Inclusion Of Lutein And Zeaxanthin İn Standard Or Omega-3 Fatty Acids Enriched Laying Hens Diets:Effects On Performance, Egg Quality Parameters, Fatty Acid Composition And Individual Carotenoids Concentration Of Egg Yolk

DOI: https://doi.org/10.21203/rs.3.rs-1259724/v1

Abstract

The effects of lutein (L) and zeaxanthin (Z) inclusion to standard or omega-3 fatty acids enriched laying hens’ diets, on performance, egg quality parameters, fatty acids and individual carotenoids of egg yolk was investigated. The dietary treatments were designed as: standard (S) diet, 50 mg/kg L plus 50 mg/kg Z (S+L/Z (50+50)) or 100 mg/kg lutein plus 100 mg/kg zeaxanthin (S+L/Z (100+100)) added to S diet, Omega-3 fatty acids enriched (OM3) diet, 50 mg/kg lutein plus 50 mg/kg zeaxanthin (OM3+L/Z (50+50) or 100 mg/kg lutein plus 100 mg/kg zeaxanthin (OM3+L/Z (100+100)) added to OM3 diet. Hens with a mean live weight of 1525.88 ± 31.19 g were divided into six treatment groups. During the experimental period, there was not any mortality rate recorded. Dietary inclusion of L/Z (50+50 or 100+100) either in S or OM3 diets did not alter performance parameters. Eggs from hens fed enriched diet (OM3) significantly increased (P<0.05) the albumen index and Haugh unit. The type of dietary fat source (F), equal dose of L/Z or F×L/Z on egg yolk lutein, unknown carotenoids and total carotenoid concentration were found to be significant. However, the F on zeaxanthin concentration was not significant (P>0.05). The yolk color a* value was significantly increased by inclusion of L/Z (50+50 or 100+100) to diets (S or OM3) (P<0.05) and this was correlated with increased (P<0.05) concentration of individual carotenoids. The major changes in egg yolk fatty acid MUFA and PUFA composition occurred when laying hen fed with OM3 diet.

Introduction

Consumer surveys have shown that a growing concern to nutritional quality of eggs during the past decade. Many consumers prefer to consume eggs enriched by natural nutrients such as polyunsaturated fatty acids (omega-3), vitamins (D, E, etc.), minerals (iyodine, zinc, iron, selenium, etc.), phenolics flavonoids, and carotenoids to improve human health. The three major omega-3 (n−3) PUFA, α-linolenic acid (18:3 n−3, ALA), eicosapentaenoic acid (20:5 n−3, EPA) and docosahexaenoic acid (22:6 n−3, DHA) have been acknowledged as beneficial applications on growth, health and immune functions of human and animal (Fraeye et al., 2012; Alagawany et al., 2019). Dietary manipulation with n-3 PUFA sources can improve those fatty acids in egg yolk, as declared in previous studies (Basmacıoğlu et al., 2003; Kralik et al., 2008; Wu et al., 2019).

Egg yolk colour is the most important attribute among the physical characteristics for egg quality on consumer perception (Rajput et al., 2012). Thus, yolk colour has higher market value. There is a variation in yolk colour due to the source of the pigmentation (natural or synthetic) and their utilization and combination between xanthophyll’s, stability, availability, feed composition, genetic and physiological status of laying hen, as well as other factors (Berkhoff et al., 2020). Layer and all other animals cannot synthesize pigments in the nova, they must take them from their diet.

The natural carotenoids lutein and zeaxanthin are liable for the orange-yellow color of the egg yolk. Egg consumption via enriched lutein and zeaxanthin (xanthophyll, carotenoids) has epidemiologically related with specific human health, including preventing macular degeneration (Mares, 2016), protecting against cardiovascular diseases (Andersen 2015), oxidative stress (Fiedor and Burda, 2014), neurodegenerative disorders (Zaheer, 2017) and cancer (Mares-Perlman et al., 2002). It has been reported in recent works the possibility of produce carotenoids enriched eggs (lutein and zeaxanthin) (Leeson and Caston, 2004; Skŕivan et al., 2015; Pitarque et al., 2019). Although the xanthophylls have used as the main source of egg yolk pigmentation for poultry industry, limited information is available on the efficiency of transport of lutein and zeaxanthin into egg and their bioavailability, and little published information is available about their combination with fish oil or flaxseed, which are ingredients commonly used in producing designer eggs (Gonzalez and Leeson, 2001; Leesen and Caston, 2004; Panaite et al., 2021).

Therefore, the main aim of this study was to inclusion of two high doses of L/Z (50+50) and L/Z (100+100) to standard or n-3 fatty acids enriched control diet on performance, egg quality parameters, fatty acid composition and individual carotenoids of egg yolk.

Materials And Methods

According to the principles of the Ege University Animal Research Ethics Committee and national law (no. 5199), the animals used in this experiment were kept in safe conditions.

Laying Hens, Experimental Diets And Management

In an open-sided housing, 108 Super-nick laying hens were placed individually in triple-deck battery cages (41×41×50 cm). Hens with a mean live weight of 1525.88 ± 31.19 g were randomly distributed to six experiment groups, each group have three replicates (6 hens/each replicate). Lutein (DSM, Nutritional Products Ltd, Switzerland, 5% lutein), zeaxanthin (OPTISHARPTM, DSM, Nutritional Products Ltd, Switzerland, 5% zeaxanthin), were added to two main standard or omega-3 fatty acids enriched basal diets. The diets were formulated according to layer hens at age of 35-40 weeks requirements reported by NRC (1994). Standard (S) basal diet contained sunflower oil (25 g/kg) and omega-3 enriched (OM3) basal diet contained flax seed (39 g/kg) and fish oil (15 g/kg) to enrich omega-3 PUFA content of egg yolk. The nutrient composition of the diets used in this experiment is shown in Table 1. Experimental diets were also analyzed for individual carotenoids (Table 2).

Table 1

Nutrient composition of standard and omega-3 fatty acids enriched control diets (g/kg)

Diet

S

OM3

Analysed composition (g/kg)

   

Dry matter

885.0

887.0

Crude protein

171.7

173.3

Ether extract

44.20

45.20

Crude fibre

18.00

16.50

Crude ash

130.3

130.5

Starch

347.3

342.5

Sugar

55.9

58.7

Calcium

37.2

38.9

Total phosphorus

5.1

6.5

Calculated composition

   

Metabolisable energy (MJ/ kg)a

11.55

11.56

Fatty acid profile (%)

   

ΣSFAb

16.84

15.82

ΣPUFAc

39.90

48.10

Σn-6FAd

39.12

45.12

Σn-3FAe

0.78

2.98

Σn-6FA/Σn-3FA ratiof

50.15

15.14

S: Standard diet, OM3: Omega-3 fatty acids enriched diet
aMetabolisable energy content of diet was estimated using the equation of Carpenter and Clegg (Leeson and Summers, 2005).
bΣSFA (Total Saturated Fatty Acid): ΣSFA = C14:0 + C16:0 + C17:0 + C18:0+ C20:0+C21:0.
cΣPUFA (Total Polyunsaturated Fatty Acid): ΣPUFA = C18:2n-6 +C18:3n-6 + C20:5n-3 + C22:6n-3.
dΣn-6FA (Total n-6 Fatty Acid): Σn-6FA = C18:2n-6 + C18:3n-6.
eΣn-3FA (Total n-3 Fatty Acid): Σn-3FA = C20:5n-3 + C22:6n-3.
fΣn-6FA/Σn-3FA ratio = Σn-6FA / Σn-3FA

Table 2

The concentration of individual and total carotenoids in experimental diets (µg/g)

   

Treatments

SEM

P-values

Diet

 

S

     

OM3

 

L/Z (mg/kg)

0

50+50

100+100

 

0

50+50

100+100

Lutein

5.28e

60.17c

98.20b

 

6.29e

36.32d

120.83a

4.65

<0.001

Zeaxanthin

4.71c

49.11b

82.97a

 

5.18c

40.45b

94.88a

3.85

<0.001

β- carotene

0.25

0.24

0.15

 

0.17

0.20

0.55

0.12

0.274

Unknown carotenoids

0.29c

0.42c

0.63ab

 

0.30c

0.46bc

0.80a

0.04

<0.001

Total carotenoids

11.54e

111.38c

183.48b

 

13.10e

78.28d

218.90a

5.18

<0.001

S: Standard diet, S+L/Z(50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to S diet, S+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to S diet, OM3: Omega-3 fatty acids enriched diet, OM3+L/Z (50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to OM3 diet and OM3+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to OM3 diet.
SEM: Standard error of the mean
P-values: Probability of a significant effect
a−eMeans with no common superscripts within the row of each classification are significantly (P<0.05) different.

Each diet was freshly prepared within a week. Water and experimental feed were supplied ad libitum. Lighting schedule was 17 h light and 7 h dark. The temperature in the henhouse was recorded between 11-16°C during the experimental period.

Sample Collection

Daily collected eggs were coded according to their treatment group. On a weekly basis, egg production (%), feed consumption (g/hen/day) and feed conversion rate (kg feed consumed/kg eggs) were calculated. Egg quality parameters (i.e egg weight, shape index, shell strength and shell thickness, yolk index, albumen index, Haugh Unit) and yolk color were measured biweekly.

The eggs were weighed (g) in a precision scale after being kept at room temperature for 24 hours. The albumen height, yolk diameter, albumen length and width of egg were measured with a digital display caliper for to calculate yolk index and albumen index. The Haugh Unit (HU) was determined by calculating with the given equation. HU = 100 × log (H + 7.5 – 1.7 × W0.37), where H is the measurement of the albumen height (mm), W is the measurement of the egg weight (g). Shell strength (kg/cm×cm) was determined by using machine with “spiral pressure system” and shell thickness (mm) was measured by using micrometer after the shells were dried for 6-8 hours in a vacuum oven set at 60°C. Evaluation of yolk colors was performed by using Chroma meter (CR-300, Minolta, Japan) daily calibrating against a white standard instrument.

Chemical Analysis

According to procedures of AOAC (1990), the diets (S or OM3) were analyzed for dry matter (code: 930.15), crude protein (code: 976.06), crude ash (code: 942.05) and ether extract (code: 920.39). Also, according to VDLUFA method (Naumann and Bassler, 1993), the diets were analyzed for sugar, starch, phosphorus and total calcium. Metabolizable energy (ME) concentration of diets was estimated by using the equation of Carpenter and Clegg (Leeson and Summers, 2005): ME, kcal/kg= 53 + 38 [(CP, %) + (2.25 × ether extract, %) + (1.1 × (starch + sugar), %)].

Analysis of carotenoid concentration of experimental diets and egg yolk

The carotenoid concentration evaluation of the experimental diets and egg yolk was performed by using Shimatzu Prominence HPLC. Diet samples were saponificated with ethanolic potassium hydroxide in the presence of pyrogallol (Leeson and Summers, 2005). For egg yolk extraction 200-300 mg egg yolk was weight and mixed in 0.7 ml 5% sodium chloride, then 1 ml ethanol was added and samples were homogenized with 2 ml hexane twice, centrifuged and hexane phase removed from tube. Then samples were evaporated under nitrogen. The final residue was re-dissolved with 1:1, v/v dichloromethane-methanol and transferred to vial. Individual carotenoids (lutein, cis-lutein, zeaxanthin, β- carotene, unknown carotenoids) were determined by peaks were identified by comparison standards (variously obtained from Sigma, Poole, UK; Hoffman-La Roche, Basel, Switzerland) (Granado et al., 1998).

Analysis of fatty acid composition of experimental diets and egg yolk

The fatty acids analysis of experimental diets and egg yolk samples extracted with a modification of the method based on Bligh and Dyer (1959) was acquired by HP (Hewlett-Packard)-Agilent/6890 GC. The conditions of chromatogram were carried out as following orders: the oven temperature was maintained at 140 ºC for 5 minutes, after waited at this temperature and reach 240 ºC with an increase of 4 ºC/min., and hold for 20 min. at this temperature and sample volume injected 1 µl.

Statistical analysis

In SPSS 13.0 program according to 2×3 factorial design was applied for statistical analysis. Differences between the treatment groups were evaluated according to Tukey’s test.

y ijl= µ + Fi + (L/Z)j + (F × (L/Z))ij + eijklwhere yijl is the observation; µ is the overall mean; Fi the type of dietary fat source (i = 1-2); (L/Z)j the equal dose of lutein and zeaxanthin (j = 1-3); (F × (L/Z))ij the interaction between the type of dietary fat source and equal dose of lutein and zeaxanthin and eijkl the residual error.

Results

Dietary inclusion of L/Z (50+50 or 100+100) either in S or OM3 diets did not alter performance parameters. During the 5 weeks experimental period, there was not any mortality rate recorded and the mean value of feed intake (FI) for laying hens fed with S and OM3 diets were 117.51 (g/hen/day) and 117.80 (g/hen/day), respectively. Moreover, no variation in results of egg production (%), feed conversion ratio (FCR) and final body weight (BW) were noted as a significance of F, L/Z or their interaction F×L/Z (Table 3). The mean egg production, FCR and final BW were 95.19 (%), 1.94 (kg feed /kg egg) and 1536.82 (g), respectively. Eggs from hens fed enriched diet (OM3) significantly increased (P<0.05) the albumen index and Haugh unit (Table 4).

Table 3

Performance of laying hens

   

Treatments

P-values

Diet

S

 

OM3

SEM2

F

L/Z

F×L/Z

L/Z (mg/kg)

0

50+50

100+100

 

0

50+50

100+100

Feed intake

(g/hen/ day)

117.51

118.73

118.92

 

117.80

112.60

117.72

1.54

0.089

0.235

0.107

Egg production

(%)

95.08

95.24

94.92

 

94.29

94.76

96.83

0.93

0.784

0.437

0.524

Feed conversion

(kg feed/kg eggs)

1.98

1.91

1.92

 

1.97

1.89

1.95

0.04

0.983

0.177

0.781

Initial body weight

(g)

1531.67

1564.67

1532.94

 

1566.67

1470.89

1488.44

31.19

0.179

0.425

0.173

Final body weight

(g)

1534.33

1567.83

1541.22

 

1589.67

1494.11

1493.78

27.96

0.339

0.268

0.099

S: Standard diet, S+L/Z(50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to S diet, S+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to S diet, OM3: Omega-3 fatty acids enriched diet, OM3+L/Z (50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to OM3 diet and OM3+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to OM3 diet.
SEM: Standard error of the mean
P-values: Probability of a significant effect are due to type of dietary fat source (F), equal dose of lutein and zeaxanthin (L/Z) and their interaction (F×L/Z).

Table 4

Egg quality parameters of laying hens

   

Treatments

P-values

Diet

S

 

OM3

Pooled SEM

F

L/Z

F×L/Z

L/Z (mg/kg)

0

50+50

100+100

 

0

50+50

100+100

Egg weight (g)

62.47

65.48

65.28

 

63.55

63.00

62.50

0.81

0.056

0.325

0.062

Shape index (%)

73.81

74.47

74.01

 

74.20

75.29

73.95

0.44

0.288

0.076

0.213

Shell strength (kg/cm2)

3.08

3.29

3.18

 

3.26

3.16

3.22

0.13

0.761

0.910

0.838

Shell thickness (mm)

0.38

0.38

0.38

 

0.39

0.38

0.38

0.004

0.107

0.334

0.102

Albumen index (%)

9.30b

9.52

9.68

 

10.58a

9.91

10.29

0.34

0.010

0.711

0.086

Yolk index (%)

44.23

45.34

45.18

 

44.45

44.96

44.76

0.44

0.601

0.182

0.417

Haugh Unit

84.88b

85.11

86.41

 

88.47a

86.55

88.06

1.28

0.040

0.545

0.242

S: Standard diet, S+L/Z(50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to S diet, S+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to S diet, OM3: Omega-3 fatty acids enriched diet, OM3+L/Z (50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to OM3 diet and OM3+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to OM3 diet.
SEM: Standard error of the mean
P-values: Probability of a significant effect are due to type of dietary fat source (F), equal dose of lutein and zeaxanthin (L/Z) and their interaction (F×L/Z).
a−bMeans with no common superscripts within the row of each classification are significantly (P<0.05) different.

Dietary inclusion of L/Z (50+50 or 100+100) to S diet increased both the lutein content (by 32.49 µg/g and 39.16 µg/g) and the zeaxanthin content (by 26.42 µg/g and 31.28 µg/g) in yolks of eggs, respectively. Also, inclusion of L/Z (50+50 or 100+100) to OM3 diet increased both the lutein content (by 31.15 µg/g and 34.96 µg/g) and the zeaxanthin content (by 26.41 µg/g and 34.46 µg/g) in yolks of eggs, respectively. The effect of the type of dietary F and equal dose of L/Z or F×L/Z on egg yolk lutein, unknown carotenoids and total carotenoid concentration were found to be significant. However, the zeaxanthin concentration was altered by the equal dose of L/Z and the interaction between F×L/Z, regardless the type of dietary fat source (Table 5). Similarly, the pigment concentration of lightness (L*) and redness (a*) values were significantly changed by the equal dose of L/Z and the interaction between F×L/Z. The yolk color a* value was significantly increased by inclusion of L/Z (50+50 or 100+100) to diets (S or OM3) (P<0.05) and this was correlated with increased (P<0.05) concentration of individual carotenoids (Table 5). The changes for yolk color a* and b* values have shown in Figure 1 and 2. The dietary treatments of S or OM3 did not vary in yolk color a* and b* over the 35-d period when compared to L/Z (50+50 or 100+100) added to these diets. Figure 1 indicated that pigment accumulation of first 5 to 7 d, after which yolk color a* produced stability when dietary L/Z (50+50 or 100+100) added to either S or OM3 diets. After stabilization (8 to 35 d) the L/Z (100+100) added to S diet had the greatest yolk pigmentation, followed by the L/Z (100+100) added to OM3 diet, L/Z (50+50) added to OM3 diet and L/Z (50+50) added to S diet.

Table 5

The concentration of (µg/g) individual carotenoids in the egg yolk measured by HPLC and Minolta L*, a*, b* pigment record

 

Results measurement by HPLC

   
 

Treatments

 

P-values

Diet

 

S

     

OM3

 

Pooled

SEM

F

L/Z

F×L/Z

L/Z (mg/kg)

0

50+50

100+100

 

0

50+50

100+100

Lutein

10.35c

42.84b

49.51a

 

9.26c

40.41b

44.22ab

1.50

0.023

<0.001

<0.001

Zeaxanthin

7.43c

33.85b

38.71ab

 

7.06c

33.47b

41.52a

1.28

0.516

<0.001

<0.001

Unknown carotenoids

1.13c

5.66ab

6.25a

 

0.92c

4.74b

5.64ab

0.22

0.003

<0.001

<0.001

Total carotenoids

21.87d

97.89bc

111.93a

 

19.87d

90.92c

105.26ab

2.90

0.036

<0.001

<0.001

 

Results measurement by Minolta

       
 

Treatments1

 

P-values

Diet

 

S

     

OM3

 

Pooled

SEM

F

L/Z

F×L/Z

L/Z (mg/kg)

0

50+50

100+100

 

0

50+50

100+100

L*

61.07a

55.63b

55.81b

 

60.80a

56.89b

55.93b

0.32

0.165

<0.001

<0.001

a*

-5.69c

2.32b

3.57a

 

-5.40c

2.49b

3.61a

0.16

0.224

<0.001

<0.001

b*

39.63ab

41.13a

41.90a

 

35.32d

37.02cd

37.91bc

0.53

<0.001

0.026

<0.001

S: Standard diet, S+L/Z(50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to S diet, S+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to S diet, OM3: Omega-3 fatty acids enriched diet, OM3+L/Z (50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to OM3 diet and OM3+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to OM3 diet.
SEM: Standard error of the mean
P-values: Probability of a significant effect are due to type of dietary fat source (F), equal dose of lutein and zeaxanthin (L/Z) and their interaction (F×L/Z).
a−cMeans with no common superscripts within the row of each classification are significantly (P<0.05) different.

Dietary inclusion of L/Z (50+50 or 100+100) either S or OM3 diets did not alter saturated fatty acids (C14:0, C16:0, C18:0) of egg yolk. However, the F and their interaction F×L/Z on total polyunsaturated fatty acid (ΣPUFA) composition of egg yolk was significant (Table 6). The major changes in egg yolk fatty acid composition when laying hen fed OM3 diet can be summarized as an increase of MUFA and PUFA. In fact, laying hen fed OM3 diet increased egg yolk fatty acid of C16:1. In addition, depending on the type of dietary F and their interaction F×L/Z, the egg yolk fatty acids of C18:1, C20:1 n-9, C18:3n-3, C20:5n-3 and C22:6n-3 was increased significantly when laying hens fed with OM3 or OM3+L/Z (50+50 or 100+100) diets. Instead, the fatty acid composition of C18:2n-6 remained constant regardless of type of dietary F or F×L/Z.

Table 6

Fatty acid composition (% of total fatty acid methyl esters) of egg yolk

   

Treatments

P-values

Diet

S

 

OM3

SEM

F

L/Z

F×(L/Z)

L/Z (mg/kg)

0

50+50

100+100

 

0

50+50

100+100

C14:0 Myristic

0.34

0.36

0.34

 

0.36

0.38

0.39

0.01

0.081

0.651

0.388

C16:0 Palmitic

25.25

23.80

23.84

 

23.87

23.00

24.41

0.43

0.159

0.059

0.058

C16:1 Palmitoleic

2.84b

2.84b

2.90b

 

3.30a

3.36a

3.43a

0.23

0.019

0.912

0.255

C18:0 Stearic

6.77

6.76

6.73

 

7.16

7.09

7.25

0.27

0.086

0.971

0.607

C18:1 Oleic

46.16abc

45.60bc

44.86c

 

47.74ab

47.82ab

48.30a

0.45

0.000

0.163

0.001

C18:2n-6 Linoleic

18.50

18.56

18.04

 

18.48

18.73

18.30

0.28

0.563

0.254

0.610

C18:3n-3 Linolenic

0.58b

0.60b

0.71b

 

2.31a

2.44a

2.49a

0.12

<0.001

0.460

<0.001

C20:1n-9 Gondoic

0.32abc

0.31bc

0.28c

 

0.33ab

0.35ab

0.36a

0.01

<0.001

0.546

0.002

C20:5n-3 EPA

0.03b

0.02b

0.02b

 

0.39a

0.39a

0.42a

0.03

<0.001

0.852

<0.001

C22:6n-3 DHA

0.13b

0.10b

0.10b

 

0.33a

0.32a

0.31a

0.02

<0.001

0.234

<0.001

ΣSFAd

32.36

30.92

30.91

 

31.40

30.46

32.04

0.50

0.820

0.097

0.138

ΣMUFAe

49.32bc

48.75c

48.04c

 

51.37ab

51.53a

52.09a

0.45

<0.001

0.120

<0.001

ΣPUFAf

19.24b

19.28b

18.87b

 

21.51a

21.88a

21.52a

0.76

<0.001

0.591

<0.001

Σn-6FAg

18.50

18.56

18.04

 

18.48

18.73

18.30

0.28

0.563

0.254

0.610

Σn-3FAh

0.74b

0.72b

0.83b

 

3.03a

3.15a

3.22a

0.23

<0.001

0.871

<0.001

Σn-6FA/Σn-3FAi

25.00a

25.78a

21.73a

 

6.10b

5.95b

5.68b

0.79

<0.001

0.098

<0.001

S: Standard diet, S+L/Z(50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to S diet, S+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to S diet, OM3: Omega-3 fatty acids enriched diet, OM3+L/Z (50+50): 50 mg/kg lutein plus 50 mg/kg zeaxanthin added to OM3 diet and OM3+L/Z (100+100): 100 mg/kg lutein plus 100 mg/kg zeaxanthin added to OM3 diet.
SEM: Standard error of the mean
P-values: Probability of a significant effect are due to type of dietary fat source (F), equal dose of lutein and zeaxanthin (L/Z) and their interaction (F×L/Z).
a−cMeans with no common superscripts within the row of each classification are significantly (P<0.05) different.
dΣSFA (Total Saturated Fatty Acid): ΣSFA = C14:0 + C16:0 + C18:0
eΣMUFA (Total Monounsaturated Fatty Acid): ΣMUFA= C16:1 + C18:1 + C20:1n-9.
fΣPUFA (Total Polyunsaturated Fatty Acid): ΣPUFA = C18:2n-6 + C18:3n-3 + C20:5n-3 + C22:6n-3.
gΣn-6FA (Total n-6 Fatty Acid): Σn-6FA= C18:2n-6.
hΣn-3FA (Total n-3 Fatty Acid): Σn-3FA = C18:3n-3 + C20:5n-3 + C22:6n-3.
iΣn-6FA/Σn-3FA = Σn-6FA / Σn-3FA.

Discussion

In this experiment, compared with the S or OM3 diets, the addition of pigments at high level L/Z (50+50 or 100+100) did not have any significant impact on the egg production, FI, FCR or final BW during the 5-week experimental period (P>0.05). Findings of these data are in line with previous studies which have been reported that pigments did not have any significant effects on feed intake or body weight (Pérez-Vendrell et al., 2001; Li et al., 2012, Karadas et al., 2016) or egg production data were not significantly affected by using different dose or type of carotenoids in laying feeds (Hasin et al., 2006; Zhang et al., 2011; Lu et al., 2013; Karadas et al., 2016). Leeson and Caston (2004) also reported that dietary 125, 250, 375, 500, 625, 750 and 1000 ppm lutein supplementation had no significant effect on egg production data is in line with the results of this experiment. However, it has been reported that inclusion of marigold flower extract at 150 mg/kg dose in hens fed provide the highest hen-day egg production (Skřivan et al., 2015) not in agreement with our results.

As seen in Table 4, the mean values for albumen index and Haugh Unit were parallel to each other, which were significantly increased (P<0.05) when laying hens fed with OM3 diet. These finding data are in line with previous studies (Al-Daraji et al., 2010; Promila et al., 2017) which have been reported that higher levels of linseed or linseed oil significantly increase these values when they added to layer diets. However, these finding data are not compatible with results of the studies (Jiang et al. 1991; Scheideler et al., 1998; Grobas et al., 2001) who found no significant effect on HU and albumen index. These differences between findings data could be due to a strain-diet interaction, as mentioned by Scheideler et al. (1998).

The accumulation of xanthophylls (lutein/zeaxanthin) in egg yolk is noteably affected by dietary factors including type and concentrations of carotenoids, type of dietary fat, extent of processing, etc. and non-dietary factors including management system and physiological status of laying hens such as stress, diseases, age and breed (Zaheer, 2017; Pitarque et al., 2019). Such dietary factors as the level and saturation of fat have important role in bioavailability of xanthophylls due to their transportation of low-density lipoprotein and high-density lipoprotein (Papadopoulos et al., 2019).

There are series of steps that are associated with the release of lutein and zeaxanthin from the dietary matrix, the transfer of lutein and zeaxanthin to micelles, the absorption of lutein and zeaxanthin by intestinal membrane, and the transport of lutein and zeaxanthin to the blood and egg yolk. There are several factors that affect the absorption, bioaccessibility or bioavailability of carotenoids (Goltz et al., 2012). The literature reported controversial data on the effect of fat source for bioavailability of carotenoids. Some researchers (Hu et al., 2000; Gleize et al., 2013) suggested in vitro studies that dietary fats rich in saturated fatty acids led to higher bioavailability of lutein and zeaxanthin. On the other hand, some researchers (Failla et al., 2014; Mashurabad et al., 2016) suggested in vitro studies that the extent of micellarization of carotenoids increased with dietary rich in unsaturated fatty acid when compared to saturated fatty acid.

In this experiment, dietary inclusion of L/Z (50+50 or 100+100) either S or OM3 diets significantly increased the individual carotenoid concentrations of egg yolks paralleled those in the feed. But interestingly there was significant different has been recorded for egg yolk lutein concentration between L/Z (50+50) and L/Z (100+100) added to S diet. However same significant was not recorded for zeaxanthin level of egg yolk. For standard egg yolk feed lutein transport to egg yolk were more efficient compare to zeaxanthin. Parallel results were recorded with 125 ppm lutein in the diet of layer even higher level of lutein is recorded in 500 ppm lutein in the diet there was not difference lutein concentration in egg yolk by different lutein inclusion of 375 to 1000 ppm (Leeson and Caston, 2004). Similar results were obtained by Steinberg et al. (2000), with maximum 120 ppm level of lutein added to layer diets. Skřivan et al. (2015) reported that the addition of marigold flower extracts (350 mg/kg) rich in lutein and zeaxanthin increased both the lutein and zeaxanthin content of egg yolk when compared to control diet, which are in agreement with our results.

These accumulation carotenoids were recorded opposite effect between L/Z (50+50 or 100+100) added to OM3 diet and it was seen that zeaxanthin more efficiently accumulate in egg yolk compare to lutein concentration (P<0.05). Leeson et al. (2007) indicated that when flaxseed added to layer diets the accumulation of lutein into the egg reduced are in agreement with our results. But this concern not seen by zeaxanthin supplement diets. Therefore, in commercial omega-3 enriched egg production could be better for zeaxanthin supplementation to layer diet.

Since zeaxanthin and lutein are both found in the retina to form yellow pigments to protect eye from light and retinal damage (Tapiero et al., 2004). But for enrichment carotenoids egg yolk as a functional food production, we should consider the cost of feed and accumulation of carotenoids in the egg yolk as well. Factors such as appropriate doses of carotenoids, storage conditions, and the combination of dietary carotenoids influence optimal health-promoting aspects of carotenoids (Merhan, 2017).

In this experiment, egg yolk colorimeter (L* and a*) measurement results (Table 5) showed that compared with the S or OM3 diet, the addition of pigments at high level L/Z (50+50) and L/Z (100+100) cause significant improvement of L* and a*. This result is in agreement with report of Alay and Karadas (2016) who investigated the same dose (10 mg/kg) of carotenoids pigments (apoester, canthaxanthin, paprika oleoresin and aztec marigold extract pigments addition to non-pigmented wheat-soybean based quail’s diet improve calorimeter results of L*, a* values compare non-supplemented control group. However, ISA brown’s corn-soybean basal diet supplemented with different concentration (120,180 and 240 ppm) of marigold extract or with 40, 60 and 80 ppm apoester did not significantly affect L* values (Sirri et al., 2007) result not in line with our L*data. But, Hy-line White strain egg yolk L* values getting decreased by higher concentration of marigold 120 and 240 ppm or 40, 60, 80 ppm supplement of apoester (Sirri et al., 2007) showed significant different results are in line with our results. Similarly, redness a* results are in line with our results since all concentrations significantly improved in both strain (Sirri et al., 2007). Our L* and a* values are in agreement with previous reports that using red and yellow sources of pigments in hen’s diets associate with increasing a* values but decreased L* values (Niu et al., 2008; Skřivan et al., 2015). Minolta b* values showed interesting results in our experiment, OM3 group significantly low values compare all other groups except OM3+L/Z (50+50) group. However, L/Z (50+50 or 100+100) added to S diet could not change b* values significantly (P>0.05), since higher concentration of pigments shaded yellowness and converted to redness. Similar results have been reported by (Sirri et al., 2007) using different concentration of Marigold extract (120, 180 and 240 ppm) or 40, 60, 80 ppm apoester added to ISA Brown’s or Hy-line white strain layer corn-soybean diets did not significantly changed yellowness (b* values). It is important underline that compare all standard groups b* values were significantly decreased when laying hens fed with OM3 diet, even this diet (OM3) supplemented with L/Z (50+50) some improved seen but reached at the level of control at the L/Z (100+100) added to OM3 diet. In commercial condition in case of omega-3 enriched eggs production b* value reduction has to be taken in consideration. Egg yolk Roche color fan score was not affected by high dose of lutein (between 250-1250 ppm) in diet of layer (Leeson and Caston, 2004) are in agreement with our results even we did not record Roche color fan score data in case of b* value. Loetscher et al. (2013) was reported as lutein and zeaxanthin are highly active as yolk colorants are in line with our results.

In this experiment, the total saturated fatty acid (SFA) composition remained constant while total PUFA and MUFA composition was higher when laying hens fed with OM3 or OM3+L/Z (50+50 or 100+100) diets. The present results are in agreement with the findings of some other studies using different doses of fish oil, flaxseed or its oil (Galobart et al., 2001; Basmacıoğlu et al., 2003; Souza et al., 2008). Also, the absorption of MUFA especially C18:1 and C20:1n-9 seems to be encouraged by the dietary OM3, OM3+L/Z (50+50) and OM3+L/Z (100+100). Selvaraj and Cherian (2004) reported that increasing the levels of dietary fat increased the circulation of fatty acids which are preferentially deposited into specific tissues. This could result in increased MUFA composition of egg yolk. However, such lutein or zeaxanthin effect has not previously reported, so the reason for high egg yolk MUFA and PUFA composition are not due to the effect of lutein and zeaxanthin.

Conclusion

1. The results of this experiment showed that enrichment of egg yolk by carotenoids and omega-3 are possible. It has been reported that zeaxanthin and lutein are both found in the retina to form yellow pigments to protect eye from light and retinal damage, consumption of carotenoids enrichment egg yolk will be benefit for human health.  

2. S+ L/Z (50+50 or 100+100) inclusion to layer diet significantly improved total carotenoids, unknown carotenoids, lutein and zeaxanthin concentration of egg yolk compare to standard diets. However, lutein transfer from diet to egg yolk was efficiently than zeaxanthin. Opposite record was seen in case of omega-3 enrichment egg yolk. Zeaxanthin accumulation was more efficiently compare to lutein. But for production of functional food as enrichment of egg yolk by omega-3 or carotenoids enrichment or both of them optimum concentration needs to be taken account for economical purpose. 

Declarations

Author contribution 

All authors contributed to the concept and design of the study. Material preparation and data collection was carried out with Burcu Aktaş and Pınar Özdemir. Laboratory analyses and stastical analyses conducted with Hatice Basmacıoğlu-Malayoğlu, Filiz Karadaş and Burcu Aktaş. The first draft of the article was written by Hatice Basmacıoğlu-Malayoğlu, and all authors have commented on previous versions of the article. All authors have read and approved the final article.

Funding No funds, grants or other support was received.

Data availability All data in this study are available. The nutrient composition of the diets used in this experiment is shown in Table 1. Experimental diets were also analyzed for individual carotenoids in Table 2. Performance of laying hens and egg quality parameters are shown in Table 3 and Table 4. The individual carotenoids in the egg yolk are shown in Table 5. Minolta L*, a*, b* pigment record also in Table 5 and Fig 1 and Fig 2. Fatty acid composition of egg yolk is included in Table 6.

Ethics approval According to the principles of the Ege University Animal Research Ethics Committee and national law (no. 5199), the animals used in this experiment were kept in safe conditions.

Conflict of interest The authors declare no competing interests.

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