Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 24 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ COMPARATIVE HISTOLOGICAL STUDY OF THE INTEGUMENT IN BUFFALO AND COW Raad Shaalan Ibrahim1 Amer Muttib Hussin2 raadhisto1982@gmail.com amerhussain42@yahoo.com 1,2 College of Vet. Med., University of Diyala and Baghdad respectively, Iraq. 3Corresponding author: raadhisto1982@gmail.com ABSTRACT This study was done to make a histological comparison between the epidermis of skin in buffaloes and cows. The samples were taken from six body areas (muzzle, neck, thorax, dorsum, abdomen and perineum). The present results revealed that, the skin in buffaloes was thicker and stronger than that of the cow in all regions of the body. Generally, the skin of buffalo and cow consist of two main layers, epidermis and dermis. The epidermis of the skin in both animals consists of four clear and prominent layers except the stratum lucidum which was not present. In buffalo, all layers of the epidermis presented high number of melanocytes in comparison with that of cow which showed few numbers in all studied regions except the muzzle region in which the number increased. The epidermis of the buffalo contained more number of epidermal ridges in comparison with that of the cow. The dermis of the skin in both animals subdivided into indistincted layers, the upper papillary and the inner reticular layers. There is no sharp demarcation between the two layers of the dermis. Key words: skin, epidermis, corneum, dermis, basement membrane, Buffalo, Cow. INTRODUCTION The integument of an animal is not only the protective cover of its body, but also it acts as a medium through which the animal is in continuous contact with its surroundings that mean sensory organ. The role played by the skin in the adaptation of the animal to its environment is manifested through various modifications of this important tissue and its accessory structures in various species living in different habitat (Mahdi, 1979). The histological structure of the skin of both buffaloes and cows consists of two main layers; the epidermis was the outermost layer of the skin. The thickness of the epidermis varies with its location; in regions where there is a heavy protective coat of hair, the epidermis is thin but in non-hairy regions, the epidermis is thicker. The epidermis layer is devoid of blood supply (Di et al., 2001). The dermis, is the connective tissue layer that supports the epidermis. Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 25 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ MATERIALS AND METHODS Samples Collection and Tissue processing Skin specimens were collected immediately after slaughtering from adult males of buffaloes and cows from the local Shu,lla abattoir. The specimens were taken from six body areas (muzzle, neck, thorax, dorsum, abdomen and perineum). Samples were fixed in 10% neutral buffered formalin for 24 hours. Specimens were dehydrated using upgrading of alcohol 50%, 60%, 70%, 80%, 90% and 100%. Xylen was used as clearing reagent. Paraffin was used for infiltration and embedding. Paraffin blocks were cut about (5-7 µm), and then stained by many kinds of stains, Harris hematoxylin and eosin stain for appearance of the general structure of the tissue, Periodic-acid Schiff (PAS) reagents for coloring the basement membrane (Luna, 1968) and Van Gieson stain for appearance of the collagen fibers. Oculometer was used for measurement thickness of corneum, folded epidermis, smooth epidermis, total epidermis (smooth and folded), diameters of melanocytes, thickness of basement membrane, total dermis. RESULTS AND DISCUSSION Histological Study The basic structure of skin ruminants (buffaloes and cows) was the same as that of mammals. A number of variations must be considered. The present study revealed that the skin of both buffaloes and cows consists of two main layers namely, the epidermis and dermis in addition to the skin appendages i.e. hair follicles, arrector pili muscle and the skin glands (sebaceous and sweat glands) (Figure 1 and 2). Figure 2. Showed genereal structure of the skin in the cow ( ) (H and E, X40) Figure 1. Showed genereal structure of the skin in the buffalo ( ) (H and E, X40) Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 26 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ EPIDERMIS The epidermis in both buffaloes and cows formed the external layer of the skin which consist of stratified squamous keratinized epithelium. It consist of four layers from bottom to top was stratum basale, stratum spinosum, stratum granulosum and stratum corneum, this was similar to the results of (Abdul Raheem et al., 2006 ; Abbasi, 2008). Stratum lucidum was not observed in both buffaloes and cows, this finding was in agreement with the findings of (Hafez et al, 1955). The deepest layer, the stratum basale, was a single layer of crowded basophilic cuboidal to columnar cells, resting on a basement membrane. This layer was characterized by high mitotic activity and was responsible for renewal of epidermis, in addition to the initial portion of stratum spinosum. Both the stratum basale and the initial portion of stratum spinosum called stratum germinativum. The cells of this layer possessed darkly stained nuclei, which may be covered by high concentration of melanocytes granules in most studied regions in buffaloes and in the muzzle region of cow (Figure 3 and 4). The diameter of melanocytes in buffalo ranges between (10-10.5 µm, ±0.2) and is larger than that of the cow (8-8.5 µm, ±0.1). The increase in concentration of melanin pigment in buffaloes leads to increase absorption of sun light which was the main reason that the buffaloes was regarded as a less tolerant animals to the heat (poor thermo regulator animal) in comparison to the cow, this was in accordance with the finding of Ali (2008) in buffalo, and Ali et al., (2002) in Awassi sheep, who stated that the exposure to ultraviolet light particularly the sun's rays stimulates a more rapid rate of melanin production and thus acts as a protection against ultraviolet radiation effects because our explanation declaired that the dark color of the buffalo skin absorbs the sun light. The cell bodies of Figure 3. Showed melanocytes in muzzle of the buffalo ( ) (H and E, X400) Figure 4. Showed melanocytes in muzzleof the cow ( ) (H and E, X400). Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 27 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ melanocytes also appeared in the supra basale layers of epidermis showing upward migration to the upper most layers. The stratum spinosum formed several cells thickness. The cells were larger and lighter than those of the stratum basale. This layer consisted of polyhedral cells containing spherical or oval nuclei toward the upper part of this layer. They exhibit numerous cytoplasmic processes or spines, which gives this layer its name. Melanin granules were showed in their cytoplasm, this was similar to the observation of Banks (1993) in sheep. The stratum spinosum had many cell layers in epidermis was varied with the epidermal thickness and species of the animal. The present study found that the number of cell layers in buffaloes ranged between 6-8 cell layers in the dorsum, 16-19 cell layers in the muzzle and in other regions it ranged between 10-12 cell layers, while in cows the number of cell layers ranged between 3-5 cell layers in the dorsum, 14-17 cell layers in the muzzle and in other regions ranged between 7-9 cell layers, this was in agreement with the findings of Mulling (2000) in cows whereas in variance with the result of Sultan (2006) in local black goat, who referred that the number of cell layers of stratum spinosum ranged between 2-4 cell layers in most of skin regions while the number of cell layers ranged between 10-16 cell layers in the muzzle. In buffaloes, the cells of the stratum spinosum firmly bound together by the cytoplasmic spines (desmosomes) giving a spine- studded appearance (Fig. 5). The spine cellular processes that extend to every adjacent cell that strongly bind the cells of this layer together and provide the principle morphological feature of this region of the epidermis. Each process ends as a desmosome, making this region of the skin highly resistance to the mechanical effects of stretching and pressing and resist abrasion, this was similar to results of Junqueria and Carneiro (2005). The inter-cellular spaces between these processes may provide conduits for substances to be secreted eventually by the keratinocytes as they approach full development while in cow these cytoplasmic spines were smaller and not obvious (Fig. 6). Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 28 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ The cells of stratum granulosum were flattened, fusiform and contain basophilic keratohyalin granules, the nuclei of this layer showed progressive signs of degeneration towards the stratum corneum, this was similar to the results of Bagi and Vtas (1983) in buffaloes. Stratum granulosum consist of 3-5 cell layers in buffaloes and 1-3 cell layers in cows, this was in variance with the findings of Al-Bideri (1995) in sheep, who stated that the stratum granulosum consist of one row of cells in the ear epidermis. Stratum granulosum was present in all body regions examined in buffalo and cow, this layer was also observed by Ali et al., (2003) in local goat and in variance with Hafez et al., (1955) who stated that the stratum granulosum appeared faintly in some skin regions in buffalo and was absent in cattle. The last layer, the upper most stratum corneum consisted of dead, horny, scale–like cells, the cytoplasm had been replaced by a protein keratin, this layer was represented by strips of highly acidophilic keratin which breaks into weakly acidophilic filaments towards the free surface, and this was in agreement with the results of Onet and Kelleher (2005) in cow. The principle difference between the epidermis of thick and thin skin was as follow, in all regions of the body, the thickness of the stratum corneum in buffalo was more than in the cow (Table 1). Table 1. The thickness of corneum in buffaloes and cows (micrometer) Region Buffaloes Mean ± SE µm Cows Mean ± SE µm Muzzle* 124.4±1.7 81.6±1.2 Neck* 20.5±0.5 9.9±0.1 Chest* 55.5±0.9 25.5±0.9 Abdomen* 22.47±0.3 10.3±0.2 Perineum* 25.2±0.9 12.5±0.2 Dorsum* 18.9±0.2 8.1±0.02 - * Represent significant differences at (P<0.05) between horizontal rows. Figure 5. Showed stratum spinosum in the buffalo ( ) (H and E, X400) Figure 6. Showed stratum spinosum in the cow ( ) (H and E, X400). Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 29 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ Stratum corneum in buffalo was dense and compact especially in the muzzle (Fig. 7) in comparison with the cow where it was loose and separate (Fig. 8). The current study declared that the highest thickness of the stratum corneum in buffalo and cow was in the muzzle region while the lowest thickness was in the dorsum; the chest, perineum, abdomen and neck comes in between these two readings because on the muzzle, where there was considerable abrasive action, the stratum corneum was thickened. The present study found that the thickness of epidermis (smooth and folded) varies in the different body regions (Table 2). Table 2. The thickness of (folded and smooth) epidermis in buffaloes and cows (micrometer) Areas Folded epidermis Smooth epidermis Buffaloes Mean ± SE µm Cows Mean ± SE µm Buffaloes Mean ± SE µm Cows Mean ± SE µm Muzzle* 1123.3±5.7 1055.4±5.1 746.52±3.1 726±2.4 Neck* 187.04±1.4 146.4±0.7 65.3±0.3 51.42±0.4 Chest* 227.5±1.5 193.2±0.2 105.9±0.9 78.97±0.5 Abdomen* 193.7±0.7 159.6±0.6 71.9±0.9 60.7±0.9 Perineum* 198.5±1.1 170.5±0.3 85.7±0.8 63.4±0.4 Dorsum* 137.3±1.2 103.5±0.4 54.1±0.6 41.4±0.6 - * Represent significant differences at (P<0.05) between horizontal rows. Also, the present study revealed that the epidermis and dermis in buffaloes was thicker than that of the cows in all regions of the body (Table 3). Figure 7. Showed stratum corneum in buffalo was dense and compact ( ) (H and E, X400) Figure 8. Showed stratum corneum in cow was loosely attach ( ) (H and E, X400) Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 30 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ Table 3. The thickness of total (epidermis and dermis) in buffaloes and cows (micrometer) Areas Total Epidermis Total Dermis Buffaloes Mean ± SE µm Cows Mean ± SE µm Buffaloes Mean ± SE µm Cows Mean ± SE µm Muzzle* 934.91±2.03 890.9±3.3 7922.5±1.8 7785.3±2.1 Neck* 126.17±0.8 99.4±0.5 8195.5±0.7 8023.9±2.7 Chest* 166.63±1.1 136.08±0.4 6846.3±2.6 6521.8±2.8 Abdomen* 132.8±0.9 110.15±0.7 6582.2±3.4 6371.8±1.9 Perineum* 142.1±0.9 116.95±0.4 7843.4±2.03 7459.3±2.1 Dorsum* 95.7±0.8 72.45±0.5 8168.3±4.3 7914.6±2 * Represent significant differences at (P<0.05) between horizontal rows. The muzzle region was the thickest in comparison with other body regions in spite of the presence of a few amount of hair follicles, this was due to the presence of interpapillary pegs which extend deeply into the dermis. Table 4 showed that the epidermal ridges has direct proportion with the thickness of the skin as it was increased in the thick skin and decreased in the thin skin so that the number of the epidermal ridges in buffalo was more than the cow. The table 4 also showed that the muzzle and abdomenal regions were registered the highest results as the muzzle region undergoes abrasive action and the abdomen holds the weight and pressure of the bowels, this was similar to the results of Abdul Raheem and Al-Hety (1999) in one humped camel. Table 4. The density of the epidermal ridges and hair follicles/mm2 in buffaloes and cows (micrometer) Areas Density of epidermal ridges Density of hair follicles Buffaloes Mean ± SE µm Cows Mean ± SE µm Buffaloes Mean ± SE µm Cows Mean ± SE µm Muzzle* 11.6±0.2 9.2±0.1 0.33±0.1 0.44±0.03 Neck* 8.4±0.1 5.7±0.2 4.3±0.2 13.5±0.2 Chest* 9.4±0.1 6.3±0.2 2.6±0.1 9.3±0.2 Abdomen* 10.2±0.1 7.8±0.1 3.8±0.1 12.6±0.1 Perineum* 6.5±0.1 4.3±0.1 3.2±0.1 11.1±0.3 Dorsum* 9.8±0.1 7.2±0.2 4.8±0.2 14.4±0.4 - * Represent significant differences at (P<0.05) between horizontal rows. The present results revealed that the dermis and epidermis were attached by the underlying basement membrane which adapt to the interdigitations between the two layers and appear as smooth in skin protected by dense coat of hair (thin Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 31 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ skin) and undulant in areas subjected to mechanical stress (thick skin), this was similar with the findings of Dellmann and Brown (1976) in different animals; Al-Bideri (2003) in adult Iraqi mammals, who said that according to the thickness of the skin there was two shapes of basement membrane (Figures 9 and 10). This study firstly stated that the basement membrane in buffaloes was thicker than in the cows, the thickness of the basement membrane in buffaloes ranges between (3.3-5µm, ±0.3SE) and in the cow range between 2-2.5 µm, ±0.1SE). DERMIS Beneath the epidermis, the dermis was a layer of connective tissue was separated from the epidermis by a basement membrane. The surface of the dermis was very irregular and has many projections (dermal papillae) that interdigite with the projections (epidermal pegs or ridges) of the epidermis. In this study, the dermis was composed of connective tissue containing collagen, elastic and reticular fibers, hair follicles, sebaceous glands, sweat glands, arrector pili muscle, blood vessels. The dermis divided into two layers, the superficial papillary and the deep reticular layers. There is no sharp demarcation between the two layers of the dermis, this was similar to the findings of Panchal et al., (1999) in indian buffalo. The papillary layer consisted of dense connective tissue and fine, delicately arranged collagenous fibers with elastic fibers. The cells of the dermis were similar to those found within other connective tissue of the body. The fibroblast, plasma cells, mast cells and macrophage are predominate, leukocytes may be found, this was in agreement with the findings of Ownby (2004) in ruminant skin. Reticular layer was the deeper and thicker layer of the dermis and composed of coarse bundles of collagenous fibers which Figure 10. Showed basement membrane in abdominal region of the cow ( ) (PAS, X400) Figure 9. Showed basement membrane in abdominal region of the buffalo ( ) (PAS, X400) Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 32 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ usually parallel to the skin surface. Elastic fibers were thick and less abundant in the deeper part of the skin, this layer have fewer connective tissue cells than does the papillary layer ,this was similar to the finding of Sultan (2006) in local black goat. The thickness of the dermis varies between buffaloes and cows. The dermis was thick in both buffaloes and cows. Table 3 showed that the thickness of the dermis in buffaloes was more than in the cows. The thickest dermal region in both of the two animals was in the neck and dorsum, the thinnest dermis was in the abdomen and chest; Muzzle and perineum come in between. The thickest dermis was the neck and dorsum due to its contain large number of connective tissue and highly vascularized, this was in accordance with the findings of Abdul Raheem et al., (2006) in cow. Generally, the current results demonstrated that in buffaloes there were numerous wide short bands of collagen fibers oriented in different directions (Figure 11), whereas in cows, the collagen fibers were less abundant in comparison with buffaloes (Figure 12). REFERENCES Abbasi, M., A. Ghazi, H. Karmi and H. Khosoravinia. 2008. Effects of sex on histological characteristics of various area of skin in an Iranian native breed of sheep. J. Anim. Vet. Adv. 4(13): 321-343. Abdul Raheem, M. H., A. M. Elias and N. S. Ahmed. 2006. The correction factor of hair density in the skin of native cattle. Iraqi. J. Vet. Med., 5(11): 445-468. Abdul Raheem, M.H., M. S. Al-Hety and N. S. Ahmed. 1999. Histological and morphological study of the skin of the one humped camel (Camelus doredarius). Iraqi J. Vet. Sci., 12:1-11. Figure 11. Showed the thicker bands of collagen fibers in the buffalo ( ) (Van Gisien, X400) Figure 12. Showed the thinner collagen fibers in the cow( ) (Van Gisien, X400) Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 33 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ Al-Bideri, A. W. 1995. Parameters of epidermocytes organization in the ear: a comparative study of some Iraqi mammals. J. Coll. Educ. Baghdad Univ., 6: 3-8. Al-Bideri, A. W. 2003. Ultrastructure and cellular organization in different anatomical sites of some adult Iraqi mammal's epidermis. M.Sc. Thesis, Al- Qadissiya University. Ali, S. A. 2008. Histological and histochemical study of the skin in local buffalo. M.Sc. Thesis, College of Veterinary Medicine, Al-Basrah University. Ali, S. H., F. H. Abdelattif and A. L. Dasher. 2002. Histological study of the skin in Awassi sheep. Al-Qadissiya J. Vet. Med. Sci., 1: 39-44. Ali, S. H., K. N. Taher and N. Sh. Ali. 2003. Histological study of skin of the local goat, number of hair follicles and some factors influencing there. Al- Qadissiya J. Vet. Med. Sci., 1: 6-8. Bagi, A. S. and K. M. Vtas. 1983. Morphological of skin surti buffaloes compared to that of other breeds of buffaloes and ox and its role in thermoregulation. Guj. Agric Univ. Res. J. 12: 564-582. Banks, W. J. 1993. "Applied Veterinary Histology" William and Wilkins, Baltimore. London, Pp: 341-371. Dellmann, H. D. and S. Brown. 1976. "Textbook of Veterinary Histology" 1st Ed. Lea and Fibger. Philadelphia., 18: 459-477. Di, W. L., E. L. Rug and D. P. Kellest. 2001. Multiple epidermal cannikins are expressed indifferent keratinocyte subpopulations including onnexin 31. J. Invest. Dermatol., 117(8): 958-994. Hafez, B. S., A. L. Badreldin and M. L. Shafei. 1955. Skin structure of Egyptian buffaloes and cattle with particular references to sweat gland. J. Agric. Sci., 49: 19-30. Junqueira, L. C. and J. Carneiro. 2005. "Basic Histology". (Text and Atlas)". 11Ed. The McGraw-Hill Companies, USA, Pp: 370-380. Luna, I. G. 1968. "Manual of Histology Staining Methods of the Armed Force Institute and Pathology". 3rd Ed. McGraw-Hill Book Company. New York. Mahdi, A. H. 1979. Morphological and histochemical studies on the skin of camel (Camelus dromedarius). M.Sc. Thesis, Baghdad University. Pp: 29- 54. Mulling, C. 2000. Three-dimensional appearance of bovine epidermal keratinocytes in different stages of differentiation revealed by cell maceration and scanning electron microscopic investigation. Folia Morphol., 59(17): 239-246. Diyala Journal of Agricultural Sciences, 10(Special Issue): 24-34, 2018 Ibrahim and Hussin 34 1st Scientific Conf. College of Vet. Med., Diyala Univ., 2018 http://www.agriculmag.uodiyala.edu.iq/ Onet, G. E. and C. Kelleher. 2005. Histological changes in bovine skin exposed to natural environmental condition. National Institute for Discovery Science. www.ascessny. Com Inids. Pp: 625-637. Ownby, C. L. 2004. Veterinary histology. VME 7123. http//www.cvm. okstate. Edu/ instruction/ mm/ curry/ Histology Reference/ index. Htm. Pp: 176- 186. Panchal, K. M., K. M. Vyas and Y. I. Vyas. 1999. Histogensis at skin and adenexa of surti buffalo. J. Vet. Anat. Ind., 11(2): 158-160. Sultan, G. A. 2006. Comparative histological, morphometrical and topographical study of the skin of local black goat. M.Sc. Thesis, College of Veterinary Medicine. Al-Mousl University. نسجية مقارنة لجلد الجاموس واألبقاردراسة رعد شعالن ابراهيم1،3 عامر متعب حسين2 1،2 كلية الطب البيطري- جامعة ديالى وجامعة بغداد على التوالي- العراق. 3 raadhisto1982@gmail.comالمسؤول عن النشر: المستخلص عينات ست جمعت. النسجية الناحية من والجاموس األبقار جلود بين للمقارنة الدراسة هذه أجريت أظهرت اهم النتائج ان سمك الجلد في . العجانيةوالبطن، و، هرالظوالصدر، والرقبة، والخطم، مناطق من عامة جلد بصورة. للدراسةالجاموس يكون اسمك واقوى من األبقار ولجميع مناطق الجسم التي خضعت ن يتتكون بشرة الجلد في كال الحيواني الجاموس واألبقار يتكون من طبقتين رئيسيتين هما البشرة واألدمة. ( لم تالحظ في Stratum lucidum، الطبقة الصافية )ومميزةمن اربع طبقات كانت جميعها واضحة الميالنين خاليا من كبيرة أعداد على الجاموس في( Epidermis) البشرة طبقات جميع احتوت. نتائجنا (Melanocytes )منطقة باستثناء درست التي المناطق جميع في قليلة بأعداد ظهرت التي باألبقار مقارنة البروزات من أكثر أعداد على في الجاموس البشرة طبقة احتوت .ت اعدادهاازدادذ إ Muzzle)) الخطم في كال الحيوانيين (Dermis) دمةاأل طبقة انقسمت. باألبقار مقارنة( Epidermal ridges) البشروية بينهما الفاصلة الحدود كانتو الشبكية الداخلية والطبقة الحليمية الخارجية الطبقة هما، ينرئيس جزئيين إلى .واضحة غير الجلد، البشرة، الطبقة المتقرنة، األدمة، الغشاء القاعدي، الجاموس، االبقار. :لكلمات المفتاحيةا