The architecture and structure of alpaca skin ( Vicugna pacos ): impacts on industry and wool trade

Background: Alpacas are reared mainly for fiber extraction, which is a highly valued product in the textile industry. For this reason, this work aims to evaluate the morphological and quantitative aspects of the light and dark alpaca skin of Huacaya and Suri alpacas, comparing the structure and architecture of the scapular, costal and lateral femoral skin. Biopsies were collected from the skin of 12 alpacas from the Pacomarca Experimental Fund, located at Puno - Peru. The samples were weighed and fixed in 10% aqueous formalin solution for histological procedures. The histological sections were stained with Hematoxylin eosin, Picrossirius red and Masson Trichromic and immunostained for types I, III and IV collagen and S100. Results: The derma presented sebaceous and sweat glands, as well as follicular groups with primary and secondary hair follicles. Each follicle had a hair called fiber, some with medulla and some without, but both surrounded by cortex and cuticle. The skin presented similar immunostaining for type I, II and IV and S100. Collagen III was detected only in the derma. The total volume of the skin, derma, follicular groups and sebaceous and sweat glands was estimated by stereology for the three body regions for both Huacaya and Suri alpacas. The total volume of skin (Vref) and total volume of follicular groups (VGF) were different for body regions. Femoral region showed higher values for VGF. Colour and breed were also different for Vref and total volume of derma (VD). Conclusion: Comparing the two breeds the femoral region presented higher fiber production. Dark animals had more derma and it was reported close relationship between total skin volume and their fractions volumes: derma, follicular groups and sweat glands.

follicles, sebaceous and sweat glands are located [11]. There is a negative correlation between follicular density and fiber thickness. The main histological characteristic of thin fibers is the larger number of secondary follicles, as it has been widely demonstrated that animals with smaller fiber thickness have a higher number of secondary follicles [12][13][14].
The fleece varies according to the body region, with the thinnest, longest and more dense in the dorsal, costal, sacral, brachial and femoral subregions. It is known that 80% of the fleece is made of delicate fibers, and 20% of the thickest fleece is on the head, as well as in the carpal and tarsal regions [15][16][17].
For these reasons, the integument of three body regions of the two varieties of alpacas (Huacaya and Suri) was evaluated to establish comparisons between them and to define which of these regions would produce the best fiber for commercial purposes.

Alpaca skin biopsies were collected from the Pacomarca Experimental Fund in
Llalli, Melgar, Puno -Peru, from Inca Tops SA. Twelve alpacas of both sexes, aged 5 to 19 months, were used (Six Huacaya variety and six Suri variety alpacas, divided into two groups for light and dark tones respectively). Skin biopsies were performed with an 8 mm punch in three body regions: scapular, costal and lateral femoral and fixed in 10% formaldehyde. They were later cut into 2mm thick fragments. Of the total fragments generated, ¼ was destined for scanning electron microscopy, ¼ for microscopic analysis, ¼ for immunohistochemical analysis and ¼ for stereological analysis. The distribution of fragments in these four sets was made following the pattern of systematic uniform random sampling (SURS).

Scanning electron microscopy
The alpaca skin fragments were washed with distilled water and then additional washes were made in ultrasonic distilled water. The fragments were dehydrated in increasing concentrations of alcohols (70%, 80% and 90%). The samples were then dehydrated in a critical point Leica EM CPD300 and placed on an aluminum disc (stub) to be gold plated in the Emitech K550 metallizer. Samples were analyzed using the LEO 435VP Scanning Electron Microscope (SEM) (Zeiss, Germany) from the Advanced Center for Diagnostic Imaging (CADI).

Light microscopy
Biopsies were dehydrated in an increasing series of ethanol concentrations (70%, 80%, 90% and 100%) followed by xylene. The fragments were then embedded in paraffin as described by [18]. Longitudinal and transverse skin sections were cut in an automatic microtome (Leica, RM2165, Germany). The sections were stained with Hematoxylin and Eosin (HE), Masson's Trichrome and Picrosirius Red. The slides were analyzed under a Fluorescence Light Microscope (FLM) (Nikon Eclipse 80i, Japan).
Those slides stained with Picrosirius Red were also observed under polarized light and images were acquired with AxioCam HRc camera (Zeiss, Germany) connected to an Olympus BX60 Microscope (Olympus, Japan).

Immunohistochemistry
The EnVision ™ Flex kit, High pH, (Link), a high-sensitivity visualization kit used together with Autostainer Link (code K8000, Agilent Dako, USA), was used. Tissue sections were deparaffinized and hydrated. The slides were submerged in Flex Target Retrieval Solution (50x) previously heated to 95 ºC in a water bath for 20 minutes, followed by cooling at room temperature for 20 minutes. The slides were washed twice for 5 minutes with Flex Wash Buffer (Agilent Dako, USA) (20x). Peroxidase-Blocking Reagent was added for 30 minutes at room temperature. Samples were washed twice with Flex Wash Buffer (Agilent Dako, USA), 5 minutes each. The sections were then incubated in a humid chamber overnight at 4°C, with the primary antibody (Collagen I clone (5D8-G9 / Col 1), GTX 60939, rabbit GeneTex brand polyclonal, at a 1:50 dilution; Collagen III (1E7-D7 / Col 3) GTX 60940, GeneTex mouse monoclonal mouse 1:50 dilution; LSBio (Life Span BioSciences Inc.) Collagen IV LS-B8763 rabbit polyclonal (IgG) at a 1: 100 dilution; S100A4 LS-B11817 rabbit polyclonal LSBio (Life Span BioSciences, Inc) at a 1: 100 dilution. For the negative control, the sections were incubated with phosphate buffered solution (PBS).

Total volume of the skin (reference volume)
The 2mm-fragments, sampled previously, were embedded in paraffin and 5µm sections were collected on slides and stained with Masson trichrome. The distance between sections was 100 µm. The total volume of the skin (Vref) was estimated using the Cavalieri Method [19,20]. The image sections were captured using a digital camera connected to the light microscope (Nikon Eclipse 80i, Japan). The following formula was applied to estimate the total volume of skin (Vref): = ∑ × ( ) × × ∑ : points hitting the whole skin fragment; a(p): area of points (square distance between points), t: section thickness; k: the distance between the sections.

Tissue shrinkage
The 8mm-biopsy-fragments were weighed using a digital precision balance (model JK3202B) and their wet weights were converted into volumes considering a tissue density of 1.06g/cm3. After the estimation of the total volume of the skin by the Cavalieri method above, the tissue shrinkage was calculated as 15%.

Total volume of the derma, follicular groups, sebaceous and sweat glands
The total volume of the derma (VD), follicular groups (VGF), sebaceous (VGSe) and sweat glands (VGSu) and the fraction volume were calculated applying the following formulae:

Scanning electron microscopy
The two constituent layers of the skin were visualized: the epidermis and the dermis. In addition, there was the presence of adipose tissue-associated hypodermis (Fig.   1a). In the dermis, it was possible to observe the hair bulbs and the follicular groups, which determine the grouping of hair follicles, surrounded by the connective tissue of the dermis ( Fig. 1b and Fig. 1c).
The fiber was observed emerging from the epidermis, with the presence of the outermost layer, the cuticle, arranged in scales ( Fig. 1d). At the junction between the epidermis and the dermis, it was possible to observe a cluster of cells irregularly arranged in the epidermis, called the basal layer. Still in the epidermis, the stratum corneum was seen, which is the last layer ( Fig. 1d and Fig. 1e). In the papillary dermis, there was a group of six fibers emerging from the same hair follicle (Fig. 1f). Adjacent to the hair follicles were the sebaceous glands ( Fig. 1g).

Histology
In transverse sections, it was seen that alpaca skin is composed of the epidermis and dermis. It was also possible to visualize the hypodermis or subcutaneous tissue as 2e-2g). In the dermis, the sebaceous gland was visible, that is a tubular gland located inside a follicular group (Fig. 2g). The hair follicles usually contained more than one hair shaft or fiber inside. They were called composite follicles, which are seen in Fig. 6h, where it was possible to verify the presence of four fibers without marrow in a single hair follicle. Primary follicles consisted of three structures: the medulla internally, the cortex and the cuticle surrounding the hair cortex. Three layers of the hair follicle were observed: the inner sheath, consisting of a cuticle, a granular epithelial layer (consisting of flattened cells), and a pale epithelial layer (outer layer of cuboid cells), followed by the outer sheath. The last layer was the vitreous membrane (Fig. 2i).
In the reticular dermis, hair bulbs surrounded by dense connective tissue were observed, which did not emerge evenly ( Fig. 2j and Fig. 2k). These were confirmed by the polarization technique, again showing the collagen fibers dispersed by the dermal tissue ( Fig. 4d and Fig. 4e). This technique allowed the differentiation of collagen fiber types, with red and orange fibers compatible with type I collagen fibers interspersed with green fibers, indicating reticular fibers or type III collagen fibers (Fig. 4f).
In both Picrosirius Red staining and polarized light analysis, follicular groups delimited by collagen fibers were observed. However, in the hair follicles only collagen fibers are observed in the outer layer. Most collagen fibers were red to orange and therefore considered collagen type I fibers (Figs. 4g-4l).

Immunohistochemistry
In the four groups of alpacas: light-fiber Huacaya (Fig. 5 a, e, i, n, r), dark-fiber Huacaya (Fig. 5 b, f, j, o, s), light-fiber Suri (Fig. 5 c, g, l, p, t) and dark-fiber Suri (Fig.   5 d, h, m, q, u), when antibodies for Collagen Type I, Collagen Type IV and S100 were used, the structures of the epidermis, some collagen fibers of the dermis, hair follicles, sebaceous glands and sweat glands were marked, which was observed for the scapular, costal and lateral femoral regions.

Quantification
Vref and VGF were significantly different for body region (Table 1), with femoral region showing higher values than scapula, but costal was not different from femoral or scapula for VGF (Table 2). There were also significant interactions between colour and breed for Vref and VD. Dark coats had significantly lower values in Huacaya than Suri breeds for both traits while VD was lower in light than dark coats within the Suri breed (Table 3).
Correlations between Vref and VD were high and medium with VGSu, while others were low (Table 4). The principal component analysis showed a strong relationship between all traits except VGSe (Fig. 6).

Discussion
In the macroscopic analysis of alpaca skin and fiber fragments, two layers of the skin were observed: the epidermis and the dermis. Inside the follicular groups containing the hair, the structure was similar to that found in other mammals. The skin is an important organ as it is a protective barrier which acts as a defense of the body to the external environment [19,20,15,23,24,11]. The set of hairs that emerge from the epidermis form the fleece [16,17], protecting animals in extremely cold climates.
Unlike other authors, it was not possible to view all epidermis layers. The basal stratum contained cubic cells. In turn, the stratum corneum of dead keratinized cells was observed, which appeared as a detached layer from the rest of the other strata of the epidermis. However, our data are in agreement with the previous cited authors [11,14,18,21,22,25,26] and the alpaca epidermis constitutes a very thin layer in the scapular, costal and lateral femoral regions, more precisely parts of the lateral regions of the thoracic limb, thorax and pelvic limb.
The dermis was also analyzed, which had two parts, the papillary dermis, closer to the epidermis and the reticular but deep dermis, near the hypodermis. The difference between both portions was not evident. In the dermis, we found glandular tissue composed of sebaceous glands and sweat glands. The sweat glands were visualized as tubular glands responsible for the thermal regulation and elimination of toxic substances.
In the present study, we evaluated the costal, scapular and lateral femoral regions, which were associated with the hair follicles. These were composed of rounded cells with spherical nuclei. In other studies, where other regions were analyzed, the authors did not find these glands in the interdigital space [27].
In the reticular dermis, the hair bulbs were surrounded by dense connective tissue with an oblique disposition. These did not to emerge uniformly. Surrounding the dermal papilla were melanocytes within the hair cortex. The hair cuticle and two root sheaths were also observed.
It was possible to separate the follicular groups of hair follicles, which could be primary or secondary, being responsible for the formation of primary and secondary hair, respectively. Hair follicles were found both inside and outside the follicular groups.
Collagen is a matrix protein, that gives rigidity and consistency to the skin, making dermis structures more stable inside the dermal tissue, serving as a basis for sustaining and nourishing hair [15,21,23,26]. Masson's trichrome stains showed bluestained collagen fibers compatible with type I collagen fibers. These bypassed the follicular groups, offering support to them as well as the other structures in the dermis. In the case of the slides stained with Picrosirius Red and observed under polarized light, the dermis was visualized, which presented fibers of red color, indicating collagen type I fibers. In the deepest dermis, the presence of red, orange and green polarized fibers was observed, the latter being type III collagen fibers, or reticular fibers, which are small and provide a supporting the whole structure.
Through scanning electron microscopy analysis, it was possible to evaluate the components present in the integument, as well as the microstructures, but higher resolutions would be needed to identify them. It was possible to observe the two layers of the skin separated by the basal layer. In the epidermal layer, we could visualize the stratum corneum. This layer is the last superficial layer of the epidermis responsible for the protective barrier [11,15,[19][20][21][22][23].
Through scanning electron microscopy, it was possible to see that there were follicles with only one hair or a set of fibers emerging from the same follicular group, as also described by [11,19,20,25]. As hairs emerge they come together to share the same follicle. This is because secondary follicles are responsible for the cover hair, which help maintain thermal regulation [16,17]. Therefore, they do not need to be very thick, just be numerous to make up the total body coat of the animal.
Scanning electron microscopy was used to determine the hair diameter of the costal region. This is the region considered as representative of the alpaca integument [3,10,23,32]. In the light and dark alpacas of the Huacaya and Suri varieties, the diameter ranged from 12.65 µm to 55.00 µm. [34]  According to Peruvian Technical Standards, for alpacas a fiber of at most 21.50 µm is the most valued fine fiber for the textile industry. This is because it provides a softer and more comfortable fiber for the production of fabrics, whereas the average fiber is 26.60 µm to 29 µm, and the thickest fiber of 31.60 µm is not used in the textile industry because they are not comfortable fibers to produce clothing.
The presence of different matrix proteins from the three body regions of the tegument of the light and dark alpacas of the Huacaya and Suri varieties was analyzed.
First, descriptions were made for the various types of collagen (I, III and IV), where the marking for type I and type IV collagen was observed throughout the epidermal and dermal tissue, together with their glandular structures, and hair follicles. In the case of collagen III, only the marking for dermis collagen fibers was observed.
The S100 protein was also observed, important for the integument, because it is present in melanocytes, cells derived from the neural crest and is also present in More follicular groups and skin volume were seen in the femoral region indicating higher fiber production in this region. In dark skinned animals, more skin volume was seen in the Suri compared to the light colored animals and Dark Suri produced more derma than light-skinned animals or Huacaya. The principal component analysis shows the close relationship between total volume of the derma (VD), follicular groups (VGF), skin volume (Vref) and sweat glands (VGSu). This indicates that for fiber production, selection for one of these traits should increase the others. This may be useful for breeders when selecting animals for reproduction in breeding schemes.