Influence of Source and Ratio of Xanthophyll Pigments on Broiler Chicken Pigmentation and Performance

Influence of Source and Ratio of Xanthophyll Pigments on Broiler Chicken Pigmentation and Performance A. M. Pe´rez-Vendrell,*,1 J. M. Herna´ndez,† L. Llaurado´,* J. Schierle,‡ and J. Brufau* *Institut de Recerca i Tecnologia Agroalimenta`ries, Department of Animal Nutrition, Centre de Mas Bove´, Apartat 415, 43280 Reus, Spain; †Roche Vitamins Europe Ltd., CH-4070 Basel, Switzerland; and ‡F. Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals Division, CH-4070 Basel, Switzerland because of dietary pigments on shanks and breast skin. Birds fed the SME-25 diet had less pigmentation than those fed equivalent quantities of a combination of SME10 + CTX. The Minolta coordinate “b” measured in breast skin was a good indicator of YX content in feed, whereas the “a” coordinate measured on the shank showed a linear relationship with the dietary CTX level (r = 0.61, P < 0.0001). The same visual color classification of chickens was achieved irrespective of the rank test performed (by shank or carcass color). Lutein and zeaxanthin from the SME-25 product had lower deposition rates in skin and fat tissues than those from the SME-10 product. This finding seems to be related to the ratio of zeaxanthin stereoisomer RR (optically active) vs. RS that was found in tissues from the SME-10 product (97.8%:2.2%), whereas with SME-25 this ratio was 16.0:84.0%. These results suggest that inclusion of only the SME-25 product could not replace the current addition of SME-10 and CTX combinations.

ABSTRACT One experiment was conducted using 960 1-d-old, sexed broilers of Ross 308 strain from 1 to 43 d to evaluate if one type of chemically isomerized marigold with 25% of xanthophylls as zeaxanthin (SME-25) could produce pigmentation equivalent to the current addition of conventional marigold with 10% of xanthophylls as zeaxanthin (SME-10) plus canthaxanthin (CTX) in practical broiler diets (maize-wheat-soybean). Birds were allocated in 32 pens, in a randomized complete block design (four blocks × four treatments). The treatments consisted of a nonpigmented control (T1), a combination of 35 ppm of yellow xanthophylls (YX) from SME-10 + 5 ppm of CTX (T2), a combination of 32 ppm of YX from SME-10 + 2 ppm of CTX (T4), and one treatment with 40 ppm of YX from a new SME-25 (T3). There were no significant treatment effects on chicken performance. All color parameters (Minolta coordinates, Roche color fan scores, Rank test) presented significant differences (P < 0.0001)

(Key words: poultry pigmentation, marigold petals, canthaxanthin, color evaluation, xanthopyll deposition rate) 2001 Poultry Science 80:320–326

et al., 1991). Carotenoids are required by the immune system, and as detoxifiers, they neutralize free radicals before they damage DNA, lipids, and proteins. Poultry cannot synthesize these compounds and must obtain carotenoids from their diets (Schiedt, 1998; Blanch, 1999). The amounts and availability of carotenoids in poultry feed ingredients fluctuate considerably. It has therefore become common practice in the poultry industry to add carotenoids to the feed to assure the necessary amount for pigmentation but also for optimal health, because a number of these compounds have vitamin A activity (Hencken, 1992). To achieve the desired color, feed producers usually combine a yellow carotenoid (apo-ester, lutein, zeaxanthin) and a red one [canthaxanthin (CTX), citranaxanthin, capsanthin, or capsorubin]. The effectiveness of the red xanthophyll, CTX, for pigmentation of egg yolks and broilers has been demonstrated by many

INTRODUCTION Pigmentation is an important factor in consumer acceptance and perceived quality of broilers (Ouart et al., 1988). The color of poultry skin is provided by carotenoid pigments present in the diet of birds that are deposited in the skin and subcutaneous fat. Carotenoids are a group of more than 500 pigments spread throughout the plant and animal kingdom. Poultry use carotenoids for pigmentation, and these substances are also involved in growth metabolism and fertility (Schiedt, 1998). Some carotenoids serve as precursors for the synthesis of vitamin A (Sklan et al., 1989; Surai and Speake, 1998), and some provide protection against damaging reactions in the body, acting as physiological antioxidants (Burton, 1989), and thus enhancing the immune response (Bendich, 1989; Prabhala

Received for publication April 11, 2000. Accepted for publication October 26, 2000. 1 To whom correspondence should be addressed: [email protected].

Abbreviation Key: CTX = canthaxanthin; SME-10 = 10% zeaxanthin; SME-25 = 25% zeaxanthin; YX = yellow xanthophylls.

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researchers (Fletcher et al., 1978; Saylor, 1986). Canthaxanthin can significantly increase the degree of pigmentation in broilers when used in diets containing yellow carotenoids (Marusich and Bauernfeind, 1981). One of the more widely used sources of yellow pigments is the flower petals of marigolds (Tagetes erecta), which contain up to 2,000 ppm of carotenoids (Tyczkowski and Hamilton, 1986). Healthy poultry absorb pigments from their diet, which are transported in blood to the subcutaneous fat tissues and skin, where they are stored. This process is impaired in birds afflicted with diseases, especially intestinal infections and parasitic infestations (Tyczkowski et al., 1991). Most consumers want a yellow bird because this implies that the bird is reasonably free of health problems (Sunde, 1992). Most of the natural carotenoids that are relevant for poultry pigmentation occur in free form, but the lutein in Tagetes petals occur mainly as diesters of palmitic and of myristic acids (Hencken, 1992). Feed carotenoids are absorbed in their free form; thus esterified hydroxycarotenoids have to be saponified before they are absorbed. Feed carotenoids occur in natural compounds in about 60 to 90% trans form and 10 to 30% cis form. When evaluated in vitro, the trans isomer is a more effective pigment than the cis isomer because of the redder hue and greater stability (Hencken, 1992). Nevertheless, several studies in eggs have shown that the cis:trans profile in yolk is quite constant, independent of the cis:trans profile of feed carotenoids, which would imply that there is no advantage of using trans-carotenoids in vivo (Hencken, 1992). Usually, plants synthesize only optically active RR carotenoids. However, pure chemical synthesis yields optically inactive, racemic carotenoids as, for instance, zeaxanthin (Hencken, 1992). Dietary carotenoids are absorbed in different sections of the intestine; whereas zeaxanthin is mainly absorbed in the ileum, the absorption of lutein takes place in the duodenum and jejunum (Tyczkowski and Hamilton, 1986). Subsequent to absorption, the carotenoids are rapidly deposited in broiler tissues (subcutaneous adipose layer, breast and shank skin, and toe-web), principally as the esterified form. Pigmenting efficiency is determined by how much of the ingested carotenoid is absorbed from the intestine and deposited in the target tissue and by its wavelength. The color (wavelength) of the carotenoid as well as its use by the bird (intestinal absorption and subsequent deposition) determines the pigmenting efficiency of a given carotenoid molecule. The wavelengths of the yellow carotenoids from marigold petals range from 445 nm (lutein) to 450 nm (zeaxanthin). The proportion lutein:zeaxanthin in natural Tagetes is 90:10, but some chemically treated Tagetes products could have a ratio up to 40:60 and thus could provide a more orange color tonality. These products with higher proportions of zeaxanthin are obtained by reacting marigold Tagetes erecta meal or its oleoresin at a controlled temperature and pressure with strongly alkaline aqueous solutions to isomerize the lutein into zeaxantin.

TABLE 1. Ingredient composition of experimental starter and finisher diets Basal diets Ingredients

Starter 0–21 d

Wheat (%) Maize (%) Lard (%) Full-fat soybean (%) Soybean 47.5% CP (%) DL-Methionine (%) Calcium carbonate (%) Dicalcium phosphate (%) Salt (%) Nicarbacin (mg/kg) Lasolacid acid (mg/kg) Mineral and vitamin premix1 (%) Calculated nutrient content Metabolizable energy (Kcal/kg) Crude protein Lysine Methionine + cystine Calcium Inorganic phosphate Total xanthophylls (mg/kg)

Finisher 21–42 d

24.15 30.00 1.00 20.53 20.31 0.19 1.09 1.99 0.31 125.00 0.00 0.40

26.99 30.00 2.00 25.86 11.61 0.09 1.04 1.69 0.31 0.00 90.00 0.40

3,100.00 22.00 1.21 0.90 1.00 0.45 5.10

3,250.00 20.00 1.10 0.75 0.90 0.40 5.10

1 Mineral-vitamin premix provided the following per kilogram of diet: vitamin A, 12,000 UI; vitamin D3, 5,000 UI; vitamin E, 30 mg; vitamin K3, 3 mg; vitamin B1, 2.2 mg; vitamin B2, 8 mg; vitamin B6, 5 mg; vitamin B12, 11 µg; folic acid, 1.5 mg; biotin, 150 µg; calcium pantotenate, 25 mg; nicotinic acid, 65 mg; Mn, 60 mg; Zn, 40 mg; I, 0.33 mg; Fe, 80 mg; Cu, 8 mg; Se, 0.15 mg; etoxiquı´n, 150 mg.

The objective of this study was to evaluate the efficacy of two types of marigold: natural SME-10 (in which the lutein:zeaxanthin ratio was 90:10) and a new formulation SME-25 (characterized by a lutein:zeaxanthin ratio of 75:25, which alone could provide a more orange color tonality) on practical broiler grower diets based on a standard diet (maize-wheat-soybean). The SME-10 was supplemented at different levels and also was added or mixed with different levels of CTX, wheeras SME-25 was used alone, because of its higher zeaxanthin proportion.

MATERIALS AND METHODS Animals, Housing, and Management The study was conducted using 960 1-d-old sexed broilers of Ross 308 strain. The birds were housed in a room with forced ventilation, automated gas heating, and programmable fluorescent lights. The experiment was conducted in a poultry barn with 32 pens, 4 m2, each in four rows of eight. In each pen, 30 1-d-old chickens were randomly distributed. The experiment lasted 6 wk. All birds had access to water and the experimental diets ad libitum. The lighting program consisted of 23 h of light during the first 4 d, 20 h of light until Day 10, and 18 h thereafter. Temperature was set at 32 to 34 C the first week, 29 to 31 C the second week, 26 to 28 C the third week, 23 to 25 C the fourth, and 20 to 22 C the fifth week. Maximum and minimum temperatures were recorded daily. Mortality was also recorded daily, and probable cause of death was noted.

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TABLE 2. Analyzed composition of experimental diets

Treatment1

Type of feed

Moisture

Ether extract

T-1 T-2 T-3 T-4

Grower Grower Grower Grower

11.71 11.35 11.52 11.55

8.46 8.69 9.61 8.35

in in in in

pellets pellets pellets pellets

Yellow xanthophyll

Crude protein

Lutein

Zeaxanthin

(%) 20.29 20.63 19.74 19.12

4.4 34.0 25.0 31.0

3.4 8.5 14.2 6.8

1 Treatments: T-1, no added xanthopylls; T-2, 35 ppm yellow xanthophylls from 10% zeaxanthin (SME-10) + 5 ppm canthaxanthin; T-3, 40 ppm yellow xanthophylls from 25% zeaxanthin (SME-25); T-4, 32 ppm yellow xanthophylls from SME-10 + 2 ppm canthaxanthin.

Diets and Feeding Program The feeding program consisted of two diets: a starter diet from 0 to 21 d and a grower diet from 21 to 43 d. The broilers received the same finisher diet during the last 5 d without Avatec威,2 (lasolacid acid) as a withdrawal period. The composition of the experimental basal diets is shown in Table 1. All ingredients except vitamins, minerals, fat, and amino acids were ground through a 3mm screen. The feed was presented as mash for the first week, and thereafter the feed was presented as pellets. During the starter period (0 to 21 d), all chickens were fed the starter basal diet with no added pigments. Pigments or carotenoids were only tested during the growing period between 21 d and the end of experiment at 43 d. The analyzed composition of the feeds and their content in yellow xanthophylls (YX) are reported in Table 2. The experiment consisted of four different dietary treatments: T-1, a control basal diet (having 5.1 ppm of YX from maize); T-2, basal diet plus 35 ppm YX from the SME-10 product and 5 ppm CTX from Carophyll威,2 Red; T-3, basal diet plus 40 ppm YX from the SME-25 product; and T-4, basal diet plus 32 ppm YX from the SME-10 product and 2 ppm CTX from Carophyll威 Red. The experimental design was a randomized complete block factorial design (4 × 2; experimental diet, sex), and therefore there were four pens of females and four of males per dietary treatment.

Test Compounds Two different products from marigold were tested in the study: the SME-10 product (characterized by a ratio of lutein:zeaxanthin of 90:10) and the SME-25 product (characterized by a ratio of lutein:zeaxanthin of 75:25). In the study, the SME-25 product was tested alone (taking into account the higher proportion of zeaxanthin), whereas the SME-10 product was assayed combined with Carophyll威 Red as a supply of CTX. These products were stored at 4 C in the dark until used. The total xanthophyll contents of maize and the experimental products, analyzed by the Association of Official

2

Hoffman-Laroche, CH-4070, Basel, Switzerland.

Analytical Chemists (AOAC, 1980) method, were maize, 17 mg/kg; SME-10 product, 30.72 g/kg; SME-25 product, 12.79 g/kg; and Carophyll威 Red, 11.52 g/kg.

Performance and Color Evaluation The birds were weighed at the beginning of the experiment. Body weight gain, feed intake, and feed-to-gain ratio were measured at 21 and 43 d of age and were averaged on a pen basis. Several modes of measuring color were used in the study: 1. Visual observation by means a color graduated visual aid (Roche color fan) (Vuilleumier, 1969). The Roche color fan was used at 29 d of age to measure the shank pigmentation of five chickens per pen, as a measure of carotenoid repletion. At 43 d of age, 15 numerically identified chickens from each pen were sent to the slaughter house, and 24 h after cooling the carcasses, the shank pigmentation was evaluated by Roche color fan scores. 2. Visual observation and ranking from deepest to lightest pigmentation (Rank Test) (Braunlich, 1974). Two rank tests were also performed at the end of the experiment (43 d) with four identified chickens from each pen; in one case the birds were ordered according the color of the entire carcass (24 h after cooling) and in the other according to shank pigmentation only. 3. Use of special photoelectric instruments that measure reflectance, expressing color in three coordinates “L”, lightness; “a”, redness; and “b”, yellowness, according to the 1976 CIE color system (Commission Internationale de l’Eclairage). The pigmentation of breast skin and shank was individually measured for 15 identified chickens from each pen 24 h after cooling the carcasses. A Minolta chroma meter CR-300 was used and it recorded the three color coordinates: “L”, “a”, and “b”. 4. Assessment of carotenoid deposition in poultry tissues, extracting the carotenoids (from plasma, toeweb, shank, or breast skin), followed by quantitative HPLC (Tyczkowki and Hamilton, 1984; Philip and Chen, 1988). Samples of abdominal fat and skin from breast were taken from two chickens of each pen in order to study the deposition of lutein and zeaxanthin in these target tissues. The tissues were exhaustively

INFLUENCE OF XANTHOPHYLLS RATIO ON BROILER PIGMENTATION

extracted with acetone and magnesium sulfate by using a Polytron homogenizer. The extract was filtered through sintered glass and was evaporated under reduced pressure at 50 C. The residue was dissolved in 60 mL ethanol, 10 mL tert-butyl methyl ether, and 5 mL 50% aqueous potassium hydroxide, and the mixture refluxed for 15 min in a water bath at 80 C. The alkaline solution was then exhaustively extracted with diethyl ether. The combined ether fractions were washed with water and evaporated under reduced pressure at 50 C. The residue was finally dissolved in the mobile phase of the HPLC. The xanthophylls were measured by a straight-phase HPLC using n-hexane:acetone (81:19) as the mobile phase at a flow rate of 1.5 mL/min. The separation of pigments was performed in a silica gel prepacked, stainlesssteel column, length 25 cm with an inner diameter of 4 mm, filled with Lichrosorb威 Si 60, 5-µm pore size (Merck) with VIS-detection at 450 nm (Weber, 1988). In order to determine the ratio of zeaxanthin RR and RS stereo-isomers in feeds and target tissues, lutein and zeaxanthin were extracted with acetone and magnesium sulfate, as described above. The extract (without derivatization) was directly analyzed by HPLC by using n-hexane:2-propanol (95:5) as the mobile phase at flow rates of 0.8 mL/min (0 to 55 min, and 66 to 75 min) and 1.5 mL/min (55 to 66 min). The separation of isomers was performed in a HP-1100 chromatograph provided with a Chiralpack威,3 AD column (250 × 4.6 mm) and with diode array detection between 300 and 600 nm.

Statistical Analysis Statistical treatment of data was conducted by analysis of variance using the general linear models procedure of SAS software (SAS 1988) as a completely randomized design. Significant differences between treatment means for each parameter recorded were separated by Duncan’s new multiple-range test with a 5% level of probability. Data from the rank test were evaluated using the rank test of SAS (1988), which calculates the mean value of the punctuation done to the animals; also the sum of all values per treatment were calculated. Regression analysis was performed to obtain the effect of CTX level in the feed on shank redness.

RESULTS AND DISCUSSION Productive Parameters Starter Period. As expected and taking into account that all birds were fed the same diet, there were no statistically significant differences between treatments; the performance of males was significantly better than of the females (body weight and average daily gain, P < 0.0001;

3

Daicel Chemical Industries Ltd., Chiyoda-ku, Tokyo 100, Japan.

323

average daily consumption, P < 0.0231; and feed efficiency, P < 0.0039). Growing Period. In this period, animals were fed the experimental diets that were only different due to the inclusion of pigments. Performance parameters obtained during this period are shown in Table 3. There were no significant differences in any productive parameter, therefore pigments added to basal diet did not modify bird performance, and only expected differences between males and females were found. No dietary treatment by sex interactions were observed.

Color Measurement The results of shank pigmentation measurements, using a color fan scale (Roche color fan score, RCF) at 29 d and at killing (43 d), are presented in Table 4 (per treatment). At 29 d of age (1 wk after pigment inclusion), statistically significant differences (P < 0.0001) were found between treatments. Birds from Treatment T-2 had the most pigmentation, and those from the control group had the least; both were statistically different in color from birds from Treatments T-3 and T-4. Therefore, 40 ppm of YX coming from the SME-25 product (25% zeaxanthin) were not enough to develop color equal to 35 ppm YX (YX) from the SME-10 product (10% zeaxanthin) + 5 ppm of CTX (T-2) but was the same as the combination 32 ppm of YX from the SME-10 product + 2 ppm CTX. At 43 d of age, the birds were killed, and the carcasses were kept refrigerated at 4 C for 24 h. Then, the pigmentation of skin of breast and shanks were determined individually on each bird with a Minolta chromameter. The Minolta color coordinates, measured on chicken shanks 24 h after killing, are also shown in Table 4. Statistically significant differences between treatments (P < 0.0001) were found for all color coordinates. Values from the shanks were higher than those taken from the skin of breast (Table 5) but followed the same pattern. The “L” values of birds fed the control diet were significantly higher. Birds fed the control diet had the lowest values of the “a” coordinate, and the shank “a” values increased in relation to the level of added canthaxantin in the feeds. These results agree with those found by Branellec (1985), who stated that zeaxanthin and canthaxantin are mainly deposited in shanks, whereas lutein has better pigmenting properties in fat. The color coordinate “b” (yellowness) measured in shanks followed the same pattern as in breast skin. Treatments T-2 and T-4 had similar values despite the different contents of total xanthophylls (35 ppm YX from SME-10 + 5 ppm CTX vs. 32 ppm YX from SME-10 + 2 ppm CTX). Color coordinate (“L”, “a”, and “b”) values recorded from breast skin are presented in Table 5. Statistically significant differences (P < 0.0001) between treatments for all color parameters were obtained. The “L” and “a” values measured in breast skin of birds fed the control diet were higher than those obtained in birds fed the other treatments. The color coordinate “b” was closely

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TABLE 3. Performance in experimental finisher period Dietary treatment1 Statistical analysis

Criteria Live performance2

Control T-1

T-2

T-3

T-4

SE

Probability

Live weight at 21 d, g Live weight at 43 d, g Males Females Feed intake, g/d Males Females

615.03 2,383.6 2,597.1 2,170.2 154.57 164.13 145.00

613.45 2,329.8 2,482.8 2,176.7 151.64 159.56 143.72

610.68 2,398.9 2,577.2 2,220.6 155.35 164.64 146.06

620.20 2,393.0 2,553.0 2,233.1 155.88 165.25 146.51

8.65 70.3

NS NS

3.72

NS

80.39 89.79 70.99

78.01 84.21 71.82

81.28 88.16 74.41

80.58 87.34 73.82

2.95

NS

0.031

NS

1.41

NS

Body weight gain, g/d Males Females Feed conversion, kg feed:kg gain Males Females Mortality, % Males Females

1.936 1.829 2.042 4.26 3.52 5.00

1.950 1.896 2.003 3.31 4.97 1.65

1.916 1.868 1.963 1.67 2.50 0.85

1.939 1.893 1.985 3.85 6.02 1.67

1 Treatments: T-1, no added xanthopylls; T-2, 35 ppm yellow xanthophylls from 10% zeaxanthin (SME-10) + 5 ppm canthaxanthin; T-3, 40 ppm yellow xanthophylls from 25% zeaxanthin (SME-25); T-4, 32 ppm yellow xanthophylls from SME-10 + 2 ppm canthaxanthin. 2 Data pooled for male and female. No interactions between treatment and sex were found for any parameter.

influenced by the total addition of pigments in the experimental feed. Control Treatment T-1 had the lowest “b” values, and Treatment T-2 had the highest. Yellow pigmentation measured in breast skin of birds fed T-3 (40 ppm of YX from SME-25) was lower than those of chickens fed the combination of Tagetes erecta SME-10 product and CTX (T-2 and T-4). Two rank tests were also performed 24 h after killing with four identified chickens from each pen. In one case the birds were ordered according to the color of the entire carcass, and in the second case, they were ordered only by shank pigmentation. The results (expressed as mean scores) are shown in Table 4 (shanks) and Table 5 (carcass). Significant differences between treatments (P < 0.0001) were found in both cases. The control treatment

had the lowest values, which were clearly different from the other treatments. In the rank test performed on shanks, within pigmented Treatment T-2 (35 ppm of YX from SME-10 + 5 ppm CTX) had the highest scores, whereas Treatment T-3 (with SME-25 added) had the lowest values. The results from the rank test showed similar results and were found by ordering entire bird carcasses by their color. The two rank tests were equivalent in expressing pigmentation, and a good linear relationship was found between scores obtained by the two methods (r = 0.9696). The lowest values obtained in shank rank test were due to the fact that the total number on shanks ordered was lower (76 shanks vs. 114 carcasses), because after carcass rank test was performed, 38 birds were kept separately to be used in a market assay.

TABLE 4. Effect of dietary treatment on broiler shank pigmentation evaluated according to different systems: Roche color fan (RCF) scores, Minolta colorimeter, or rank test Dietary treatment1 Control T-1

Criteria 2

RCF Scores at 29 d RCF Scores at 43 d2 Shank rank test3 Minolta color coordinates L, lightness a, redness b, yellowness

c

Statistical analysis T-2

T-3 a

T-4 b

b

SE

Probability

2.10 2.16c 13.00c

7.00 8.89a 58.71a

5.90 7.36b 42.13b

5.40 8.00b 49.79b

0.30 0.22 2.39

0.0001 0.0001 0.0001

77.541a 1.745d 35.930c

73.740c 6.886a 64.832a

74.889b 3.463c 60.824b

74.434bc 4.424b 65.028a

0.277 0.239 0.519

0.0001 0.0001 0.0001

a–d Means within rows with no common superscripts differ significantly (P < 0.05). Data pooled over male and female. No interactions between treatment and sex were found for any parameter. 1 Treatments: T-1, no added xanthopylls; T-2, 35 ppm yellow xanthophylls from 10% zeaxanthin (SME-10) + 5 ppm canthaxanthin; T-3, 40 ppm yellow xanthophylls from 25% zeaxanthin (SME-25); T-4, 32 ppm yellow xanthophylls from SME-10 + 2 ppm canthaxanthin. 2 Range of values between 1 and 15. 3 Mean value of treatment scores. Scores ranged from 1 to 76.

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INFLUENCE OF XANTHOPHYLLS RATIO ON BROILER PIGMENTATION TABLE 5. Effect of dietary treatment on broiler breast skin pigmentation evaluated according to different systems: Roche color fan (RCF) scores, Minolta colorimeter, or rank test Dietary treatment1 Control T-1

Criteria 2

c

Carcass rank test Minolta color coordinates L, lightness a, redness b, yellowness

Statistical analysis T-2

T-3 a

T-4 b

ab

SE

Probability

15.93

78.22

64.75

74.94

3.89

0.0001

69.061a 1.880a 9.309d

67.236b 1.100b 25.638a

66.945b 0.748b 21.686c

67.564b 0.658b 24.178b

0.342 0.197 0.391

0.0001 0.0001 0.0001

a–c Means within rows with no common superscripts differ significantly (P < 0.05). Data pooled over male and female. No interactions between treatment and sex were found for any parameter. 1 Treatments: T-1, no added xanthopylls; T-2, 35 ppm yellow xanthophylls from 10% zeaxanthin (SME-10) + 5 ppm canthaxanthin; T-3, 40 ppm yellow xanthophylls from 25% zeaxanthin (SME-25); T-4, 32 ppm yellow xanthophylls from SME-10 + 2 ppm canthaxanthin. 2 Mean value of treatment scores. Scores ranged from 1 to 114.

In addition to their tint, the deposition rate of feed carotenoids in target tissues (percentage of consumed carotenoid that was found in target tissue) was the main factor that determined the pigmenting efficiency of the carotenoids. Carotenoids with a higher deposition rate contributed to the pigmentation to a greater extent than those with the lower rate of deposition (Huyghebaert, 1991). The results of deposition rates of lutein and zeaxanthin in breast skin and abdominal fat obtained in the present study are presented in Table 6. In the experimental diet T-3, 14.2 ppm of zeaxanthin were found due to the addition of SME-25 against 8.5 ppm of zeaxantin found in diet T-2 provided by the SME-10 product. Nevertheless, the deposition rate of lutein and zeaxanthin from the normal SME-10 product was higher than that deposited by these carotenoids when provided by the SME-25 product in breast skin and abdominal fat (Table 6), decreasing the difference between treatments in their xanthophyll pigments present in the target tissues evaluated (breast skin and fat). In addition, the individual stereo-isomers of zeaxanthin show different deposition rates (Hencken, 1992). With few exceptions, plants synthesize only optically active RR (3R, 3′R) carotenoids. However, pure chemical synthesis yields optically inactive, racemic carotenoids, as in the case of zeaxanthin. The two marigold products tested presented different ratios of zeaxanthin isomers; the SME-10 product showed a ratio of stereo-

isomers RR (optically active) vs. RS of 97.8:2.20%, whereas this ratio analyzed in SME-25 product was 16.0:84.0%, explaining the lower pigmenting capacity observed for this product. These results agree with those reported by Schiedt (1998) and Allen et al. (1998), who found that the racemic (3RS, 3′RS) isomers of zeaxanthin were absorbed to a lower extent than the (3R,3′R) enantiomer. The results reported herein suggest that the performance of birds was not affected by inclusion of different amounts of the tested pigments in feeds. The highest chicken pigmentation was obtained by inclusion of 35 ppm of YX from SME-10 product + 5 ppm of CTX. Birds fed the experimental diet with only isomerized SME-25 product added had less pigmentation than those fed diet with equivalent quantities of a combination of SME-10 + CTX. The color coordinate “b” measured in breast skin with the Minolta equipment was a good indicator of the yellow xanthophyll content present in feed, whereas the “a” coordinate measured in the shank showed a linear relationship with the level of CTX added to feeds. The same visual classification of chickens is achieved irrespective of the rank test performed (ordering by shank color or by entire carcass color). In the breast skin and abdominal fat, the deposition rate of lutein and zeaxanthin present in the SME-25 product was lower than those found in the SME-10 product. Accordingly, the higher content of zeaxanthin in the diet supplemented with SME-25 (14.2 ppm), relative to those supplemented with combinations

TABLE 6. Deposition of lutein and zeaxanthin on abdominal fat and breast skin of broiler chickens Carotenoid content (ppm) Pigment levels in feed2

Breast skin

Abdominal fat

Deposition ratio feed:skin

Deposition ratio feed:abdominal fat

Dietary treatment1

Lutein

Zeaxanthin

Lutein

Zeaxanthin

Lutein

Zeaxanthin

Lutein

Zeaxanthin

Lutein

Zeaxanthin

T-2 T-3 T-4

34.0 25.0 31.0

8.5 14.2 6.8

4.23 2.18 3.53

0.83 1.13 0.58

2.93 1.75 2.43

0.45 0.58 0.33

1:0.12 1:0.09 1:0.12

1:0.10 1:0.08 1:0.09

1:0.09 1:0.07 1:0.08

1:0.05 1:0.04 1:0.05

1 Treatments: T-1, no added xanthopylls; T-2, 35 ppm yellow xanthophylls from 10% zeaxanthin (SME-10) + 5 ppm canthaxanthin; T-3, 40 ppm yellow xanthophylls from 25% zeaxanthin (SME-25); T-4, 32 ppm yellow xanthophylls from SME-10 + 2 ppm canthaxanthin. 2 Analyzed contents of experimental feeds.

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of SME-10 + CTX (8.5 and 6.8 ppm), caused relatively higher contents of this carotenoid in breast skin and abdominal fat. However, the pigmentation of birds fed the diet supplemented with SME-25 was less compared to that of birds fed diets with SME-10 + CTX, irrespective of test used for evaluation (rank test, RFC scores, or Minolta color coordinates).

ACKNOWLEDGMENTS The authors thank Roche Vitamins Europe Ltd. for their financial and technical support to this study.

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