**1. Introduction**

Humic substances (HS) have a complex organic matter structure, which, among other things, reduces the toxic effect of herbicides, heavy metals, and soil-polluting radionuclides [1–4]. Moreover, it has been shown that HS transfer micronutrients, especially iron from soil to plants, and improve microbial populations in soils [5]. Active components of HS consist of humic acid, humus, ulmic acid, fulvic acid, humin, and certain microelements [6,7]. HS have primarily been used to stimulate plant growth [8], but they were also successfully applied in poultry nutrition, e.g., in various forms (natural and acidified HS) and concentrations (0.25% of HS extract, 0.6%, 0.8%, and 1.0% HS) [9–12].

Recently, trends in agriculture and animal feed applications are aimed at studying the impacts of various feed supplements targeting the enhanced health and productivity of the

**Citation:** Mudro ˇnová, D.; Karaffová, V.; Semjon, B.; Nad', P.; Košˇcová, J.; Bartkovský, M.; Makiš, A.; Buj ˇnák, L.; Nagy, J.; Mojžišová, J.; et al. Effects of Dietary Supplementation of Humic Substances on Production Parameters, Immune Status and Gut Microbiota of Laying Hens. *Agriculture* **2021**, *11*, 744. https://doi.org/10.3390/ agriculture11080744

Academic Editor: Maria Grazia Cappai

Received: 12 July 2021 Accepted: 4 August 2021 Published: 6 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

animals to produce healthy and safe food [13]. Egg quality and production are the most important economic factors in layer hen industry because worldwide egg consumption has been increasing in last decade [14]. Laying hen nutrition could be considered as one of the critical factors for optimizing egg quality in terms of nutritional composition or the economic aspects of whole egg production [15]. Novel nutritional approaches in poultry nutrition represent appropriate strategies for the improvement of animal feed conversion, mainly via feed ingredients or additives. Natural nutritional substances, such as antioxidants, vitamins, trace elements, etc., are well known for their positive impact on health and disease prevention in laying hens [16], while the use of HS in the diets of laying hens as feed additive is substantially new issue [17]. The productive performance and egg quality in layers could be affected by hen nutrition [18], and because of that, we assumed the improvement of the observed variables with HS supplementation in the diets of laying hens.

Nagaraju et al. [19] presented that the addition of humic acids to antibiotic-free feed improved the performance and immune status of broilers. Specifically, modulation of immune functions by means of HS also enables the support of health and production parameters. The effect of HS on the body's immune system is related to the ability of these substances to form relatively strong complexes with carbohydrates [20]. Consequently, these complexes make it possible to produce glycoproteins in the body, which are defined by the ability to bind to NK cells and T lymphocytes in order to act as modulators and to ensure subsequent communication between these cells. Thus, the ability of HS to affect the immune system lies in the regulation of immune activity and the prevention of excessive activity [21].

In addition, HS are also able to support the formation of a protective mucous film on the intestine epithelium, which has an impact on gu<sup>t</sup> health [22]. Similarly, Islam et al. [23] reported positive effect of HS on farm animal health through the modification of the ecosystem in the intestine. HS allows for the better utilization of nutrients and improves not only gu<sup>t</sup> health but also the quality of laid eggs [24]. Arpášová et al. [24] observed that the addition of humic acid in 0.5% concentration led to significantly higher egg production with an increased egg albumen index. Moreover, HS with supplementation with probiotic products based on lactobacilli significantly increased the egg albumen index and Haugh units. Sopoliga et al. [25] reported increased hatchability in pheasant laying hens after the intake of humic substances in comparison to hens fed with the conventional feed mixture.

However, scientific studies on the effect of HS supplementation in the diets laying hens on egg production and quality that are also supported by the immune parameters are limited. For this reason, our study was focused on the influence of 0.5% HS supplementation on the production parameters of eggshell mineral quality, immune parameters (relative expression of IgA, IGF-2, MUC-2 gene in cecum; activity of phagocytes, percentage of selected lymphocyte subpopulations (IgM+, CD3+, CD4+, CD8a+) in the peripheral blood), and the number of lactic acid bacteria and enterobacteria in the intestinal contents in laying hens.

#### **2. Materials and Methods**
