Analysis of the content of chemical elements in horse hooves

Analysis of the content of chemical elements in horse hooves | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of the content of chemical elements in horse hooves Karolina Zaworska, Agnieszka Drożdżyńska, Wojciech Juzwa, Maria Nabzdyk, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2867145/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract The research was aimed at analysing the content of chemical elements in horse hooves and exploring their influence on hoof brittleness and the formation of laminitis. Moreover, the activity of the biofilm cells covering the horse's hoof was assessed and its microscopic structure was explored. The material used for the research was the hooves of Polish-Konik horses and sport horses. In order to determine the content of chemical elements, the method of atomic absorption spectrometry was used. The activity of biofilm cells was assessed using flow cytometry. The research showed significant differences in the content of potassium, manganese, iron and aluminum, as well as high zinc deficiencies. For this reason, it is recommended to add the preparations for horses with poor hoof quality with potassium and zinc, and to limit the consumption of manganese and aluminum. Assessment of the activity of the biofilm cells showed that it is mainly composed of dead cells, however the activity differed depending on the type of horse. The microscopic structure analysis shows differences in the number and size of the hoof tubules. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Horses, belonging to solipeds, have been present in human life for thousands of years. Their domestication dates back to around 3,500 BC (Outram et al. 2009 ). Since then, horses have been used by humans incl. for transport, economic or sports purposes. Among horses, there are many breeds and types that are diverse in terms of morphology, as well as physiology and biochemistry (Komosa et al. 2006 ). The hoof is the finger organ of ungulates, characterized by a keratinized epidermis. The horn wall of the hoof is hard at the same time and flexible, and the organ itself is light. The horn flask constructed in this way provides protection of the finger when it comes into contact with the ground (Kolstrug et al. 2012). Due to the multitude of factors to which the hoof is exposed, it faces many challenges, such as preventing mechanical or thermal injuries, as well as receiving stimuli from the ground (Królak et al. 2019). A common problem in horses is the brittleness and brittleness of the hoof horn. This ailment can lead to orthopedic diseases such as fractures of the hoof wall (Strzelec et al. 2017 ). This problem mainly concerns horses of sports breeds, as they are the ones most vulnerable to mechanical damage. Another ailment that horses often struggle with is laminitis (Latin: laminitis ), i.e. diffuse hoof inflammation. Laminitis is classified as a group of diseases due to the presence of comorbidities (Witkowska et al. 2016 ). There are many causes of laminitis, including improper diet or mechanical damage to the hoof (Bergsten 2003 ). It is suspected that both of the above-mentioned diseases may be caused by disturbances in the elemental composition of the hooves. The general mineral composition of horse hooves is known, but there are differences in the hoof types of horses (Sargentini et al. 2015 ). This work aims to determine whether the content of elements and bacterial biofilm in horse's hoofs affect their fragility and whether they are tied with the formation of laminitis. Material and Methods Material The hooves of Polish and sports horses were investigated. The control sample consisted of 10 representatives of Polish horses from the Sieraków stable, while the research sample consisted of 5 sports horses from the Brzeźno stable. Animals of one breed, coming from the same stable, were fed in the same way. The hooves were collected by a blacksmith using a traditional hoof shearing procedure. The obtained samples are taken from the hoof wall. Methods Atomic Absorption Spectometry In the aim to carried out the analyzes of chemical elements by Atomic Absorption Spectometry hooves were placed in portions of 600–750 mg in plastic Falcon tubes. Tubes were labeled as appropriate and stored in a chill (three portions of all hooves were stored). Hooves mineralization were performed by addition of 750 µl of mineralization solution consisting of nitric acid (V) and hydrogen peroxide in the proportion 2:1 to all samples. In the next stage, the samples were placed in an oven for 5 hours at a temperature of 150°C. After the samples had cooled down, to the vassels were added 24.2 ml of distilled water. The diluted samples were then transferred to previously prepared and appropriately described 50 ml Falcon tubes. To determine the chemical elements in the horses' hooves, an AAnalyst 600 spectrometer with the use of atomization in a graphite furnace and Zeeman background correlation was used. The determinations of 13 chemical elements both macro (calcium, potassium, magnesium, and sodium) and micro-elements (iron, copper, zinc, aluminum, chromium, and manganese) were made using the external standard method. WinLab32 for AA (version 7.3.0.0697) was used to analyze the samples. Lamps adapted to the study of specific chemical elements were used for the analysis. Graphite cuvettes were used. The standards for the investigated minerals were made of PerkinElmer Pure VIII, which contained each element at a concentration of 1000 µg/l. The Atomic Absorption Modifier Solution reagents for the preparation of modifiers contained magnesium nitrate (V) and palladium nitrate (V), respectively. The concentration of Mg and Pd in ​​the preparations dedicated to these elements was equal 10,000 mg / l. The final volume of the analyzed sample was 20 µl for elements that do not require the addition of a modifier and 25 µl for elements for which a modifier is required to be determined. Both before and after the analysis of the samples, a calibration curve for the indicated element was prepared, and the concentration of the standard as well as the purity of water and an empty graphite cuvette were checked. Flow cytometry For flow cytometry hooves samples were ground in a mortar with liquid nitrogen. After grinding the hooves to powder, 1% PBS solution was added in a volume of 1 ml for horses no 1–12 and 1.5 ml for horses no 13–15 and rubbed to liquefy. The samples were centrifuged (2000 rpm, 5 min). Than 300 µl of supernatant was collected, which was filtered through a 100 µm Nylon Net Filter, into newly prepared Eppendorf tubes. Then the samples were centrifuged for (30 s; 2000 rpm) and 250 µl of supernatant was collected. In order to determine the activity of the hoof biofilm cells, 0.5 µl of Kalcein AM dyes was added, which staining active cells for green, and BD 660 Horizon that staining dead cells for red. Subsequently, the samples were mixed in a mini-vortex and left for 10 minutes in the dark. After incubation the samples were remixed in a mini-vortex and then cell activity was determined by flow cytometry. The analysis was performed on a Becton Dickinson brand BD FACSAria ™ III cell sorter. The results of the research were analyzed in order to compare the activity of the biofilm covering the hooves of Polish horses and sports horses. Microscopic preparations In order to study the microscopic structure of the horse's finger organ, preparations were made on the basis of cut hooves. The tests were observed under a Zeiss light microscope at a magnification of 400x. On the basis of the photos taken of the microscope slides, hooves tubes in the field of view were calculated for each horse. The results were averaged and then analyzed to compare the structure of the hoof wall of Polish horses and sports horses. Statistical analysis From the calibration curve, the concentration of each element in the test sample was calculated. On the basis of the obtained results, the mean content of individual elements in 1 g of the hoof (µg / g) was determined together with the standard deviation. In order to determine the significance of differences in the content of minerals in the hooves of Polish and sports horses, the Student's t-test was performed for independent samples. The level of significance was α = 0.8. The number of degrees of freedom k was 13. The critical value was read from the t-Student distribution table, and then the critical area was determined. Results Atomic Absorption Spectometry In order to determine the content of test elements in the hooves (macro- as well as microelements), the method of atomic absorption spectometry was used. The average values ​​of the content of the examined elements for sports and Polish horses are presented in Figs. 1 and 2. As a result of assessment of macroelements concentration, it was observed that that the hooves of Polish horses are richer in potassium and calcium in relation to the hooves of sports horses. However, there were no significant differences in the content of sodium and magnesium (p < 0.05). The analyzes of microelements showed that Polish horses' hooves reflected higher zinc and copper concentrations, while sports horses' hooves are characterized by a higher content of manganese, iron, aluminum and chromium (Fig. 2a, 2b). Furthermore, to insightfully determine differences in the content of elements, the critical area was determined in the ranges (-∞; -0.259) ∪ (0.295; + ∞). The critical area included the values ​​calculated for potassium, manganese, iron and aluminum, therefore the differences in the content of these elements in the hoofs of Polish and sport horses are statistically significant. Flow cytometry The method of flow cytometry was used to determine the activity of the biofilm covering the horses' hooves (Fig. 3, Fig. 5). Figures 3 shows the stained cells of the biofilm covering the hooves of Polish horses. Independently whether single cells or cell aggregates were tested, the biofilm stained mostly red. Therefore, it was found that the hooves of the Polish horses were covered with a biofilm characterized mainly by dead (inactive) cells. The Fig. 4 shows the activity distribution of all cells present in the biofilm of the Polish horse Poeta (horse no 1). Cells were classified according to activity into active, medium activity, and dead cells. It was observed that the biofilm of the Polish horses consisted mainly of from dead cells. On the other hand, active cells were the least numerous group. The activity of cell aggregates building the biofilm covering the hooves of sport horses is shown in Fig. 5. A comparable number of green stained active cells and red glowing dead cells were observed. The activity of single biofilm cells differed from that of aggregates. Indeed, single cells were stained primarily green and therefore active. Figure 6 shows the activity distribution of cells covering the biofilm of sports horses for the example of horse 14 (no 263). It has been observed that most of the biofilm are dead cells, while the number of active and mid-active cells is comparable. Generally, the research showed that hoof biofilm was mainly composed of dead cells, but their percentage was greater in the hooves of Polish horses (73.26%) than in the hooves of sports horses (58.65%). Cells with medium activity created a hoof biofilm at a similar level in the case of both breeds of horses (18.85% in the case of Polish horses and 16.83% in the case of sports horses). The lowest percentage of biofilm cells were active cells. However, they had a higher percentage in the hoofs of sports horses (9.45%) than in the hooves of Polish horses (0.27%). Microscopic structure of the hoof Figure 7 shows a photo of a microscopic specimen of a Polish horse's hoof. The tubes in one field of view at a magnification of 400x are observed (horse no 3, name: Tasak 142). Tubular cores were observed to be of similar diameter and surrounded by a thick layer of keratinocytes. The adipoid substance constituted about half of the microscopic image. In a microscopic specimen of a sport horse's hoof (horse no 14), it was observed that the cores of the tubules forming the hoof had different sizes in diameter, and the tubes were surrounded by a relatively thick layer of keratinocytes. On the other hand, the intermarital substance constituted about half of the microscopic image (Fig. 8). Finally, the mean number of tubes observed under the microscope in one field of view at a magnification of 400x for each horse is summarized (Fig. 9). In the speciments from the hoofs of Polish horses, the number of tubes was between 20–29, with an average of 24. On the other hand, in the case of sports horses, the number of tubules observed on the microscopic image was 24–46, with an average of 34. Discussion Research on the content of the chemical elements in hooves has been the subject of research by many scientific teams. So far, research has focused on warmblooded horses' hooves (Lancaster et al. 2013 ; Sargentini et al. 2015 ). Studies on the basis of flow cytometry showed differences between the activity of biofilm on the hooves of Polish and sports horses. Sport horses' biofilm cells were more active. Another difference between Polish primitive horse and sport horses was the number of hoof tubese. So far research has focused on density of the hoof tubese. Lancaster et al. ( 2013 ) indicated that in the case of racing and wild horses. In the case of the sport horses the number of the hoof tubese was greater in comparison to Polish horses. However, the hoof tubese of Polish horses were characterized by a larger diameter. Furthermore, in the case of the sports horses the hoof tubese was smaller. However, in both cases, the hoof tubes made up half the field of view. The differences in the activity of biofilm cells hooves of different horse types may indicate that the microflora of the hooves of polish horses may be different that the hooves of sports horses. Similarly, our research found some differences in the number of hoof tubes. In the case of sport horses the number of the tubes was higher than the Polish horses. The previous research was aimed at determining the density of the hoof tubes in individual layers of the hoof wall. Lancaster et al. ( 2013 ) indicate that tube density was similar for wild and race horses, but lower than for recreational horses. Our research indicated that despite the differences in the mean number of hoof tubes in the field of view, the tube density is similar. In the case of polish horses, the tubes had a larger diameter. In the case of sports horses hooves the number of tubes was higher, however the tubes was smaller. It should be noted that in both cases, the hoof tubes took up a half of the image in one field of view. This observation is in line with the previous research BY Lancaster et al. ( 2013 ) Regardless of the breed, the biofilm of horses consists mainly of dead cells, however, the cells of the biofilm of sport horses are more active. This condition can negatively affect the quality of the hoof due to the metabolites produced by the microorganisms. Summarizing, the hooves of Polish and sports horses differed in the number of tubes observed in one field of view, as well as in size. However, the tubular density was similar. Statistically significant differences in the content of potassium, manganese and aluminum affect positively to fragility of sports horses hooves and may predispose to cracks and laminitis. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding Not applicable Authors' contributions Conceptualization, K.S.-L.; methodology, A.D., K.S.-L. K.D-M., W.J.; validation, K.S.-L., T.U., M.N.; formal analysis, K.S.-L., A.G., W.J..; investigation, K.S.-L., T.U., A.D., W.J., K.D.-M., M.N.; resources, K.S.-L..; data curation, K.S.-L.; writing—original draft preparation, K.S.-L..; writing—review and editing, K.S.-L.; visualization, K.S.-L.; supervision, K.S.-L.; project administration, K.S.-L.. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable Authors' information (optional) Not applicable References Bergsten C, Causes (2003) Risk Factors, and Prevention of Laminitis and Related Claw Lesions. Acta Vet Scand 44(1):157 de Souza AF, Schade J, Laus R, Moreira MA, Muller TR, Fonteque JH (2019) Differences in mineral concentrations on hooves of horses, mules and donkeys. Rev Brasil de Ciência Vet 26:31–39 Kolstrung R, Ciesielczuk K, Silmanowicz P (2012) The rate of hoof wall growth of Małopolska horses in the spring and summer season. (In Polish: Tempo przyrastania ścian kopyt koni małopolskich w sezonie wiosenno-letnim).Med. Wet.,4 Komosa M, Molinski K, Godynicki S (2006) The Variability of Cranial Morphology in Modern Horses. Zoolog Sci 23(3):289–298 Królak K, Łojek J, Albera-Łojek A Influence of the keeping system on the growth of the hoof capsule of horses. Zootechnical news (In Polish: Wpływ systemu utrzymania na przyrost puszki kopytowej koni.Wiadomości zootechniczne, 2019, R. LVII( 3 ),63–72 Lancaster LS, Bowker RM, Mauer WA (2013) Equine hoof wall tubule density and morphology. J Vet Med Sci 75(6):773–778 Outram AK, Stear NA, Bendrey R, Olsen S, Kasparov A, Zaibert V, Thorpe N, Evershed RP (2009) The Earliest Horse Harnessing and Milking Science 323(5919):1332–1335 Sargentini C, Tocci R, Pezzati A, Benvenuti D, Martini A (2015) Morphological, chemical-physical and mineralogical characteristics on hoof of Anglo-Arabian and Maremmano Horses and discriminant analysis (PCA) on mineral content. Global J of Animal Sci 3(4):164–171 Strzelec K, Liss M, Jaroszyńska K, Janczarek I, Górski K (2017) Orthopedic diseases of racing and sport horses. (In Polish: Schorzenia ortopedyczne koni wyścigowych i sportowych). 4:16–21 Witkowska O, Turło A, Michlik K, Cywińska A (2016) Horse laminitis - etiopathogenesis, symptoms and treatment. Veterinary Life. In Polish: Ochwat koni – etiopatogeneza, objawy i leczenie. Życie Weterynaryjne 91(4):231–235 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2867145","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":473167769,"identity":"04b10713-dc7e-46f6-a3f1-034c4f7297d4","order_by":0,"name":"Karolina Zaworska","email":"","orcid":"","institution":"Poznan University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Zaworska","suffix":""},{"id":473167772,"identity":"cfef2243-e312-4399-86f5-bfcde0e79eeb","order_by":1,"name":"Agnieszka Drożdżyńska","email":"","orcid":"","institution":"Poznan University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Drożdżyńska","suffix":""},{"id":473167773,"identity":"352128fa-ec5b-4ba5-bef6-72eb28aa5e29","order_by":2,"name":"Wojciech Juzwa","email":"","orcid":"","institution":"Poznan University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Juzwa","suffix":""},{"id":473202791,"identity":"ec3dec07-aa4f-469a-9d81-c047eb47059f","order_by":3,"name":"Maria Nabzdyk","email":"","orcid":"","institution":"Poznan University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Nabzdyk","suffix":""},{"id":473202792,"identity":"7384b7be-8f1a-408b-a971-2a69cc405508","order_by":4,"name":"Katarzyna Serwańska-Leja","email":"data:image/png;base64,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","orcid":"","institution":"Poznan University of Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Serwańska-Leja","suffix":""}],"badges":[],"createdAt":"2023-04-27 07:14:20","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-2867145/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2867145/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85312598,"identity":"2454c11b-4d6b-426c-9d86-c30bceceed81","added_by":"auto","created_at":"2025-06-24 13:54:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23931,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the content of macroelements in the hooves of Polish horses (horses 1-10) and sports horses (horses 11-15) per 1g of hoof (μg / g)\u003c/p\u003e","description":"","filename":"Onlinefig.1..png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/f82e4eb440f118f5b1b539b4.png"},{"id":85312596,"identity":"89084884-8853-49fb-bea3-80d745cba437","added_by":"auto","created_at":"2025-06-24 13:54:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35686,"visible":true,"origin":"","legend":"\u003cp\u003ea Comparison of the content of microelements present in the hoof at a level of \u0026lt;15 µg/g, in the hooves of Polish horses and sports horses per 1g of hoof (µg/g)\u003c/p\u003e\n\u003cp\u003eb Comparison of the content of micronutrients present in the hoof at a level\u0026gt; 15 µg / g, in the hooves of Polish horses and sports horses per 1g of hoof (µg/g)\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/277aabfdc9921dd8d58f6db6.png"},{"id":85313343,"identity":"5869ea27-e964-4210-bc61-e7866ef88f4f","added_by":"auto","created_at":"2025-06-24 14:02:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105699,"visible":true,"origin":"","legend":"\u003cp\u003eActivity of cell aggregates forming a biofilm covering the hooves of Polish horses on the example of horse 1 (name: Poeta)\u003c/p\u003e","description":"","filename":"Onlinefig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/ab7fe5b3ec30275b48d8f8c4.png"},{"id":85313342,"identity":"7e622771-d0f6-43d1-9e36-61544d291abe","added_by":"auto","created_at":"2025-06-24 14:02:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56891,"visible":true,"origin":"","legend":"\u003cp\u003eActivity of biofilm cells covering the hooves of Polish horses on the example of studied horse no. 1 (Poet) divided into active cells (active), mid-active and dead depending on the intensity of the dyes glow\u003c/p\u003e","description":"","filename":"Onlinefig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/137e94612ee016642badb4fb.png"},{"id":85314365,"identity":"85eccd3d-600a-4f66-8efc-a25625120852","added_by":"auto","created_at":"2025-06-24 14:10:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81234,"visible":true,"origin":"","legend":"\u003cp\u003eThe activity of cell aggregates forming the biofilm covering the hooves of sports horses on the example of a horse 14 (no 263)\u003c/p\u003e","description":"","filename":"Onlinefig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/5905f12dd7c0ad4be22e3694.png"},{"id":85314702,"identity":"f130c94a-608f-47bc-9b6b-c43244e9439f","added_by":"auto","created_at":"2025-06-24 14:18:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60371,"visible":true,"origin":"","legend":"\u003cp\u003eActivity of biofilm cells covering the hooves of sport horses on the example of horse 14 (no 263) with division into active cells (active), mid-active and dead depending on the intensity of the dyes glow\u003c/p\u003e","description":"","filename":"Onlinefig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/c03eafddd7777ba6d30356b5.png"},{"id":85312601,"identity":"42005053-af01-41f3-9fc3-7ee1bc91e4ac","added_by":"auto","created_at":"2025-06-24 13:54:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":155320,"visible":true,"origin":"","legend":"\u003cp\u003eTubes in the hoof of Polish horse (horse 3, name: Tasak 142) (magnification: 400x).\u003c/p\u003e","description":"","filename":"Onlinefig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/261bb191ca26995b5219fe56.png"},{"id":85313344,"identity":"864a4f4b-8953-4f61-9861-1036e97b43d0","added_by":"auto","created_at":"2025-06-24 14:02:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":75402,"visible":true,"origin":"","legend":"\u003cp\u003eTubes in the hoof of sport horse (horse no. 14) (magnification: 400x).\u003c/p\u003e","description":"","filename":"Onlinefig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/579279cbd8aa82a444cad777.png"},{"id":85312600,"identity":"221f8af0-3361-4d4f-8fe2-c1fee2776b38","added_by":"auto","created_at":"2025-06-24 13:54:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":25195,"visible":true,"origin":"","legend":"\u003cp\u003eAverage number of tubes observed under the microscope, in one field of view with 400x magnification, in the hooves of Polish horses (horses 1-10) and sports horses (horses 11-15)\u003c/p\u003e","description":"","filename":"Onlinefig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/875fec0d28a3e89b3c27d356.png"},{"id":85314703,"identity":"4a710576-56f1-402f-b886-769fd2d68d4b","added_by":"auto","created_at":"2025-06-24 14:18:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1173138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2867145/v2/4424047c-24ae-4053-bb99-b6b76bcaf8de.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Analysis of the content of chemical elements in horse hooves","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHorses, belonging to solipeds, have been present in human life for thousands of years. Their domestication dates back to around 3,500 BC (Outram et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Since then, horses have been used by humans incl. for transport, economic or sports purposes. Among horses, there are many breeds and types that are diverse in terms of morphology, as well as physiology and biochemistry (Komosa et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe hoof is the finger organ of ungulates, characterized by a keratinized epidermis. The horn wall of the hoof is hard at the same time and flexible, and the organ itself is light. The horn flask constructed in this way provides protection of the finger when it comes into contact with the ground (Kolstrug et al. 2012). Due to the multitude of factors to which the hoof is exposed, it faces many challenges, such as preventing mechanical or thermal injuries, as well as receiving stimuli from the ground (Kr\u0026oacute;lak et al. 2019). A common problem in horses is the brittleness and brittleness of the hoof horn. This ailment can lead to orthopedic diseases such as fractures of the hoof wall (Strzelec et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This problem mainly concerns horses of sports breeds, as they are the ones most vulnerable to mechanical damage.\u003c/p\u003e \u003cp\u003eAnother ailment that horses often struggle with is laminitis (Latin: \u003cem\u003elaminitis\u003c/em\u003e), i.e. diffuse hoof inflammation. \u003cem\u003eLaminitis\u003c/em\u003e is classified as a group of diseases due to the presence of comorbidities (Witkowska et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). There are many causes of laminitis, including improper diet or mechanical damage to the hoof (Bergsten \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). It is suspected that both of the above-mentioned diseases may be caused by disturbances in the elemental composition of the hooves. The general mineral composition of horse hooves is known, but there are differences in the hoof types of horses (Sargentini et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis work aims to determine whether the content of elements and bacterial biofilm in horse's hoofs affect their fragility and whether they are tied with the formation of laminitis.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003eThe hooves of Polish and sports horses were investigated. The control sample consisted of 10 representatives of Polish horses from the Sierak\u0026oacute;w stable, while the research sample consisted of 5 sports horses from the Brzeźno stable. Animals of one breed, coming from the same stable, were fed in the same way. The hooves were collected by a blacksmith using a traditional hoof shearing procedure. The obtained samples are taken from the hoof wall.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAtomic Absorption Spectometry\u003c/h2\u003e \u003cp\u003eIn the aim to carried out the analyzes of chemical elements by Atomic Absorption Spectometry hooves were placed in portions of 600\u0026ndash;750 mg in plastic Falcon tubes. Tubes were labeled as appropriate and stored in a chill (three portions of all hooves were stored). Hooves mineralization were performed by addition of 750 \u0026micro;l of mineralization solution consisting of nitric acid (V) and hydrogen peroxide in the proportion 2:1 to all samples. In the next stage, the samples were placed in an oven for 5 hours at a temperature of 150\u0026deg;C. After the samples had cooled down, to the vassels were added 24.2 ml of distilled water. The diluted samples were then transferred to previously prepared and appropriately described 50 ml Falcon tubes.\u003c/p\u003e \u003cp\u003eTo determine the chemical elements in the horses' hooves, an AAnalyst 600 spectrometer with the use of atomization in a graphite furnace and Zeeman background correlation was used.\u003c/p\u003e \u003cp\u003eThe determinations of 13 chemical elements both macro (calcium, potassium, magnesium, and sodium) and micro-elements (iron, copper, zinc, aluminum, chromium, and manganese) were made using the external standard method. WinLab32 for AA (version 7.3.0.0697) was used to analyze the samples. Lamps adapted to the study of specific chemical elements were used for the analysis. Graphite cuvettes were used. The standards for the investigated minerals were made of PerkinElmer Pure VIII, which contained each element at a concentration of 1000 \u0026micro;g/l. The Atomic Absorption Modifier Solution reagents for the preparation of modifiers contained magnesium nitrate (V) and palladium nitrate (V), respectively. The concentration of Mg and Pd in ​​the preparations dedicated to these elements was equal 10,000 mg / l.\u003c/p\u003e \u003cp\u003eThe final volume of the analyzed sample was 20 \u0026micro;l for elements that do not require the addition of a modifier and 25 \u0026micro;l for elements for which a modifier is required to be determined. Both before and after the analysis of the samples, a calibration curve for the indicated element was prepared, and the concentration of the standard as well as the purity of water and an empty graphite cuvette were checked.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFor flow cytometry hooves samples were ground in a mortar with liquid nitrogen. After grinding the hooves to powder, 1% PBS solution was added in a volume of 1 ml for horses no 1\u0026ndash;12 and 1.5 ml for horses no 13\u0026ndash;15 and rubbed to liquefy. The samples were centrifuged (2000 rpm, 5 min). Than 300 \u0026micro;l of supernatant was collected, which was filtered through a 100 \u0026micro;m Nylon Net Filter, into newly prepared Eppendorf tubes. Then the samples were centrifuged for (30 s; 2000 rpm) and 250 \u0026micro;l of supernatant was collected. In order to determine the activity of the hoof biofilm cells, 0.5 \u0026micro;l of Kalcein AM dyes was added, which staining active cells for green, and BD 660 Horizon that staining dead cells for red. Subsequently, the samples were mixed in a mini-vortex and left for 10 minutes in the dark. After incubation the samples were remixed in a mini-vortex and then cell activity was determined by flow cytometry. The analysis was performed on a Becton Dickinson brand BD FACSAria \u0026trade; III cell sorter. The results of the research were analyzed in order to compare the activity of the biofilm covering the hooves of Polish horses and sports horses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic preparations\u003c/h2\u003e \u003cp\u003eIn order to study the microscopic structure of the horse's finger organ, preparations were made on the basis of cut hooves. The tests were observed under a Zeiss light microscope at a magnification of 400x. On the basis of the photos taken of the microscope slides, hooves tubes in the field of view were calculated for each horse. The results were averaged and then analyzed to compare the structure of the hoof wall of Polish horses and sports horses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFrom the calibration curve, the concentration of each element in the test sample was calculated. On the basis of the obtained results, the mean content of individual elements in 1 g of the hoof (\u0026micro;g / g) was determined together with the standard deviation. In order to determine the significance of differences in the content of minerals in the hooves of Polish and sports horses, the Student's t-test was performed for independent samples. The level of significance was α\u0026thinsp;=\u0026thinsp;0.8. The number of degrees of freedom k was 13. The critical value was read from the t-Student distribution table, and then the critical area was determined.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAtomic Absorption Spectometry\u003c/h2\u003e \u003cp\u003eIn order to determine the content of test elements in the hooves (macro- as well as microelements), the method of atomic absorption spectometry was used. The average values ​​of the content of the examined elements for sports and Polish horses are presented in Figs.\u0026nbsp;1 and 2. As a result of assessment of macroelements concentration, it was observed that that the hooves of Polish horses are richer in potassium and calcium in relation to the hooves of sports horses. However, there were no significant differences in the content of sodium and magnesium (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analyzes of microelements showed that Polish horses' hooves reflected higher zinc and copper concentrations, while sports horses' hooves are characterized by a higher content of manganese, iron, aluminum and chromium (Fig.\u0026nbsp;2a, 2b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, to insightfully determine differences in the content of elements, the critical area was determined in the ranges (-\u0026infin;; -0.259) \u0026cup; (0.295; + \u0026infin;). The critical area included the values ​​calculated for potassium, manganese, iron and aluminum, therefore the differences in the content of these elements in the hoofs of Polish and sport horses are statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eThe method of flow cytometry was used to determine the activity of the biofilm covering the horses' hooves (Fig.\u0026nbsp;3, Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eFigures 3 shows the stained cells of the biofilm covering the hooves of Polish horses. Independently whether single cells or cell aggregates were tested, the biofilm stained mostly red. Therefore, it was found that the hooves of the Polish horses were covered with a biofilm characterized mainly by dead (inactive) cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;4 shows the activity distribution of all cells present in the biofilm of the Polish horse Poeta (horse no 1). Cells were classified according to activity into active, medium activity, and dead cells. It was observed that the biofilm of the Polish horses consisted mainly of from dead cells. On the other hand, active cells were the least numerous group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe activity of cell aggregates building the biofilm covering the hooves of sport horses is shown in Fig.\u0026nbsp;5. A comparable number of green stained active cells and red glowing dead cells were observed. The activity of single biofilm cells differed from that of aggregates. Indeed, single cells were stained primarily green and therefore active.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;6 shows the activity distribution of cells covering the biofilm of sports horses for the example of horse 14 (no 263). It has been observed that most of the biofilm are dead cells, while the number of active and mid-active cells is comparable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenerally, the research showed that hoof biofilm was mainly composed of dead cells, but their percentage was greater in the hooves of Polish horses (73.26%) than in the hooves of sports horses (58.65%). Cells with medium activity created a hoof biofilm at a similar level in the case of both breeds of horses (18.85% in the case of Polish horses and 16.83% in the case of sports horses). The lowest percentage of biofilm cells were active cells. However, they had a higher percentage in the hoofs of sports horses (9.45%) than in the hooves of Polish horses (0.27%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic structure of the hoof\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;7 shows a photo of a microscopic specimen of a Polish horse's hoof. The tubes in one field of view at a magnification of 400x are observed (horse no 3, name: Tasak 142). Tubular cores were observed to be of similar diameter and surrounded by a thick layer of keratinocytes. The adipoid substance constituted about half of the microscopic image.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a microscopic specimen of a sport horse's hoof (horse no 14), it was observed that the cores of the tubules forming the hoof had different sizes in diameter, and the tubes were surrounded by a relatively thick layer of keratinocytes. On the other hand, the intermarital substance constituted about half of the microscopic image (Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, the mean number of tubes observed under the microscope in one field of view at a magnification of 400x for each horse is summarized (Fig.\u0026nbsp;9). In the speciments from the hoofs of Polish horses, the number of tubes was between 20\u0026ndash;29, with an average of 24. On the other hand, in the case of sports horses, the number of tubules observed on the microscopic image was 24\u0026ndash;46, with an average of 34.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eResearch on the content of the chemical elements in hooves has been the subject of research by many scientific teams. So far, research has focused on warmblooded horses' hooves (Lancaster et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sargentini et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Studies on the basis of flow cytometry showed differences between the activity of biofilm on the hooves of Polish and sports horses. Sport horses' biofilm cells were more active. Another difference between Polish primitive horse and sport horses was the number of hoof tubese. So far research has focused on density of the hoof tubese. Lancaster et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) indicated that in the case of racing and wild horses. In the case of the sport horses the number of the hoof tubese was greater in comparison to Polish horses. However, the hoof tubese of Polish horses were characterized by a larger diameter. Furthermore, in the case of the sports horses the hoof tubese was smaller. However, in both cases, the hoof tubes made up half the field of view. The differences in the activity of biofilm cells hooves of different horse types may indicate that the microflora of the hooves of polish horses may be different that the hooves of sports horses. Similarly, our research found some differences in the number of hoof tubes. In the case of sport horses the number of the tubes was higher than the Polish horses. The previous research was aimed at determining the density of the hoof tubes in individual layers of the hoof wall. Lancaster et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) indicate that tube density was similar for wild and race horses, but lower than for recreational horses. Our research indicated that despite the differences in the mean number of hoof tubes in the field of view, the tube density is similar. In the case of polish horses, the tubes had a larger diameter. In the case of sports horses hooves the number of tubes was higher, however the tubes was smaller. It should be noted that in both cases, the hoof tubes took up a half of the image in one field of view. This observation is in line with the previous research BY Lancaster et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) Regardless of the breed, the biofilm of horses consists mainly of dead cells, however, the cells of the biofilm of sport horses are more active. This condition can negatively affect the quality of the hoof due to the metabolites produced by the microorganisms. Summarizing, the hooves of Polish and sports horses differed in the number of tubes observed in one field of view, as well as in size. However, the tubular density was similar. Statistically significant differences in the content of potassium, manganese and aluminum affect positively to fragility of sports horses hooves and may predispose to cracks and laminitis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eConceptualization, K.S.-L.; methodology, A.D., K.S.-L. K.D-M., W.J.; validation, K.S.-L., T.U., M.N.; formal analysis, K.S.-L., A.G., W.J..; investigation, K.S.-L., T.U., A.D., W.J., K.D.-M., M.N.; resources, K.S.-L..; data curation, K.S.-L.; writing\u0026mdash;original draft preparation, K.S.-L..; writing\u0026mdash;review and editing, K.S.-L.; visualization, K.S.-L.; supervision, K.S.-L.; project administration, K.S.-L.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; information (optional)\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBergsten C, Causes (2003) Risk Factors, and Prevention of Laminitis and Related Claw Lesions. Acta Vet Scand 44(1):157\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Souza AF, Schade J, Laus R, Moreira MA, Muller TR, Fonteque JH (2019) Differences in mineral concentrations on hooves of horses, mules and donkeys. Rev Brasil de Ci\u0026ecirc;ncia Vet 26:31\u0026ndash;39\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolstrung R, Ciesielczuk K, Silmanowicz P (2012) The rate of hoof wall growth of Małopolska horses in the spring and summer season. (In Polish: Tempo przyrastania ścian kopyt koni małopolskich w sezonie wiosenno-letnim).Med. Wet.,4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomosa M, Molinski K, Godynicki S (2006) The Variability of Cranial Morphology in Modern Horses. Zoolog Sci 23(3):289\u0026ndash;298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026oacute;lak K, Łojek J, Albera-Łojek A Influence of the keeping system on the growth of the hoof capsule of horses. Zootechnical news (In Polish: Wpływ systemu utrzymania na przyrost puszki kopytowej koni.Wiadomości zootechniczne, 2019, R. LVII(\u003cem\u003e3\u003c/em\u003e),63\u0026ndash;72\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLancaster LS, Bowker RM, Mauer WA (2013) Equine hoof wall tubule density and morphology. J Vet Med Sci 75(6):773\u0026ndash;778\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOutram AK, Stear NA, Bendrey R, Olsen S, Kasparov A, Zaibert V, Thorpe N, Evershed RP (2009) The Earliest Horse Harnessing and Milking Science 323(5919):1332\u0026ndash;1335\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSargentini C, Tocci R, Pezzati A, Benvenuti D, Martini A (2015) Morphological, chemical-physical and mineralogical characteristics on hoof of Anglo-Arabian and Maremmano Horses and discriminant analysis (PCA) on mineral content. Global J of Animal Sci 3(4):164\u0026ndash;171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrzelec K, Liss M, Jaroszyńska K, Janczarek I, G\u0026oacute;rski K (2017) Orthopedic diseases of racing and sport horses. (In Polish: Schorzenia ortopedyczne koni wyścigowych i sportowych). 4:16\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWitkowska O, Turło A, Michlik K, Cywińska A (2016) Horse laminitis - etiopathogenesis, symptoms and treatment. Veterinary Life. In Polish: Ochwat koni \u0026ndash; etiopatogeneza, objawy i leczenie. Życie Weterynaryjne 91(4):231\u0026ndash;235\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2867145/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2867145/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe research was aimed at analysing the content of chemical elements in horse hooves and exploring their influence on hoof brittleness and the formation of laminitis. Moreover, the activity of the biofilm cells covering the horse's hoof was assessed and its microscopic structure was explored. The material used for the research was the hooves of Polish-Konik horses and sport horses. In order to determine the content of chemical elements, the method of atomic absorption spectrometry was used. The activity of biofilm cells was assessed using flow cytometry. The research showed significant differences in the content of potassium, manganese, iron and aluminum, as well as high zinc deficiencies. For this reason, it is recommended to add the preparations for horses with poor hoof quality with potassium and zinc, and to limit the consumption of manganese and aluminum. Assessment of the activity of the biofilm cells showed that it is mainly composed of dead cells, however the activity differed depending on the type of horse. The microscopic structure analysis shows differences in the number and size of the hoof tubules.\u003c/p\u003e","manuscriptTitle":"Analysis of the content of chemical elements in horse hooves","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-06-24 13:54:21","doi":"10.21203/rs.3.rs-2867145/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-05-04 20:09:27","doi":"10.21203/rs.3.rs-2867145/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f475174-e1d8-4888-887c-6e33ba46ac1d","owner":[],"postedDate":"June 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-27T10:29:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-24 13:54:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2867145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2867145","identity":"rs-2867145","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}