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Effects of Vitamin D3-Enriched Diet on Egg YolkVitamin D3 Content and Yolk QualityLinxing Yao, Tong Wang, Michael Persia, Ronald L. Horst, and Mallory Higgins

Abstract: A 40-wk experiment was conducted using Hy-Line W-36 laying hens (19-wk old) to investigate the impact offeeding cholecalciferol-enriched diets on egg yolk quality. Feeds were enriched with 4 cholecalciferol levels, 9700 (diet 2),17200 (diet 3), 24700 (diet 4), and 102200 (diet 5) IU/kg feed. The control (diet 1) contained 2200 IU cholecalciferol/kgfeed. Eggs from each replicate group of enriched diets were collected daily and the yolks were pooled into 2-d periodduring the first 2 wk. During weeks 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, and 40, pooled samples were generated bydaily collection of 3 consecutive days of egg production. The cholecalciferol content of egg yolk from the enriched dietsincreased rapidly during the first 3 wk. The peak cholecalciferol concentrations in egg yolk that occurred at week 3 were865, 1641, 2411, and 34815 IU/100 g egg yolk (wet basis) from diet 2 to 5. The average cholecalciferol concentrationin yolk during weeks 3 to 40 and the deposition rate of cholecalciferol during the first 3 wk were both linearly increasedwith the dietary cholecalciferol level when the feed contained no more than 24700 IU/kg cholecalciferol. Egg yolklipid profile (total lipid content, fatty acid composition, phospholipid composition, and unsaponifiables), physical andfunctional properties (yolk viscosity and emulsifying property), and sensory quality of hard-boiled egg yolk were notaffected by the cholecalciferol enrichment in the feed.

Keywords: cholecalciferol, egg yolk, fortification, transfer efficiency, vitamin D3

Practical Application: A linear dose-response relationship between dietary vitamin D3 level and egg yolk vitamin D3

content was established at relatively low enrichment levels. Such relationship can be used to formulate feed to achieve atarget egg vitamin D level. High vitamin D yolk showed no difference from the conventional yolk in other compositional,functional, and sensory properties.

IntroductionVitamin D promotes calcium absorption and regulates serum

calcium and phosphorus concentration to maintain normal bonehealth (Morris 2005). Although vitamin D can be produced en-dogenously in the skin under sunlight, the exposure to ultravioletlight also poses skin cancer risk (American Cancer Society 2009a).Public awareness of the negative effects of sun exposure and the useof effective means of skin protection from sun exposure (AmericanCancer Society 2009b) may have had certain unintended effectson vitamin D status, contributing to an increased dietary vitaminD need. In addition, people who have limited sunlight exposurein winter and in northern latitude also are at high risk of vitaminD deficiency. Three quarters of U.S. teens and adults are deficientin vitamin D, and such deficiency is associated with osteomala-cia (weak muscles and bones), reduced immune functions, andincreased inflammation (Lite 2009).

The recently released Dietary Reference Intakes for calcium andvitamin D increased the recommended daily intake of vitamin Dfor children and adults from 200 to 600 IU a day, and from 600 to800 IU a day for elder people. The safe upper limit on daily intake

MS 20121189 Submitted 8/29/2012, Accepted 12/3/2012. Authors Yao andWang are with Dept. of Food Science and Human Nutrition, Iowa State Univ.,Ames, IA 50011, U.S.A. Authors Persia and Higgins are with Dept. of AnimalScience, Iowa State Univ., Ames, IA 50011, U.S.A. Author Horst is with HeartlandAssays, LLC, Ames, IA 50010, U.S.A. Direct inquiries to author Wang (E-mail:tongwang@iastate.edu).

has been increased from 2000 IU to today’s 4000 IU for most agegroups (Institute of Medicine 2010). Few natural foods containsignificant amount of vitamin D, which necessitates fortificationof the diet or specific foods with vitamin D to satisfy the currentrecommendations. For instance, fortified milk with vitamin D isan important dietary source of vitamin D; however, it supplies lessthan 50% of the current daily value. Another important naturalfoodstuff rich in vitamin D is egg. According to USDA nationalnutrient database, one large egg (about 50 g) contains approxi-mately 41 IU vitamin D (concentrated in the yolk). However, thenormal consumption level of eggs still cannot provide adequateamount of vitamin D to meet the daily requirement. Researchhas shown that vitamin D level in eggs can be readily increasedby feeding the laying hens with vitamin-D-enriched diet becauseof the efficient transfer of lipid and lipid soluble molecules fromthe diet to eggs (Mattila and others 1999, 2003, 2004). Since eggsare versatile food or ingredient and are well accepted in most cul-tures, production of a high vitamin D egg may provide a uniqueopportunity to enhance human vitamin D consumption withoutgreatly altering food consumption patterns.

A study by Mattila and others (2003) showed that supplementa-tion of laying hen diets with 12000 IU of vitamin D3 (or cholecal-ciferol)/kg feed resulted in eggs that contained more than 6 timesthe normal amount of vitamin D. Although this report demon-strated transfer of vitamin D, it only evaluated a single feedingdose and the 24-wk study suggested that after prolonged feed-ing, transfer of vitamin D to the egg might be down regulated.Morrisey and others (1977) reported that 40000 IU/kg feed was

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Table 1–Laying hen basal diets.a

Diet forc Diet for Diet forIngredients hen weeks hen weeks hen weeks(% Basis) 18 to 38c 39 to 53 54 to 59

Corn 48.94 51.97 55.35DDGSb 10.00 10.05 10.05Soybean meal 48 24.03 21.74 19.97Animal-vegetable fat blend 4.90 4.55 3.09Salt 0.40 0.40 0.33DL methionine 0.15 0.11 0.02Unical S 4.60 4.62 4.71Shell and bone builder 4.60 4.62 4.71Dicalcium phosphate 1.78 1.81 1.53Choline chloride 60 0.10 0.10 0.10Vitamin/mineral premix 0.50 0.50 0.50Bio-Lys – – 0.13Threonine – 0.03 –Calculated crude protein 18.43% 17.50% 16.96%

aCholecalciferol was added to base diets in order to obtain 5 levels of dietarycholecalciferol (2200, 9700, 17200, 24700, and 102200 IU/kg feed). The basal diet(control) contained 2200 IU/kg feed.bDried distillers’ grains with solubles.cThis week is hen’s age, not treatment week.

the maximal concentration of supplemental vitamin D in layinghen diets that would not cause negative hen health effects and that400000 IU/kg feed (the next level after 40000 IU/kg evaluated)resulted in hen renal tubular calcification.

The objectives of this study were to determine the dose-responserelationship between the laying hen dietary vitamin D3 level (5levels) and the resultant egg yolk vitamin D3 concentration over alonger laying cycle than what was done previously, and to evaluatethe egg yolk quality as affected by this fortification. Three dietswere designed with vitamin D3 below the recommended safe doseof 40000, and one diet below the toxic dose of 400000 IU/kg,but above the previously demonstrated safe dose. The effects ofsuch vitamin D3 fortification on hen performance including eggproduction, feed intake, and feed conversion, and on egg physicalqualities and bird physiological changes were examined as well andthe results will be presented in another paper.

Materials and Methods

Feeding experimentThe composition of the phase feeding basal diets is given in

Table 1. All diets were changed at the same time. Diets werebased on corn-soybean meal-dried distiller’s grains with solubles(DDGSs) and were formulated to meet or exceed the nutrientrequirements (National Research Council 1994). All diets werecreated from a common basal diet with the only difference beingthe amount of vitamin D3 supplemented. The cholecalciferol inthe 5 experimental diets were 2200 (control, or diet 1), 9700(control + 7500, or diet 2), 17200 (control + 15000, or diet3), 24700 (control + 22500, or diet 4), and 102200 (control +100000, or diet 5) IU/kg feed. Experimental diets were generatedevery 2 wk to minimize vitamin D3 degradation during storage.Feed consumption was monitored weekly. Diets were fed to layinghens (Hy-Line W-36, 19-wk-old) starting at the onset of theegg laying cycle and continued for the duration of the layingcycle. Each diet was fed to 3 consecutive pens of 3 hens perpen (9 birds as one experimental unit) with a total of 8 replicategroups. Therefore, 72 hens were used for each treatment. Henswere housed in 68 sq in per birdcage (with stocking density of438.7 cm2) and arranged in a random complete block design.

Hens were provided with 13 h of light initially and lighting hourswere increased 0.5 h per wk until 16 h of lighting was reached andmaintained for the duration of the experiment. House temperaturewas set to 75 ˚F and feed and water were provided ad libitum.

Eggs from each replicate group were collected daily and theyolk were pooled into 2-d periods during the first 2 wk, and thenwith 3 consecutive days pooled during the weeks of 3, 4, 6, 8, 12,16, 20, 24, 28, 32, 36, and 40. Because the cholecalciferol level inyolks from the control diet was expected to be unchanged duringthe entire feeding cycle, only samples from weeks 1, 2, 8, and 36were assayed to lower analytical cost.

Fresh eggs (about 8 eggs per treatment) were split to 4 pairs, andthe 2 egg yolks in each pair were pooled to result in 4 replicateyolk samples per treatment for various analyses. The fresh eggswere stored at 45 ◦F for no more than 3 d before the eggs werebroken. The yolk was manually separated from the white. For thedetermination of cholecalciferol concentration, the pooled eggyolk was homogenized using a spatula, vacuum packed, and thenstored under −26 ◦C until analysis. For other analyses includingtotal lipid, phospholipid, fatty acid composition, total unsaponifi-ables, yolk physical and functional properties, and sensory evalua-tion of the egg yolk, fresh eggs that were collected in one singleday between weeks 12 and 25 were used. The procedure to poolthe egg yolks to generate 4 replicates per treatment was the sameas described above.

Determination of cholecalciferol concentration in egg yolkThe cholecalciferol concentration in the egg yolk was deter-

mined by a commercial laboratory, Heartland Assay LLC (Ames,Iowa, U.S.A.) following a method of Phillips and others (2008).The assay procedures included saponification, solvent extraction,purification by a semipreparative normal-phase high-performanceliquid chromatography (HPLC), and quantification with a reverse-phase HPLC. Ergocalciferol (D2) was used as the internal standardfor quantification.

Calculation of cholecalciferol transfer efficiencyThe efficiency of vitamin D3 transfer in eggs as a function

of dietary intake is expressed as cholecalciferol transfer efficiency(Naber 1993; Walker and others 2012): transfer efficiency (%) =100 × [yolk cholecalciferol concentration (IU/g) × yolk mass(g) × egg production (% day−1 per hen)]/[feed cholecalciferolconcentration (IU/g) × feed intake (g/d per hen)]. The averagedaily feed intake of 100 g/d per hen (weeks 3 to 40), the averageegg mass of 53 g per egg (week 3 to 40), and the average eggyolk mass of 28% (or 15 g) of the egg (with shell) were used inthe calculation of transfer efficiency. The determination of theseproduction data is described in details in another report underpreparation. The transfer efficiency at week 3 and its average valueduring weeks 3 to 40 for each enrichment level were obtained.

Egg yolk lipid analysisThe crude lipids of egg yolk were extracted by using the mod-

ified Folch method (Christie and Han 2010). Briefly, 3 g of eggyolk was extracted twice with CHCl3-MeOH (2 : 1, v/v). Theliquid extract was washed with 0.74% KCl. The desolventizedegg yolk lipids were then dissolved in CHCl3 and stored at −26◦C in the dark until analysis. The phospholipid composition ofegg yolk lipids was determined by 31P NMR following a proce-dure described by Yao and Jung (2010). The yolk lipids (15 mg)were converted to fatty acid methyl esters (FAMEs) with 1 mL of

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1 M sodium methoxide in methanol. The resulting FAMEs wereextracted with hexane (3 mL) and washed with water (10 mL),and then 1 μL of FAMEs was injected to a GC (Hewlett-Packard5890 Series II, Pa., U.S.A.) equipped with a SPB-2330 fused silicacolumn (15 m × 0.25 mm × 0.20 μm, Supelco, Bellefonte, Pa.).The injector and flame ionization detector were set at 230 ◦C, andthe oven temperature was programmed from 100 to 220 ◦C at arate of 10 ◦C/min. The carrier gas flow rate was 3.4 mL/min, andthe split ratio was 50 : 1.

The unsaponifiable matters of egg yolk were determined byfollowing AOCS Official Method Ca 6b-53 using 5 g of yolksample per replicate.

Egg yolk viscosityThe dynamic viscosity of fresh egg yolks was measured with a

RS150 RheoStress rheometer (Haake, N.J., U.S.A.) coupled witha TC81 Peltier thermal controller. Careful separation of egg yolkfrom the white was done to not break the yolk membrane. Theyolk was rolled in a filter paper to remove any remaining albumen.Approximately 10 drops of well-mixed egg yolk were used foreach measurement. A parallel plate sensor with 33-mm diameterwas used. Samples were placed between the plate and attachment(PP35T) with a gap of 1.0 mm. The temperature was maintainedat 23 ◦C. At the selected shear rate of 60 s−1, apparent viscosityof the yolk was measured. Duplicate measurements were done foreach yolk sample.

Egg yolk emulsification propertiesThe methods of Wang and Wang (2009) were used. Egg yolk

(1.5 g) was suspended in 25 mL of DI water and soybean oil wasadded at a constant rate. The amount of oil added until phaseinversion was used to calculate the emulsification capacity (EC, goil/g yolk). Emulsion stability (ES) was measured and calculatedby dividing the nonseparated volume by total volume after 1 d ofstanding of the emulsion at 23 ◦C.

Sensory evaluationA triangle test (difference analysis) was performed to detect

possible difference in the overall sensory quality of hard-boiledfresh egg yolk from diet 1, 2, and 5 and store-bought (SB) eggs(Sparboe white eggs from conventional production) from a localgrocery store in Ames, IA, U.S.A. The eggs were put in boilingwater and boiled for 10 min, removed and naturally cooled atambient temperature for 1 h, and then stored in refrigerator (4 ˚C)for 1 d before the sensory test. Whole eggs were cut into halvesand the halves were served to the panelists at ambient temperature.Limited by the number of triangles that one taster can evaluate ata time, 4 sets of triangles were designed: SB against diet 1, diet 1against diet 2, diet 1 against diet 5, and diet 2 against diet 5. Wehypothesized that there would not be any detectable differenceamong all treatments, so we included the eggs with the lowest andhighest enrichment levels. Sixty-eight untrained tasters, including31 females, 33 males, and 4 unidentified, were recruited with anaverage age of 25. Each taster was served with 3 randomly chosentriangles at a time, and was asked to evaluate the yolk and identifyan odd sample out of the 3 in 1 triangle. The difference in eachpair of treatments was examined by comparing the actual numberof correct answers with the number necessary to establish 5%significance for the testing (Larmond 1977).

Statistical analysisThe effect of diet on the yolk lipids, phospholipids, fatty

acid composition, unsaponifiables, and yolk physical and func-tional properties were examined by GLM procedure using SAS(Version 9.1, SAS Institute Inc., Cary, N.C., U.S.A.) at a sig-nificance level of α = 0.05. The difference of the cholecalcif-erol concentrations in egg yolk of the 5 diets was examinedby GLM after the logarithm transformation. The relationshipof cholecalciferol content in egg yolk and D3 enrichment levelwas fitted in a generalized linear model with gamma distribu-tion and identity link, and the likelihood-ratio test for lack offit was conducted using R. The estimated deposition rate ofcholecalciferol in yolk in response to dietary level (within thefirst 3 wk) was obtained by fitting the following linear model:Yi j k = (β0,i + β1,i X j ) · I

(Xj ≤ 3

) + εi j k,where Yi j k denotes thekth replicate (k = 1, 2, . . . ni j ) of the measured cholecalciferolconcentration in yolk from the ith diet (i = 1, 2, . . . , 5) at thejth time point ( j = 1, 2, . . . , 15). I (. . .) is an indicator functionwhose value is 1 if the condition in the parenthesis was satis-fied and 0 otherwise. Measurement time points are denoted byXj ∈ {0.3, 0.6, 0.9, . . . , 36, 40}. εi j k , which models the varia-tions, is assumed to be independent and follow a location-scaledistribution with location and homogeneous scale equal to 0 andσ 2, respectively. The 95% percentile bootstrap intervals (Davisonand Hinkley 1997) for the estimated rates were computed by stan-dard parametric bootstrap method (10000 samplings were drawn).

Results and Discussion

Effect of the dietary cholecalciferol level on the transfer ofcholecalciferol to egg yolk

Cholecalciferol content of egg yolk from the control (diet 1)was 119 ± 31 IU/100 g egg yolk (or 17.7 IU per egg), which wasalmost unchanged during the entire experimental period. Chole-calciferol concentrations in the yolk from diet 2 to 5 increasedrapidly during the first 3 wk, and then remained relatively sta-ble for another 5 wk. After 8-wk feeding, a gradual reductionof cholecalciferol concentrations in the yolks from diet 2 to 5 oc-curred and then reached a steady state at weeks 16 to 25 (Figure 1).Toward the end of hen laying cycle, the cholecalciferol

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Figure 1–Effect of dietary cholecalciferol level on the cholecalciferol con-tent in egg yolk during a 40-wk feeding period (diet 1 (control), 2200IU cholecalciferol/kg of feed; diet 2, 9700 IU cholecalciferol/kg of feed;diet 3, 17200 IU cholecalciferol/kg of feed; diet 4, 24700 IU cholecalcif-erol/kg of feed; diet 5, 102200 IU cholecalciferol/kg of feed). The errorbars represent standard deviations (n = 4).

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concentrations in the yolk from diet 2, 3, and 5 slightly increased tothe levels equivalent to those in week 4. The peak values of chole-calciferol concentration in egg yolk from diet 2 to 5 occurred atweek 3 and were 865 ± 90, 1641 ± 91, 2411 ± 291, and 34815 ±3560 IU/100 g egg yolk, respectively. These concentrations of the5 diets at week 3 were significantly different with P < 0.0001.

The study of Mattila and others (2003) with much shorter feed-ing duration compared to ours showed that after 8 to 13 d feeding,cholecalciferol concentrations in the yolk reached a peak value ofabout 1216 IU/100 g egg yolk, based on a diet with 11200 to12000 IU vitamin D3/kg feed. Even though the feed concentra-tions used in our study were different from this earlier study, ourdata indicate the similar yolk cholecalciferol concentration at theequivalent feeding level. The high-cholecalciferol diets evaluatedin the present study demonstrate that up to 160 time, higher chole-calciferol concentration than that of typical value of 218 IU/100 gyolk (USDA survey) can be obtained through feeding the vitaminD3-enriched diets, although such a high concentration may notbe needed.

The decrease of cholecalciferol concentrations, which occurredduring weeks 8 to 16 in this study, was also reported by Mattila andothers (2003). The reason for the decreasing trend was suggestedto be caused by the instability of vitamin D in the feed or theheterogeneity of the feed (Mattila and others 2003). Since the feedswere mixed every 2 wk in our study, the reduction or slight increaselater on of cholecalciferol in yolk could not be fully explained byits degradation in feed. Further study is needed to understand themechanism causing such reduction.

The transfer of cholecalciferol from the feed into egg yolk wasvery efficient and responsive to the enrichment level in the feed.Figure 2 shows that cholecalciferol content in yolk over weeks 3to 40 feeding period increased with the D3 level in the feed, inagreement with other reports (Kawazoe and others 1996, 1997;Mattila and others 1999). A generalized linear model was fitted tothe data of diet 1 to 4 under the assumption of independence of themeasurement of cholecalciferol content from different samplingtime and diet. The linear relationship between cholecalciferol inyolk and diet was confirmed with a likelihood-ratio test, which

Figure 2–The relationship of cholecalciferol content in yolk and dietarycholecalciferol level in the feed of diet 1 to 5. The data of week 3 to 40from diet 1 to 4 were fitted in a generalized linear model with gammadistribution and identity link. The likelihood-ratio test for the lack of fitgave P = 0.985 (n = 4). The presented linear equation applies to diet 1 to4, not to diet 5.

gave a P value of 0.958. The highest D3 level (diet 5) was notin the linear relationship of diet 1 to 4. This observation is, ingeneral, consistent with cholecalciferol transfer efficiency of the 5diets, as shown in Figure 3. Cholecalciferol transfer efficiency ofdiet 5 was about 41% to 47%, more than 3-fold of those of diet 2to 4, and 5-fold of the control.

It is known that plasma cholecalciferol is deposited into yolk byforming a complex with a vitamin D binding protein (DBP) andthe deposition of vitamin D in egg yolk is believed to meet theneeds of development of chick embryo, such as in regulating yolkcalcium mobilization (Fraser and Emtage 1976; Tuan and Suyama1996). Fraser and Emtage (1976) reported that the binding capac-ity of DBP was about 1.9 μg/15 mL yolk (or 507 IU/100 mLyolk), similar to the cholecalciferol concentration in the yolk ofdiet 2 in the present study. White and Whitehead (1987) studiedthe deposition of biotin in egg yolk and found that biotin transferefficiency from plasma to yolk was lower at low biotin plasmaconcentration than it was at higher concentrations. Its depositionin yolk was dependent on the concentration of biotin bindingprotein (BBP), and the production of BBP was regulated by sexhormones and biotin availability, meaning that higher dietary levelwould lead to higher production of BBP. However, at very highdietary level when exceeding the binding capacity of BBP, therewas an influx of biotin deposition in yolk. The extra biotin wasthought to be bound to a second site on BBP or associated withlipids. The example of biotin deposition in yolk gave insight intothe possible reason of why very high dietary cholecalciferol levelled to a nonlinear increase of cholecalciferol concentration in yolk.However, the deposition of riboflavin in yolk was limited by theamount of riboflavin-binding protein that was independent of di-etary riboflavin (White and others 1986). Vitamin deposition inegg yolk in response to dietary vitamin level was a function of indi-vidual vitamin as suggested by Naber (1993). Further investigationis needed to understand the specific regulation of cholecalciferoldeposition in egg yolk.

Diet 2 to 4 had similar transfer efficiency of about 11% to 14%,significantly higher than the control diet (7% to 8%) (Figure 3).Naber (1993) compared transfer efficiency of vitamin D with otherfat or water soluble vitamins and categorized vitamin D as havingmedian-level transfer efficiency, 15% to 25%, which was at thesame level as vitamin E; carotenoids, biotin, and riboflavin arethose with high transfer efficiency, more than 40%. To compare

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Figure 3–Effect of dietary cholecalciferol level on egg cholecalciferol trans-fer efficiency. The error bars are standard deviations (n = 4). The differentletters denote significant difference at α = 0.05. The statistical differencesamong treatments for “Average of week 3 to 40” and “week 3” are thesame.

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Table 2–The estimated deposition rates of cholecalciferol in eggyolk during week 0 to 3 and their confidence intervals.

Estimated 95% BootstrapDiet rate percentile intervals

1 − 2.31 − 15.87 11.212 108.94 78.75 139.273 177.22 113.73 239.984 309.03 203.18 411.525 5344.92 3925.40 6763.15

Table 3–Phospholipid composition (weight%, relative) of eggyolk lipids as determined by 31P NMR.a,b

Diet

Phospholipidc 1 2 3 4 5

LPE 1.3 ± 0.1 1.2 ± 0.3 1.3 ± 0.3 1.4 ± 0.6 1.1 ± 0.2PE 18.9 ± 1.5 18.8 ± 1.0 18.7 ± 0.7 18.9 ± 0.7 18.3 ± 1.1SPH 1.9 ± 0.5 2.2 ± 0.5 2.1 ± 0.4 2.4 ± 0.7 2.2 ± 0.7PC 78.0 ± 1.9 77.8 ± 1.2 77.9 ± 0.6 77.3 ± 1.0 78.4 ± 0.6

aValues are means ± standard deviations from 4 samples.bThe means of each phospholipid had no significant difference among the diets at 0.05significance level (n = 4).cLPE, lysophosphatidylethanolamine; PE, phosphatidylethanolamine; SPH,sphingomyelin; PC, phosphatidylcholine.

with other recent vitamin D feeding studies, we estimated chole-calciferol transfer efficiency using the data reported by others. The3 feeding studies conducted by Mattila and others (1999, 2003,2004) had 8% and 15% transfer efficiency from diets with 2496and 8640 IU/kg cholecalciferol, 14% transfer efficiency from adiet containing 12000 IU/kg, and 11% to 12% transfer efficiencyfrom diets with 2500, 6000, and 15000 IU/kg. Park and others(2005) had 1% to 7% transfer efficiency from diets with 2000 to20000 IU /kg of vitamin D3 but their results might be compli-cated by the co-fortification of vitamin K and iron. The transferefficiency of our diet 1 to 4 was within the “typical range” of thereported vitamin D feeding studies.

The estimated deposition rates of cholecalciferol in yolk duringweek 0 to 3 are presented in Table 2. A reasonable linear rela-tionship was found between the deposition rates of diet 1 to 4and dietary cholecalciferol levels. However, the rates of diet 1 to 4were not significantly different based on the confidence intervalsobtained from bootstrap method, as shown in Table 2. Addition-ally, there was a striking difference in the rate of diet 5 comparedwith those of diet 1 to 4. Diet 5 promoted much higher rate ofcholecalciferol deposition in the first 3 wk, which was more than18 times of other diets.

Yolk lipid composition as affected by vitamin D3enrichment

We hypothesized that vitamin D enrichment would not changelipid class and fatty acid compositions so that this aspect of eggquality would not be altered by this feeding practice. No sig-nificant difference in phospholipid composition was found amongthe 5 treatments with P > 0.05 (Table 3). Phosphatidylcholine wasthe major phospholipid and it composed of 78% of total phospho-lipids, followed by phosphatidylethanolamine (18% to 19%). Thetwo minor phospholipids were lysophosphatidylethanolamine andsphingomyelin, representing about 3% of total phospholipids. Theresult of phospholipid composition agreed with the literature val-ues (Sotirhos and others 1986).

The lipid fatty acid composition of yolk from vitamin D-enriched diets was the same as that of the control (Table 4) and

not different from literature values (Walker and others 2012). Theyolk lipid content calculated from the GC-FAME analysis wasabout 77% of the total lipids for the 5 diets. The presence of highpercentage of phospholipids in yolk lipids explains the differencefrom the 100% value because the polar functional groups on phos-pholipids were not converted to methyl esters (Walker and others2012).

Unsaponifiable matters of egg yolk lipids of the 5 diet treatmentswere about the same, 1.1% of dry yolk lipids on average (Table 5).Unsaponifiable matters include carotenoids, cholesterol, vitaminD, and other sterols, of which cholesterol is the major component.Elevated cholecalciferol content in egg yolk did not significantlyincrease the concentration of unsaponifables in the egg yolk.

Yolk physical, functional, and sensory properties asaffected vitamin D3 enrichment

Any change in viscosity of egg yolk would influence the physicaland functional properties of the food products that contain eggyolk as an ingredient (Severa and others 2010). Egg yolk is a non-Newtonian fluid in that its viscosity is dependent on shear rate.We first examined the yolk apparent viscosity as a function of shearrate as previously described (Buxmann and others 2010; Walkerand others 2012) and found that there was no difference in theflow index and consistency index among the 5 diets. Then, theapparent viscosity of the yolk at a selected shear rate of 60 s−1

was measured and is presented in Table 5. The apparent viscosityof the yolks from the 5 diets was not significantly different withP > 0.05. Thus, the rheological properties of the yolk were notaffected by the vitamin D3 enrichment in the diet.

The emulsification properties of the yolk were also measuredbecause of the slight difference observed by Walker and others(2012), studying egg enrichment with tocopherols and carotenoidsby feeding. For vitamin D enrichment, no significant differences inemulsification capacity and emulsification stability were observedamong all treatments, with the mean values of 59.5 g oil/g yolkand 49.2%, respectively. These values are similar to those reportedearlier (Walker and others 2012).

A descriptive sensory evaluation was conducted using an un-trained panel and it showed no difference in the appearance, tex-ture, and flavor profile of hard-boiled egg yolks from the 5 diettreatments (data not shown). The triangle difference test was de-signed to detect any overall sensory difference in the egg yolk witha large number of untrained tasters. The results of the triangle testconfirmed that the overall yolk sensory properties from controldiet was not different from SB, the lowest (diet 2) and highest(diet 5) levels of vitamin D-enriched diets were not different fromthe control diet and not different from each other at 5% level ofsignificance.

Mattila and others (2003) also reported that supplementationof cholecalciferol in the diet of laying hens did not affect thephysical, functional, and sensory properties of egg yolk. Similarly,carotenoids- and tocopherols-enriched diet had no significant ef-fects on physical and functional properties of egg yolk (Walker andothers 2012).

Hen performanceHen performance data and other physiological examination re-

sults are presented in another report (manuscript in preparation). Ingeneral, hen performance, including egg production, feed intake,feed conversion, and egg physical qualities were not significantlyaffected by the 5 dietary treatments.

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Table 4–Fatty acid composition (weight%, relative) of egg yolk lipids.a,b

Diet

Fatty acid 1 2 3 4 5

Myristic acid 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0Palmitic acid 27.7 ± 0.4 27.4 ± 0.4 27.5 ± 0.9 27.7 ± 0.5 27.4 ± 0.4Palmitoleic acid 2.5 ± 0.2 2.3 ± 0.4 2.2 ± 0.3 2.5 ± 0.1 2.4 ± 0.2Stearic acid 10.2 ± 0.4 10.3 ± 0.5 10.4 ± 0.2 10.3 ± 0.3 10.2 ± 0.1Oleic acid 39.0 ± 0.8 39.3 ± 0.5 39.1 ± 0.5 39.4 ± 0.6 38.6 ± 0.4Linoleic acid 19.4 ± 1.3 19.6 ± 0.9 19.6 ± 0.9 19.0 ± 1.2 20.2 ± 0.3Linolenic acid 0.6 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 0.6 ± 0.0Arachidic acid 0.3 ± 0.1 0.3 ± 0.0 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1Total in yolk lipids 78.09 ± 3.49 74.85 ± 4.32 75.01 ± 2.33 78.73 ± 8.68 76.47 ± 2.74

aValues are means ± standard deviations.bThe means of each phospholipid had no significant difference among the diets at 0.05 significance level (n = 4).

Table 5–Moisture content, unsaponifiable matters, and viscosityof egg yolksa,b

Diet

1 2 3 4 5

Moisture (%) 50.0 ± 0.1 49.9 ± 0.4 50.1 ± 0.3 50.3 ± 0.5 49.8 ± 0.3Unsaponifiable

matters (% ofyolk lipid)

1.1 ± 0.1 1.2 ± 0.3 1.0 ± 0.1 1.1 ± 0.1 1.1 ± 0.1

Viscosity (Pa s) 1.3 ± 0.1 1.3 ± 0.2 1.3 ± 0.2 1.2 ± 0.2 1.3 ± 0.2

aValues are means ± standard deviations.bThe means of each parameter had no significant difference among the diets at 0.05significance level (n = 4).

ConclusionThe deposition of vitamin D3 in eggs increased rapidly in the

first 3 wk, and then the level was relatively stable over a period of40 wk. Egg yolk vitamin D3 content increased with diet enrich-ment level. The results suggest that increasing the vitamin D3 inhen diets does not adversely affect egg quality and by such a di-etary fortification, egg vitamin D content can be readily enriched,resulting in a value-added and nutritious egg.

AcknowledgmentFinancial support for this study was provided by American Egg

Board. We thank Wen Zhou for statistical analysis and ChristineFedler for providing assistance on sensory tests. We also appreciateProfessors Hongwei Xin, Donald C. Beitz, and Susan J. Lamontfor their contribution to the experiment planning.

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