Body color intensity of red-colored koi carp Cyprinus carpio and goldfish Carassius auratus auratus varieties were measured to evaluate the effect of paprika used as a feed additive. Digital photos of the experimental fish were processed and analyzed by using special software. The red, green and blue (RGB) values of images were recorded and grayscale values of R, G and B were analyzed. The RGB values seem to play different roles in the development of the visible 'redness' of the two species. In most cases the B values decreased continuously during the administration of the paprika as a carotenoid feed additive, which seemed to have no effect on this process. The G values remained unchanged or decreased slightly as redness increased due to paprika feeding. The R values had a tendency to increase due to paprika feeding but significant differences can be expected only after 4 weeks at the feeding conditions applied here. Both the initial rate of redness and genetic background are thought to influence the rate of red color intensity change, which was observed to be different in the two fish species studied here

Hancz, Csaba

Horn, Péter

Magyary, István

Molnár, Tamás Gergely

Sato, S.

English

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UNCORRECTED PROOF FISHERIES   SCIENCE   2003;  69 : 1156–1159 Blackwell Science, LtdOxford, UKFISFisheries Science0919-92682003 Blackwell Science Asia Pty Ltd696December 2003740Color of goldfish and koi carpC Hancz et al.10.1046/j.0919-9268.2003.00740.xOriginal Article11561159BEES SGML *Corresponding author: Tel: 81-22-717-8740.Fax: 81-22-717-8743. Email: nobuhiko@bios.tohoku.ac.jpReceived 24 September 2002. Accepted 18 July 2003. Evaluation of color intensity enhanced by paprika as feed additive in goldfish and koi carp using computer-assisted image analysis Csaba  HANCZ, 1   István  MAGYARY, 1   Tamás  MOLNÁR, 1   Shoh  SATO, 3   Péter  HORN 1  AND Nobuhiko  TANIGUCHI 2 * 1 University of Kaposvár, Faculty of Animal Science, Department of Small Animal Production, H-7400 Kaposvár, Hungary,  2 Tohoku University, Laboratory of Applied Population Genetics, Graduate School of Agriculture, Sendai, Miyagi 981-8555 and  3 Niigata Prefectural Inland Water Fisheries Experiment Station, Nagaoka, Niigata 940-1137, Japan ABSTRACT: Body color intensity of red-colored koi carp  Cyprinus carpio  and goldfish  Carassiusauratus auratus  varieties were measured to evaluate the effect of paprika used as a feed additive.Digital photos of the experimental fish were processed and analyzed by using special software. Thered, green and blue (RGB) values of images were recorded and grayscale values of R, G and B wereanalyzed. The RGB values seem to play different roles in the development of the visible ‘redness’ ofthe two species. In most cases the B values decreased continuously during the administration of thepaprika as a carotenoid feed additive, which seemed to have no effect on this process. The G valuesremained unchanged or decreased slightly as redness increased due to paprika feeding. The R valueshad a tendency to increase due to paprika feeding but significant differences can be expected onlyafter 4 weeks at the feeding conditions applied here. Both the initial rate of redness and geneticbackground are thought to influence the rate of red color intensity change, which was observed tobe different in the two fish species studied here. KEY WORDS: computer-assisted analysis, digital imaging, goldfish, nishikigoi, ornamentalkoi carp, paprika, red color intensity. INTRODUCTION The popularity of Japanese ornamental carp,  Cyp-rinus carpio  (‘koi carp’ or ‘nishikigoi’) and the Chi-nese goldfish,  Carassius auratus auratus  is growingworldwide due to the increasing number of hobby-ists (e.g. garden pond keepers). Consequently, thedemand for more attractive and valuable koi carpand goldfish varieties is increasing as well. How-ever, these high-quality fish are not produced effi-ciently. Selective breeding of higher quality koicarp and goldfish is based mainly on traditionaland empiric ways, lacking a standard evaluationsystem. A quantitative evaluation system should beset up in order to make the selective breeding onbody color of koi carp and goldfish more efficient.The application of up-to-date digital photographyand computer-assisted image processing and anal-ysis may become a powerful tool for selectivebreeding of ornamental fish, including the appre-ciation of ornamental carp and gold fish.The beautiful yellow, orange and red colorsfound in the skin of ornamental fish are the resultof a group of carotenoid pigments. Fish, in thesame way as other animals, must ingest caro-tenoids as precursors for vitamin A, as well asdepositing pigments. Because animals are incapa-ble of a de novo synthesis of carotenoids, thesecompounds must be supplied in the diet. Fish fallinto three broad groups according to their abilityto transform these pigments: (i) those that canmake no changes and can only deposit the pig-ment; (ii) those that can make some changesincluding a conversion of  b -carotene to astaxan-thin; and (iii) those that can make astaxanthin butnot from  b -carotene. 1 According to Katayama  et al ., goldfish and fancyred carp (koi carp) can convert lutein, zeaxantin (orintermediate) to astaxanthin but  b -carotene is notan efficient precursor of astaxanthin. 2  Astaxanthincan be stored directly in these species. Carotenoidsare commonly used feed additives for goldfish,UNCORRECTED PROOF Color of goldfish and koi carp FISHERIES SCIENCE 1157carp and other species. There are synthetically pro-duced  b -carotene, canthaxanthin, and astaxanthinor natural materials such as carrot, palm oil, mari-gold,  Dunaliella ,  Spirulina , krill and crustaceanmeals. Recently red paprika, containing highamounts of capsanthin, has been attracting muchattention also in aquacultural circles. Tsushima et al . investigated the accumulation of pigmentsfrom paprika in the integument of goldfishexposed to direct and indirect sunlight. 3  Consider-ing the results of Morimoto  et al . on paprika-fedfancy red carp, 4  Tsushima  et al . drew the followingconclusions: 3  (i) paprika was effective for pigmen-tation of goldfish and fancy red carp and its effectwas increased remarkably by sunlight; and (ii) thegoldfish and the fancy carp accumulated capsan-thin and oxidatively metabolized capsanthin to4-ketocapsanthin.The application of computer-assisted imageanalysis in ornamental fish breeding has been verylimited so far. Taniguchi  et al . reported on theapplication of an image analyzer (Shimadzu,Tokyo, Japan) for measuring the area of red andblack spots in ploidy-manipulated fancy carp. 5  Thedifferent colors recognized with normal visualactivity are the result of a mixture of light of differ-ent wavelengths. It has been determined that cer-tain wavelengths of red (R), green (G) and blue (B),when combined with each other in various propor-tions (intensities) will produce a wider range ofcolors than any other combinations of the threecolors. These colors are thus referred as the pri-mary colors of light. 6  The ‘true color’ digital imagesconsist of 24 bits (3 bytes): 1 byte red, 1 byte green,1 byte blue (RGB) in each pixel (dot). The color ofa certain pixel is determined by the so-called ‘gray-scale’ values (256 shades, 8 bits) of R, G and B.In the present study we measured the effects ofpaprika on the red color intensity of goldfish andornamental koi carp using standard light condi-tions, digital photos and a special software incomputer-assisted image analysis. MATERIALS AND METHODS Kohaku koi carp (red markings on a white back-ground) and red telescope goldfish were usedduring the experiments. Thirteen kohaku koi carpand 35 goldfish were kept per treatment unit (aer-ated plastic tank of 150 L, working in recirculationsystem). Experimental fish in both species wererandomly sampled from larger populations. Thetwo control groups were fed on common carpfeed fabricated by Feed Full (Hernad, Hungary)containing 28% protein and 5.4 mg/kg caro-tenoid. The two experimental groups were fed oncolor-enhanced feed containing paprika powder(6.38% mass/mass), 28% protein and 171 mg/kgcarotenoid.The daily amount of feed was 3% of the totalbody weight. Feed consumption was measuredand calculated for the whole investigation period.Fish were weighed at the taking of digital photos.Average initial weights of koi carp and goldfishwere 69.1 g and 2.1 g, respectively. The duration ofthe experiment was 62 days for koi carp and27 days for goldfish. Fish in the tanks were exposedto natural photoperiod.Digital photos were taken for computer-assistedimage analysis under standard light conditions(lamp type: Philips TL-D Special 90; two tubes ofF95, color temperature 5300 ∞ K; one tube of F33,color temperature 4100 ∞ K), by using a digitalcamera (Olympus C-2000 Zoom; Olympus Optical,Tokyo, Japan). Photos of koi carp were taken ondays 0, 37 and 62; photos of goldfish were taken ondays 0 and 27, when 10 randomly chosen fish pergroup were photographed. Digital photos wereprocessed using special software for koi carpappreciation designed and created with the contri-bution of the University of Kaposvar and Pictron inHungary. Grayscale values (0–255) of R, G and Bcomponents of the RGB scale were measuredwithin a 30  ¥  30 pixel area of the red-colored dorsalbody surface.The experimental data were analyzed statisti-cally using  SPSS  8.0 program (SPSS, Chicago, IL,USA). The effect of feeding time on RGB values wasevaluated by one-way  ANOVA , control and paprikaseparately, where means were compared by theStudent–Neuman–Keuls range test. Differencesbetween the control and paprika-added groupmeans at the same photographing time werecompared by independent sample  t -tests. RESULTS The control group of koi carp had a 0.50 g dailygain and 2.1 g/g of feed conversion ratio. Corre-sponding values of the paprika-fed group were0.26 g and 4.2 g/g. Goldfish had a low rate of weightgain: 0.07 g/day (control group) and 0.09 g/day(‘paprika’ group), respectively. Feed conversionwas 1.94 g/g for the control and 2.28 g/g for thepaprika group.The difference in red color intensity between thecontrol and paprika groups was clearly visible tothe naked eye in goldfish after 1 month and in koicarp after 2 months of paprika feeding. The initiallyless reddish goldfish had a more rapid and appar-ent improvement in red color intensity in thepaprika group.UNCORRECTED PROOF 1158 FISHERIES SCIENCE C Hancz  et al. Results detected in the comparison of RGB val-ues of each sampling of koi carp within control andpaprika groups using one-way  ANOVA  are as fol-lows. A significant decrease in B values was foundin the control group of koi carp at the second andthird samplings when compared to B values of day0, whereas B values in the paprika group decreasedsignificantly only at the third sampling. Signifi-cantly higher R values were found in the paprikagroup of koi carp at the third sampling. Changes ofG values in both groups at all samplings were notsignificant. Results derived from the comparisonof control and paprika groups of koi carp usingindependent sample  t -tests showed significantlylower G values for the paprika group at the thirdsampling (Table 1).Results achieved with the comparison of RGBvalues of each sampling of goldfish within controland paprika groups are as follows. Significantlylower R values were detected in the control groupat second sampling. In contrast, the R values in thepaprika group increased somewhat (non-signifi-cantly). Significantly lower B values could be foundin the paprika group at second sampling. Resultsderived from the comparison of control andpaprika groups of goldfish using independent sam-ple  t -tests showed a significant increase of R valuesand a significant decrease of B values at secondsampling (Table 2). DISCUSSION Data of the growth rate and feed conversion calcu-lated for the whole experimental period indicatethat 6.4% (m/m) paprika addition negativelyaffects feed consumption of koi carp. This phe-nomenon was not observed in the case of goldfish.The RGB values seem to play different roles inthe development of the visible ‘redness’ of the twospecies. In most of the cases the B values decreasedcontinuously in artificial keeping and feeding con-ditions and the paprika as a carotenoid feed addi-tive seems to have no effect on this process. The Gvalues remain unchanged or decreased slightly asredness increased due to paprika feeding. The Rvalues had a tendency to increase due to paprikafeeding but significant differences can be expectedonly after 4 weeks at the feeding rate and caro-tenoid concentration we applied. Both the initialrate of redness and genetic background arethought to influence the rate of red color intensitychange, which could explain the differencesbetween the two studied fish species. Our resultsare in accordance with Morimoto  et al . 4  and Tsush-ima  et al ., 3  proving the efficacy of paprika as a feedadditive in goldfish and koi carp.The RGB values are proposed to be used forearlier detection of diet effect or fine phenotypicdifferences in future investigations. Significantimprovement is needed for following the develop-ment of colors and a precise differentiation of colorintensity and patterns within and between variet-ies in both species is essential. The methodologyused in these experiments can serve as a promisingbase for testing the genetic potential of coloration.Because our software was proved to be applicablein the measurement of further characteristics (e.g.shape and distribution of patterns) of ornamentalkoi carp, it is planned to be applied in studies onthe inheritance of these characteristics. REFERENCES 1. Simpson KL. Use of carotenoids in fish feeds. In:  Proceedingsof World Symposium on Finfish Nutrition and Feed Technol-ogy, Hamburg, 20–23 June 1978 , Volume 11. Heinemann,Berlin. XXX; 415–424.1 Table 1 Changes of RGB grayscale values in koi carpSampling RGBControl Paprika Mean SD Mean SD1 R 213.2 14.1 206.1 a 8.9G 69.1 20.6 60.7 11.8B 19.1 a 5.4 18.0 a 8.42 R 216.4 12.6 206.9 a 10.2G 76.2 11.7 69.4 13.9B 7.7 b 4.1 12.5 a 7.53 R 215.0 9.2 222.2 b 7.4G 86.4* 12.8 62.0* 15.0B 0.8 c 1.2 2.0 b 1.8 n   =  10 randomly chosen fish per experimental unit.R, red; G, green; B, blue (grayscale values).* P   <  0.05 (control vs paprika,  t -test);  a,b,c P   <  0.05 (comparisonof RGB means of samplings using  ANOVA ). Table 2 Changes of RGB graycale values in goldfishSampling RGBControl Paprika Mean SD Mean SD1 R 195.5 a 12.6 197.7 9.5G 91.1 12.2 90.8 13.7B 30.4 14.7 31.5 a 12.82 R 173.5 b * 10.1 204.4* 13.0G 98.6 18.0 84.0 15.4B 35.2* 15.1 5.4 b * 5.1 n   =  10 randomly chosen fish per experimental unit.R, red; G, green; B, blue (grayscale values).* P   <  0.05 (control vs paprika,  t -test);  a,b,c P   <  0.05 (comparisonof RGB means of samplings using  ANOVA ).UNCORRECTED PROOF Color of goldfish and koi carp FISHERIES SCIENCE 1159 2. Katayama T, Shintani K, Chichester CO. The biosynthesis ofastaxanthin.  Comp. Biochem. Physiol.  1973;  448 : 253–257.3. Tsushima M, Nemoto H, Matsuno T. The accumulation ofpigments from paprika in the integument of goldfish Carassius auratus .  Fish. Sci.  1999;  64 : 656–657.4. Morimoto T, Amano T, Okamoto M. XXX. In: Takeda M,Watanabe T (eds).  Proceedings of the Third International2 Symposium on Feeding and Nutrition In Fish, 28 August - 1September, Toba, Japan  1989; 379–385.5. Taniguchi N, Kijima A, Tamura T, Takegami K, Yamasaki I.Color, growth and maturation in ploidy-manipulated fancycarp.  Aquaculture  1986;  57 : 321–328.6. Gonzales RC, Wintz P.  Digital Image Processing . Addison-Wesley, Reading, Massachusetts. 1987.UNCORRECTED PROOF AUTHOR QUERY FORM Dear AuthorDuring the preparation of your manuscript, the questions listed below have arisen. Please answer  all  the queries (marking any other corrections on the proof enclosed) and return this form with your proofs. Query Query Reply no. 1 Au: please provide year of publication. 2 Au: please provide title of paper.   Fisheries Science Volume 69, 2003BSA article no: 740

Evaluation of color intensity enhanced by paprika as feed additive in goldfish and koi carp using computer-assisted image analysis

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