A microbial muramidase improves growth performance and reduces inflammatory cell infiltration in the intestine of broilers chickens under Eimeria and Clostridium perfringens challenge.

The objective of the present studies was to evaluate muramidase (MUR) supplementation in broilers under Eimeria and/or Clostridium perfringens challenge. For this, 2 experiments were conducted. Experiment 1. A total of 256 one-day old male Cobb 500 chicks were placed in battery cages in a completely randomized design, with 5 treatment groups, 7 replicate cages per treatment and 8 birds per cage. The treatments were: nonchallenged control (NC), challenged control (CC), CC + MUR at 25,000 or 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC). Challenge consisted of 15× the recommended dose of coccidiosis vaccine at placement, and Clostridium perfringens (108 CFU/bird) inoculation at 10, 11, and 12 d. Macro and microscopic evaluation, immunohistochemistry, and gene expression were evaluated at 7, 14, 21, and 28 d of age. Experiment 2. A total of 1,120 one-day old male Cobb 500 chicks were placed in floor pens with fresh litter in a completely randomized design, with 4 treatment groups, 8 replicate pens per treatment, and 35 birds per pen. The treatments were: Control, supplementation of MUR at 25,000 or 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin). At 10, 11, and 12 d of the experiment all the birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (0.5 mL containing 106 CFU/bird). It was observed that in Experiment 1 MUR supplementation reduced the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds, downregulated IL-8 and upregulated IL-10 expression. In Experiment 2, MUR linearly improved the growth performance of the birds, increased breast meat yield, and improved absorption capacity. MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model that may explain the improved growth performance of supplemented birds.

Effect of microbial muramidase supplementation in diets formulated with different fiber profiles for broiler chickens raised under various coccidiosis management programs.

The objective of the present study was to determine the effects of muramidase (MUR) supplemented to diets formulated with different fiber sources (inert or fermentable) on the growth performance and intestinal parameters of broiler chickens raised under different coccidiosis management programs. A total of 2,208 male Ross 308 broilers were housed in 96 floor pens and distributed into a 2 × 3 × 2 factorial arrangement in a completely randomized block design with 2 sources of fiber (inert or fermentable fiber), 3 coccidiosis management programs (none, vaccine, or Salinomycin), and with or without supplementation of MUR at 35,000 LSU(F)/kg of diet. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were calculated for each feeding phase (d 0-14, d 14-28, d 28-36) and from d 0 to 36. On d 17 and d 31, samples were taken to analyze several parameters. The experimental data were analyzed with 3-way ANOVA considering the main effect of fiber source, coccidiosis program, inclusion of MUR, and their interactions using JMP 16.2. 16S rDNA sequencing of the ileal and cecal content was carried out to analyze the diversity, composition, and predictive function of the microbiota. From d 0 to 36, BWG increased (P = 0.05) by 2.5% in birds supplemented with Salinomycin (P = 0.04), and by 2.2% with MUR supplementation (P = 0.02). Salinomycin and MUR improved FCR (P < 0.0001) when compared to nonsupplemented birds. The supplementation of MUR, regardless of the coccidiosis management program, reduced the intestinal viscosity (P = 0.03). On d 31, the highest blood concentration of carotenoids was observed in chickens fed diets supplemented with Salinomycin. MUR led to significant changes in the diversity, composition, and predictive function of the ileal microbiota, mainly on d 31. The results observed herein further explain the positive effects of MUR on the growth performance of broiler chickens.

Effect of benzoic acid with or without a Bacillus-based direct-fed microbial on the performance and carcass merit of grow-finish pigs.

Two hundred and forty barrows and gilts (DNA 600 × 241, DNA Genetics, Columbus, NE) with an initial body weight (BW) of 35.5 ± 4.2 kg were sorted into split-sex pens, blocked by initial body weight, and randomly allocated to one of three dietary treatments with eight pigs per pen and ten pens per treatment. Dietary treatments included a standard diet (CON), CON plus 0.3% benzoic acid (BA; VevoVitall, DSM Nutritional Products, Parsippany, NJ), and CON plus 0.3% BA and 0.025% Bacillus-based direct-fed microbial (BA+DFM; PureGro, DSM Nutritional Products, Parsippany, NJ). The experimental diets were fed in four feeding phases. Pigs were weighed and feed intake measured at the beginning and end of each phase for the calculation of average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (G:F). In addition, ultra-sound was utilized at the conclusion of the trial on day 81 for measurements of backfat and loin eye area. Data were analyzed as repeated measures in SAS 9.4 (SAS Institute, Cary, NC) with fixed effects of treatment, phase, sex, and block included in the model. Pen was the experimental unit, and results were considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10. Overall, pigs fed BA had increased ADFI compared to pigs fed CON (2.88 vs. 2.75 kg, P = 0.015), while pigs fed BA + DFM had similar ADFI compared to pigs fed CON or BA (P ≥ 0.279). There was a tendency for an effect of dietary treatment on ADG (P = 0.063), where pigs fed BA tended to grow faster than pigs fed CON (1.11 vs. 1.08 kg, P = 0.051); however, there were no differences in feed efficiency between treatments (P = 0.450). Additionally, there was no evidence of an effect of dietary treatment on pig BF or LEA (P ≥ 0.334). In conclusion, supplementing 0.3% benzoic acid to grow-finish pigs stimulated feed intake, but did not affect efficiency, or carcass merit.

Evaluation of a Novel Precision Biotic on Enterohepatic Health Markers and Growth Performance of Broiler Chickens under Enteric Challenge.

This study evaluated the supplementation of a precision biotic (PB) on the enterohepatic health markers and growth performance of broiler chickens undergoing an enteric challenge. In the first study, three treatments were used: Unchallenged Control (UC); Challenged Control (CC; dietary challenge and 10× dose of coccidia vaccine); and a challenged group supplemented with PB (1.3 kg/ton). In the second study, three treatments were used: control diet, diet supplemented with Avilamycin (10 ppm), and a diet supplemented with PB (0.9 kg/ton). All the birds were exposed to natural challenge composed by dietary formulation and reused litter from a coccidiosis positive flock. In Trial 1, PB decreased ileal histological damage, increased villi length, and the expression of SLC5A8 in ileal tissue versus CC; it reduced ileal expression of IL-1ß compared to both UC and CC treatments. PB increased the expression of cell cycling gene markers CCNA2 and CDK2 in the ileum compared to CC. In Trial 2, PB improved the growth performance, intestinal lesion scores and intestinal morphology of broiler chickens. These results indicate that birds supplemented with PB are more resilient to enteric challenges, probably by its action in modulating microbiome metabolic pathways related to nitrogen metabolism and protein utilization.

A multi-omics approach to elucidate the mechanisms of action of a dietary muramidase administered to broiler chickens.

A novel dietary muramidase has been shown to have positive effects on broiler chickens. However, very little is known about its mechanisms of action. The present multi-omics investigation sought to address this knowledge gap. A total of 2,340 day-old male broilers were assigned to 3 groups (12 replicates each) fed, from 0 to 42 d, a basal diet (control group-CON) or the basal diet supplemented with muramidase at 25,000 (low-dose group-MUL) or 45,000 LSU(F)/kg feed (high-dose group-MUH). MUH significantly outperformed CON in terms of cumulative feed intake (4,798 vs 4,705 g), body weight (2,906 vs 2,775 g), and feed conversion ratio (1.686 vs 1.729), while MUL exhibited intermediate performance. At caecal level, MUH showed the lowest alpha diversity, a significantly different beta diversity, a reduction in Firmicutes, and a rise in Bacteroidetes, especially compared with MUL. MUH also exhibited a considerable decrease in Clostridiaceae and an overrepresentation of Bacteroidaceae and Lactobacillaceae. At blood level, MUH had lower hypoxanthine-probably due to its drop at caecal level-histidine, and uracil, while greater pyruvate, 2-oxoglutarate, and glucose. This study sheds light on the mode of action of this muramidase and lays the groundwork for future investigations on its effects on the intestinal ecosystem and systemic metabolism of broiler chickens.

Effect of dietary crude protein level on growth performance, blood characteristics, and indicators of intestinal health in weanling pigs.

An experiment was conducted to test the hypothesis that reducing crude protein (CP) in starter diets for pigs reduces post-weaning diarrhea and improves intestinal health. In total, 180 weanling pigs were allotted to 3 diets containing 22, 19, or 16% CP. Fecal scores were visually assessed every other day. Blood samples were collected from 1 pig per pen on days 1, 6, 13, 20, and 27, and 1 pig per pen was euthanized on day 12. Results indicated that reducing dietary CP reduced (P < 0.01) overall average daily gain, gain to feed ratio, final body weight, and fecal scores of pigs. Pigs fed the 16% CP diet had reduced (P < 0.01) serum albumin compared with pigs fed other diets. Blood urea nitrogen, haptoglobin, interleukin-1ß, and interleukin-6 concentrations in serum were greatest (P < 0.01) on day 13, whereas tumor necrosis factor-α and interleukin-10 concentrations were greatest (P < 0.01) on day 6. Villus height in the jejunum increased (P < 0.05) and crypt depth in the ileum was reduced (P < 0.01) if the 19% CP diet was fed to pigs compared with the 22% CP diet. A reduction (P < 0.05) in mRNA abundance of interferon-γ, chemokine ligand 10, occludin, trefoil factor-2, trefoil factor-3, and mucin 2 was observed when pigs were fed diets with 16% CP. In conclusion, reducing CP in diets for weanling pigs reduces fecal score and expression of genes associated with inflammation.

Dietary Oxidative Distress: A Review of Nutritional Challenges as Models for Poultry, Swine and Fish.

The redox system is essential for maintaining cellular homeostasis. When redox homeostasis is disrupted through an increase of reactive oxygen species or a decrease of antioxidants, oxidative distress occurs resulting in multiple tissue and systemic responses and damage. Poultry, swine and fish, raised in commercial conditions, are exposed to different stressors that can affect their productivity. Some dietary stressors can generate oxidative distress and alter the health status and subsequent productive performance of commercial farm animals. For several years, researchers used different dietary stressors to describe the multiple and detrimental effects of oxidative distress in animals. Some of these dietary challenge models, including oxidized fats and oils, exposure to excess heavy metals, soybean meal, protein or amino acids, and feeding diets contaminated with mycotoxins are discussed in this review. A better understanding of the oxidative distress mechanisms associated with dietary stressors allows for improved understanding and evaluation of feed additives as mitigators of oxidative distress.

A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens.

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Dietary muramidase degrades bacterial peptidoglycan to NOD-activating muramyl dipeptides and reduces duodenal inflammation in broiler chickens.

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

Evaluation of dietary supplementation of a novel microbial muramidase on gastrointestinal functionality and growth performance in broiler chickens.

This study was conducted to assess the effect of dietary supplementation of Muramidase 007 to broiler chickens on gastrointestinal functionality, evaluating growth performance, apparent ileal digestibility, intestinal histomorphology, vitamin A in plasma and cecal microbiota. A total of 480 one-day male chicks (Ross 308) were distributed in 16 pens allocated in 2 experimental diets: the control diet (CTR) without feed enzymes, coccidiostat or growth promoters, and the experimental diet (MUR): CTR supplemented with 35,000 units (LSU(F))/kg of the Muramidase 007. Digesta and tissue samples were obtained on days 9 and 36 of the study. A lower feed conversion ratio was observed in the MUR treatment. Apparent ileal digestibility of DM, organic matter and energy were improved by Muramidase 007. It was also observed that MUR improved digestibility of total fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids, and content of vitamin A in plasma at day 9 (P < 0.05). Histomorphological analysis of jejunum samples revealed no differences in the villus height or crypt depth; but a higher number of goblet cells and intraepithelial lymphocytes at day 36 with MUR. No differences were observed in plate counts of enterobacteria or Lactobacillus along the gastrointestinal tract, neither on the cecal short-chain fatty acids. An statistical trend was observed for reduction of cecal clostridia at day 9 for MUR. Analysis of cecal microbiota structure by 16S rRNA gene sequencing revealed relevant changes correlated to age. At day 9, broilers receiving MUR showed decreased alpha diversity compared to CTR that was not detected at day 36. Changes in specific taxonomic groups with an increase in Lactobacillus genus were identified. In conclusion, evaluation of the variables in this study indicates that dietary Muramidase 007 contributes to improve feed conversation ratio and gastrointestinal function in broiler chickens. Effects could have been mediated by slight shifts observed in the intestinal microbiota. More studies are guaranteed to fully understand the mechanisms involved.

Safety and efficacy evaluation of a novel dietary muramidase for swine.

The safety of a novel microbial muramidase (Muramidase 007) as a feed additive for swine was evaluated in a target animal safety study (Experiment 1). Forty weanling pigs were allotted to 4 dietary treatments: T1 control group, and 3 groups receiving Muramidase 007 in increasing doses: T2 65,000 (1X), T3 325,000 (5X) and T4 650,000 (10X) LSU(F)/kg feed. The efficacy of Muramidase 007 on growth performance was evaluated in a feeding experiment (Experiment 2). A total of 288 piglets were allotted to two groups: T1 control group and T2 receiving Muramidase 007 at 50,000 (LSU(F)/kg feed. In Experiment 1, no growth depression of pigs was observed. No adverse effects of Muramidase 007 were observed for any of the hematology and serum chemistry parameters measured or on pig health status. Post-mortem evaluation showed no adverse effects due to Muramidase 007 supplementation in the gross pathology or in the histological examination. In Experiment 2, Muramidase 007 significantly increased overall (d 0-42) average daily gain (ADG) and tended to improve overall average daily feed intake (ADFI) and day 42 body weight of nursery pigs and had no effect on feed conversion ratio (FCR). Overall, results of these studies show that there were no adverse effects of Muramidase 007 compared to the control group.