Histological Characteristics of Hair Follicle Pattern in Indian Bison (bos Gaurus), Black Buck (antelope Cervicapra) and Nilgai (boselaphus Tragocamelus)

Introduction Indian Wildlife Protection Act (1972) it is an endangered animal per Schedule-I and also it is included in The gaur (Bos gaurus), is also called Indian bison, Appendix I of the Conservation on International trade the largest and tallest species of wild cattle and are in in Endangered Species of Wild Fauna and Flora danger of extinction, because people are hunting them (CITES) and categorized as Vulnerable by the for meat, horns for decorative items and internal organs International Union for Conservation of Nature and for traditional medicinal purposes [1]. The number of Natural Resources (IUCN) [3, 4]. The black buck wild gaurs decreased dramatically due to the loss of (Antelope cervicapra) is one of 26 species of suitable habitat, hunting or hybridization with domestic mammals, which have been declared endangered and is cattle [2]. Gaurs were once distributed throughout the the only living species of the genus Antelope [5]. forested tracts of India and South Nepal, east to Poaching and habitat destruction have seriously Vietnam and south to Malaya but today they are confined affected the population of the Black Bucks. It is usually to just over a hundred existing and 27 proposed hunted for its skin, flesh and also for sports. As its range Protected Areas in India [6]. The species is listed under has decreased sharply during the 20th century, it has the IUCN Red List as vulnerable and according to been classified as near threatened by IUCN since 2003 th [3]. During the 18 century to first half of the 20th centuries, blackbucks were the most hunted wild animal all over India. The nilgai (Boselaphus tragocamelus) Abstract Background and Aim: Indian bison, Black buck and Nilgai are in danger of extinction because people are hunting them for their meat and horns for sports, trophies etc. These animals are also protected under International Union for Conservation of Nature and Natural Resources (IUCN) and Conservation on International trade in Endangered Species of Wild Fauna and Flora (CITES). Taking note of the vulnerability of these animals to extinction, we characterized the hair follicle pattern of these animals for future reference. This study included a total of 30 skin samples comprised of 8 samples of bison, 10 samples of black buck and 12 samples of nilgai.


Introduction
Indian Wildlife Protection Act (1972) it is an endangered animal per Schedule-I and also it is included in The gaur (Bos gaurus), is also called Indian bison, Appendix I of the Conservation on International trade the largest and tallest species of wild cattle and are in in Endangered Species of Wild Fauna and Flora danger of extinction, because people are hunting them (CITES) and categorized as Vulnerable by the for meat, horns for decorative items and internal organs International Union for Conservation of Nature and for traditional medicinal purposes [1].The number of Natural Resources (IUCN) [3,4].The black buck wild gaurs decreased dramatically due to the loss of (Antelope cervicapra) is one of 26 species of suitable habitat, hunting or hybridization with domestic mammals, which have been declared endangered and is cattle [2].Gaurs were once distributed throughout the the only living species of the genus Antelope [5].forested tracts of India and South Nepal, east to Poaching and habitat destruction have seriously Vietnam and south to Malaya but today they are confined affected the population of the Black Bucks.It is usually to just over a hundred existing and 27 proposed hunted for its skin, flesh and also for sports.As its range Protected Areas in India [6].The species is listed under has decreased sharply during the 20th century, it has the IUCN Red List as vulnerable and according to been classified as near threatened by IUCN since 2003 th [3].During the 18 century to first half of the 20th centuries, blackbucks were the most hunted wild animal all over India.The nilgai (Boselaphus tragocamelus)

Histological characteristics of hair follicle pattern in Indian bison (Bos gaurus), Black buck (Antelope cervicapra) and Nilgai (Boselaphus tragocamelus)
has become extinct in Bangladesh, it is the only Weigerts resorcine fuchsine stain for elastic fibres was member of genus Boselaphus and the main threat to done as follows.The sections were deparaffinized in xyline and hydrated to distilled water.Following that, this species is the loss of habitat due to deforestation sections were stained with Weigert's hematoxylin and human population growth [5].Half of the top 20 (A&B solutions were mixed in equal quantities to countries for numbers of threatened species are in Asia.make working solution) then washed well in water to Habitat loss, affecting over 2000 mammal species, is intensify the stain and treated with resorcin-fuchsin the greatest threat globally [7].The arrangement of hair solution for 30 minutes and rinsed in 95 % alcohol.follicles in the domestic animals such as cattle, sheep, Tissues were then washed in water and kept in Van goat, dog and cat and wild carnivores such as Bengal Gieson's solution for a minute, dehydrated in 95% tiger, leopard, lion and sloth bear have been described alcohol, absolute alcohol and cleared in xyline (two and indicated that they have species specific arrangechanges) then mounted with D.P.X mountant.ment of hair follicles [8,9,10].Review of literature has Masson's Trichrome stain for collagen fibres was not shown any detail on the architecture of hair follicle carried out by deparaffinizing in xyline and hydrated to in wild herbivore animals.Keeping this in view, the distilled water and mordant in bouin's solution present study has been conducted on the original skin overnight at room temperature and then washed in of wild herbivores such as Indian gaur, black buck and running water until yellow colour disappears.This was nilgai.
followed by rinsing the tissues in distilled water, This study will help in the identification of fake treated with biebrich scarlet-acid solution (biebrich skin from the original herbivore skins which are sold at scarlet, aqueous 1%-90 ml; acid fuchsin, aqueous 1%an exorbitant price by the poachers in the international 10 ml and glacial acetic acid, 1ml ) for 2 minutes and market.

Materials and Methods
tungstic acid (phosphormolybdic acid 5gm and phosphotungstic acid 5gm were dissolved in 200 ml of Ethical approval: Permission obtained from Principal distilled water) solution for 15 minutes then sections Chief Conservator of Forest (Wildlife) and Chief were directly treated with aniline blue solution for 3 Wildlife Warden, Karnataka State, Govt. of Karnataka minutes and rinsed in distilled water.Tissues were dated 12.09.2012(No.C1/WL/CR-49/2012-13).
transferred to glacial acetic acid solution for 3-5 The study of morphological features of hair minutes and dehydrated in 95% alcohol, absolute alcohol follicle pattern of the skin of bison, black buck and and cleared in xyline (two changes) then mounted with nilgai were undertaken in the Department of Anatomy D.P.X mountant [13].and Histology, Veterinary College, Bangalore.The Gomori's staining method for reticulum was done samples were collected from Sri Jayachamarajendra by deparaffinizing in xyline and hydrated to distilled Zoological Gardens, Mysore and Bannerghatta water, oxidised in potassium permanganate solution Biological Park, Bangalore, Karnataka.A total of 30 for 1 minute, washed in tap water for 2 minutes, then samples of which 8 samples from bison, 10 samples differentiated with potassium meta bisulfate solution from black buck and 12 samples from nilgai were taken for 1 minute.Then tissue sections were washed in tap for the present study.These skin specimens were water for 2 minutes, sensitized in ferric ammonium obtained from animals that died of natural causes.
sulphate for 1 minute and washed in tap water for 2 During post mortem from the lateral abdomen and leg minutes along with 2 changes of distilled water.regions, the skin samples were cut into 2×2 cm size and Following that, tissues were impregnated in the silver fixed in 10% Neutral buffered formalin for a minimum solution for 1 minute, rinsed in distilled water, reduced of 48 hours [11].
in formalin solution for 3 minutes, washed in tap water Further, they were processed by manual tissue for 3 minutes, then toned in gold chloride solution for processor and dehydrated in increasing concentration 10 minutes, rinsed in distilled water, after this tissues of ethanol and cleared in benzene and embedded in were reduced in potassium meta bisulfate solution for 1 o minute, fixed in sodium thiosulfate solution for a paraffin wax with ceresin (60 C melting point).The processed tissues were horizontally sectioned in Microm minute, washed in tap water for 2 minutes.Finally HM 325 rotary microtome at 6-7 µm thickness.The tissues were dehydrated in 95% alcohol, absolute sections were stained with H&E Phloxine for alcohol and cleared in xyline (two changes) then morphological studies [12] by deparaffinizing the mounted with D.P.X mountant, [13].tissue samples in xylene I & II and hydrated to distilled Because of various thickness of the skin of wild water by decreasing grade of alcohol (Absolute I, animals, before routine histological technique, fixed Absolute II, 95 %, 70% and 50%), then sections were skin samples were washed in running water for stained with Mayer"s haematoxylin solution for 5 overnight and immersed in 4% aqueous solution of minutes and washed in running water for 15 minutes phenol for 3-5 days for softening of skin samples such and rinsed in distilled water.Sections were stained with as black buck and nilgai and 6% aqueous solution of eosine-phloxine solution for 1 minute, dehydrated in phenol for thick samples such as bison for 8-10 days.95% alcohol, Absolute I, Absolute II and cleared in The protocol has been modified as specified in xyline I & II then mounted with D.P.X mountant.

Results
follicle there was linear arrangement of primary and secondary follicles observed.Each primary hair Histological structure of the skin of wild follicle was supported by 2-4 secondary hair follicles ruminants like bison, black buck and nilgai were (Figure -5) and the primary hair follicle was always studied under light microscope.In the bison, present at the centre when it was present with 2 compound hair follicles were uniformly distributed secondary hair follicles (Figure -6).Sebaceous glands (Figure -1) and were rectangular in shape comprising of were associated with each hair follicle and numerous primary hair follicle associated with 3-4 secondary hair coiled tubular sweat glands were also present in the follicles together with their sebaceous glands (Figure -dermis of skin (Figure -7).Elastic fibres were 2).In addition, uniformly distributed coiled tubular uniformly distributed among the collagen fibres and sweat glands were also noticed which were lined by also around the hair follicles.The smooth muscle fibres simple cuboidal epithelium.The lining cells of sweat were sparsely distributed between the collagen fibres glands exhibited secretary blebs on their surface, and the reticular fibres were absent.suggesting apocrine mode of secretion (Figure -3).The In nilgai, the compound hair follicles were collagen fibres were abundant compared to smooth densely distributed in the dermis of skin.The primary muscle fibre distribution in the dermis.The capillary hair follicles were bilaterally surrounded by 2-3 plexus and fine arterioles and venules comprising the secondary hair follicles and each primary and vascular component of the dermis were also noted.
secondary hair follicle was associated with sebaceous Evenly distributed fine elastic fibres were present and glands (Figure -8).The dermal connective tissue reticular fibres were not observed within the comprised of bundles of collagen and smooth muscle connective tissue stroma of dermis.
fibres separating the hair follicle.The fine strands of In black buck, the compound hair follicles were elastic fibres and reticular fibres were present in the densely distributed and were arranged linearly in the connective tissue (Figure -9).dermis of skin (Figure -4).Within the compound

Discussion
of Raini goats were described [18].The simple tubular sweat glands in the spotted deer, cattle and goat which The compound hair follicle with a primary hair were located at the junction of dermis and hypodermis follicle associated with two to three secondary hair with merocrine mode of secretion have been described follicles in spotted deer which were arranged in a [9].However sebaceous glands are associated with parallel fashion to each other and the sebaceous glands secondary hair follicle deep in the dermis of goat skin surrounding the primary hair follicle alone was seen in [19] but both sebaceous and sweat glands associated spotted deer, cattle and goat [9].However, in the with hair follicle in red kandhari cows have been present study the primary hair follicle was associated reported [20].However, no sweat glands were with 3-4 secondary follicles in bison and bilaterally observed in nilgai skin in the present study.The dermal surrounded 2-3 secondary hair follicles in nilgai which connective tissue comprised of bundles of collagen, were uniformly distributed in the skin but not arranged elastic and smooth muscle fibres in all the wild animals in parallel fashion as observed in spotted deer.All the in the present study but reticular fibres were not hair follicles were associated with sebaceous glands.In observed in all the animals except nilgai.In black buck, the compound hair follicles were densely distributed and were arranged linearly as observed in hippopotamus skin, reticular dermis showed very thick spotted deer and each primary hair follicle was bundles of coarse collagen fibres arranged in a more supported by 2-4 secondary hair follicles with regular pattern and subdermal glands were also sebaceous glands.The primary hair follicle was always observed [21].present at the centre when it was present with 2 Conclusion secondary hair follicles.However in Iraqi goats, The histological study of Indian bison, black buck sebaceous glands were associated with all primary and and nilgai skins were established.The above some secondary follicles [14].
observations and description of hair follicle pattern Both simple and compound hair follicle and the will be useful in the identification of the wild ruminants dorsal and lateral aspects of the body presented high skin which are poached by the poachers.Observation density of hair follicles with one or two primary hair made by this study will help wildlife students, follicles surrounded by multiple smaller secondary pathologists and researchers for academic purposes follicles and oriented at a sharp oblique angle in llama and solving the vetero-legal cases.skin [15] but there were no oblique orientation of the hair follicle in the wild ruminants in present study.

Authors' contributions
However in the present study there was only one YBS and RVP designed the study and initiated the primary hair follicle situated in the compound hair research.MNB and VR carried out collection and follicle.European cattle having primary hair follicle preservation of samples from the national park and zoo with no secondary hair follicle has also been reported during the study, KVJ helped in standardi-sation of [16].Presence of primary hair follicle, secondary hair procedure, YBS and HDN helped in preparation of follicle and sebaceous glands were reported in nonwoolly Indian goats [17] but their distribution pattern tissue blocks and photography.RVP and KVJ helped was not described.
in draft and revision of manuscript, interpretation of The coiled tubular sweat glands which were lined results, All authors read and approved the final by simple cuboidal epithelium with secretory blebs on manuscript.their surface suggested apocrine mode of secretion in

Figure- 1 :Figure- 2 :Figure- 3 :Figure- 4 :Figure- 5 :Figure- 6 :
Figure-1: Photomicrograph of horizontal section of Indian bison skin showing uniformly distributed Compound hair follicles.Masson's Trichrome (X40) Figure-2: Photomicrograph of horizontal section of Indian bison skin showing rectangular shape compound hair follicle comprising of primary hair follicle associated with 3-4 secondary hair follicles together with their sebaceous glands.Masson's Trichrome (X40) Figure-3: Photomicrograph of horizontal section of Indian bison skin showing coiled tubular sweat glands exhibited secretary blebs on their cell line surface suggesting apocrine mode of secretion.H and E (X 400)

Figure- 7 :Figure- 8 :Figure- 9 :
Figure-7: Photomicrograph of horizontal section of black buck skin showing coiled tubular sweat glands in the dermis of skin.Masson's Trichrome (X 40) Figure-8: Photomicrograph of horizontal section of nilgai skin showing the primary hair follicles with bilaterally surrounded 2-3 secondary hair follicles and sebaceous glands.H & E (X 40) Figure-9: Photomicrograph of horizontal section of nilgai skin showing reticular fibres in the connective tissue.Gomori's (X 100)