British Poultry Science

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Effects of dietary fat source on lutein, zeaxanthin

and total carotenoids content of the egg yolk in
laying hens during the early laying period

G. A. Papadopoulos, S. Chalvatzi, J. Kopecký, G. Arsenos & P. D. Fortomaris

To cite this article: G. A. Papadopoulos, S. Chalvatzi, J. Kopecký, G. Arsenos & P. D. Fortomaris

(2019) Effects of dietary fat source on lutein, zeaxanthin and total carotenoids content of the egg
yolk in laying hens during the early laying period, British Poultry Science, 60:4, 431-438, DOI:
10.1080/00071668.2019.1614526

To link to this article: https://doi.org/10.1080/00071668.2019.1614526

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May 2019.
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BRITISH POULTRY SCIENCE
2019, VOL. 60, NO. 4, 431–438
https://doi.org/10.1080/00071668.2019.1614526

Effects of dietary fat source on lutein, zeaxanthin and total carotenoids content of
the egg yolk in laying hens during the early laying period
G. A. Papadopoulosa, S. Chalvatzia, J. Kopeckýb, G. Arsenosa and P. D. Fortomarisa
a
Laboratory of Animal Husbandry, Faculty of Veterinary Medicine, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki,
Greece; bInstitute of Microbiology, Academy of Sciences of the Czech Republic, Třeboň, Czech Republic

ABSTRACT ARTICLE HISTORY

1. The study was conducted to investigate the effects of different dietary levels of an unsaturated or Received 4 August 2018
saturated fat source and the effects of different dietary energy levels in laying hen diets on the Accepted 21 February 2019
carotenoid content of the egg yolk. KEYWORDS
2. Seventy-two ISA Brown laying hens aged 22 weeks old were allocated, for a 10 week period, to Carotenoids; laying hens;
three treatments: control diet (HE group) with a 3.4% supplemented fat containing 2.1% soybean lutein; saturated fat;
and 1.3% palm oil as fat sources and a ratio of unsaturated to saturated fatty acid (u/s) of 3.33; unsaturated fat
a lowered energy diet with 2.4% supplemented fat containing 1.4% soybean and 1.0% palm oil and
u/s of 3.41 (LE+high u/s); a lowered energy diet with 2.4% supplemented fat containing 0.5%
soybean and 1.9% palm oil and a u/s of 2.39 (LE+low u/s). A marigold plant extract supplement was
used as a source of lutein.
3. Performance parameters (feed consumption, feed conversion, body weight), egg production rate
and egg quality parameters were similar between treatments. Dietary fat digestibility at the middle
of the study period was not different. Egg yolk total fat content was similar in all treatments at the
middle and at the end of the study period.
4. Lutein, zeaxanthin, cis-lutein and total carotenoids content were significantly lower in eggs
produced from the LE+low u/s group compared to those from the HE and the LE+high u/s groups
(P < 0.01 for all parameters).
5. It was concluded that feeding laying hens with a diet containing 1% less supplemented fat and
a lower u/s ratio compared to a control diet and to a diet with 1% less supplemented fat with
a higher u/s ratio resulted in a significant reduction of carotenoid expression in the egg yolk at the
end of experimental period. Performance and egg quality parameters were not affected by
treatments.

Introduction Summers 2005). Therefore, the balance of saturated to

unsaturated fatty acids can be a criterion in designing fat
Lutein and zeaxanthin are xanthophyll carotenoids that are
blends for poultry diets (Leeson and Summers 2005). It is
related to specific human health benefits, including prevent-
known that the fatty acid profile of the egg yolk depends on
ing macular degeneration (Leeson and Caston 2004; Zaheer
the type of dietary fat (Lemahieu et al. 2015). Hence, sup-
2017), protecting against cardiovascular diseases (Andersen
plemented fat may influence the bioavailability of carote-
2015; Gammone et al. 2015), oxidative stress (Fiedor and
noids (Failla et al. 2014). It has been shown that adding
Burda 2014), neurodegenerative disorders (Calabrese et al.
flaxseed to the diets of laying hens decreased yolk lutein
2010; Nolan et al. 2015) and cancer (Mares-Perlman et al.
content (Leeson and Caston 2004). Elsewhere, micellarisa-
2002). Lutein and zeaxanthin constitute the main carote-
tion and cellular uptake of β-carotene was increased in vivo
noids in egg yolks (Widomska and Subczynski 2014).
by lipids rich in unsaturated fatty acids (Failla et al. 2014).
Specific feed additives have been previously used to modify
Pérez-Bonilla et al. (2011) reported that yolk colour was
egg yolk lutein and carotenoids content (Steinberg et al.
higher for hens fed with lard rather than those fed soy oil or
2000; Sirri et al. 2007; Leeson et al. 2007; Kotrbaček et al.,
acidulated vegetable soap stocks. Hence, it could be
2013; Skřivan et al. 2015).
hypothesised that carotenoid composition of egg yolks
Carotenoids are fat-soluble compounds. Their absorp-
may be affected by feeding diets differing in fat saturation.
tion involves solubilisation in bile salts and incorporation
Combining a more saturated profile fat source as blend with
into micelles in the small intestine (Roodenburg et al. 2000;
a more unsaturated oil could be beneficial, due to potential
Failla et al. 2014). Because dietary fats are essential for
fatty acid synergism (Leeson and Summers 2005).
micelle formation, they are associated with carotenoid
Competition with the human food industry usually makes
absorption (Roodenburg et al. 2000; Failla et al. 2014). Fat
certain fats and oils uneconomic for use in animal feeds,
absorption depends on bile salt supply and on the balance
and as a result poultry diets are formulated to a least cost
of unsaturated to saturated fatty acids (Leeson and

CONTACT G. A. Papadopoulos geopaps@vet.auth.gr Laboratory of Animal Husbandry, Faculty of Veterinary Medicine, School of Health Sciences,
Aristotle University of Thessaloniki, PO BOX 393, 54124, Greece
Affiliation where the research was conducted: Research and analysis of samples was conducted within the experimental facility of the Laboratory of Animal
Husbandry of the Faculty of Veterinary Medicine, School of Health Sciences, Aristotle University of Thessaloniki, Greece and the Institute of Microbiology,
Academy of Sciences of the Czech Republic, Třeboň, Czech Republic
© 2019 British Poultry Science Ltd
432 G. A. PAPADOPOULOS ET AL.
output (Leeson and Summers 2005). Thus, combining dif-
ferent levels of fat sources can have a practical relevance.
The objective of the present study was to test whether
laying hen diets, differing in the inclusion level and type of
supplemented fat, could have an impact on the concentra-
tion of lutein, zeaxanthin and total carotenoids in the egg
yolk. To investigate this, blends of an unsaturated fat source
(soybean oil) and of a saturated fat source (palm oil) were
used to produce diets with a higher or lower unsaturated to
saturated fatty acid ratio. To further evaluate whether the
quantity of supplemented fat may impact egg yolk para-
meters, two low-energy diets were formulated containing
1% less supplemented fat than a standard diet.
Materials and methods
Ethical statement
Experimental procedures were approved by the Ethical
Committee branch of the Research Committee of Aristotle
University of Thessaloniki, Greece (approval number 70
770/2015). The animal phase of the experiment was
designed considering all welfare requirements described by
Good Farming Practice Guidelines (Directive 2010/63/EC;
Commission recommendation 2007/526/EC).
Birds, housing and diets
A total of 72 ISA Brown laying pullets at the age of
16 weeks, obtained from B.A.K Farm (Registration number:
1EL 54 081, 57 200 Assiros, Thessaloniki, Greece), were
randomly allocated to the three experimental diet groups.
Each group consisted of 24 birds and was subdivided into
12 replicates each containing two hens. All birds were kept
into three-tier battery cages (41 × 41 × 50 cm). Cages were
equipped with trough-type feeders and nipple drinkers,
with ad-libitum provision of feed and water. During the
first 6 weeks all hens were offered the same diet formulated
according to birds’ age nutrient requirements (Isa Brown
Nutrition Management Guide, Version L7121-2, 2018).
After this period of acclimatisation, the hens then received
the experimental diets for a 10-week period from the age of
22 weeks. Specifically, the control group (HE group) was fed
a high energy diet while the two other groups (LE+ high u/s
and LE+low u/s groups) were fed a low energy diet with
high or low unsaturated/saturated ratio, respectively.
A combination of soybean and palm oil in varying levels
were used to formulate the diets. All diets were supplemen-
ted at a ratio of 0.3% with a marigold extract supplement
(Oro Glo Layer DryTM; declared composition: 1.56% total
xanthophylls of which 88% was lutein; Kemin Europa NV,
Herentals, Belgium). The main ingredients and the nutrient
composition of the experimental diets are shown in Table 1.
Performance of laying hens and egg quality parameters
Throughout the 10-week experimental period, egg produc-
tion and feed intake was recorded on a daily basis. Feed
refusals and egg weight were recorded on a weekly basis, in
order to calculate daily feed intake and egg mass produc-
tion. Feed conversion ratio was expressed as kg of feed
per kg of eggs produced. The body weights of hens were
measured at the start and the end of the experimental
period. At the age of 22, 25, 28 and 31 weeks of age, 12
eggs per treatment (one egg from each cage-replicate) were
randomly collected for measurement of quality parameters.
At the age of 26 and 31 weeks, 24 eggs per treatment (two
eggs from each cage-replicate) were collected, which were
subsequently broken, and yolks were separated from albu-
men and the shell for analysis. The egg yolk samples were
subsequently frozen in at −80°C. Half of the egg yolk sam-
ples were analysed for carotenoid (12 per treatment and per
time point) and fatty acid composition (12 per treatment
and per time point). The weight of whole egg, shell, yolk
and albumen were measured with a precision electronic
scale (Navigator TM, N2B110, OHAUS Corporation). Yolk
colour was measured using on Roche Colour Yolk Fan scale
while Haugh units were measured using designated equip-
ment by the EQM York Electronics Center (Egg Quality
Microprocessor, Technical Services & Supplies Ltd.,
Dunnington, York, UK).
Fat digestibility and chemical analysis of feed
A titanium dioxide tracer (TiO2; E171 titanium dioxide,
IMCD Benelux N.V., Mechelen, Belgium) was added as
digestibility marker in the experimental diets. The level of
supplementation was 0.3% and diets were offered to hens
starting from 26 weeks of age. Plastic trays were placed
under each cage at the fifth day of initiation of the titanium
supplemented feed for excreta collection, which lasted until
the end of that week. Excreta samples, free of feed and
feathers, were placed in plastic bags and stored in
a freezer at −20°C until further analysis. Feed samples
from each pen were collected. Feed and excreta moisture,
nitrogen and fat content were determined according to the
methods of the Association of Official Analytical Chemists
(AOAC, 2003). Gross energy of feed was calculated using an
adiabatic bomb calorimeter (IKA® Calorimeter System
C 5000 Control, IKA-Werke GmbH & Co. KG, Staufen,
Germany). Determination of titanium dioxide was per-
formed by inductively coupled plasma optical emission
spectrometry following the procedure of van Bussel et al.
(2010). Digestibility of fat was calculated according to
equation:
% Digestibility ¼ ð1  ð%Þ
 fat in excretað%Þ markerexcreta ð%Þ
 fat inð%ÞÞ  100
Fatty acid composition analysis of eggs and fat sources
Fat determination was done according to AOAC (1995). For
fat extraction 5 g of yolk from 5 or 6 eggs was homogenised,
dried at 105°C and extracted at room temperature (25°C)
with diethylether. The solvent was removed in a rotary
evaporator (40–50°C). A quantity of 0.1 g extracted fat
was trans-esterified with methanolic solution of potassium
hydroxide at room temperature as described in regulation
EEC 2568/91 Annex X § 3. Gas chromatography of fatty
acids methyl esters was conducted as described by
Sinanoglou et al. (2011) with an internal standard of lauric
acid methyl ester (Aldrich 234 591) and a GC Agilent
(7890A). Both quantitative and qualitative analysis were
performed on an Agilent 7890 A gas chromatograph
 BRITISH POULTRY SCIENCE 433

Table 1. Main ingredients and nutrient composition of the experimental diets and retention times. Reference material used for identifica-
fed to laying hens from 22 to 31 weeks of age.
tion of peaks was Sigma-Aldrich Fame mix C4-C24 18 919-
Treatmentsa
1AMP. The fatty acid composition of the fat sources used in
LE+high LE+low the present study was determined using the same method as
Item HE u/s u/s
for the egg yolk lipids (Table 2).
Ingredients (g/kg)
Corn 406 406 406
Soybean meal 185 185 185
Wheat 150 150 150 Egg yolk carotenoid composition analysis
Limestone 89 89 89
Sunflower meal 85 85 85 All chemicals used were of analytical grade and were purchased
Wheat bran 27 37 37 from Sigma-Aldrich. Organic solvents for pigment extraction
Soybean oil 21 14 5
Palm oil 13 10 19 and HPLC analyses were obtained from Analytika (Czech
Monocalcium phosphate 11 11 11 Republic). All solutions were prepared using reverse-osmosis
Vitamin and mineral premixb 5 5 5 deionised water (Ultrapur, Watrex, Prague, Czech Republic).
Sodium chloride 2 2 2
Sodium bicarbonate 2 2 2 Nitrogen and helium (99.999% for both) were obtained from
DL Methionine 1 1 1 Linde Gas (Czech Republic). All samples were filtered through
Lutein supplement 3 3 3 0.45 μm PTFE membrane discs (Sartorius) prior to HPLC
Nutrient Compositionc
Crude Protein (g/kg) 169.5 171.0 171.0 analysis.
Crude Fat (g/kg) 55.0 45.0 45.0 The frozen egg yolks were lyophilised and subsequently
Crude Fibre (g/kg) 40.0 41.0 41.0 homogenised in a mortar. Afterwards, 500 mg of each dry egg
ME (MJ/kg) 11.41 11.05 11.05
Ratio Unsaturated to Saturated fatty acids 3.33 3.41 2.39 yolk sample were weighed and extracted three times with cold
Calcium (g/kg) 37.0 38.0 38.0 100% acetone. All three extracts were centrifuged, combined,
Total phosphorus (g/kg) 7.0 7.0 7.0 clarified using 0.45 μm PTFE filters and filled up to a total
Lysine (g/kg) 8.0 8.0 8.0
Methionine (g/kg) 4.0 4.0 4.0 volume of 15 ml before pigment analysis. All work was per-
Methionine + cysteine (g/kg) 7.0 8.0 8.0 formed under dim light to prevent degradation. The total car-
Threonine (g/kg) 6.0 6.0 6.0 otenoid content was determined spectrophotometrically using
Analysed Compositiond
Dry matter (g/kg) 913.1 914.4 918.3 the equation according to Lichtenthaler and Wellburn (1983).
Crude Protein (g/kg) 158.2 150.1 157.7 The extracts were further analysed by the high-
Crude Fat (g/kg) 36.5 33.3 33.0 performance liquid chromatography (HPLC) using the
Gross Energy (MJ/kg) 14.62 14.20 14.21
Analysed Carotenoid Composition Agilent 1100 Series system (Agilent Technologies Inc.,
Lutein (μg/g) 81.1 91.4 81.0 Palo Alto, CA, USA) equipped with a UV-VIS diode-array
cis-Lutein (μg/g) 6.8 8.0 5.7 detector (Agilent DAD 61315B) and an in-line Agilent 1100
Total carotenoids (μg/g) 87.9 99.4 86.7
a Series Ion Trap SL mass spectrometer with electrospray
Laying hens were fed a high energy diet (HE) or a low energy diet (LE)
differing in the unsaturated to saturated fatty acids ratio (LE+high u/s: high ionisation source (ESI). The operating parameters for the
unsaturated to saturated fatty acids ratio; LE+low u/s: low unsaturated to mass spectrometer were as follows: the spray needle voltage
saturated fatty acids ratio). was set at 4.5 kV, nebulising gas nitrogen (50 psi), drying
b
Provided per kg diet: Retinyl acetate: 4.2 mg; Cholecalciferol: 0.1 mg; α-
tocopherol acetate: 31.25 mg; Menadione: 5.0 mg; Cyanocobalamin: gas nitrogen (10 l/min) and. drying temperature 325°C. ESI
0.025 mg; folic acid: 1.0 mg; Choline chloride:: 450 mg; Pantothenic acid: capillary voltage was 230 V and helium was used as aux-
12.5 mg; Riboflavin, 6.25 mg; Nicotinic acid: 43.75 mg; Thiamin: 3.0 mg; iliary gas (15 ml/min). Collision energy 50% of 5 V for MS/
D-biotin: 0.1 mg; Pyridoxine: 5.0 mg; Manganese: 125 mg; Zinc: 112 mg;
Iron: 62 mg; Copper: 10 mg; Iodine: 1.0 mg; Selenium: 0.15 mg. MS experiments was used. The scan range at full scan mode
c
Calculated value was 50–2200 m/z and scan speed 13,000 m/z per sec. MS2
d
expressed on dry matter basis was performed on the selected precursor ion (isolation
width 2.0 m/z units) by using the helium present in the
trap as the collision gas. The amplitude of the supplemen-
tary radio-frequency (RF) applied across the end caps was
(Agilent Technologies, Palo Alto, CA) equipped with set during a HPLC run to a fixed value suitable for the
a flame ionisation detector. DB-23 capillary column
(60 m × 0.25 mm i.d.0.15 pm film) (50%-(cyanopropyl)-
methylpolysiloxane; Agilent Technologies. Catalogue No.: Table 2. Analysed fatty acid composition of soybean and palm oil used in the
experimental diets.
122–2361) was used. The analysis was split injection, with
Item (in % of total fatty acids) Soybean oil Palm oil
a split ratio was 1:2. Helium was used as the carrier gas. The
Lauric acid (C12:0) 0.00 0.57
injector and detector temperature were 250°C and 260°C, Myristic acid (C14:0) 0.21 1.30
respectively. The temperature was programmed at 100°C for Palmitic acid (C16:0) 11.45 43.19
0 min, raised from 100 to 150°C by a rate of 10°Cmin and Palmitoleic acid (C16:1) 0.22 0.27
Stearic acid (C18:0) 4.33 4.06
held constant at 150°C for 0 min. Then it was raised from Oleic acid (C18:1) 23.83 37.82
150 to 195°C at a rate of 2°C min−1, and was held constant Linoleic acid (C18:2) 49.06 10.28
at 195°C for 5 min. It was then raised from 195 to 210°C at Linolenic acid (C18:3) 5.06 0.48
Arachidatic acid (C20:0) 0.70 0.49
a rate of 1°C min−1, and was held constant at 210°C for Eicosanoic acid (C20:1) 1.13 0.48
0 min; and was finally raised from 210 to 240°C at a rate of Behanatic acid (C22:0) 0.79 0.22
10°C min−1 and was held constant at 240°C for 5 min. The Lignoceratic acid (C24:0) 0.34 0.00
Nervonatic acid (C24:1) 1.38 0.00
duration of the analysis was 55.5 min (Loukas et al. 2010). Total Saturated 17.82 49.40
The injection volume was 1.0 pi. The Hewlett-Packard Total Unsaturated 82.18 50.60
Chem Station Software was used to calculate peak areas Unsaturated: Saturated 4.61 1.02
434 G. A. PAPADOPOULOS ET AL.

fragmentation (ranging from 0.8 to 1.4 V). All full scan and
CID ion mass spectra represent averages of 7–10 scans.
Pigments were separated using a modified method of van
Heukelem and Thomas (2001) on the Phenomenex Luna 3μ C8
100Å column with binary solvent system (0 min 100% A, 20 min
100% B, 25 min 100% B, 27 min 100% A, 30 min 100% A; A: 70%
methanol + 28 mM ammonium acetate, B: methanol). Injection
volume was 20 μl. The peak assignment was based on compar-
ison of spectral characteristic with the known retention beha-
viour of carotenoids in a reverse phase system and confirmed by
characteristic mass spectral ions.
The analysis of feed for carotenoid levels was done by
a chemical procedure similar to the analysis of egg yolks.
Briefly, sample weight of 1 g, was homogenised in a mortar
and extracted three times with cold 100% acetone. All three
extracts (sampled from the three experimental diets) were cen-
trifuged, combined, clarified using 0.45 µm PTFE filters and
filled up to the total volume of 30 ml before pigment analysis
(spectrophotometrically and HPLC).
Statistical analysis
Data was analysed using the Statistical Package for the Social
Sciences (SPSS) software (SPSS 22.0 Version, Chicago, IL, USA).
Statistical significance was considered at P < 0.05. Results are
presented as mean ± standard deviation (SD). The zootechnical
performance of laying hens and egg quality parameters data was
analysed by one-way ANOVA of General Linear Model proce-
dure of SPSS, and post-hoc comparisons between treatments
were made by Tukey’s test. Statistical evaluation of fatty acid
and carotenoids composition of egg yolks was evaluated by
GLM repeated measures analysis. Sampling period (designated
period 1 and 2) was the within subject factor and treatment (HE,
LE+ high u/s and LE+low u/s) was the ‘between-subject’ factor.
Differences were considered significant at P < 0.05 and signifi-
cant differences between means were tested using the
Bonferroni’s test.
Results
Performance of laying hens and egg quality parameters
The mean values of laying hens’ performance and egg
quality parameters are shown in Table 3. Dietary treatments
had no effect on all the tested parameters.
Fat digestibility
Digestibility of dietary fat was estimated for all treatments
during the 26th week of age of layers. The digestibility of fat
in the HE, LE+ high u/s and LE+low u/s groups was 87.7%
(±7.49), 85.8% (±7.12) and 87.4% (±5.22), respectively.
Differences between treatments were not significant
(P = 0.791).
Fatty acid composition of eggs
Fatty acid and total fat content of egg yolks obtained from
laying hens at the 26th and 31st week of age (Period 1 and
Period 2, respectively) are shown in Table 4.
Results were presented as a treatment effect including
both periods, and as a period-time effect including all treat-
ments. Egg yolk total fat content was not different between
treatments at any stage of the experiment. Egg total fats
were higher in Period 1 compared to Period 2 (time effect;
P = 0.032). Oleic and total monounsaturated fatty acids
were lower in the LE+high u/s group compared to the HE
and LE+low u/s in Period 1, Period 2 and in both Periods
(P = 0.008, P = 0.038, P < 0.001, respectively, for oleic;
P = 0.002, P = 0.012, P < 0.001, respectively, for monoun-
saturated). Oleic content was higher in eggs collected during
Period 2 than Period 1 (time effect; P = 0.011). Linoleic acid,
total polyunsaturated and n-6 fatty acids were higher com-
pared to the HE and LE+low u/s groups in Period 2
(P = 0.006, P = 0.005, P = 0.005, respectively) and for
both periods (treatment effect; P = 0.002, P = 0.002,
P = 0.002, respectively). The n-3 fatty acid content was
higher in the LE+high u/s and HE group compared to the
LE+low u/s group during Period 2 (P = 0.015) and for both
periods (treatment effect; P = 0.002). Moreover, n-3 fatty
acids were increased in Period 2 than Period 1 (time effect;
P < 0.001).
Egg yolk carotenoid composition
Egg yolk carotenoid content of eggs obtained from laying
hens at the 26th and 31st week of age (Period 1 and Period
2, respectively) are shown in Table 5.
Results were presented as a treatment effect including
both periods, and as a period-time effect including all treat-
ments. Dietary treatments had no significant effect on the

Table 3. Performance and egg quality parameters of laying hens (n = 72) supplemented with diets differing in the unsaturated to saturated fatty acid ratio
(mean value ± standard deviation).
Treatmentsa,f
Item HE LE+high u/s LE+low u/s P-value
Feed consumption (g/d)b 107.1 ± 4.41 109.2 ± 4.96 108.8 ± 5.10 0.634
Feed conversion (g/g)b 1.94 ± 0.157 1.93 ± 0.215 1.88 ± 0.127 0.692
Baseline body weight (kg)d 1.67 ± 0.142 1.69 ± 0.125 1.69 ± 0.123 0.861
Final body weight (kg)e 1.77 ± 0.145 1.76 ± 0.117 1.78 ± 0.122 0.832
Egg production rate (%)b 96.3 ± 6.03 96.6 ± 7.45 97.5 ± 3.87 0.886
Egg weight (g)b 57.6 ± 1.84 58.6 ± 2.12 59.1 ± 2.03 0.201
Yolk weight (g)c 14.6 ± 1.18 14.5 ± 0.62 15.1 ± 1.18 0.267
Albumen weight (g)c 37.6 ± 1.18 38.3 ± 2.41 39.2 ± 1.73 0.155
Shell weight (g)c 5.5 ± 0.54 5.6 ± 0.26 5.9 ± 0.32 0.082
Haugh Unitsc 94.8 ± 4.19 94.9 ± 4.74 94.5 ± 4.98 0.972
Yolk colourc 12.0 ± 0.31 12.2 ± 0.37 12.3 ± 0.22 0.085
a
Laying hens were fed a high energy diet (HE) or a low energy diet (LE) differing in the unsaturated to saturated fatty acids ratio (LE+high u/s: high unsaturated
to saturated fatty acids ratio; LE+low u/s: low unsaturated to saturated fatty acids ratio).
b
Average values of measurements performed weekly from 22 weeks up to 31 weeks of age.
c
Average values of measurements performed at 22 (start of the experimental period), 25, 28, and 31 weeks of age (end of the experimental period).
d
Estimated at 22 weeks of age (start of the experimental period).
e
Estimated at 31 weeks of age (end of the experimental period).
f
Data represent means based on 12 replicates per treatment, with two hens per replicate.
 BRITISH POULTRY SCIENCE 435

Table 4. Egg yolk fatty acid composition from eggs collected from laying hens at 26th week of age (Period 1) and 31st
week of age (Period 2) [in % of total fatty acids] (mean value ± standard deviation).
Item (%) Treatments1,2
Period 1 HE LE+high u/s LE+low u/s P-value
Total fats 31.3 ± 1.42 32.2 ± 0.69 32.1 ± 0.95 0.484
Palmitic (16:0) 24.0 ± 1.02 24.5 ± 1.04 24.7 ± 0.96 0.676
Oleic (18:1n-9) 44.6 ± 0.81a 42.6 ± 0.47b 44.4 ± 0.90a 0.008
Linoleic (18:2n-6) 14.6 ± 0.92 15.8 ± 1.22 14.3 ± 1.03 0.136
Monounsaturated 52.4 ± 0.71a 49.9 ± 0.10b 51.9 ± 0.91a 0.002
Polyunsaturated 15.1 ± 0.95 16.5 ± 1.27 14.8 ± 1.13 0.125
Saturated 31.6 ± 1.14 32.5 ± 1.13 32.4 ± 1.22 0.541
n-3 0.4 ± 0.04 0.5 ± 0.05 0.4 ± 0.07 0.087
n-6 14.7 ± 0.90 16.1 ± 1.22 14.4 ± 1.07 0.129
Period 2
Total fats 31.0 ± 0.62 30.9 ± 1.10 31.3 ± 1.22 0.855
Palmitic (16:0) 24.2 ± 0.70 23.9 ± 0.26 24.8 ± 0.27 0.066
Oleic (18:1n-9) 45.4 ± 0.59a 43.8 ± 0.65b 45.3 ± 1.05a 0.038
Linoleic (18:2n-6) 14.4 ± 1.05a 16.0 ± 0.46b 13.7 ± 0.67a 0.006
Monounsaturated 52.4 ± 0.79a 50.4 ± 0.70b 52.2 ± 0.90a 0.012
Polyunsaturated 15.4 ± 1.01a 17.1 ± 0.59b 14.8 ± 0.64a 0.005
Saturated 31.4 ± 0.71 31.5 ± 0.30 32.3 ± 0.40 0.061
n-3 0.7 ± 0.05a 0.7 ± 0.10a 0.5 ± 0.02b 0.015
n-6 14.7 ± 0.97a 16.4 ± 0.50b 14.2 ± 0.62a 0.005
Period 1 and 2
Total fats 31.2 ± 1.03 31.5 ± 1.09 31.7 ± 1.10 0.587
Palmitic (16:0) 24.1 ± 0.81 24.2 ± 0.75 24.7 ± 0.65 0.245
Oleic (18:1n-9) 45.0 ± 0.76a 43.2 ± 0.80b 44.8 ± 1.00a <0.001
Linoleic (18:2n-6) 14.5 ± 0.92a 15.9 ± 0.85b 14.0 ± 0.86a 0.002
Monounsaturated 52.4 ± 0.70a 50.2 ± 0.52b 52.0 ± 0.85a <0.001
Polyunsaturated 15.3 ± 0.92a 16.8 ± 0.97b 14.8 ± 0.85a 0.002
Saturated 31.5 ± 0.89 32.0 ± 0.93 32.4 ± 0.85 0.209
n-3 0.6 ± 0.17a 0.6 ± 0.16a 0.5 ± 0.11b 0.002
n-6 14.7 ± 0.87a 16.2 ± 0.88b 14.3 ± 0.82a 0.002
All treatments
Period 1 Period 2

Total fats 31.9 ± 1.10 31.1 ± 1.05 0.032

Palmitic (16:0) 24.4 ± 0.95 24.4 ± 0.56 0.831
Oleic (18:1n-9) 43.9 ± 1.15a 44.8 ± 1.03b 0.011
Linoleic (18:2n-6) 14.9 ± 1.20 14.7 ± 1.22 0.656
Monounsaturated 51.4 ± 1.25 51.6 ± 1.18 0.481
Polyunsaturated 15.5 ± 1.28 15.8 ± 1.25 0.502
Saturated 32.2 ± 1.13 31.7 ± 0.62 0.222
n-3 0.4 ± 0.06a 0.7 ± 0.09b <0.001
n-6 15.1 ± 1.22 15.1 ± 1.18 0.955
a-c
Mean values in the same row with different letters indicate significant difference between the 3 treatments (P < 0.05).
1
Laying hens were fed a high energy diet (HE) or a low energy diet (LE) differing in the unsaturated to saturated fatty
acids ratio (LE+high u/s: high unsaturated to saturated fatty acids ratio; LE+low u/s: low unsaturated to saturated
fatty acids ratio).
2
Data represent means based on 12 replicates per treatment.

levels of egg yolk carotenoids in Period 1. However, signifi- Discussion

cant differences between treatments were detected for
In earlier studies, a single fat source was used at variable
Period 2 and for both Periods 1 and 2 (an overall treatment
levels ranging from 1.8% up to 5.5% (Breithaupt et al. 2003;
effect). Specifically, lutein, zeaxanthin, cis-lutein and total
Sirri et al. 2007). Elsewhere, a combination of fat sources
carotenoids content were significantly lower in the LE+low
have been tested in laying hens’ diets e.g. 4% or 2% as
u/s group compared to the HE and the LE+high u/s groups
a mixture of animal and vegetable fat (Leeson and Caston
for Period 2 (P = 0.004, P = 0.003, P = 0.003 and P = 0.003,
2004; Leeson et al. 2007, respectively). In the current experi-
respectively). Similarly, a significant treatment effect was
ment, a combination of soybean and palm oil was used,
detected in both periods for lutein, zeaxanthin, cis-lutein
while the total inclusion level in the diet was 1% more in the
and total carotenoids content, being lower in the LE+low u/
HE compared to LE+high u/s and LE+low u/s groups (3.4%
s group compared to the other two treatments (P = 0.001,
and 2.4%, respectively). Despite differences in energy levels
P = 0.003, P = 0.002 and P = 0.001, respectively).
between treatments, performance indicators were not
Additionally, an overall treatment effect was noted for α-
affected and total fat levels in the egg yolk did not differ.
and β-carotene levels, which were lower in the LE+low u/s
Laying hens tolerated the imposed diet changes without any
group compared to HE and the LE+high u/s groups
penalty in performance. These findings agree with those
(P = 0.022 and P = 0.037, respectively). An overall period-
reported by Huang et al. (2018), in which feeding laying
time effect was revealed for β-carotene levels being lower in
hens with lower metabolisable energy diets were compared
Period 1 than Period 2 (P = 0.033).
436 G. A. PAPADOPOULOS ET AL.

Table 5. Carotenoid content in egg yolk from eggs collected from laying hens at 26th week of age (Period 1) and 31st week of age (Period 2) (mean value ±
standard deviation).
Treatments1,2
HE LE+high u/s LE+low u/s P-value
Period 1
Lutein [μg/g] 73.6 ± 6.69 77.8 ± 7.85 72.4 ± 5.22 0.509
Zeaxanthin [μg/g] 7.46 ± 0.59 7.62 ± 1.03 7.31 ± 0.73 0.867
cis-Lutein [μg/g] 18.7 ± 1.24 19.5 ± 1.82 18.6 ± 1.92 0.733
α-Carotene [μg/g] 2.9 ± 0.23 3.0 ± 0.57 2.5 ± 0.47 0.279
β-Carotene [μg/g] 4.0 ± 0.38 3.5 ± 0.52 3.7 ± 0.56 0.346
Total Carotenoids [μg/g] 110.3 ± 8.51 115.5 ± 11.63 108.5 ± 8.73 0.598
Period 2
Lutein [μg/g] 74.9 ± 4.96a 81.0 ± 12.20a 49.2 ± 17.05b 0.004
Zeaxanthin [μg/g] 8.1 ± 0.22a 8.2 ± 1.49a 5.2 ± 0.85b 0.003
cis-Lutein [μg/g] 21.1 ± 1.45a 19.7 ± 2.01a 13.1 ± 3.58b 0.003
α-Carotene [μg/g] 3.3 ± 0.61 3.7 ± 0.77 2.6 ± 0.31 0.083
β-Carotene [μg/g] 4.9 ± 0.70 4.6 ± 1.03 3.5 ± 0.47 0.064
Total Carotenoids [μg/g] 116.4 ± 7.30a 121.8 ± 16.25a 76.3 ± 24.84b 0.003
Period 1 and 2
Lutein [μg/g] 74.3 ± 5.50a 79.5 ± 9.65a 60.8 ± 14.90b 0.001
Zeaxanthin [μg/g] 7.8 ± 0.55a 7.9 ± 1.22a 6.2 ± 1.35b 0.003
cis-Lutein [μg/g] 19.9 ± 1.79a 19.6 ± 1.78a 15.8 ± 3.96b 0.002
α-Carotene [μg/g] 3.2 ± 0.48a 3.4 ± 0.72a 2.6 ± 0.37b 0.022
β-Carotene [μg/g] 4.5 ± 0.69a 4.1 ± 0.96a 3.6 ± 0.49b 0.037
Total Carotenoids [μg/g] 113.3 ± 8.02a 118.6 ± 13.50a 92.4 ± 21.41b 0.001
Periods
All treatments 1 2
Lutein [μg/g] 74.6 ± 6.51 68.4 ± 17.05 0.091
Zeaxanthin [μg/g] 7.4 ± 0.73 7.2 ± 1.73 0.434
cis-Lutein [μg/g] 18.9 ± 1.58 18.0 ± 4.31 0.297
α-Carotene [μg/g] 2.9 ± 0.47 3.2 ± 0.72 0.102
β-Carotene [μg/g] 3.8 ± 0.51a 4.4 ± 0.94b 0.033
Total Carotenoids [μg/g] 111.5 ± 9.31 104.8 ± 24.84 0.201
a-b
Mean values in the same row with different letters indicate significant difference between the 3 treatments (P < 0.05).
1
Laying hens were fed a high energy diet (HE) or a low energy diet (LE) differing in the unsaturated to saturated fatty acids ratio (LE+high u/s: high unsaturated
to saturated fatty acids ratio; LE+low u/s: low unsaturated to saturated fatty acids ratio).
2
Data represent means based on 12 replicates per treatment.

to a control, and these treatments did not have any effect on
feed intake, FCR, egg production and egg weights.
Lutein, zeaxanthin and carotenoid levels were analysed
in the egg yolk at two 5-week intervals. The first was at the
completion of the 5th and the second at the 10th experi-
mental week. The timepoint where change in egg yolk
carotenoids was seen due to dietary changes differed
between studies. Breithaupt et al. (2003) showed that sup-
plementation of lutein and carotenoid sources increased
plasma carotenoid levels and egg yolk colour index within
one week. In other trials, 4 weeks were needed to reach
a plateau of lutein, zeaxanthin and total carotenoids levels
in the egg yolk (Kotrbáček et al. 2013). In the present study,
in both sampling periods, lutein was the predominant car-
otenoid in the egg yolk, in agreement with previous reports
(Steinberg et al. 2000; Surai and Sparks 2001). In the 5th
week of the experimental period, no differences were found
between treatments. Remarkably, lutein, zeaxanthin and
total carotenoids concentration decreased at the 10th week
in the LE+low u/s ratio group. The change in carotenoids
levels did not affect total egg yolk fat levels, which remained
similar between groups. Thus, carotenoid levels in the egg
yolk may be modulated by a different mechanism than the
one responsible for yolk fatty acids. In support of this
notion, fat digestibility of feed did not differ between treat-
ments at the middle of the experiment. Nevertheless, fat
digestibility was not estimated at the end of the experimen-
tal period, which would have been useful to conduct. Thus,
it can only be speculated whether or not differences would
have occurred at that time point as well. Furthermore, it
may be difficult to estimate when the decline of the egg yolk
carotenoids after the 5th week group occurred. According
to published information, this is the first report which
showed that lutein and zeaxanthin content in the egg yolk
changes due to dietary fat source profile, i.e. a low ratio of
unsaturated to saturated fatty acids could decrease of lutein,
zeaxanthin and total carotenoids. Notably, this decrease did
not correspond to any changes in egg yolk colour as mea-
sured by the Roche colour fan. In contrast, Roche colour
index of egg yolks were greater in laying hens supplemented
with a saturated fat source (lard) than those supplemented
with unsaturated fat sources (soybean oil and acidulated
vegetable oil soap stocks) (Pérez-Bonilla et al. 2011). It
was hypothesised that dietary pigments were more stable
in the presence of a saturated fat source than in the presence
of unsaturated fat. Nevertheless, no data on lutein, zeax-
anthin or carotenoid levels in the egg yolk were provided in
the latter study.
In quails, the lowest egg yolk carotenoid was found
together with a higher score in the Roche colour fan and
was suggested that carotenoid profile may be more impor-
tant for yolk coloration than the quantity of total carote-
noids in the egg yolk (Karadas et al. 2006). The latter
findings are in agreement with the ones reported herein.
Overall, yolk colour index may not always correspond to the
total carotenoid content of the egg yolk. Meanwhile, no
differences between treatments occurred for other quality
parameters of eggs, such as weight or shell thickness.

Several factors affect the absorption, bioaccessibility or
bioavailability of carotenoids (Goltz et al. 2012). In humans,
consumption of diets rich in unsaturated fatty acids led to
higher plasma lutein response (Roodenburg et al. 2000).
Elsewhere, a mono-unsaturated lipid source rather than
a saturated one improved the absorption of polar carote-
noids (Goltz et al. 2012). Recently, in vitro studies have
shown that carotenoid micellariasation was improved in
the case of unsaturated fat sources compared to saturated
ones (Failla et al. 2014; Mashurabad et al. 2017). On the
other hand, consumption of saturated rich lipid sources by
women promoted carotenoid absorption (Hu et al. 2000). It
has been demonstrated in vitro that dietary fats rich in
saturated fatty acids led to higher bioavailability of lutein
and zeaxanthin (Gleize et al. 2013). In the current study,
results from the fat digestibility analysis did not show any
differences between treatments. Thus, it could be hypothe-
sised that fat absorption was similar for all groups.
However, fat digestibility was only estimated in the middle
of the experiment, but not at the end of the experimental
period. It cannot be excluded that differences between
groups for lutein, zeaxanthin and rest carotenoids, could
have been due to differences in dietary fat digestibility. This
field warrants further investigation.
From the findings of the current trial it may be difficult
to attribute the effects on lutein and zeaxanthin levels in the
egg yolk solely to the mode of action of either the poly-
unsaturated or the saturated fatty acids. Interestingly, lutein
and zeaxanthin levels were similar in both sampling periods
between the HE and the LE+high u/s group. Notably, the LE
+high u/s contained 0.7% less soybean oil and similar
amount of palm oil compared to the HE group. Thus,
decreasing the quantity of the polyunsaturated fat source
may not be as important as maintaining a dietary u/s ratio
above 3.3, which was approximately the ratio used in the
HE and LE+high u/s diets. However, under the combina-
tions used, a higher inclusion of palm oil was accompanied
by a decrease in the soybean oil, which resulted in a dietary
U/S ratio equal to 2.4. This dietary u/s ratio led to decreased
lutein, zeaxanthin and total carotenoids levels after 10 weeks
of feeding. Thus, it can be proposed that a higher inclusion
of a saturated fat source did not promote lutein and zeax-
anthin bioavailability. However, the underlying mechanism
of the observed effects of dietary fat sources on lutein and
zeaxanthin levels is not clear and requires further investiga-
tion involving other combinations of energy levels and fat
sources.
Conclusions
The present study showed that feeding laying hens with
diets enriched in saturated fatty acids and with a low dietary
u/s ratio, may reduce lutein, zeaxanthin and total carote-
noids levels in egg yolk during the early laying period. In
contrast, feeding diets with a higher content of polyunsatu-
rated fatty acids and less saturated, did not affect egg yolk
carotenoids. Under the tested dietary combinations, the
energy reduction of diets by removing 1% of supplemented
fat may not be as detrimental to maintain egg yolk carote-
noids as compared to the type of supplemented fat. Further
research is warranted towards investigating the effects of an
unaltered energy diet more enriched in saturated fats on egg
yolk carotenoid levels.
BRITISH POULTRY SCIENCE 437
Acknowledgments
The authors would like to thank Professor George Zachariadis from the
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle
University, for technical assistance and providing the equipment for
analysis of titanium in samples. The authors also thank the Assistant
Professor Ioannis Karapanagiotidis from the Department of Icthyology
and Aquatic Environment of University of Thessaly in Volos for provi-
sion of the adiabatic bomb calorimeter. Finally, the authors would like to
thank Professor Ioannis Amvrosiadis and Assistant Professor Aikaterini
Papavergou of the Laboratory of Food Technology of Animal Origin,
Faculty of Veterinary Medicine, Aristotle University, for providing us
access to perform chemical analysis.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was funded by Kemin Europa N.V. (project number
92358; Research Committee of Aristotle University, Thessaloniki,
Greece). Kemin Europa N.V. provided the supplement Oro Glo
Layer (Kemin Europa NV, Herentals, Belgium). There was no further
involvement of the funding source in study design; in the collection,
analysis and interpretation of data; in the writing of the report; and in
the decision to submit the article for publication.
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