The hen's egg yolk: a source of

biologically active substances

C. HARTMANN' and M. WILHELMSON2
'Swedish University of Agriculture Sciences, Department of Animal
Breeding and Genetics, Funbo-Lovsta, 755 97 Uppsala and
*Immunsystem I M S AB, Uppsala Science Park, 751 83 Uppsala,
Sweden

The egg yolk contains several substances of potential therapeutic value, some
of which are already used in commercial products. A number of scientific
papers dealing with their properties and applications have been published. In
this review we present some current and possible applications for egg yolk
lipids, lipoprotein, phosvitin, sialic acid and antibodies (IgY). Moreover, we
discuss the possibilities of increasing the yield of yolk and simultaneously the
yield of these substances by both management and breeding approaches.

Keywords: Biologically active substances; breeding; egg yolk; management

Introduction
For a long time man has made use of the high nutritional value of the egg yolk.
It has also been used for non-food purposes, such as in the preparation of leather.
Today the egg yolk is still an important source of nutrients and is also used for
non-food purposes. For example, it has been shown to be a useful source of
biologically active substances, some of which are used commercially as
alternative egg products. Industry has shown great interest in using specific
substances from the egg yolk and in recent years a considerable number of
scientific papers dealing with biologically active substances originating from the
egg yolk have appeared. However, although information is comprehensive, it
tends to be scattered.
When large numbers of eggs are processed industrially it may be worthwhile to
consider how their composition may be improved in order to achieve a more
profitable raw material. This could involve improving both the quantity and
quality of the egg components. Over 30 years ago Marion et aZ. (1965) stated that
"a fairly large amount of literature is now available on component parts of eggs
from GaZZus domesticus". In that paper the effects of breeding, egg size, age and
0 World's Poultry Science Association 2001
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Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
season on egg composition were investigated. During the decades that have
passed since then, more knowledge has accumulated on the genetic and
environmental factors influencing egg composition.
The purpose of this paper is to bring together and review the recent scientific
literature dealing with applied aspects and possible applications for biologically
active substances originating from the egg yolk, as well as current knowledge on
factors influencing yolk production.

Biologically active substances in egg yolk

It appears that the egg processing industry will develop along two paths - food
production and the production of non-food products that will add to the profit of
the egg industry (Ros, 1998). The yolk fat industry has noticed an increasing
interest in egg yolk lipids for a whole range of technological and nutritional
applications from a variety of industries (Schneider, 1999). In the following
section some of the current and possible future applications for egg yolk lipids,
lipoprotein, phosvitin, sialic acid and antibodies (IgY) are reviewed.

EGG YOLK LIPIDS

For many years the pharmaceutical industry has made use of the favourable
emulsifying properties (e.g. emulsion stability) of yolk phospholipids. A well
established product on the market is the parenteral fat emulsion Intralipid@
which, in addition, is used as a carrier of fat soluble drugs (HAkansson, personal
communication, 1995).
Long chain polyunsaturated fatty acids of the n-6 and n-3 series have proved to
be important for the development of the human fetal nervous system, a
development which largely takes place during the last 3 months of pregnancy
(Jensen et al., 1992). In normal pregnancies the mother’s circulation provides the
fatty acids required by the placenta, whereas in the case of premature births the
necessary fatty acids must come from the infant’s dietary formulae. Egg yolk is a
rich source of long chain polyunsaturated fatty acids of the n-6 series and is
therefore used as a source of these in the preparation of dietary formulae for
premature babies (Siewert, personal communication, 1998).
Alzheimer’s disease is characterised by a number of structural and neuro-
pharmacological changes, some of which include a reduction in the neuro-
transmitter acetylcholine, the precursor of which is choline. Masuda et al. (1998)
studied the effect of administration of egg yolk phosphatidylcholine conjugated
with vitamin B,, to rats with nucleus basalis magnocellularis lesions. The treatment
improved memory retention and increased the acetylcholineconcentrations in the
frontal cortex. Sanada et al. (1997) assessed the possible clinical benefits of a
similar treatment in humans suffering from Alzheimer’s disease. Oral administra-
tion increased the concentrations of choline and vitamin B,, in the serum, but any
effects on an improvement of mental and intellectual functions were less clear.
Food products containing unsaturated fatty acids are sensitive to oxidation
which results in the formation of lipid peroxide and subsequent undesirable
effects. To prevent oxidation, antioxidants are commonly added to the foodstuffs.
However, because the safety of synthetic antioxidants is increasingly being
questioned, research on potent natural antioxidants is receiving a lot of attention.
Ethanol extracted yolk lipid showed antioxidative effects on the lipids
docosahexaenoic acid (DHA) and squalene. The higher the purity of the egg yolk
lipid, the lower was the degree of oxidation in the DHA oil. The outcome of

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 Biologically active subsfances in yolk: C. Harfmann and M . Wilhelmson
hydrogenating phosphatidylcholine and phosphatidylethanolamine extracted
from yolk gave rise to the suggestion that the degree of unsaturation of the fatty
acyl chain containing phospholipid would be closely associated with its
antioxidative capability. A higher degree of unsaturation coincided with stronger
antioxidant activity (Sugino et al., 1997)
The main constituent of liposomes (aqueous compartments enclosed by a lipid
bilayer) is phosphatidylcholine, the most abundant phospholipid in egg yolk.
Liposomes can be used for a variety of purposes. The most common source of
phospholipids for liposome production is soya beans. However, egg yolk
phospholipids have several advantageous characteristics, such as high entrap-
ment efficiency and stability, which justify their role in liposome production (Sim,
1994). Egg yolk liposomes have been used several times to model membranes
(Gabrielska and Gruszecki, 1996; Tyurin-Kuz’min, 1997; Wodall et al., 1997) and
have also been used as drug carriers in experiments. Timofeev et al. (1994)
observed a decrease in acute toxicity as well as therapeutic effects when the drug
diamidine, used in the treatment of babesiasis, was encapsulated in egg yolk
phosphatidylcholine liposomes rather than applying the free drug by itself.
Administration of the acetylcholinesterase inhibitor 9-amino-1, 2, 3, 4-tetra-
hydroacridine (THA) encapsulated in liposomes prepared from egg yolk
phosphatidylcholine reduced both drug toxicity and its side effects in mice, and
the effective concentration of THA in the brain was prolonged (Kobayashi et al.,
1996).
Egg yolk phospholipids are commonly extracted using organic solvents, the
safety of which has been questioned. This has stimulated attempts to find other
methods of extraction. Sim (1994) used aqueous ethanol to extract phospholipids,
egg oil and lipid-free yolk protein from both fresh yolk and yolk powder. Juneja
et al. (1994a) investigated a method for large scale extraction of phospholipids:
500 kg yolk extracted with acetone and ethanol yielded 37.5 kg phospholipids
with a phosphatidylcholine content of 80.8%. Supercritical carbon dioxide
techniques for extraction of phospholipids are also under investigation (Ryhanen,
personal communication, 1999).

PHOSVITIN
The significant amounts of unsaturated fatty acids and iron in egg yolk greatly
increase its susceptibility to lipid oxidation. Nevertheless, lipid oxidation does not
occur rapidly. Greengaard et al. (1964) found that 5.96mg of 6.53mg iron/100g
egg yolk was bound to the phosphoprotein phosvitin. The iron was not easily
removed (Albrighet et al., 1984) and this means that the iron is not readily
available as a catalyst for oxidation. It has therefore been suggested that phosvitin
is the natural antioxidant in egg yolk.
Lu and Baker (1986) added phosvitin, iron and copper to a yolk phospholipid
emulsion. Phosvitin was found to prevent iron catalysed oxidation. Addition of
freeze dried egg yolk to an egg phospholipid emulsion also reduced oxidation
under certain conditions (Lu and Baker, 1987). When adding egg yolk (l%,2%
and 3%) or phosvitin (0.0625%)to patties made from turkey neck or drumstick
meat, Lu (1987) observed a significant decrease in the degree of oxidation in the
neck meat patties, irrespective of treatment, but not in those prepared with the
drumstick meat. There was generally no difference between the treatments in
reducing oxidation.
Yamamoto et al. (1990) found that whole egg yolk, as well as the granule, had
antioxidant activity on a linoleic acid emulsion, whereas low density lipoprotein

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Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
(LDL) and phosvitin had a lower activity. By adding lipoprotein to the phosvitin
the antioxidative effect was improved. The granule also had antioxidative effects
on non-emulsified linoleic acid and lard; the start of oxidation was delayed by 12
days in the linoleic acid and no oxidation at all was observed in the lard
(Yamamoto et al., 1991).By adding 1.5%of a yolk high density lipoprotein (HDL)
fraction to linoleic acid, oxidation was inhibited (Yamamoto and Omori, 1994).
At typical cooking and sterilisation temperatures phosvitin is denaturated
(Chung and Ferrier, 1995). By conjugating phosvitin and galactomannan
Nakamura et al. (1998)obtained an antioxidant which could withstand 15 minutes
in an autoclave (121°C at 1.2 atmospheres). Egg yolk phosvitin has been further
found to possess emulsifying properties (Sattar Kahn et al., 1998) which were also
improved by conjugating phosvitin with galactomannan (Nakamura et al., 1998).

LIPOPROTEINS
It is generally accepted that the growth of virtually all types of cells in culture
requires the presence of serum in the medium. However, it is possible and
sometimes advantageous to grow cells without the presence of serum. The serum-
free environment provides the opportunity to carry out studies on cells in the
absence of unwanted serum components. Furthermore, the serum-free environ-
ment enables nutritional studies and studies on the release of cellular products in
a controlled environment to be investigated (Barnes and Sato, 1980).
Egg yolk added to a serum-free environment supported the proliferation of
several types of mammalian cells. Optimal growth promoting effects were
observed at concentrations of 7.5-10% egg yolk volume/volume (Fuji and
Gospodarowicz, 1983). Murikami et al. (1988) attributed the growth promoting
effect of the egg yolk to a very low density lipoprotein, YLP-pl7.5, which they
separated from egg yolk lipoprotein. The original lipoprotein had a growth
promoting effect on several mammalian cells. It also activated cell growth in the
presence of insulin, transferrin, ethanolamine and selenite (ITES). YLP-pl7.5
added to a serum-free eRDF medium supplemented with ITES significantly
accelerated the growth of mouse myeloma cells. Several human cells were also
successfully grown on the medium. Nakama and Yamada (1993) grew hepato-
cellular carcinoma cells on a serum-free medium to which growth factors,
hormones, nutrients and egg yolk LDL had been added. Shinohara et al. (1993)
showed that an LDL-rich yolk fraction had growth promoting effects on human-
human hybridoma cells. It also promoted IgM secretion from these cells.

SIALJC ACID
Sialic acid is the generic term for approximately 30 derivatives (Juneja et al.,
1991) of neuraminic acid which have an acyl group on the amino nitrogen (Itoh
et al., 1990).The most widely distributed sialic acid in nature is Neu5Ac. The wide
occurrence of sialic acids in exposed positions of molecules and cells, their strong
electronegative charge and the existence of multiple tissue-specific forms suggests
that they are involved in cellular functions. Furthermore, the enzymatic removal
of sialic acids leads to changes in the biological behaviour of cells and molecules
(Schauer, 1985).There are hopes that sialic acid can provide a base for a new class
of drugs.
Eggs are a natural source of Neu5Ac (Itoh et al., 1990) which is mainly present
in the yolk, yolk membrane and chalaza. The highest concentration was found in
the chalaza (2.4% of dry matter) followed by the yolk membrane (1.8%)and the
yolk (0.2%) Uuneja et al., 1994b). Juneja et aI. (1994b) extracted Neu5Ac from a

16 World’s Poultry Science Journal, Vol. 57, March 2001

 Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
mixture of yolk membrane and chalaza, 800kg raw material yielding 300g
Neu5Ac, and 500 kg yolk (from which the lipid had been removed) yielding 500 g
Neu5Ac. Itoh et al. (1990) extracted 47.3 mg and 174.8mg Neu5Ac from 1OOg
chalaza and yolk membrane, respectively.
Leucocyte (L-), platelet (P-) and endothelial (E-) selectins comprise a family of
transmembrane glycoproteins whose binding to carbohydrates is fundamental to
their function in cell adhesion. Each of the selectins can bind to sialyl lewis X
(Slex), a sialylated and fucosylated tetrasaccharide found as a terminal moiety of
sugar chains on glycoproteins and glycolipids expressed by a variety of cell types
such as neutrophils and monocytes (Nelson et al., 1993).It has been suggested that
sialic acid derivatives such as Slex can be used as anti-inflammatory drugs
because they bind to selectins. The anti-inflammatory properties of Slex have been
demonstrated in clinical trials. Infusion of Slex drastically reduced lung injury in
rats induced by cobra venom and diminished the tissue accumulation of
neutrophils (Mulligan et al., 1993). Han et al. (1995) studied the role of Slex on
tissue injury in rabbit ears caused by a temporary decrease in blood supply.
Groups treated with Slex developed less oedema than saline treated controls. No
tissue death was observed in rabbits treated with Slex either immediately upon
recovery of the blood supply or after a delay of 1 hour. However, delaying Slex
treatment for 4-12 hours resulted in tissue loss. Lin et al. (1995) synthesised a
dimeric Slex glycopeptide enzymatically from sialic acid prepared from egg yolk.
The dimeric Slex was as active as monomeric Slex in inhibiting E-selectin. The
authors suggested that eggs could be a useful source of sialic acid for the
synthesis of Slex.
Sialidase is an enzyme which is found on the surface of the influenza virus and
binds to sialic acid on cell surfaces. The enzyme is essential for the replication of
the virus and allows the release of the virus from infected cells (Itzstein et al.,
1993). An antiviral effect of the sialic acid analogue zanamivir (GG167) was
revealed as it was distributed directly to the respiratory tract in humans (Hayden
et al., 1997). The favourable properties of zanamivir (GG167) are now commer-
cially used in the neuramidase inhibitor Relenza@(http:\ \www.biota.com.au).
Rotavirus can also be inhibited by sialic acid. Koketsu et al. (1995) found
favourable effects after oral administration of a sialyloligosaccharide extracted
from egg yolk (from which the lipid had been removed) to 6-day old mice which
had been inoculated with rotavirus. After 1, 3 and 5 days of treatment the mice
showed a significantly lower incidence of diarrhoea than their control counter-
parts. It was confirmed that sialic acid was the active substance.

CHICKEN ANTIBODIES (IgY)

The application of specific antibodies for the detection, quantification and
isolation of various molecules in food and biological fluids is revolutionising
nutritional, chemical and biological research. At present the major source of
antibodies is from serum from different immunised mammals. However, egg yolk
also contains antibodies, IgY. The benefits of IgY compared with mammalian IgG
- which include its continuous production, cheap maintenance costs of hens, the
lower susceptibility of hens to disease and the lack of any need for bleeding - are
receiving growing attention. The hen’s higher production of antibodies is
demonstrated by the results of Gottstein and Hemmler (1985).Hens and rabbits
were inoculated with Echinococcus granulosus. During a 6-week period a hen
produced 298 mg specific antibodies compared with only 17mg from a rabbit
over the same period.

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Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
IgYs have been produced against a variety of antigens. The long evolutionary
distance between birds and mammals endows IgY with useful properties (Larsson
et al., 1993) such as a high specificity against mammalian antigens and human Fc
receptors. In addition, IgY does not activate human complement factors. The
phylogenetic difference between birds and mammals further facilitates the IgY
response to antigens which have a weak antigenic character in mammals such as
human insulin and RNA polymerases. IgY has also proved to have other useful
properties. It did not bind to protein A or protein G, which are present on the
surface of streptococci and staphylococci (Larsson and Sjoquist, 1988). Likewise,
IgY did not bind to rheumatic factor (Larsson and Sjoquist, 1988).
There is consistent evidence that IgY can alleviate disease. Hatta et al. (1993)
inoculated suckling mice with human rotavirus (HRV) 1, 3, 9 and 24 hours after
oral administration of anti-HRV IgY. No incidence of diarrhoea was observed in
the mice administered with IgY 1 hour before inoculation. In the other three
groups the incidence of diarrhoea was 27.3%, 41.7% and 93.3%, respectively. The
incidence in the control group was 83.3%.Administration of anti-bovine rotavirus
IgY to calves revealed positive effects on growth (Kuroki et al., 1997). The
emergence of E. coli infections in inoculated calves was also successfully
prevented by administration of IgY (Ikemori et al., 1992). Piglets fed with spray
dried IgY as Protimax@,a commercially available feed supplement, had a lower
mortality rate and a reduced occurrence and duration of diarrhoea caused by E .
coli serotype K88 than a control group (Mroz et al., 1999).
Streptococcus mutans serotype c is the major aetiological agent of human dental
caries. Volunteers who rinsed with anti-S. mutans IgY showed a tendency to have
a lower ratio of S mutans to total streptococci in the plaque (Hatta et al., 1997).
Promising results, in terms of failing incidence of lung infection, have been
achieved with anti-pseudomonas IgY treatment of patients suffering from cystic
fibrosis (Kollberg, personal communication, 1997). Because pathogens have little
chance to develop resistance against IgY, it might become a useful supplement to
antibiotics in the future.
Moreover, IgY has been shown to inhibit various bacteria in in vitro
experiments. Sugita-Konishi et al. (1996) found that IgY inhibited the in vitro
growth of Pseudomonas aeruginosa, the production of Staphylococcus aureus
enterotoxin A and the adhesion of Salmonella enteriditis to cultured human
intestinal cells. An additional possible field of application for IgY is as a protective
food agent. Schimzu et al. (1988) studied anti-E. coli IgY and concluded that
bacterial cells were aggregated and in vitro growth was prevented. In vitro
experiments have also shown that IgY can be used to detect cancer cells. When a
specific pathogen-free hen was immunised with an antigen which had been
purified from human stomach cancer cells, IgY could specifically recognise the
gastrointestinal cancer cells but not the normal cells or tissues (Yang et aI.,
1997).

How to improve yolk production

The previous section has shown that the egg yolk contains several biologically
active substances which have great potential in the field of medicine. In this
section factors which influence different yolk traits and consequently the
production of substances with therapeutic properties are considered. The
relationship between yolk production and egg production will only be touched on
briefly.

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 Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
IMPROVING YOLK PRODUCTION BY FEEDING AND MANAGEMENT
The fact that the fatty acid composition of the yolk can be altered by means of
the fat source in the diet is well established. For example, Jiang et al. (1991)
showed that, when hens were fed high oleic acid sunflower seed, full flax seed or
high linoleic acid sunflower seed, the fatty acids present in the diet were
recovered as fatty acids in the yolk. The increase in oleic acid was entirely directed
towards the triglycerides whereas the increase in linoleic and arachidonic acids
after feeding high linoleic acid sunflower seed was evenly distributed. The
increase in linolenic acid after feeding flax seed was mainly in the triglycerides.
Long chain n-3 fatty acids were only deposited in the phospholipids. Scheideler
et al. (1999) observed that the increase in yolk lipids as a response to feeding n-6
fatty acids differed between commercial hybrids, suggesting the response was
under genetic control.
Yolk weight is also affected by the diet. Feeding diets containing flax seed
reduced yolk weight whereas yolk percentage was not significantly affected
(Scheideler et al., 1998). By increasing the fat content in the diet, Whitehead et al.
(1991) found that yolk weights increased when treatment was started at a young
age. The group fed the low fat diet showed a lower increase in yolk weights at
22-32 weeks of age than the group fed on the high fat diet. Yolk weights increased
by 2.83 g and 3.57 g in the two groups, respectively. No response to fat content in
the diet was seen in older hens. The results of ad libitum versus restricted feeding
during rearing on the effect of the dietary fat content on the proportion of yolk
were equivocal (Petersen et al., 1986a, b).
Results from studies investigating the effect of dietary protein on yolk
production are difficult to interpret. Akbar et al. (1983) found that an increased
concentration of dietary protein increased yolk weight and the proportion of yolk
in a group of hens producing large eggs, whereas there was no response in the
groups producing small or medium sized eggs. By increasing the daily lysine
intake from 638mg/hen to 828mg/hen at 23 weeks of age, an increase in yolk
weight and yolk protein was established but there was no response in yolk solids.
Neither a delay in the start of treatment to 42 weeks of age nor a further increase
in lysine intake produced any response in yolk weight, yolk protein or yolk solids
(Prochaska et al., 1996).An increase in the daily methionine intake from 413 mg/
hen to 507mg/hen to 556mg/hen at 29 weeks of age yielded no response in total
solids but yolk crude protein increased (Shafer et al., 1998).
Artificial 1ight:dark cycles are essential for efficient egg production, but the
impact of the lighting programme on yolk production is not obvious. Lakshma-
nan et al. (1992)compared layers kept under a 28 h lighting programme with birds
under a 24h programme. Yolk weights were little affected. The effect of the
1ight:dark cycle during rearing has also been studied. A shorter day increased the
proportion of yolk as a consequence of a decrease in egg weight, whereas no effect
of day length could be established for yolk weight (Petersen et al., 1986a). A
decrease in day length restricted to the period around the start of lay had a less
pronounced effect on the proportion of yolk (Petersen et al., 1986b).
In general, information regarding the effect of nutritional manipulation and
management factors on yolk production seems to be fairly ambiguous.

IMPROVING YOLK PRODUCTION BY BREEDING

If a substantial additive genetic variation exists in a certain yolk trait, breeding
ought to be a viable alternative to alter that trait. Do yolk traits such as yolk weight,
its proportion or chemical composition show additive genetic variation?

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Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
Genetic variation in yolk traits. It is a well established fact that both the
weight of the yolk and its proportion is dependent on bird species (Sotherland
and Rahn, 1987).There is also evidence of variation in yolk traits within species,
of which Gallus domesticus has been well studied. Akbar et al. (1983) showed that
mean yolk weight during the period 24-75 weeks of age was significantlyaffected
by strain. At 30 weeks of age the yolk weight of several native Spanish breeds
ranged from 15.94g to 17.36g (Campo, 1995).Overall mean yolk weights during
the period 26-61 weeks of age in three selection lines were found to be 15.49g,
17.18g and 15.87g, respectively (Ambrosen and Rotenberg, 1981).Yolk weights of
hens aged 52 weeks, with a subsequent increase in yolk weight, were 19.00g and
20.67g in lines selected on the bases of egg number and egg weight, respectively
(Rotenberg and Srarensen, 1978).
Significant differences between strains have also been found for the proportion
of yolk (Marion et al., 1965; Akbar et al., 1983). Marion et al. (1965) studied three
lines selected for egg production traits and found that the proportion of yolk
ranged from 28.54% to 30.75%.In a more recent study with two different hybrids
the proportions of yolk were 26.0% and 28.3% (Scheideler et al., 1999).Sometimes
the proportion of yolk is expressed as the ratio of yolk to albumen. This trait also
shows strain differences (Campo, 1995; Ahn et al., 1997).
The number of reports on strain differences in both yolk weight and yolk
proportion ought to be sufficient evidence that genetic variation exists. There is
less agreement regarding the effect of strain on the concentration of dry matter in
the yolk, which is usually reported to be about 52% (Marion et al., 1965;Ambrosen
and Rotenberg, 1981). However, there are deviations from this value (Ahn et al.,
1997; Scheideler et al., 1999). Significant strain differences in the concentration of
dry matter have been reported (Ambrosen and Rotenberg, 1981; Ahn et al., 19971,
but the opposite has also been found (Leclercq et al., 1985; Scheideler et al.,
1999).
Some comparative studies on the egg composition of different strains of birds
have included the chemical composition of the yolk. As for the concentration of
dry matter, both significant and non-significant strain differences have been
reported for total lipid concentration (Rotenberg and Srarensen, 1978; Ahn et al.,
1997; Scheideler et al., 1998).
At present, yolk phospholipids are probably the substances most used for the
production of alternative non-food egg products. As far as total lipids are
concerned, information on the effects of strain on phospholipid concentration is
unclear with both differences (Ambrosen and Rotenberg, 1981) and no difference
(Marion et al., 1964; Leclercq et al., 1985) having been found. Ambrosen and
Rotenberg (1981) reported that the average phospholipid concentration during
the period 26-61 weeks of age was 10.80g, 11.02 g and 11.11 g/lOOg yolk in three
selection lines.
Yolk cholesterol has also been subject to comparisons between animals with
different genetic backgrounds and, likewise, differences have been established in
some studies (Rotenberg and Smensen, 1978; Ambrosen and Rotenberg, 1981)but
not in others (Leclerq et al., 1985).The concentration of cholesterol in the yolk is
approximately 1200-1300 mg/100 g yolk (Ambrosen and Rotenberg, 1981).
Leclercq et al. (1985) revealed strain differences in fatty acid composition. The
hens of a fat broiler line subjected to ad libitum feeding produced yolks with
higher concentrations of myristic and palmitoleic acids but lower concentrations
of palmitic, stearic, linoleic and arachidonic acids than the hens of the lean
line.

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 Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
Table 1 Heritability estimates for yolk weight, yolk proportion, yolk dry matter concentration
and total yolk dry matter

Yolk dry matter Total yolk

Yolk weight Yolk concentration dry matter
(g) (%) (%) (g)

Hill et al. (1966) 0.39 - 0.52 0.40

Jaff6 (1964) 0.43 0.20 - -
Rodda et al. (1977) 0.50 - 0.50 0.6
Mennicken et al. (1996) 0.57 0.50 0.29 -
Hartmann et al. (2000) 0.22 0.38 - -

With regard to single yolk proteins, few comparisons between strains of birds
have been presented. Rotenberg and S~rensen(1978) and Ambrosen and
Rotenberg (1981) have both, however, reported differences in the total protein
concentration. It is a well established fact that almost all yolk proteins appear in
more than one molecular form, but again little is known about strain differences.
Rao and Mahadevan (1983) studied the lipovitellin composition in two breeds
and found a higher concentration of a-lipovitellin than P-lipovitellin in the yolks
of eggs from White Plymouth Rock hens and vice versa in the yolks of eggs from
White Leghorns. Li et al. (1998)concluded that both the concentration and activity
of y-livetin (i.e. IgY) obtained from yolks of hens of different genetic origin was
the same.
Actual estimates of the additive genetic variation in terms of heritabilities for
different yolk traits have also been presented (Table 1). The size of these
heritability estimates is worth noting, especially the intermediate sized heritabil-
ity of the dry matter concentration in the yolk which is an essential trait in the
context of extraction of biologically active substances from the yolk. Kreuzer et al.
(1995) presented a repeatability of 0.4-0.6 for emulsion stability which suggests
genetic variation in the chemical composition of the yolk. Ambrosen and
Rotenberg (1981)estimated that the heritability of the phospholipid concentration
was between 0.05 and 0.14.

Direct response t o selection. Only a few reports have studied the effect of
selection on different yolk traits, the most comprehensive of which is probably the
one by Miyoshi and Mitsumoto (1980). In this long term divergent selection
experiment a response in the yolkalbumen was obtained after one round of
selection. Hartmann et al. (2000) found that one round of divergent selection was
enough to produce a response in yolk proportion. Furthermore, a sustained effect
of selection was observed over the entire laying period. During the 1970s two
selection experiments on cholesterol concentration were reported (Cunningham et
al., 1974; Marks and Washburn, 1977). The former found an indication of a
separation of the population whereas the latter found that upwards selection but
not downwards selection yielded a response.

Indirect response to selection. As early as 1964 Jaffk was considering what

would happen to the yolk when selecting for larger eggs. This is relevant in the
context of breeding for yolk traits. Comparisons between lines selected for the
size of the egg, either directly or as egg number, have shown that yolk weights

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Biologically active substances in yolk: C . Hartmann and M . Wilhelmson
Table 2 Genetic correlations between egg weight and various yolk traits

Trait Correlation Authors

Yolk weight (g) 0.71 Hill e f al. (1966)

0.54 to 0.62 Rodda ef al. (1977)
0.47 Hartmann et al. (2000)
Yolk 70 -0.28 to -0.10 Rodda et al. (1977)
-0.43 Tawfik et al. (1981)
-0.30 Mennicken et al. (1996)
-0.51 Hartmann et al. (2000)
Yolk dry matter concentration (%) 0.03 Hill et al. (1966)
0.15 to 0.33 Rodda e t a / . (1977)
Total yolk dry matter (g) 0.63 Hill et al. (1966)
0.57 to 0.62 Rodda e f al. (1977)

are higher in large eggs than in small eggs (Marion et al., 1965; Rotenberg and
Serrensen, 1978; Ambrosen and Rotenberg, 1981). Marion et al. (1965) reported
that average yolk size in the large egg selection line was 18.2g whereas in the
low egg weight line it was only 16.4 g. Several authors have presented data
which show a positive genetic correlation between the weight of the yolk and
the egg (Table 2).
However, the relative yolk weight (i.e. the proportion of yolk) is larger in small
eggs. Marion et al. (1965) found that the average proportion of yolk in their low
egg weight line was 30.75% compared with 28.54% in the high egg weight line.
Hartmann et al. (2000) observed a decrease in egg weight when selecting on the
basis of high yolk proportion and vice versa when selecting on the basis of low
yolk proportion. Estimates of the genetic correlation between yolk proportion and
egg weight confirm the negative relationships seen in the phenotypes (Table 2).
Furthermore, it can be seen in Table 3 that there is a positive genetic relationship
between the absolute and relative weights of the yolk. The estimated genetic
correlation between the concentration of dry matter in the yolk and egg weight
was fairly low, whereas the corresponding correlation with total dry matter was
intermediate (Table 2).

Table 3 Genetic correlations between yolk weight and various yolk traits
~~~

Trait Correlation Author

Yolk dry matter concentration (%) 0.11 to 0.38 Rodda et al. (1977)
-0.07 Hill et al. (1966)
0.09 Mennicken et al. (1996)
Total yolk dry matter (g) 0.99 to 1.00 Rodda et al. (1977)
0.28 Hill et al. (1966)
Yolk percentage (%) 0.52 Hartmann et al. (2000)
Yolk/albumen solids ratio 0.56 to 0.63 Rodda et al. (1977)
~~~~~

22 World’s Poultry Science Journal, Vol. 57, March 2001

 Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
What happens to the dry matter content if yolk size is increased? Ambrosen
and Rotenberg (1981) reported phenotypic correlations between yolk weight
and yolk composition. Yolk weight was negatively correlated with total lipid,
phospholipid and cholesterol concentration, whereas the correlations with dry
matter and protein concentration were positive. Genetic correlations between
yolk weight and dry matter concentration indicate a positive value (Table 3).
Rodda et al. (1977) presented positive genetic correlations (0.26-0.49) between
the concentration of dry matter and the total dry matter in the yolk. It has also
been reported that the concentration of dry matter in the yolk is positively
correlated (0.21) with the concentration of dry matter in the albumen (Men-
nicken et al., 1996).
Rotenberg and Ssrensen (1978) and Li et al. (1998) showed that total output of
a certain substance was larger from a large yolk. A yolk from a large egg, which
would coincide with a large yolk, contained more total lipid, phospholipid,
cholesterol and triglyceride per egg than the yolk of a smaller egg. However, two
rounds of selection on egg weight, either directly as egg weight or indirectly as
egg number, yielded no differences in lipid production per hen day (Rotenberg
and Ssrensen, 1978). Li et al. (1998) concluded that a large yolk contained more
IgY than a small yolk.
Efficient egg production includes several more traits than just the weight of
the egg, especially traits such as egg mass and feed conversion. In general, egg
production traits appear to be genetically negatively correlated with yolk
proportion. The genetic correlation between the proportion of yolk and egg
mass was -0.3 (Tawfik et al., 1981; Mennicken et al., 1996) and the corresponding
correlation with egg number was -0.06 (Mennicken et al., 1996). Negative
genetic correlations were also found between yolk weight and egg production
rate and number of eggs per hen housed (Rodda et al., 1977). The yolk dry
matter constitutes about 60% of the total egg dry matter and it could therefore
be expected that there is a relationship between feed intake and traits relating to
yolk deposition. Mennicken et al. (1996) estimated the genetic correlation
between feed intake and the proportion of yolk to be 0.16 and the correlation
between the proportion of yolk and body weight to be 0.21. Anthony et al.
(1989a, b) found that the concentration of dry matter in the yolk was higher in
a high body weight line than in a low body weight line. Selection for high feed
efficiency resulted in hens producing more polyunsaturated fatty acids (PUFA)
and thereby the ratio between the PUFA and the saturated fatty acids increased
(Zaky et al., 1996).

Conclusions and reflections

The egg yolk contains several substances which have great therapeutic potential.
Some are already used in commercial products and many scientific papers on
their properties and applications have been published. There is also evidence that,
by the application of both management and breeding approaches, it is possible to
influence yolk traits such as yolk weight, yolk proportion and yolk composition,
and thus to enhance the yield of these substances.
In general, information concerning the effects of nutritional manipulation and
housing on yolk production is fairly ambiguous. However, the effect of age at the
onset of treatment appears to be considerable. Moreover, there is strong evidence
of the existence of additive genetic variation in several yolk traits, a synergy
which would make breeding a viable alternative to the modification of yolk

World’s Poultry Science Journal, Vol. 57, March 2001 23

Biologically active substances in yolk: C . Hartmann and M . Wilkelmson
production. The feasibility of breeding is further accentuated by properties such
as an accumulation of favourable genes over rounds of selection and conse-
quently a continuous improvement in the trait. A selection response and a
correlated selection response have been obtained for yolk proportion and yolk
weight, respectively. With regard to the chemical composition of yolk, there are
indications of genetic effects but the results are less convincing than for yolk
weight and yolk proportion.
A relevant question to ask in the context of breeding for an increased yield of
a certain substance is “which is the most suitable selection trait?” There are two
likely scenarios which should be kept in mind when answering this question:
whether the eggs and yolks are exclusively produced for extraction of a certain
substance or whether they have a wider field of application. In the first case
selection for a specific substance may be the attractive option. The complexity
of the molecular structure of the egg yolk, however, renders chemical analysis
difficult. It makes the analysis both tedious and costly and may end up in a too
random variation. In addition, the demands of the chicken embryo for a
balanced nutritional yolk composition might restrict selection response. Another
option would be to select for the concentration of dry matter in the yolk.
However, it would seem that the concentration of dry matter in the yolk is
positively correlated with yolk weight (Table 31, i.e. selection for yolk weight is
likely to be followed by an increased concentration of dry matter as well as dry
matter yield. This correlated increase in yolk dry matter yield has given rise to
the suggestion of using yolk weight as a selection trait (Hill et al., 1966; Rodda
et al., 1977). Use of yolk weight or yolk proportion as selection traits would not
only increase the yield of a certain substance but also of the yolk in general.
Furthermore, the higher dry matter content of these eggs would make them
desirable to the food processing industry. It is likely that selection for an
increased yolk yield would affect egg production, but in this review we chose
to confine ourselves to yolk traits. In any breeding programme, however, the
effect of selection for an increased yolk yield on egg production needs to be
considered.
The uncertainty about the effect of different management factors should not
entirely rule out the possibilities of using these to increase yolk yield. Scheideler
et al. (1998) found strain X diet interactions in yolk lipid deposition. Thus, the
ambiguity of results from studies of the effects of management on yolk traits
might to some extent be caused by genotype X environment interactions,
interactions which Scheideler e f al. (1998) suggest should be utilised in the
construction of a modified fat egg. Perhaps similar interactions, used in
constructing eggs with required compositions, exist for other yolk
components.
Thus, the egg yolk contains biologically active substances of commercial
interest. Feeding, management as well as breeding can be used to alter yolk
production. Breeding is an appealing alternative because of the presence of
additive genetic variation in both yolk weight and yolk proportion and,
furthermore, selection would yield a continuous improvement in the selected trait
over successive rounds of selection. The effects of feeding and management are
more limited to a particular period of time. If breeding is to be used to increase
yolk yield, the optimal design of the breeding programme must be decided for
each specific case and, if possible, breeding should be combined with a
management or feeding system which allows the full genetic potential to be
expressed.

24 World’s Poultry Science Journal, Vol. 57, March 2001

 Biologically active substances in yolk: C. Hartmann and M . Wilhelmson
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28 Worlds Poultry Science Journal, Vol. 57, March 2001