APPLICATION OF NATURAL PIGMENTS IN POULTRY FEED FOR BETTER EGG YOLKS PIGMENTATION
Humans and other animals have used eggs as a food source for millennia. Eggs are an important and well-balanced source of essential nutrients. Bird eggs (for example, chicken eggs) consist of a protective eggshell, albumen (egg white), and vitellus (egg yolk), contained within various thin membranes.
Egg yolks and whole eggs are a good source of protein and choline. For this reason, the United States Department of Agriculture (USDA) categorises eggs as Meat. Chicken eggs that are produced commercially for eating have not been fertilised by a rooster and, therefore, cannot develop into embryo and finally a chick. It is possible to enrich table eggs with minerals (e.g. iron and iodine), antioxidants (e.g. selenium, vitamin E) or omega-3 fatty acids and vitamins, by adding these as components of the hen diet. There are some enriched eggs available in Australia but this represents a niche market for eggs, as only a small percentage of consumers purchase such eggs.
Chicken Egg biology
Nature’s role for the egg is as an incubation chamber for the developing chick. For an egg to develop into a chick, it should have been fertilised by a rooster (also called a cockerel). Once the fertilised egg is laid, it must be kept warm and this may be done either naturally by the hen sitting on the egg, or artificially by placing the egg into an incubator.
The egg contains all the nutrients that the developing chick needs during the three weeks of incubation period and also for the first couple of days after hatch. Water vapour and gases such as oxygen and carbon dioxide are able to move across the egg shell through small openings called pores. The developing chick starts off as a single fertilised cell on the surface of the yolk and progressively grows, using up the yolk, some of the albumen and some calcium from the inside of the egg shell. In the final stages of development, the chick takes up the last bits of yolk into its own digestive system.
Eggs vary in colour
The production of the bird egg consists of a series of steps that occur as the egg enters and passes along the hen’s reproductive tract (oviduct). The yolk of the egg enters the top of the oviduct and passes into the infundibulum where it spends about 15 minutes. A membrane is added around the yolk and, if the hen has been inseminated, fertilisation occurs in this section of the oviduct.
The yolk then spends about three hours in the magnum where the egg white is formed and then one hour in the isthmus where the shell membrane is laid down. The main part of the egg shell is formed in the tubular shell gland and the shell gland pouch which takes about 20 hours. The egg shell is sometimes referred to as a bio-ceramic because it is made up of calcium carbonate with an organic matrix running through it.
Composition of the chicken egg
The physical composition on the avian egg can be seen in Table 1, below. The egg is comprised of 32-35% yolk, 52-58% albumen and 9-14% shell. Composition of the albumen and yolk are further presented in the image gallery above (Cross-sectional diagram of an avian egg).
| Egg components | ||
| Nutrient | Albumen | Yolk |
| Protein | 9.7 – 10.6 | 15.7 – 16.6 |
| Lipid | 0.03 | 31.8 – 35.5 |
| Carbohydrate | 0.4 – 0.9 | 0.2 – 1.0 |
| Water | 84.3 – 88.8 | 48 |
| Element | ||
| Sulphur | 0.195 | 0.016 |
| Potassium | 0.145 – 0.167 | 0.112 – 0.360 |
| Sodium | 0.161 – 0.169 | 0.070 – 0.093 |
| Phosphorus | 0.018 | 0.543 – 0.980 |
| Calcium | 0.008 – 0.02 | 0.121 – 0.262 |
| Magnesium | 0.009 | 0.032 – 0.128 |
| Iron | 0.0009 | 0.0053 – 0.011 |
Adapted from Li-Chan, ECY, Powrie, WD and Nakai, S (1995) The chemistry of eggs and egg products, In Egg Science and Technology, WJ Stadelman and OJ Cotterill (Eds), The Haworth Press Inc, New York.
Yolk
A raw egg yolk provides the lipids and proteins that are required for embryonic growth. The yolk is comprised of 33% lipid, 17% protein, and small amounts of minerals, vitamins and carbohydrates. The lipid composition of the egg yolk consists of various lipids and fatty acids, of which the triglycerides
represent more than two third of the total lipid content (Table 2). Yolk pigmentation is mainly a result from the direct transfer of pigments from feed and/or feed additives, but there is also some endogenous pigment used to colour the yolk. It has been speculated that this endogenous pigment is from body tissues, but the mechanism of storage or release of the pigment is not fully understood.
Table 2. Lipid composition of the egg yolk
| Lipid Composition | Percentage |
| Triglycerides | 63 |
| Phospholipids | 31 |
| Cholesterol | 4 |
| Other (including fat soluble vitamins) | 2 |
| Fatty Acid Composition | Percentage |
| Saturated fatty acids | 35 – 45 |
| Unsaturated fatty acids | 55 – 65 |
| Monounsaturated fatty acids | 35 – 50 |
| Linoleic acid | 10 – 20 |
| Polyunsaturated fatty acids | 3 – 5 |
Adapted from Larbier, M and Leclercq, B (1994) Nutrition and feeding of poultry, Nottingham University Press, Nottingham.
A raw egg yolk
Egg Yolk Colour
Eggs are one of the most popular foods in the market. Everyone, rich or poor, partakes in the consumption of the superfood. The smell of egg might be off-putting to some, but anyone who has eaten it can tell you eggs-actly how delicious it is. There are so many ways to prepare an egg, so there is a recipe to fulfil everyone’s eggs-pectations. The key source of nutrition in an egg is the yolk, which contains most of the fats, calories, vitamins and minerals. We usually associate the yolk of an egg as being deep orange. Indians would know that more often than not, this is not the case. Most eggs we buy in the market are light orange or pale yellow.
In Australia, people like a yolk that is bright yellow-orange. Europeans like their egg yolks even darker whereas, in North America, paler yolks are the norm. Fifty years ago, most people had a few chickens in the backyard, even if they lived in a city. These chickens would often be fed only on table scraps and what they could scavenge in the back yard. They would eat many types of plants which are a source of a group of pigments known as carotenoids. These get laid down in the egg yolk and give it a bright yellow-orange colour.
As far as is known, these pigments do not contribute to the nutritional value of the egg. This is because the pigments (carotenoids) are the precursor of vitamin A, however, in the human body, they are not converted to vitamin A. When egg production became more intensive and laying hens were fed a complete diet, egg producers added carotenoids to the poultry feed to produce the bright-coloured yolks that people were accustomed to from the back-yard hen days. In the commercial egg industry in Australia, most egg producers use a synthetic form of carotenoid. It is possible to obtain natural pigments, which are produced from plant products such as marigold flower petals or red capsicums, but these are more expensive than the synthetic pigments. The grains fed to chickens contain some carotenoids particularly from corn (maize) which is commonly used in poultry diets in North America. In commercial free-range production, it is usual to add carotenoids to the feed also.
Albumen
The albumen consists chiefly of proteins, including ovalbumin, ovotransferrin, ovomucoid, ovoglobulin, lysozyme and ovomucin (See Table 3). The various roles of these proteins include inhibition of protein break down and maintenance of the viscosity of the thick albumen. The function of the albumen is to protect the embryo (or yolk) from attack by microorganisms and also to serve as a source of water, protein and minerals for the embryo.
There are four distinct layers of albumen that have surrounded the yolk by the time the egg is laid. The innermost layer is known as the chalaziferous layer (attached to the yolk) comprising 2.7% of the total albumen, followed by the inner thin layer (16% total albumen), the middle thick layer (50% total albumen) and the outer thin layer (25% total albumen). The majority of the modifications to the albumen occurs during the first 6-8 hours (2-3 hours in modern layers), after which the developing egg enters the shell gland and undergoes a process called ‘plumping’. During this process, a uterine secretion, which is mostly water with some minerals such as sodium, calcium and potassium, is pumped into the egg.
The color of the egg yolk
As in other foods, the egg color is one of the most important aspects for consumers, especially the color of the yolk. It is an important organoleptic aspect for the acceptance of this product that the consumer relates to its quality. Both the color of the yolk and its homogeneity (within the yolk and between different eggs) are important.
The color of the egg yolk is determined by the type and profile of carotenoids present in the feed and their intestinal absorption. Carotenoids are a fat-soluble group of yellow, red and orange pigments. They can be divided into two large groups: carotenes and xanthophylls.
More than 600 different types of carotenoids have been described. Xanthophylls are more important in egg coloration than carotenes.
Egg quality has different meanings, according to the perspectives of egg producers, consumers and processors. Most of the consumers are interested in shelf life, external appearance, and sensorial qualities, such as eggshell and yolk color (Faitarone et al. 2016). Usually, yellow corn is used in laying hens diets as the essential grain providing energy, which has xanthophyll particularly of lutein and zeaxanthin; however, this amount of xanthophyll is not enough to provide yolk color and meet the demands of market and it does not enhance the red pigmentation of yolk properly (Rowghani et al. 2006; Faitarone et al. 2016). The contents of egg components can be changed by adding specific ingredients in order to enrich the quality of yolk. Egg yolk color is known to be influenced mostly by the diet of the hens because birds are not able to synthesize pigments of yolk, but they can store the pigments obtained from the diet (Englmaierova et al. 2014). It is the result of the deposition and colouring capacity of oxycarotenoids, called xan thophylls, in the egg yolk. Therefore, the color and intensity of yolk depend on xanthophyll and its bioavailability in diet, metabolism and deposition of carotenoids in the target tissue (Breithaupt, 2007; Moeini et al. 2013; Englmaierova et al. 2014). Some of the xanthophylls such as lutein and zeaxanthin are present in common feedstuffs such corn, grains of dried distillers or alfalfa (Santos-Bocanegra et al. 2004). Egg yolk color is considered as a quality indicator and plays an important role in egg acceptance by the consumers (Faitarone et al. 2016). The preferable color intensity of yolk is different among countries. From the viewpoint of consumers, darker yolks are demanded (Englmaierova et al. 2014). In Iran, the orange color is more desirable (above index 10 on Roche yolk color fan, (RYCF)), which has the higher price and is famous as golden yolk egg at the market. Hence, most of the poultry producers in this country use different kinds of synthetic pigments in laying hens diet to produce orange yolk color. Changes in yolk color can be observed when supplemental pigments (synthetic or natural) sources are added to the diet. The influence and deposition rate of synthetic pigments including Apo-8-ester are several times stronger than natural pigments for production of orange yolk color (Roche Vitamins and Fine Chemicals, 1988; Nys, 2000). However, excessive use of the permissible value of some synthetic pigments, such as canthaxanthin in poultry diets, may result in the formation of crystals in the retina of the human eye. Therefore, synthetic canthaxanthin is categorized as a potentially hazardous substance for human health in European Union (Breithaupt, 2007). Nowadays, due to more attention to the importance of feed safety and public concerns for utilization of synthetic additives, there is a demand for discovering new plant extracts for substituting natural carotenoid, delay lipid oxidation and reducing yolk cholesterol content in eggs. Beneficial activity of such natural extracted sources, like the marigold flower (Tagetes erectus), is related to the content of various secondary metabolites such as polyphenols, carotenoids, xanthophyll, triterpenes and essential oils (Ariana et al. 2011; Lokaewmanee et al. 2011). One of the most important indexes of egg quality is the stability of egg yolk lipids against oxidation during the storage period. This is because lipid oxidation affects food quality, aroma, taste, and nutritional value, in addition to producing toxic compounds (Faitarone et al. 2016). Malondialdehyde (MDA) is a major degradation product of lipid hydroperoxides, which can be used as a marker for assessing the extent of lipid peroxidation. This component has attracted much concern since its mutagenic and carcinogenic property is shown (Botsoglou et al. 1994). A simple and fast method used to assess lipid oxidation in food is the thiobarbituric acid (TBA) test. This test meas ures the level of MDA, which is a secondary oxidation product. The reaction of MDA with 2-thiobarbituric acid, forming a coloured complex, which is measurable by spectrophotometrically at λ= 532 nm. Results are expressed as mg MDA per kg sample or frequently named as “TBA value” (Botsoglou et al. 1994; Osawa et al. 2005). It has been indicated that in addition to producing the optimal egg yolk color natural xanthophyll (such as lutein and zeaxanthin) in laying hens diet, has antioxidant properties.
Illustration 2. Egg yolks with different color shades.
- What does the color of the yolk depend on?
The color of the egg yolk depends on numerous factors that can be divided into primary factors, those that depend on the type and concentration of carotenoids, and secondary factors, those that depend on the animal.
Illustration 3. Astaxanthin, red pigment used in animal feed.
Primary factors include digestibility, metabolism, transference to the egg and deposition percentage of carotenoids administered with the diet. Food disbalances, such as vitamin deficiencies, can also affect the color of the yolk. Some of these carotenoids are precursors of vitamin A, metabolizing to this vitamin when there are deficiencies and reducing the quantity that is deposited in the yolk.
The composition of the diet affects the absorption of these pigments, for example, diets rich in fats favor their absorption. The percentage of deposition also shows great variations between the different carotenoids, from 14% fir astaxanthin to 40% for canthaxanthin.
Secondary factors include age, lineage, health status, as well as animal management. Studies show that all those factors that affect digestive health such as mycotoxins, aflatoxins or ochratoxins have a noticeable impact on the absorption of pigments and diseases, such as coccidiosis or the Newcastle disease.
Maintaining the intestinal health of the animals, for example, by using intestinal conditioner pronutrients, is key for an adequate and maintained pigment absorption throughout the laying phase.
Intestinal conditioner pronutrients are active molecules of botanical origin that act at a metagenetic level on intestinal cells. They stimulate the synthesis of functional proteins and increase the renewal of enterocytes, thus favoring the intestinal physiological status and nutrient absorption, including pigments present in feed.
- How to get the desired coloration by the consumer
A key point that egg producers should keep in mind is that there is no coloring capable of meeting the needs of all markets. There is a wide variation in the colors demanded by different countries and regions, ranging from pale yellow, for example, in Switzerland or Canada, to an intense reddish yellow, as demanded, for example, in the Japanese market.
Such preferences are usually determined by geographical and cultural differences. A factor that also influences consumer preferences for one or another color is the availability of raw materials to feed of hens. For example, in Tanzania, sorghum is used in feed and, since it contains a lower proportion of carotenoids than maize, yolks of a lighter color are preferred.
In order to evaluate the color of the egg yolk, a wide variety of scales have developed in recent decades. However, the scale of La Roche, created by the La Roche Vitamins laboratory, is the most accepted one. This scale relates a given color of yolk to a numerical value on a scale of 1-15, from lowest to highest color intensity.
Illustration 4. La Roche scale for the determination of the color of the egg yolk.
The preferences between the different European countries can be classified following this scale. In Germany, the Netherlands, Spain and Belgium, consumers prefer more orange colors, with values between 13-14 on the scale of La Roche, while countries such as Ireland, Sweden or the north of England prefer paler colors, with values between 8-9. There are also countries looking for intermediate colors such as northern France, the South of England and Finland, who demand colors with values between 11-12 in the La Roche scale.
Table 1. Yolk color preferences in European countries according to the La Roche scale.
Certain raw materials present in poultry diets contain natural pigments, such as maize or lucerne, although they are not in a quantity enough to obtain the color demanded by most countries. This is why concentrated extracts from certain plants such as marigold and paprika are used. The use of pronutrients also contributes to the absorption of the pigments in these raw materials.
To get the desired yolk color in each country, it is necessary to add pigments to the diet of birds. As each country, and region has different preferences in terms of yolk color, the dose of pigments should be adjusted for each individual case.
- Types of pigments
These pigments can have a natural origin, such as concentrated extracts from certain plants such as marigold or paprika, or a synthetic one. The current trend is to standardize the doses of natural pigment needed for different yolk colors.
There are 6 main types of carotenoids, three that provide yellow color: zeaxanthin, lutein and apo-ester, and other three that provide red color: canthaxanthin, astaxanthin and capsanthin. Different combinations of these pigments allow to obtain different yolk colors, for example, the combination of 2- 4 mg/kg canthaxanthin and 10-20 mg/kg of zeaxanthin allows to obtain eggs with a yolk color between 12 and 15. We must keep in mind that, for values greater than 10 on the La Roche scale, it is necessary to add a red pigment.
Carotenoids are natural compounds present in animals and plants; with yellow, orange and red typically associated with birds. It is well known that birds are not able to synthesise carotenoids, and thus these essential compounds must come from their diet. Carotenoids are mainly found in the egg yolk, the skin and fat, the liver, and the feathers. They are essential not only for pigmentation, but also for immunomodulation as antioxidants, evidenced by the high disappearance rate of carotenoids from the blood stream during immune stress periods, and reduced pigmentation throughout the body.
Carotenoids were first discovered in carrots, from which in 1831 a compound that was to be named ‘beta-carotene’ was isolated. Since then, we have identified in the carotenoids family more than 600 molecules. The differences in carotenoid profiles in the various tissues are determined by the processes of absorption and transportation, and by the metabolic capacity to modify dietary carotenoids.
Colour is one of the most important factors affecting consumer choices through sensory evaluation of foods. In most countries, golden yolks are preferred because good colour has been traditionally associated with high health. Also, eggs used in recipes can influence the colour of pasta, bakery products and sauces. In addition, although consumer preference for broiler skin colour varies according to culture and region, here also, good health is generally associated with golden skin colour.
Did you know that more than 90 % of the sensory perception of humans is realised by the eye? In other species also, visual stimuli are important. This particularly applies to birds and may have to do with the fact that here other stimuli are less well developed. Contrary to mammals, smell and taste stimuli play a minor role. Not without reason, nature thus probably saw to it that only male birds have magnificent feathers and a loudly coloured comb and wattle. The colourful plumage and shiny red comb of cocks can act as a deterrent to possible rivals when trying to win the favour of hens and, at the same time, should attract the attention of the other sex. Here colours serve for reproduction in the wider sense. In this context, colours are of subordinate importance in humans. When choosing food, however, colours and appearance play an important role. These two parameters are relevant in judging the quality of food. Only a small number of further processed products is produced without colourants which demonstrates how important colour is for humans. For marketing of poultry products, appearance and colour have a central role in judging quality. Consumers rate the yolk of an egg as inferior if the dose of oxycarotenoids is insufficient. In regions where maize is traditionally grown, chicks with white skin are rarely marketed and when buying young hens the yellow colour of legs and beak are a quality criterion. In the following, the factors which influence the pigmentation of egg yolk and skin should be considered. Oxycarotenoids are responsible for the pigmentation of egg yolk and skin as well as of legs, beak, comb and feathers and as poultry cannot produce these substances, they must be added via the feed.
Different carotenoids
The most important carotenoids in modern poultry production include lutein, zeaxanthin, and canthaxanthin. Less frequent ones include citranaxanthin (only in eggs), apo-carotene-ester, and the infrequently used capsanthin. Under commercial conditions, carotenoids are provided by the ingredients mentioned in Table 1: yellow corn and derivates such as gluten and distillers grains; alfalfa and concentrate; nature-identical or nature-extracted carotenoids (flowers like marigold/tagetes or paprika). Yellow carotenoids mainly originate in vegetable sources. For example, lutein and zeaxanthin are found in corn and its derivatives. Lutein is the main carotenoid in alfalfa and tagetes. Paprika extracts provide red carotenoids, mainly capsanthin and capsorubin, but they also contain lutein and zeaxanthin. Apo–ester, canthaxanthin, and citranaxanthin are commercially available sources of nature-identical carotenoids. Today, due to its high pigmentation efficacy compared to citranaxanthin, canthaxanthin is the preferred red xanthophyll in poultry production.
Oxycarotenoid source
In nature, oxycarotenoids are found in many raw materials including various components of poultry feed. Choice of raw materials used in poultry feed is therefore a major influencing factor in pigmentation. Maize, wheat and barley which are major feed components can cause significant variations in egg yolk pigmentation (see Table 1). The visual colour assessment was made using a Hoffmann La Roche yolk fan. The maizecontaining feed led to a fan value of 10, whereas wheat and barley showed extremely pale yolks with a fan value of 4. The reason for these differences, of course, is due to the different oxycarotenoid contents in the raw materials used.
Oxycarotenoid content of components
Table 2 provides an overview of the most important feed components that affect pigmentation. The most important oxycarotenoid source for layer and broiler feeds is maize together with its by-products. Especially high fan values can be found in the high-protein maize by-product, which contain a high percentage of the outer part of the maize kernel. Furthermore, alfalfa and grass meal are also used. It is known from the literature and practical experience that certain sources of peas and rape may supply colourants. Because of problems relating to egg taint, rape should not be used in brown layers. Very often in practice, when optimising feed, “standard table values” are used and this is also the case for pigments. In reality, fluctuations may occur which may be due to difference in strains, different growing conditions, harvesting methods, harvesting times, storage conditions, time spent in storage, etc. When there are high levels of materials in the feed formula the real oxycarotenoid content should be analysed and, if necessary, changed in the matrix.
Influence of storage on the colourant contents
Oxycarotenoids are fat-related substances and therefore very sensitive to oxidation. This means that a certain loss of activity is unavoidable in the course of storage. Figure 1 shows this with the example of an alfalfa concentrate. Directly after drying, the xanthophyll content is approx. 440 mg/kg. After 2 months of storage the value has already reduced by 23 % to 340 mg/kg. After 4 months of storage the loss of pigments amounted to 30 %. Compared to the values analysed in the fresh material, a reduction of more than 50 % may be expected for maize, maize by-products, alfalfa and grass meal when the storage period is up to 12 months. These losses in activity must be considered when formulating feed in order to avoid complaints – especially self-evident in the case of broiler skin pigmentation.
Heat treatment
Many feed additives show reduced activity due to the influence of heat during the feed production process (e.g. hot steam pelleting, expanding). Oxycarotenoids are no exception. Thus, in case of doubt, the contents should be analysed.
Supplementation of colourants
So far only raw materials have been mentioned as sources of pigments. However, egg yolk colour demanded by the consumer cannot be achieved with the usual components because this depends on the colouring effect of the oxycarotenoids included in the raw materials. All the raw materials mentioned up to now contain mainly the pure yellow-colouring lutein. Maize additionally contains the orange-colouring zeaxanthin. Even with high inclusion rates in the feed the Roche fan value cannot be raised over 10. For a more intensive yolk colour, therefore, colourants must be added. Similar circumstances apply to skin pigmentation. The high oxycarotenoid level required in the feed necessary to reach a sufficient degree of yellowness in the epidermis cannot be achieved with the usual feed components. Yellow colourants, and in some areas, red colourants must be added. The following table shows the usual colouring additives (Table 3). If the yellow degree of egg yolk or skin needs to be intensified, the synthetic apo-ester or standardised marigold products may alternatively be used. For the intensification of the red colour, two synthetic products – can thaxanthin and citranaxanthin – are available. Standardised paprika products may be a natural alternative. With regard to the pigment content, all colourants should be standardised with a fixed value. Because of the presence of added antioxidants, pigment activity can normally be guaranteed for one year. As the inclusion rate of pigments in feed is low, a homogeneous mixing with all the other feed components is extremely important for good colour efficiency. It is also fundamental, for marigold and paprika products, to choose the correct carrier to ensure stability and optimum mixing in the feed.
Saponification
The biological efficiency of pigments from vegetable sources depends on whether they are available in esterified or in free form. In nature, pigments in many plants are almost solely attached to fatty acids as the ester. Pigment molecule and fatty acid molecules have to be split to enable them to pass through the intestinal wall in poultry. This happens by means of saponification which more or less takes place in the intestinal tract of poultry. Many efficiency trials, however, have shown that the use of presaponified pigments is advantageous for pigmentation. Figure 2 illustrates this with an example of paprika. Hamilton (1990) investigated the deposition of esterified and saponified paprika pigments by measuring the red colour of the egg yolk photometrically. Saponification seems to accelerate the deposition of pigments. This is indicated by the steeper slope of the red curve plotted for the saponified paprika product. Saponification achieves a stronger shade of red. This is due to improved resorption of the paprika products released through saponification and is confirmed by analysis of the yolk pigment content. Twice as much of the saponified paprika product was stored as the esterified product (16 % versus 8 %). Similar results are also valid for marigold pigments. At the same inclusion level related to pigment activity, a marigold colourant attached to fatty acids certainly gives a less intensive skin pigmentation in broilers than a saponified product. This means that, besides the absolute pigment value, the degree of saponification is also important for choosing natural pigment supplements.
Effect of incorrect feed mixtures on pigmentation
With regard to pigment supplementation, the possibility of incorrect mixtures cannot be completely excluded. Their effects can be demonstrated using the example of yolk pigmentation. Although a mature hen lays one egg almost every day, the egg, and in particular the yolk, takes 15 to 21 days to form. The yolk mass grows by having new layers laid around it. These layers can be clearly seen in hard boiled eggs in the form of so-called concentric rings. Due to this process, any change in the feed’s pigment content and therefore any incorrect blend can be ascertained very quickly. Hatzipanagiotou and Hartfiel (1984) demonstrated this method in a trial with laying hens Note the upper part first. After a depletion period the birds were given a feed supplemented with 20 ppm of synthetic red pigment. Eggs were collected over a ten day period, the first egg at the start of the pigment supplementation of the feed. All eggs were then boiled and the colour of the yolk judged visually. A slight reddening of the yolk can clearly be seen as early as the second day after the addition of pigment, present as a narrow outer layer. From day 9 onwards, the yolk appears to have taken on a uniform colouring. The withdrawal of colour components corresponds to the accumulation of oxycarotenoids in the yolk mass. When omitting the red pigment from the feed, the yolk appears pale on the outer layers after a few days. As described above, it is possible to identify an incorrect blend in the case of egg yolk pigmentation within a few days. If the feed is changed immediately the loss might be limited to 6 to 8 eggs per hen. An incorrect blend cannot be recognised so quickly in the case of skin pigmentation although leg colour may be a hint for colour deviations. With regard to the skin colour of broilers, mistakes due to incorrect blends cannot usually be completely rectified during the course of the fattening period.
Feed intake
Feed intake and thus oxycarotenoid intake have an important influence on pigmentation. The energy content of the ration plays a significant role, particularly in the laying hen. An increased energy content in the feed normally leads to a decreased feed intake and in such cases all relevant nutrients and active substances (including colourants) must be adjusted. During higher ambient temperatures (e.g. in summer time) a decreased feed intake may also occur. Here corres ponding steps – as mentioned above – can be used to counteract the situation. A precondition for optimum feed intake is a good feed structure. Particularly high amounts of fine particles lead to depression of intake in laying hens. The proportion of particles under 0.5 mm should amount to less than 19 %. If the pigment supplement is among these fine particles, which the hen tends not to eat, this must lead to problems with pigmentation. Attention must be paid to a good pellet quality in fattening poultry so that the animals take in enough feed. As already mentioned in the introduction smell and taste play a minor role in poultry. However, under certain conditions these reactions can also be seen in poultry. e.g. with the use of bitter constituents such as medication or the use of acids. The higher the acidity, the higher is the negative influence on feed and hence water intake. A decreased feed intake can partially be seen at 0.5 to 1.0 % acid use. This particularly applies to fumaric acid and their acetates (decreasing acid-effect: fumaric acid>formic acid>acetic acid>propionic acid). Rancid fats may also lead to reduced feed intake. As is well known, feed and water intake are closely related. A restricted water intake normally leads to a restricted feed intake. In the case of decreased feed intake, the watering system should always be checked (e.g. hydraulic pressure). Furthermore mycotoxins (mainly vomitoxin) as well as amino acids (tryptophan) and amino acid imbalances may influence feed intake.
Additional wheat feeding
In recent years many broiler producers use whole-wheat kernels which they provide together with supplementary broiler feed (a kind of a concentrate) in the later part of the fattening period. A basic precondition for successful “wheat feeding” is homogenous mixing with the concentrate feed. Furthermore, it is important that no separation occurs in the feeding system on the farm. To achieve optimum skin pigmentation, it must be ensured that the pigment quantity taken in with the feed is the same as that from complete feeding. The wheat, therefore, should only be administered together with supplementary feed. Colourants, as well as other nutrients and active sub stances (e.g. coccidiostats), must be added at the correct level based on the quantity of supplemental wheat. But also in case of an optimum composition of the supplementary feed non-uniform results in skin pigmentation – but also in live weight and with regard to fat deposition in the carcass – may occur. The reason: Chickens are able to eat selectively and in particular they prefer wheat grains. If they have the possibility to choose they will eat whole wheat rather than pelleted supplementary feed. This may cause problems, particularly if there is not enough trough area available, because the first birds will increasingly take in wheat leaving the leftovers for the following chicks.
Fat and fat quality
The resorption of fat-soluble oxycarotenoids is influenced by the fat included in the feed. Soybean oil and lard increase the oxycarotenoid deposition in the egg linearly up to a dose of 5 %. Using for example 6 % soybean oil in the feed, the citranaxanthin dosage can be decreased from 6 ppm to 4 ppm compared with the control without oil supplementation, without any change in the egg yolk pigmentation. The use of long chain, polyunsaturated fatty acids has a positive effect on the oxycarotenoid deposition, however, the use of long chain saturated fatty acids should be avoided. The contradictory results found in the literature with the use of long-chain polyunsaturated fatty acids have to do with the fact that these fatty acids have a considerably higher tendency to oxidation. In the intestinal tract oxidised fatty acids react with the oxycarotenoids and destroy them resulting in less colourants being accumulated in the yolk and skin. The relation between fat quality and deposition of colourants was demonstrated by Oertel and Hartfiel (1981; Figure 4). The peroxide value in the feed increased over a 77-day storage period especially with soybean oil and lard, but less so with tallow. The canthaxanthin content of the egg yolk took a contrary course – shown here as a change in comparison to the initial value. In the fist two weeks soybean oil causes a rapid and considerable colourant increase in the egg yolk. Afterwards the pigment content in the egg yolk decreases continuously – particularly with use of soybean oil. Thus the fat quality in poultry feed should be regarded as especially important. Oxidised fats may considerably reduce the deposition of colourants and as both fat oxidation and temperature have a negative effect it is sensible to exercise caution during the summer months.
Antioxidants
Because of fat oxidation, antioxidants must be taken into consideration. Oxidation processes can be retarded or stopped using antioxidants and hence fat quality maintained for a longer time. Figure 5 shows the results of a stability test with the aforementioned alfalfa concentrate. A non-treated control was investigated with two further treatments, ethoxyquin and Loxidan (an antioxidant mixture) supplemented directly during the production. In contrast to the negative control, with a pigment loss of 30 % within 4 months of storage, the decrease with the ethoxiquin stabilisation was 14 %, and in case of Loxidan treatment only 10 %. A trial by Harms, who administered ethoxyquin to laying hens via the drinking water, shows that ethoxyquin develops its protective effect mainly in the intestinal tract (Table 4). Feed intake was unchanged by treatments and yolk colour spectrum – judged by the wave length – was identical. Ethoxyquin given via the drinking water led to a significantly more intense yolk colour compared to untreated water. Synthetically produced antioxidants react with the decomposition products of the fat oxidation in the intestine and thus also protect other feed additives which are endangered by fat oxidation. Besides oxycarotenoids antioxidants can also protect vitamin E against oxidation. Because of the acetate form vitamin E feed supplements are protected against oxidation during feed storage. However, in the intestinal tract vitamin E is split into free tocopherol and acetate. If oxidised fats are present in the intestinal tract any free tocopherol can act as an antioxidant and can be used up. As a result, less tocopherol is absorbed through the intestinal wall and its important function, as a biological antioxidant in the organism, is reduced. In extreme cases vitamin E deficiency symptoms may occur. In addition the negative effects regarding stability of poultry and poultry products during storage must be considered.
Vitamin E as a natural antioxidant
As already mentioned with regard to fat quality, tocopherols also act as antioxidants and their supplementation in the feed therefore has a positive effect on pigmentation. Tocopherols may protect the oxycarotenoids in the intestinal tract against oxidation but with the result that less tocopherols are absorbed as a biological antioxidant.
Calcium
The calcium content in feed is repeatedly mentioned in the literature in connection with egg yolk pigmentation. High calcium levels negatively influence yolk colour. If the calcium content is raised from 2.5 % to 3.5 % in layer feed it is necessary to include 1.7 ppm citraxanthin instead of 1.0 ppm to achieve the same yolk pigmentation. In a further trial an increase of the calcium content from 3 % to 4 % led to a decreased yolk colour of one on the Roche fan scale. There are also references in the literature to a reduction of feed intake with an increased calcium content in the ration which will result in a reduced oxycarotenoid intake. Thus the calcium content in the feed should not be adjusted higher than is necessary.
Vitamin A
We return to the vitamins. In high doses, vitamin A disturbs the absorption of oxycarotenoids because both compete for the same transport mechanism. Several years ago the feed industry used this fact to include high amounts of vitamin A (up to 100,000 IU/kg) in broiler feed. A white carcass could be produced even though high amounts of maize were used – at that time maize was very cheap. Legislation was introduced to limit vitamin A during the fattening because of the possible risk of excessive vitamin A contents in foodstuffs of animal origin (e.g. liver, liver products). Incorrect mixtures with high vitamin A contents – or also massive doses of vitamins administered via the drinking water – can be a reason for insufficient pigmentation of skin and yolk. A 3-fold overdose – 36,000 IU instead of 12,000 IU vitamin A/kg feed – leads to a decreased colourant concentration in the tissue of toes and blood plasma of almost 50 %. The content of colourant in the liver decreases by about 30 %, accompanied by a significant increase of the vitamin A concentration.
Growth promoters
There are different findings concerning the influence of growth promoters on pigmentation. Positive effects can be seen in some trials, whereas other trials show no difference. Some authors assume, that performance promoters may improve the absorption of pigments by exerting a positive effect on intestinal health especially in situations of subclinical diseases of the intestine.
Feed ingredients with an unfavourable effect on pigmentation
It is known that barley, as a component in poultry feed, has a negative effect on pigmentation. This is explained by the fact that barley contains non-starch-polysaccharides (beta-glucan), which cause a higher viscosity of the intestinal content and therefore unfavourably influence the digestion and resporption of pigments. Similarly, wheat with a high pentosan content may lead to similar effects. Supplementation with enzymes (beta-glucanase, xylanase etc.) leads to a positive effect on yolk pigmentation (Figure 6). In a trial with 68 % barley, carried out in Spain, an improvement of 0.2 according to the Roche scale was registered with very high initial fan values (13). With a significantly lower initial fan value (6 – 9), the effect of enzyme supplementation was much more evident in the Australian trial and which also applied in the wheat ration. Australian wheat is characterised by its high amounts of soluble pentosans.
Mycotoxins can also unfavourably influence the pigmentation of yolk and carcass. Ochratoxin in particular, as well as aflatoxin and fusarium-toxin must be mentioned in this connection. The oxycarotenoid metabolism can be changed by the presence of mycotoxins as follows: • Dilution of oxycarotenoids in the intestine • Reduced resorption via the intestinal wall • Reduced transport in the serum • Changed storage in the liver • Changed deposition in the tissues. As the aforementioned mycotoxins hamper fat transport in general, an indirect influence on the fat soluble oxycarotenoids can be expected. Ochratoxin and aflatoxin may be a cause for increased meat and blood spots in eggs as well as bleeding in the carcass. Aflatoxin, and also T2-toxin (source is maize), may cause intestinal resorption irritations. Fat metabolism is especially affected by a decreased lipase and bile production. Fat is excreted via the faeces. Another feed ingredient with a negative effect on yolk pigmentation is gossypol from cottonseed. Gossypol forms complexes with iron, which lead to undesirable greenish to dark spots in the yolk.
Health
Some poultry diseases have a specially negative effect on pigmentation of yolk and carcass. Coccidiosis out breaks occur more frequently in alternative housing systems affecting broilers but also laying hens. Even a light coccidiosis without obvious symptoms may cause reduced skin pigmentation. The coccidia type, as well as the gravity of the disease and the respective intestinal area affected, are decisive factors effecting colour. Coccidia, which settle in the anterior intestine have a stronger effect on pigmentation than, for example, caecal coccidia. The effect of an infection of the small intestine with Eimeria acervulina is demonstrated in the following Table (6). Already, 3 days after the infection, the contents of lutein and canthaxanthin in the serum and in the liver have dropped dramatically. Five days after the infection canthaxanthin is hardly detectable in serum and liver and in the toe web only 30 % of the initial values were found. These figures illustrate clearly the effect of coccidiosis on pigmentation. There are several other diseases that show a direct influence on pigmentation. Diseases of the intestine, Contagious Avian Coryza, Newcastle Disease as well as helminthosis belong to this category. Similarly, Fatty Liver Syndrome in laying hens may lead to alterations in pigmentation. Thus, it can be said that a consistent colouring of carcass and egg yolk may be regarded as an indicator of good health and good practical hygienic conditions.
Coccidiostats and helminthica
It is known that the use of nicarbazin or piperacin causes spots in egg yolks.
Influence of the housing system
The influence of different housing systems, which is often mentioned in the literature, mainly depends on differences in feed intake and hygienic status. A trial carried out in the USA (Allen, 1993) shows, how the hygienic status may in flu ence pigmentation. An additional fumigation of the broiler house with formaldehyde resulted in higher lutein contents in the blood plasma and in the toe web of 3 week-old broilers at both pigment doses than when classical wet cleaning was used. Also, body weight was significantly increased with a formaldehyde treatment (see Table 7) A number of studies on the influence of daylight on pigmentation have been undertaken. Thus daylight causes a more intense pigmentation of yolk and skin with the skin mainly showing a shift from yellow to orange. The increased feed intake due to daylight is not the reason for this. Possible reasons may either be a pigment change in the feed influenced by light (isomerization of betacarotene to zeaxanthin) or a change in the pigment metabolism of the animal (transformation of zeaxanthin to astaxanthin). Remember the cock with its red comb, the colour of which is caused by astaxanthin and which would not be found in animals that were housed without daylight. With regard to the increased interest in alternative housing systems and the claims of the animal welfare this subject is becoming more important.
Factors which influence pigmentation Genetic Factors
- Oxycarotenoid source • Oxycarotenoid content • Storage period and storage conditions of raw components and feed • Heat treatment of the feed • Pigment additives • Saponification of natural pigment supplements • Effect of incorrect mixtures regarding pigments • Feed intake Energy content of the ration Ambient temperature Feed structure (esp. in meal feed) Pellet quality Taste and smell Water intake Light (-programme) • Additional feeding with wheat • Fat and fat quality • Antioxidants • Vitamin E as antioxidant • Calcium • Vitamin A • Growth promoters • Feed ingredients with negative effect NSP Mycotoxins • Helth status • Housing • Genetic factors
Today mostly hybrid layers and broilers are used and genetic effects on pigmentation are not seen. It is known that the epidermis of certain breeds of fattening poultry cannot be coloured but now only hybrids are used in economical poultry breeding and these are able to be pigmented due to crossing with Asian breeds. Thus genetic factors have no influence on changes in pigmentation. Research work has not shown whether differences in the intensity of skin pigmentation exist between colourable races at similar feeding. However, influences due to different feed conversions are possible, which would also influence the oxycarotenoid intake.
Assessing the value of the egg
The quality characteristics of eggs are usually divided into external features, such as:
- egg weight
· egg shape
· shell structure
· shell crack resistance
· dynamic shell resistance
· shell color
and internal characteristics, including:
- albumen weight
· Haugh unit (a measure of egg protein quality)
· yolk height,
· yolk diameter,
· albumen pH,
· yolk pH
· yolk color
For consumers, yolk color is probably the most important criterion for egg quality. Higher color intensity often is taken as indicating the good health of the laying hen.
Depending on the region or on the culture, people prefer more yellow or more orange yolks. In countries with traditional corn feeding, e.g., Mexico, they often like a deep yellow. In Northern Europe, consumers prefer a lighter yellow; in Southern Europe, more gold-orange yolks (see table 1).
| Country | Yolk color fan value* |
| Belgium | 12-13 |
| Denmark | 9-10 |
| Finland | 9-10 |
| France | 11-12 |
| Germany | 11-14 |
| Greece | 11 |
| Italy | 12-13 |
| Netherlands | 7-9 |
| Austria | 12-14 |
| Portugal | 12-14 |
| Spain | 11-14 |
| Sweden | 9-10 |
| United Kingdom | 10-11 |
Table 1. Egg pigmentation preferences – variation across European countries
* Values range from 1 (very pale yellow) to 16 (intense orange)
Egg yolk color is achieved via feed
The typical color of the yolk depends on pigments that are ingested with the feed. Corn and alfalfa meal provide the yellow pigments lutein and zeaxanthin, belonging to the xanthophylls, a sub-group of carotenoids. The golden-orange color is provided by red pigments from chili or paprika (Grashorn, 2008). Egg yolks start changing color about 48 h after the application of xanthophylls.
To reach an optimal yolk coloration in egg production, diets should be supplemented with yellow and red xanthophylls. Yellow xanthophylls achieve a correct yellow base coloration. The main yellow pigments used in poultry feeding are apoester, a synthetic carotenoid, and saponified marigold extracts, a natural alternative containing lutein and zeaxanthin. For the redness, paprika or chili offer natural sources; canthaxanthin is a nature-identical red xanthophyll.
For a long time, synthetic colorants were the substances of choice in the poultry industry because they provide consistently predictable results and high product stability. However, consumers’ preferences concerning food have shifted; demand favors natural over synthetic food ingredients. Moreover, current EU regulations restrict these synthetic molecules’ inclusion level due to their potentially harmful effects on human health if applied in excessive doses.
| Carotenoid | Maximum inclusion level |
| Apoester (ethyl ester of β-apo-8’-carotenoic acid) | 5 ppm |
| Canthaxanthin (β,β-Carotene-4,4′-dione) | 8 ppm |
Table 2. Maximum concentration allowed in feed for poultry production
Fortunately, there is already a natural, highly efficient option to replace apoester.
Lutein: a natural colorant, antioxidant, and provider of health benefits
One of these natural compounds is lutein, a lipophilic pigment. It is extracted from marigold petals, which contain up to 8.5 mg/g wet weight. Lutein is always accompanied by its isomer zeaxanthin.
Lutein – the yolk colorant
The use of xanthophylls such as lutein and zeaxanthin enables producers to safely control the color of the egg yolk and the broiler skin. In poultry, the carotenoids are deposited in high quantities in the epidermis, the fatty tissue, and the egg yolk. According to Grashorn (2016), between 4.4-23 % of dietary lutein and 23 % of dietary zeaxanthin are deposited in the egg yolk.
Lutein – the antioxidant protects the egg lipids
Another critical characteristic of lutein is its antioxidant effect. Egg yolks contain a high fat content. Therefore, they are very susceptible to lipid oxidation. Lutein, acting as an antioxidant, can prevent or at least limit lipid oxidation during egg processing. Kljak et al. (2021) compared different sources of pigments (basil, calendula, dandelion, marigold, and an industrial product containing canthaxanthin) concerning their antioxidant capacity. In this trial, marigold improved the yolks’ oxidative stability by 75 % compared to the control, with canthaxanthin showing no antioxidant effect. Kljak et al. attributed this effect to the carotenoids in the marigold extract.
Lutein – a value-added ingredient
Lutein and its isomer are nutritionally valuable and, therefore, welcome ingredients of the eggs. Once more, due to their antioxidant effects, they play an essential role in preventing and reducing cataracts and age-related eye dysfunctionalities in humans and animals (Landrum & Bone, 2001; Wang et al., 2016).
However, the amounts of antioxidant pigments in a standard egg are not very high (approx. 400 µg/egg). Compared to the total amount of antioxidants ingested, their importance for humans is only limited (Grashorn, 2008). The situation is different for functional eggs, which are widely sold in certain English-speaking countries. These eggs are enriched with n-3 fatty acids and with antioxidants such as ß-carotene (ca. 150 IE/egg).
Colour determination
Yolk colour can be determined basically by two methods. An objective method uses a mechanical equipment (for example, MiniScan XE HunterLab, Minolta colourimeter CR 300) that expresses colouration in the so-called HunterLab scale (L, lightness; a, redness; and b, yellowness). A simple but subjective method involves the DSM Yolk Colour Fan that expresses results in a 1 to 15 scale by means of comparison with calibrated cards. Both systems are well correlated but because the DSM Fan is more economical and readily available it has become the preferred method in most parts of the world. With regards to the products containing eggs, both methods are suitable, but for scores higher than 15, the DSM Fan is insufficient. For this purpose, a new and fast method based on spectrophotometry can determine carotenoid content expressed as beta-carotene equivalents (BCE): the iCheck Egg. This fast method is perfectly correlated with the equivalent standardised method carried out in laboratory.
Skin colour
Compared to yolks which have homogeneous colour, broiler skin is much more difficult to assess because carcass colour is more heterogeneous. Colour can be assessed by the DSM Broiler Fan, expressed in a 101-110 scale, or by a colourimeter, as above. The results can vary according to the protocol. Ideally, measurements must be carried out on the fatty part of the breast collected after chilling. Sex (female birds have darker skin colour) and genotype are also important factors of variability.
In a French survey, the yellow value was more useful to distinguish yellow broilers, whereas the red value was more variable and did not depend on chicken type. Again, as the DSM Fan allows for a better ranking and is more economical and readily available, it has become the preferred method for many abattoirs. Finally, in markets where the shank colour is of importance when the broilers are sold with the feet, the red colour is the most important, with canthaxanthin being the most efficient compound for shank pigmentation.
Carotenoids products
Synthetic products are standardised and more stable than extracted products. For example, in one study, apo-carotene-ester concentration in feed samples showed very little variation from expected values, whereas it was 30% below expectations for marigold extracts. Esterification and saponification usually helps to produce better stable tagetes and paprika products; the same as spray-drying protection, but there is a wide variation in the quality of the coating. For example, the stability of various red carotenoids products in premixes was checked after three months of storage and recovery varied considerably: 66% to 92% for canthaxanthin, 76% for citranaxanthin, and 39% for capsanthin from paprika. Carotenoids are very unstable on their isomeric forms; the trans-configuration is the most common, found in the yolk, but the cis-configuration (less efficient for pigmentation) can be produced when heat, light, or oxygen damage occurs. Good coating usually prevents such damage.
In table eggs, from hens raised under traditional commercial conditions, six carotenoids are present at the greatest concentration. These include lutein, zeaxanthin, canthaxanthin, citranaxanthin, apo-caroten-ester, and cryptoxanthin. In contrast, organic eggs usually contain only lutein, zeaxanthin, and cryptoxanthin, as regulations do not allow synthetic carotenoid in the feed. Apo-carotene-ester is the most deposited yellow carotenoid in egg yolk with a transfer efficiency from feed to egg of about 55% (as compared to only 17% for marigold carotenoids). On the other hand, canthaxanthin is the most efficiently deposited red carotenoid in egg yolk, with an average deposition rate of about 40%. Interestingly, in the last years, new canthaxanthin commercial products have appeared on the European market. In a relevant study, the deposition rate for three such new products was 30, 27 and 24%, as compared with a traditional cantha-xanthin product that showed a 35% deposition rate. It should be noted here that bioavailability of formulated products can be influenced by many factors, including matrix type and structure, formulation particle size, formation of non-absorbable complexes with matrix components, and degradability of carrier.
Colour deposition in the skin
In broilers, zeaxanthin influences the yellow value in all tissues, more noticeably in the abdominal fat, with lutein and zeaxanthin being deposited in skin and fat with a rate of 8-12% and 4-9%, respectively. In broilers, the deposition of the isomerised tagetes (enriched in zeaxanthin) is lower than the standard tagetes (mostly lutein), whereas contents of apo-carotene-ester increases in skin and abdominal fat with increasing supplementation of this carotenoid, reflecting a linear increase over the entire dose range (not common among other carotenoids).
It has been determined that 1 ppm of apo-carotene-ester is equivalent to about 2 ppm of lutein-zeaxanthin (from tagetes). Canthaxanthin is the dominating red carotenoid in pigmented broilers, whereas paprika receives little attention because of its low efficiency (requiring two to three times the amount of paprika xanthophylls compared to canthaxanthin).
Feed recommendations
There are two components of the pigmentation process. The first refers to the saturation phase and involves the deposition of yellow carotenoids to create the yellow base (about 7 on the DSM Yolk Fan). Then the addition of red carotenoids is called colour phase and changes the hue in a more orange colour. For table eggs, quantities of yellow and red carotenoids can be added depending on market requirements (Table 2). Feed supplementation should be implemented for at least 10 days to ensure a constant colour. For processed eggs, the requirement for yellow base is higher. When specification is above 20 ppm BCE (above 15 on the DSM Fan), it is better recommended to formulate according to the BCE value. High concentrations of apo-carotene-ester in the feed are frequently necessary to fulfil the requirements such as pasta production.
Regarding pigmented broilers, the amount of required feed carotenoids is higher than for table eggs, because of lower deposition rates. The most common practice is based on a lutein-zeaxanthin and canthaxanthin mix, with apo-carotene-ester added to ensure colour homogeneity. Feed supplementation should be done for three weeks minimum with no withdrawal before slaughter.
Source & Reference–
By Catherine Hamelin and Ulrich Altemueller , DSM Nutritional Products Europe
https://ew-nutrition.com/appetizing-eggs-natural-pigmentation/
https://www.veterinariadigital.com/en/articulos/the-egg-yolk-color-and-pigments/
https://www.poultryhub.org/anatomy-and-physiology/the-avian-egg
COMPILED BY-DR. R. BHASKAR,POULTRY EXPERT, CHANDIGARH
