*3.1. Production Parameters*

Dietary supplementation with 0.5% HS additives had a significant effect on the following laying performance variables recorded in week 29: laying rate, daily egg mass, egg weight, and FCR, compared to the control group (*p* < 0.05) (Table 4).


**Table 4.** The results of layer production indices in week 29 (means ± SD).

C: control group; H: layers fed a diet enriched with supplementation of 0.5% HS. a,b Means not sharing the same superscripts in the same row are significantly different.

## *3.2. Eggshell Mineral Analysis*

When analyzing experimental eggshell samples, higher concentrations of minerals were determined, compared to the results from the control group. A significant increase was observed in the contents of copper (*p* < 0.001), phosphorus (*p* < 0.01), calcium, manganese, and zinc (*p* < 0.05) (Table 5).


**Table 5.** The results of eggshell mineral analysis (means ± SD).

C: control group; H: layers fed a diet enriched with supplementation of 0.5% HS. a,b Means not sharing the same superscripts in the same row are significantly different.

#### *3.3. The Relative Expression of Selected Genes*

The relative expression for the IgA gene was significantly upregulated in the humic group compared to the control group (*p* < 0.05). Similarly, MUC-2 gene expression was upregulated, but it was not significant in H group, compared to the control. On the contrary, the relative expression for the IGF-2 gene was not influenced by the addition of HS (Figure 1).

**Figure 1.** Relative expression of IgA (**a**), IGF-2 (**b**), and MUC-2 (**c**) genes in cecum of laying hens treated with 0.5% HS. Results at each time point are the median of 2−<sup>Δ</sup>Cq. C: control group; H: layers fed a diet enriched with supplementation of 0.5% HS. Columns marked with stars are significantly different from control: \* *p* < 0.05.

#### *3.4. Cellular Immune Response*

In testing the effect of HS on cellular immunity, the phagocyte activity, which represents the innate component of the immune response and the representation of the selected lymphocyte population in the blood as a parameter of the acquired immune response, was monitored.

The addition of HS to the feed of laying hens significantly increased the percentage of active phagocytes as well as the engulfing capacity (Figure 2).

The results from the phenotyping of the blood lymphocytes showed that HS stimulate the differentiation of the B lymphocytes when a significantly higher proportion of IgM+ lymphocytes was recorded (Figure 3a). This finding is also supported by the increased expression of IgA genes in the gut. In contrast, the proportion of total T cells (CD3+) was thus reduced (Figure 3b). There was no statistically significant effect of HS on the T cell subpopulations; either CD4+ (Figure 3c) or CD4-CD8+ (Figure 3d) was observed.

**Figure 2.** Influence of 0.5% HS on phagocyte activity in layer blood evaluated as (**a**) percentage of active phagocytes (phagocytic activity) and (**b**) engulfing capacity of the phagocytes (expressed as mean fluorescence intensity—MFI). C: control group; H: layers fed a diet enriched with supplementation of 0.5% HS. Columns marked with stars are significantly different from control: \* *p* < 0.05; \*\*\* *p* < 0.001.

**Figure 3.** Percentage of (**a**) IgM+, (**b**) CD3+, (**c**) CD4+, and (**d**) CD4-CD8+ lymphocytes in the blood of laying hens receiving 0.5% HS. C: control group; H: layers fed a diet enriched with supplementation of 0.5% HS. Columns marked with stars are significantly different from control: \* *p* < 0.05; \*\* *p* < 0.01.
