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Basic nutritional investigation
Free-range farming: A natural alternative to produce
vitamin D-enriched eggs
Julia Kühn, Alexandra Schutkowski Ph.D., Holger Kluge Ph.D.,
Frank Hirche Ph.D., Gabriele I. Stangl Ph.D.
*
Institute of Agricultural and Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany
article info
Article history:
Received 5 August 2013
Accepted 8 October 2013
Keywords:
Free-range farming
Natural sunlight
Vitamin D
Eggs
Hen
abstract
Objective: Food-based strategies need to be developed to improve the vitamin D status of in-
dividuals. Recent studies identified ultraviolet B irradiation as an efficient method to enrich
mushrooms and eggs with vitamin D. The aim of this study was to determine whether free-range
farming of hens could provide a valuable method to produce vitamin D-enriched eggs.
Methods: Laying hens were randomly assigned to three groups of 33 to 34 animals each, and were
kept either indoors (indoor group), outdoors (outdoor group), or with an indoor/outdoor option
(indoor/outdoor group) over 4 wk.
Results: The study shows that the vitamin D
3
content of egg yolk was three- to fourfold higher in
the groups that were exposed to sunlight (outdoor and indoor/outdoor groups) compared with the
indoor group (P<0.001). Egg yolk from the outdoor group revealed the highest vitamin D
3
content, which averaged 14.3
m
g/100 g dry matter (DM), followed by that from the indoor/outdoor
group (11.3
m
g/100 g DM). Yolk from indoor eggs contained only 3.8
m
g vitamin D/100 g DM. The
25-hydroxyvitamin D (25[OH]D
3
) content of egg yolk was also influenced by sunlight exposure,
although less pronounced than the vitamin D content (P<0.05). In contrast, free-range eggs
randomly acquired from supermarkets had relatively low vitamin D contents.
Conclusion: Free-range farming offers an efficient alternative to fortify eggs with vitamin D, pro-
vided that farming conditions are sufficiently attractive for hens to range outside.
Ó2014 Elsevier Inc. All rights reserved.
Introduction
The main source of vitamin D in humans is the conversion of
7-dehydrocholesterol (7-DHC) in the epidermis to previtamin D
3
,
which isomerizes to form vitamin D
3
. Seasonal and local varia-
tions of ultraviolet (UV) radiation, increasing indoor activities,
but also public awareness of the hazardous effects of sun expo-
sure that lead to sun avoidance strategies or the use of sun-
screens limit the endogenous synthesis of vitamin D
3
.Asa
consequence, the need for vitamin D from food sources is on
the rise.
In contrast to vitamin D from sunlight, dietary vitamin D is
available during any season and offers a reliable source of the
vitamin for individuals who do not have access to sunlight
exposure. Oily fish is the most important dietary source of
vitamin D [1], whereas most other foods from animal origin
contain very low amounts and do not significantly contribute to
an improvement of vitamin D status. A recent study was able to
ascertain UVB exposure of hens as a highly effective approach to
increase the vitamin D content in eggs [2]. Data from that study
showed that UVB radiation increased the vitamin D content
much stronger than feeding the maximum permissible dosages
of dietary vitamin D
3
.
Currently, three farming systems for laying hens are
frequently implemented in practice: Free-range farming, floor
management, and small-group systems, whereby mixed forms of
these housing systems are also common. In free-range systems,
hens have the possibility to go outside and become exposed to
natural sunlight, whereas floor management and small-group
systems operate with UV-free light regimens. Therefore, we hy-
pothesized that eggs from free-range hens might contain higher
vitamin D contents than barn eggs. The present study aimed to
investigate the vitamin D content of eggs in response to different
housing conditions.
GIS, JK, and AS conceived and designed the experiment. JK, AS, and HK per-
formed the experiment. JK and FH analyzed the data. JK, GIS, and AS wrote the
article.
*Corresponding author: Tel.: þ49-345-5522707; fax: þ49-345-5527124.
E-mail address: Gabriele.stangl@landw.uni-halle.de (G. I. Stangl).
0899-9007/$ - see front matter Ó2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nut.2013.10.002
Contents lists available at ScienceDirect
Nutrition
journal homepage: www.nutritionjrnl.com
Nutrition 30 (2014) 481–484
Materials and methods
Animals and treatment
The study was conducted with 101 25-wk-old laying hens (Lohmann silver).
The hens were randomly assigned to three groups. Group 1 (n ¼34, indoor
group) was kept indoors without anyexposure to natural sun light over the entire
study period; group 2 (n ¼34, outdoor group) was placed outside for 9 h/d from
0700 h to 1600 h. Group 3 (n ¼33, indoor/outdoor group) could choose freely
between inside and outside (outside option from 0700 h to 1600 h). The free-
range area was partly covered by UV-permeable acrylic glass to provide rain
protection. All hens received a standard feed with 2500 IU vitamin D/kg (Deuka,
Könnern, Germany). Feed and water were offered ad libitum. The indoor group
was fed inside the barn; the outdoor and indoor/outdoor groups were provided
with feed and water, both indoors and outdoors.
Study protocol
The study was performed from August 22 to September 19, 2012 at the
experimental facility of the Martin-Luther-University Halle-Wittenberg (51
latitude). Before the experiment, all hens were housed in a barn system for 2 wk.
The experimental period lasted 4 wk. Eggs were collected at the beginning of the
study (basal) and after the fourth week (final) of the experiment. To study the
choice behavior of the indoor/outdoor group, hens were monitored on three
randomly selected days during the experimental period.
Additionally, free-range and barn eggs were collected randomly from su-
permarkets and analyzed for their vitamin D content. To ensure that the com-
mercial free-range eggs were produced under similar weather and sunlight
conditions as the experimental eggs, we acquired eggs from two different hen
farms located near the experimental facility. The eggs were collected in
September and January to determine out seasonal influence on the vitamin D.
Sun light exposure conditions and sunshine duration
The outdoor enclosure conditions (location, incidence of sun light) were ar-
ranged to ensure an optimal UVB exposure from 1000 h to 1600 h. The UV
permeability of the coverage was >90% (manufacturer’s specification). Sunshine
duration, defined as the time during which the solar irradiance exceeds 120 W/
m
2
, was recorded daily.
Analysis of eggshell thickness and stability
Eggshell stability was determined by an electronically controlled breaking
strength tester (Messtechnik Gutsch, Nauendorf, Germany). Eggshell thickness
was measured using a micrometer screw with an accuracy of 10.0
m
m. Shell
fragments were collected from the equatorial area and mechanically purified
from organic materials.
Analysis of vitamin D
3
, 25-hydroxyvitamin D
3
, and 7-DHC contents in egg yolk
Pooled egg yolk samples (of three yolks each) of each group were used for the
analysis of vitamin D metabolites. Commercial eggs were analyzed individually.
Vitamin D
3
, 25-hydroxyvitamin D
3
(25[OH]D
3
) and 7-DHC were determined by
liquid chromatography-tandem mass spectrometry, as described previously [2].
Statistical analysis
Statistical analyzes were performed using SPSS Statistics version 20 (IBM,
Armonk, NY, USA). Differences in the vitamin D content of eggs from basal to final
were analyzed by a paired ttest. Differences between the three groups of hens
were analyzed by one-way analysis of variances (ANOVA). Vitamin D content of
the purchased eggs was compared by two-way ANOVAwith the factors “farming”
and “season,”and the interaction between these two factors. When two-way
ANOVA revealed significance for one of these factors, a post hoc comparison
was performed. In case of variance homogeneity, means of the four groups were
compared by Tukey’s test, or in case of variance heterogeneity, by Games-Howell
test. Means were considered significantly different at P<0.05.
Results
Sunshine duration and behavior of hens
The average weekly daily sunshine durations were 6.6, 5.5,
5.9, and 7 h, respectively (Fig. 1). Observations from three
randomly selected days showed that hens from the indoor/out-
door group spent most of their time outside the barn. Thus, we
assumed that UVB exposure of this group was comparable to that
of the outdoor group.
Egg weight and eggshell quality
Egg weight, eggshell thickness, and stability did not differ
between the three groups of hens at basal (Table 1). In all groups
of hens, egg weights significantly increased from basal to final
(P<0.05). The housing systems did not reveal any effect on final
egg weights and eggshell stability (Table 1). The eggshell thick-
ness increased from basal to final in the outdoor and indoor/
outdoor groups (P<0.05), but not in the indoor group (Table 1).
Final eggshell thickness was highest in the outdoor group, lowest
in the indoor group, and showed intermediate values in the in-
door/outdoor group.
Vitamin D content of egg yolk in response to the housing system
Analysis of vitamin D metabolites in pooled egg yolks from the
treatment groups was performed at basal and after 4 wk of the
experiment (Fig. 2). Egg yolk data did not reveal any differences
in basal contents of vitamin D
3
, 25(OH)D
3
, and 7-DHC between
the three groups of hens. At the end of the experiment, the
vitamin D
3
content of egg yolk was three- to fourfold higher in
the sunlight-exposed groups (outdoor group and indoor/outdoor
group) than in the indoor group (P<0.001; Fig. 2A). Egg yolks
from the outdoor group revealed the highest vitamin D
3
content,
which averaged 14.3
m
g/100 g dry matter (DM), followed by yolk
from the indoor/outdoor group with 11.3
m
g/100 g DM. Egg yolk
0.0
2.0
4.0
6.0
8.0
Aug. 22 Aug. 29 Sept. 5 Sept. 12 Sept. 19
Sunshi ne duration
(h/d)
Fig. 1. Sunshine duration at the trial site (data averaged to weekly sunshine
duration).
Table 1
Egg weights and eggshell quality in response to the housing system
Indoor
group
Outdoor
group
Indoor/outdoor
group
Egg weight (g)
Basal 54.3 3.1 52.7 2.4 52.9 1.9
Final 62.2 4.8
*
59.8 1.9
*
59.3 2.6
*
Eggshell thickness (
m
m)
Basal 352 17 337 20 333 14
Final 362 14
b
380 16
*,a
368 13
*,a,b
Eggshell stability (N)
Basal 39.3 7.7 36.8 5.1 38.1 4.4
Final 46.5 7.2 49.6 7.5
*
49.3 9.1
*
Values are presented as mean SD (n ¼7 to 11)
Means not sharing the same superscripts (a,b) are significantly different
(P<0.05, Tukey’s test).
*
Means are significantly different compared with basal (P<0.05, paired
ttest).
J. Kühn et al. / Nutrition 30 (2014) 481–484482
of the indoor group revealed the lowest vitamin D levels. The
content of 25(OH)D
3
in egg yolk also increased in response to
sunlight exposure, although less pronounced than the vitamin D
3
content (P<0.05; Fig. 2B). The 7-DHC contents of eggs were not
influenced by the housing system (Fig. 2C).
Vitamin D content of commercial eggs
Vitamin D data did not show a consistent picture of free-range
and barn eggs. Regardless of the housing system and the season,
the vitamin D
3
content of commercial eggs from farm A was
low and ranged between 2 and 4
m
g/100 g DM (Fig. 3A). Also,
free-range eggs from farm B were not characterized by higher
0
5
10
15
20
Vitamin D3(µg/100 g DM)
A
Indoor
Outdoor
Indoor/outdoor
Basal
Final
‡
‡
†
†
*
0
2
4
6
25(OH)D
3
(µg/100 g DM)
B
Indoor
Outdoor
Indoor/outdoor
Basal
Final
†
†
‡
‡
0
100
200
300
400
500
7-DHC (µg/100 g DM)
C
Indoor
Outdoor
Indoor/outdoor
Basal Final
Fig. 2. Content of (A) vitamin D
3
, (B) 25(OH)D
3
, and (C) 7-dehydrocholesterol (7-
DHC) in dry matter (DM) of pooled egg yolks from three hens each (n ¼7to11,
resulting from inconsistent numbers of laid eggs). Values are expressed as
mean SD. Data were analyzed by one-way ANOVA using multiple comparison
procedures to analyze means of the groups (Tukey’s test or Games-Howell test).
*P<0.05,
y
P<0.001,
z
significantly different from basal (P<0.05, paired ttest).
0
3
6
9
12
Vitamin D 3(µg/100 g DM)
Free -range
Barn
Summ er Sum mer
Winter
Winter
Farm A Farm B
2WA f arming,
far ming x season:
P< 0.01
0
2
4
6
25(OH)D
3
(µg/100 g DM)
Free-range
Barn
Summer Summer Winter
Winter
Farm A
Farm B
0
100
200
300
400
500
7-DH C (µg/100 g DM)
Free-range
Barn
Summ er Summer
Winter
Farm A
Farm B
Winter
2WA f arming:
P < 0.001
A
B
C
‡
††
Fig. 3. Content of (A) vitamin D
3
, (B) 25(OH)D
3
, and (C) 7-dehydrocholesterol (7-
DHC) in dry matter (DM) of yolks from commercial eggs (n ¼6 per set). Values
are expressed as mean SD. Data were analyzed by two-way ANOVA (2 WA) with
the factors “farming”and “season”and the interaction between these factors,
respectively. Tukey’s test and Games-Howell test were applied to compare means:
y
P<0.001,
z
P<0.01.
J. Kühn et al. / Nutrition 30 (2014) 481–484 483
vitamin D content compared with barn eggs (Fig. 3A). The yolk
content of 25(OH)D
3
did not differ between the commercial eggs
analyzed (Fig. 3B). The 7-DHC content was not different between
eggs from farm A, but was higher in free-range eggs than in barn
eggs from farm B (P<0.05; Fig. 3C).
Discussion
UV radiation is essential for endogenous vitamin D synthesis
in humans and animals [3]. Recent data show that eggs from
hens exposed for 3 h to artificial UVB radiation contained sixfold
higher amounts of vitamin D
3
than eggs from non-exposed an-
imals [2]. Here, we show that free-range versus barn-rearing
systems for laying hens could offer an appropriate and cheap
alternative to fortify eggs with vitamin D. Eggs from free-range
hens that spent 9 h/d outdoors contained on average fourfold
higher amounts of vitamin D
3
and 45% higher amounts of 25(OH)
D
3
than barn eggs. However, we need to acknowledge that the
findings are specific to the season (August 22 to September 19)
and latitude (51
, Central Germany) under which the experiment
was conducted. It is to be expected that the efficacy of free-range
systems to fortify animal-based foods such as eggs, largely
depend on weather, season, and the latitude of the area where
the animals are housed [4,5]. We further found that eggs from
chickens with the inside/outside option contained markedly
more vitamin D
3
than the indoor group, although the vitamin D
3
contents of the outdoor group were not fully reached. Although
such eggs may not cover the daily vitamin D recommendation,
the idea of vitamin D bio-addition by sunlight could be used for
other animal housing systems to improve the vitamin D content
of meat or milk. The fact that the vitamin D content of milk could
be increased by sunlight exposure of cows [6,7] indicates the
effectiveness of the free-range system in production of other
vitamin D-enriched food.
Owing to the efficient bio-addition of eggs with vitamin D by
natural sunlight exposure of hens, we hypothesized that com-
mercial free-range eggs produced in summer should contain
higher amounts of vitamin D than barn eggs or free-range eggs
from winter. However, the vitamin D analysis did not support our
hypothesis. After visiting the farms, we assume that the free-
range housing conditions are crucial for the choice behavior of
the chickens. An important aspect that could influence the
movement of chickens in and out of the hen house is the offered
space for feeding [8]. We observed that chickens in a free-range
husbandry avoided moving outside if they received their feed
exclusively inside the barn. Besides the feeding points, habitat
preferences of free-range chickens are also important. One study
observed that the number of chickens found to be ranging
outside was positively correlated with the amount of tree cover
the range area contained [9]. Thus, the efficiency of sunlight
exposure to produce vitamin D-enriched eggs also depends on
the habitat conditions.
Another interesting finding from this study was an increase in
the stability of eggshells from basal to final in the sunlight-
exposed groups, but not in the indoor group. Eggshell quality
could be affected by diverse factors [10]. Most of the studies did
not found any effect of the housing system on eggshell quality
[11–13] , but it is still a subject of controversy whether UVB ra-
diation could improve the eggshell quality.
Conclusion
Data show that the free-range farming system could be an
appropriate method to fortify eggs with vitamin D, provided
free-range farming conditions are sufficiently attractive for the
hens to range outside.
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