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* Institute of Animal Nutrition, School of Veterinary Medicine Hannover, Hannover D30173, Germany, Institute of Nutrition, Veterinary University of Vienna, Vienna A1210, Austria, ** German Wool Research Institute, Aachen D52062, Germany, and Royal Canin, Aimargues F30470, France
3 To whom correspondence should be addressed. E-mail: juergen.zentek{at}vu-wien.ac.at.
KEY WORDS: • dogs • hair color • pigmentation • UV light • humidity
Hair color is genetically determined. Pigmentation depends mainly on the presence and proportions of reddish eumelanin and black phaeomelanin and the density and distribution of the melanins in the hair cortex and medulla (1). In aged dogs, hair color tends to fade. However, discoloration of the hair coat can also be a problem of practical importance in younger dogs with the discrete appearance of cream- or reddish-colored hairs in formerly white or black dogs. Presently the etiology for this is not obvious, and nutritional and other exogenous influences must be considered among genetic factors. For example, protein malnutrition induces disturbances in hair growth and quality (2). Tyrosine and phenylalanine deficiencies can induce hair discoloration (3). Reddish phaeomelanin and black eumelanin are synthesized from tyrosine by the copper-dependent enzyme tyrosinase, which catalyzes the formation of 3,4-dihydroxyphenylalanine (DOPA) from tyrosine. DOPA is oxidized to DOPA quinone, and this is metabolized further to eumelanin or phaeomelanin (4,5). Although not tested specifically in canines, cystine, methionine, and arginine deficiencies are reported to induce hair discoloration (6). Trace-element deficiencies or imbalances also affect hair quality (7). Suboptimal zinc levels induce graying of hair, and copper deficiency causes fading of brown- or black-pigmented hair (8). Other trace elements such as iron and iodine can affect hair color as well as vitamins A, B-2 and B-6, pantothenic, folic, and nicotinic acids, and biotin (9,10).
In addition to nutrition, other exogenous factors have the potential to affect hair pigmentation. Photoyellowing or photobleaching is induced by UV irradiation with shorter (
300 nm) or longer (398 nm) wavelengths (11). The photochemical effects on hair color are strongly dependent on the presence of melanins and chromophores in the hair (12). Phaeomelanin is more sensitive than eumelanin (13). Humidity may increase the effects of UV light (11). UV light and oxygen affect not only the melanins but also the amino acids and fatty acids in the hair and on the cuticle. Amino acids can be destroyed or polymerized. The aromatic amino acids are more sensitive than other amino acids (11).
The present study investigated the presence of melanins in canine hair and the influence of defined exposure to UV light or a combination of high temperature and humidity on hair color in dogs.
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MATERIALS AND METHODS |
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The presence and the character of pigments in the hair samples were studied using light microscopy (Aristoplan, Leitz, Wetzlar, Germany) (1,14). Hair samples (bundles of 10–20 hairs) were paraffin embedded, and longitudinal sections were made with a microtome. A self-made microtome was used for cross sectioning of the hairs. For this, a hair bundle was fixed in the microtome and sectioned by a shaving blade at a total length of 5 µm. Before being sectioned, the hairs were fixed in a glue mixture that consisted of (in mL) 50 methyl acetate, 50 ethyl acetate, 32 ethylcellulose (Merck, Darmstadt, Germany), and 8 dioxylsebacetate (Pfalz and Bauer, Stamford, U.K.). Cross sections were investigated at 400-fold magnification. Anise and cedar oils (Sigma Fluka, Deisenhofen, Germany) were used as immersion oils. The occurrence of eumelanins and phaeomelanins was assessed from one longitudinal and four cross-sectional areas of each hair sample. Black pigments were considered to be eumelanin and reddish-brown pigments to be phaeomelanin. Mixtures of both pigments were classified as mixed type. Results of the longitudinal and cross-sectional studies were combined for the final classification. All images were captured by a video camera (JVC, Tokyo, Japan) and analyzed by SigmaScan Pro image-analysis software (Jandel, San Rafael, CA).
Spectral analysis of hair color was performed using a reflectometer (Datacolor 3890, Dietlikon, Switzerland) (15). The reflectometer light source emits standardized light (D 65, daylight). The light is reflected from the hair sample and measured by a photomultiplier. Sample color and brightness values were assessed after calibration with white and black standards by the CIEL color-measurement system. The CIEL*a*b* system measures the lightness (L* value) on a numerical scale where white = 100 and black = 0. Color is characterized by the a* (red to green) and b* (yellow to blue) values. A combination of these three coordinates describes the color of an object. The data were analyzed by a computer adapted to the camera system. Samples investigated using this system consisted of bundles of 50–100 hairs in the horizontal position that appeared to be white, cream, reddish, or red. Three independent samples were studied for each color, and each sample was measured at four different locations. The final data are the averages of the four measurements.
Brightness of the hair was measured by quantification of light reflection from a 50–100-hair sample in the horizontal position illuminated by two 25-W photo lamps (Kaiser, Buchen, Germany). Reflected light was captured by a video camera (PC 10.5f, Sony, Japan) and analyzed by image-analysis software as described above. The brightness was characterized on a 255-step graded color scale where 0 = black and 255 = white. The computerized system averaged the gray color of each pixel and from that calculated the average value.
The influence of UV light was investigated by irradiation of hair samples for a total of 50 h (16). A standard UV illuminator (Hanseatic, Hamburg, Germany) was adapted to a photo stand, and 9 reddish, 21 cream, and 7 white horizontally positioned, single hairs were irradiated. This procedure allowed us to standardize the irradiation intensity for each hair sample. The brightness was measured individually for each hair before and after UV irradiation with the same image-analysis system as described above. The absolute brightness values of the individual hair samples were low compared with the brightness color measurements of the hair bundles. Effects of temperature and humidity were investigated after 14 white-hair samples were fixed in a glass dessicator with the lower reservoir filled with water. Samples were placed in a temperature-controlled oven at 70°C for 360 h. Hair brightness was measured with the picture-analysis system and transformed to a gray scale as previously described.
Statistics
All data were processed using Excel 9.0 (Microsoft). Brightness values of the hair samples initially and after exposure to UV light, humidity, and temperature were compared by t test. Probability values of <0.05 were taken as significant.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0 U in white hair and 7.6 U in red hair. The b* values were, numerically, 6 U in white hair and 16 U in red hair (Table 2).
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UV exposure induced increases in brightness of the hair samples. The brightness values of the individually evaluated hair samples increased after 50 h of UV irradiation from 155.8 ± 34.1 to 207.6 ± 53.0 U (n = 7) in the white-hair samples, from 78.4 ± 9.7 to 91.2 ± 9.0 U in cream hair (P < 0.001, n = 21), and from 56.1 ± 5.3 to 70.0 ± 4.4 in reddish hair (P < 0.001, n = 9; t test).
Exposure to heat in a humid environment reduced the brightness of white hair significantly from 202 ± 13 to 162 ± 28 U after 15 d. This decrease was observed within 24 h after the beginning of the experiment and did not change further until termination of the experiment after 360 h (Table 3). In another experiment performed with reddish hair from one dog, the brightness decreased after exposure to humidity and high temperature from 136 to 94 U and increased substantially to 202 U after >91 h of UV radiation.
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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10–20 hairs), in a few cases, pigment occurrence might have been underestimated: hair pigment may have been missed due to its unequal distribution along the hair shaft. All of the red and the majority of the reddish hair samples contained pigments, which were mainly reddish pigments classified as phaeomelanins. Therefore, it may be speculated that hair discoloration is caused by changes in pigment quality, density, or distribution. However, changes in hair color are often faint and difficult to assess visually. Objective measurements of hair color or brightness are needed especially for the scientific evaluation of hair quality in dogs. For this purpose, two different methods were employed in this study. The brightness measurement was made possible by quantification of reflected light from a hair bundle that was illuminated by a standardized light source that was then transformed to a gray scale. The hair-color assessment using the CIEL*a*b* system was suitable to discriminate white, cream, or reddish hair. This method allows us to study even faint color changes and may be useful for identification of influencing factors. In the present study, UV light as well as increased temperature and humidity had measurable effects on hair color. These factors were tested because they are important especially for dogs kept outdoors. The UV-light exposure resulted in brightening of the coat color. Temperature and humidity induced darkening of the hair color only in the first 24 h. The effects of UV light are more prominent with UV-A wavelengths and are described as photobleaching or photoyellowing in wool. Light wavelengths >398 nm induce bleaching and wavelengths <340 nm induce yellowing (11). The UV-A light used in our study had a wavelength of 320–400 nm and could have induced both yellowing and bleaching. Visually only bleaching occurred, and yellowing was not assessed. In nonpigmented hair, the bleaching can be explained by damage of the yellowish chromophores. This differs from the bleaching mechanism in pigmented hair. In pigmented hair, UV light can exert deleterious effects on melanin. Eumelanin seems to be more resistant to UV effects compared to the reddish phaeomelanin. The latter can be affected even by visible light. The sensitivity of phaeomelanin to light influences can be explained by its chemical structure. Approximately 87% of this melanin type is protein, and the rest is a chromophore (17) that is sensitive to irradiation. The higher resistance of black hair to UV irradiation has been demonstrated for human hair samples and could be related to the higher eumelanin concentration (18,19). Photochemical effects do not relate only to melanins, but have also been described for amino acids in hair. Cystine, methionine, and aromatic amino acids can be destroyed by UV light (6). Cystine is oxidized, and the resulting product is cysteic acid. Tryptophan levels decrease during irradiation, and this decrease indicates the extent of UV exposure of hair (20). Light absorption by aromatic amino acids can induce the destruction of the aliphatic amino acids glycine and alanine. The levels of free-radical, oxidation, and condensation products increase under those conditions (11). Although not studied for canine hair, it can be expected that humidity and UV irradiation have additive effects (19). The question remains whether hair color can be affected by changes in the hair cuticula. Destruction of the cuticle can affect hair gloss and can also appear visually as a color change.
Conclusion
The different methods used in this study to assess hair color demonstrated the effects of UV light, humidity, and temperature on color and brightness of canine hair samples. These procedures could be useful tools for assessing coat quality more objectively compared with visual systems.
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FOOTNOTES |
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2 This work was supported by Royal Canin, Aimargues, France.
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LITERATURE CITED |
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1. Cesarini, J. P. (1991) Haarmelanin und Haarfarbe. In: Haar und Haarkrankheiten (Orfanos, C. E.), pp. 137–166. Gustav Fischer Verlag, Stuttgart.
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13. Hoting, E., Zimmermann, M. & Höcker, H. (1995) Photochemical alterations in human hair, part II: analysis of melanin. J. Soc. Cosmet. Chem. 46: 181–190.
14. Brunner, H. & Comann, B. (1974) The Identification of Mammalian Hair, pp. 1–18. Inkata Press, Melbourne, Australia.
15. Bohnert, M., Vogt, S. & Weinmann, W. (1998) Farbmetrische Untersuchungen der menschlichen Kopfhaare. Rechtsmedizin 8: 207–211.
16. Ruetsch, S., Kamath, Y. & Weigmann, H. D. (2000) Photodegradation of human hair. J. Cosmet. Sci. 51: 103–125.
17. Chedekel, M. R., Smith, S. K., Post, P. W., Pokora, A. & Vessell, D. L. (1978) Photodestruction of pheomelanin: role of oxygen. Proc. Natl. Acad. Sci. USA 75: 5395–5399.
18. Ruetsch, S. B., Yang, B. & Kamath, Y. K. (2003) Chemical and photo-oxidative hair damage studied by dye diffusion and electrophoresis. J. Cosmet. Sci. 54: 379–394.[Medline]
19. Ruetsch, S. B., Kamath, Y. K. & Weigmann, H. D. (2000) Photodegradation of human hair: A SEM study. J. Cosmet. Sci. 51: 103–125.
20. Gao, T. & Bedell, A. (2001) Ultraviolet damage on natural gray hair and its photoprotection. J. Cosmet. Sci. 52: 103–118.[Medline]
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