Introduction

Today’s streamlining of production processes is placed on the primary rungs of society, but this targeted production process also brings with it considerable negative impacts on reproduction, breeding, welfare of horses1,2.

Environmental authorities are paying more and more attention to heavy metal contamination. There is an enormous potential for implementing animals as bioindicators of environmental contamination. Because of their close closeness to humans, same habitats, and increased organismal similarities, animals like horses may be valuable as markers of environmental pollution3,4.

Contaminants, also known as risk factors, can vary in characteristics based where they came from5,6,7. Physical contamination is caused by ubiquitous ionizing and non-ionizing radiation8. Toxic metals are examples of chemical pollutants. Toxic metals are difficult to degrade from the body, in their essence they also tend to accumulate in various organs and, last but not least, impair their functions. Most of the risk elements (As, Pb, Hg, Cd) are extremely toxic, and even their low concentrations can cause disturbances in both humans and animals9,10,11. Poisoning by toxic metals can be acute or develop over time. Skin issues, respiratory issues, convulsions, dyspepsia, or significant central nervous system malfunctions are frequently present after poisoning by toxic metals12.

Concentration of elements in horse blood, which are accumulated from feed and environment play a key role in their health. Elements can be classified as macro (Ca, K, Mg, and P), micro/trace (Al, As, Cd, Cu, Fe, Hg, Pb, Se and Zn)m essential (Cu, Fe, Zn, etc.) and non-essential (As, Cd, and Pb), however some of them are toxic to body in extensive concentrations13,14. The source of toxic elements in feed can originate from air emissions, various industrial, urban environments or different contamination of feed. Optimal concentrations of chemical elements are necessary for biological processes in animals as well humans15. While urine, feces, hair, kidneys, and liver are among the specimens that can be used to investigate an animal’s mineral status, blood is still the most commonly used biological material in practical applications to discover toxicity or deficiencies16,17,18.

Based on the reliability and accessibility of the specimen, blood as the sole substrate was made since hematological variables are thought to be reliable indicators of metal contamination. They are influenced by environmental changes and can be obtained less invasively than for example kidneys and liver. Furthermore, regular hematological analysis enables to observe the animal’s health without the need to sacrifice the animal18.

Oxidative stress markers are another crucial tool for determining environmental pollution of trace element poisoning19,20. Antioxidant defense plays a crucial function in protecting organisms from metal-induced oxidative stress since oxidative damage is typically linked to the generation of reactive oxygen species (ROS) by metals21.

The current study’s goal was to identify the differences of haematological, biochemical parameters, markers of oxidative status and concentration of chemical elements (Al, Cd, Cu, Fe, Li, Sr, Zn, Ni, Pb, Mo, Mn, Ba, Co, Cr, Se and Sb) in blood of horses stabled in two diverse localities in Slovak Republic. Alterations of the health status of horses in the study might be the result of environmental contamination as a comparison of natural location in The National Stud Farm Topoľčianky and city centre location of the Experimental Centre, Institute of Animal Husbandry, SUA in Nitra.

Materials and methods

Blood samples were obtained from horses (n = 21) from two localities in the Slovak Republic during May. In The National Stud Farm Topoľčianky, farm Krásny majer were obtained blood from 11 stallions, consisting of the breeds 6 Lipizzan, 3 Slovak warmblood, 2 Holsteiner, located in a natural location and from the Experimental Centre at Institute of Animal Husbandry, SUA in Nitra from 4 stallions, 5 geldings and 1 mare, consisting of the breeds 3 Slovak warmblood, 4 Czech warmblood, 3 Holsteiner, located in the city centre. All horses were with weight ± 550 kg and in age 4–15 years, housed in stalls (3 × 3.5 m) and were fed a complete feed mixture three times a day (3 kg of oat per horse per day and 1 kg of supplementary compound feed per horse per day) and two times a day with hay (10 kg of hay per horse per day). Water was available ad libitum using waterers. The exercise of horses was ensured by riding exercise once a day and by treadmill exercise or by exercise in the field (Experimental Centre at Institute of Animal Husbandry, SUA in Nitra), exercise of horses from The National Stud Farm Topoľčianky were provided by horse walker or exercise in the field.

Blood collection

Hemos H-02 tubes were used to draw blood from the vena jugularis (10 mL from each horse) and transported in portable refrigerator to the laboratory of Applied Biology, SUA in Nitra. Collected blood was centrifuged for 20 min at 3000 g and thusly obtained serum was then kept at − 20 °C until the realization of oxidative stress and biochemical analyses. For haematological examination, an aliquot (2 mL) of the uncoagulated blood (diluted with EDTA in tubes) was examined within 1 h after collection22,23,24.

Ethical statement

All methods were performed in accordance with the relevant guidelines and regulations. Every horse was bred and carefully handled in compliance with European Community guidelines m. 86/609/ EEC concerning the safety of animals used in experiments, following with the ethical guidelines of the Animal Protection Regulation of the Slovak Republic RD 377/12. Experimental protocol no. 178/2002 was approved by the committee at SUA in Nitra, Slovak Republic.

ARRIVE statement

Animal care protocols were realized in accordance with ARRIVE guidelines25.

Haematological analyses

An automatic haematology analyzer Abacus Vet5 (Diatron MI LDT, Budapest, Hungary) was used to analyse the parameters in whole blood as per the manufacturer indications and the subsequent parameters were noted: WBC—Total count of leukocytes; LYM—Total count of lymphocytes; MID—Mid-size population of monocytes, basophils, eosinophils, blasts, and other immature cells; GRA—Total count of granulocytes; RBC—Total count of erythrocytes; HGB—Haemoglobin; HCT—Haematocrit; MCH—Mean corpuscular haemoglobin; MCHC—Mean corpuscular haemoglobin concentration; PLT—Total count of platelets22.

Biochemical analyses

Total proteins, glucose, urea, phosphorus (P), magnesium (Mg) and calcium (Ca) were analysed using standard commercial kits from DiaSys (Diagnostic Systems GmbH, Holzheim, Germany) on the semi-automated clinical chemistry analyzer Randox RX Monza (Randox Laboratories, Crumlin, UK). Some of the mineral profile potassium (K), sodium (Na) and chloride (Cl) ions were measured using the EasyLytePlus automatic analyser (The Hague, Netherlands) provided with an ion-selective electrode24.

Total oxidant status

The basis for the total oxidant status (TOS) investigation is the oxidation of ferrous ions-o-dianisidine complexes to ferric ions by the sample’s oxidants. The glycerol molecules in the reaction solution facilitated the oxidation reaction process. Consequently, in the reaction solution’s acidic environment, the ferric ions and xylenol orange produced a colorful complex. The total number of oxidant molecules in the sample is represented by the color intensity, which is quantifiable spectrophotometrically. The assay is calibrated using hydrogen peroxide, and the results are expressed as μmol H2O2/L26. First and second reaction solutions (TOS R1 and TOS R2) were freshly prepared. The TOS R1 consisted of 150 μmol xylenol orange disodium salt (Sigma-Aldrich, St. Louis, MO, USA), 140 mmol sodium chloride (Sigma-Aldrich, St. Louis, MO, USA), and 1.35 mol glycerol (Centralchem, Bratislava, Slovakia) in 25 mmol H2SO4 (Sigma-Aldrich, St. Louis, MO, USA). The TOS R2 consisted of 5 mmol ferrous ammonium sulfate hexahydrate (Centralchem, Bratislava, Slovakia), and 10 mmol o-dianisidine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in 25 sulfuric acid (Sigma-Aldrich, St. Louis, MO, USA). Standards (H2O2) and the plasma samples were transferred (35 μL) in pairs to a 96-well plate. After adding 225 μL of TOS R1, the reference reading at 560 nm using the Glomax Multi + Detection System plate reader (Promega, Madison, WI, USA) was realised. Following a 10-min incubation period, 11 μL of TOS R2 was pipetted into each well. Three minutes later, the absorbance was determined using spectrophotometry at the identical wavelength27.

Ferric reducing ability of plasma

Based on the Benzie and Strain28 method, the ferric reducing ability of plasma (FRAP) was determined. The FRAP reagent consists of 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine; Sigma-Aldrich, St. Louis, MO, USA) solution in 40 mmol/L HCl (Centralchem, Bratislava, Slovakia) and 5 mL of 20 mmol/L ferric chloride (Centralchem, Bratislava, Slovakia) and 50 mL of 0.3 mmol/L acetate buffer (pH = 3.6; Centralchem, Bratislava, Slovakia). A Multiskan FC spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland) was used to measure the absorbance of a reagent blank at 593 nm after samples (100 μL) were combined with 3 mL of FRAP reagent. After the samples and standards were co-incubated with the reaction solution for 4 min, the absorbance measurement was repeated. Each sample’s final value was determined using the FRAP equation ΔA = M2 − M129.

Superoxide dismutase

Using a RANDOX assay kit RANSOD (Randox Laboratories, Crumlin, UK), superoxide dismutase (SOD) was measured on the Randox RX Monza (Randox Laboratories, Crumlin, UK) in accordance with the producer’s instructions. The method for measuring SOD employs xanthine and xanthine oxidase (XOD) to generate superoxide radicals, which react with 2-(4-iodophentyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye. SOD activity was measured by the inhibition degree of this reaction at 505 nm and characterized as U/mL of total protein30.

Total antioxidant status

The total antioxidant status (TAS) of a sample is based on each antioxidant’s capacity to neutralize a prooxidant molecule. The TAS Randox (Randox Laboratories, Crumlin, UK) assay follows an incubation of ABTS (2,2′-Azino-di-[3-ethylbenzthiazoline sulphonate]) with a peroxidase (metmyoglobin) and H2O2 to produce the ABTS + radical. This has a rather consistent blue–green color, which may be detected at 600 nm. To a degree proportional to their concentration, antioxidants in the sample inhibit this color generation. Genesys 10 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to measure TAS and following was defined as mM/L of total protein31.

Glutathione peroxidase

The commercially available RANSEL kit (Randox Laboratories, Crumlin, UK) and the Randox RX Monza analyzer (Randox Laboratories, Crumlin, UK) were used to measure the activity of GPx. The decrease in absorbance was measured at 340 nm. The measurement of GPx was based on catalyzation of glutathione with cumene hydroperoxide. With glutathione reductase and nicotinamide adenine dinucleotide phosphate present, the oxidated glutathione immediately changed into its reduced form with the oxidation of NADPH into NADP+. The Gpx activity was stated as U/L of total protein32.

Detection of chemical elements

Using an inductively coupled plasma emission spectrophotometer (ICP Thermo iCAP 7000 Dual, Thermo Fisher Scientific, Waltham, MA, USA), the chemical elements (Al, Cd, Cu, Fe, Li, Sr, Zn, Ni, Pb, Mo, Mn, Ba, Co, Cr, Se and Sb) present in horse blood serum were quantified. For this procedure, all the chemicals used had high purity grade to minimize contamination. Samples (0.5 mL) were digested in the Ethos UP high-performance microwave digestion system (Milestone. Srl, Sorisole, BG, Italy) in 5 mL HNO3 solution (TraceSELECT®, Honeywell Fluka, Morris Plains, NJ, USA) and 1 mL of H2O2 (30% for trace analysis, Merck, Darmstadt, Germany). All samples were mineralized in compliance with the manufacturer’s guidelines, along with a blind sample. The process involves heating and cooling phases. The samples were warmed to 200 °C continuously during the heating phase, which takes 15 min. The temperature was maintained at that level for an additional 15 min, and for the final 15 min, the samples were actively cooled down to 50 °C. After that, the samples were filtered through Sartorius filtration discs (class 390) (Sartorius AG in Goettingen, Germany), and placed into a volumetric flask. Ultraclean water (purity, with a resistivity of 18.2 MΩ.cm) was then added to the flask until 50 mL of solution were obtained, or until additional dilution was required. Dilution was taken into account in the final results compilation. Multielement standard solution V for ICP (CRM–ERM CE278K, Sigma-Aldrich Production GmbH, Buchs, Switzerland) was utilized in the calibration. The validity of the whole procedure was checked by processing of duplicate samples against the certified reference material30,33. Limits of quantification (LOQs) for all assessed elements are highlighted in Table 1.

Table 1 The LOQs for each measured chemical element.
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Statistical analysis

GraphPad 8 (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical analyses. The mean, standard deviation (SD) and coefficient of variation (CV%) calculations were part of the descriptive analysis. All collected data were exanimated for normal Gaussian distribution using a D’Agostino–Pearson normality test and Shapiro–Wilk normality test. The level of significance for the comparative analysis was set at ***(p < 0.001); **(p < 0.01); *(p < 0.05). Pearson correlation between markers of chemical elements, haematological and biochemical parameters was carried out in order to suggest a potential chemical element’s mode of action. Furthermore, the relationship between oxidative status markers and chemical elements was also evaluated using Pearson correlation. Significance was determined at p < 0.05 (a) and at p < 0.01 (A). Heatmaps with clustering were performed to visualize interactions (Pearson correlations coefficients-r)30.

Results

All results of selected haematological parameters of blood are presented in the Table 2. Mean results of white blood cells were 6.78 109/L in The National Stud Farm in Topoľčianky, respectively 6.34 109/L in the Experimental Centre at Institute of Animal Husbandry, SUA Nitra. Mean values of red blood cells were 8.59 1012/L and 8.41 1012/L. No significant changes were observed in the Table 2 between haematological paraments.

Table 2 Haematological parameters of horses blood from two locations.
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Mean concentrations of blood biochemical parameters are reported in Table 3. Significant change was observed in the Na (p < 0.05) concentration.

Table 3 Biochemical blood serum parameters of horses from two locations.
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Values of oxidative status markers are listed in Table 4. Significant change was observed in the TOS (p < 0.05) concentration in blood samples collected from horses in Topoľčianky and Nitra (Table 4).

Table 4 Oxidative status markers of horses blood from two locations.
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Based on the values in Table 5, chemical elements fit into the following order: Fe > Zn > Al > Cu > Cd > Sr > Li. The highest concentration of all measured chemical elements was Fe (383.95 mg/L and 403.61 mg/L, respectively). The lowest measured chemical element was represented by Li (6.11 µg/L and 3.95 µg/L, respectively). Chemical elements Ni, Pb, Mo, Mn, Ba, Co, Cr, Se and Sb, were also analysed in our samples, but their concentrations were found to be below the instrumental detection limits (Table 5).

Table 5 Chemical elements concentrations in horses blood serum from two locations.
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Correlation analysis between chemical elements, haematological and biochemical parameters in The National Stud Farm Topoľčianky are listed in Fig. 1. The negative correlation between Cd and GRA, HGB, HCT (p < 0.01) and RBC (p < 0.05), respectively Cl (p < 0.05) were observed. A significant (p < 0.05) interaction between Cu and MCH was detected. A negative correlation (p < 0.05) was detected among Fe and GRA, RBC, HGB, HCT and Cl. The statistically significant association (p < 0.05) was found between Li and GRA, Sr and RBC as well as with Cl, furthermore among Zn and glucose and Cl.

Fig. 1
figure 1

Correlations: chemical elements versus haematological and biochemical parameters, The National Stud Farm Topoľčianky. a—significant at p < 0.05; A—significant at p < 0.01. WBC total white blood cell count, LYM lymphocytes count, MID cell population of middle dimensions including monocytes and eosinophils, GRA granulocytes count, RBC red blood cell count, HGB hemoglobin, HCT hematocrit, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, PLT Total count of platelets.

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Relationship between Al and LYM, and HCT showed significant positive correlation (p < 0.05). The statistically significant correlation (p < 0.05) was observed among Fe and Ca, Li and K as well as with Cl. A positive correlation (p < 0.01) was recorded between Zn and total proteins (Fig. 2).

Fig. 2
figure 2

Correlations: chemical elements vs. haematological and biochemical parameters, Experimental Centre, Institute of Animal Husbandry, SUA in Nitra. a—significant at p < 0.05; A—significant at p < 0.01. WBC total white blood cell count, LYM lymphocytes count, MID cell population of middle dimensions including monocytes and eosinophils, GRA granulocytes count, RBC red blood cell count, HGB hemoglobin, HCT hematocrit, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, PLT Total count of platelets.

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Correlation analysis of oxidative status markers and chemical elements proved significantly (p < 0.05) negative correlation between TOS and Cd displayed in Fig. 3. A significant positive correlation was revealed between Cd and TOS and GPx in samples from horse blood located in the Experimental Centre, Institute of Animal Husbandry, SUA in Nitra (Fig. 4).

Fig. 3
figure 3

Correlations: oxidative status markers vs. chemical elements, The National Stud Farm Topoľčianky. a—significant at p < 0.05; A—significant at p < 0.01. TOS total oxidant status, FRAP ferric reducing ability of plasma, SOD superoxide dismutase, GPx glutathione peroxidase, TAS total antioxidant status.

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Fig. 4
figure 4

Correlations: oxidative status markers vs. chemical elements, Experimental Centre, Institute of Animal Husbandry, SUA in Nitra. a—significant at p < 0.05; A—significant at p < 0.01. TOS total oxidant status, FRAP ferric reducing ability of plasma, SOD superoxide dismutase, GPx glutathione peroxidase, TAS total antioxidant status.

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Discussion

The amount of heavy metals in the environment has increased as a result of human-caused environmental pollution34. Based on this knowledge, it is crucial to track the amounts of heavy metals in crops35 and the environment36, as well as their effects animals. The main purpose was to determine whether different environments, natural environment of The National Stud Farm in Topoľčianky and the Experimental Centre at Institute of Animal Husbandry, SUA Nitra, which is located in the city centre can effect haematological, biochemical parameters, markers of oxidative status and concentration of chemical elements.

Haematological and biochemical analyses of blood are used in practice as a basic indicator of animal health status23. Pritchard et al.37 in their work defined the reference values of the haematological profile of horses like—WBC 6.0–12.0 109/L and RBC RBC 8.5–12.5 1012/L, among others. From the obtained haematological parameters, we can conclude that the results were in normal reference values during the blood collection period. Present results from haematological examinations are also comparable with data of other authors38.

Present results agree with those declared by Wright et al.39, who obtained very similar results of horse blood serum biochemistry parameters (Ca, Cl, Na, K, glucose, total proteins, urea). Isović et al.40 measured concentrations of total proteins in sport horses before physical activity, what corresponds to our set up of experiment. Their findings showed 72.10 g/L in males, respectively 73.80 g/L in females, which are higher than ours, recording 58.24 g/L (The National Stud Farm Topoľčianky) and 60.65 g/L (Experimental Centre at Institute of Animal Husbandry). However, glucose level detected in our study was similar to the results claimed in the aforementioned study.

Investigation of blood oxidant/antioxidant markers and their effect depending on horse breeds were analysed in study of Kirschvink et al.41. Study observed antioxidant enzymes SOD and GPx, in 4–6 years old horses, achieving results 1268 ± 58 iu/g HB, 275 ± 7 iu/g HB, respectively, while horses older than 6 years had respective SOD and GPx levels at 1023 ± 67 iu/g HB, 276 ± 10 iu/g HB. We have acquired results of SOD (0.02 U/mL TP and 0.01 U/mL TP, respectively) and GPx (32.68 U/L TP and 36.90 U/L TP in Topoľčianky and Nitra, respectively. As Molinari et al.42 reported in their study no significant effect of breed, age and sex were detected on oxidative stress parameters of horses.

TOS levels detected in our horses blood serum samples were lower than those reported by Fazio et al.43 with a concentration 1295.38 ± 394.20 μM, who collected blood in the morning hours before traiting of horses as we realised in our study. Pourmohammad et al.44 observed, that level of FRAP in Arabian horses before exercise was 590.6 ± 18.1 μmol/L, while we observed lower levels in both locations (163.30 µM Fe2+/g TP and 168.80 µM Fe2+/g TP, respectively) and SOD level 1757.0 ± 134.2 U/g Hb in comparison with our results (0.02 U/mL TP and 0.01 U/mL TP, respectively).

Both selected stables are located within 40 kms from nuclear power plant Mochovce, which can be a source of different heavy metals, for example Cd45, moreover, the Nitra region has serious environmental issues because of the long-standing importance of the chemical, coal-mining, and energy industries46. Previous work47 studied certain elements such as Cd, Cu, Pb and Zn in soil from the Nitra region.

Results in the study of Mendiola et al.48, who studied heavy metals in the different biological samples (seminal plasma, blood plasma, whole blood) show no correlation with each other. Furthermore, it is very interesting to evaluate concentrations of heavy metals and other chemical elements in other tissues to understand the different bioaccumulation of individual selected elements in animals27,49,50,51. Ingested cadmium induces the synthesis of metallothioneine, a cysteine-rich protein that can carry cadmium to organs such as the liver and kidneys, and binds to metabolites in tissues, resulting in cell damage52. To a lesser extent, free Cd accumulates in the liver and decreases glutathione synthesis and encourages oxidative cell damage that causes a large amount of necrosis and apoptosis in the hepatocytes53. In addition to interfering with the metabolism of calcium and vitamin D, cadmium accumulation can cause bone demineralization, which can result in bone loss. However, these toxicities are very rare, since most species absorb just around 2% of ingested cadmium enterally, the majority of cadmium binds to enterocyte metallothionein and as enterocytes ripen, it is exfoliated and excreted in the faeces54. Our results confirm negative effect of Cd on haematological parameters as well as on oxidative markers.

In study of Nava et al.3 who monitored environmental risk of trace elements from horse blood from different Sicily areas and their impact on health and welfare, however our main topic of study was to compare two different locations and correlation of oxidative status markers with chemical elements as well as with haematological and biological parameters. Studied horse whole blood elements from different Sicily areas, with a concentration of Al (0.058–0.442 mg/L), Fe (0.504–18.762 mg/L), Zn (0.050–0.174 mg/L). The levels were lower than the results obtained in our study, however levels of Sr (0.193–1.012 mg/L) were higher than observed in both locations in our study.

Giannetto et al.55 states in his study that Ni concentrations determined in the blood of animals can be considered low, also in our study these values were below detection limits. However, this element is often present in horse feed and hay, so it is important to determine the levels of Ni in horses to evaluate the state of the health status of animal and rule out potential Ni intoxication.

The mean concentrations of Cu (688.7 µg/L and 763.10 µg/L) and Zn (1.99 mg/L and 2.10 mg/L) in the blood serum samples of this research were higher than those observed in horse blood serum from Brazil shown by Maia et al.4. Another study by Souza et al.56 from Brazil examined levels of Cu and Zn in horse blood serum from two different localities (non-industrial and industrial area). Concentrations of Cu and Zn were higher than observed by Maia et al.4, however lower than obtained in our study.

Horses located near the industrialized area of Milazzo and horses located in Nebrodi mountains, which is non-industrialized part of Sicily had higher concentrations of Li (0.056 mg/kg and 0.054 mg/kg) and Sr (3.803 mg/kg and 3.770 mg/kg) in blood, compared to what we detected in our study14.

Brummer-Holder et al.57 reported a range of whole blood Fe values (439.90 ± 48.9 mg/L) for horses. It is similar to what we have reported here, however, the horse whole blood Cu values (1.543 ± 0.52 mg/L) were lower than we detected in blood serum.

Impact of essential and risk elements on the health status of domestic animals are not often studied. As a result, the information gathered might be used as a control indication to identify specific hazardous risks associated with the presence of risk elements on the health of animals.

Conclusions

The acquisition of new knowledge in the field of biochemistry, haematology, molecular markers and their correlation with toxicants is crucial nowadays, especially with rapidly evolving industrial production. Results of this study demonstrate that the negative correlation between Cd and GRA, HGB and HCT was observed in The National Stud Farm Topoľčianky and a positive correlation was recorded between Zn and total proteins in the Experimental Centre at Institute of Animal Husbandry, SUA Nitra. The obtained results show the differing significant association between chemical elements in horse blood and haematological, biochemical parameters as well as oxidative status markers. This highlights the significance of environmental effect on animal health. In order to increase knowledge of the environmental burden and its impact on health status of horses, in the future we recommend increasing the number of locations.