THE CORRELATION OF ELEMENTAL COMPOSITION AND MORPHOLOGICAL PROPERTIES OF THE HORSES HAIR AFTER 110 DAYS OF FEEDING WITH HIGH QUALITY COMMERCIAL FOOD ENRICHED WITH ZN AND CU ORGANIC FORMS

Krzysztof Marycz¹, Eberhard Moll², Wojciech Zawadzki³, Jakub Nicpoń^4
1 Electron Microscope Laboratory, Departament of Animal Hygiene and Ichthyology, Wrocław University of Environmental and Life Sciences, Poland
² Műhle Ebert Dielheim, Germany
³ Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
⁴ Department and Clinic of Veterinary Surgery, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

The objective of this study was to evaluate the impact of high quality commercial food enriched with trace minerals, particularly Zn and Cu on hair coat of yearling thoroughbred horses. In the following research horses for 110 days were fed with St Hippolit diet with 5% higher Zn and Cu contents comparing to standard high concentrated foals feed. The additional advantage of the fodder was the contents of organic forms of mentioned trace elements which has been supposed to markedly enable their absorption from the gastrointestinal tract. Elemental composition of the hairs and their ultrastructure were examined by means of SEM/EDS at the beginning and at the end of the study. In the course of the following research. after period of feeding with Fohlengold both morphological features of the hair coat and hairs elemental composition significantly improved. That point at the positive impact of high quality, Zn and Cu enriched diet on hair coat quality. Moreover marked correlation between elemental composition of the hair and their morphological properties were observed which show of possible SEM/EDS application in estimating elemental status of the organism.

Key words: trace elements, hair, horse, SEM/EDS, feeding.

INTRODUCTION

The first years of a horse's life is fundamental for the later development. Incorrect or imbalanced nutrition during this period may lead, among others,to incorrect skeletal formation, disturbances of immune system function and poor hair coat condition. Colostrum is initially the most important nutritional component for the foal but concurrently young horse is able to digest solid food just one month after birth. This includes meadow grass, hay, but also high quality concentrated feed.

Animals included into the following research were young, thoroughbred, sport racing horses. Thoroughbreds are known for their agility and speed. On the other hand they are extremely fragile, prone to many diseases and demanding as far as nutrition process is concerned [6]. To achieve those high requirements well balanced, high quality diet seems to be a key breeding factor.

It is well known not only from the field of veterinary but also human medicine that diet has a great impact on quality and morphology of the skin and its most apparent appendages – hair. A lot of food components such as proteins, lipids and trace elements are important as far as skin structure and function are concerned [9]. The requirement for trace minerals in young, growing animals are often higher than those of mature animals because of the increased need for skeletal growth and other organs (including skin) development. Trace elements are often supplemented in horse diets due to the relatively low level and availability of many minerals in feedstuffs, particularly those derived from plants. Still deficiencies in trace minerals do in fact occur more frequently than is usually recognized.  Sub-clinical deficiency is far more wide spread than acute deficiencies and can lead to reduced growth rates, loss of feed efficiency, depressed immune system and poor hair coat condition. Animals short of essential elements can show depraved appetite symptoms. These deficiency indicators can include licking wood and stones, eating soil or bark. There are seven trace elements that have shown to be needed to be supplemented. These elements are, Copper(Cu), Cobalt(Co), Zinc(Zn), Iodine(J), Iron(Fe), Manganese(Mn) and Selenium(Se). Moreover there are increasing evidences that many minerals and trace minerals such as Sodium, Chloride, zinc, copper, selenium, and magnesium are needed at higher levels to improve the horse's immune system and generally help to maximize equine health.[8] Not only the quantity and quality of mentioned diet components but also their chemical formulation influences their correct absorption from the gastrointestinal tract and conversion by the animal [4]. Many supplemental sources of trace minerals exist, including inorganic sulfates, oxides, and carbonates, as well as organic chelates, polysaccharide complexes, and proteinates. It was reported that chelated minerals may be more effective in meeting the needs of the rapidly growing hoof of young horses and that retention of copper and zinc were improved when organic sources of these minerals were fed to yearling horses [10].

MATERIAL AND METHODS

Horses
Twelve out of thirty eights yearlings, thoroughbred male horses were encounter into this research. The examined animal group was chosen because they were in similar physical condition, of approximately the same weight and all had mild deficiency anemia. The accuracy of the horse choice was assessed through a blind examination  carried out by two trained veterinary doctors. Examined horses macroscopically exhibited bad coat quality namely the hair coat was weak, shine-less, breakable and rough. Besides having mild underweight they clinically exhibited no other symptoms of any disease. All horses lived in in Golejewko (central-west part of Poland). The animals were kept in a stable with an access to open paddock and meadow spending the majority of the day outside. The following experiment was kept from the end of the November for 110 days. During the experiment animals had no additional sources of food outside the stable. The group of twelve examined horses was farther divided into two 6-individuals subgroups namely experimental and control one. The first subgroup was fed with 700g 44- ingredients manufactured high-quality muesli Fohlengold St-Hippolyt made by Muhle Ebot Dilheim and additionally  hay ad libitum. Control horses received only hay and oats. All animals were fed tree times a day.

At the beginning of the experiment 20 hairs from mane, 20 from tail and 20 from trunk were collected from each horse and they were analyzed by means of SEM-EDS in order to described they ultrastructural appearance and elemental composition. A standardized method of obtaining a sample from the same anatomical areas was developed. Clippings were removed using sterile gloves and there was no contact with the ground or any other surface which might have provided contamination.The hairs were washed and degreased in demineralised water with detergent, then rinsed tree times and dried. Then hairs were covered with gold and observed in Scanning electron microscope LEO ZEISS 435 Fp (Oberkohen). The following features of hair morphology were described: the appearance of the hair cuticle including size and shape of the keratin scales, the medulla cellular structure, medulla width, diameter of each hair shaft and the general appearance of the hair shafts. The analogical examination was performed after 110 days -at the end of the research. Additionally every part of the hair was analyzed regarding elemental content using microroentgenographic detector Roentec at the beginning and at the end of the experiment.

Collection of peripheral blood
From the horse population examined (12 individuals) samples of peripheral blood were collected into EDTA tubes and coagulation tubes by jugular venipuncture and the blood biochemistry was performed including Zn and Cu levels. Complete blood count (CBC) was also done both at the beginning and at the end of the experiment. Blood examination was carry out by two laboratories namely SBL Klinik Czech Republic and Laboratory at Wroclaw University of Environmental and Life Sciences.

Skin biopsy
Skin 7-mm specimens were taken using local anesthesia (lidokaine injectable 2%) from the right side of the horse's neck. Specimens were biopsied using a scalpel blade. Each specimen was fixed immediately after sampling in 4% neutral buffered formalin and later, paraffin embedded 4 µm sections were stained with H & E.

The cross sections of each specimen was examined by means of light microscope in order to measure the width of horse epidermis at the beginning and at the end of the research.

RESULTS

There was significant distinctness concerning hair coat appearance, hair ultrastructure, blood and hairs elemental composition and width of the epidermis observed between control horse group and horses eating manufactured high-quality muesli with high levels of Zn and Cu organic forms. To show how structural characteristic of the hair coat correlates with its elemental composition authors described appearance of the hair shafts, ultrastructure of hair medulla an cuticle cells and medulla/hair shaft diameter ratio. Both guard and soft hairs were analyzed as far as general hair shaft shape and appearance of cuticle surface are concerned. Only cross sections of guard hairs were performed and examined farther to describe cellular structure of the medulla and the medulla/hair shaft diameter ratio. At the beginning of the experiment horses from experimental and control group had a lot of ultrastructural defects concerning the hair shaft, hair cuticle and the structure of the medulla. In the examined hair samples one could appreciate flattened or twisted shape of the hair shaft and ragged endings of the hair which was probably responsible for dull appearance and rough texture of the hair coat. Numerous ruptures of the keratin cuticle scales ranging from shallow to deep in their nature and irregular distribution of the keratin scales were noted (Phot. 1). In case of rough hairs from mane and tail even greater abnormalities were observed. They were connected with serious damage of cuticle and cortex of the hair exposing medulla fragments. It could be the reason for mechanical weakness of the hair but also potentially promote bacterial and fungal infections. Analyzing the cross sections of guard hairs from mane and tail of horses at the beginning of research medulla was very thin comparing to diameter of the hair shaft. Additionally medulla cells were irregularly distributed and did not tightly adhere to each other (Phot. 2). Quite contrary observations were made at the and of experiment with respect to experimental horses group. Medulla cells were regularly distributed along the hair shaft and the medulla/hair diameter  ratios were in majority of hairs relatively high (Phot. 3, 4). Mentioned positive alternations of hair structure were not observed in control horses group.

Phot. 1. The hair from horse tail with deep cuticle and cortex cracking

Phot. 2. The surface of the hair from the tail with proper cuticle structure

Phot. 3. Cross-section of the mane hair with loose and irregular medulla structure and low medulla/hair shaft diameter ratio

Phot. 4. Cross-section of the mane hair with regular medulla cells architecture and high medulla/hair shaft diameter ratio

Also with respect to average epidermis width en improvement was observed in case of experimental horses; namely epidermis after feeding with Fohlengold was much thicker than at the beginning of the research. Quite opposite situation took place in case of control horse group.

Morphological examination of the blood indicated mild anemia in all horses at the beginning of the research and marked improvement of red blood cells parameters at the end of feeding program with respect to experimental group. As far as blood elemental analysis is concerned the Cu and Zn levels were significantly higher in case of experimental horses after the period of supplementation with FohlenGold muesli compared to serum Cu and Zn content in control horses (Fig. 1).

Fig. 1. Cu and Zn levels in serum of control (Serie 2) and experimental (Serie 1) horses after the period of supplementation with FohlenGold muesli

Fig. 2. Contents of Si, S, Mg, in hair of experimental (serie 1) and control group (serie 2), measured by means of method X-ray SEM [%] at the end of the research program

Average concentration of Cu, Zn, S, Si, Mg, Co, Fe and P in examined hair of experimental horses before the research was lover then at the and of the supplementation program. With reference to control horses the all mentioned minerals concentrations decreased during the course of the experiment (Fig. 2).

DISCUSSION

Humans began to horses around 4000 BC and since then equine population has become more and more dependent on human being. Horses have to cope with environment conditions created by breeders whose demands have been concentrated both on exterior and efficiency of the animals. The one of most potent environmental factor with enormous impact on animal condition is nutrition. It is essential to fulfill quantitative and qualitative horse nutritional requirements with respect to its age, level of activity and health status. Well balanced diet consisting of adequate proportions of carbohydrates, lipids (including essential fatty acids), proteins, minerals and vitamins enable to keep homeostasis of the organism and good general physical condition. The minerals in foods do not contribute directly to energy needs but are important as body regulators and as constituents of many vital substances like enzymes, vitamins and amino acids. Major minerals such as calcium, phosphorus, iron, sodium, potassium, and chloride ions and other essential minerals as copper, cobalt, manganese, zinc, magnesium, fluorine, and iodine significantly affect metabolic homeostasis [7]. Skin is the largest and most visible organ of the mammals body. The skin barrier function seems to be the most obvious but one can appreciate that skin together with the hair coat also acts as a mirror reflecting general health or internal pathological dysfunctions of the organism [11]. The impact of unsaturated fatty acids on impermeability of lipid epidermal layer and appearance of hair coat is well documented. Also protein malnutrition induces disturbances in hair growth and quality [2]. As it was indicated microelements play particular role in the formation of skin and hair structure. Minerals influence hair and skin quality at many different levels and Zn and Cu seems to be specially important as far as thoroughbred horses high coat quality is concerned. Authors of the following paper examined the influence of Fohlengold horse food on the hair morphology and condition, because of several reasons. Primarily this high quality, well balanced fodder containing 5% higher amounts of some trace elements including Zn and Cu comparing to others high quality horse food. Moreover in Folengold classic both Zn and Cu have organic chemical formulation which seems to have a grate impact on their absorption and utilization by the horse.

In examined though hair of experimental horses average concentration of Cu and Zn but also S, Si, Mg, Co, Fe, P before the research was lower then at the and of the supplementation. With reference to control horse group eating only hay and oats decrease of mentioned element concentrations was noticed. This might indicate that there is a relationship between dietary intake of specific elements and their concentration in the hair.

Zinc is extremely important trace element involved in many enzyme functions and formation of the body proteins. There is strong connection between Zn diet content and the proper outlook of the skin and hair coat most desirable feature of animal exterior. Even mild deficiency may result in really bad hair and skin quality. Zn regulates the activity of sebaceous glands and the healing of the wounds. High calcium, protein and fitynians level in diet significantly multiply Zn requirement. Moreover adequate copper and zinc ratio should be provided in food (Zn:Cu 3-4: 1) [1]. Suboptimal zinc levels induce graying of hair, prolonged wounds healing, parakeratosis, susceptibility to infections and developmental orthopedic diseases in young horses. In case of examined hair at the beginning of the following research low Zn concentration could be responsible for structural hair cuticle and medulla defects both in experimental and control horses.

Copper is trace element required for the absorption of iron from gastro intestinal tract, skin metabolism and in general connective tissue metabolism [5]. Aids in the formation of the hemoglobin and red blood cells, works with vitamin C and zinc to form elastin and collagen and is involved in the healing process and hair and skin pigmentation. Deficiency may result in general weakness, anemia and fading of brown- or black-pigmented hair. Copper deficiency in foals have been suggested to be a reason for certain developmental orthopedic diseases. In case of experimental horses rise of hair and blood Cu concentration after feeding with high quality fodder may be the reason of hair coat quality improvement and anemia correction respectively.

Iron is essential part of haemoglobin and it is involved in oxidative processes. Its deficiency may result in anemia. Feeding with high quality well balanced diet in the course of the following research could be the reason of anemia correction in experimental horse group.

Also sulphur and silicon are essential trace elements required for skin and hair proper structure and function. Sulfur is a biotin, chondroitin sulfate, insulin and heparin component. It makes up almost 0.15% of his horse body weight. Two very important for skin metabolism amino acids:cystine and methionine, both contain this mineral [3].

Trace element analysis of hair has been described as additional source of information on nutritional status of the organism. The levels of minerals in the hair can correlate with the mineral level in the body's organs. The following study indicates that changes in nutritional regimes have an impact on mineral composition of the equine hairs. At the same time it was observed that elemental composition correlates with hair morphological properties of the hair such as cuticle and medulla structure and medulla/hair diameter ratio. Taking into consideration impaired hair structure and impoverished hair elemental composition at the beginning of the experiment and significant improvement of mentioned parameters in case of the experimental horse group at the end of the research it might be possible to correlate dietary mineral deficiencies with the functionality of keratin tissue.

To determined the mode of elements assimilation from the given food source elemental composition of the horse hairs was evaluated by means of scanning microscopy (SEM-EDS) Mentioned analysis as a fast, noninvasive and nondestructive method may be treated as screening diagnostic tool to determine the correct program of diet and supplementation for each individual's specific needs connected with breed or technological group.

CONCLUSIONS

Mentioned analysis as a fast, noninvasive and nondestructive method may be treated as screening diagnostic tool to determine the correct program of diet and supplementation for each individual's specific needs connected with breed or technological group. After 110 days of suplemmentation with high quality, Zn and Cu enriched diet positive impact of on hair coat quality was observed. Moreover marked correlation between elemental composition of the hair and their morphological properties were notised which show of possible SEM/EDS application in estimating elemental status of the organism.

REFERENCES

1. Bishop Y., 2004. The Veterinary Formulary Sixth edition Edited, Pharmaceutical Press, 429.

2. Busch-Kschiewan K., Zentek J, Wortmann F.J., Biourge V., 2004. UV Light, Temperature and Humidity Effects on White Hair Color in Dogs, J. Nutr. 134, 2053-2055.

3. Committee on Animal Nutrition, National Research Council, Nutrient Requirements of Horses, Fifth Revised Edition, 1989, 14.

4. Dobrzański Z., Korczyński M., Chojnacka K., Górecki H., Opaliński S., 2008. Influence of organic forms of copper, manganese and iron on bioaccumulation of these metals and zinc in laying hens, J. Elementol. 13, 309-319.

5. Frape D., 2004. Equine Nutrition and Feeding, 3rd Edition, Wiley-Blackwell, 53.

6. Frape D.L., Cantab L., House A., 1989. Nutrition and the growth and racing performance of thoroughbred horses, Proceedings of the Nutrition Society 48, 141-152.

7. Harper F., 2005. Minerals for Horses Part I: Major Minerals, Horse Express.

8. Lewis L.D., Knight A., Lewis B.L., Lewis C., 1995. Equine Clinical Nutrition: Feeding and Care, Wiley-Blackwell, 25.

9. Lewis L.D., Knight A., Lewis B.L., Lewis C., 1996. Feeding and care of the horse, Wiley-Blackwell, 18.

10. Naile T.L., Cooper S.R., Freeman D.W., Krehbiel C.R., 2003. Effects of Trace Mineral Source on Growth and Mineral Balance in Yearling Horses, Animal Science Research Report

11. Scott D.W, Miller W.H, Griffin C.E., 2001. Muller & Kirk's Small Animal Dermatology, 6th edn. Philadelphia: W.B. Saunders Company.

 

Accepted for print: 1.07.2009

---

Krzysztof Marycz
Electron Microscope Laboratory,
Departament of Animal Hygiene and Ichthyology,
Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 5b, 50-375 Wrocław, Poland
Phone: +48 71 320 58 88
email: krzysztofmarycz@interia.pl

Eberhard Moll
Műhle Ebert Dielheim, Germany

email: moll@muehle-ebert.de

Wojciech Zawadzki
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland
Phone: +48 71 320 5401
email: wojciech.zawadzki@up.wroc.pl

Jakub Nicpoń
Department and Clinic of Veterinary Surgery,
Wrocław University of Environmental and Life Sciences, Poland
51 Grunwaldzki Square, 50-366 Wroclaw, Poland
email: nickub@poczta.onet.pl

---

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

---

Main  -  Issues  -  How to Submit  -  From the Publisher  -  Search  -  Subscription