IMPACTS OF HUMIC ACIDS IN NUTRITION ON HAEMATOLOGICAL AND BIOCHEMICAL PARAMETERS OF BROWN HARES

and biochemical blood tests. evaluated the subsequent analyses within individual samplings as well as by factorial ANOVA. standard feed + 1% of Humac Natur AFM – humic acids, enriched with green feed – clover-grass mixture). We found a statistically significant difference between the control group and the EG2 group in the RBC parameter ( P < 0.05) after three months of consumption. We found a statistically significant decrease in urea levels in both experimental groups compared to the control group ( P < 0.001), as well as a decrease in urea concentration in the EG2 group compared to the EG1 group ( P < 0.01); decrease in cholesterol levels in the experimental groups compared to the control, and between EG1 and the control group, this decrease was statistically significant ( P < 0.05); and we recorded a statistically significant increase of triglycerides in the EG2 group compared to the other groups ( P < 0.01). Based on the obtained results, we can evaluate the use of 1% humic acid as a feed additive as safe for feeding hares.


INTRODUCTION
In recent times, the trend has been to use various organic compounds in animal feed that would otherwise be unused. They are mainly products considered to be by-products or waste products from various activities of industry and agriculture Humic substances are natural organic substances resulting from the decomposition of mainly plant but also animal residues. Humic substances are subject to further decomposition with difficulty and are contained in large quantities in soil, peat, coal and some waters (Steelink, 1964;Hayes, 1997). Humic substances are mainly used for plant nutrition. Although it is not a classic type of fertilizer, humus enables an easier intake of nutrients, stimulates the formation of root hairs, thanks to which the plant absorbs water and nutrients better, supports photosynthesis and improves soil properties (Steelink, 1964;Jindo et al., 2012). They stimulate plant growth to a degree comparable to the phytohormone auxin (Scaglia et al., 2016). The above-mentioned properties subsequently resulted in the possible use of humic acids in animal nutrition. First, these substances were tested mainly on poultry, where the use recorded quite decent results. Many studies talk about improved growth, carcass traits, higher and better-quality egg laying, or a positive effect on reproduction ( , 2018). Basically, we can talk about studies that either deal with monitoring the impact of a natural bioactive substance on quality indicators as well as increasing the production of economically interesting parameters for breeders; or studies of this type are supplemented with selected individual health parameters, such as the body's immune response, selected haematological or biochemical parameters, or markers of oxidative stress. In general, it would be appropriate and necessary to determine the possible negative/positive effect of natural substances, where we include humic acids, on the health status of the individual in commonly used concentrations. The objective of our study was to test if the humic acids can negatively/positively affect wild animal (brown hares) health status. As the main indicators for this evaluation, we chose haematological and biochemical blood tests. We evaluated the subsequent analyses within individual samplings as well as by factorial ANOVA.

Experimental design
In this study, we used 24 brown hares (12 males and 12 females) in the age of 12-24 months. All hares were fed a standard chow diet (0-11KKZ for rabbits and hares) on ad libitum basis to the beginning of the experiment. As part of the 6month experiment, we planned three blood samplings (at the beginning of the experiment and then after three and six months). Animals were divided into three groups at the beginning of the experiment (n = 8/group): CG (control group, 0-11KKZ standard diet without additives), EG1 group (experimental group 1, received 0-10KK/D standard feed + 1% of Humac Natur AFMhumic acids), and EG2 group (experimental group 2, received 0-10KK/D standard feed + 1% of Humac Natur AFMhumic acids, enriched with green feedclover-grass mixture). The composition of feed mixtures was: nitrogenous substances (150-175 g/kg), crude fat (35-90 g/kg), crude fiber (160-170 g/kg), ash matter (60-90 g/kg), Humic substances are natural organic substances resulting from the decomposition of mainly plant but also animal residues. The objective of our study was to test if the humic acids can negatively/positively affect brown hares health status. As the main indicators for this evaluation, we chose haematological and biochemical blood tests. In this study, we used 24 brown hares (12 males and 12 females) in the age of 12-24 months. As part of the 6-month experiment, we planned three blood samplings (at the beginning of the experiment and then after three and six months). Animals were divided into three groups at the beginning of the experiment (n = 8/group): CG (control group, 0-11KKZ standard diet without additives), EG1 group (experimental group 1, received 0-10KK/D standard feed + 1% of Humac Natur AFMhumic acids), and EG2 group (experimental group 2, received 0-10KK/D standard feed + 1% of Humac Natur AFMhumic acids, enriched with green feedclover-grass mixture). We found a statistically significant difference between the control group and the EG2 group in the RBC parameter (P < 0.05) after three months of consumption. We found a statistically significant decrease in urea levels in both experimental groups compared to the control group (P < 0.001), as well as a decrease in urea concentration in the EG2 group compared to the EG1 group (P < 0.01); decrease in cholesterol levels in the experimental groups compared to the control, and between EG1 and the control group, this decrease was statistically significant (P < 0.05); and we recorded a statistically significant increase of triglycerides in the EG2 group compared to the other groups (P < 0.01). Based on the obtained results, we can evaluate the use of 1% humic acid as a feed additive as safe for feeding hares.
ARTICLE INFO lysine (9 g/kg), calcium (7.5-12 g/kg), sodium (1.5 g/kg), phosphorus (4.5-7.0 g/kg), vitamin A (6900-9800 IU/kg), vitamin D3 (1000-1870 IU/kg), iron (74-110 mg/kg), manganese (108-120 mg/kg), zinc (79-90 mg/kg), copper (14 mg/kg), selenium (0.25-0.28mg/kg), iodine (1.1 mg/kg). The animals were housed in individual cages per pair. The dimensions of the cage were: floor plan 2 x 1.8 m, height 1 m. The cage was divided into 3 parts. The front feeding part was 2 x 1 m in size, the two rear parts (asylum part) were 0.8 x 1 m in size. The structure of the cage was wooden, the floor was made of plastic grates and the roof was thermally insulated. The conditions of animal care, manipulations and use corresponded with the instructions of the Ethics Committee of the Slovak University of Agriculture in Nitra.

Blood sampling
All animals were healthy and in good health condition. We carried out the control sampling on March 13th, followed by the sampling on June 14th and the final sampling on September 18th. Blood samples were taken by a qualified veterinarian from vena auricularis centralis and placed into two tubes. Samples for biochemical assessment were placed into tubes without additive, and tubes containing EDTA as an anticoagulant, were used for the haematological analysis. Coagulated blood samples were centrifuged at 1006 x g for 20 min and obtained blood serum was stored at -20 °C until further analyses at the Institute of Applied Biology (SUA in Nitra).

Statistical analysis
The obtained data were subjected to statistical analysis using the STATGRAPHICS Centurion© (StatPoint Technologies, Inc., Warrenton, VA, USA). The data were checked for normality using a Kolmogorov-Smirnov normality test before the statistical analyses. The effect of the humic acids on the haematology and serum biochemistry parameters was analysed using the analyses of variance (ANOVA) followed by Tukey's multiple comparison test (the means and standard errors are reported). All obtained data was also analysed using factorial ANOVA, with effects of the group (different diet), duration of administration and gender (P values are reported). The results of the analyses were considered significant at P < 0.05; P < 0.01 and P < 0.001.

RESULTS
The data obtained by blood and blood serum analyses after three months of the experiment are presented in Tables 1 and 2. Based on the statistical analysis, we found a statistically significant difference between the control group and the EG2 group in the RBC parameter (P < 0.05) for the haematological examination. During the biochemical examination, we noted a statistically significant increase in the cholesterol content in the EG2 group compared to the EG1 group (P < 0.05). No significant differences in other parameters were observed between the control and experimental groups. Data obtained by blood and blood serum analyses after six months of the experiment are presented in Tables 3 and 4. Based on the statistical analysis, we found no statistically significant difference between the control group and the experimental groups for the haematological parameters. In biochemical analyses, we noted several statistically significant differences. We recorded a statistically significant decrease in urea levels in both experimental groups compared to the control group (P < 0.001), as well as a decrease in urea concentration in the EG2 group compared to the EG1 group (P < 0.01). We noted a decrease in cholesterol levels in the experimental groups compared to the control, and between EG1 and the control group, this decrease was statistically significant (P < 0.05). Triglyceride levels were relatively balanced in the control and EG1 groups, but in the EG2 group we recorded a statistically significant increase compared to the other groups (P < 0.01). The other monitored markers were relatively balanced. We then subjected the entire set of results obtained during the entire length of the experiment to a multifactor ANOVA. We used group, duration, and gender as main effects. We also observed statistical dependence for interactions between observed main effects. The results of this analysis are presented in Table 5. We recorded a statistically significant influence of the main effect "group" for RBC, PDWc, urea, ALT, cholesterol, and triglycerides. We recorded a statistically significant influence of the main characteristic "duration" for WBC, MID, GRA, MCH, MCHC, RDWc, PLT, PCTCa, glucose, AST, ALT, and triglycerides. For the last main effect "gender", we noted an influence on the parameters WBC, MID, GRA, LYM%, RBC, HGB, HCT, MCHC, RDWc, Ca, Mg, urea, TP, albumin, globulin, and A/G ratio. By analysing the interactions of the monitored main effects, we noted the impact on the parameters LYM%, GRA%, MCV, MCH, MCHC, RDWc, MPV, Mg, urea, TP, AST, ALT, triglycerides, globulin, and A/G ratio, as presented in table 5.    Legend: bold values are significant; * The means within a row with *sign differ significantly (P < 0.05); a,b The means within a row with different superscript letters differ significantly (P < 0.01); A,B The means within a row with different superscript letters differ significantly (P < 0.001)

DISCUSSION
Our basic intention was a strict evaluation of the health status of individuals after the application of 1% humic acids as a feed additive. The main and very important health assessment is a haematological blood test. In both sampling, we can talk about the minimal influence of humic acids on the haematological parameters of the tested animals. We recorded a change in only one parameter, namely a decrease in RBC in experimental group 2. However, this change was also within the physiologically normal range. Galip et al. (2010) tested humic acid supplemented diets (5 g/day and 10 g/day) during 22 days in rams. Their results have a similar tendency to ours in several haematological parameters, such as a decrease in lymphocyte content, a partial increase in granulocytes, a decrease in erythrocyte content, or an increase in mean corpuscular haemoglobin (with no significant effect on blood haematology, except significant effect on eosinophils level). Other studies where the haematological examination was carried out were most often used on poultry, where the authors confirmed a significant influence again in a similar tendency; a decrease in haemoglobin, a decrease in the content of red blood cells, but in the case of white blood cells, the results were relatively uneven in the experimental groups ( Their results confirm the positive effects in this issue as well, as the ratio of live births and weaned leverets was higher than in the control group. After an overall evaluation, we can talk about the possible safety of using this supplement in animal nutrition at presented concentration. When combining humic acid additions with clover-grass mixture (EG2), we recorded more contradictory results. Based on the evaluation of the results, we would not recommend such feeding. Basically, we can talk about a negative impact on haematological parameters (especially white blood cells) and cholesterol content, when combined standard feed + 1% of Humac Natur with green feeding. On the other hand, the group tested purely with humic acids showed a very good health status, and many previous studies on different species of animals refer to really positive effects from the stabilization of breeding, improvement of performance characteristics, as well as many proactive effects and improved reproductive characteristics.

CONCLUSION
Based on our results, we can talk about several conclusions. In the first case, we noticed a minimal impact on animal health after three or six months of feeding the supplement into the feed. Rather, we can talk about signs of positive effects (e.g., decrease in cholesterol content, balanced haematological parameters). In the second case, we cannot ignore the results of the factor analysis, where the effect of the time of administration of the additive is clear, as well as the effect of gender.
In the latter case, we do not recommend combining prepared feeds containing humic acids with green feeding. In this experimental group, there are rather indications of a negative character. Based on the obtained results, we can evaluate the use of 1% humic acid as a feed additive as safe for feeding hares.
From the point of view of determining the effects of bioactive substances and the subsequent possible application in the physiology of animals, it is necessary to determine the protective or toxic effect, as well as their mutual interactions due to the elimination of health risks. Due to the wide variability of bioactive and bioprotective substances, it is difficult to include all active substances and examine their importance and effects. Therefore, we recommend continuing testing humic substances on other animal species as well as at higher concentrations.