Evaluation of dietary supplementation of a novel microbial muramidase on gastrointestinal functionality and growth performance in broiler chickens Mounira Sais,∗ Ana C. Barroeta,∗ Paola López-Colom,∗ Miquel Nofrarías,† Natàlia Majó,† ,‡ Rual Lopez-Ulibarri,§ Estefanía Pérez Calvo,# and Susana M. Martín-Orúe∗ ,1 ∗ Animal Nutrition and Welfare Service. Animal and Food Science Department, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; † IRTA, Centre de Recerca en Sanitat Animal (CReSA, UAB-IRTA), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; ‡ Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; § DSM Nutritional Products Ltd, Nutrition Innovation Center - ANH, 4303 Kaiseraugst, Switzerland; and # DSM Nutritional Products Ltd, Nutrition Innovation Center - ANH, 68128 Village Neuf, France ABSTRACT This study was conducted to assess the differences in the villus height or crypt depth; but a effect of dietary supplementation of Muramidase 007 to higher number of goblet cells and intraepithelial lym- broiler chickens on gastrointestinal functionality, eval- phocytes at day 36 with MUR. No differences were uating growth performance, apparent ileal digestibility, observed in plate counts of enterobacteria or Lacto- intestinal histomorphology, vitamin A in plasma and bacillus along the gastrointestinal tract, neither on the cecal microbiota. A total of 480 one-day male chicks cecal short-chain fatty acids. An statistical trend was (Ross 308) were distributed in 16 pens allocated in observed for reduction of cecal clostridia at day 9 for 2 experimental diets: the control diet (CTR) without MUR. Analysis of cecal microbiota structure by 16S feed enzymes, coccidiostat or growth promoters, and rRNA gene sequencing revealed relevant changes corre- the experimental diet (MUR): CTR supplemented with lated to age. At day 9, broilers receiving MUR showed 35,000 units (LSU(F))/kg of the Muramidase 007. Di- decreased alpha diversity compared to CTR that was gesta and tissue samples were obtained on days 9 and 36 not detected at day 36. Changes in specific taxonomic of the study. A lower feed conversion ratio was observed groups with an increase in Lactobacillus genus were in the MUR treatment. Apparent ileal digestibility of identified. In conclusion, evaluation of the variables in DM, organic matter and energy were improved by Mu- this study indicates that dietary Muramidase 007 con- ramidase 007. It was also observed that MUR improved tributes to improve feed conversation ratio and gas- digestibility of total fatty acids, mono-unsaturated fatty trointestinal function in broiler chickens. Effects could acids and poly-unsaturated fatty acids, and content have been mediated by slight shifts observed in the of vitamin A in plasma at day 9 (P < 0.05). Histo- intestinal microbiota. More studies are guaranteed to morphological analysis of jejunum samples revealed no fully understand the mechanisms involved. Key words: muramidase, broiler, ileal digestibility, 16S rRNA gene, microbiota 2020 Poultry Science 99:235–245 http://dx.doi.org/10.3382/ps/pez466 INTRODUCTION Kapell et al., 2012). In addition, an effective functional- ity of the gastrointestinal tract (GI) is very important A fast growth rate and high feed efficiency are the in determining animal performance. Optimal GI func- 2 main targets in modern poultry production. For op- tionality has been recently defined as a steady state timum performance of birds, important factors affect- where the microbiome and the intestinal tract (host) ing gastrointestinal health like genetic potential of the exist in symbiotic equilibrium, allowing the mainte- birds, quality of the diets, environmental conditions, nance of key physiological functions resulting in im- and disease outbreaks, should be taken into consid- proved health and welfare and performance (Celi et al., eration (Yegani and Korver, 2008). Actually, in the 2017). Among all components involved in GI functional- modern poultry industry, multi-environment selection ity and health, the composition and metabolic activity is employed by the breeding companies (Neeteson, 2010; of the GI tract microbiota have a significant impact on the health of the host due to their influence on nutri- ent absorption, intestinal physiology, immune response, and consequently, resistance to pathogen colonization Received December 5, 2018. Accepted August 5, 2019. (Apajalahti et al., 1998; Lu et al., 2003; Santos et al., 1 Corresponding author: susana.martin@uab.cat 2008). © 2019 The Authors. Published by Elsevier on behalf of Poultry Science Association Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 235 236 SAIS ET AL. While probiotics, essential oils, organic acids, or pre- conditions strictly conformed to European Union biotics are used to help stabilizing the gut microbiota, Guidelines (Directive 2010/63/EU). little attention has been paid so far on the massive turnover and dead of the bacterial populations resulting in high amounts of bacterial cell wall fragments, con- Muramidase taining peptidoglycans (PGN), that take place in the The novel muramidase used in this study was selected GI tract (Johnson et al., 2013). PGNs are the mayor through a discovery program. The gene coding for this structural components of the cell wall, uniquely found muramidase (denominated Muramidase 007) from the in bacteria, and considered as conserved products of fungus Acremonium alcalophilum (strain 114.92) was bacterial metabolism and activity modulators in the GI expressed in an industrial production host, produced tract (Humann and Lenz, 2009). in enough amounts and characterized as described by Muramidase (EC 3.2.1.17), also known as lysozyme Lichtenberg et al. (2017). This product was produced or N-acetylmuramidase, belongs to the family of glyco- by Novozymes A/S (Bagsvaerd, Denmark). syl hydrolytic enzymes. Muramidase cleaves the β -1,4- glycosidic linkages between N-acetylmuramic acid and N-acetyl glucosamine, the basic elements in the carbo- Location and Housing hydrate backbone of PGN from bacterial cell walls. The Animals were housed in one single room with 16 most studied muramidase is the one abundantly found floor pens (8 pens (1.5 m × 1 m) at each side of in hen egg white, however, many muramidases are nat- the room). The environmental conditions (lighting pro- urally found in a great variety of animal secretions, gram, temperature, relative humidity and ventilation plants, or micro-organisms. Muramidases are ubiqui- rates) were controlled accordingly to the Ross broiler tous and occur naturally in the GI tract of animals management guidelines. Animals disposed of nipple (Sahoo et al., 2012). drinkers (3 drinkers/pen) and manual pan feeders Studies with nursery pig using diets supplemented (1 pan/pen). Litter material was wood shavings. with muramidase (isolated from hen egg white) re- sulted in ameliorated growth performance probably due to improvements on histomorphology of small in- Animals and Diets testine (Oliver and Wells, 2013, 2015). Contributing to better gut integrity as well as stronger nonspe- A total of 480 one-day-old male broiler chickens (Ross cific immune response could also explain the perfor- 308), were obtained from a local hatchery (Pondex mance improvements (Oliver and Wells, 2015; Long SAU, Lleida, Spain), already vaccinated in ovo against et al., 2016). Recently, it was demonstrated that di- Gumboro and Marek diseases. Additionally, chicks were etary supplementation of a novel microbial murami- vaccinated against coccidiosis (coarse spray; Hypracox, dase (Muramidase 007) in broiler diets improved the Amer, Spain) and infectious bronchitis (fine spray) at performance variables, increased the number of lac- day 1 after hatching. The birds were individually wing- tobacilli without disturbing the rest of the func- tagged, weighted and randomly distributed in 16 pens tional microbiota in the caecum of broilers, while (30 chicks per pen). proven safe for the chickens in a tolerance study Each pen was allocated to one of 2 experimental (Lichtenberg et al., 2017). Moreover (Goodarzi Boroo- treatments: A control diet (CTR) or CTR diet sup- jeni et al., 2019) supplementing increasing dosis of this plemented with Muramidase 007 (MUR) at 35,000 mu- muramidase reported improvements in feed conversion ramidase units (LSU(F))/kg. During the experimental ratio (FCR) and apparent ileal digestibility (AID) of period the birds received a feeding program of 2 phases: nutrients. a starter diet from 0 to 21 d (in crumble form) and a The present study aimed to evaluate the impact of grower diet from 21 to 36 d (in pellet form). Diets were dietary supplementation of Muramidase 007 on the GI formulated to meet or exceed FEDNA (2008) require- functionality of broiler chickens by measuring AID of ments, as it figures in Table 1. Basal diets did not con- nutrients, intestinal histomorphology, vitamin A con- tain any feed enzymes (other than Muramidase 007 in tent in plasma, microbial ecosystem and metabolism, the experimental diet), coccidiostats, veterinary antibi- and animal performance variables. otics or any other growth promoters. In addition, tita- nium dioxide (TiO2 ) was added as indigestible marker at 0.5%. MATERIALS AND METHODS Experimental Procedures The in vivo experimental procedure of this study was performed at the Experimental Unit of the Universi- Individual weight of the animals and feed residuals tat Autònoma de Barcelona (UAB) and received prior (by pen) were registered at days 0 (arrival), 9 (first approval from the Animal and Human Experimental sampling), 21 (change of diet), and 36 (final day-second Ethical Committee of this Institution. The treatment, sampling) to monitor individual body weight (BW) as management, housing, husbandry and slaughtering well as the feed intake along the study. From these EVALUATING A DIETARY NOVEL MURAMIDASE IN BROILERS 237 Table 1. Ingredient and nutrient composition of basal diets. kept frozen at -20°C, freeze-dried, grounded, and kept Ingredients (%, as-fed basis) Starter Grower at 5°C until analyzed. Pooled sample of blood was sampled via venipuncture Soybean meal 48 34.97 27.23 (2 or 3 animals on 9 d and individual bird on 36 d Maize/Corn 23.71 31.30 Wheat 20.00 20.00 from each pen) and was taken in heparinized tubes for Rye 12.00 12.00 vitamin A analyses. Soybean oil 4.86 - Dicalcium phosphate 1.79 1.47 Fat 5 FYSFEED1 - 5.54 Analytical Methods Calcium carbonate 0.92 0.80 Sodium chloride 0.40 0.36 AID AID was calculated using the index marker for a Mineral-vitamin premix2 0.30 0.30 DL-methionine 0.27 0.23 100% recovery. Analytical determinations of feeds and L-lysine 0.17 0.19 digesta were performed accordingly to the Association Choline chloride 0.04 - of Official Agricultural Chemists (AOAC) International L-threonine 0.04 0.05 Ethoxyquin 66%3 0.02 0.02 methods (2005): dry matter (Method 934.01) by desic- TiO2 0.50 0.50 cating the sample in an air-forced oven at 103°C for Carophyll Yellow4 0.006 0.006 24 h. Organic matter (OM) content was gravimetri- Calculated nutrients (%, DM basis) cally measured by placing samples in a muffle furnace at Calcium 0.95 0.80 550°C for 4 h, (ash, Method 942.05), crude fat (Method Digestible phosphorus 0.45 0.38 Digestible lysine 1.20 1.02 2003.05 for feed and with previous acid hydrolysis, fol- Digestible methionine + cysteine 0.87 0.76 lowing method 954.02 of the AOAC for excreta) and Digestible threonine 0.76 0.66 gross energy content (IKA-Kalorimeter system C4000, Analyzed nutrients (%, DM basis) CTR5 MUR6 CTR5 MUR6 Staufen, Germany). The fatty acid (FA) content of the Dry Matter 91.49 91.26 90.27 90.35 feed and digestive content was determined according to Crude protein 24.10 23.48 20.07 20.23 Crude fat 6.60 6.36 7.08 7.07 the method of Sukhija and Palmquist (1988). This an- Crude fiber 3.02 3.58 2.87 2.91 alytic procedure consists of a direct transesterification Ash 6.03 6.17 5.56 5.42 (the lipid extraction and FA methylation is achieved in Gross energy (kcal/kg) 4157 4103 4096 4090 only one step). Samples were incubated at 70°C with Muramidase activity (LSU(F)/kg) 1089 33,025 962 28,879 methanolic hydrochloric acid (a mixture of methanol 1 Fat 5 FYSFEED (SYSFEED, Granollers, Spain) is a commercial and acetyl chloride) for the methylation. After the source of animal fat obtained by mechanical pressing after cooking of extraction and methylation potassium carbonate and slaughterhouse by-products coming from animals declared fit from hu- man consumption before and after slaughter. toluene were added to separate the organic layer. Non- 2 One kg premix contains vitamin A (all-trans retinol/retynil adecanoic acid (C19; Sigma-Aldrich Chemical Co., St. acetate/retynil palmitate/β -carotene): 3333,334 IU; vitamin D3 : Louis, MO) was used as internal standard. The final 1600,000 IU; vitamin E (dl-α-tocopheryl acetate/dl-α-tocopherol/d-α- tocopherol): 15,000 IU; vitamin K3 : 1,000 mg; vitamin B1 : 1,000 mg; extract was injected in a gas chromatograph (HP6890, vitamin B2 : 3,000 mg; vitamin B6 : 1,500 mg: vitamin B12 : 13.3 mg; folic Agilent Technologies, Waldbronn, Germany) following acid: 600 mg; biotin: 50 mg; calcium pantothenate: 5,500 mg; nicotinic the method conditions previously described by Corti- acid: 17,000 mg; Mn: 30,000 mg; Zn: 22,000 mg; I: 400 mg; Fe: 18,000 mg; Cu: 4,000 mg; Se: 60 mg; ethoxyquin: 1,333 mg; fumaric acid: 25,0000 mg; nas et al. (2004). FAs were identified by matching their and malic acid: 20,000 mg. retention times with those of their relative standards 3 4 Ethoxyquin antioxidant produced by ITPSA (Barcelona, Spain). (Supelco 37 component FAME Mix, Sigma-Aldrich Co., Carophyl yellow (10% Apo-ester; DSM Nutritional Products Ltd, Kaiseraugst, Switzerland). St. Louis, MO) and quantified by internal normaliza- 5 CTR: Control diet. tion. Nonadecanoic acid was used for the calibration 6 MUR: Muramidase supplemented diet at 35,000 LSU (F)/kg. curves and quantification of FA. Total FAs (TFA) con- tent was calculated as the sum of individual FAs. Ni- values the average daily feed intake (ADFI), average trogen was analyzed by the DUMAS method. Analysis daily weight gain (ADG), and the FCR were calculated. of Ti of the Titanium dioxide (TiO2 ) marker was per- At 9 d of live, 21 birds per cage were randomly se- formed by atomic absorption spectroscopy (AAS). The lected and euthanized by decapitation, and represen- AID of dry matter (DM), OM, nitrogen (N), FA, and tative samples of mid-jejunum tissue, ileal digesta and energy (E) was calculated as follows: blood, were taken. At 35 d of age, the remaining birds    were euthanized, and the same procedure was repeated. AID (%) = 1 − [Ntr]M /[Ntr]D Jejunal tissue was collected from 1 bird per pen for histological analysis. Content from different intestinal ×([Ti]D /[Ti]M )] × 100 segments: crop, ileum and cecum were collected; indi- vidually from 2 birds per pen, 1 for classical micro- Where [Ntr]M is the nutrient content in the ileal di- biology and the other for microbiota (1 cecum) and gesta, [Ntr]D is the nutrient content in the diet, [Ti]M short-chain fatty acids (SCFA) analysis (other ceca). is the concentration of Ti in the ileal digesta, and [Ti]D Moreover, representative sample of terminal ileum con- is the concentration of Ti in the diet. tent of each cage was taken (21 birds at 9 d; 7 birds Vitamin A Plasma proteins were precipitated with at 36 d) for AID analyses. Digesta was homogenized, ethanol. Retinol was extracted from the aqueous 238 SAIS ET AL. suspension with n-hexane/butylated hydroxytoluene Statistical Analysis solution. After centrifugation, an aliquot of the organic phase was dried, re-dissolved in solvent solution and in- The results are expressed as means with their stan- jected into a reversed-phase (C18) HPLC system. The dard errors unless otherwise stated. Data were analyzed detection was performed with a fluorescence detector. with ANOVA using the GLM procedure of SAS and the Classical Microbiology and Jejunal Histomor- following statistical model: phology Colony forming units (cfu) count of enter- obacteria, lactobacilli and clostridia, in the content Yij = μ + Bi + εi of crop, ileum and cecum was performed by selec- tive growth medium. Enterobacteria were counted af- ter 24 to 48 h of incubation in MacConkey agar. Lac- Where Yij is the dependent variable, µ is the overall tic acid bacteria were determined in MRS agar and mean, Bi is the additive effect, and ɛi is the residual clostridia in SPS Agar used for isolation and enumera- error effect. tion of sulphite-reducing clostridia. To evaluate possible When frequencies were analyzed, the Fisher’s exact changes in the microbiota metabolism, the concentra- test was used. For the microbiological data, the plate tion of SCFA was analyzed in cecal samples by gas chro- counts values were log-transformed. All statistical anal- matography following the method of Jouany (1982). yses were performed using the Statistical Analysis Soft- Histological analysis was performed in tissue sam- ware SAS version 9.2 (SAS Institute Inc., Cary, NC). ples from jejunum fixed by immersion in 10% forma- The pen was considered as the experimental unit for all lin, paraffin-embedded, and sections stained with hema- the variables analyzed except for classical microbiology toxylin/eosin. Measurements analyzed included villus and histology. In those analyses, the bird was consid- height (μm), crypt depth (μm), villus: crypt ratio, vil- ered the unit since only 1 bird per pen was sampled. lus width (μm), villus surface (mm2 ), intraepithelial The α level used for the determination of significance lymphocytes (IEL) (number cells/100 μm), goblet cells for all the analysis was P = 0.05. The statistical trend number (GC) (cells/100 μm), and number of mitoses was also considered for P-values > 0.05 and < 0.10. (cells/100 μm). Biostatistics of quality filtered sequences of the 16S Molecular Microbiology DNA from cecal samples rRNA gene were performed using an open source soft- was extracted using the QIAamp DNA Stool Mini Kit ware (R v.3.4.0; R Core Team, 2013) after importing (Qiagen, Toronto, Canada) according to the manufac- the OTU table to R using the phyloseq package. Di- turer’s instructions. The global structure, dynamics and versity and ordination analysis (non-multidimensional functionality of the cecal microbial populations was an- scaling, NMDS) were performed using the vegan pack- alyzed by high throughput sequencing, targeting V3-V4 age at OTU level. Richness and alpha diversity were cal- region of 16S rRNA, using the kit MiSeq® Reagent Kit culated with raw counts while beta diversity and ordi- v2 (500 cycle) (MiSeq, Illumina, San Diego, CA). nation analysis were obtained with relative abundances. Primers used in the construction of libraries were the Alpha diversity consisted of calculation of three related following (generating amplicons of putative 460 bp): indexes: Shannon, Simpson and Simpson inverse. For F-5 -TCGTCGGCAGCGTCAGATGTGTATAAGA beta diversity, betadiver() function based on Whittaker GACAGCCTACGGGNGGCWGCAG index was used. To compare any differential effects from R-5 -GTCTCGTGGGCTCGGAGATGTGTATAAG treatments, an ANOVA analysis was performed for al- AGACAGGACTACHVGGGTATCTAATCC pha and beta diversities. Bray-curtis distance matrix Sequence reads of 16S rRNA gene generated from was calculated from the dissimilarities between pairs MiSeq Illumina® system were processed on QIIME of samples and the relative position of each sample v.1.9.1. The quality filter of already demultiplexed se- was projected and analyzed in the NMDS plot. Finally, quences was performed at a maximum unacceptable read counts were normalized with the metagenomeSeq Phred quality score of Q20. Resulting reads were clus- package, that uses a cumulative-sum scaling where raw tered to operational taxonomic unit (OTU) using uclust counts are divided by the cumulative sum counts into algorithm with 97% sequence similarity and subsam- a particular quantile, by which differential abundance pling pick open reference method (Rideout et al., 2014) analysis under a zero-inflated Gaussian model could at 10% of sequences subsampled. Representative se- be performed. To achieve that, taxa were aggregated quences were assigned to taxonomy against bacterial at Phylum and Genus level and expressed as log10 16S GreenGenes v.13.8 reference database at a 90% (n + 1). confidence threshold and sequence alignment and phy- logenetic tree building were obtained through uclust RESULTS and FastTree. Thereafter, chimeric sequences were re- moved with ChimeraSlayer with default settings and The trial was successfully carried out and animals further quality filtering consisted in removing single- showed good health along the whole study. Mortality tons and OTUs with relative abundance across all sam- was very low along the trial being 0.83% and 1.25% for ples below 0.005% as recommended by Bokulich et al. CTR and MUR, respectively with no difference between (2013). treatments. EVALUATING A DIETARY NOVEL MURAMIDASE IN BROILERS 239 Table 2. Performance of broiler chickens fed a control diet (CTR) modified the villus height or crypt depth, neither the or the same diet supplemented with Muramidase 007 (MUR) villus/crypt ratio. Increases (P < 0.05) were detected during the starter and grower periods. with the MUR treatment at day 36 in IEL and GC, Treatments1 regardless if they were expressed in absolute or relative Variables CTR2 MUR3 SEM P-value terms. Regarding possible changes induced by the experi- BW (g) mental diets on cecal profile and concentration of to- 0d 42 42 0.1 0.438 9d 259 259 2.7 1.000 tal SCFA, no statistically significant differences among 21 d 1005 1030 11.1 0.134 treatments on the profile, concentration of SCFA or 36 d 2787 2844 27.9 0.172 their molar percentage at any of the sampling days were ADG (g/d) detected (data not shown). 0 to 21 d 45.7 47.1 0.61 0.137 21 to 36 d 118.8 119.4 1.60 0.820 Regarding possible effects on microbiota, neither lac- 0 to 36 d 76.2 77.1 0.75 0.415 tobacilli nor enterobacteria (log cfu/g fresh matter) ADFI (g/d) were modified by the experimental treatments in any 0 to 21 d 63.9 62.1 1.09 0.277 gastrointestinal section and any sampling day (Table 5). 21 to 36 d 172.0 165.3 3.22 0.162 0 to 36 d 108.9 105.1 1.34 0.063 For clostridia, as plate counts were in some animals be- FCR4 (g feed:g BW gain) low detection level of the method, a frequency anal- 0 to 21 d 1.40 1.32 0.04 0.128 ysis was performed (Table 6). By this mean, it could 21 to 36 d 1.45 1.39 0.03 0.207 be seen that MUR tended to decrease the number of 0 to 36 d 1.43 1.36 0.02 0.045 animals with detectable clostridia in cecum at day 9 1 Data are means for 8 replicate pens with 30 birds from days 0 to 9 (P = 0.060). and 9 birds from days 9 to 36. The analysis of the cecal microbiota profile by se- 2 CTR: Control diet. 3 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. quencing of the V3-V4 region of the 16S rRNA gene 4 Feed Conversion Ratio. revealed relevant structural changes related to the de- velopment of the cecal microbiota with age. Figure 1 Table 2 shows the evolution of BW, ADG, ADFI, shows the community structure based on Bray-Curtis and FCR along the study. There were no differences distance matrix with OTUs. A clear impact of the age between treatments in the BW or in the ADG although was observed on cecal microbiota structure being clus- final BW showed numerical values (P = 0.172) with the tered closer microbial structures of day 9 than those addition of MUR. Moreover, animals receiving MUR in of day 36, coincident with numerical higher beta di- the diet showed higher ADFI (P = 0.060) and higher versity value for the later (data not shown). Interest- FCR (P = 0.050) when all the experimental period was ingly, at day 36 a group of animals from both treat- considered. ments clustered separately to the rest (4 from CTR The AID (%) of E, DM, OM, N, TFA, saturated fatty treatment and 6 from MUR treatment) with a pro- acids (SFA), mono-unsaturated fatty acids, (MUFA), file closer to those animals of day 9, suggesting that and poly-unsaturated fatty acids (PUFA) estimated us- these animals had not evolved to a mature ecosystem. ing TiO2 as digestibility marker are shown in Table 3. Nonetheless, animals of day 9 presented higher rich- The inclusion of MUR in the diets increased the di- ness range (600 to 1000 OTU) than those from day gestibility of E at day 36 (P < 0.001) and shown nu- 36 (250 to 800 OTU), despite similar number of reads merical higher values at day 9 (P = 0.120). The in- between both ages. Phyla distribution was similar be- creased amount of digested energy could be the result tween ages, being Firmicutes (60 and 55% for 9 and of the observed increase in the DM and OM digestibil- 36 d, respectively) and Bacteroidetes (26 and 35% for 9 ity at day 36 (more than 4 percent; P < 0.001). Di- and 36 d, respectively) the main phyla registered. When gestibility of N showed an increase of more than 2% analyzing differences in relative abundance of genera at day 36, although differences did not reach statisti- (Tables 7 and 8), it was registered a relevant increase in cal significance (P = 0.095). Digestibility of TFA was the percentage of Bacteroides with age (from 7 to 29%). higher with MUR but the effects were only evidenced Regarding to changes promoted by experimental diets, at day 9 (P = 0.049). This increase in the digestibil- animals receiving MUR showed a decrease in alpha di- ity with the inclusion of MUR in the diets was seen versity (Simpson Index (P = 0.052); Shannon index in all studied fractions: SFA (P = 0.08), MUFA and (P = 0.091); Simpson inverse index (P = 0.001)) but no PUFA. differences were detected in beta diversity. At day 36 no Regarding plasma vitamin A concentration, it was changes were detected in alpha or beta diversity. When found an increase in total retinol at day 9 with the sup- differences in abundance of taxa promoted by diets were plementation of MUR (MUR: 615.6 ng/mL vs. CTR: analyzed, no changes were seen in phyla at day 9, nor 521.4 ng/mL; P = 0.040). At day 36, both diets showed at day 36. Only minor changes were found in genera higher values, without differences (CTR: 775.5 ng/mL at day 36 (Table 8) with an increase in Lactobacillus and MUR: 740.9 ng/mL; P = 0.556). (3.28 ± 2.319% vs. 4.90 ± 1.087%) and cc-115 (0.01 Histomorphological observations in jejunum are ± 0.003% vs 0.15 ± 0.046%) genera when analyzed as shown in Table 4. None of the experimental diets normalized counts (P < 0.05). 240 SAIS ET AL. Table 3. Apparent ileal digestibility (%) of energy and nutrients in broiler chickens fed a control diet (CTR) or the same diet supplemented with Muramidase 007 (MUR) during the starter and grower periods. Treatments2 Apparent ileal digest. (%)1 Day CTR3 MUR4 SEM P-value Energy 9 71.2 72.4 0.52 0.123 36 71.8 75.6 0.50 <0.001 Dry Matter 9 68.4 69.6 0.45 0.085 36 68.1 72.3 0.49 <0.001 Organic Matter 9 69.7 70.9 0.44 0.077 36 69.8 74.0 0.47 <0.001 Nitrogen 9 82.2 82.9 0.40 0.264 36 75.2 77.5 0.91 0.095 Total fatty acids (TFA) 9 86.5 89.1 0.85 0.049 36 88.9 90.3 0.98 0.324 Saturated fatty acids (SFA) 9 61.5 67.6 2.29 0.080 36 83.9 85.1 1.37 0.530 Mono-unsaturated fatty acids (MUFA) 9 82.4 86.3 1.29 0.048 36 91.3 92.5 0.96 0.407 Poly-unsaturated fatty acids (PUFA) 9 95.6 96.7 0.26 0.012 36 90.6 92.2 0.85 0.208 1 Apparent ileal digestibility was estimated using TiO2 as digestibility marker. 2 Data are means for 8 replicate pens with 19 pooled birds from days 9 and 7 pooled birds from day 36. 3 CTR: Control diet. 4 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. Table 4. Histomorphometry of jejunum in broiler chickens fed a Table 5. Plate counts (log cfu/g fresh matter) for different micro- control diet (CTR) or the same diet supplemented with Murami- bial groups in crop, ileum, and cecum digesta in broiler chickens dase 007 (MUR) during the starter and grower periods. fed a control diet (CTR) or the same diet supplemented with Muramidase 007 (MUR) during the starter and grower periods. Treatment1 Treatment1 Variables Day CTR2 MUR3 SEM P-value Section Day Microbial group CTR2 MUR3 SEM P-value Villus height (µm) 9 442.3 444.1 15.60 0.936 36 1027.8 996.3 38.50 0.572 Crop 9 Lactobacillus 8.1 7.9 0.75 0.799 Crypt depth (µm) 9 147.5 157.6 6.67 0.303 Enterobacteria 6.5 6.9 1.08 0.810 36 185.3 170.5 6.08 0.108 36 Lactobacillus 7.5 7.6 0.35 0.805 Enterobacteria 4.5 5.3 0.37 0.176 Villus:crypt ratio (µm: µm) 9 3.1 2.9 0.13 0.288 36 5.7 6.1 0.23 0.258 Ileum 9 Lactobacillus 8.1 8.3 0.60 0.885 4 Enterobacteria 5.3 5.8 0.75 0.645 IEL (cells) 9 15.4 18.0 2.59 0.495 36 Lactobacillus 9.5 8.8 0.40 0.201 36 46.0 59.6 2.49 0.002 Enterobacteria 7.1 6.4 0.49 0.296 5 GC (cells) 9 22.1 23.6 1.61 0.500 Cecum 9 Lactobacillus 11.0 9.9 0.62 0.219 36 57.6 72.7 2.96 0.003 Enterobacteria 11.3 11.4 0.37 0.815 1 n = 8 animals. 36 Lactobacillus 10.5 11.3 0.42 0.224 2 CTR: Control diet. Enterobacteria 11.3 11.1 0.38 0.821 3 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. 1 4 n = 8 animals. IEL: intraepithelial lymphocytes. 2 5 CTR: Control diet. GC: goblet cells regardless they were expressed in absolute or relative 3 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. terms. DISCUSSION els. Other studies have also reported similar effects on Effect on Growth Performance and AID growth performance when other muramidases, from dif- ferent origins, were included on broiler diets. Humphrey In the present study, the inclusion of the Murami- et al. (2002) found that chickens fed a conventional diet dase 007 in broiler chicken diets, throughout the whole containing 10% modified rice expressing lysozyme had growing period, had a relevant impact on their FCR at an improved feed efficiency (P = 0.01). Liu et al. (2010) the end of the trial. FCR improved from 1.43 to 1.36 g reported that supplemental hen egg white lysozyme feed: g BW gain (P = 0.045) at day 36. These data (HEWL) significantly improved FCR and tended to in- agree with those obtained by Lichtenberg et al. (2017) crease weight gain (P = 0.082). Abdel-Latif et al. (2017) and Goodarzi Boroojeni et al. (2019), who reported a also reported that BW gain and FCR were significantly significant improvement in BW gain and FCR when improved when HEWL was included in poultry diets Muramidase 007 was included at different dose lev- (P < 0.05). However, Gong et al. (2016) reported no EVALUATING A DIETARY NOVEL MURAMIDASE IN BROILERS 241 Table 6. Number of animals with detectable number of clostridia effect found on growth variables by the inclusion of along the gastrointestinal tract (minimum level of detection 10 HEWL on broiler diets (P > 0.05). cfu/g). Broiler chickens were fed a control diet (CTR) or the same diet supplemented with Muramidase 007 (MUR) during This improvement observed in FCR is consistent with the starter and grower periods. the improvement reported in ileal apparent digestibil- ity of energy, DM, and OM that also was reported by Treatment1 Goodarzi Boroojeni et al. (2019) for this muramidase. Clostridium (+) Day CTR2 MUR3 P-value It can be hypothesized that an improved digestibility Crop 9 4/8 (4.4) 5/8 (5.3) 0.343 of nutrients could be due to a better intestinal absorp- 36 4/8 (4.4) 6/8 (6.2) 0.245 tion (Noy and Skylan, 1995). However, attending to the Ileum 9 7/8 (7.1) 3/8 (3.5) 0.056 histomorphological effect of the diets, we were not able 36 6/8 (6.2) 6/8 (6.2) 0.431 to show changes in the height of jejunal villi or crypt Cecum 9 7/8 (7.1) 5/8 (5.3) 0.246 depth despite Goodarzi Boroojeni et al. (2019) were 36 5/8 (5.3) 4/8 (4.4) 0.343 able to find an increase in ileal villus length to crypt 1 2 n = 8 animals. depth ratio with this same muramidase. However, at CTR: Control diet. 3 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. the end of the study, we observed an increment on the number of GC in the jejunum of chickens fed the MUR diet. Mucus constitutes a protective barrier against Figure 1. Community structure based on Bray-Curtis distance matrix. Non-metric multidimensional scaling (NMDS) of operational taxonomic unit (OTU) relative abundances in broiler chickens fed a control diet (CTR) or the same diet supplemented with Muramidase 007 (MUR) during the starter and grower periods. Data are clustered by the age of the animal (sampling days at 9 and 36 d of life) and the experimental diets (CTR diet and MUR diet). Black: CTR day 9; blue: MUR day 9; white; CTR day 36; and green: MUR day 36. 242 SAIS ET AL. Table 7. Microbiota composition at the genus level for the 15 colonization offering attachment site and carbon source most abundant genera in cecal digesta of broilers of 9 d of live for resident bacteria. Both, bacteria and the host can in- receiving control diet (CTR) or the same diet supplemented with Muramidase (MUR). Results are expressed as relative abundance fluence the composition for the mucus layer, and mucus (% ± SE) by diet. No statistical differences were found between can turn in a way to the host to modulate its indigenous experimental diets. microbiota although mucus-derived components could also offer cues for adaptation and pathogenesis of some Treatment1 bacteria (Sicard et al., 2017). Some authors also have 2 Genus CTR MUR3 described stimulation of mucin productions by molec- Bacteroides 7.45 ± 0.601 5.89 ± 0.572 ular patterns such as lipopolysaccharide (Petersson et Ruminococcus 3.98 ± 0.465 7.48 ± 2.114 al., 2011) relating mucin production and inflammation. Oscillospira 4.13 ± 0.420 4.82 ± 0.491 Prevotella 4.34 ± 0.350 3.35 ± 0.340 Taking all this in consideration is therefore simplistic Lactobacillus 3.28 ± 1.267 2.88 ± 0.887 to draw conclusions just from an observed increase in Parabacteroides 3.23 ± 0.266 2.58 ± 0.259 GC. Akkermansia 1.99 ± 0.974 1.07 ± 0.197 Ruminococcus 1.54 ± 0.207 1.19 ± 0.151 Our results show that inclusion of the Muramidase Coprococcus 1.44 ± 0.172 1.94 ± 0.608 007 increased ileal apparent digestibility of fat (TFA, Anaerotruncus 0.58 ± 0.206 0.60 ± 0.236 SFA (P = 0.08), MUFA and PUFA), particularly at Enhydrobacter 0.72 ± 0.071 0.59 ± 0.075 Clostridium 0.40 ± 0.173 0.30 ± 0.063 day 9 suggesting an early development of intestinal ca- Bifidobacterium 0.72 ± 0.067 0.51 ± 0.061 pacity to digest fat nutrient. Accordingly, plasma levels Dorea 0.45 ± 0.101 0.41 ± 0.094 of vitamin A also were increased at day 9 with Mu- Actinobacillus 0.57 ± 0.058 0.45 ± 0.057 ramidase 007, being another indication of a better di- 1 n = 21 for CTR, and n = 24 for MUR. gestion and absorption of fats. In poultry, the jejunum 2 CTR: Control diet. is the major site of digestion and absorption of fat, with 3 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. the digestion continuing in the upper ileum (Ravindran Table 8. Microbiota composition at the genus level for the 15 et al., 2016). It has been proposed that he immature most abundant genera in cecal digesta of broilers of 36 d of live digestive system of young chickens could lead to the receiving control diet (CTR) or the same diet supplemented with misuse of nutrients (Kaczmarek et al., 2014) and par- Muramidase 007 (MUR). Results are expressed as relative abun- ticularly at first ages when chickens show an impaired dance (% ± SE) by diet. digestion of fat. According to Kussaibati et al. (1982), Treatment1 the intestinal microbiota would be responsible for the 2 decreased apparent fecal digestibility of vegetable lipids Genus CTR MUR3 in chickens younger than 3 wk. This reduction in fat Bacteroides 32.67 ± 2.253 24.41 ± 1.801 digestibility would be the result of the deconjugation Oscillospira 4.21 ± 0.409 4.90 ± 0.316 Lactobacillus 3.28b ± 2.319 4.90a ± 1.087 of the bile salts by certain bacterial species, in partic- Akkermansia 3.83 ± 0.714 2.08 ± 0.370 ular lactobacilli. Since the conjugated bile salts serve Sutterella 2.22 ± 0.290 1.96 ± 0.323 for the formation of micelles, their low concentration Ruminococcus 1.46 ± 0.210 2.64 ± 0.396 Megamonas 0.92 ± 0.574 0.69 ± 0.261 would reduce lipid solubilization and thus their absorp- Ruminococcus 1.28 ± 0.126 1.26 ± 0.096 tion, in particular those containing long chain satu- Prevotella 0.89 ± 0.332 1.47 ± 0.430 rated fatty acids (Gabriel et al., 2005). In this regard, Coprococcus 0.64 ± 0.113 1.67 ± 0.408 Parabacteroides 0.65 ± 0.242 0.99 ± 0.275 the analysis of the cecal structure by the 16S rRNA Anaerotruncus 0.73 ± 0.261 0.41 ± 0.116 gene sequencing revealed important structural changes Blautia 0.18 ± 0.026 0.44 ± 0.162 in the microbiota between days 9 and 36 with signifi- Bifidobacterium 0.15 ± 0.060 0.21 ± 0.061 Dorea 0.20 ± 0.035 0.16 ± 0.018 cant increases with age in the members of Bacteroides genus. 1 n = 24 for CTR and n = 25 for MUR. 2 CTR: Control diet. 3 MUR: Muramidase supplemented at 35,000 LSU (F)/kg. a,b Means within a row lacking a common superscript differ (P < 0.05). Effects on Intestinal Architecture and Immune Response microorganisms, physical and chemical attacks, and has the role of lubrication of the digestive tract (Deplancke As we have seen before, improvements in AID of nu- and Gaskins, 2001) it is considered as a major compo- trients were not related to changes in intestinal his- nent of innate immunity. We could therefore hypoth- tomorphometry in terms of villus: crypt ratio. How- esize that the increase observed in GC, and presum- ever, significant changes in the number of GC and IEL ably in the mucus layer density, could have facilitated were found with supplementation of Muramidase 007 a better nutrient digestion and absorption, explaining at day 36 that could reflect an intestinal response con- the improved efficiency of feed utilization. However it ditioned to differential luminal stimuli. Changes in the is truth that the role of intestinal mucus is complex microbiota, or an increase in PGNs from a higher bac- and still unknown, and that a higher numbers of goblet terial turnover, or other metabolites, could be behind cells not necessarily means a higher amount of mucus. these effects. Lichtenberg et al. (2017) showed higher Mucus layer is known to provide a niche for bacterial number of Lactobacillus in the ceca of the broilers EVALUATING A DIETARY NOVEL MURAMIDASE IN BROILERS 243 supplemented with Muramidase 007 compared to the Whereas variation between individuals has been consid- un-supplemented broilers. In the present study, we were ered to decrease with the age of the animals (Crhanova unable to demonstrate quantitative changes in lacto- et al., 2011), in the present study we found an increase bacilli or enterobacteria by plate counts among treat- of beta diversity with the age of animals accordingly to ments. However, analysis by 16S rRNA gene sequenc- what has been described by other authors (Ballou et al., ing showed a decrease in alpha diversity at day 9 and 2016). Nevertheless, dynamics and structure of intesti- also an increase in the relative abundance of sequences nal microbiota is not only affected by age but also by from the Lactobacillus genus supplementing Murami- other factors like diet, housing, litter, or climate (Kers dase 007. Therefore, it cannot be discarded that some et al., 2018). changes in minor microbial groups or in microbiota Including exogenous muramidase into chicken diets, structure could have induced those changes in the local depending on dose and specificity, could have an im- immune response. pact on the intestinal microbiota considering that in the Changes promoted in IEL numbers were quantita- gut, natural endogenous muramidase is part of the pri- tively low, being within the physiological range and mary protective factors in epithelial secretions (Harder could reflect an activation of the intestinal mucosal et al., 2007; Dave et al., 2016). HEWL is more effec- immune response of the animals without inducing tive against gram positive bacteria, but also has been inflammation. IEL are considered part of the gut- demonstrated to kill gram negative bacteria in vitro associated lymphoid tissue and provide a first line (Masschalck and Michiels, 2003). In vitro experiments of defense against intestinal microbial invasion. These with transgenic human lysozyme have shown activ- frontline lymphocytes residing within the epithelial ity against several gastrointestinal pathogens, including layer are incredibly heterogeneous regarding their func- Clostridium perfringens (Brundige et al., 2008). Liu et tion and phenotype. IEL subsets can provide immune al. (2010) showed that the administration of HEWL surveillance at the epithelial barrier to prevent or im- in broiler diets reduced C. perfringens, Escherichia coli pair infection in the intestine either through innate- and lactobacilli populations in ileal digesta of birds with like mechanisms or as antigen-specific effectors or and/or without and C. perfringens challenge. In the memory CD8αβ+ T cells. However, we are only re- current study, the plate count results for Lactobacillus cently starting to unravel the complex and sophis- and enterobacteria did not show a significant difference ticated network of mucosal immune responses and with the inclusion of the microbial muramidase, in any, function and regulation of these cells (Sheridan and crop, ileum or cecum samples. However, a statistical Lefrançois, 2011). trend for a decrease was observed for Clostridium in Similarly, goblet cells contribute to the protection of the ileum (P = 0.06) at day 9, suggesting a possible the intestinal epithelium by the production and main- impact of the additive in the microbiota structure at tenance of the protective mucus blanket by synthesiz- early age, reducing the growth of this particular mi- ing and secreting high-molecular-weight glycoproteins crobial group. Moreover, the 16 rRNA gene sequencing known as mucins. Changes in GC functions and in the also was able to show that at day 9 animals receiving chemical composition of intestinal mucus have been de- Muramidase 007 showed lower alpha diversity although scribed in response to a broad range of luminal insults, we were not able to detect changes in particular taxo- including alterations of the normal microbiota (Mon- nomic groups. Nevertheless, such variations were small tagne et al., 2004; Sicard et al., 2017). Available data and could be the indirect consequence of the modifi- indicate that intestinal microbes may affect GC dy- cation of the microenvironment in the GI tract by the namics and the mucus layer directly via the local re- hydrolysis products of the muramidase activity or by lease of bioactive factors or indirectly via activation indirect effects associated to the increased digestibility of host immune cells (Deplancke and Gaskins, 2001). of nutrients. Changes promoted by Muramidase 007, directly or in- Differences between results of these studies could be directly, in intestinal ecosystem or in the release of explained by the different types (origin) of the murami- bioactive factors could therefore be behind the observed dases evaluated: HEWL, microbial, etc., with different effects. properties in vitro and in vivo. In summary, results of this study demonstrate the positive effects of the inclusion of Muramidase 007 Effects on Gastrointestinal Microbial in the diet of broiler chickens resulting in increased groups and Cecal Fermentation feed efficiency and improved FCR. This improvement can be explained by a significant improvement in Firmicutes and Bacteroidetes were the two main bac- the ileal digestibility of energy and nutrients. Effects terial phyla in the cecum of broilers representing more on cecal microbiota and their metabolisms seems to than 80% of total sequencing. These values are in ac- be minimal, despite this some changes could be de- cordance to values described by other authors using 16S tected in particular microbial genera, like an increase rRNA gene sequencing methodology (Kers et al., 2018). in Lactobacillus. 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