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Production of first grade ostrich skins
Raw ostrich skins produced in Australia are stored for periods between a few weeks to one year. At present, 20% of the raw skins are tanned in Australia, whilst the remaining 80% are sold off-shore to Korea and South Africa. These skins are sent chilled and salted and are stored between two months and one year prior to tanning.

Raw skins are sold ungraded off-shore and fetch a gross return of $US125-140. The average return for finished skins is $US240 minus the tanning costs of $US60, thus proving a premium of $US40-55 over selling as raw skins. Consequently as a result of the value-added return from sale of the finished skins significant gains are to be made by increasing the number of skins tanned on-shore. At present, only a few farms, representing 10-20% of Australia’s output, produce 90% of their raw skins as 1st or 2nd grade. Based on current trends in the Australian Ostrich Industry, which has moved from the breeder phase into a commercial industry, there will be a substantial increase in the number of raw skins produced per annum. Therefore, to promote the expansion of the ostrich skin industry, it is essential to maximise production of 1st grade skins demanded by local and overseas markets.

Rationale for this study
The development of optimal short and long-term storage conditions prior to tanning is crucial in achieving 1st and 2nd grade finished skins. Factors contributing to downgrading of the skins include dark patches, rough texture and poor plumping of the feather follicles. These factors are considered to result from deterioration during storage, and in particular to be due to damage inflicted on the collagen fibres by microbial organisms. However, the link between storage conditions of raw skins and skin quality after tanning is largely anecdotal. There is scant scientific data on the effects of different storage conditions and storage duration on skin quality in the ostrich and the emu.

Hide salt is currently used as the main preservative of raw skins during storage. Concern is growing at local and state government levels over the potential environmental impact of high effluent salt released from tanneries. Consequently, the effects of storage without salting on skin quality were investigated.

Aims & methodology
The aim of this project was to promote the growth and economic sustainability of the Australian Ostrich Industry by optimising storage conditions of the raw skins. Anecdotal evidence suggests that cold storage, pre-treatment with a bactericide/fungicide and salting minimises skin deterioration during storage. This work aimed to evaluate the effects of these treatments on the structure of the raw skins and to examine the relationship between changes in structure and skin quality after tanning.

A multidisciplinary approach was taken to investigate the effects of different storage regimes on skin quality. Skin quality was assessed by a detailed examination of the structure of the skin using light and scanning electron microscopy, measurement of skin thickness, tear strength and grading after tanning. Two storage durations were included; these were four and nineteen weeks. A pilot study was undertaken to investigate firstly the time course of microbial flora on raw skins during long-term storage and secondly, the potential of these organisms to degrade collagen. The project was designed to address the following questions.
 

• What structures comprise ostrich skin? How do these structures give the skin its unique properties?
• What is the structure of filoplumes and bristle hairs in ostrich skin? What is the distribution of these structures in individual skins? What do the distributions of filoplumes and bristle hairs suggest about their genetic heritability?
• What changes occur within the skin during different storage regimes? How do these changes contribute to downgrading of the skin?
• How does temperature and bactericide/fungicide treatment affect skin quality during shortterm storage?
• How does temperature, bactericide/fungicide treatment and salting during long-term storage affect skin quality?
• What is the relationship between the thickness of raw skin, skin thickness after tanning and skin quality?
• How does salting and temperature during storage affect the growth of microbial flora on the skins? What is the potential of the microbial flora to degrade collagen in ostrich skin?

Relationship between skin structure & skin quality
Light and electron microscopy revealed that the surface of ostrich skin consists of a thin epidermis comprised of two to three layers of cells covered by keratin, the stratum corneum. In live birds keratin would provide protection from abrasion, assist in control of water loss and act as a physical barrier to invasion by bacteria and other microbial organisms. As the epidermis is removed during liming it is not a component of tanned skin. However, in raw skins it may act as a barrier to physical damage of the underlying dermis during handling and also to serve to inhibit colonisation of the connective tissue by microbial flora during storage.

The main component of ostrich skin was the dermis comprised predominantly of collagen fibres. As in volant (flying) birds, the dermis consisted of two layers, a superficial thin grain and an extensive corium. Both these layers consisted of three-dimensional arrays of collagen fibres orientated perpendicular to one another and predominantly aligned parallel to the surface of the skin. Verhoeff and van Gieson staining revealed very few elastic fibres scattered between the collagen. These fibres were predominantly found either in small bundles deep within the dermis or associated with the attachment of smooth muscles to the feather follicles. The paucity of elastic fibres suggests that they minimally contribute to the elasticity of the skin and that the strength and flexibility of the skin is provided by the collagen fibres and by their cross-weave pattern within the dermis. Therefore it is the integrity of the collagen fibres that is crucial to skin quality.

The finding that the grain formed an extremely thin superficial layer of the dermis is consistent with the skin of the emu. Similarly to emu skin, collagen fibres within the grain layer were more compact and thinner compared to those in the adjacent corium. In addition, the grain and corium layers were separated by a narrow band of loose connective tissue. This band of tissue is likely to provide little resistance to shearing forces during processing of the skins and would account for the lamination and loose grain defects prevalent in tanned skin of the ostrich and the emu.

Both the band of loose connective tissue and the overlying grain layer contained numerous blood vessels. The high vascularity near the surface of the skin would explain why the skin is readily bruised during transport and handling of the ostriches prior to slaughter.

Filoplumes & bristle hairs
Macroscopically filoplumes in the ostrich resembled late immature filoplumes of the domestic chicken. Filoplumes consisted of a single rachis surrounded by two to 12 barbs extending eight to 12 millimetres in length. Barbules were present along the extent of the shaft of the barbs. Distally the barbules of each barb were slightly longer than those along the shaft giving a tuft-like appearance at the tips. Bristle hairs had a single distally tapered rachis extending three to five mm in length.

Scanning electron microscopy revealed a cluster of two to four short barbs at the base of each bristle hair with an absence of barbs at the tip of the rachis. Four to six filoplumes were arranged in a semicircular pattern at the base of the feather follicles in all skins. Bristle hairs were interspersed between the filoplumes. The filoplumes and bristle hairs at the base of the contour feathers were restricted to the caudal side of each feather follicle. In a live bird, the contour feathers are angled in a cranial to caudal direction thus they would obscure the filoplumes and bristle hairs at the base of the feathers.

Both filoplumes and bristle hairs were present in the interfollicular space of many skins. Their distribution in individual skins ranged from sparse to covering the entire crown area. Filoplumes and bristle hairs had identical distributions for any given skin. This finding supports the notion that these traits may be genetically linked. As such, filoplumes and bristle hairs may be expressed from two separate gene sets or be different phenotypic expressions of the same pleiotropic gene set. In the current study skins were examined from different flocks separated by a period of two years. Our finding of these miniature feathers at the base of all skins examined, raises the possibility that all ostriches may have filoplumes and bristle hairs at the base of the follicles and that it is only the extent of their distribution between the follicles that is subject to genetic variation.

After tanning the pattern of tiny discrete pinholes paralleled the distribution of filoplumes and bristle hairs found in individual skins. This indicates that the pinholes are the openings of the miniature follicles of the filoplumes and bristle hairs. In support of this notion, light microscopy revealed that the tiny follicles of the filoplumes and bristle hairs had a structure similar to the follicles of the contour feathers with an inner lining of epidermal cells surrounded by a band of collagen. This histology accounts for the discrete plumping of the skin immediately surrounding the pinholes at the base of the follicles of the contour feathers as well as the pinholes in the interfollicular space after tanning. Thus, the plumping of the follicles of the contour feathers is augmented by the presence of the pinholes at their base.

Short-term storage: effects of temperature & bactericide
The effect of storage temperature and bactericide pre-treatment on skin quality was examined following four weeks of storage. Changes in raw skin thickness or deterioration of the general tissue structure after storage using different treatment regimes were compared to the tear strength and thickness of ostrich crusts.

Pre-treatment with bactericide Busan 85® prevented a change in thickness of the raw ostrich skins stored at either 4-6°C or 22°C for four weeks. Without bactericide pre-treatment cold stored skins became significantly thinner (p=0.05) whereas skins stored at room temperature showed a significant increase in thickness (p=0.049). This differential response in skin thickness with storage temperature may reflect a time course of degradation, with the rate of degradation inhibited at colder temperatures.

Denaturation of the collagen fibres would likely result in a thicker connective tissue layer comprised of a decreased density of the collagen fibres as well as an increased diameter of individual fibres. As this change was prevented with bactericide treatment it is suggested that any loss in the integrity of the collagen fibres is likely to be caused by bacterial infection rather than lysosomal autolysis.

Light microscopy revealed all skins had a similar histological appearance with no differences in general tissue architecture before and after storage. Viewed by scanning electron microscopy the collagen fibres showed slight disruption in the alignment of their fibrils as well as increased separation of fibrils after storage. These changes were observed to a similar extent among the treatments. Loss of the ordered array of collagen fibrils after storage suggests some loss of integrity of individual collagen fibres.

No differences were detected in tear strength of the crusts between the storage groups in either perpendicular (p=0.08) or parallel (p=0.6) orientations. Similarly, no differences in thickness of the ostrich crusts were found among the different treatment groups (p=0.42).

The data indicate that bactericide pre-treatment is essential to prevent changes in the thickness of the raw salted ostrich skins stored for four weeks. Although microscopy revealed some deterioration of the raw salted skins following short-term storage without bactericide pre-treatment, this had no observable affect on skin quality, tear strength or thickness of the ostrich crusts. Therefore, salted skins may be stored either at room temperature or at 4-6°C for a period of four weeks without compromising skin quality after tanning.

Long-term storage: effects of temperature, bactericide & salt
The effects of temperature, bactericide pre-treatment and salting on the preservation of raw ostrich skins during storage for 19 weeks were determined. Changes in skin thickness and/or deterioration of the general tissue structure after storage were compared to both the tear strength and thickness of the tanned skins. Skin quality was determined by grading after chrome-tanning. The effects of different storage treatments on the structural and physical attributes of the skin were determined.

Bactericide/fungicide pre-treatment with either Diamoll® C or sodium hypochlorite had no effect on the thickness of salted skins after two weeks of storage at 4-6ºC (p=0.26). In contrast, unsalted skins pre-treated with Diamoll® C and similarly stored for two weeks, were significantly thicker compared to skins pre-treated with sodium hypochlorite (p=0.047). Unsalted skins stored at 4-6°C became significantly thinner after 14 weeks storage (p=0.037). Salted skins stored at 4-6°C for 14 weeks became thicker by an average of 171 µm while salted skins stored at 22°C became thinner by an average of 934 µm.

Light microscopy revealed no histological differences between the groups after two weeks of storage.

After 14 weeks however, salted skins stored at 22ºC had greater inter-collagenous spaces within the corium layer compared to skins stored salted at 4-6°C. In contrast, the corium of skins stored unsalted for 14 weeks appeared as an irregularly stained mass with the spaces between the individual fibres often obliterated. Scanning electron microscopy revealed pitting of the keratin in all skins after 19 weeks of storage. Skins from all treatments showed splaying of collagen fibrils within the grain and corium layers. As the extent of splaying was highly variable within a given skin sample, it was not possible to relate storage treatment to the extent of loss of structural integrity of the collagen fibres.

No differences were observed in tear strength among the treatment groups in either perpendicular (p=0.12) or parallel (p=0.59) orientations. After 19 weeks storage, no differences were observed in tear strength between skins cold-stored with and without salt or between salted skins stored at 4-6ºC and 22ºC. No differences were found in thickness of the tanned skins among the groups when measurements of skin samples taken parallel and perpendicular to the backbone were analysed jointly (p=0.14). No significant association was found between tear strength and thickness of the raw skins after 19 weeks of storage (p=0.12).

Cold storage without salting for 19 weeks markedly decreased the grade of the tanned skins as a result of bacterial damage and flattened follicles. In contrast no difference in grading was observed between salted skins stored for 19 weeks at either 4-6ºC or 22ºC.

The data suggests that salted skins may be stored for up to 19 weeks either at room temperature or at 4-6°C without compromising skin quality. In contrast, cold storage for 19 weeks in the absence of salt is insufficient in preventing significant skin damage.

Pilot study of microbial flora on skins during storage
Microbial flora were isolated from skins stored under different treatment regimes over a storage period of four months. The effects of hide salt, temperature, antimicrobial pre-treatment, and ultraviolet irradiation on the type and persistence of microbial flora were determined. As an indication of their potential to degrade collagen, representative micro-organisms isolated from the skins were subsequently assessed for their ability to liquefy gelatin in vitro. Selected isolates showing varying degrees of gelatinase activity were reapplied to ostrich skin swatches for 45 days to evaluate their ability to degrade collagen in situ. Skin deterioration was determined by qualitative assessment of general tissue structure and the organisation of collagen fibres using scanning electron microscopy.

The work revealed clear differences between the types of micro-organisms colonising the salted and non-salted skins. Non-salted skins showed heavy growth of mixed Gram-negative bacilli. Grampositive bacteria also colonised the non-salted skins but in fewer numbers. In contrast, no Gramnegative organisms were isolated from the salted skins. Staphylococcus, Micrococcus and Bacillus species were cultured from swabs of the salted and unsalted skins. In addition to these Gram-positive bacteria, two fungi, Zygomycete sp. and Alternaria sp. were isolated from the salted skins stored at 4- 6°C.

Gram-negative bacteria persisted in the non-salted skins throughout the entire storage period.

Treatment with 1% sodium hypochlorite and ultraviolet radiation did not prevent the growth of microbial flora on these skins. In contrast, Gram-negative bacteria were never isolated from skins that were salted for the entire storage period. Furthermore, Gram-negative bacteria colonising the initially non-salted skins were not found beyond two weeks of salting. This inhibition of growth of Gramnegative bacteria with salting was independent of storage temperature.

Assessment of the ability of selected Gram-positive and Gram-negative bacteria as well as yeast isolates to liquefy 15% porcine gelatin in vitro revealed varying degrees of gelatinase activity.

Seventy-two percent of the selected isolates from non-salted skins and 33% of isolates from salted skins showed gelatinase activity. In addition, four out of five of the selected Gram-negative isolates liquefied gelatin. This supports the notion that Gram-negative bacteria are particularly effective at liquefying gelatin and that the microbial flora of non-salted skins are likely to possess greater proteolytic activity than that of salted skins. No correlation was found between the ability of a particular isolate to liquefy gelatin in vitro and their ability to degrade collagen in situ.

In summary raw ostrich skins are colonised, in addition to fungi, by several species of Gram-positive and Gram-negative bacteria. Gram-negative bacteria were found exclusively on unsalted skins.

Although isolates of either Gram-positive or Gram-negative bacteria were able to liquefy gelatin in vitro, only skins that were stored unsalted were downgraded as a result of bacterial damage. These findings suggest that bacteria responsible for collagen degradation in ostrich skin are Gram-negative and that their growth is inhibited by salting.

Implications

• The skin of the ostrich and the emu are structurally very similar. In both species the organisation of the grain and corium layers and the numerous blood vessels just beneath the surface of the skin accounts for their susceptibility to lamination and bruising.
• The strength and flexibility of skin in the ostrich, as in the emu, is derived almost exclusively from the three-dimensional cross-weave arrangement of collagen fibres.
• Filoplumes and bristle hairs form a semi-circular pattern at the base of the feather follicles in every skin. Their density and distribution between the feather follicles is highly variable among individual birds.
• The data implies all ostriches farmed in Australia have filoplumes and bristle hairs at the base of their follicles and it is their distribution between the follicles that is subject to genetic variation.
• A time course of change in skin thickness occurs with storage duration. Initially the raw skins become thicker followed by eventual thinning. It is predicted that bacteria cause minor disruption of the collagen fibres within the first few days of slaughter. This is followed by dehydration with continued storage.
• Collagen fibres are highly resilient to disruption of their internal organisation. The ultrastructure of the raw skins, in particular collagen fibres, and tear strength after tanning are minimally affected by either storage duration up to five months, salting, storage temperature or bactericide/fungicide treatment.
• Salted skins may be either cold stored or stored at room temperature for four weeks without compromising skin quality after tanning. Bactericide/fungicide pre-treatment of salted skins prior to storage does not improve skin quality.
• For long term storage of several months salting is essential to prevent downgrading of the skins by bacterial damage. Bactericide/fungicide treatment of the raw skins is not effective in preventing bacterial damage with long term storage. For salted skins, cold storage provides no advantage over storage at room temperature.
• In the current study, there was no correlation between the thickness of the raw skins, skin thickness after tanning and skin quality.
• Several species of Gram-positive and Gram-negative bacteria were isolated from the skins.

Gram-positive bacteria colonised salted skins stored at either room temperature or at 4-6°C.

Salting totally inhibited the growth of Gram-negative bacteria. The data suggest that bacteria responsible for collagen degradation in ostrich skin are Gram-negative.

Recommendations
Recommendations for storage are designed to achieve optimal quality of tanned ostrich skin.

• Hide salt is essential to prevent bacterial growth for storage beyond 24 hours.
•  Optimally skins should be tanned within 24 hours of slaughter. To avoid salting, skins should be soaked in chilled water containing an antibacterial agent immediately after slaughter prior to transport to the tannery.
• Raw salted skins may be stored for up to four weeks without pre-treatment with bactericide/fungicide. For long-term storage, either pre-treatment with bactericide/fungicide, treatment with ultraviolet radiation or periodic soaking in sodium hypochlorite during storage is not effective in preventing bacterial growth.
• Dilute sodium hypochlorite is an effective anti-bacterial agent for short-term storage. This needs to be removed by soaking prior to tanning as it inhibits even uptake of dye in the finishing process.
• For salted skins there is no advantage in storage at 4-6°C compared to room temperature. This effect is independent of storage duration.

Future work

• Determine in all flocks within Australia the distribution of bristle hairs and filoplumes in ostrich skin by examination of the raw skins after slaughter as well as scoring of filoplumes in the live bird.
• Further classify the types of Gram-negative bacteria able to colonise raw ostrich skin.
• Determine the ability of these Gram-negative bacteria to liquefy gelatin derived from ostrich skin.
• Re-apply the identified isolates to ostrich skin immediately after slaughter to accurately assess the ability of the isolates to break down collagen in situ. The integrity of collagen fibres need to be examined by both scanning and transmission electron microscopy and the data obtained compared to skin quality and physical properties after tanning.
• Examine the effectiveness of potassium chloride as a preservative of raw ostrich and emu skin.

At present potassium chloride is cost ineffective compared to hide salt (98% sodium chloride).

In contrast to hide salt, which is highly detrimental to the environment, potassium chloride is considered not harmful to the environment and has been shown in other types of skins to be an effective preservative.

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Last updated: July 2006
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http://www.rirdc.gov.au/reports/NAP/06-054sum.html