*Article* **Effects of Dietary Supplementation of Humic Substances on Production Parameters, Immune Status and Gut Microbiota of Laying Hens**

**Dagmar Mudro ˇnová 1, Viera Karaffová 2,\*, Boris Semjon 3, Pavel Nad' 4, Jana Košˇcová 1, Martin Bartkovský 3, Andrej Makiš 3, Lukáš Buj ˇnák 4, Jozef Nagy 3, Jana Mojžišová 5 and Slavomír Marcinˇcák 3,\***


**Abstract:** Despite the fact that humic substances (HS) have been frequently studied in relation to their effects on livestock health, studies on their influence on egg production and quality, immunity, and intestinal microbiota of laying hens are limited. In this study, the influence of 0.5% HS supplementation on the specific 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 in the peripheral blood), and number of lactic acid bacteria and enterobacteria in the intestinal contents in laying hens was tested. The addition of 0.5% HS to the laying hen feed had a positive effect on egg laying rate, daily egg mass, egg weight, feed conversion and eggshell quality and also had an immunostimulatory effect manifested by increased phagocyte activity and B cell response. Concurrently, an increase in the number of enterobacteria in the intestinal contents and a decrease in the proportion of T lymphocytes (*p* < 0.05) was observed, which can be considered as a negative effect of HS. The results confirmed that HS can be used for the improvement of egg production and targeted immunostimulation, but their effect on the intestinal microbiota and T lymphocytes should be studied in more detail.

**Keywords:** humic substance; egg; immune answer; lymphocyte; intestinal microbiota
