# Assessment of Postbiotic, Mundticin-like Substance EM 41/3 Application in Broiler Rabbits
## 2 Materials and Methods
### Experimental Design and Sampling  
A total of 48 rabbits (meat lines M91 and P91) after weaning (in age 35 days), of both sexes (equal male-to-female ratio per treatment), were divided into an experimental group (EG) and a control group (CG), with 24 animals in each. At the start of the experiment, the average body weight of rabbits was 1041 g (EG)-1078 g (CG). The rabbits were placed in the menagerie at the National Agricultural and Food Centre (NAFC) in Nitra-Luzianky (Slovakia), with which we have co-operated almost for 20 years. The guidelines stated in the Guide for the Care and Use of Laboratory Animals approved by the Slovak Veterinary and Food Administration and Ethical Commissions of both institutions (permission code: SK CH 17016 and SK U 18016) were accepted for care and experimental procedures. The animals were fed a commercial diet for growing rabbits (SIGI Trade, Dvory nad Zitavou, Slovakia), with the following nutritional values: dry matter 896.79 g/kg, crude fiber 140.29 g/kg, fat 23.84 g/kg, N-substances 172.83 g/kg, ash 93.68 g/kg, organic matter 803.11 g/kg, and starch 167.66 g/kg. The diet was supplemented with minerals in the following amounts: magnesium\(-\)3.27 g/kg, sodium\(-\)1.25 g/kg, kalium\(-\)13.22 g/kg, iron\(-\)626.97 mg/kg, and zinc\(-\)173.02 mg/kg. The energy value of the diet reached 11.22 MJ/kg. Broiler rabbits were kept in standard cages (type D-KV-72; 0.61 m \(\times\) 0.34 cm \(\times\) 0.33 m; Kovobel company, DomaZlice, Czech Republic), with two animals per cage. A cycle of 16 h light and 8 h dark was applied throughout the experiment. The temperature and humidity in the menagerie were recorded continuously by a digital thermograph positioned at the same level as the cages. The heating and ventilation systems allowed the menagerie an air temperature maintained at 16 \(\pm\) 4 \({}^{\circ}\)C and a relative humidity of approximately 70 \(\pm\) 5% throughout the experiment as previously reported by [PERSON] et al. [12]. The rabbits had ad libitum access to drinking water.  
In addition, animals in the EG were administered Mundticin-like substance (MLS) EM 41/3 in drinking water (activity concentration 25,600 AU/mL), 50 \(\mu\)L for each animal per day over 21 days (3 weeks). The rabbits in the CG were only administered a commercial diet. The duration of the experiment was 42 days. Fecal sampling was performed at day 0/1 (mixed samples of all animals, \(n\) = 10, before MLS application), at day 21 (mixed sample of all animals, \(n\) = 5, 3 weeks of MLS application), and at day 42 (mixed samples of animals, \(n\) = 5, 3 weeks of MLS cessation). In addition, at days 21 and 42, caecum (\(n\) = 4) and appendix (\(n\) = 4) were also sampled after the rabbits were slaughtered as previously described by [PERSON] et al. [12], with one rabbit/one replicate. They were selected based on daily weight measurements to ensure similar weights of animals. Moreover, fecal samples were also taken to test hydrolytic (enzymatic) activity.  
_Musculus longissimus thoracis and lumborum_ (MLTL) was separated by removing skin, connective tissue, chilled and stored at 4 \({}^{\circ}\)C for 24 h until analysis. The rabbits were also regularly weighted. Rabbit blood (_vena auricularis_) was sampled into Eppendorf tubes (with and without heparin according to parameter analyzed) at days 0/1, 21 and 42.