Effects of blue lupin (Lupinus angustifolius) in organic layer diets and supplementation with foraging material on egg production and some egg quality parameters

Effects of Blue Lupin (Lupinus angustifolius) in Organic Layer Diets and Supplementation with Foraging Material on Egg Production and Some Egg Quality Parameters M. Hammershøj*,1 and S. Steenfeldt† *Department of Food Science; and †Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, P.O. Box 50, DK-8830, Tjele, Denmark 15% lupin treatments, respectively. Feeding the 25% lupin diet significantly reduced diet intake to ∼91 g, and increased supplement intake to 118 g for the treatment with foraging material. Eggs from treatments with foraging material had significantly higher sulfur-like aftertaste in sensory evaluation. Yolk color became significantly lighter and more yellow with lupin content, but darker and less greenish with foraging material. Increased lupin levels decreased albumen DM, whereas foraging material had no effect. Inclusion of 25% lupin in layer diets is only recommended when supplying some methionine source, as egg production and quality parameters are dramatically impaired. However, supplement of foraging material significantly improves egg production.

(Key words: laying hen, egg production, lupin, sensory evaluation, yolk color) 2005 Poultry Science 84:723–733

Lupin (Lupinus spp.) is a nitrogen-fixing plant, which is a great advantage in organic farming due to the recycling of the nitrogen in the system to the subsequent crop (Doyle et al., 1988). Moreover, most modern varieties of L. angustifolius contain low concentrations of alkaloids (Milford and Shield, 1996; Petterson, 2000). Lupin has a high protein content (Petterson, 2000) and could be a good supplement to soybeans, which are presently used as the main protein source in organic poultry feed. However, like most legume proteins, the content of TSAA is low (Alloui et al., 1994; Petterson, 2000). This could make it difficult to optimize diets with adequate amounts of the essential amino acids, thereby limiting the inclusion level of lupin in layer diets. The high level of the nonstarch polysaccharides (NSP) in lupins, almost twice as high as in other protein-rich plants ˚ man, 1993; Bach-Knudsen, 1997), could be (Daveby and A another factor restricting its use in poultry diets. In organic egg production, the free-range keeping of hens can, in some flocks, present problems with higher

INTRODUCTION Organic egg production has expanded markedly during the last 15 yr, and in 2001, the market share in Denmark of organically produced hen eggs was 16% of all eggs in retail (Det Danske Fjerkræra˚d / Danish Poultry Council, 2002). Beginning in 2005, the feed for organic egg production within the European Union must be 100% organic [Commission Regulation (EC) 2277, 2003]. Additionally, it will no longer be permitted to add synthetically produced amino acids to organic diets. Therefore, it becomes essential to carefully balance feed components for organic egg production to obtain high productivity and egg quality, as nutritionally poor ingredients cannot be compensated for by supplementation with synthetic ingredients. Hence, the demand for alternative protein sources, which supply the laying hen with the necessary nutrients, is increasing, especially with respect to essential amino acids.

 2005 Poultry Science Association, Inc. Received for publication April 29, 2004. Accepted for publication December 8, 2004. 1 To whom correspondence should be addressed: marianne. [email protected].

Abbreviation Key: Diets A, B, C = experimental diets containing 0, 15, and 25% blue lupin, respectively, with (p) or without (m) foraging material as supplement; GC-MS = gas chromatography-mass spectrometry; NCP = noncellulosic polysaccharides; NSP = nonstarch polysaccharides; RYC = Roche yolk color.

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ABSTRACT Six groups of organically fed hens were studied for egg production, feed parameters, and egg quality from 20 to 31 wk of age. Treatments were 3 diets (0, 15, and 25% blue lupin) with or without supplements of foraging material (whole carrots and corn silage). Increased lupin content increased nonstarch polysaccharides content and reduced methionine content below the hens’ requirement. Egg production at 31 wk was lower with the 25% lupin diet without (69%) and with foraging material (76%) compared with the other diets (∼90%). Egg weight was highest with the 0% lupin diet (64 g), and 15% lupin diet (60 g), whereas the 25% lupin diet without and with foraging material resulted in egg weights of 58 and 56 g, respectively. Feed intakes were ∼113 g of diet/ hen per d and 113 g of supplement/hen per d in 0 and

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MATERIALS AND METHODS Diets and Treatments The lupin (Lupinus angustifolius) and naked oats (Avena nuda) used were the cultivars Prima and Mozart, respectively, which were grown in the southern part of Denmark in 2001. Prima is the most widespread lupin cultivar grown in Denmark and is considered a low-alkaloid variety. The composition of the 3 experimental diets (A, B, and C) used in the study is given in Table 1. Inclusion of lupin as a protein source and naked oats primarily as an energy source was made mostly at the expense of the protein sources soybeans and sunflower meal, but the wheat content was also reduced, and peas were excluded in diets B and C. The diets were formulated to contain the same protein content (172 g/kg) and ME (2,630 kcal/kg). No essential amino acids were added to the experimental diets, because their use is prohibited in diets for layers in organic egg production. We decided to exclude fishmeal because

TABLE 1. The composition of experimental diets (g/kg) Ingredients Wheat Barley Naked oats Lupin seeds Peas Soybeans, toasted, whole Sunflower meal Potato protein concentrate Alfalfa-meal Rape seed oil Calcium carbonate Monocalcium phosphate Sodium bicarbonate Sodium chloride Vitamin and mineral premix1 Betaine Grindazym2 Calculated content Protein (N × 6.25) Fat Lysine Methionine Threonine Calcium Phosphorus, total ME, kcal/kg

Diet A

Diet B

Diet C

468.4 50.0 — — 50.0 189.7 72.0 25.0 26.6 10.0 90.3 10.7 3.0 1.0 2.5 0.5 0.3

435.8 50.0 75.0 147.8 — 100.0 — 42.7 22.1 18.6 89.9 11.0 3.0 0.9 2.5 0.5 0.3

309.9 50.0 125.0 250.0 — 50.0 — 39.5 20.4 47.9 89.5 10.6 3.0 0.9 2.5 0.5 0.3

172.0 67.0 8.7 2.8 6.5 38.0 5.6 2,630

172.0 64.0 8.8 2.5 6.6 38.0 5.2 2,630

172.0 80.0 8.7 2.3 6.7 38.0 5.3 2,630

1 The vitamin and mineral premix provided per kilogram of diet: vitamin A, 13,000 IU; vitamin D3, 3,000 IU; vitamin E, 30 mg; vitamin K3, 4.5 mg; thiamin, 1 mg; riboflavin, 5 mg; vitamin B6, 3 mg; D-pantothenic acid, 5 mg; niacin, 50 mg; betaine anhydrate, 680 mg; folic acid, 2 mg; biotin, 0.1 mg; B12, 0.03 mg; Fe, 25 mg; Zn, 60 mg; Mn, 100 mg; Cu, 5 mg; I, 0.5 mg; Se, 0.3 mg. 2 Xylanase (EC 3.2.1.8), 12 U/kg of diet; β-glucanase (EC 3.2.1.4), 5 U/kg of diet.

use of this product in organic farming has been questioned due to the risk of contamination with dioxin, and the possible contamination with prohibited meat-and-bone meal. Due to these constraints, it was not possible to obtain similar levels of methionine in the diets because the Met content is low in lupin and naked oats. Potato protein concentrate was included as a main protein and amino acid source instead of fishmeal, but as the content of saponins such as α-chaconine and α-solanine, which are toxic, can be high in potato products (Becker and Weltring, 1998), a maximum level around 4% in the diet is recommended. The protein content in potato protein concentrate was given to be 700 g/kg, and the content of Met is given to be 17 g/ kg, which corresponds with previously reported levels of 16.5 to18.5 g of Met/kg protein (Rexen, 1976). The 3 experimental diets were fed to laying hens with or without access to supplements of whole plant corn silage (26.5% DM) and cleaned, uncooked roots of carrots (11% DM) giving 6 treatments subsequently referred to as Am, Ap, Bm, Bp, Cm, Cp, where m = without (minus) supplement and p = with (plus) supplement. The diets had a very coarse texture and were provided ad libitum in crumble form.

Birds, Housing, and Experimental Design One-day-old ISA Brown chickens were obtained from a commercial hatchery and reared under identical conditions

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incidences of feather pecking that in severe cases may lead to cannibalism. One way to minimize feather pecking and cannibalism in free-range laying hen flocks could be supplementation of foraging material (Wechsler and HuberEicher, 1998), in which time spent feeding increases compared with normal pelleted diets. For laying hens, the usage of lupin as a feed component together with foraging material must be without detrimental productivity and egg quality effects. Egg production level together with the taste and appearance of the eggs should be maintained or improved if organically produced feedstuffs are to find application. However, very limited knowledge exists concerning how such feed and foraging materials affect egg quality parameters such as taste, aroma components, and egg appearance. Other studies have demonstrated that various feed components such as rapeseed (Goh et al., 1979; Pearson et al., 1980), fish products (Koehler and Bearse, 1975; Hammershøj, 1995), grape seeds (Tallarico et al., 2002), and lupins (Vogt et al., 1983) induce unwanted taste and off flavors in hen eggs. One of the most important quality parameters for the consumer buying retail eggs is yolk color. Hence, with the requirement of 100% organic feed components in organic production, it is crucial to have feed components with sufficient natural content of xanthophylls to ensure the preferred level of yolk color. Corn contains lutein and zeazanthin (Adams, 1985) at concentrations of 20 to 25 mg of xanthophyll/kg (Sikder et al., 1998), and carrot meal contains 54 to 65 mg of xanthophyll/kg (Sikder et al., 1998). Corn silage (grains and cobs) has a positive effect on yolk color when included in diets for laying hens (Jeroch, 1986). The aim of this experiment was to study the effect of organic layer diets containing different levels of blue lupin together with supplements of foraging material on egg production, sensory evaluation, aroma components, yolk color, and albumen DM. We decided to use corn silage and whole carrots as supplements to supply pigments, which were expected to affect yolk color.

BLUE LUPIN IN ORGANIC EGG PRODUCTION

Chemical Analysis of Diets, Ingredients, and Foraging Material Dry matter content was determined by drying at 105°C for 8 h. Protein (N × 6.25) was determined by the Kjeldahl method (AOAC, 1990a) using a Kjell-Foss 16200 autoanalyzer; energy was analyzed by a LECO AC 300 automated calorimeter system 789-500.2 Ash was analyzed according to method 923.03 (AOAC, 1990b), and fat (hydrochloric acid-fat) was extracted with diethyl ether after acid-hydrolysis (Stoldt, 1952). Amino acids were analyzed as previously described (Mason et al., 1980). The sugars (glucose,

2

LECO, St. Joseph, MI. Sigma-Aldrich, St. Louis, MO. 4 Varian Analytical Instruments, Walnut Creek, CA. 5 Agilent Technologies, Palo Alto, CA. 3

fructose, and sucrose) and oligosaccharides (raffinose, stachyose, and versbascose) were extracted with 50% (vol/ vol) ethanol at 60°C and quantified by gas-liquid chromatography (Bach-Knudsen and Li, 1991). Starch was analyzed by the enzymatic-colorimetric method (BachKnudsen, 1997). Total NSP and their constituent sugars were determined as alditol acetates by gas-liquid chromatography for neutral sugars and by colorimetric method for uronic acids using a modification of the Uppsala procedure (Bach-Knudsen, 1997; Theander et al., 1994). Cellulose was determined as the difference in glucose content of NSP when the swelling step with 12 M sulfuric acid was included and omitted, respectively, and the content of cellulose, noncellulosic (NCP), and soluble NSP was calculated as previously described (Bach-Knudsen, 1997). Klason lignin was measured gravimetrically as the residue obtained after the treatment with 12 M sulfuric acid (Theander et al., 1994). All analyses were performed in duplicate.

Sensory Evaluation of Eggs Forty eggs from each Am, Ap, Cm, and Cp treatment were collected at 20 wk of age for 5 initial training sessions of the sensory panel in order to define descriptors of aroma and taste. These descriptors were quantified by a 15-point unstructured scale and used in 2 test evaluations of eggs (40/treatment) collected at 22 and 30 wk of hen age. The sensory panel had 10 judge members, and each judge evaluated 3 replicates per treatment i.e. a total of 30 evaluations per treatment were obtained per session. Eggs were collected 10 d before the tests and stored at 5°C. Five shell eggs per 750 mL of water were heated at 97°C for 15 min, transferred to cold water for 10 min, peeled, divided into halves, kept in 100-mL closed plastic containers for 45 min at 20°C, and served as coded samples for the sensory panel.

Volatile Aroma Compounds of Eggs by Gas Chromatography-Mass Spectrometry Six eggs per treatment were collected from hens fed Am, Ap, Cm, and Cp at 20 and 30 wk of age (i.e., 48 eggs total) and analyzed for volatile aroma compounds. The egg yolk and albumen were separated within 6 h of collection and each fraction was homogenized by stirring. Samples of 3 g were transferred to 10-mL sterilized glass vials and stored at −80°C until analysis. Before analysis, samples were heated for 10 min at 90°C followed by absorption with a PDMS/carboxen3 fiber for 10 min at room temperature (22 ± 1°C). The fiber desorbed at the injection-port of a Varian Star 3400 CX GC-MS4 at 250°C during the total analysis time of 29.33 min. The injection was split-less, i.e., the total sample volume of 1 µL was analyzed. The gas chromatography-mass spectrometry (GC-MS) analysis was run on an HP-5MS column,5 30 m long, 0.25 mm diameter, and 0.25 µm film thickness. Temperature was held at 35°C for 10 min, then increased by 15°C/min to 250°C, and held constant for 5 min. Helium was used as carrier gas at 35°C with flow of 41.2 cm/s, and detection area was 30 to 300 m/z.

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in floor pens covered with wood shavings. The floor pens were situated indoors, in an environmentally controlled house. From 8 wk of age, half of the pullets were fed small amounts of carrots and corn silage to allow adaptation to the forage materials used as supplements in the experiment. The birds were not beak trimmed. All birds were fed a commercial starter and grower diet until 16 wk of age, when 900 pullets were moved from the rearing house and placed in 30 identical indoor floor pens, with 30 hens per 8.9-m2 pen or 3.4 hens/m2. Wood shavings were used as litter. Each pen was provided with a round feeder (34 cm diameter), nipple drinkers, and 9 single nests (28-cm wide) in a battery, and the birds had access to perches. Thin wooden walls, which allowed auditory but not visual contact between hens from separate pens and treatments, separated adjacent pens. The lighting program was 12L:12D at 16 wk and gradually increased to 16L:8D at 31 wk. From 16 to 17 wk of age, the birds were fed a commercial layer diet, and were introduced to the experimental treatments from wk 18. The 6 experimental treatments were randomized among 30 pens, i.e. 5 replicates of each, and recording of data started at 20 wk of age. In the experimental period from 20 to 31 wk of age, the “plus supplement” pens received corn silage and carrots, at 35 and 100 g/hen per d, respectively. This amount approximated ad libitum consumption based on calculations from a pilot experiment (unpublished data). The supplements were unprocessed and were weighed before given fresh each morning in a wooden box (height: 40 cm, length: 50 cm, width: 40 cm) after leftovers (mainly corn silage) from the previous day were removed and weighed. Moisture loss of foraging material during the day was not recorded, but was estimated to be low, as the majority of the foraging material was eaten during the light period of the day. All data were collected per pen. Eggs were collected and recorded daily per pen and one day’s collection was weighed each week. Feed consumption of the layer diets was recorded every other week. Mortality was recorded daily, and the BW of the hens was recorded before (17 wk) and after (31 wk) the experimental period.

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Egg Yolk Color and Albumen DM Content

Statistical Analysis The experiment was conducted as a randomized complete block design with the single pen representing the experimental unit (replicate). Data were subjected to statistical analysis by the GLM procedure.7 For each feeding treatment, variance homogeneity and data distribution were analyzed by the Bartlett’s test (Bartlett, 1937) and the Probit analysis (Blæsild and Granfeldt, 1995), respectively, leading to no exclusion of outlier values. The data were distributed by the normality function; hence, data were not transformed. The model for analysis was: Yijl = ai + bj + cij + eijl where a = main effect of diet i (A, B, C); b = main effect of supplement j (m, p); c = interactive effect between diet and supplement; and e = replicate l (1,....5). Where no interactive effects between the class variables were found, the cij was excluded from the model. For the sensory evaluation, the score data were analyzed by a similar model, however, with diet i (A,C). The LS-Means were calculated and differences regarded as significant at minimum 95%level (P ≤ 0.05). Differences were classified by the RyanEinot-Gabriel-Welsch (REGW) multiple range test.7

RESULTS Chemical Analysis of Diets and Foraging Material The results of chemical analyses of the ingredients lupin, soybean, naked oats, and the 3 experimental diets are given in Table 2, with amino acid contents in Table 3. The total content of NCP was high in lupin, being 30.4% of DM with the galactose residue amounting to 15.3% of DM (50% of NCP), showing that galactose is the main monomer in

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Minolta Co. Ltd., Osaka, Japan. Version 8.01, SAS Institute, Inc., Cary, NC.

7

Hen Performance The main effect of diets was highly significant for all parameters, whereas the main effect of supplements was less significant (Tables 4 and 5). Laying hen performance during the period from 20 to 31 wk was markedly lower (P ≤ 0.0001) for hens fed diets with 25% lupin compared with groups fed 0 or 15% lupin diets (Table 4). Hens fed diet Cp improved performance (P ≤ 0.05) compared with hens fed diet Cm, but performance was still considerably lower than for the A and B groups. Access to supplementa-

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At 20 and 30 wk of hen age, 5 eggs were collected from each treatment and analyzed for yolk color with a Minolta Chroma Meter CR-3006 using the CIE (Commission Internationale de L’Enclairage) scale (D65). The L*, a*, and b* values reflect lightness (0 = black, 100 = white), redness (−100 = green, 100 = red), and yellowness (−100 = blue, 100 = yellow) of the samples, respectively. The instrument was calibrated against a white standard plate with L* = 92.3, a* = 0.33, and b* = 0.1. The albumen was removed from the yolk by cutting into the thick part of the albumen before yolk color measurement. The DM content (g/kg) of the egg albumen was analyzed in 5 replicates by weighing 3 mL of albumen into a porcelain vial. The samples were dried for 18 h at 98°C in an oven and reweighed after equilibration to room temperature (22 ± 1°C).

lupin, followed by arabinose, xylose, and uronic acid. In addition, approximately 44% of the NCP were soluble. The cellulose content was 12% of DM, which resulted in a total content of NSP of 42.6% of DM. The content of NSP was much lower in the soybean (16.6% of DM) and naked oats (11.3% of DM), where cellulose and the NCP-residue galactose differed from lupin to a large extent. The lignin content was low in all 3 ingredients. The amino acid profile in lupin was very good regarding the requirements of the hen, but the content of Met was low, compared with soybean (Table 3). As expected with a cereal, the content of Met was low in naked oats, but comparable to the content obtained in lupin, which elucidated the low content of this important amino acid in lupin. The chemical analyses of the 3 diets (Tables 2 and 3) were in agreement with the expected values given in Table 1. The protein content was almost identical between the 3 diets, x = 20.8% of DM (18.8% “as fed”), and a little higher than the calculated values. The same tendency was obtained with regard to the fat content. The NSP content in the diets was influenced by the increased inclusion of lupin, reflecting the high NSP content in this ingredient, as the highest content was found in diet C (18.1% of DM). The content of the amino acids in the diets (Table 3) showed that the requirement for most of the amino acids was covered. The content of Lys and Met in diet A was a little higher than calculated, whereas the content of these in diet B and C was close to the expected values, confirming that diet C was probably deficient in Met. The NSP content of the corn silage was high, 43% DM. The main part of the NSP was insoluble (40% DM, ∼93% of the total NSP content). In addition, the lignin content was 10% DM, resulting in a total content of dietary fiber (NSP + lignin) of 53% DM, which is relatively high. The protein content was 10% DM, the starch content 12.3% DM, and the DM content was 26.5%. The DM content in carrots is very low, being 11% in the carrots used in the present experiment. The content of NSP is much lower (24% DM), and in contrast to the silage, the soluble NSP constitute as much as 56% of the total NSP content. There are only trace levels of starch in carrots, but the sugar content is as high as 45% DM (sum of glucose, sucrose, and fructose). The protein content was 7% DM. For both corn silage and carrots, the essential amino acids content was very low, i.e., Lys was 2.57 and 2.65 g/kg of DM, respectively, and Met was 1.30 and 0.84 g/kg of DM, respectively.

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BLUE LUPIN IN ORGANIC EGG PRODUCTION TABLE 2. Chemical composition (g/kg of DM) of lupin, soybean, naked oats, and 3 experimental diets1 (A, B, and C) Constituent

Lupin 887.7 44.6 324.1 57.0 5.6 4.5 5.7 122 304 426 134 292 5 431 4,713

Naked oats

929.4 58.0 386.9 242.8 37.5 3.4 5.4

Diet A

Diet B

Diet C

907.7 141.9 208.4 93.9 329.8 42.6 6.8

902.8 142.5 205.9 84.6 352.0 45.4 6.8

899.1 146.7 208.8 98.9 339.0 45.5 6.4

889.4 27.5 131.6 80.9 588.2 1.1 4.9 <1 113 113 61 52 19 132 4,613

43 123 166 37 129 18 184 5,712

42 82 124 29 95 34 158 4,385

42 106 148 43 105 24 172 4,246

61 120 181 63 118 22 203 4,488

1

Diets A, B, and C contain 0, 15, and 25% lupin, respectively. Nonstarch polysaccharides (cellulose + NCP). 3 Noncellulosic polysaccharides are the sum of the monosaccharides: rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, and uronic acids. 4 Soluble noncellulosic polysaccharides. 5 Insoluble nonstarch polysaccharides. 6 Dietary fiber = NSP + lignin. 2

tion did not change the number of eggs produced by hens fed diets A or B. The hens fed diet B produced lower egg mass than those fed diet A when no access to supplement was given, whereas no significant difference in egg mass was found between these groups when the hens were fed corn silage and carrots in addition to the layer diets (Table 4). No difference was observed in feed intake between the groups fed diets Am, Ap, Bm, or Bp. Feeding diet Cm or Cp resulted in a lower intake of the layer diet (P ≤ 0.0001) and a higher intake of the supplements (P ≤ 0.05) compared with the other groups. On an as-fed basis, the intake of the supplement amounted to 50% or more of the total feed intake per day (Table 4). However, on a DM basis, the

intake of supplements amounted to 16, 15, and 20% of total feed intake for diets A, B, and C, respectively. Due to the high water content in the carrots, corn silage counted for approximately one-third of the total DM intake of the supplements. The feed conversion, in terms of grams of layer feed/grams of egg, obtained with diet A was better than with the B and C diets. However, consumption of corn silage and carrots together with the lupin-based diet C improved feed conversion of the layer diet (P ≤ 0.0001). Consumption of supplements together with diet A and B only had a minor positive effect on feed efficiency. Significant interactions between diet type (A, B, C) and access to supplements were observed for egg number and egg mass, i.e., consumption of supplements had a significantly posi-

TABLE 3. Content of amino acids (g/kg of DM) in lupin, soybean, naked oats, and the 3 experimental diets (A, B, and C) Amino acids

Lupin

Soybean

Naked oats

Diet A

Diet B

Diet C

Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

10.89 35.86 33.24 4.96 66.54 13.31 8.84 13.60 21.44 15.10 2.15 12.51 12.82 17.05 11.25 —1 13.10

16.77 28.66 45.62 6.43 67.51 16.51 10.44 18.34 28.40 23.18 5.59 18.92 19.36 20.83 15.60 —1 19.37

7.44 10.84 12.95 5.14 29.86 8.05 3.54 6.01 11.18 6.62 2.69 7.88 8.41 8.27 5.47 —1 8.44

8.78 13.33 19.40 3.57 38.75 9.18 5.16 9.39 15.15 10.70 3.43 10.12 12.36 10.16 7.64 7.08 9.98

8.37 14.39 19.07 3.55 38.65 8.90 5.09 9.27 15.25 10.28 2.88 9.68 11.93 10.15 7.57 7.30 9.84

7.94 16.25 18.61 3.82 40.36 8.87 5.09 8.97 14.83 9.59 2.63 9.26 11.11 10.39 7.35 7.44 9.17

1

Not measured.

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Dry matter Ash Protein (6.25 × N) Fat Starch Calcium Phosphorus Nonstarch polysaccharides2 Cellulose NCP3 Total NSP S-NCP4 I-NSP5 Klason lignin Dietary fiber6 Gross energy (kcal/kg of DM)

Soybean

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HAMMERSHØJ AND STEENFELDT TABLE 4. The effect of experimental diets with or without supplements on laying hen performance (20 to 31 wk)1 Treatment (T)2

Parameters

Am

SEM

T2

D3

S4

D×S

54.45b 2.83c 33.70c

1.17 0.09 1.03

0.0001 0.0001 0.0001

0.0001 0.0001 0.0001

0.0146 0.0868 0.0868

0.0169 0.0144 0.0144

90.48b — — —

92.20b 118.22a 93.12 25.10

0.19 1.00 0.50 0.84

0.0001 0.0492 0.3991 0.0788

0.0001

0.7624

0.2672

2.99a —

2.73bc 3.52a

0.04 0.12

0.0001 0.0001

0.0001

0.0196

0.1183

4.74 23.18 1.65

0.0001 0.0001

0.0001 0.0001

0.0001 0.0022

0.5027 0.3252

Ap

Bm

Bp

Cm

Cp

63.13a 3.65ab 43.41ab

61.19a 3.42b 40.73b

63.45a 3.53ab 42.04ab

48.25c 2.54d 30.29d

115.94a 113.56b 92.64 20.92 2.76b 2.71b

108.48a 112.99b 91.44 21.55

113.35a — — —

2.50c 2.61b 1,519 1,957b 28.9ab

2.78ab — 1,532 1,945b 27.0b

1,498 1,874c 25.1b

1,517 1,778d 17.2c

1,519 1,671e 10.0d

Means within each column with different letters differ significantly (P < 0.05). Values are means of 5 replicates. 2 T = effect of treatments (Am, Ap, Bm, Bp, Cm, Cp). Diets A, B, and C contain 0, 15, and 25% lupin, respectively, each with (p) or without (m) foraging material as supplement. 3 D = effect of diet (lupin inclusion level, A (0% lupin), B (15% lupin), C (25% lupin). 4 S = effect of supplement, with (p) or without (m) access to corn silage and carrots. a-e 1

tive effect on these parameters with diet C (the highest lupin and naked oats content), whereas no effect was seen with diets A and B. During the experimental period from 20 to 31 wk of age, there was no significant difference in egg production (%) among hens fed diets Am, Ap, Bm, and Bp (Table 5). At 24 to 25 wk of age, egg production values reached maximum and remained at that level for the rest of the experimental period. During the first 2 wk, the egg production of hens fed diets Cm or Cp was comparable with that for the other diets, but from 24 to 25 wk of age until 31 wk, the increase in egg production for these 2 groups was

significantly (P ≤ 0.0001) lower compared with the other diets. The largest egg weights were obtained with diets Am and Ap at 30 to 31 wk of age (Table 5). Access to corn silage and carrots did not have any effect on egg production with diets A or B, but improved egg production significantly (P < 0.05) with diet C from 28 to 31 wk (Table 5). In contrast, the supplements had a negative influence on the egg weight for all diets at the age of 24 to 31 wk, but was only significant for diet A at 28 to 29 wk and diet C at 30 to 31 wk (Table 5). A significant interaction was found between diet and supplement for egg production during

TABLE 5. The effect of experimental diets with or without supplements on laying hen performance1 Treatment (T)2

P-value

Am

Ap

Bm

Bp

Cm

Cp

SEM

T

D

S4

D*S

Hen-day egg production, % 20 to 21 wk 22 to 23 wk 24 to 25 wk 26 to 27 wk 28 to 29 wk 30 to 31 wk

20.62 75.33ab 91.91a 91.91a 88.11a 90.22a

21.67 73.86ab 87.43a 89.62a 89.38a 89.00a

18.86 74.91ab 88.81a 84.31a 82.74a 87.47a

20.31 76.27a 88.95a 91.53a 85.25a 90.88a

15.43 63.35b 76.83b 63.04b 56.94c 69.02c

22.52 69.03ab 77.87b 71.60b 71.85b 76.07b

1.02 1.41 1.46 2.30 2.35 1.71

0.4296 0.0460 0.0002 0.0001 0.0001 0.0001

0.6742 0.0092 0.0001 0.0001 0.0001 0.0001

0.1301 0.4633 0.5489 0.0674 0.0095 0.0232

0.4142 0.5076 0.4212 0.1426 0.0346 0.0446

Egg weight, g 20 to 21 wk 22 to23 wk 24to 25 wk 26 to 27 wk 28 to 29 wk 30 to 31 wk

45.43ab 52.21a 57.22a 59.80a 61.69a 64.38a

46.91ab 51.97a 56.55a 58.87a 60.12b 62.71a

43.98b 50.60b 54.89b 56.92b 58.10c 60.59b

43.49b 51.16ab 53.91b 56.45b 58.06c 60.06b

44.25ab 49.25c 51.07c 52.12c 54.93d 57.94c

48.79a 48.79c 50.62c 52.19c 53.98d 55.86d

0.50 0.27 0.49 0.59 0.52 0.55

0.0068 0.0001 0.0001 0.0001 0.0001 0.0001

0.0191 0.0001 0.0001 0.0001 0.0001 0.0001

0.0318 0.8674 0.0382 0.2521 0.0253 0.0009

0.0553 0.3030 0.7947 0.7054 0.1821 0.2423

Parameters

2

3

Means within each column with different letters differ significantly (P < 0.05). Values are means of 5 replicates. 2 T = effect of treatments (Am, Ap, Bm, Bp, Cm, Cp). Diets A, B, and C contain 0, 15, and 25% lupin, respectively, each with (p) or without (m) foraging material as supplement. 3 D = effect of diet (lupin inclusion level, A (0% lupin), B (15% lupin), C (25% lupin). 4 S = effect of supplement, with (p) or without (m) access to corn silage and carrots. a-d 1

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Total eggs (n/hen) 64.13a Egg mass (kg/hen) 3.76a Egg mass (g/hen per d) 44.81a Feed intake (as-fed; g/hen per d) Layer feed 115.08a Supplement, total — Carrots — Corn silage — Feed conversion (g of feed/g of egg) Layer feed 2.57bc Supplement, total — BW (g) 17 wk 1,501 31 wk 2,026a Relative BW gain, wk 17 to 31 (%) 35.0a

P-value

BLUE LUPIN IN ORGANIC EGG PRODUCTION

729

foraging material) were (P ≤ 0.05) less dry in textural attribute compared with all other treatment (data not given). The sensory panel found no further differences.

Aroma Compound Analysis

Yolk Color FIGURE 1. Sensory evaluation score of egg yolks from Am (control diet), Ap (control diet + foraging material), Cm (25% lupin), and Cp (25% lupin + foraging material) on the attribute “sulfur-like taste”. Scale ranged from 0 to 15 points. a,bBars with different letters are significantly different (P < 0.05).

wk 28 to 29 and wk 30 to 31, caused by a significant, positive effect of supplement with the C diet only. The average BW at 17 wk of age was 1,514 g, with no significant difference between the groups (Table 4). It was observed that consumption of supplements during the 12wk experiment subsequently had a significantly negative effect on the BW in all groups. The relative BW gain was especially low with diet C compared with the other groups. Mortality was generally low during the 12-wk experimental period, with group Cm being the exception. Mortality was 2% (Am), 0% (Ap), 0% (Bm), 1% (Bp), 4% (Cm), and 1% (Cp), and not significantly different. Cannibalism was observed to be the main cause of death, and access to foraging material seemed to have a positive effect on mortality in the groups fed diet C, indicating that the time spent on eating corn silage and carrots could be one explanation for reducing mortality caused by cannibalism.

Sensory Evaluation During the training sessions of the sensory panel, no distinct differences in albumen aroma or taste were found; hence, only the yolks were tested. The descriptors that were found to describe the aroma and taste of egg yolk were: fresh boiled egg, old boiled egg, sweet/corn, and butter. For the aftertaste of the samples, the descriptors were: sulfur-like and water of boiled fish. The textural attributes were: dry and gritty/mealy. An aftertaste described as sulfur-like was higher (P ≤ 0.05) in egg yolk samples from hens fed foraging material, but was unaffected by the level of lupin and naked oats in diet (Figure 1). Eggs from treatment Ap (no lupin, plus

There were significant dietary effects on all yolk color parameters (Figure 2). Lightness (L*) increased with increasing content of lupin and naked oats in the diet; however, supplementing foraging material counteracted this effect and yolks of these treatments became darker (Figure 3A). The a* (red-green) parameter was mainly affected by the supplement of foraging material, where increased intake resulted in less greenish yolks (lower negative a* values), whereas yolk a* of treatments without foraging material (Am, Bm, and Cm) were similar (Figure 2B). The yellowness (b*) of the yolk increased with increased lupin and naked oats content of diets and was not significantly affected by foraging material (Figure 2C). Hen age affected color values L* and b* significantly (Table 6), as the yolk lightness and yellowness increased. This indicates that the dietary effects may be time dependent regarding the deposition of yellow pigments into the yolk. No significant interactions of age and diet were found.

Albumen DM A significant dietary effect was observed for albumen DM content (Figure 3). Increasing levels of lupin and naked oats in diets resulted in decreased albumen DM content, whereas the supplement of foraging material had no significant effect on this parameter. Furthermore, the effect of age on albumen DM (Table 6) was insignificant.

DISCUSSION The performance of the hens fed the 15% lupin diet was comparable with hens fed the 0% lupin diet, whereas there was a significant negative effect on performance, egg weight, and daily egg mass of hens fed the 25% lupin diet, which was probably due to a significantly decreased feed intake. White lupin (L. albus) up to a 30% inclusion level in diets for layers resulted in good egg production (Prinsloo

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As the sensory panel found the largest differences in the yolk, it was decided to analyze only yolk samples by GCMS. The GC-MS analysis of volatile aroma compounds from the heated yolks showed only a few small peaks (intensity <100 kCounts; data not shown). Forty-eight samples were analyzed and the amount of volatile aroma compounds was found to be very small, probably due to coincidence with the detection limit of the apparatus. No differences among the treatments were detected. There was a trend towards a slightly higher intensity of the volatile compounds in Cp samples; however, it was insignificant. We did not identify sulfide compounds in any of the samples.

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et al., 1992), whereas others found a lower egg mass for hens fed diets with 25 and 30% white lupin compared with hens on a lupin-free diet (Watkins and Mirosh, 1987). In contrast with our results, the use of 25% lupin (L. angustifolius) in layer diets resulted in high performance in a 40-wk experiment, where hen-day egg production was higher than 90% and feed intake was approximately 120 g/hen per d (Perez-Maldonado et al., 1999). The content of the NSP fraction of 42.6% DM of the lupin in our study was comparable with some other studies (Gdala and Buraczewska, 1996; Bach-Knudsen, 1997; Petterson, 2000; Steenfeldt et al., 2003), but higher than the NSP content analyzed in the lupin (cultivar Gungurru) used by Perez-Maldonado et al. (1999). The higher NSP content in C diets could be one explanation for the lower feed intake here. In other studies, the feed intake was less influenced by increased lupin level, and Prinsloo et al. (1992) found that the feed intake tended to increase with increasing dietary lupin. Because the hens fed diet Cp had a high intake of the supplements, in spite of a high fiber content, the low feed intake of the layer diet and the resulting low production may be associated with other factors and not solely caused by a high fiber content. The poorer performance and lower BW gain obtained with 25% inclusion of lupin may be due to the inadequate FIGURE 2. Egg yolk color expressed by A) L*= lightness, B) a*= red/ green value and C) b*= yellow/blue value as a function of dietary treatments (n = 10): Am = control diet, Ap = control diet + foraging material, Bm = 15% lupin, Bp = 15% lupin + foraging material, Cm = 25% lupin, Cp = 25% lupin + foraging material. The foraging material was whole carrots and corn silage. a,b,cBars with different letters differ significantly at the given level within each diagram.

TABLE 6. Least square means of egg albumen DM and yolk color parameters L* (lightness), a* (red/green value), and b*(yellow/ blue value) as function of hen age (n = 30) Hen age (wk) 20 30 F-test

DM (g/kg)

Yolk L*

Yolk a*

Yolk b*

124.9 123.0 NS

62.31 64.72 P < 0.05

−2.62 −2.88 NS

52.54 55.21 P < 0.05

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FIGURE 3. Egg albumen DM content (g/kg) as function of dietary treatment (n = 10). Am = control diet, Ap = control diet + foraging material, Bm = 15% lupin, Bp = 15% lupin + foraging material, Cm = 25% lupin, Cp = 25% lupin + foraging material. a,bBars with different letters are significantly different (P < 0.001).

BLUE LUPIN IN ORGANIC EGG PRODUCTION

dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide were identified in eggs (MacLeod and Cave, 1975; Warren, 1994). These compounds have odors of “sulfurous, bad eggs, pungent, drains” (MacLeod and Cave, 1975), which may correspond with the present score for “sulfurlike” taste of egg yolks. Sulfides, thiopenes, trithiolane, and thiirane were previously identified in eggs (Warren, 1994), and were characterized as having strong odors with low sensory threshold values, which may be detected by sensory evaluation but not by GC-MS. The reason why foraging material causes a sulfur-like taste in eggs may be because it favors gram-positive flora, which predominate in the ceca and colon. In fact, these microorganisms were recently found to significantly contribute to the formation of malodorous compounds, which can lead to tainted eggs (Zentek and Kamphues, 2002). Some dietary components are known to affect the microflora of poultry, e.g., pentosans, β-glucans, cellulose, oligosaccharides, and lignin, which are not digested by endogenous enzymes (Ferket, 1991). Recently, it was found that the high content of 450 g/kg of DM of NSP (127 g/kg of DM cellulose and 9 g/ kg of DM lignin) in blue lupin depressed the digestibility in broilers by 10% (Steenfeldt et al., 2003). Even though the content of such indigestible components in laying hen diets may induce a “sulfur-like” taste of eggs (via intestinal microorganisms), the maximum level in diets to avoid such an effect is unknown and is not reported in the literature. Furthermore, different indigestible components may have different effects on the microflora. Hence, further studies are needed to elucidate the effects of indigestible components on intestinal microflora composition and egg taste and flavor. Inclusion of 87.6% naked oats in hen diets gave a significantly higher sensory score for sulfurous aroma in eggs than an oat-free diet (Cave et al., 1992). We found no effect of the lupin and naked oats diets on egg sensory evaluation, which could be due to the lower level of naked oats (12.5%). However, in a previous study, 16% lupin caused a bitter taste in eggs, but 8% lupin had no effect (Vogt et al., 1983). Yolk color is important to consumers and is one of the main parameters by which the quality of an egg is judged, even though color has nothing to do with nutritive value, freshness, or cooking characteristics. The color of egg yolk originates from yellow (e.g., lutein, zeaxanthin, apo-ester) and red (e.g., canthaxanthin, citraxanthin, astaxanthin) carotenoids or xanthophylls in the hen’s diet (Beardsworth and Hernandez, 2004). Corn and alfalfa are well known sources of carotenoids as well as carrots that contain αand β-carotene. The amount of xanthophylls on a DM basis ranges from 54 to 65 mg/kg in carrots and 20 to 25 mg/ kg in corn (Sikder et al., 1998). We chose the L*, a*, b* color system for analysis of yolk color (Oziemblowski and Grashorn, 1997) instead of the 15-grade Roche Yolk Color (RYC) fan to enhance objectivity. Lupin in hen diets was previously found to decrease yolk color at 16% inclusion (Vogt et al., 1987), or to have no effect with inclusions of 15% white lupin (Lupinus albus) meal (Quarantelli et al., 1993) or 30% sweet lupin (Prinsloo et al., 1992). Our study indicated that yellowness

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supply of Met. The consequence of low intake of Met, which is a methyl group donor, is reduced metabolism in animals (Pourreza and Smith, 1988), which affects protein synthesis for body growth and egg production. Research has shown that several parameters such as albumen solids (Shafer et al., 1998), egg performance (Keshavarz and Jackson, 1992; Harms and Russell, 1996a), egg size (Keshavarz and Jackson, 1992; Hsu et al., 1998), and BW (Pourreza and Smith, 1988; Keshavarz and Jackson, 1992; Harms and Russell, 1996a) are negatively affected by decreased dietary Met content and inadequate Met intake of the hen. The present low level of Met could therefore be limiting for optimum performance. The dietary Met requirement for production of 1 g of egg content (albumen and yolk) is ∼5.4 to 5.6 mg (Harms and Russell, 1996a,b). The high intake of supplements in the present study could be an attempt by the hens to cover their requirement for essential amino acids. However, the low protein concentration in both of the supplements, combined with a very high content of insoluble fiber in the corn silage, would make it physically difficult to obtain a sufficient nutrient intake. The Met intake ranged from 214 to 358 mg/hen per d (data not shown), and the recommended requirement is 300 mg/hen per d (NRC, 1994). Intake of sufficient protein, Lys, and Met during the laying period is essential for optimal egg production (egg number, egg weight, egg mass, and feed efficiency) (Al-Bustany and Elwinger, 1987; Hammershøj and Kjaer, 1999). In addition, feeding pullets with a low-Met and low-Lys diet in the starter/grower phase reduced development of immature BW and delayed onset of production, negatively influencing the egg production during the first 6 wk of lay (Halle, 2002). In our study, the dietary Lys content was not a limiting factor for egg production and quality, as daily intakes were 780 to 1118 mg, i.e. the recommendation of 690 mg/hen per d (NRC, 1994) was fulfilled. Another consequence of the low dietary Met content in the present diets Cm and Cp is the significantly lower DM content of albumen, which is likely to be an effect enhanced by the lower feed intake. A high DM content of the egg albumen is important when, for example, heating eggs to obtain a strong gel (Hammershøj et al., 2001), or processing eggs into dried powder products. Previous experiments have shown increased Met intake from 325 to 423 mg/hen per d to significantly increased albumen DM from 116 to 119 g/kg (Shafer et al., 1996). The protein intake decreased simultaneously from 16.6 to 14.8 g/hen per d; therefore, the observed increase in albumen DM was not due to increased protein intake (Shafer et al., 1996). Within the present Met intake range (214 to 358 mg/hen per d) there was a positive second-order correlation with the albumen DM with R2 = 0.767 (not shown). The hens on diets Cm and Cp may, in the long-term, be deficient in Met, as their requirement is not met. There were no differences in the volatile aroma compounds of yolks between treatments, and no volatile sulfur compounds were identified. Nevertheless, “sulfur-like” taste was significantly higher in the sensory evaluation of yolks from treatments with foraging material. Previously,

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ACKNOWLEDGMENTS The authors thank technician Kirsten Balthzersen, Danish Institute of Agricultural Sciences (DIAS) for daily hen management together with feed and egg registration, research scientist Mogens T. Jensen (DIAS) for performing the GC-MS analysis, and Ph.D. student Lisbeth Wienberg, The Royal Veterinary and Agricultural University, Copenhagen, for management of the sensory evaluation. Financial support was provided by the Directorate for Food, Fisheries and Agro Business.

REFERENCES Adams, C. A. 1985. Pigmenters and poultry feeds. The Feed Compounder 5:12–14. Al-Bustany, Z., and K. Elwinger. 1987. Shell and interior quality and chemical composition of eggs from hens of different strains and ages fed different dietary lysine levels. Acta Agric. Scand. 37:175–187. Alloui, O., S. Smulikowska, M. Chibowska, and B. Pastuszewska. 1994. The nutritive value of lupin seeds (L. luteus, L. angustifolius and L. albus) for broiler chickens as affected by variety and enzyme supplementation. J. Anim. Feed Sci. 3:215–227. AOAC. 1990a. Nitrogen (total) in fertilizers. Kjeldahl method no. 978.02. Pages 18–19 in Official Methods of Analysis. Association of Official Analytical Chemists, Arlington, VA. AOAC. 1990b. Official Methods of Analysis. Association of Official Analytical Chemists, Arlington, VA. Bach-Knudsen, K. E. 1997. Carbohydrate and lignin contents of plant materials used in animal feeding. Anim. Feed Sci. Technol. 67:319–338. Bach-Knudsen, K. E., and B. W. Li. 1991. Determination of oligosaccharides in protein-rich feedstuffs by gas-liquid chromatography and high-performance liquid chromatography. J. Agric. Food Chem. 39:689–694. Bartlett, M. S. 1937. Properties of sufficiency and statistical tests. Proc. R. Soc. Lond. 160:268–282. Beardsworth, P. M., and J. M. Hernandez. 2004. Yolk colour–An important egg quality attribute. Int. Poult. Prod. 12:17–18. Becker, P., and K. M. Weltring. 1998. Purification and characterization of alpha-chaconinase of Gibberella pulicaris. FEMS Microbiol. Lett. 167:197–202. Blæsild, P., and J. Granfeldt. 1995. Statistik for biologer og geologer. Dept. of Theoretical Statistics, University of Aarhus, Denmark. (In Danish). Cave, N. A., L. M. Poste, G. Butler, E. E. Farnworth, and V. D. Burrows. 1992. Effect of dietary level of naked oats (Avena nuda) on internal and sensory quality of eggs and on yolk lipid composition. Can. J. Anim. Sci. 72:147–153. Commission Regulation 2277. 2003. Amending annexes I and II to Council Regulation (EEC) 2092/91 on organic production of agricultural products and indications referring there to agricultural products and foodstuffs. Off. J. Eur. Union L336 46:68–74. ˚ man. 1993. Chemical composition of Daveby, Y. D., and P. A certain dehulled legume seeds and their hulls with special reference to carbohydrates. Swed. J. Agric. Res. 23:133–139. Det Danske Fjerkræra˚d / Danish Poultry Council. 2002. Beretning 2002. 1–200. (In Danish). Doyle, A. D., K. J. Moore, and D. F. Herridge. 1988. The narrowleafed lupin (Lupinus angustifolius L.) as a nitrogen-fixing rotation crop for cereal production. III. Residual effects of lupins on subsequent cereal crops. Aust. J. Agric. Res. 39:1029–1037. Ferket, P. R. 1991. Effect of diet on gut microflora of poultry. Zootecn. Int. 7-8:44–49. Francesch, M., V. A. Perez, M. Almirall, G. E. Esteve, J. Brufau, A. F. B. v. d. Poel, J. Huisman, and H. S. Saini. 1993. Utilisation

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(b*) of yolk increased in diet Cm, although not significantly, compared with diet Am, which supports findings from a study using inclusion of 20% lupin (Francesch et al., 1993). Inclusion of naked oats up to 80% in diets for hens was found to decrease RYC values significantly from 7.4 to 3.7 (Cave et al., 1992). The present supplement of foraging material had the most significant effect on yolk redness (higher a*), and darker (lower L*) and more yellow (higher b*) yolks were obtained. This is consistent with a study where 8% carrot meal in layer diets significantly increased RYC score from ∼2 to ∼4 (Sikder et al., 1998). A diet of 74% corn silage (corn and cob) increased RYC score from 5.4 to 7.2 compared with a 68% corn-wheat-barley grain diet (Jeroch, 1986), which is believed to be a direct result of the xanthophylls in corn (Adams, 1985). The subsequent increase in lupin and naked oats in diets A to C significantly increased yolk redness (a*), which could be a response of the corresponding lower feed intake of layer diets, together with a significantly higher intake of the foraging material in treatment Cp than in Ap and Bp. The only other dietary source of carotenoids in the present study is alfalfa meal. The alfalfa content, and thereby pigment contribution, decreases slightly from diet A to diet C; hence, the treatment effects on yolk color are even stronger. No standards are given for acceptable or required yolk color in organic eggs, which makes it difficult to provide guidance of pigment level in organic layer diets. A European survey recently showed that consumers prefer the darkest yolk colors presented to them, albeit with differences between countries (Beardsworth and Hernandez, 2004), as consumer perception to yolk color is generally linked to geographical location, culture, and traditions. In conclusion, lupin and naked oats are potential protein sources in laying hen diets together with supplements of foraging material. The present diets were formulated to fulfil the regulations of Danish organic egg production, where dietary inclusion of synthetic amino acids is prohibited. Fishmeal (<5%) is still permitted in organic layer diets as an important amino acid source; however, a ban on fishmeal in future organic feed is expected. The exclusion of fishmeal probably makes it impossible to formulate diets using 25% lupin with the required amount of Met for egg production. It is therefore recommended that fishmeal or other acceptable sources of sulfur amino acids are used because egg production and egg quality can be dramatically impaired. Supplementation with foraging material significantly improves egg production, but deteriorates the sensory evaluation of egg yolks. Whole carrots and corn silage as foraging materials improve yolk color. Further studies are required to ensure high productivity and quality of organic eggs with focus on improving dietary Met supply in organic egg production, ways to increase the DM content of the egg albumen, supplement the diet with natural yolk color pigments, and reduce the risk of development of sulfur taste. It would be valuable for the organic egg producer if future results could provide recommendations with respect to diet formulation, level, and source of supplements.

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