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Environmental, nutritional and neuroendocrine regulation of skin coloration in the red porgy (pargus pargus), towards the development of natural hue in culture populations

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This study revealed that a crucial factor for the observed difference in skin colour between wild and (non-carotenoid fed) reared red porgy is due to differences in the expression of cerebral alpha 2 and beta adrenoceptors between wild and cultured fish. In most cases, down regulation of á2 and â adrenoceptors found in cultured teleost brain is possibly related to stress mechanisms due to the specific cultured environmental conditions. However, specific differences in hypothalamic (TA) and thalamic (TPp) areas, known to participate in the melanin-concentrating hormone system, indicate a possible role of central adrenoceptors in plasticity mechanisms of functional brain circuits regulating colour alterations. Parts of the results are of scientific interest exclusively between partners for the present and TIP will help partners to clarify and detail our strategy in future project applications. Development of know how and methodologies for determining noradrenergic neurotransmitter receptors and their function in pagrus skin and brain.
The MSH release mechanisms from the pituitary of both wild and reared red porgy was identified. Dopamine (DA) and Melanocyte-concentrating hormone (MCH) have an inhibitory effect on the release of MSH from the pituitary of red porgy. DA shows a biphasic response, in which the 10{-11} concentration has a slightly stimulatory effect while the other concentrations are inhibitory. The concentrations measured in wild fish show a similar response. MCH shows an inhibition of the release at all concentrations except at very low concentrations, when no response is visible. Wild fish pituitaries show a similar range of inhibition. CRH has no effect at most concentrations, except for very high concentrations when a strong stimulatory effect can be observed. In wild fish, the effects are exactly the same. TRH shows stimulation at all concentrations tested. At low concentrations, this effect is fairly small, while at high concentrations a strong stimulatory effect is visible. A similar result was obtained with the concentrations tested for wild fish. However, in wild fish the effect is about 40-50% lower than in cultured fish. For all secretagogues tested and for both the highest and lowest concentrations, mono-acetylated á-MSH was the dominant form. Concerning the role of husbandry stress on skin colour results show that there was no significant effect of background colour, lighting spectrum & intensity and stocking density on the circulating stress indicators determined (cortisol, áMSH, glucose, lactate, osmolality, electrolytes). In addition, there was no correlation between chromaticity parameters and the used stress indexes.
This study clearly showed that: - Colour reversibility in wild and cultured red porgies from red to dark skin and the opposite as a consequence of dietary carotenoid ingestion take about 100 days, although some colour changes can be observed after only 50 days of feeding. - Diesther astaxanthin contained in natural ingredients when used in formula feeds for the red porgy give closer fish skin coloration to that of the wild fish. - Krill meal either hydrolyzed or not, is a good source of carotenoids for the red porgy, resulting in increased redness and yellowness but not skin lightness. Inclusion levels under 30% are highly enough to obtain a skin colour similar to that of the wild specimens. - Shrimp waste meal because of its high availability and cheaper price is a good alternative to be included in diets for the red porgy. Inclusion levels of shrimp waste meal, which represents up to 40mg astaxanthin per Kg diet are high enough to obtain a skin colour of the fish closer to that of the wild ones. - Although canthaxanthin is absorved by the red porgy its not produce any colour improvement in fish skin when is included in the diet up to 100mg/Kg diet. - Colour appearance similar to that of the wild fish can be achieved by feeding the adequate formula feed between 2 to 3 months before harvesting of the fish. - Body composition is affected by dietary carotenoid addition, being this effect different for the different carotenoid sources used in the formula feed.
The compensation for the immune depression caused by husbandry procedures is based in the adaptation of cultures to more friendly environment for this species (darker environment and low intensity light) and to adaptations in the diet (see partner ICCM). In terms of immunocompetence the most suitable development in the red porgy for aqua culturists should be the use of deeper cages for on growing and an adapted diet. In addition, an effort on commercialisation and publicity regarding the quality of red porgy would give benefits for the increase of the production of red porgy.
Our objectives were, in the principal markets for red porgy - Greece, Italy, Spain and France - to assess the acceptability of the farmed product to those responsible for purchasing it for the main distribution channels. Since the farmed fish remains visibly different from the wild one, the task was in fact twofold: - To assess the degree of assimilation and acceptability for the trade buyer of the farmed fish along side of the wild one of the same name. - As a consequence, to establish the extent to which the farmed fish - a "new" red porgy, which is no longer red, nor necessarily the same size as the wild fish - would be accepted and/or welcomed by the trade. In so doing, we have ascertained the optimal form, colour and size for this, and the applicable price ranges for sale to the public. The result of this was to show us what farmed red porgy's appearance and qualities should be in order to obtain maximum acceptance in its main distribution networks, and the extent to which these can be valorised in the price.
Post mortem changes in skin chromaticity parameters were observed in all treatments. In carotenoid fed fish there was a significant decrease in brightness (L) and in ECI from Day 3 to day 7 of storage on ice. This decrease is also observed in wild fish stored under similar conditions. The use of skin colour as an index of freshness was verified by correlation with established freshness indexes.
Interpretation of the significance of coloration patterns and colour changes in fish is complicated by the lack of data on skin chromaticity parameters, and by difficulties in expressing colour differences in a way that correlate more with our understanding of the three dimensional nature of colour. The measurement of chromaticity parameters provides a means of quantifying colour. Based on the opponent colours theory of vision, the CIELAB scales provide single values (L, a, b) that can be used to express the red (+a) or green (-a), and yellow (+b) or blue (-b). Comparisons between specimens can be easily expressed as colour differences in terms of £GL, £Ga, and £Gb. A total colour difference can also be calculated from theses parameters, but cannot distinguish whether the differences are lightness (£GL) changes only or more severe hue changes or differences in saturation of a colour. Therefore, an alternative method for the expression of colour differences that correlate more with our basic understanding of the three dimensional nature of colour has been developed (CIE £GL £GC £GH) that has added the concept of hue-angle (hab). Hue is determined by the dominant wavelength and is the name of a colour as found in its pure state in the spectrum. Thus, hue is the attribute of colour perception by which an object is judged to be red (0„a), yellow (90„a), green (180„a), blue (270„a), and so forth. Chroma refers to the intensity or purity of hue, regardless of how light or dark it is, ranges from neutral to brilliant, and is a measure of how much gray and white light is mixed in with the ¡¥pure¡¦ focal colour. However, for comparisons between different specimens the mean hue has to be calculated. Most of the published literature on fish skin or fillet colour performs hue comparison between different samples with the classical statistical approach, without asking whether there is, in fact, a mean direction among the population of data that were sampled. Therefore, for testing significance of the mean hue and for multisample testing of hue the nature of the variable, being from circular distributions should be considered. Data from circular distributions may not be analysed using conventional statistics for reasons stemming from the arbitrariness of the zero point on the circular scale. Statistical methods for describing and analysing data from circular distributions are relatively new and are still undergoing development. These methods were applied in the present work both for descriptive statistics and hypothesis testing for the hue variable. This approach offered also the possibility to develop a new parameter, the Entire Colour Index, which by definition is a combined variable of Hue and Chroma, and provide a good approximation of the actual colour.
There is an interesting potential for aquaculture as the results of the project have provided a way to solve the principal difficulty for the industrial commercialisation of red porgy, i.e., the maintenance of the natural colour. This consists in two aspects: - Specific adapted feeds supplemented with carotenoids. - Recommendations regarding husbandry conditions. Therefore, it is recommended: - To culture fish in stocking densities up to 10 kg/m³. - To use grey or light blue background weaning, pre-growing or on-growing (in the case of indoor close systems) tanks. - To use (in the case of indoor close systems) low light intensities and blue lighting spectrum. - To avoid water temperatures above 21°C. Optimum rearing temperature 17-19°C. - To use shadow nets (in the case of sea-cages) to decrease the sun light intensity. - To use feeds supplemented with appropriate carotenoids. Different formula feeds have been developed to improve the colour appearance - To use the adequate formula feed between 2 to 3 months before harvesting of the fish.
The work undertaken gave the opportunity to define the coloration pattern of wild red porgy and to characterize the major difference in skin colour between reared ans wild fish. It was found that: - There is a significant dorsoventral gradient in skin brightness in both wild and reared fish, with the dorsal area being less bright than the ventral one. A similar gradient was also observed in melanophore density (number of melanophores/mm² skin) and in the skin melanin content. - There is a significant difference in all chromaticity parameters between wild and reared red porgies especially in the dorsal body area and the tail. Wild fish display a bright pinkish hue (mean dorsal: L=60, Hue=66°, Chroma=11; mean ventral: L=60, Hue=78°, Chroma=12) while cultured fish display a dark gray colour (mean dorsal: L=35, Hue=34°, Chroma=3). Thus, the basic difference between wild and reared fish is mainly to the amount of light the colour reflects or transmits (L) and to its saturation (Chroma) and to a less extend in hue (the attribute of colour as found in its pure state in the spectrum). Colour differences were also found in wild fish of different origin, indicating possible differences due to habitat or size/age. The basis of the difference in skin colour between wild and (non-carotenoid fed) reared red porgy is due to: - Differences in the type of dermal chromatophores; wild fish skin contain melanophores, iridophores and xanthophores while reared fish skin only melanophores and iridophores; - Differences in the size and shape of melanophores; melanophores of reared individuals were in the dispersed state; - Differences in skin melanin content; reared specimens contained approximately 3 times higher melanin content than wild fish; - Differences in the amount and type of carotenoid present in the skin; Wild red porgy contained predominantly astaxanthin diester, astaxanthin monoester and free astaxanthin (approximately 50% of the total carotenoids, contributing to the red hue) and unidentified “yellow” carotenoids (approximately 47% of the total carotenoids, contributing to the yellow hue). Reared fish contain either none or an extremely weak “yellow” band.
The role of husbandry conditions in skin colour was investigated, and in particular the effect of background colour, lighting spectrum, light intensity and stocking density. This study shows that: - Background, lighting spectrum, light intensity and stocking density have an important effect on skin chromaticity parameters. White background increases skin brightness in levels similar to that of wild fish but reduces skin chroma. Blue lighting spectrum increases skin brightness in dark background (black and red) held fish but did not show any significant effect on hue or chroma. Low light intensity resulted also in higher L in fish held under dark backgrounds. However, fish placed in complete darkness show a decreased L value. Stocking density affected significantly L, Hue and ECI. In particular, fish held under dark backgrounds and high stocking density showed lower L and ECI values than fish held at low stocking density. - There was no effect of background, lighting spectra, light intensity and stocking density on the number of melanophores per mm² of skin. - Higher skin melanin content was observed in red porgies held under dark backgrounds, high light intensity and high stocking density compared to fish held under low light intensity and low stocking density. - The results altogether indicate that the observed differences in skin lightness are related to differences in the motility of skin chromatophores; melanophores in pale fish are aggregated while in control fish dispersed. Concerning the neuroendocrine regulation of this motility, melanophore aggregation is not mediated through circulating cortisol or MSH, but through changes in plasma catecholamines.

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