Biofortified Orange Maize Enhances Î²-Cryptoxanthin Concentrations

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Biofortified Orange Maize Enhances beta-Cryptoxanthin Concentrations in Egg Yolks of Laying Hens Better than Tangerine Peel Fortificant Emily Heying, Jacob Tanumihardjo, Vedran Vasic, Mark E. Cook, Natalia Palacios, and Sherry A. Tanumihardjo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5037195 • Publication Date (Web): 13 Nov 2014 Downloaded from http://pubs.acs.org on November 16, 2014

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Journal of Agricultural and Food Chemistry

Yolk from orange maize-fed hen

Yolk from white maize-fed hen

ACS Paragon Plus Environment


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Biofortified Orange Maize Enhances β-Cryptoxanthin Concentrations in Egg Yolks of

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Laying Hens Better than Tangerine Peel Fortificant1

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Emily K. Heying†, Jacob P. Tanumihardjo†, Vedran Vasic†, Mark Cook†,‡, Natalia Palacios-

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Rojas#, and Sherry A. Tanumihardjo†,*

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†

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Department of Nutritional Sciences, Madison WI 53706. ‡Department of Animal Sciences,

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Madison, WI 53706. #International Maize and Wheat Improvement Center (CIMMYT),

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University of Wisconsin-Madison, Interdepartmental Graduate Program in Nutritional Science,

Texcoco, Mexico

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Online supporting information: Supporting Table S1 and Figures S1-4

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Running Title: Biofortified maize enhances yolk β-cryptoxanthin

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EKH, JPT, VV, MC, NP-R, and SAT have no conflicts of interest.

Part of this work was presented at the Experimental Biology Meeting in 2014, San Diego, CA.

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*

To whom correspondence should be addressed. E-mail: [email protected]

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Abbreviations: VA, vitamin A


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ABSTRACT: The xanthophyll β-cryptoxanthin provides vitamin A and has other purported

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health benefits. Laying hens deposit xanthophyll carotenoids into egg yolk. Hens (n = 8/group)

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were fed conventional-bred high β-cryptoxanthin biofortified (orange) maize, tangerine peel-

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fortified white maize, lutein-fortified yellow maize, or white maize for 40 d to investigate yolk

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color changes using L*a*b* scales, yolk carotenoid enhancement, and hen vitamin A status.

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Yolks from hens fed orange maize had scores indicating a darker, orange color and mean higher

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β-cryptoxanthin, zeaxanthin, and β-carotene concentrations (8.43 + 1.82, 23.1 + 4.8, 0.16 + 0.08

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nmol/g, respectively) than other treatments (P < 0.0001). Yolk retinol concentrations (mean:

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14.4 + 3.42 nmol/g) were similar among groups and decreased with time (P < 0.0001). Hens fed

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orange maize had higher liver retinol (0.53 + 0.20 µmol/g liver) than other groups (P < 0.0001).

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β-Cryptoxanthin-biofortified eggs could be another choice for consumers, providing enhanced

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color through a provitamin A carotenoid and supporting eggs’ status as a functional food.

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KEYWORDS: biofortification, chicken liver, chickens, functional food, retinol, xanthophyll,

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zeaxanthin



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INTRODUCTION

Over 700 carotenoids exist in nature and about 50 are precursors of vitamin A (VA),

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commonly referred to as provitamin A carotenoids. The provitamin A xanthophyll, β-

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cryptoxanthin, is a bipolar, oxygenated molecule that is highly bioavailable from supplements

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and food. β-Cryptoxanthin is found in citrus fruit, such as tangerines and oranges, papaya, and in

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some pumpkins.1,2 Theoretically, β-cryptoxanthin yields one molecule of retinol upon central

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cleavage, while the hydrocarbon β-carotene yields two retinol molecules. For a mixed diet, the

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Institute of Medicine suggests using a bioconversion factor of 24 µg β-cryptoxanthin to 1 µg

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retinol, which is derived theoretically from the estimated 12 µg all-trans-β-carotene to 1 µg

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retinol.3 However, β-cryptoxanthin was comparable to all-trans-β-carotene in raising the VA

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status of Mongolian gerbils, possibly due to the polarity of the structure increasing absorption

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and micellarization.4,5 Furthermore, β-cryptoxanthin has apparent higher bioavailability than β-

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carotene in humans consuming mixed foods.6 In addition to contributing VA, dietary β-

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cryptoxanthin may have other purported health benefits, such as antioxidant properties7 and

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decreasing the risk of cancer2 and cardiovascular diseases.8 It also may aid in bone health by

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stimulating osteoblast formation and inhibiting bone resorption.9

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In addition to orange fruits, β-cryptoxanthin is found in low amounts in yellow maize,

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which is used for human food in some countries and animal feed around the world.10

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Conventional-bred maize biofortifed with β-carotene and β-cryptoxanthin (commonly referred to

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as orange maize) supports VA status in humans11-13 and animals.10,14 Orange maize also has the

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potential to impact other aspects of the food chain, such as egg yolk produced by feeding

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biofortified orange maize to laying hens.

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Xanthophyll carotenoids play a unique role in poultry. They are responsible for laying hen skin color and egg yolk15, which is the main reason to enrich feeds with carotenoids.16


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Chicken feed is often fortified with the non-provitamin A xanthophyll lutein as colorant to

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produce the deep yellow egg yolk preferred by some consumers with little regard to enhanced

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nutritional value.17,18 In several countries, including Mexico, China, and Bangladesh, intensely

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colored chicken skin and egg yolks are perceived as healthier products, which becomes an

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economic driver in the poultry industry.19 Egg yolks high in lutein and zeaxanthin20,21 provide an

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optimal digestible lipid matrix for carotenoid absorption22, but do not enhance the VA value. Hens are efficient converters of β-carotene to VA and absorb very little intact.23,24 When

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chickens were fed high β-cryptoxanthin maize, β-cryptoxanthin increased in eggs whereas high

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β-carotene maize feed resulted in no change in yolk β-carotene.25 Furthermore, β-cryptoxanthin

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had a higher efficiency of deposition in yolk than lutein or zeaxanthin.26 This evidence makes β-

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cryptoxanthin a candidate for use as an egg yolk colorant. The bipolar nature of β-cryptoxanthin

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likely allows it to accumulate in yolk better than the non-polar hydrocarbon β-carotene.25 β-

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Cryptoxanthin enhancement of eggs could favorably impact dietary intake of β-cryptoxanthin.

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The source of β-cryptoxanthin may impact the bioavailability of β-cryptoxanthin in hens

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for egg yolk deposition. The objective of this study was to comprehensively compare the source

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of β-cryptoxanthin in hens’ feed, i.e., orange maize or tangerine peel, and the impact on egg yolk

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color, skin color, carotenoid concentrations, and VA status of the laying hens compared with the

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industry-recommended lutein standard.

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MATERIALS AND METHODS

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Maize, Feed Preparation, and Analysis. Four different maize feeds were prepared. The

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high β-cryptoxanthin (orange) maize genotype was conventionally bred and obtained from the

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International Maize and Wheat Improvement Center/HarvestPlus maize provitamin A



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biofortification project and was grown in Mexico at Agua Fria, Puebla (20°32´N, 97°28´ W; 110

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m above sea level). Ears were harvested and dried, and grain was stored at -20oC before

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shipping to University of Wisconsin-Madison. The kernels were stored at -24oC for the duration

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of the study. Kernels were ground to a fine powder before hen feed preparation using a C&N

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Hammermill No. 8 (Christy-Norris Ltd., Ipswich, UK). Feed composition was identical to Liu et

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al. with the only source of VA through the orange and yellow maize25 or tangerine peel.

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Tangerine peel was freeze-dried for 48 h, finely ground, and stored at -80°C until preparation of

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the tangerine-fortified white maize feed. The white maize was sourced locally (DeLong Co.,

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Inc., Clinton, WI) and approximately 40 g ground freeze-dried tangerine peel was added per 1 kg

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feed, which was based on the β-cryptoxanthin concentration of the orange maize feed (5.64 +

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0.39 nmol/g feed). Concentrations were based on saponified β-cryptoxanthin because 75 to 94%

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of β-cryptoxanthin is esterified in tangerine peel27, but only 1-5% of xanthophylls are esterified

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in maize.28,29 Lutein ORO GLO-20 (graciously donated by Kemin Industries, Inc., Des Moines,

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IA) was added to the yellow maize (DeLong Co., Inc., Clinton, WI) at 0.05%, according to

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industry recommendations.30

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Feeds were mixed and analyzed weekly, and stored at -20⁰C to prevent carotenoid

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degradation. Carotenoid composition of maize and feeds (Table 1) was determined using an

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adapted saponification method.10,29 This ensured that the carotenyl esters present in the tangerine

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peel were hydrolyzed to the free form31, which was subsequently quantified in the tissues. After

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preheating 0.6 g maize feed suspended in 6 mL ethanol at 85 oC for 5 min, saponification was

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done with addition of 500 µL 80:20 potassium hydroxide:water (w:v) for 10 additional

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min. Water (3 mL) was added to aid phase separation during hexane extraction (3 X 3 mL

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hexane). The hexane extracts were dried, reconstituted in 500 µL 50:50


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methanol:dichloroethane, and 50 µL aliquots were analyzed by HPLC using a YMCTM C30

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column (4.6 x 250 mm).4 A 40-min multistep, binary gradient [A: 92:8 methanol:water (10 mM

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ammonium acetate) and B: methyl tertiary butyl ether] was run at 2 mL/min (Supporting Table

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S1). Extraction efficiencies ranged from 90 + 7 to 95 + 3% for the different maize feeds.

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Hens: All animal procedures were approved by the College of Agricultural and Life

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Sciences Animal Care and Use Committee, University of Wisconsin-Madison. Single Comb

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White Leghorns (n = 32), 28 wk into their laying cycle, were individually housed in metal

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battery cages under a 16-h light:8-h dark cycle. The hens were checked daily to ensure adequate

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supply of ad libitum feed and water and to monitor health status. Hens were given

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approximately 110 g feed/d, but total feed intake was not measured. Hens were killed by CO2

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fixation on intervention d 50. Hens were weighed before blood, liver, and breast skin were

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harvested.

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Experimental Design. Laying hens were fed a white-maize feed for a 10-d depletion period.

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For the next 40 d, hens were divided into 4 treatments based on maize genotype and fortificant (n

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= 8/treatment): high β-cryptoxanthin biofortified orange maize (orange), tangerine-fortified

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white maize (tangerine), lutein-fortified yellow maize (yellow), and white maize only (white) as

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a negative control. Eggs were collected every day and subjected to colorization every other day.

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Upon breaking the shell, yolks were separated from the whites and color was analyzed using a

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portable Konica Minolta colorimeter (Chroma Meter CR-300, Konica Minolta Sensing

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Americas, Inc., Ramsey, NJ) using the complementary color model. Color space (often referred

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to as L*a*b*) was used for assessment of changes in yolk color over the course of the study (n =

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6-8/treatment and collection time). The L-scale represents lightness, with a value of 0-100 (L =

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0 indicates darkest, L = 100 lightest). The a-scale represents redness on a scale from -60 (green),



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to +60 (red). The b-scale represents yellowness on a scale from -60 (blue) to +60 (yellow).

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After color analysis, yolks were transferred into black conical tubes and stored at -80°C.

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Analysis of Egg Yolk. Egg yolk samples from every 4 d were prepared for carotenoid and

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retinol equivalent composition analysis using an adaptation from a previously published

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method.22 Yolk (0.3 g) was mixed with 1 mL deionized water using a vortex, followed by 3 mL

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ethanol (0.1% butylated hydroxytoluene as antioxidant). Potassium hydroxide:water (500 µL,

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50:50, w:v) was added and the mixture was saponified for 30 min at 60oC. One mL cold

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deionized water was added to quench the reaction and 200 µL C-23-apo-carotenol was added as

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internal standard. The mixture was extracted with 4 mL 9:1 hexane:ethyl acetate (v:v), and then

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twice more with 3 mL. The organic layers were pooled, dried under nitrogen, and reconstituted

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in 200 µL 75:25 methanol:dichloroethane (v:v); 4 µL was injected onto a Waters Ultra

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Performance Liquid Chromatograph (UPLC) equipped with an ACQUITY HSS C18 1.8 µm, 2.1

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x 150 mm column at 35oC (Milford, MA). Solvent A was 90:10 water:isopropanol (10 mM

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ammonium acetate) and solvent B was 80:20 acetonitrile:isopropanol at 0.45 mL/min with a total

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runtime of 25 min. The gradient started with 30% solvent A and 70% solvent B switching to 5%

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A and 95% B from 0-10 min, with a change to 1% A and 99% B at 12 min and holding until

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reverse conditions from 21.5 to 25 min. Retention times are noted in Supporting Table S1.

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All-trans-β-carotene (537 g/mol; Sigma-Aldrich, St. Louis, MO), β-cryptoxanthin (553

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g/mol; CaroteNature, GmbH, Lupsingen, Switzerland), lutein (569 g/mol; GNC, Inc., Pittsburg,

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PA), and zeaxanthin (569 g/mol; GNC, Inc., Pittsburg, PA) were quantified using HPLC-purified

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standards. Provitamin A equivalents were the sum of β-cryptoxanthin, α-carotene, and twice the

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β-carotene due to the theoretical yield of retinol from the chemical structures.


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Analysis of Chicken Livers, Serum, and Skin. Livers were prepared and analyzed for

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carotenoids, retinol, and retinyl esters using an adaptation of published methods.32 Serum was

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prepared and analyzed for carotenoids and retinol and retinyl esters using previously published

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methods.10 Retinol and retinyl esters were quantified at 325 nm and carotenoids were quantified

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at 450 nm. Chicken skin was colorized with the colorimeter.25 Skin (1 g) was ground with 4-5 g

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sodium sulfate, 150 µL β-apo-8’-carotenal was added as internal standard, and the mixture was

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extracted with 25 mL dichloromethane. A 5 mL aliquot was dried under nitrogen, suspended in

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750 µL ethanol (0.1% butylated hydroxytoluene), and saponified with 300 µL potassium

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hydroxide:water (50:50, w:v) for 15 min at 45°C. After saponification, 1 mL deionized water

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was added to quench the reaction and the mixture was extracted 3 times with 2 mL hexanes.

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Organic layers were pooled and dried under nitrogen, reconstituted in 150 µL

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methanol:dichloroethane (50:50, v:v); 75 µL was injected onto a Waters Resolve C30 5 µm, 3.9 x

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300-mm reversed-phase column (Waters, Milford, MA) equipped with a guard column. The

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HPLC system consisted of a 1525 binary pump, a 717 autosampler, and a 996 photodiode array

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detector (Waters). Solvents and run-time were similar to published methods32 (Supporting Table

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S1).

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Statistical analysis. Values are means + SD. Data were analyzed using Statistical Analysis

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System software (SAS version 9.2, SAS Institute, Cary, NC). A repeated measures two-way

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ANOVA with mixed effects was used with PROC MIXED for egg color analysis and carotenoid

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and retinol concentrations of saponified egg yolk. The overall differences in color were

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evaluated using equation 1:33

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Equation 1: ∆E =

(∆L)2 + (∆a)2 + (∆b)2



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The influence of treatment on liver VA stores and serum retinol concentrations were

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evaluated using one-way ANOVA for the hens. LSMEANS was used to determine differences

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between treatments. Main treatment effects for feed type and time, and the interaction of

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treatment by time were considered significant at P < 0.05.

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RESULTS Carotenoid Concentration of Feeds. By design, orange and tangerine feeds did not differ

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in total β-cryptoxanthin concentration determined after saponification, although they presumably

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did differ in percentage of free versus esterified β-cryptoxanthin. The theoretical value of

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provitamin A equivalents were different due to the endogenous β-carotene in the orange maize

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(Table 1). Much lower amounts of provitamin A equivalents were present in the yellow and

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white maize feeds (Table 1). The yellow feed had four times more lutein than the orange feed.

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Zeaxanthin was similar in orange and yellow feeds, and it was more than four times higher than

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the tangerine and white feeds.

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Egg and Hen Weight. Egg weights (53.4 + 2.80 g) among treatment groups across all time

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periods did not differ (Supporting Figure S1). However, egg weight increased over time as the

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hens aged (P < 0.0001). A paired t-test comparing eggs at d 10 (end of washout period) and d 50

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(end of treatment phase) found a significant increase in egg weight (5.07 + 3.31 g) from hens in

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all treatment groups (P < 0.0001). A treatment by time interaction occurred (P = 0.02) where

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egg weight increase in the tangerine group (3.1 + 3.3 g) was less than the other treatment groups

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(5.2 + 3.2 g). Egg production differed among treatment groups (P < 0.01), but was almost one

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egg/d. Egg percent production (eggs/d on treatment X 100) were 93.0 + 5.3%, 90.8 + 7.2%, 97.2

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+ 2.4%, and 99.0 + 3.6% for hens fed the orange, tangerine, yellow, and white treatments,


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respectively, where yellow and white groups produced more eggs than orange and tangerine

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groups. One hen in the white group was euthanized early due to a prolapsed follicle. Hen

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weight at kill (1.40 + 0.11 kg) did not differ among treatment groups.

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Color Assessment of Egg Yolk. The L-scales for the yolks were different among

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treatments, time, and an interaction existed (P < 0.0001 for all; Supporting Figure S2).

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Although the L-scale remained relatively flat over time, yolks from all hens were significantly

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lighter on d 10 at the end of the washout period with VA-depleted white maize than d 0 at the

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beginning of the study (P < 0.0001). Similar to the L-scale, the a-scale responded to treatment,

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time, and an interaction existed (P < 0.0001) (Supporting Figure S3). The yolks from orange

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and yellow groups had similar a-scale values throughout the treatment phase of the study and

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were significantly higher than the tangerine and white groups’ yolks (P < 0.0001). Similar to the

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other scales, the b-scale differed by treatment, time, and an interaction between the two existed

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(P < 0.0001; Supporting Figure S4). After the initial washout period, the b-scale remained

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relatively flat. Total color differences (Table 2) were significant by treatment group (P = 0.006),

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as well as overall between treatment groups (P < 0.011). The orange and yellow groups’ yolks

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had similar values as did the tangerine and white groups’ yolks.

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Egg Yolk Carotenoid and Retinol Concentrations. Carotenoid concentrations in yolks

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from the orange group, but not other treatment groups, increased until d 24 and then stabilized (P

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< 0.0001) (Table 3). β-Cryptoxanthin concentrations were significantly different among

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treatment groups, increased with time, and a treatment by time interaction occurred (all P
2 times between d 16 and 24 and plateaued throughout the remainder of the study.

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As expected, lutein concentration was significantly higher in eggs from chickens fed yellow

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maize than that of any other group, but also increased with time across all groups and a treatment

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by time interaction occurred (all P < 0.0001). Yolks from the yellow group were the only yolks

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to reach lutein concentrations similar to the beginning of the washout period. Zeaxanthin was

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significantly higher in yolks from the orange group throughout the treatment period, but

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decreased with time across all groups and a treatment by time interaction occurred (all P
tangerine = yellow = white groups. Treatment also affected

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liver carotenoid concentrations (Figure 2A). β-Cryptoxanthin, zeaxanthin, and β-carotene

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concentrations of free carotenoids and total carotenoid liver reserves were highest in chickens

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from the orange group (Figure 2A, 2B, all P < 0.0001). Lutein concentration and total liver

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lutein reserves were highest in the yellow group. However, zeaxanthin was consistently the most


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abundant carotenoid in liver, except for in the tangerine group where β-cryptoxanthin was

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similar to zeaxanthin (Figures 2A, 2B). α-Carotene was not detected in the chicken liver. Hen serum retinol concentrations differed among treatment groups (P < 0.0001) and were

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highest in the tangerine group (Figure 1C). Serum retinol concentrations did not follow the

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same pattern as the retinol concentrations in the liver. All hens had serum retinol concentrations

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>1.0 µmol/L. Hen serum β-cryptoxanthin and zeaxanthin concentrations were highest in the

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orange group (P < 0.0001). Serum lutein was highest in the yellow group (P < 0.0001) (Figure

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2C). While serum lutein, zeaxanthin, and β-cryptoxanthin concentrations reflected those in the

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liver, β-carotene was not detectable in any group. In contrast, liver β-carotene concentrations

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were detectable and highest in the orange group compared with other groups. Chicken Skin Colorization and Carotenoids. No differences were observed among the

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skin from any of the feeding groups for the L-, a-, and b-color scales. Lutein and zeaxanthin

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were highest in the yellow group’s skin, but no significant differences existed (Table 5, P =

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0.09, P = 0.38, respectively). Skin β-cryptoxanthin concentration was low compared with lutein

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and zeaxanthin, but was significantly higher in the orange group than other groups (Table 5, P =

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0.002).

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DISCUSSION This study evaluated the impact of β-cryptoxanthin from biofortified orange maize or

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tangerine peel powder on egg yolk color and VA status of laying hens fed for 40 d. The orange

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feed resulted in deeper orange yolk, which was visible to the naked eye, significantly higher

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concentrations of β-cryptoxanthin (~2 times higher) and zeaxanthin (~4 times higher), and

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differentiated color from the tangerine group on all color scales, even though both feeds



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contained similar concentrations of β-cryptoxanthin. Eggs from hens that were fed orange maize

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became significantly darker on the L-scale, redder on the a-scale, and more yellow on the b-

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scale. Total color difference was small between white and tangerine and between orange and

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yellow, which was expected because they were similar on each color-scale measurement.

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Hens consuming the orange maize had the highest liver VA stores, which were 1.5 times

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higher than the tangerine group and 2.6 times higher than the yellow group. Tangerine peel was

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chosen as a fortificant because it is considered a waste product in juice production and therefore

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could be repurposed in chicken feed manufacturing. Considering the difference in theoretical

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retinol equivalents between the orange maize and tangerine peel feeds, it is likely that the β-

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cryptoxanthin from the tangerine peel was serving as a major source of VA for the hen itself,

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because other provitamin A carotenoids were absent in the tangerine peel feed. Follow-up

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studies should equalize maize feeds for provitamin A equivalents to determine if β-cryptoxanthin

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is less bioavailable from the tangerine peel matrix compared with orange maize, and to

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investigate different levels of tangerine fortificant to maximize the β-cryptoxanthin response in

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the egg yolk in addition to meeting the hen’s VA needs. In addition, the degree of fatty acid

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esterification of the β-cryptoxanthin in the tangerine peel should be determined to investigate

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whether this has any effect on bioavailability in the chicken.31

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In addition to this study, two prior studies fed biofortified maize feeds to laying hens that

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resulted in enhanced β-cryptoxanthin of the yolks.25,26 The β-cryptoxanthin concentration in egg

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yolks from this study, on d 28, were twice that (8.66 + 0.53 nmol/g) of a previous study (4.20

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nmol/g), yet similar β-cryptoxanthin concentrations were fed (4.71 nmol/g and 5.64 nmol/g,

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respectively).25 Furthermore, a recent study in which biofortified β-cryptoxanthin maize was fed

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to laying hens for 140 d with a concentration of 9.8 nmol/g, found a mean egg yolk β-


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cryptoxanthin concentration of 10.1 nmol/g after freeze drying.26 Raw egg yolk is approximately

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50% water34, therefore the prior two study values are similar. Interaction between zeaxanthin

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and β-cryptoxanthin for uptake into the egg yolk may contribute to these discrepancies.

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However, yolk concentration of β-cryptoxanthin did mirror theoretical provitamin A intake in

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this study. β-Carotene in the orange maize likely met the VA requirements of the hens, which

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allowed for more β-cryptoxanthin to be deposited into the egg yolk than utilized for VA needs.

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Unlike the prior studies, this study also evaluated the VA statuses of the hens, which

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were impacted by the level of β-cryptoxanthin and other provitamin A carotenoids. Currently,

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there is no carotenoid requirement for poultry, although dietary carotenoids contribute to

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plumage, beak, skin, and egg yolk color.15 Consequences of VA deficiency in laying hens are

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decreased egg production and yolk VA concentration, and lesions in the upper respiratory tract

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(esophagus) and ovarian follicle.35 The National Research Council’s VA requirement for mature

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Leghorn laying hens is 300 IU VA/d (90 µg/d).34 According to international standards and

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theoretical VA concentrations in the feeds, all hens except those on the white maize feed met 300

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IU/day. Assuming they ate ~100 g feed/day, hens fed orange maize theoretically consumed 1555

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IU/d, the tangerine group consumed 828 IU/d, the yellow group consumed 309 IU/d, and the

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white group consumed 102 IU/d. Although they were theoretically consuming one third the VA

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requirement, hens fed the white maize had adequate liver reserves [0.17 µmol/g liver compared

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with 0.1 µmol/g liver37] after 50 d treatment. Retinol concentration in the eggs was not impacted

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by the level of provitamin A equivalents in the feed during this short-term study, which indicates

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highly regulated deposition of VA into the yolk. Hens fed orange maize had higher liver VA

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concentrations and total reserves than other groups. Using biofortified orange maize or tangerine

311

fortificant could eliminate the need for addition of preformed retinyl palmitate to feed.



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312

According to serum retinol and liver VA concentrations, hens on all treatments were VA-

313

adequate meeting the cutoffs of 0.7 µmol retinol/L serum and 0.1 µmol VA/g liver, respectively.

314

The orange feed was higher in β-carotene than other groups. Hens are efficient at

315

cleaving β-carotene to retinol, most likely contributing to the increased VA concentration and

316

reserves in hen liver; however, liver β-carotene concentrations were significantly higher in hens

317

fed orange maize, indicating that some absorption of intact β-carotene did occur in this study.

318

Hens fed the orange maize also had higher liver β-cryptoxanthin concentrations than other

319

groups, reflective of β-cryptoxanthin concentrations in the feed. Lutein and zeaxanthin

320

accumulation in liver reflected feed intake, similar to results found when feeding high lutein and

321

zeaxanthin feeds to Leghorn chicks.38 Lutein concentration was highest in the yellow group’s

322

livers. Zeaxanthin was highest in the orange group and greater than the β-cryptoxanthin

323

concentration. Furthermore, the ratio of liver β-cryptoxanthin to lutein (2.6) was higher than the

324

ratio in the orange feed (0.9), which may indicate a bias against accumulation of lutein in the

325

chicken liver. Differences among animal models in carotenoid metabolism are interesting. A

326

common model used for provitamin A carotenoid metabolism is Mongolian gerbils, but they

327

have the limitation of only absorbing and storing very small amounts of lutein and

328

zeaxanthin.39,40 Chickens appear to be a better model for studying xanthophyll carotenoid

329

bioavailability. Similar to gerbils, serum concentrations of β-carotene were not detectable,

330

which may be because chickens have higher HDL than LDL.41 β-Carotene is predominantly

331

carried by LDL, whereas lutein and zeaxanthin are carried by HDL.42

332

Chicken skin color is strongly associated with dietary concentration of carotenoids, 43 but

333

there were no color differences among treatment groups in this study using L*a*b* scales. β-

334

Cryptoxanthin was present in small amounts in chicken skin and was significantly higher in


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335

chickens fed orange maize. Even though the lutein concentration in the yellow group was

336

extremely high, lutein concentrations did not differ in the skin. However, the hens used in this

337

study were young and early in their laying cycle. Hens in peak egg production withdraw

338

xanthophyll carotenoids from skin and deposit them in egg yolk. Increased deposition of

339

xanthophyll carotenoids into skin is probably best studied in non-egg-laying chickens.

340

In order to be effective, biofortified foods must be accepted by targeted populations.

341

Consumers have documented egg yolk preferences. Consumers in Germany, Belgium,

342

Netherlands, and Spain prefer an orange-colored yolk, whereas those in Ireland and Sweden

343

prefer a lighter yolk.43 Dietary sources of β-cryptoxanthin are few and most humans obtain it

344

from orange citrus fruits. Although β-cryptoxanthin concentration varies by fruit source, a mean

345

value for orange juice is ~200 µg/100 mL.44 Thus, two biofortifed egg yolks (e.g. 30 g yolk with

346

5 µg β-cryptoxanthin/g) could provide about 75% of that amount or provide more than a medium

347

fresh orange, which contains ~60 mL juice.

348

The consumption of white staple foods by both humans and livestock in developing

349

countries provides no VA. Many countries prefer nutrient-poor white maize to meet their daily

350

calorie needs.45 The introduction of provitamin A biofortified crops, such as orange maize, has

351

the potential to raise xanthophyll intakes in humans and animals.4,10-14 Due to the xanthophyll

352

profile of maize, it should be especially targeted, because it is the primary ingredient in many

353

livestock feeds, such as chickens, swine, and cattle. By substituting biofortified orange maize,

354

which has enhanced β-cryptoxanthin, lutein, and zeaxanthin, for white or yellow maize in

355

chicken feed, the need for VA or lutein-fortified feeds could be diverted lowering the cost for

356

hen husbandry and improving the nutritional profile of eggs for human consumption.



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The introduction of biofortified maize as a human food and an animal feed in developed

357 358

and developing countries could have economic and health considerations for all. Eggs are a

359

target for provitamin A carotenoid biofortification through feeding orange maize to laying hens,

360

which could further eggs’ status as a functional food with enhanced nutrition.46 Furthermore,

361

lutein and zeaxanthin, which are also enhanced in many genotypes of biofortified maize, may

362

contribute to decreased risk of macular degeneration.47 Thus, future studies should determine the

363

impact of the complete carotenoid profile of the biofortified egg on human health.

364 365

Acknowledgements

366

The authors would like to thank James Claus, University of Wisconsin-Madison, for guidance

367

and use of the colorimeter. The authors thank Michael Grahn, Chris Davis, and Shellen Goltz

368

for assisting with sample analysis and guidance during this study. The authors also thank Chris

369

Davis for assisting us with the preparation of Supporting Table S1.

370 371

372

Corresponding Author

373

*Mailing address: 1415 Linden Drive, UW-Madison, Madison, WI 53706. Phone: 608-265-

374

0792. Email: [email protected]. Reprints will not be available.

AUTHOR INFORMATION

375 376

Authors’ Contributions

377

EKH conducted research, analyzed samples, analyzed data, and wrote the first draft of the

378

manuscript. JPT and VV were responsible for the day-to-day care of the hens, colorized the

379

eggs, and analyzed samples. MC was responsible for animal care training and oversight of hen


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380

husbandry. NP-R and SAT designed the research. SAT provided input for the statistical

381

analysis and revised the manuscript. All authors read and approved the final manuscript. The

382

authors have no conflicts of interest to declare.

383 384

Funding

385

This research was supported by MASAGRO (Modernizacion Sustentable de la Agricultura), a

386

program of SAGARPA-Mexico in collaboration with the International Center of Wheat and

387

Maize Improvement and USDA Hatch WIS01528 and WIS01804.

388 389

Notes

390

None of the authors had any financial interest in the work or a conflict of interest with the

391

sponsors of this study.

392 393

Supporting Information Available: Supporting Table S1 and Figures S1-4. This material is

394

available free of charge via the Internet at http://pubs.acs.org.

REFERENCES

(1) Arscott, S. A. Food Sources of Carotenoids. In: Carotenoids and Human Health; Tanumihardjo, S. A., Ed., New York: Springer Science and Business Media. 2013, pp. 21– 28. (2) Liu, C.; Bronson, R. T.; Russell, R. M.; Wang, W. D. β-Cryptoxanthin supplementation prevents cigarette smoke-induced lung inflammation, oxidative damage, and squamous metaplasia in ferrets. Am. Assoc. Cancer Res. 2011, 4, 1255–1266.



Page 20 of 35

19

(3) Institute of Medicine. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. National Academy Press: Washington, DC. 2001, pp. 65–126. (4) Davis, C.; Jing, H.; Howe, J. A.; Rocheford, T.; Tanumihardjo, S. A. β-Cryptoxanthin from supplements or carotenoid-enhanced maize maintains liver vitamin A in Mongolian gerbils (Meriones unguiculatus) better than or equal to β-carotene supplements. Br. J. Nutr. 2008, 100, 786–793. (5) Dhuique-Mayer, C.; Borel, P.; Reboul, E.; Caporiccio, B.; Besancon, P.; Amiot, M. J. βCryptoxanthin from citrus juices: assessment of bioaccessibility using an in vitro/Caco-2 cell culture model. Br. J. Nutr. 2007, 97, 883–890. (6) Burri, B. J.; Chang, J. S. T.; Neidlinger, T. R. β-Cryptoxanthin and α-carotene rich foods have greater apparent bioavailability than β-carotene-rich foods in Western diets. Br. J. Nutr. 2011, 105, 212–219. (7) Fu, H.; Xie, B.; Fan, G.; Ma, S.; Zhu, X.; Pan, S. Effect of esterification with fatty acid of βcryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method. J. Food Chem. 2010, 122, 602–609. (8) Ciccone, M. M.; Cortese, F.; Gesualdo, M.; Carbonara, S.; Zito, A.; Ricci, G.; De Pascalis, F.; Scicchitano, P.; Riccioni, G. Dietary intake of carotenoids and their antioxidant and antiinflammatory effects in cardiovascular care. Mediators Inflamm. 2013, 2013, 1–11 (Article ID 782137). (9) Yamaguchi, M. Role of carotenoid β-cryptoxanthin in bone homeostasis. J. Biomed. Sci. 2012, 19, 36 (http://www.jbiomedsci.com/content/19/1/36).


Page 21 of 35


20

(10)

Howe, J. A.; Tanumihardjo, S. A. Carotenoid-biofortified maize maintains adequate

vitamin A status in Mongolian gerbils. J. Nutr. 2006, 136, 2562–2567. (11)

Li, S.; Nugroho, A.; Rocheford, T.; White, W. S. Vitamin A equivalence of the β-

carotene in β-carotene-biofortified maize porridge consumed by women. Am. J. Clin. Nutr. 2010, 92, 1105–1112. (12)

Muzhingi, T.; Gadaga, T. H.; Siwela, A. H.; Grusak, M. A.; Russell, R. M.; Tang, G.

Yellow maize with high β-carotene is an effective source of vitamin A in healthy Zimbabwean men. Am. J. Clin. Nutr. 2011, 94, 510–519. (13)

Gannon, B.; Kaliwile, C.; Arscott, S. A.; Schmaelzle, S.; Masi, C.; Chileshe, J.;

Kalungwana, N.; Mosonda, M.; Pixley, K.; Masi, C.; Tanumihardjo, S. A. Biofortified orange maize is as efficacious as a vitamin A supplement in Zambian children even in the presence of high liver reserves of vitamin A: a community-based, randomized placebocontrolled trial. Am. J. Clin. Nutr. (In press). (14)

Heying, E. K.; Grahn, M.; Pixley, K. V.; Rocheford, T.; Tanumihardjo, S. A. High-

provitamin A carotenoid (orange) maize increases hepatic vitamin A reserves of offspring in a vitamin A-depleted sow-piglet model during lactation. J. Nutr. 2013, 143, 1141–1146. (15)

Goodwin, T. W. Avian Carotenoids. In: Carotenoids, Their Comparative Biochemistry.

Chemical Publishing Co. New York, NY. 1954, pp. 259–268. (16)

Hencken, H. Chemical and physiological behavior of feed carotenoids and their effects

on pigmentation. Poult. Sci. 1992, 71, 711–717. (17)

Castaneda, M. P.; Hirschler, E. M.; Sams, A. R. Skin pigmentation evaluation in broilers

fed natural and synthetic pigments. Poult. Sci. 2005, 84, 143–147.



Page 22 of 35

21

(18)

Perez-Vendrell, J.; Hernández, M.; Llauradó, L.; Schierle, J.; Brufau, J. Influence of

source and ratio of xanthophyll pigments on broiler chicken pigmentation and performance. Poult. Sci. 2001, 80, 320–326. (19)

Martinez, P. M.; Cortes, C. A.; Avila, G. E. Evaluation of three pigment levels of

marigold petals (Tagetes erecta) on skin pigmentation of broiler chicken. Tec. Pecu. Mex. 2004, 42, 105-111. (20)

Schaeffer, J. L.; Tyczkowski, J. K.; Parkhurst, C. R.; Hamilton, P. B. Carotenoid

composition of serum and egg yolks of hens fed diets varying in carotenoid composition. Poult. Sci. 1988, 67, 608–614. (21)

Mangels, A. R.; Holden, J. M.; Beecher, G. R.; Forman, M. R.; Lanza, E. Carotenoid

contents of fruits and vegetables: an evaluation of analytic data. J. Am. Diet. Assoc. 1993, 93, 284–296. (22)

Handelman, G. J.; Nightingale, Z. D.; Lichtenstein, A. H.; Schaefer, E. J.; Blumberg, J.

B. Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk. Am. J. Clin. Nutr. 1999, 70, 247–251. (23)

Poor, C. L.; Miller, S. D.; Fahey, G, C.; Easter, R. A.; Erdman, J. W., Jr. Animal models

for carotenoid utilization studies: evaluation of the chick and the pig. Nutr. Rep. Int. 1987, 36, 229–234. (24)

Surai, P. F.; Noble, R. C.; Speake, B. K. Tissue-specific differences in antioxidant

distribution and susceptibility to lipid peroxidation during development of the chick embryo. Biochim. Biophys. Acta. 1996, 1304, 1–10.


Page 23 of 35


22

(25)

Liu, Y. Q.; Davis, C. R.; Schmaelzle, S. T.; Rocheford, T.; Cook, M. E.; Tanumihardjo,

S. A. β-Cryptoxanthin biofortified maize (Zea mays) increases β-cryptoxanthin concentration and enhances the color of chicken egg yolk. Poult. Sci. 2012, 91, 432–438. (26)

Burt, A. J.; Caston, L.; Leeson, S.; Shelp, B. J.; Lee, E. A. Development and utilization

of high carotenoid maize germplasm: proof of concept. Crop Sci. 2013, 53, 554–563. (27)

Breithaupt, D. E.; Bamedi, A. Carotenoid esters in vegetables and fruits: a screening with

emphasis on beta-cryptoxanthin esters. J. Agric. Food Chem. 2001, 49, 2064–2070. (28)

Blessin, C. W. Carotenoids of corn and sorghum. Cereal Chem. 1962, 39, 236–242.

(29)

Howe, J. A.; Tanumihardjo, S. A. Evaluation of analytical methods for carotenoid

extraction from biofortified maize (Zea mays sp.). J. Agric. Food Chem. 2006, 54, 7992– 7997. (30)

Kemin Industries. Utilization of dry Kem GLO® brand and dry ORO GLO® brand for

pigmentation of egg yolks as quantitated through fan score comparison and reflectance colorimeter readings. 2000. (31)

Pérez-Gálvez, A.; Mínguez-Mosquera, M. I. Esterification of xanthophylls and its effect

on chemical behavior and bioavailability of carotenoids in the human. Nutr. Res. 2005, 25, 631–640. (32)

Tanumihardjo, S. A.; Howe, J. A. Twice the amount of α-carotene isolated from carrots

is as effective as β-carotene in maintaining the vitamin A status of Mongolian gerbils. J. Nutr. 2005, 135, 2622–2626. (33)

Interpretation of color data. Technical service report Number 79. PolyOne Corp., 2005.



Page 24 of 35

23

(34)

U.S. Department of Agriculture, Agricultural Research Service. 2013. USDA National

Nutrient Database for Standard Reference, Release 26. Nutrient Data Laboratory Home Page, http://www.ars.usda.gov/ba/bhnrc/ndl. (Accessed in June 2014). (35)

Bermudez, A. J.; Swayne, D. E.; Squires, M. W.; Radin, M. J. Effects of vitamin A

deficiency on the reproductive system of mature White Leghorn hens. Avian Diseases. 1993, 37, 274–283. (36)

National Research Council. Nutrient Requirements of Laboratory Animals. 4th ed.

Washington, DC: National Academy Press; 1995. (37)

Tanumihardjo, S. A. Vitamin A: biomarkers of nutrition for development. Am. J. Clin.

Nutr. 2011, 94, S658–S665. (38)

Wang, Y.; Illingworth, D. R.; Connor, S. L.; Duell, P. B.; Connor, W. E. Competitive

inhibition of carotenoid transport and tissue concentrations by high dose supplements of lutein, zeaxanthin, and beta-carotene. Eur. J. Nutr. 2010, 49, 327–336. (39)

Molldrem, K.; Tanumihardjo, S. A. Lutein supplements are not bioavailable in the

Mongolian gerbil while consuming a diet with or without cranberries. Int. J. Vitam. Nutr. Res. 2004, 74, 153–160. (40)

Escaron, A. L.; Tanumihardjo, S. A. Absorption and transit of lutein and beta-carotene

supplements in the Mongolian gerbil (Meriones unguiculatus). Int. J. Vitam. Nutr. Res. 2006, 76, 315–323. (41)

Arshad MS, Anjum FM, Khan MI, Shahid M. Wheat germ oil and α-lipoic acid

predominantly improve the lipid profile of broiler meat. J. Agric. Food Chem. 2013, 61, 11158-11165.


Page 25 of 35


24

(42)

Clevidence, B. A.; Bieri, J. G. Association of carotenoids with human plasma

lipoproteins. Methods Enzymol. 1993, 214, 33–46. (43)

Tarique, T. M.; Yang, S.; Mohsina, Z.; Qiu, J.; Zhao, Y.; Gang, C.; Ailiang, C. Role of

carotenoids in poultry industry in China: a review. J. Nat. Sci. Res. 2013, 3, 111–121. (44)

Stinco, C. M.; Fernández-Vázquez, R.; Escudero-Gilete, M. L.; Heredia, F. J.; Meléndez-

Martínez, A. J.; Vicario, I. M. Effect of orange juice's processing on the color, particle size, and bioaccessibility of carotenoids. J. Agric. Food Chem. 2012, 60, 1447-55. (45)

Nuss, E. T.; Tanumihardjo, S. A. Maize: A paramount staple crop in the context of global

nutrition. Compr. Rev. Food Sci. Food Saf. 2010, 9, 417-436. (46)

Applegate, E. Introduction: Nutritional and functional roles of eggs in the diet. J. Am.

Coll. Nutr. 2000, 19, 495S–498S. (47)

Vishwanathan, R.; Johnson, E. J. Lutein and zeaxanthin and eye disease. In: Carotenoids

and Human Health; Tanumihardjo, S. A., Ed., New York: Springer Science and Business Media. 2013, pp. 215–235.



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Table 1. Carotenoid and Theoretical Retinol Feed Concentrations (nmol/g feed)A Prepared for Laying Hens using a Combination of Different maize Genotypes and Fortificants TreatmentB Carotenoid

Orange

Tangerine

Yellow

White

Lutein

6.2 + 0.32b

4.40 + 0.48b

24.8 + 5.83a

4.0 + 0.89b

Zeaxanthin

3.42 + 0.57a

0.72 + 0.09c

2.14 + 0.73b

0.68 + 0.27c

β-Cryptoxanthin

5.64 + 0.39a

4.85 + 0.75a

0.79 + 0.03b

0.13 + 0.06c

α-Carotene

0.24 + 0.11a

0.13 + 0.03bc

0.20 + 0.05ab

0.06 + 0.03c

9-cis-β-carotene

0.93 + 0.13a

0.20 + 0.07b

0.23 + 0.02b

0.10 + 0.01c

All-trans-β-carotene

3.37 + 0.20a

0.87 + 0.29b

0.60 + 0.03c

0.27 + 0.03d

13-cis-β-carotene

1.03 + 0.30a

0.22 + 0.07b

0.33 + 0.04b

0.15 + 0.06b

Theoretical retinol

16.3 + 1.36a

8.69 + 1.71b

3.24 + 0.26c

1.07 + 0.33c

A

Concentrations are mean + SD; n = 27 for white maize; n = 21 for other treatment feeds. All

carotenoid and theoretical retinol differed by treatment using one-way ANOVA (All P < 0.0001). Lowercase superscript letters in a row designate differences between feeds (tested with LSMEANS at P < 0.05). B

Treatment groups are designated by color regarding maize and/or fortification. Orange = high

β-cryptoxanthin biofortified maize, Tangerine = white maize + tangerine-peel fortificant, Yellow = yellow maize + lutein fortificant, White = white maize only.


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Table 2. Color Scale Readings (L*a*b*) on Select Days and Total Color Difference of Egg Yolks Assessed by ∆E. Hens were fed Orange, Tangerine-fortified White, Lutein-fortified Yellow, or Unfortified White Maize for 40 d Color scale measurementsA,B Day 10

Day 50

60.2 + 2.63

58.3 + 0.94

a

-3.27 + 0.56

2.79 + 0.72

b

38.4 + 3.43

44.6 + 2.46

Tangerine L

61.1 + 2.00

61.9 + 2.40

a

-3.50 + 0.36

-2.80 + 0.64

b

37.5 + 1.57

38.1 + 2.70

L

61.3 + 1.07

57.8 + 1.73

a

-3.09 + 0.55

1.96 + 0.61

b

38.5 + 2.70

46.1 + 1.40

L

62.7 + 1.16

61.2 + 2.50

a

-3.43 + 0.57

-4.03 + 0.43

b

39.1 + 5.26

36.7 + 1.47

Within treatment groupB,D

∆EE

OrangeC

Yellow

White

L

Orange

9.46 + 2.36a

Tangerine

4.15 + 2.38b

Yellow

8.59 + 2.72a

White

4.72 + 5.16b

Between treatment group

∆EE



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means F Orange vs. tangerine

10.3

Orange vs. yellow

1.36

Orange vs. white

10.8

Tangerine vs. yellow

9.74

Tangerine vs. white

1.62

Yellow vs. white

10.2

A

All L*a*b scale measurements can be found in Supporting Figures S2-4.

B

Values are means + SD; n = 8/treatment group.

C

Treatment groups: Orange = high β-cryptoxanthin maize, Tangerine = white maize + tangerine

peel fortificant, Yellow = yellow maize + lutein fortificant, White = white maize only. D

The treatment effect within a group is significant (P = 0.006); means with lowercase

superscript letters are significantly different using LSMEANS at P < 0.05. E

∆E is a measure of change in L-, a-, and b-scales from the beginning (d 10) to end (d 50) of the

experimental feeding period (see Equation 1). F

Means of each group on d 50 of the experiment were compared between groups for ∆E; P

Biofortified Orange Maize Enhances Î²-Cryptoxanthin Concentrations

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