*2.1. Experimental Design*

The animal protocol for this research was approved by the Ethical Committee for Animal Care and Use of University of Veterinary Medicine and Pharmacy in Košice (the Slovak Republic) and the Food Administration of the Slovak Republic (approved the experimental protocol number 3040/14-221). All procedures in this study were performed in accordance with the principles of the European Directive on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (European Parliament and Council, 2010). The experimental diets were composed of commercial basal feed mixture with naturally occurring biogenic and heterogeneous organic substances as a HS supplement in accordance with regulation 68/2013 from 16 January 2013 of the European Union Commission,

which states that the use of Leonardite is allowed as a source of HS as a feed component in animal diets.

A total of sixty laying hens of the Lohman Brown classic hybrid (Eggro-farm Ltd., Košická Polianka, Slovakia) in the 17th week of life were divided into two groups: the control (C) group and experimental humic (H) group. Each treatment group consisted of 30 birds (average weight 1.36 ± 0.15 kg). The control group (C) was fed with a basal feed mixture (De Heus, Buˇcovice, Czech Republic) without HS supplementation during the trial. The experimental group (H) was fed with basic feed mixture supplemented with 0.5% dietary natural HS (Humac Ltd. Košice, Slovakia). The diet of the experimental group (H) was prepared daily and was enriched with HS at a concentration of 0.5%. The supplement was applied to the surface of the basal diet. The nutritional composition of the basal feed mixtures is presented in Table 1. The dietary natural HS supplement used in the experiment was ground and physically purified Leonardite without chemical treatment. It contained natural HS with more than 65% of humic acid without acid salts [12]. Laying hens were housed in floor pens with deep wood untreated litter in the form of being free range in secure and controlled microclimate conditions according to the Lohmann Brown Classic Management Guide [26]. The diet of the laying hens was formulated according to the recommended nutrient content for Lohmann Brown Classic hens [26]. The chemical compositions of the fermented feed and diets were determined for dry matter, crude protein, crude fat, crude fibre, starch, and total phosphorus according to the EC Commission Regulation 152/2009 [27] and Semjon et al. [28].


**Table 1.** Components and nutritional composition of laying hen diet.

\* Premix of amino acids, vitamins, trace elements (per kg): lysine 140 155 g; DL-methionine 180 g; vit. A 1,200,000 IU; D3 500,000 IU; E 2000 mg; pantothenic acid 1800 mg; niacin 6000 156 mg; choline 60 mg; B6 500 mg; B12 1.8 mg; folic acid 200 mg; copper 1100 mg; iron 8400 mg; zinc 8000 mg; 157 manganese 12,000 mg; iodine 110 mg; selenium 40 mg.

Laying hens were fed once a day with daily prepared diets. During weeks 17 and 18, they were fed 75 g; in week 19, they were fed 81 g; from week 20 to the laying phase, they were fed 93 g; and during the laying phase, they were fed 100 g of diet/layer/day. Access of the layers to water was ad libitum. The lightening program from the 17th week of age was set to a lighting period of at least 10 h, taking the natural day length into account, and it was increased by one hour every week up to 14 h until 21 weeks of age and remained stable from that point on [26]. Sufficient ventilation to ensure good litter condition was set. Their health and weight were monitored continuously. The experiment was finished when the laying hens were 29 weeks of age, at which point twelve hens were randomly selected from both groups. These animals were used for blood collection, and after euthanization by cervical dislocation, they were carcassed and sampled for further laboratory tests.

### *2.2. Production Parameters Screening*

Daily egg production, egg weight and feed intake were recorded daily throughout the trial. The feed conversation ratio (FCR) was expressed as each kilogram of feed consumed per kilogram of egg produced by the batch. The laying rate (%) was calculated as the number of laid eggs to number of laying hens by batch per day. For the presented experiment, the number of eggs produced and the consumption of feed per animal, egg weight (g), FCR, and laying rate in week 29 were recorded.

## *2.3. Egg Shell Analysis*

The mineral composition of the eggshells was analyzed according to the procedure of Skalická et al. [29]. Eggshell samples were immediately frozen and stored at −20 ◦C until they were analyzed. The samples were digested in a MLS 1200 MEGA (Milestone Microwave Laboratory System, Shelton, CT, USA) microwave oven using a mixture of 5 mL HNO3 and 1 mL HCl per 1 g of sample. The digested samples were analyzed for the presence of Ca, Mg, K, Na, Cu, Zn, and Mn using an atomic absorption spectrometer (Unicam Solar 939, Cambridge, UK). The phosphorus (P) content in the eggshell samples was determined spectrophotometrically [30]. A total of 18 egg samples from the C and H experimental groups were collected in week 29 and were subjected to eggshell analysis. Eggshell analysis was performed in triplicate and was expressed as the mean and the standard deviation

#### *2.4. Homogenization of Cecum and Isolation of Total RNA of IgA, IGF-2 and MUC-2 Genes*

Tissue samples (cecum) were cut into approximately 20 mg pieces and were immediately placed in RNA Later solution (Qiagen, West Sussex, UK). They were stored at −70 ◦C before RNA purification, as described in Karaffová et al. [31]. A total of 12 cecum samples from each experimental group of laying hens were collected after slaughtering and, the samples were subjected to analysis.

#### *2.5. Relative Expression of IgA, IGF-2 and MUC-2 Genes in Quantitative Real-Time PCR (qRT-PCR)*

The mRNA levels of selected genes were determined. In addition, the mRNA relative expression for reference gene coding GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was selected based on expression stability using BestKeeper software (Pfaffl, Germany). The primer sequences, optimal annealing temperature, and time for each primer used for qRT-PCR are listed in Table 2. All primer sets allowed DNA amplification efficiencies between 94% and 100%.

The amplification and detection of specific products were performed using the CFX 96 RT system (Bio-Rad, Hercules, CA, USA) and the Maxima SYBR Green qPCR Master Mix (Thermo Scientific, Waltham, MA, USA). Subsequent qRT-PCR to detect the relative expression of mRNA in the selected parameters was conducted over 38 cycles under the following conditions: initial denaturation at 94 ◦C for 3 min, subsequent denaturation at 93 ◦C for 45 s, and annealing (Table 2) and extension for 10 min at 72 ◦C. A melting curve from 50 ◦C to 95 ◦C with readings at every 0.5 ◦C was produced for each individual qRT- PCR plate. Analysis was performed after every run to ensure a single amplified product for each reaction. All reactions for real-time PCR were done in duplicate, and the mean values of the duplicates were used for subsequent analysis. We also confirmed that the efficiency of the target gene amplification including GAPDH was essentially 100% in the exponential phase of the reaction, where the quantification cycle (Cq) was calculated. The Cq values of the studied genes were normalised to an average Cq value of the reference genes (ΔCq), and the relative expression of each gene was calculated as 2−<sup>Δ</sup>Cq.


**Table 2.** List of primers used in RT-PCR for IgA, MUC-2, and IGF-2 mRNA detection in layer hens.

## *2.6. Phagocyte Activity Testing*

The percentage of active phagocytes as well as the engulfing capacity of the phagocytes was determined using a commercial Phagotest® assay (Glycotope Biotechnology, Heidelberg, Germany). The test was performed according to the manufacturer's instructions and were performed using fresh heparinized blood [36].

#### *2.7. Identification of Lymphocyte Subpopulations*

For the identification of selected lymphocyte subpopulations, mononuclear cells were isolated from 600 μL of heparinized blood diluted 1:1 with phosphate buffer saline (PBS; MP Biomedicals, Illkirch, France), which was carefully overlaid on the 2.5 mL of separation solution LSM 1077 (PAA Laboratories GmbH, Pashing, Austria). Mononuclear cells were obtained from the interphase between the separation solution and the plasma after centrifugation at 600× *g* for 30 min. The obtained cells were washed twice with PBS through centrifugation at 250× *g* for 5 min. The concentration of the mononuclears was determined after staining with Türck's solution in a Bürker chamber and was adjusted to 5 × 10<sup>5</sup> cells in 50 μL.

To identify selected subpopulations of lymphocytes, direct immunostaining using two combinations of conjugated mouse anti-chicken monoclonal antibodies (Southern Biotech, Birmingham, AL, USA): CD4/CD8a/CD45 and CD3/IgM was used according to the specifications given in Table 3. The cells were incubated with antibodies for 20 min in the dark at laboratory temperature. The cells were then washed twice with 1 mL PBS (250× *g* for 5 min) and were resuspended in 100 μL of PBS for subsequent cytometric analysis.

**Table 3.** Specification and amounts of used mouse anti-chicken monoclonal antibodies.


#### *2.8. Flow Cytometric Analysis*

Phagocytic activity analysis as well as the identification of lymphocyte subpopulations was performed on a six colour BD FACSCantoTM flow cytometer (Becton Dickinson

Biosciences, San Diego, CA, USA) using BD FACS DivaTM software. The position of the analysed cells was gated in FSC vs. SSC dot plots. Granulocytes and monocytes were gated for phagocytic activity analysis. Bacterial aggregates were excluded from further analysis based on the low DNA content in the red fluorescence histogram (FL-2). The percentage of active phagocytes and the mean fluorescence intensity were determined in the green fluorescence histogram (FL-1).

Gated lymphocytes were used for the identification of lymphocyte subpopulations, while contaminating chicken thrombocytes were differentiated from lymphocytes based on their higher side scatter profiles [37]. CD3+ lymphocytes represent T lymphocytes, and IgM+ cells are a subpopulation of B lymphocytes. CD4+CD8a- and CD4+CD8alow/mid subpopulations were counted together as a representative of the T helper lymphocytes. The CD4-CD8a+ subpopulation was evaluated as T cytotoxic cells. Proportions of lymphocytes are expressed in percentage.

## *2.9. Intestinal Bacteria Analysis*

In the contents of the small intestine and caecum were analysed to determine the number of lactic acid bacteria and enterobacteria using the plate count method after a 10-fold dilution in saline. MRS agar plates (HiMedia, Karnataka, India) that had been anaerobically incubated (GasPak system, Becton Dickinson, San Diego, CA, USA) for 48 h at 37 ◦C were used to determine the number of lactic acid bacteria. Enterobacteria were counted on Endo agar plates (HiMedia, Karnataka, India) after a 24 h incubation period at 37 ◦C under aerobic conditions. The bacterial counts are expressed in log10 of colony forming units per gram of content (log10 cfu·g<sup>−</sup>1) ± standard deviation.
