
Treatment groups1 l Low m High
12.6 13.2 0.162 0.06 0.985 0.00
AMEn (MJ/kg DM) 12.98 2 12.96 6 2 7
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0.68 0.71 0.010 0.10 0.701 0.01

DMR 0.705 1 0.696 7 4 7 6
0.70 0.74 0.009 0.05 0.844 0.00
OMR 0.729 6 0.725 3 5 8 7
0.57 0.61 0.014 0.17 0.889 0.04
NR 0.592 1 0.577 3 0 5 5
0.81 0.84 0.010 0.09 0.050 0.18
FR 0.815 1 0.834 3 4 9 7
0.074 0.42 0.408 0.42
SA (µg/g) 1.88 2.00 1.91 1.96 3 2
0.22 0.10 0.871 0.22

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SA total (µg/24h) 32.8 37.1 31.8 34.0 8 5

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Control: control diet, low (25,000 LSU(F)/kg muramidase), medium (35,000 LSU(F)/kg

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muramidase), high (45,000 LSU(F)/kg muramidase). Data are means of 24 replicate pens
with 2 birds per pen. P value describes significance between treatments determined by

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ANOVA. Linear (L) and quadratic (Q) effects of dietary treatment. Results are statistically
significant when P<0.05.

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