**4. Discussion**

HS are a heterogeneous group of natural organic molecules that are part of soil and water organic matter. These are substances with a whole range of positive health effects. In our work, we focused on the effect of the administration of HS on cellular immune response, the protection of intestine in poultry as well as on specific productive parameters.

Dietary supplementation with HS additives had a significant effect on the laying rate, daily egg mass, egg weight, and feed conversion, which indicate the ability of HS to stabilize the intestinal microbiota, thus ensuring improved nutrient utilization in feed [38,39]. These findings are consistent with our results. Similarly, the beneficial effect of feeding humates in a 0.25% concentration during broiler fattening was observed in Kocabagli et al. [40]. In this study, the supplementation of humates at the time of fattening (day 22–42) significantly improved feed conversion as well as increased the body weight of the broilers compared to the control group and broilers supplemented with HS over the time period of 0–21 days. However, some studies sugges<sup>t</sup> conflicting results. For example, a study in 2006 by Rath et al. [41] reported that HS in 0.5 and 1% concentrations have a negative effect on the broiler growth performance.

The importance of minerals on eggshell quality is unquestionable. The eggshell consists of 94.85% of minerals, of which calcium carbonate, magnesium carbonate, and calcium phosphate have the highest proportions. Therefore, phosphorus and calcium are the most important elements for the formation of the outer shell of an egg. In addition, eggshell contains 4.15% organic matter and water in a proportion of less than 2% [42]. According to the achieved results, the increase in the mineral content in the eggshell was favorably influenced by the supplementationHS in the diet of laying hens. Moreover, the administration of 0.5% HS product (Humac Natur AFM) contains up to 42,278 mg·kg−<sup>1</sup> of calcium and 5111 mg·kg−<sup>1</sup> of magnesium [20]. The increase in the mineral content may also be related to the relationships among the elements or the HS components. The absorption of calcium, manganese, iron, and phosphorus decreases with higher levels of HS in the diet [41]. Reasons for this could be the chelating effects of HS that are influenced by their large number of carboxylic acid side chains [43]. Moreover, HS have the ability to bind to elements from the environment and can also release these elements after changes in external conditions. This property, in the case of action in the gastrointestinal tract, affects the eggshell quality [44]. Interestingly, the results obtained by Ergin et al. [45] suggested that addition of humic acids (30 mg·kg−<sup>1</sup> diet) enhanced eggshell strength without affecting feed efficiency and egg production. Likewise, Tancho [46] concluded that eggshell thickness was increased in hens fed with humates at levels of 1 and 2 <sup>g</sup>·kg−1.

In order to achieve maximum production, good health is essential, which is conditioned by the state of the immune system. Generally, the largest immune organ is the

gut-associated lymphoid tissue (GALT), and the first line of defense is non-specific barriers, including mucin and other components. The mucus layer also acts as a medium for molecule transport between luminal content and enterocytes. We observed a nonsignificant increase in relative mRNA expression for MUC-2 but a significant increase in IgA gene expression, which is consistent with the finding that humic acids may aid in the formation of a protective film on the mucus epithelium of the gastrointestinal tract, which protects against infectious agents and toxins, thereby also improving animal feed utilization [23]. In addition, the main function of IgA is to neutralize antigens on the mucosal surfaces. On the other hand, our results showed that HS in a 0.5% concentration did not affect IGF-2 gene expression. This fact is interesting from the point of view that IGF-2 plays a very important role in the postnatal development of the organism and mediates most of growth effects; thus, it has significant effects on the skeletal and muscular systems. In our previous study, we observed the same effect of 0.8% HS on the relative expression of IGF-2 in broilers [20]. On the contrary, Weber et al. [47] reported an increase of serum IGF-1 in pigs treated with Menefee humate (0.25% of humic acid). In tune with previous studies and our knowledge, we suppose that the influence of HS on the expression of growth factors is dependent on the concentration of the HS that is administered.

As in our previous study, where we administered 0.8% HS to broilers, even now, after the application of 0.5% HS to laying hens, we observed a significant increase in both the percentage of active phagocytes and their engulfing capacity [20]. Sanmiguel and Rondón [48] suggested that this effect on phagocytes is time dependent. They administered 0.1% and 0.2% HS to laying hens and found that the phagocytic index was elevated after 8 and 30 days of application, but subsequently (on 60th day), phagocytic index significantly decreased and was lower than in the control group. Although the mode of action of HS on phagocytosis has not been fully explained, studies with human neutrophils have shown that HS stimulate their adhesion abilities and superoxide production, and they are able to mediate intracellular signal transduction leading to NF-κB induction, which is crucial for the transcription of many proinflammatory genes (e.g., IL-2, IL-8, MCP-1, TNFα, GM-CSF) [49]. Similarly, Riede et al. [50] confirmed the stimulatory effect of HS on the oxidative burst of human neutrophils but without activating chemotaxis. The authors hypothesize that HS contain chemical structures that can be recognized by the neutrophils and can activate them.

Because works on the effect of HS on individual lymphocyte subpopulations are rare, we examined the proportions of IgM+ lymphocytes, T lymphocytes, and T helper and T cytotoxic cells in our study. According to a study by Luthala [51], in chickens, the CD8αα homodimer may be expressed on peripheral CD4+ cells, but expression of the CD8αβ heterodimer only occurs on cytotoxic T cells. In this type of expression, chicken and mammalian lymphocytes differ significantly. Functionally, a subpopulation of CD4+CD8+ T cells in chickens is reported as T helper and/or regulatory lymphocytes. These findings were confirmed by both the expression of the CD25 molecule (IL-2 receptor—typical for regulatory cells), which was detected on part of the cells from the CD4+CD8- as well as the CD4+CD8+ subpopulations and by their cytokine profiles [52]. For these reasons, even in our study, CD4+CD8+ lymphocytes with low and medium expression of the CD8 molecule were included in the T helper cell subpopulation.

We found that the addition of 0.5% HS to the feed of laying hens increased the proportion of IgM+ lymphocytes and thus decreased the proportion of T lymphocytes, while the proportion of T helper and T cytotoxic cells was not affected. The increased percentage of the IgM+ lymphocyte subpopulation is consistent with the finding of increased gene expression for IgA in the gut. Zhang et al. [38] (2020) also noted significantly elevated serum IgM as well as IgG levels after the administration of 0.1 and 0.5% HS to laying hens, which confirms the activation of B cells. Similarly, Salah et al. [53] reported an increase in the serum gamma globulin fraction containing most of immunoglobulins after 5 days of humic acid application to broiler chickens. Interestingly, when we administered 0.8% HS to broiler chickens from day 11 of life to the end of fattening (day 38) in the previous

experiment, we observed a significantly higher percentage of T cells as well as T helper cells (CD4+), but the gene expression for IgA was not affected [20]. Based on the above results, we assume that the obtained cellular immune response depends not only on the used concentration and the duration of HS application but also depends on the category of poultry to which they are applied.

The exact mechanism of action by which HS affect specific subpopulations of immune cells has not ye<sup>t</sup> been elucidated, but their effect on cytokine production has been confirmed. Vetvicka et al. [54] reported a significant increase in the production of IL-5 and IL-6 in mice intraperitoneally treated with HS. These cytokines stimulate B lymphocyte differentiation and growth and increase the secretion of immunoglobulins. The theory that explains the immunomodulatory potential of HS is that humates are able to form complexes with carbohydrates, amino acids, and peptides. Currently such complexes are considered to be biologically active and can subsequently bind to the surface of T lymphocytes and NK cells, thereby regulating their functions, including cytokine production. Subsequently, cytokines influence further immune reactions [50,55].

Regarding the effect of HS on the intestinal microbiota, the results of different authors vary considerably. While some have reported a decrease in enterobacteria [20,39], others have observed an increase [56]. Similarly, different results were observed for other bacterial species. In our case, there was an increase in the numbers of *E. coli* in the intestine and in the cecum, but the number of lactic acid bacteria was not affected. Such a trend can be considered as negative. These results are consistent with in vitro microbiological tests that have shown that various species of *Lactobacillus* spp. and different strains of *E. coli* also respond differently to HS (unpublished data). Similar results were obtained by Buzoleva and Sidorenko [57], who found that the multiplication of enterobacteria in the presence of HS depends on both the type of bacterium and the HS composition.
