# Assessment of Postbiotic, Mundticin-like Substance EM 41/3 Application in Broiler Rabbits
## 2 Materials and Methods
### Microbiota Analyses  
Microbiota were checked in feces, caecum and appendix. To count microbiota in feces, sampling was provided at day 0/1 (before MLS EM 41/3 application), then at day 21 (after 3 weeks of MLS application) and finally at day 42 (3 weeks of MLS cessation). In the case of caecum and appendix, sampling was performed at days 21 and 42. For microbiota enumeration, the standard dilution microbiological method (ISO, International Organization for Standardization) was used with the selective media (ISO). Samples of feces, caecum and appendix (one g) were mixed with Ringer solution (ratio 1:9, Merck, Darmstadt, Germany, pH 7.0) and treated using Stomacher-Masticator (IUL Instruments, Barcelona, Spain). The appropriate dilutions were spread on M-Enterococcus agar (Becton and Dickinson, Difco, Detroit, MI, USA) to count enterococci. Mannitol salt agar (MSA, Difco) was used to enumerate staphylococci. To count coliform bacteria, Mac Conkey agar (Oxoid, Basingstoke, UK) was used. Amyloidic streptococci were counted on M17 agar (Difco) enriched with starch. The agar plates were incubated at 37 \({}^{\circ}\)C for 24-48 h. Bacterial counts were expressed in colony forming unit per gram (CFU/g) log 10 \(\pm\) SD. After re-isolation of colonies from different agar media, colonies were treated with MLS EM 41/3 using the agar spot test [11]. Inhibitory activity was expressed in arbitrary units per milliliter (AU/mL). Phagocytic Activity Analysis, Glutathione-Peroxidase (GPx) Evaluation, and Biochemistry in Blood Serum  
For phagocytic activity (PA), blood was sampled from _vena auricularis_ into Eppendorf tubes with micro-spheric hydrophilic (MSH) particles and heparin [13]. Sampling was performed at day 0/1 (\(n\) = 8), at day 21 (\(n\) = 8), and at day 42 (\(n\) = 8), meaning there were 8 mixed blood samples from animals. A volume 50 \(\upmu\)L of MSH particle suspension (ARTIM, Prague, Czech Republic) was mixed with 100 \(\upmu\)L of blood in an Eppendorf tube and incubated at 37 \({}^{\circ}\)C for 1 h. Blood smears were prepared and stained with May-Gruenwald and Giemsa-Romanowski stains. To validate PA, the direct microscopic counting procedure was performed with the calculation of the number of white cells containing at least three engulfed particles per 100 white cells (monocyte/granulocytes). PA was expressed in percentage (%). The index of phagocytic activity was also involved in analysis (IPA). GPx (glutathione-peroxidase) activity (\(n\) = 8, meaning at the start of the experiment, at days 21 and 42, in each of the 8 mixed blood samples of all animals from each group) was determined by the colorimetric method (Spectrophotometer UV-2550 Shimadzu San Dimas, CA, USA) using the commercial kit Randox RS504 (Randox Laboratory Ltd., Crumlin, UK) after blood sampling in the tube with heparin. For biochemistry, blood samples were taken into Eppendorf tubes. They were centrifuged (3000\(\times\) g, 30 min) and delivered to the SK-Lab company Lucenec (Slovakia) for analysis. In this analysis, validated methods were used. The following parameters were analyzed: total proteins (TP in g/L), albumin (g/L), creatinine (\(\upmu\)mol/L), alanine aminotransferase (ALT in \(\upmu\)kat/L), aspartate transferase (AST in \(\upmu\)kat/L), alkalic phosphatase (ALP in \(\upmu\)kat/L), glucose (mmol/L), cholesterol (mmol/L), triglycerides (mmol/L), sodium (Na), kalium (K), chlorides (CL) calcium (Ca), phosphorus (P), and magnesium (Mg) in mmol/L. Reference values for rabbits are summarized in Table 4.  
Hydrolytic Activity, Jejund Morphometry Growth Performance, Organic Acids in Chyme, and Quality of Meat  
Hydrolytic activities in feces (amyloidytic, cellulolytic, xylanolytic, pectinolytic and inulolytic expressed in \(\upmu\)mol/g/DM/min meaning in micromol per gram of dry matter per minute) were processed as previously described by [PERSON] et al. [14]. The enzymes were extracted using the procedure of [PERSON] and [PERSON] [15] and they were measured according the procedure of [PERSON] et al. [16].