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. 2023 Nov 17;103(2):103286. doi: 10.1016/j.psj.2023.103286

Enrichment efficiency of lutein in eggs and its function in improving fatty liver hemorrhagic syndrome in aged laying hens

Dieudonné M Dansou 1, Han Chen 1, Yanan Yu 1, Youyou Yang 1, Isabelle N Tchana 1, Liyuan Zhao 1, Chaohua Tang 1, Qingyu Zhao 1, Yuchang Qin 1, Junmin Zhang 1,1
PMCID: PMC10762472  PMID: 38100949

Abstract

In this study, we evaluated the enrichment efficiency of lutein in eggs and its function in preventing fatty liver hemorrhagic syndrome (FLHS) in aged laying hens. Five groups of laying hens (65 wk old) were fed basal diets supplemented with 0, 30, 60, 90, or 120 mg/kg of lutein. The supplementation period lasted 12 wk followed by 2 wk of lutein depletion in feed. The results revealed that lutein efficiently enriched the egg yolks and improved their color with a significant increase in relative redness (P < 0.001). Lutein accumulation increased in the egg yolk until day 10, then depletion reached a minimum level after 14 d. Overall, zeaxanthin content in all the groups was similar throughout the experimental period. However, triglycerides and total cholesterol were significantly decreased in the liver (P < 0.05) but not significantly different in the serum (P > 0.05). In the serum, the lipid metabolism enzyme acetyl-CoA synthetase was significantly reduced (P < 0.05), whereas dipeptidyl-peptidase 4 was not significantly different (P > 0.05), and there was no statistical difference of either enzyme in the liver (P > 0.05). Regarding oxidation and inflammation-related indexes, malondialdehyde, tumor necrosis factors alpha, interleukin-6, and interleukin-1 beta were decreased, whereas superoxide dismutase and total antioxidant capacity increased in the liver (P < 0.001). The function of lutein for the same indexes in serum was limited. It was concluded that lutein efficiently enriched the egg yolk of old laying hens to improve their color and reached the highest level on day 10 without being subject to a significant conversion into zeaxanthin. At the same time, lutein prevented liver steatosis in aged laying hens by exerting strong antioxidant and anti-inflammatory functions, but also through the modulation of lipid metabolism, which may contribute to reducing the incidence of FLHS in poultry.

Key Words: old laying hen, lutein, enrichment efficiency, egg, fatty liver hemorrhagic syndrome

INTRODUCTION

The consumption of carotenoids such as lutein and zeaxanthin gained interest with the discovery that they accumulate in the retina and are important in protecting the macula against degeneration (Thurnham, 2007). Low levels of lutein and zeaxanthin in the eyes were associated with age-related macular degeneration and cataracts. Additionally, a low amount of lutein and zeaxanthin consumed in the diet is associated with a risk for various diseases, including cancer and heart disease (Ribaya-Mercado and Blumberg, 2004). Humans cannot synthesize lutein and zeaxanthin. Rather, they are acquired in the diet by consuming fruits, green leafy vegetables, and eggs (Wenzel et al., 2006). The natural micellar matrix (triglycerides, phospholipids, and cholesterol) found in egg yolk, offers a vehicle for the effective absorption of egg-derived carotenoids in humans (Vishwanathan et al., 2009). “Designer eggs” are a way to increase egg-derived carotenoid consumption by humans, and involve the manipulation of chicken feed to improve levels of a particular carotenoid in the eggs, thereby, providing humans with eggs that have a higher nutritional value.

Research has demonstrated that after supplementation, lutein accumulates in eggs to a maximum level within 14 d of consumption and declines to a minimum level during the same amount of time (Wu et al., 2009; Bunduc et al., 2016). However, during absorption and transportation to organs, lutein can be converted into the stereoisomer zeaxanthin (EFSA, 2009). Moreover, the yellowish coloration of egg yolk, an important parameter in egg marketing, is closely related to lutein content in egg yolks. However, multiple parameters, including age, can influence the absorption and distribution of carotenoids in organs and eggs. This can affect the amount of carotenoids accumulated in the eggs following addition in diet and then the enrichment efficiency of carotenoids (Dansou et al., 2023).

Aging can also affect the health status of laying hens, and fatty liver hemorrhagic syndrome (FLHS) is one of the most common metabolic diseases in laying hens during the late laying period. FLHS can significantly decrease egg production and induce sudden death, resulting in major economic losses for the poultry industry (Lv et al., 2018). The pathogenesis of FLHS remains unclear. However, the 2 “hits” theory is a more recognized explanation. The first “hit” starts with the excessive deposition of triglycerides (TG) in the liver and the inhibition of fatty acid oxidation destroying the homeostasis of lipid metabolism. After that, in the second “hit,” oxidative stress and insulin resistance induce excessive reactive oxygen species (ROS) in the liver that further aggravates the destruction of reticulin fibers (Whitehead, 1979; Miao et al., 2021). Chickens with FLHS exhibit hepatic steatosis, and those with more severe cases develop blood clots and liver rupture leading to death (Lin et al., 2021). Lutein with its antioxidant and anti-inflammatory properties can presumably help to prevent fatty liver in laying hens. In rats, it was found that lutein could prevent the degenerative conditions of the liver by decreasing the accumulation of free cholesterol, and attenuating lipid peroxidation and the production of pro-inflammatory cytokines (Sindhu et al., 2010). Another study suggested that lutein supplementation may protect against hepatic lipid accumulation and insulin resistance induced by a high-fat diet (Qiu et al., 2015). Murillo et al. (2016) used a nanoemulsion of lutein (3.5 mg/d) in a hypercholesterolemic diet administered to guinea pigs for 6 wk and observed an increase in the concentrations of this carotenoid in plasma and liver, in addition to a decrease in liver total cholesterol (TC), plasma alanine aminotransferase (ALT) activity, and a 24% decrease in hepatic steatosis. In addition, lutein increased the level of glutathione in hepatic cells and decreased malondialdehyde (MDA) (El-Kholy et al., 2017).

We could find no study about lutein deposition in aged laying hens or its function in protecting old laying hens against FLHS. The purpose of this study was therefore to assess the enrichment of lutein in eggs and to evaluate whether it functioned to prevent liver fat accumulation in aged laying hens. We hypothesized that the age of the laying hens could affect the enrichment efficiency of lutein in eggs, and lutein could contribute to improving the lipidosis status of the aged laying hens. Therefore, we evaluated the productive performance and the quality of the eggs laid, then focused on the accumulation of lutein and its depletion in eggs as well as its effect on the color of the eggs and conversion into zeaxanthin. Finally, the lipidosis, antioxidative, and anti-inflammatory status of the laying hens were explored.

MATERIALS AND METHODS

Experimental Design

The Animal Welfare and Use Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China, approved the experimental procedures. Six hundred Hy-Line Brown laying hens (65 wk old) were raised in a 3-tier battery mode with 3 laying hens per cage. The animals were randomly split into 5 groups with 8 replications, each with 15 laying hens per replicate. The chicken house was cleaned daily and continually ventilated. The indoor physical parameters were maintained at a humidity of 55%, temperature of 21°C, photoperiod of 14 h light and 10 h dark, and light intensity of 20 Lx.

Throughout the feeding trial, the birds had free access to feed and water and were not subject to any medication except for routine vaccinations. The basal diet composition and nutritional components are presented in Supplementary Table 1. To provide 0, 30, 60, 90, and 120 mg/kg of lutein, groups were fed the basal diet supplemented with lutein extract at 0% (control group, L0), 0.094% (L1), 0.188% (L2), 0.282% (L3), and 0.376% (L4). The lutein extract was from marigold flowers (Chenguang Biotechnology Group Co., Ltd., Hebei, China) that contained 3.2% lutein. Lutein content was measured in the formulated diets, and the results are presented in Table 1. To balance the nutritional component and especially lutein content in all the groups at the start of the experiment, the chickens were fed for 2 wk with the basal diet before 12 wk of feeding with lutein-supplemented diets. This period was followed by another 2 wk of feeding with the basal diet to test lutein depletion in eggs. Therefore, the whole trial lasted 16 wk. The 14 wk from lutein supplementation to the end of the depletion is considered the experimental period.

Table 1.

Lutein and zeaxanthin content in the formulated diets.

Items L01 L11 L21 L31 L41
Calculated lutein (mg/kg)2 0 30 60 90 120
Measured lutein (mg/kg)3 5.5 ± 0.1 34.5 ± 1.5 63.4 ± 2.5 94.2 ± 3.8 132.6 ± 5.1
Measured zeaxanthin (mg/kg)3 8.2 ± 0.3 14.5 ± 0.8 13.8 ± 0.9 16.4 ± 1.7 17.6 ± 1.2
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1

L0: control diet; L1, L2, L3, and L4: control diet supplemented with lutein at 30, 60, 90, and 120 mg/kg feed, respectively.

2

Lutein was supplemented to the control group diet to provide 0, 30, 60, 90, and 120 mg/kg feed.

3

Lutein and zeaxanthin contents are measured values of 8 replicates of feed samples per group after feed formulation.

Productive Performance of Laying Hens

Daily performance data such as the number of laying hens, hen-day egg production, egg weight, and damaged eggs (abnormal and broken eggs) were recorded. Every 2 wk, total feed consumption was determined. The productive performance of the laying hens, such as laying rate, average egg weight, daily egg mass, average daily feed intake, and feed conversion ratio was calculated for weeks 1 to 6 and weeks 7 to 12 of the experimental period.

Egg Physical Quality and Egg Yolk Color

The egg quality analysis was performed as described by Dansou et al. (2021b). Briefly, on weeks 6 and 12, 3 fresh eggs were collected per replicate, and parameters such as eggshell strength was tested using an egg force reader (ORKA Food Technology Co., Ltd., Ramat HaSharon, Israel), while albumen height and Haugh unit were tested with an egg quality analyzer (ORKA Food Technology Co., Ltd.). Egg shape, yolk proportion, shell proportion, and albumen proportion were also measured and calculated. At the same time, the egg yolk color scores were determined according to the International Commission on Illumination L*a*b* model (CIE L*a*b*) using a precision colorimeter analyzer CR-400 (Konica Minolta Inc., Chiyoda, Japan). L*, a*, and b* correspond to relative lightness, redness, and yellowness, respectively. The values determined for 3 eggs per replicate were averaged to get the value per replicate, and the 3 egg yolks per replicate were completely mixed and kept at −80°C for further analysis.

Lutein and Zeaxanthin Determination

Lutein and zeaxanthin content in feed and egg yolk samples were determined with reference to the method developed by Miao et al. (2023) with some modifications. First, an extraction solvent was prepared with methanol and dichloromethane at a concentration of 1:1 (v/v), and 1% of butylated hydroxytoluene was added to the extraction solvent to avoid the oxidation of lutein and zeaxanthin. Then, 0.5 ± 0.05 g of feed or lyophilized egg yolk sample was weighed into a 15 mL centrifuge tube, and 8 mL of the extraction solvent was added. Next, the mixture was vortexed at 2,500 rpm for 30 min using a multitube vortex apparatus (Yooning, Hangzhou, China) and then centrifuged at 4,000 rpm for 10 min at 4°C. Finally, 1 mL of the supernatant was filtered and transferred into amber injection vials.

Supercritical fluid chromatography (Agilent Technologies, Palo Alto, CA) fitted with a diode array detector (DAD) and Metasil C30 column (250 mm × 4.6 mm, 5 μm particle size, Phenomenex, Tianjin, China) was used to analyze lutein and zeaxanthin. The mobile phase was supercritical CO2 (mobile phase A) and isopropanol (mobile phase B, the organic modifier). The chromatographic conditions were set as follows: gradient (0–10 min, 0%–30% of B; 10–12 min, 30% of B; 12–13 min, 30%–40% of B); column temperature (35°C); flow rate (3.0 mL/min); injection volume (10 μL); BPR pressure (160 bar); BPR temperature (60°C); detection wavelength (full wavelength).

Lutein and zeaxanthin standards were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China) and other chemicals (all HPLC grade) from Thermo Fisher Scientific (Pittsburgh, PA). The standards were tested at concentrations of 0.5, 1, 2.5, 5, 10, 20, 30, and 40 ppm to make the calibration curves with a coefficient of determination (R2) equal to or higher than 0.999.

Biochemistry, Oxidation, and Inflammation Indexes in Serum and Liver

On week 12 of the experiment, after 8 h of fasting, 8 laying hens per group (1 laying hen per replicate) were selected. Blood samples were collected between 8 am and 10 am from the branchial wing veins and placed into ethylenediaminetetraacetic acid-coated tubes (Jiangsu Kangjian Medical Supplies Co., Ltd., Taizhou, China). Blood serum was prepared by centrifuging at 4,000 rpm, 4°C for 10 min using an H1850R centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China). After the laying hens were humanely slaughtered, a section of the liver (right lobe for all samples) was removed and stored at −80°C for further analysis.

Biochemical indexes such as aspartate aminotransferase (AST) and ALT were tested. In addition, oxidation- and inflammation-related indexes, including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), MDA, interleukin-6 (IL-6), tumors necrosis factors alpha (TNF-α), and interleukin-1 beta (IL-1β) were measured in the serum and liver, using commercial chicken-specific ELISA kits (Nanjing Jiancheng Technology Co., Ltd., Nanjing, China). The procedures were conducted with strict adherence to the manufacturer's operation manual.

Lipidosis Status in Serum and Liver

To evaluate the effect of lutein on the lipidosis status of the liver, total concentrations of serum and liver dipeptidyl-peptidase 4 (DPP4) and acetoacetyl-CoA synthetase (AACS) were tested using commercial chicken-specific ELISA kits (BlueGene Biotech, Shanghai, China), following the manufacturer's procedures. Likewise, serum and liver TG and TC were measured using commercial chicken-specific ELISA kits (Nanjing Jiancheng Technology Co., Ltd.).

Liver Histology

On the day the animals were slaughtered, a 1 × 1 × 1 cm square (from the lower left lobe) of liver samples was quickly cut and fixed into a 4% paraformaldehyde solution for over 24 h. After that, samples from 3 groups of laying hens (L0, L60, and L120) were subject to hematoxylin and eosin (H&E) and Oil Red O (ORO) staining for the assessment of liver necropsy and lipid accumulation.

For H&E staining, first, the tissues were trimmed with a scalpel and dehydrated in successive organic solutions. Then, the wax-soaked tissue was placed in the embedding machine and cooled to −20°C. The wax block was removed from the embedding box and trimmed after the wax solidified. The trimmed wax block was placed on the paraffin microtome and sliced to a thickness of 4 μm. Next, the paraffin sections were dewaxed. Then, the slices were placed into Harris hematoxylin stain for 3 to 8 min, followed by tap water washing, 1% hydrochloric acid alcohol differentiation for a few seconds, tap water rinse, 0.6% ammonia back blue, and running water rinse. Next, the slices were put into an eosin staining solution for 1 to 3 min. Finally, the slices were dehydrated in successive organic compounds and examined under the microscope at 200 × magnification.

For ORO staining, ORO (0.5 g) was dissolved in 100 mL of propylene glycol. Then, 30 ml of the stock solution was diluted with 20 ml of distilled water and filtered to prepare the ORO working solution. Next, the fixed tissues were trimmed and rinsed in distilled water, placed in absolute propylene glycol for 2 min, stained in ORO working solution for 15 min, and differentiated in 85% propylene glycol solution for 1 min and rinsed with 2 changes of distilled water. After that, the samples were counterstained in Mayer's hematoxylin for 1 min, rinsed thoroughly in 3 changes of distilled water, and mounted in a warmed glycerin jelly solution. Consequently, the lipids become red, while the nuclei are dark blue.

Calculations

Lutein and zeaxanthin concentrations in samples were determined with an Agilent MassHunter Workstation Quantitative Analysis for QQQ (Version 10.1, Agilent Technologies). The calculated concentrations were reported by sample weights and solvent volumes using Excel. Based on the measured lutein content in egg yolks and feeds and the productive performance data between weeks 4 and 6 and weeks 10 and 12 of the experiment, the efficiency rate of lutein in egg yolks on week 6 and week 12 were determined and calculated as:

Luteinefficiencyrate=luteinconcentrationinyolk×yolkweight×eggproductionaveragefeedintake×luteinconcentrationinthediet×100

Statistical Analysis

IBM SPSS Statistics 20 statistical package (SPSS Inc., Chicago, IL) was used to analyze the data subject to one-way analysis of variance (ANOVA). Multiple comparisons of means were made using Tukey's test. In addition, correlation (Spearman's correlation) and regression (linear and quadratic) tests were performed in order to study the relationship between lutein content in diets, content in egg yolks, and color score of egg yolks. The original data were used to perform the tests after the homoscedasticity of the variances were confirmed with Levene's test. Except for the correlation, the statistical tests were conducted for each time point separately during the experiment. PRISM 8.0 statistical software (GraphPad Software Inc., La Jolla, CA) was used to draw the graphs. The results are significant at P < 0.05 and presented as mean ± standard deviation.

RESULTS

Productive Performance of Laying Hens

The productive performance of laying hens was calculated on weeks 6 and 12 of the experiment and presented in Supplementary Table 2. The results revealed that the supplementation of lutein did not affect the productive performance of laying hens. This was demonstrated by the nonsignificant difference (P > 0.05) between the different groups at both testing times, on either week 6 or week 12.

Egg Physical Quality

Similar to the productive performance of laying hens, lutein did not significantly affect the physical quality of eggs (P > 0.05) collected and tested on week 6 and week 12 of the experiment. The results are presented in Supplementary Table 3.

Egg Yolk Color

Results from egg yolk color tests, shown in Figure 1, revealed that lutein supplementation has contributed to the improvement of the egg yolk coloration on week 6 and week 12. Overall, an increase in the yellowness of the lutein-supplemented groups was observed compared to the control group egg yolks (P < 0.001). The redness of the yolks also increased significantly and a dose-related trend was observed in all the groups (P < 0.001) at both times of the tests (Figure 1, Supplementary Table 4). However, the increase of redness on week 12 was less than in week 6 for all the groups.

Figure 1.

Figure 1

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Egg yolk color scores on weeks 6 and 12. L0: control diet; L1, L2, L3, and L4: control diet supplemented with lutein at 30, 60, 90, and 120 mg/kg feed, respectively. L*: relative lightness; a*: relative redness; b*: relative yellowness. Data are presented as means ± standard deviation (n = 8); different superscript letters within a time of test indicate significant differences between groups (P < 0.05). Analysis of variance (ANOVA) was conducted for each time point separately.

Concentration of Lutein and Zeaxanthin and Efficiency Rate of Lutein in Eggs

Lutein content in egg yolks was measured on weeks 0 (at the beginning of supplementation), 6, and 12 of the experiment for all the groups. As lutein is susceptible to conversion into zeaxanthin, the zeaxanthin content in egg yolks was also tested. The concentrations of lutein and zeaxanthin are presented in Figure 2. The highest lutein content was found in the L4 group whereas the lowest content was in the control group L0. The results were 16.9 ± 1.4 (0.03 ± 0.002), 63.1 ± 8.6 (0.11 ± 0.02), 108.2 ± 8.5 (0.19 ± 0.02), 125.2 ± 8.2 (0.22 ± 0.01), and 144.1 ± 14.0 µg/g fresh egg yolk (0.25 ± 0.02 μmol/g), for L0, L1, L2, L3, and L4, respectively on week 6. On week 12 they were 18.3 ± 3.0 (0.03 ± 0.01), 65.2 ± 7.5 (0.11 ± 0.01), 100.7 ± 3.0 (0.18 ± 0.01), 127.8 ± 8.9 (0.22 ± 0.02), and 148.7 ± 12.5 µg/g fresh egg yolk (0.26 ± 0.02 μmol/g), for L0, L1, L2, L3, and L4, respectively. A dose-dependent increase of lutein in egg yolk following a quadratic regression was observed (P < 0.001) as shown in Supplementary Figure S1. Regarding zeaxanthin concentration, except in week 12 when yolk content in L3 and L4 was significantly higher than L0, the results for week 6 were not significantly different (P > 0.05).

Figure 2.

Figure 2

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Lutein and zeaxanthin concentration in egg yolks on weeks 0, 6, and 12. L0: control diet; L1, L2, L3, and L4: control diet supplemented with lutein at 30, 60, 90, and 120 mg/kg feed, respectively. Data are presented as means ± standard deviation (n = 8); different superscript letters within a time of the test indicate significant differences between groups (P < 0.05). Analysis of variance (ANOVA) was conducted for each time point separately.

The efficiency rate of lutein is shown in Figure 3. The results indicate that in opposition to lutein concentration, the dose-related trend of lutein efficiency rate in eggs was reversed. The higher the lutein extract dosage, the lower the efficiency rate in eggs. The efficiency rate gradually decreased from 38.4% in the L0 group to 13.6% in the L4 group, and 37.9% in the L0 group to 12.3% in the L4 group on weeks 6 and 12, respectively. Moreover, on closer observation, the difference between L1 and L2 on either week 6 (0.8%) or week 12 (2.4%) was smaller than the difference between L3 and L4 on week 6 (3.7%) or wk 12 (2.8%), respectively.

Figure 3.

Figure 3

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Efficiency rate of lutein in egg yolks on weeks 6 and 12. L0: control diet; L1, L2, L3, and L4: control diet supplemented with lutein at 30, 60, 90, and 120 mg/kg feed, respectively. Data are presented as means ± standard deviation (n = 8); different superscript letters within a time of the test indicate significant differences between groups (P < 0.05). Lutein efficiency rate = (lutein concentration in yolk × yolk weight × egg production)/(average feed intake × lutein concentration in the diet) × 100. The measured lutein contents in diets were used. Analysis of variance (ANOVA) was conducted for each time point separately.

Accumulation and Depletion of Lutein in Eggs

To study the accumulation and depletion of lutein in eggs and the influence of the dosage, the egg yolk color and lutein content in the egg yolks from the L1 (lowest supplementation, 30 mg/kg feed) and L4 (highest supplementation, 120 mg/kg feed) groups were measured on days 0, 2, 4, 6, 8, 10, 12, and 14, and weeks 6 and 12 of supplementation and days 2, 4, 6, 8, 10, 12, and 14 following depletion. The results are shown in Figure 4. Regarding trends in lutein, there was an increase until day 10 of supplementation, followed by a decrease on day 14. Yet, there were subsequent increases on weeks 6 and 12 of supplementation. Following the depletion of lutein in feeds from the end of week 12, lutein in eggs constantly decreased and reached a minimum value of 18.7 ± 2.2 µg/g fresh egg yolk (32.8 ± 3.9 µmol) in the L1 eggs on day 12 and 19.0 ± 2.8 µg/g fresh egg yolk (33.4 ± 4.9 µmol) in the L4 eggs on day 14 after depletion. Overall, the trends in the increase and decrease of lutein in the L4 group were more pronounced than in the L1 group but the dosage did not affect the day of maximum accumulation. The results of the a* scores of the egg yolk confirmed these trends. Moreover, the correlation test between the lutein content in egg yolks and the yolk scores revealed a stronger relationship between lutein and redness compared to the lightness and yellowness (Supplementary Figure S2). However, a comparative analysis of lutein content in egg yolks and egg yolk color a* scores revealed a difference between the accumulation and the depletion period. Although the lutein content at the beginning of the accumulation was similar to that at the end of the depletion period, the egg yolk color score at the end of the depletion was substantially lower than the score at the beginning. The color of egg yolks was not proportional to the level of lutein in the eggs during the depletion period.

Figure 4.

Figure 4

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Lutein accumulation and depletion trend in eggs (A) and egg yolk color trend (B). L1: control diet supplemented with lutein at 30 mg/kg feed; L4: control diet supplemented with lutein at 120 mg/kg feed. A0, A2, A4, A6, A8, A10, A12, A14: lutein content in egg yolks tested on days 0, 2, 4, 6, 8, 10, 12, and 14 of supplementation, respectively; w6, w12: lutein content in egg yolks tested on weeks 6 and 12 of supplementation, respectively; D2, D4, D6, D8, D10, D12, and D14: lutein content in egg yolks tested on days 2, 4, 6, 8, 10, 12, and 14 after supplementation period, respectively. Bars correspond to standard deviation.

Lipid Indexes in Liver and Serum

The function of lutein in lipid accumulation in the liver and the enzymes involved in lipid metabolism have also been studied. The results are shown in Figure 5. We found that lutein contributed to a significant reduction in AACS in serum (P < 0.05) but not in the liver (P > 0.05). With regards to DPP4 however, there was no significant difference between the groups in either the serum (P > 0.05) or liver (P = 0.07). TG and TC tests showed a significant decrease (P < 0.05) in the liver. The decreases were more pronounced in L2 and L4 groups. Nevertheless, the decreases in serum were not significantly different for either TC (P > 0.05) or TG (P = 0.06).

Figure 5.

Figure 5

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Lipid-related indexes in serum and liver on week 12. L0: control diet; L1, L2, L3, and L4: control diet supplemented with lutein at 30, 60, 90, and 120 mg/kg feed, respectively. Data are presented as means ± standard deviation (n = 8); different superscript letters indicate a significant difference between groups (P < 0.05). Abbreviations: TG, triglycerides; TC, total cholesterol; DPP4, dipeptidyl-peptidase 4; AACS, acetyl-CoA synthetase.

Biochemistry and Cytokines in Serum and Liver

To investigate the function of lutein in the modulation of liver function parameters related to lipidosis in aged laying hens, several indexes were tested in serum and liver and are shown in Figure 6. The results revealed a significant difference (P < 0.05) in ALT serum enzyme activity between the groups. However, only L1 was significantly different from L0. There was no difference (P > 0.05) in AST activity in serum between the groups. Regarding the antioxidant parameters, lutein contributed to significant improvements in the antioxidant condition of the laying hens. Overall, T-AOC in the liver and SOD in the serum and liver significantly increased (P < 0.05) in the lutein-supplemented groups. However, GSH-Px presented an increased trend but a nonsignificant difference between the groups in serum (P = 0.05) and liver (P = 0.09). On the contrary, MDA was significantly reduced (P < 0.05) in both liver and serum for all the lutein-supplemented groups. Regarding cytokines, lutein was found to significantly reduce (P < 0.05) the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the liver, with L4 presenting the lowest values. In serum, there was no difference in the levels of TNF-α and IL-1β between the groups (P > 0.05). IL-6 was significantly different in serum but the lowest value was observed in L2.

Figure 6.

Figure 6

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Oxidation and inflammation-related indexes in serum and liver on week 12. L0: control diet; L1, L2, L3, and L4: control diet supplemented with lutein at 30, 60, 90, and 120 mg/kg feed, respectively. Data are presented as means ± standard deviation (n = 8); different superscript letters indicate a significant difference between groups (P < 0.05). Abbreviations: MDA, malondialdehyde; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; T-AOC, total antioxidant capacity; TNF-α, tumors necrosis factors alpha; IL-6, interleukin-6; IL-1β, interleukin-1 beta.

Liver Histology

To confirm a reduction in hepatic fat accumulation by the function of lutein as revealed by the lipid index tests, ORO staining of liver samples from the L0, L2, and L4 groups was performed. The results are shown in Figure 7. Compared with the L0 group, the L2 and L4 groups showed a less prominent accumulation of lipids in hepatic cells. Lutein supplementation contributed to reduced lipid accumulation.

Figure 7.

Figure 7

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Oil Red O (ORO) staining of the liver samples on week 12. Magnification: 200 × . Red arrow: indicates nuclei in blue; blue arrow: indicates fats in red. L0: control diet; L2 and L4: control diet supplemented with lutein at 60 and 120 mg/kg feed, respectively.

To evaluate the function of lutein in improving liver condition in relationship with the inflammatory parameters, liver histological changes were evaluated with H&E staining in L0, L2, and L4 groups. As shown in Figure 8, the H&E staining of liver samples showed that the L0 group had obvious pathological changes, and some birds had large-scale hepatocyte steatosis. The structure of liver tissues was mild or moderately abnormal, hepatocyte steatosis was present, and a large number of fat vacuoles of different sizes could be seen in the cytoplasm. A small amount of inflammatory cell infiltration was seen in the liver parenchyma. However, some samples presented a complete structure of liver cells, a rounded nucleus, rich cytoplasm, and no necrosis or degeneration. In contrast, the results for the L2 and L4 groups are similar and appear to be less damaged. Some liver cells have mild edema, cell swelling, and light cytoplasmic staining. Yet, there is no obvious inflammatory cell infiltration in the liver parenchyma. Overall, the livers in the L0 group are more damaged than those in the L2 and L4 groups.

Figure 8.

Figure 8

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Liver hematoxylin and eosin (H&E) staining on week 12. Magnification: 200 × . Yellow arrow: fat vacuoles in the cytoplasm; red arrow: inflammatory cell infiltration in liver parenchyma; blue arrow: mild edema in cells. L0: control diet; L2 and L4: control diet supplemented with lutein at 60 and 120 mg/kg feed, respectively.

DISCUSSION

Productive Performance of Laying Hens and Egg Quality

Age or health status could harm intestinal health and functioning capacity, leading to a decline in laying performance and egg quality (Obianwuna et al., 2022). The effects of carotenoids on the productive performance of laying hens have been explored in former studies. However, the findings of these studies are inconsistent. Although the positive effects of lutein on the productive performance of laying hens were revealed by Englmaierová et al. (2013) and Wen et al. (2021) found that lutein did not affect the performance of laying hens. A previous study demonstrated that astaxanthin also does not affect the productive performance of laying hens (Dansou et al., 2021a). The positive function of carotenoids on the productive performance of laying hens may simply be related to the improvement of the physiological conditions of laying hens due to the antioxidant and anti-inflammatory function of carotenoids. Oxidation stress could hamper the health status of the birds and, therefore, their production, whereas carotenoids, including lutein, could contribute to an improvement. In the current study, the laying hens in all the groups still have a highly productive performance regardless of their oxidative condition. Therefore, the function of lutein on the productive performance of the birds might be intricate.

Previous studies by Lokaewmanee et al. (2011) and Shahsavari (2014) showed that lutein-derived by-products did not influence the quality of eggs. However, some natural products containing carotenoids such as lycopene and astaxanthin, have positively affected yolk and egg weights (Conradie et al., 2018; Orhan et al., 2021). Similarly, Englmaierová et al. (2013) found that lutein improved eggshell thickness and strength. Nonetheless, the direct function of lutein on egg physical quality, specifically on the eggshell thickness, was not demonstrated in their study. In the current study, we cannot confirm the function of lutein on egg quality either because the physical quality of the eggs was not significantly different among the groups.

Lutein Enrichment in Eggs

The primary use of carotenoids in the poultry industry is for the enhancement of meat and egg yolk color. Lutein and zeaxanthin mainly give egg yolk its yellow color. Our results revealed that the b* score is significantly superior in all the lutein-supplemented groups compared to the control group. Meanwhile, the opposite trend was observed with the L* score. However, the a* score is the most affected by lutein with a quadratic growth in all the groups. Lokaewmanee et al. (2011) also reported a linear trend regarding the a* score in the lutein-supplemented groups. This demonstrated that at a certain level of supplementation (30 mg/kg under the current experimental conditions), lutein intensified the redness of egg yolks rather than the yellowness, leading to a dark yellow coloration.

The significant increase in the redness of the egg yolks was a reflection of increased lutein in the egg yolks as revealed by the dose-response relationship between lutein supplementation in diets and content in egg yolks. A strong correlation between lutein content and the redness score of egg yolks, but not between the content and either lightness or yellowness was also demonstrated by Islam et al. (2017). However, despite the increase in the lutein concentration of egg yolks in each group, the efficiency rate gradually decreased. Previous studies have shown a downtrend between dose supplementation and carotenoid efficiency. Lutein content in egg yolks improved remarkably when the diets of laying hens (30 wk old) were supplemented with up to 500 ppm (500 mg/kg) of lutein; however, the transfer efficiency of lutein into egg yolk lowered as dietary lutein increased, declining from 10% with 125 ppm (125 mg/kg) in the diet to 2 to 3% with a lutein level of 500 ppm (Leeson and Caston 2004). This downtrend was reported for other carotenoids such as lycopene and canthaxanthin (Olson et al., 2008; Johnson-Dahl et al., 2017). The explanation for this may be the limited absorption capacity of carotenoids at high doses leading to an excessive excretion, and the aging state of laying hens seems to not hamper this trend.

Carotenoid accumulation into egg yolks is generally effective within 6 to 14 d after consumption (Dansou et al., 2023). Previous studies mainly considered the trend of egg yolk color to determine the maximum accumulation of carotenoid in the egg yolk of laying hens which varied from 7 to 13 d (Nelson et al., 1990; Karadas et al., 2006; Walker et al., 2012; Ortiz et al., 2021). Bunduc et al. (2016) tested the accumulation of lutein in the eggs of 58-wk-old layers over an experiment period of 37 d and found an increase of lutein for up to 14 d. Our simultaneous analysis of lutein concentration in egg yolks and measurement of the redness score for up to 14 d of supplementation for the groups fed 30 and 120 mg/kg feed confirm these trends. Lutein accumulated in egg yolks until day 10, regardless of the supplementation dose. The current results also showed that carotenoid depletion was reached after 14 d. In an experiment by Moreno et al. (2020), a minimal dose of carotenoid content in egg yolks was achieved after 12 d of washout after feeding laying hens with a diet depleted in carotenoid. Wu et al. (2009) took 14 d to achieve a carotenoid content in egg yolks that was lower than the detection limit of the testing method. However, while comparing the trend of lutein content in the egg yolks to the egg yolk color scores during the depletion period, it was found that the color score is not reflective of the lutein content in egg yolks. Therefore, the testing of lutein concentration would be more accurate in a proper depletion experiment of lutein in eggs.

Lutein and zeaxanthin are isomeric and differ only by the position of a single, double bond, such that all double bonds in zeaxanthin are conjugated (Mohd Hatta and Othman, 2020). Interconversion between lutein and zeaxanthin was reported in the quail retina (Bhosale et al., 2007), and human and monkey retinas (Arunkumar et al., 2020). Moreover, interconversion between lutein and its esterified forms were observed in chickens (Tyczkowski & Hamilton, 1986). Therefore, it was presumed that lutein might be converted into zeaxanthin following supplementation in the diet. However, the evaluation of the real conversion rate of lutein into zeaxanthin in egg yolks could not be determined under the current experimental conditions.

Lutein Function to Improve Lipidosis Status of Aged Laying Hens

One of the most prevalent metabolic disorders in laying hens is FLHS, characterized by an abnormal accumulation of fat in the liver which can reduce egg production and, in the most severe cases, result in the sudden death of the birds (Lv et al., 2018). Due to their relatively simple lymphatic systems, chickens are susceptible to accumulating fat in their liver because dietary fat flows directly into the portal vein (Davis et al., 2016). However, even though increased fatty acid flow to the liver is still a significant factor influencing liver steatoses, de novo lipogenesis is thought to be a key contributor to the development of fatty liver (Nassir and Ibdah, 2014). In chickens, the major site of de novo lipogenesis is also the liver, which increases the sensitivity of laying hens to store fat as they age (Davis et al., 2016; Huang et al., 2021; Lin et al., 2021). AACS is an enzyme involved in lipogenesis using ketone bodies to synthesize cholesterol and fatty acids (Hasegawa et al., 2018). Elevated levels of AACS and DPP4 (a serine protease that cleaves a variety of substrates, including incretin hormones, chemokines, growth factors, and neuropeptides), were found to be associated with FLHS in laying hens (Tsai et al., 2017).

Our current results showed that lutein contributed to a decrease in the expression of AACS in serum and liver with L4 presenting the best results. A decrease of TG and TC followed this decrease of AACS in the liver and serum. In addition, the ORO staining results indicated less prominent fat accumulation in the livers of the L2 and L4 groups of chickens. Moreover, H&E staining showed lipid droplets in the L0 group of livers. Lutein supplementation has been found to induce fat loss in rats fed a high-fat diet, decreased serum TC and hepatic TC and TG levels, and decreased hepatic levels of lipid accumulation (Qiu et al., 2015). Also, adding lutein to the feed of laying hens (18 wk old) fed a diet containing 10% flaxseed significantly reduced fatty liver scores (Leeson et al., 2007). Therefore, our results confirmed these previous findings. However, it is important to note that the decrease was statistically different for AACS but not for TG and TC in serum. Meanwhile, AACS was not significantly reduced in the liver whereas TG and TC were significantly decreased. Such discrepancies might occur because the decrease of TG and TC in the liver is not exclusively linked to a reduction in AACS.

We found that lutein did not significantly affect the level of DPP4 in either liver or serum. This may confirm that even though lutein contributes to reducing the enzymes involved in lipid metabolism, lutein function to reduce TG and TC in the liver might also be the consequence of other mechanisms; most probably, oxidative and anti-inflammatory functions considering the antioxidative and anti-inflammatory properties of lutein.

Antioxidative and Anti-inflammatory Functions of Lutein in Aged Laying Hens

Excess fat accumulation in the liver can result in lipotoxicity, leading to mitochondrial dysfunction and endoplasmic reticulum stress, but extramitochondrial oxidation is also enhanced. Mitochondrial dysfunction results in ROS overproduction, impairing the antioxidant balance and leading to lipid peroxidation, which triggers lipotoxicity in the liver and then stimulates fatty liver development (Nassir and Ibdah, 2014; Marra and Svegliati-Baroni, 2018; Tsai et al., 2020). Antioxidant protection systems contain nonenzymatic and enzymatic components such as SOD and GSH-Px that detoxify ROS (Arroyave-Ospina et al., 2021). Our results revealed that lutein significantly decreased lipid peroxidation (measured by MDA) in both serum and liver and improved the activity of T-AOC in the liver and SOD activity in the liver and serum. It was previously demonstrated in guinea pigs that lutein can prevent the degenerative conditions of the liver by decreasing the accumulation of free cholesterol, and by attenuating lipid peroxidation and the production of pro-inflammatory cytokines (Kim et al., 2012). In humans, oxidative stress also triggers the production of inflammatory cytokines, causing inflammation which results in the development of nonalcoholic fatty liver disease (Nassir and Ibdah, 2014). In mice, lutein downregulates oxidative stress markers (ROS, lipid peroxidation, and protein carbonyls), liver markers (ALT, AST, ALP, and LDH), and inflammatory cytokines (TNF-α, IL-1β, and IL-6) (Du et al., 2015). In our current study, ALT and AST were not descriptive of lutein function or liver misfunction in the chickens. However, TNF-α, IL-6, and IL-1β were significantly reduced in the livers of lutein-supplemented groups, with the L4 group presenting the best reduction. At the same time, the H&E staining revealed that the L2 and L4 groups presented less damage than the L0 group. Nonetheless, in the serum, the activity of these cytokines was mitigated as there was no decrease dependent on the supplementation of lutein in the diet. This is reflected in the nonsignificant difference of TG and TC in the serum. Therefore, the preventive function of lutein in liver steatosis of aged chickens might occur mainly by acting as an antioxidant and anti-inflammation agent.

CONCLUSION

The results from the current study have shown that lutein could efficiently be enriched in the eggs of aged laying hens. Ten d after consumption, the lutein reached a maximum level of content, improving the relative redness of the eggs without significant conversion into zeaxanthin or consequence on other physical parameters of the eggs. Moreover, lutein could prevent FLHS in older laying hens through the modulation of lipid metabolism but, most importantly, antioxidative and anti-inflammatory functions.

Acknowledgments

ACKNOWLEDGMENTS

This work was supported by the China Agriculture Research Systems (CARS-40-K11), the Beijing Agriculture Innovation Consortium (BAIC06-2023-G05), and the Chinese Academy of Agricultural Science and Technology Innovation Project (ASTIP-IAS-12).

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the present study.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2023.103286.

Appendix. Supplementary materials

mmc1.docx (467.4KB, docx)

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