Dietary supplementation with lycopene can effectively enhance egg quality and antioxidant function in laying hens

Dietary supplementation with lycopene can effectively enhance egg quality and antioxidant function in laying hens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dietary supplementation with lycopene can effectively enhance egg quality and antioxidant function in laying hens Mengxian Chen, Junxing Pan, Yang Song, Yu Ding, Zheng Xu, Xueqi Yu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8414745/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The strategic focus on improving the quality of eggs, a foundational and nutritious food, offers a straightforward method to elevate the nutritional value of diets. However, studies on strategies to improve egg quality remain limited. Lycopene, a lipophilic carotenoid derived from red fruits and vegetables, enhances growth performance, lowers blood lipid levels, and improves antioxidant capacity. Therefore, in this study, a total of 360 laying hens were randomly divided into four groups. The CON group was fed a basic diet, the LYC group supplemented with 120 mg/kg lycopene, whereas the CCBA group supplemented with 60 mg/kg β-carotene, 250 mg/kg curcumin, 250 mg/kg allicin, and 500 mg/kg sodium butyrate, the CLYC group replaced β-carotene with lycopene, while other ingredients remained the same. For the yolk score, the LYC group significantly increased at days 14, 21, 28, 35, and 42 compared to the CON group ( P< 0.05 ). The UFA and Vitamin content are significantly elevated in the LYC group compared to the CON group ( P<0.05 ). The result of lipid metabolism genes was shown, the relative expression of ACC , LXR-α , and PPAR-α significantly increased in the CLYC and CCBA groups compared with the CON group ( P< 0.05 ), the relative expression of LXR-α in the LYC group remarkedly higher than the CON group (P< 0.05). Additionally, the heatmap analysis showed that Turicibacter and Veillonella were enriched in the LYC group. The results of this study indicated that lycopene supplementation significantly improved egg quality, hepatic lipid metabolism, and systemic immunity in laying hens. Furthermore, analysis of gut revealed that lycopene increased ileal goblet cell amount and IgA production, while enriching beneficial microbiota. Lycopene Egg quantity Growth performance Intestinal barrier Lipid metabolism Microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Growing health consciousness has led to increased demand for health-promoting, safe, and sustainably produced foods. As societies develop, dietary priorities are shifting from sufficiency to quantity. Eggs, as a low-cost and nutritious food staple, are central to this transition. Therefore, improving egg quantity is crucial for better satisfying human nutritional needs. As natural antioxidants, phytochemicals possess distinct advantages over synthetic alternatives, including natural abundance, potent antioxidant activity, and low toxicity. Among common plant-derived foods, flavonoids and carotenoids represent two major antioxidant phytochemicals that have attracted considerable attention from both the scientific community and the general public [ 1 ]. Nutrition exerts profound and multifaceted effects on animal immune function and overall health status, encompassing both nutrient metabolism at the host-microbiota interface and complex microbiota-host system interactions [ 2 , 3 ]. Carotenoids are a class of yellow, orange, or red polyene compounds ubiquitously distributed in nature, with representative members including β-carotene, lycopene, and astaxanthin. Accumulating evidence has demonstrated their multifaceted biological functions, particularly in cancer chemoprevention, antioxidation, immunomodulation, and pigmentation [ 4 , 5 ]. Lycopene, a lipophilic carotenoid derived from red fruits and vegetables, enhances growth performance, lowers blood lipid levels, and improves antioxidant capacity and reproductive efficiency [ 6 ]. Primarily located within cell membranes and lipoproteins, it is most concentrated in low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL). Previous studies demonstrate that lycopene neutralizes free radicals, mitigates lipid peroxidation, and exerts hepatoprotective effects. It achieves this by regulating superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), thereby scavenging excess reactive oxygen species (ROS) produced in vivo [ 7 , 8 ]. Furthermore, dietary supplementation of lycopene in rats fed a high-fat diet significantly reduced serum and brain levels of total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C), and oxidized LDL (ox-LDL) [ 9 ]. However, studies on the use of natural plant extracts to enhance egg quality, particularly its polyunsaturated fatty acid content, remain limited. Furthermore, research on the effects of lycopene on egg quality and lipid metabolism remains limited to date. Therefore, this study aimed to investigate the impact of dietary lycopene supplementation on egg quality and hepatic lipid metabolism in laying hens. Additionally, our previous research demonstrated that a compound feed addictive containing β-carotene significantly enhanced breeder hen intestinal microbiota. To evaluate whether lycopene alone elicits comparable microbiota effects to β-carotene, we further examined its influence on microbiota parameters. Materials and Methods Ethics statement All animal experiments complied with the ARRIVE guidelines. This animal experimental protocol was implemented under the supervision of the Chinese Guidelines for Animal Welfare and Experimental Protocol and was approved by the Animal Ethics Committee, Jilin Agriculture University (NO.2017050001). The stocking densities for each housing system were established in accordance with standard industry and regulatory guidelines. Specifically, the conventional cage system maintained a density of approximately 9 birds/m², consistent with common commercial practices for intensive quail production. These free-range densities were designed to comply with, or exceed, established organic and free-range poultry welfare standards, including USDA Organic regulations and EU Council Regulation 889/2008. Experiment preparation Food-grade carotene powder and tomato powder were purchased from Shanxi Mixianer Biotechnology Co., Ltd., China, and Chenguang Biotechnology Group Co., Ltd., China, respectively. The β-carotene content in the carotene powder was quantitatively analyzed by Pony Testing Group, China, using high performance liquid chromatography (HPLC). Briefly, samples underwent saponification to liberate carotenoids into their free forms, followed by extraction with petroleum ether and dichloromethane to a fixed volume. The extracts were then separated external standard calibration curve (β-carotene standard, ≥ 97% purity, Sigma-Aldrich). Similarly, the lycopene content in tomato powder was determined by Chenguang Biotechnology Group, China, employing identical analytical methodology. Animal management The experimental Jinghong NO.1 laying hens were obtained from Changchun Hefeng Co., Ltd., China. Three hundred and sixty healthy 25-week-old laying hens were randomly divided into four groups (n = 90 per group, with three replicates of 30 hens each), showing no significant initial weight differences. The laying hens were kept in cages (120 cm× 60 cm×60 cm) equipped with two nipple drinkers and one 100 cm-long feed. The four groups were the control group (CON), the lycopene treatment group (LYC), the lycopene complex treatment group (CLYC), and the β-carotene complex treatment group (CCBA), respectively. After one week acclimation period, the hens in the CON group were fed a basal diet, the LYC group hens were supplemented with 120 mg/kg lycopene, the CLYC group hens were supplemented with 60 mg/kg lycopene, 250 mg/kg curcumin, 250 mg/kg allicin, and 500 mg/kg sodium butyrate, the CCBA group hens supplemented with 60 mg/kg β-carotene, 250 mg/kg curcumin, 250 mg/kg allicin, and 500 mg/kg sodium butyrate for 6 weeks. Daily feed intake and egg production were recorded for each group throughout the trial period. Egg quality parameters, including egg weight, yolk weight, yolk score, yolk weight, Protein weight, and yolk score were measured weekly (n = 5 for each group per time point). The experiment was conducted for 42 days. On day 21, blood samples were collected via venipuncture, and fresh fecal samples were obtained for subsequent analysis (n = 10 per group). At the end of the experiment, blood was collected. Body weights, liver, spleen, and intestinal weight were recorded. The following instruments were used for measurements: MA2 protein height analyzer (Beijing Heng Odd Instrument Co., Ltd., China), Roche colorimetric (Nanjing Mingao Instrument Equipment Co., Ltd., China), and a precision electronic balance (accuracy ± 0.001g; Mettler-Toledo International Inc). The compositions and nutrition levels of the basal diets are shown in Supplementary Table 1. All laying hens are fasted for 4 h to facilitate anesthetic absorption. They are then deeply anesthetized via intramuscular injection of ketamine and xylazine. Once the absence of pedal reflex was confirmed, euthanasia was performed by an intravenous injection of an overdose of sodium pentobarbital into the wing vein. Death was confirmed by the absence of corneal reflex and cessation of breathing. Growth performance and egg nutrition test The daily feed intake (ADFI) and egg production (ADE) were recorded for each group. The feed conversion ratio (FCR) was calculated (FCR = ADFI/ADE). Laying rate was determined by dividing (FCR = ADE/Total hen number) ×100%. Organ indices were computed as (organ weight / body weight) × 100%. Chemical analysis was conducted by Huace Testing (China) using validated methods: Vitamin A was quantified via HPLC-UV (325nm) following saponification and purification; β-carotene was analyzed by HLPC-UV/Vis (450nm) after organic solvent extraction; and polyunsaturated fatty acids (PUFAs) were determined as fatty acid methyl esters (FAMEs) by DC-FID following lipid extraction and methylation, with all analyses employing external standard quantification. Specially, vitamin A analysis utilized a C18 column (4.6tamin ×250 mm, 5 µm) separation after solid phase extraction, β-carotene was separated on a C30 column (4.6 ×250 mm, 5 µm) following silica gel purification, and PUFA analysis employing an HP-88 capillary column (100 m× 0.25 mm ×0.2 µm) with reference to a 37-component FAME mix. Quantification of Antioxidant Compounds in Egg Yolk and Serum Blood samples (n = 10 for each group) were collected and maintained at 4°C for 30 min prior to serum separation by centrifugation (3500g, 15 min). For egg white analysis, 0.5 mL aliquots were homogenized with 1mL of Dulbecco’s phosphate-buffered saline (D-PBS), supplemented with 52.52 mg polyethylene glycol 8000 (PEG8000), and centrifuged (14,000g, 10 min) to collect the supernatant fractions. Egg yolk samples (0.5 mL) were processed similarly using D-PBS homogenization followed by chloroform extraction (1.5 mL) and centrifugation (1000g, 30 min). Analyte concentrations in both matrices were normalized to sample volume (µg/mL). Oxidative stress markers, including Malondialdehyde (MDA), Catalase (CAT), Superoxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC), and Glutathione Peroxidase (GSH-Px) were analyzed using standardized assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., China) following manufacturer specifications. For egg yolk antioxidant assessments, reducing power was determined by physiological saline extraction, while 2,2-diphenyl-1-picrydrazyl (DPPH) radical scavenging activity was measured using ethanol extraction methodology [ 10 , 11 ]. Serum levels of High-density lipoprotein (HDL), Low-density lipoprotein (LDL), Total Cholesterol (TC), and Triglyceride (TG) were quantified using standardized assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., China) following manufacturer specifications, with triplicate measurements per sample. Histological observation of the intestine and the number of goblet cells Ileal, hepatic, and splenic tissues (n = 10 for each group) from laying hens were fixed in 4% paraformaldehyde solution, dehydrated through an ethanol series, embedded in paraffin, and sectioned at 5 µm thickness using a rotary microtome. Tissue sections were stained with hematoxylin and eosin (Soleberg Biotechnology, Beijing, China) for histological examination. Morphometric analysis was performed using an Olympus BX53 light microscope (Olympus corporation, Japan), with villus height and crypt depth measurements using ImageJ software (National Institutes of Health, USA). Goblet cells were quantified following Periodic Acid Schiff (PAS) staining (Leagene Biotechnology Co., Ltd., China) using standard histochemical protocols. Immunohistochemical measurement Splenic, hepatic, and ileal sections (5 µm) on slides were treated with xylene, different concentration gradients of alcohol, and washed in PBS (PH = 7.4 for 3 min). Antigen retrieval was performed using boiling sodium citrate antigen buffer for 20 min. After cooling to room temperature, the slides were incubated with the pre-oxygenase blocking solution (Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) at 37°C for 10 min. After washing in PBS, the slides were incubated overnight at 4°C with mouse anti-chicken immunoglobulin A (IgA) primary antibodies (1:100, Monoclonal, Southern Biotech, Cat. No.8330-01, U.S.A.). The slides treated with PBS instead of the primary antibody served as the negative control. After being exposed to biotinylated secondary antibody goat anti-mouse IgG (Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) for 20 min at 37°C, slices were incubated with streptavidin (Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) for 10 min at 37°C. Under dark conditions. Under dark conditions, the slices were immersed in diaminobenozidine hydrochloride (DAB, Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) to develop the immunoreaction. This reaction was quenched by immersion in distilled water after the brown staining developed. Slices were counter-stained with H&E followed by hydrochloric acid alcohol color separation. They were finally washed with tap water, dehydrated in ethanol, and cleared using xylene. Sections on the slide were viewed under the light microscope (Olympus, Tokyo, Japan), and any IgA-positive areas in the five different microscope fields for each tissue were measured with Image J (National Institutes of Health, Bethesda, MD U.S.A) and the average calculated. RNA extraction and real-time fluorescence quantitative analysis Total RNA was extracted from the ileum, liver, and spleen (n = 10 for each group) using Trizol reagent, with concentration and purity assessed by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). Following reverse transcription using a High-Capacity cDNA kit, qPCR analysis was performed in triplicate on a CFX Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR Green chemistry. Tissue-specific reference genes were employed: β-actin for liver ( ACC , ACO , LXR-α , PPAR-α , and SCD ), and spleen ( TNF-α , IFN-γ , IL-1β , IL-2 , and IL-10 ). Relative gene expression was calculated using 2 −△△Ct , with all primers (Sangong Biotechnology, Shanghai, China) demonstrating 90–110% amplification efficiency (sequences in Supplementary Table 2). Sequencing analysis of bacterial colonies and statistical analysis On day 21 of the experiment, total microbial RNA was extracted from hen faces (n = 5 for each group). Sequencing analysis of bacterial colonies was performed by analyzing the KEGG database and the MetaCyc database. Finally, the metagenomeSeq method was combined with calling the fitFeatureModel function to use a zero-included log-normal model to fit the distribution of each pathway/group, and the results of this model were used to determine the significance of the difference. Group distributions use the results of this model to discriminate the significance of the difference. Data were analyzed for significance using SPSS, and the mean ± standard error was plotted using Prism software. P -values less than 0.05 were considered statistically significant, and P -values less than 0.01 were considered highly statistically significant. * In the figure, lower case letters in the table indicate a P -value less than 0.05, and ** in the figure and uppercase letters indicate a P-value less than 0.01. Results and Discussion Effects of lycopene on growth performance As shown in the Table 3 , no significant changes in ADFI, ADE, and FCR were observed in the four groups during the first two weeks of the experiment. At days 21, 35, and 42, ADE was significantly increased in the three treatment groups compared with CON group ( P < 0.05 ). At days 28 and 42, ADFI was remarkedly higher in CLYC group compared with CON group ( P < 0.05 ). And ADFI was significantly increased in the CCBA group compared with CON group, at day 42 ( P < 0.05 ). Mokhtar et al [ 12 ] found that supplementing broiler diets with 100 mg/kg lycopene significantly improved growth performance. Sarker et al [ 13 ] reported that dietary supplementation of lycopene improved the average daily weight gain in broilers. Although we supplemented lycopene to the laying hen diet, our findings same with those reported in previous studies. Table 3 Effects of lycopene on growth performance. Growth performance Items ADFI ADE FCR 7 d CON 108.31 ± 1.02 25.71 ± 1.91 4.22 ± 0.15 LYC 109.71 ± 2.25 25.99 ± 0.81 4.22 ± 0.11 CLYC 112.59 ± 1.23 27.33 ± 0.83 4.12 ± 0.10 CCBA 110.26 ± 2.62 26.52 ± 0.29 4.16 ± 0.13 14 d CON 119.53 ± 1.37 24.83 ± 1.21 4.83 ± 0.19 LYC 118.47 ± 1.26 26.59 ± 0.79 4.47 ± 0.24 CLYC 119.13 ± 1.23 26.62 ± 0.85 4.48 ± 0.14 CCBA 119.71 ± 1.81 26.19 ± 0.62 4.58 ± 0.16 21 d CON 129.47 ± 4.38 25.23 ± 1.10 b 5.16 ± 0.38 LYC 129.69 ± 2.24 26.53 ± 0.21 a 4.89 ± 0.10 CLYC 133.92 ± 1.00 27.62 ± 0.72 a 4.86 ± 0.16 CCBA 132.33 ± 3.78 27.33 ± 0.29 a 4.84 ± 0.17 28 d CON 123.33 ± 2.12 b 24.67 ± 1.13 5.01 ± 0.17 LYC 127.22 ± 1.79 ab 25.30 ± 0.61 5.04 ± 0.15 CLYC 130.32 ± 2.04 a 26.05 ± 1.07 5.02 ± 0.16 CCBA 127.75 ± 1.41 ab 25.95 ± 0.50 4.93 ± 0.14 35 d CON 128.45 ± 1.56 25.84 ± 1.10 b 4.99 ± 0.21 LYC 128.13 ± 1.77 26.81 ± 1.19 a 4.78 ± 0.15 CLYC 132.67 ± 1.82 27.48 ± 0.41 a 4.83 ± 0.16 CCBA 126.94 ± 2.26 26.29 ± 0.58 a 4.83 ± 0.11 42 d CON 128.51 ± 1.41 b 25.78 ± 1.24 b 5.01 ± 0.24 LYC 129.95 ± 1.32 ab 26.22 ± 0.60 a 4.96 ± 0.13 CLYC 134.22 ± 1.49 a 27.67 ± 0.54 a 4.86 ± 0.11 CCBA 130.72 ± 1.58 a 26.33 ± 0.96 a 4.98 ± 0.17 Effects of lycopene on basic physiological parameters and egg quantity of eggs on 21 d and 42 d As shown in the Fig. 1 A, egg weight has significantly increased on days 21, 28, 35, and 42 ( P < 0.05 ), the yolk weight has significantly increased on days 21, 35 and 42 ( P < 0.05 ), and yolk score has significantly increased on days 14, 21, 28, 35, and 42 ( P < 0.05 ). In the Fig. 1 B, organ indices analysis revealed significantly increased liver and ovary indices in the LYC and CCBA groups than in the CON group ( P 0.05 ). In the Fig. 1 D, the content of β-carotene in the serum was shown on days 42, LYC, CLYC, and CCBA groups β-carotene content has significantly increased compared with CON group ( P < 0.05 ). In the Fig. 1 C, the β-carotene content in the serum was shown on day 42. Compared to the CON group, the LYC, CCBA, and CLYC groups showed a significant improvement ( P < 0.05 ). The β-carotene, UFA, and vitamin A content in the LYC and CCBA groups significantly increased than those in the CON and CLYC groups ( P < 0.05 ), and the α-linolenic acid, Linolenic acid, and Arachidonic acid content in the LYC group remarkedly higher than those in the CLYC group ( P < 0.05 ). In the Fig. 1 D, the nutritional compounds content in the eggs was shown on day 42. The content of β-carotene and UFA in the LYC and CCBA groups significantly increased compared to the CON and CLYC groups ( P < 0.05 ), the content of vitamin A in the LYC, CLY, and CCBA groups remarkedly higher than that in the CON group ( P < 0.05 ), and CLYC group also significantly increased than in the LYC group ( P < 0.05 ). It is plausible that this nutritional improvement contributes to the documented protective effects of UFAs on the intestinal epithelium, which are underpinned by the reinforcement of tight junctions and the inhibition of necroptosis [ 14 , 15 ]. Moreover, the observed significant increase in the vitamin content of these eggs suggests that their consumption could contribute to improved human health due to this nutritional enhancement. The research by An et al [ 16 ] demonstrated that dietary supplementation with either 20 mg/kg lycopene or 1.7% ketchup for 28 days significantly increased egg weight and egg production in Hy-line Brown laying hens. Similarly, Ma et al. [ 17 ] reported that supplementation with 10, 20, and 30 mg/kg lycopene enhanced the ADG of broilers. Based on the experimental data, egg weight in the CLYC group was significantly higher than that in the CON group at days 21, 28, and 42. Additionally, the yolk color score in the LYC group was significantly increased compared to the CON group after day 14. Olson et al. [ 18 ] demonstrated that dietary lycopene supplementation enhanced yolk color and immune function, which is consistent with our experimental findings. These results indicate that dietary lycopene supplementation can significantly increase egg quality and improve growth performance in laying hens. Effects of lycopene on intestinal microbiota on day 21 In the Fig. 2 , microbiota composition was analyzed. The fecal microbiota composition was analyzed across four experimental groups. In the Fig. 2 A, at the phylum level, Firmicutes represented the dominant phylum in all groups, followed by Fusobacteria and Proteobacteria . At the genus level, Lactobacillus was the most abundant genus, with Streptococcus and Clostridiaceae_Clostridium showing secondary dominance. In the Fig. 2 B, Veen diagram analysis demonstrated both unique and shared microbial species among groups. The CON group contains 733 unique species, while the LYC, CLYC, and CCBA groups contain 296, 513, and 372 unique species, respectively. Intersection analysis revealed that CON shared 253, 238, and 248 species with LYC, CLYC, and CCBA groups, respectively. The LYC group shared 181 and 188 species with the CLYC and CCBA groups, respectively. While CLYC and CCBA groups shared 206 species. As shown in Fig. 2 C and D, alpha diversity showed no significant difference among groups ( P > 0.05 ). However, NMDS analysis revealed distinct clustering patterns, with CLYC and CCBA groups demonstrating particularly similar microbiota profiles (Stress = 0.0704). In the Fig. 2 E, the heat map of species variability among groups. Six phyla ( Ruminococcus , Phascolarctobacterium , Faecalibacterium , Desulfovibrio , Bacteroides , and Oscillospira ) were significantly enriched in the CON group, four phyla ( Turicibacter , Gallibacterium , Campylobacter , and Veillonella ) were significantly enriched in the LYC group, two phyla ( Clostridium and Escherichia ) alone were significantly enriched in the CLYC group, and one phyla ( Enbacterium ) was significantly enriched in the CCBA group. Two phyla ( Enterichia and SMB53 ) were significantly enriched in the LYC and CCBA groups, four phyla ( Streptococcus , Megamonas , Candidatus Arthromitus , and Prevotella ) were significantly enriched in the CON and CLYC groups, two phyla ( Lactobacillus and Megamonas ) were significantly enriched in the CON and CCBA groups, and two phyla ( Megamonas and Clostridium ) were significantly enriched in the CLYC and CCBA groups. The metabolic pathway statistics, differential analysis of metabolic pathways is shown in the Fig. 2 F, improvements were observed in the following pathways: amino acid biosynthesis, cofactor, prosthetic, group, electron, carroer, and vitamin biosynthesis, fatty acid and lipid biosynthesis, nucleoside and nucleotide biosythesis, carbothydrate degradation, carboxylate degration, fermentation and glycolysis . Compared with the CON group, the LYC group presented highly significant differences in the following pathway: P101-PWY ( P < 0.01 ) and CRNFORCAT-PWY ( P 0.05 ). The intestinal microbiota plays a pivotal role in regulating host physiological homeostasis, including promoting intestinal tissue development, enhancing immune responsiveness, and facilitating nutrient metabolism and absorption [ 19 , 20 ]. In this study, analysis revealed that the LYC group exhibited decreased Firmicutes abundance but no significant change in the Firmicutes / Bacteroidetes (F/B) ratio compared to the CON group. Previous studies have established that deviations in the gut F/B ratio, whether an increase or decrease, can be indicative of health risks [ 21 , 22 ]. In addition, as previous studies associate a reduced F/B ratio with inhibited short-chain fatty acids (SCFAs) production, altered cholesterol metabolism, and elevated cardiovascular risk [ 23 , 24 ]. In present study, however, lycopene supplementation did not significantly alter this ratio. As one of the predominant genus in the laying hens' gut microbiota, Lactobacillus produces acetic acid, which has a well-documented beneficial effect on gut health [ 25 , 26 ]. Heatmap analysis of the gut microbiota revealed a significant enrichment of Turicibacter and Veillonella in the LYC group. Notably, both are short-chain fatty acid producing genera, with Turicibacter being a known producer of butyrate and Veillonella of acetate. As both taxa generate SCFAs maintain intestinal barrier integrity, suppress inflammation, and promote microbial homeostasis, these enrichments may functionally contribute to lycopene’s beneficial effects [ 27 , 28 ]. Furthermore, metabolism pathway analysis revealed significant enrichment of the 101-PWY and CRNFORCAT-PWY pathways in the LYC group. Consistent with established literature, 101-PWY known as ‘Pyruvate Fermentation to Butanoate I,’ is a canonical microbial metabolic pathway responsible for the biosynthesis of butyrate, a primary metabolite from pyruvate. CRNFORCAT-PWY facilitates the bacterial degradation of creatine, wherein creatine is dismantled to sarcosine and urea. Sarcosine is further metabolized, ultimately generating glycine and acetate, while urea is hydrolyzed to release ammonia, providing a nitrogen source. Previous studies have demonstrated that butyrate specifically upregulates MUC gene expression in intestinal goblet cells, notably MUC3, via histone deacetylase (HDAC) inhibition [ 29 ]. Acetic acid, which participated in cellular metabolic pathways, maintains intestinal integrity, and regulates lipid and carbohydrate metabolism [ 30 ]. Therefore, we are fully convinced that the addition of lycopene to the diet not only promotes hepatic lipid metabolism but also promotes intestinal health. Effects of lycopene on antioxidant and metabolism factors in serum and eggs on day 42 As shown in the Fig. 3 A, the content of CAT has a significant increase in the CLYC and CCBA groups than that in the CON group ( P < 0.05 ). Compared to the CON group, the content of GSH-Px and SOD was remarkedly higher than that in the CON group ( P < 0.01 ), and the content of SOD in the CCBA group also significantly increased than that in the CON group ( P < 0.01 ). The correlation analysis is displayed in Fig. 3 B. Piselman-related analysis of antioxidant substances in serum and eggs shows that the CAT level in the serum was strongly correlated with SOD and RP in eggs, the GSH-Px level was strongly correlated with DPPH, T-AOC, and SOD in eggs, and the SOD level in serum was strongly correlated with DPPH, T-AOC, and SOD in eggs. TG level in the CON group remarkedly higher than LYC, CLYC, and CCBA groups ( P < 0.05 ), as shown in the Fig. 3 C. HDL levels were higher in both the LYC and CCBA groups compared to the CON group. Furthermore, LDL levels were lower in all three treatment groups relative to the CON group, although this decrease did not reach statistical significance. Additionally, TG content was significantly lower in all three treatment groups compared to the CON group. Previous studies have shown that lycopene reduces the production of pro-inflammatory cytokines and decreases inflammatory damage in both hypercholesterolemic and high-fat diet-induced obese rats [ 31 , 32 ]. In addition, previous studies demonstrated that dietary lycopene supplementation significantly reduced serum and brain total TC, TG, and LDL-C levels in high-fat diet-fed rats, findings consistent with the results of the present study [ 9 , 33 ]. Effects of lycopene on the goblet cell number and IgA-positive expression of the ileum The number of goblet cells of the ileum among the four groups was shown in the Fig. 4 A. In the CCBA group, the number of goblet cells significantly increased compared with the CON group ( P < 0.05 ). In the Fig. 4 B, the immunohistochemical results of ileal IgA were shown. Compared with the CON group, IgA-positive expression was significantly increased in the LYC, CLYC, and CCBA groups ( P < 0.05 ). Based on observed increases in ileal goblet cell numbers and IgA-positive expression in hens, dietary lycopene supplementation appears to enhance ileal physiological function. These results are in line with our previous research which also demonstrated that[ 34 ]. The effects of lycopene on hepatic PCNA expression and lipid metabolism As shown in the Fig. 5 , the effects of lycopene on liver and the relative expression of lipid metabolic genes were shown. In the Fig. 5 A, the PCNA staining in the liver of the LYC group significantly increased compared with the CON group ( P < 0.05 ). In the Fig. 5 B, the relative expression of lipid metabolic genes was shown. The relative expression of ACC , LXR-α , and PPAR-α significantly increased in the CLYC and CCBA groups compared with CON group (P < 0.05). The relative expression of LXR-α in the LYC group was remarkedly higher compared with CON group (P < 0.05). In the LYC group, ACC and LXR-α expression were significantly upregulated compared to the CON group. Although other lipid metabolic genes demonstrated non-significant upward trends in both LYC and CLYC groups, these changes did not reach statistical significance. Collectively, these findings indicate that dietary lycopene supplementation modulates hepatic lipid metabolic pathways. The effects of lycopene on splenic PCNA expression and inflammatory genes As shown in the Fig. 6 , the effects of lycopene on spleen and the relative expression of inflammatory genes were shown. In the Fig. 6 A, the PCNA staining in the spleen showed no significant difference among the four groups ( P > 0.05 ). In the Fig. 6 B, the relative expression of inflammatory genes was shown. The relative expression of TNF-α, IL-1β, and IL-2 significantly increased in the CON group compared with LYC, CLYC, and CCBA groups ( P < 0.05 ). The relative expression of IFN-γ and IL-10 remarkedly higher in the CCBA group compared with CON group ( P < 0.05 ), and the relative expression of IL-10 also significantly increased in the CLYC group compared with CON group ( P < 0.05 ). As the largest peripheral immune organ, the spleen plays an indispensable role in immune cell growth and differentiation. Research investigating LYC effects on splenic immune function has gained increasing attention. Recent studies demonstrated that LYC counteracts aflatoxin-induced immunosuppression in mice by inhibiting splenic oxidative stress and apoptosis [ 35 ]. Consequently, we assessed the expression of inflammation factors in splenic tissue. The results demonstrated significantly reduced expression of these factors in all treatment groups compared to the CON group, consistent with previous findings. Conclusions In conclusion, based on the present study, we demonstrated that the replacement of β-carotene by lycopene in the additive package can improve the antioxidant, lipid metabolism function, and egg quality of laying hens. The immune function was also examined, which modulates gut microbiota and enhances intestinal barrier function, which is associated with increasing intestinal IgA levels. This makes a strong case for promoting the use of lycopene in poultry production, and offers a reference basis for delivering egg products of higher quality, greater nutritional value, enhanced safety, and improved health benefits. Declarations Acknowledgments: The authors would like to thank Yuan Gao, Huali Shi, Zhongbo Sun, Qi Lin, Qiyue Wu, and Shuai Zhang from Jilin Agriculture University for their assistance with the administration of the experiment. Contributions: Conceptualization, M.C., Y.D., J.P., J.L., Y.S., X.Y., M.W. and X.Z.; methodology, M.C., J.P., Y.S., J.L., Y.D., and X.Z.; formal analysis, M.C., Y.S. and X.Z.; resource, M.C., Y.S.; data curation, M.C., and J.P.; writing——original draft preparation, M.C.; Y.S.; writing ——review and editing, M.C., J.P., Y.S., and X.Z.; project administration, M.C., J.P., Y.S. X.Y. Y.L., W.S. and X.Z.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (No. 32472995). Ethics approval and consent to participate : This study was approved by the Experimental Animal Ethical Committee of Jilin agriculture university. 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Microbiome 11(1):41.http://10.1186/s40168-023-01490-5 Xu F, Wang P, Yao Q, Shao B, Yu H, Yu K, et al. (2019) Lycopene alleviates AFB(1)-induced immunosuppression by inhibiting oxidative stress and apoptosis in the spleen of mice. Food Funct 10(7):3868-3879.http://10.1039/c8fo02300j Additional Declarations No competing interests reported. Supplementary Files Experimentaldesignprocessdiagram.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":120258,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of lycopene on growth performance and egg quantity. The Fig 1 A is egg quantity, including egg weight, Yolk weight, and Yolk score. The hen organ index is shown in Fig 1 B. The nutrition compounds in eggs on days 21 and 42 are shown in Fig 1 D and E, respectively, which include β-carotene, UFA, Vitamin A, α-linolenic acid, Linolenic acid, and Arachidonic acid. In the Fig 1 C, the content of β-carotene in serum on day 42.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/f65af879923bf762b597ba38.png"},{"id":98931837,"identity":"be745b78-2578-4f49-9b61-881fe7e650fd","added_by":"auto","created_at":"2025-12-24 09:07:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199198,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of lycopene on intestinal microbiota on 21 d. The top 20 microbial composition of four groups in the feces at the phylum and gene level are shown in Fig 2A. The different microbial composition of four groups was analyzed by Vene and NMDS analysis, as shown in Fig 2B and D. Alpha-diversity analysis of the feces microbiota is shown in Fig 2C, including Chao 1, Goods_coverage, Simpson, Pielou_e, Observed_species, and Shannon. The richness of microbiota among the four groups was shown by the heat map in Fig 2E. Microbiota with significant differences at the genus level in the feces microbiota of the four groups and metabolic pathway expression were shown in Fig 2F and H. The significant difference in metabolic pathway expression in the four groups is shown in Fig 2G.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/1914b8bf9a7bbe0105dbc279.png"},{"id":98931839,"identity":"5d360963-5b44-41cd-98fa-cb2c227610c4","added_by":"auto","created_at":"2025-12-24 09:07:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82654,"visible":true,"origin":"","legend":"\u003cp\u003eThe serum and egg antioxidant substances were shown in Fig 3A. The serum, including CAT, GSH-Px, MDA, and SOD, and the egg, including DPPH, RP, SOD, and T-AOC. The Fig 3B was antioxidant substances Spearman-related analysis between hen and egg. The Fig 3C was metabolism compounds in serum of hens. n=5.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/cce3584c88526baaec27695a.png"},{"id":99310307,"identity":"7aa38155-3cad-442e-b4ec-0242d8e1a9dd","added_by":"auto","created_at":"2025-12-31 16:12:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":270041,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of lycopene on goblet cells and IgA-positive expression of the ileum. The number of goblet cells observed under 10 fields for each group, n=30. The PCNA area was observed under 15 fields for each group, n=15.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/bf713c0964ac39ed500d7b90.png"},{"id":99310306,"identity":"541bc791-fa81-450f-9702-8db202be8547","added_by":"auto","created_at":"2025-12-31 16:12:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234774,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of lycopene on hepatic PCNA expression and lipid metabolic genes. n=5.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/78557623249bd18de23147fd.png"},{"id":99310183,"identity":"d6d262f7-ecb2-4a74-aff2-c7a8e9a6ba6f","added_by":"auto","created_at":"2025-12-31 16:12:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":257821,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of lycopene on splenic PCNA expression and inflammatory genes. n=5.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/1d8c274cb722168ef23b4c70.png"},{"id":99787847,"identity":"c266ddd9-2b0e-4976-aeeb-1d1684cfa3f8","added_by":"auto","created_at":"2026-01-08 12:38:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2199227,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/acfc6124-385e-40c3-9d8b-c198ae068ab7.pdf"},{"id":99310262,"identity":"f8a6801d-cc4e-43af-abbf-c85b52b23236","added_by":"auto","created_at":"2025-12-31 16:12:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":662204,"visible":true,"origin":"","legend":"","description":"","filename":"Experimentaldesignprocessdiagram.docx","url":"https://assets-eu.researchsquare.com/files/rs-8414745/v1/ed70215f1dfc3de4bde312d6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary supplementation with lycopene can effectively enhance egg quality and antioxidant function in laying hens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrowing health consciousness has led to increased demand for health-promoting, safe, and sustainably produced foods. As societies develop, dietary priorities are shifting from sufficiency to quantity. Eggs, as a low-cost and nutritious food staple, are central to this transition. Therefore, improving egg quantity is crucial for better satisfying human nutritional needs. As natural antioxidants, phytochemicals possess distinct advantages over synthetic alternatives, including natural abundance, potent antioxidant activity, and low toxicity. Among common plant-derived foods, flavonoids and carotenoids represent two major antioxidant phytochemicals that have attracted considerable attention from both the scientific community and the general public [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nutrition exerts profound and multifaceted effects on animal immune function and overall health status, encompassing both nutrient metabolism at the host-microbiota interface and complex microbiota-host system interactions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Carotenoids are a class of yellow, orange, or red polyene compounds ubiquitously distributed in nature, with representative members including β-carotene, lycopene, and astaxanthin. Accumulating evidence has demonstrated their multifaceted biological functions, particularly in cancer chemoprevention, antioxidation, immunomodulation, and pigmentation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLycopene, a lipophilic carotenoid derived from red fruits and vegetables, enhances growth performance, lowers blood lipid levels, and improves antioxidant capacity and reproductive efficiency [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Primarily located within cell membranes and lipoproteins, it is most concentrated in low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL). Previous studies demonstrate that lycopene neutralizes free radicals, mitigates lipid peroxidation, and exerts hepatoprotective effects. It achieves this by regulating superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), thereby scavenging excess reactive oxygen species (ROS) produced in vivo [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, dietary supplementation of lycopene in rats fed a high-fat diet significantly reduced serum and brain levels of total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C), and oxidized LDL (ox-LDL) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, studies on the use of natural plant extracts to enhance egg quality, particularly its polyunsaturated fatty acid content, remain limited. Furthermore, research on the effects of lycopene on egg quality and lipid metabolism remains limited to date. Therefore, this study aimed to investigate the impact of dietary lycopene supplementation on egg quality and hepatic lipid metabolism in laying hens. Additionally, our previous research demonstrated that a compound feed addictive containing β-carotene significantly enhanced breeder hen intestinal microbiota. To evaluate whether lycopene alone elicits comparable microbiota effects to β-carotene, we further examined its influence on microbiota parameters.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e All animal experiments complied with the ARRIVE guidelines. This animal experimental protocol was implemented under the supervision of the Chinese Guidelines for Animal Welfare and Experimental Protocol and was approved by the Animal Ethics Committee, Jilin Agriculture University (NO.2017050001). The stocking densities for each housing system were established in accordance with standard industry and regulatory guidelines. Specifically, the conventional cage system maintained a density of approximately 9 birds/m\u0026sup2;, consistent with common commercial practices for intensive quail production. These free-range densities were designed to comply with, or exceed, established organic and free-range poultry welfare standards, including USDA Organic regulations and EU Council Regulation 889/2008.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperiment preparation\u003c/h3\u003e\n\u003cp\u003eFood-grade carotene powder and tomato powder were purchased from Shanxi Mixianer Biotechnology Co., Ltd., China, and Chenguang Biotechnology Group Co., Ltd., China, respectively. The β-carotene content in the carotene powder was quantitatively analyzed by Pony Testing Group, China, using high performance liquid chromatography (HPLC). Briefly, samples underwent saponification to liberate carotenoids into their free forms, followed by extraction with petroleum ether and dichloromethane to a fixed volume. The extracts were then separated external standard calibration curve (β-carotene standard, \u0026ge;\u0026thinsp;97% purity, Sigma-Aldrich). Similarly, the lycopene content in tomato powder was determined by Chenguang Biotechnology Group, China, employing identical analytical methodology.\u003c/p\u003e\n\u003ch3\u003eAnimal management\u003c/h3\u003e\n\u003cp\u003eThe experimental Jinghong NO.1 laying hens were obtained from Changchun Hefeng Co., Ltd., China. Three hundred and sixty healthy 25-week-old laying hens were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;90 per group, with three replicates of 30 hens each), showing no significant initial weight differences. The laying hens were kept in cages (120 cm\u0026times; 60 cm\u0026times;60 cm) equipped with two nipple drinkers and one 100 cm-long feed. The four groups were the control group (CON), the lycopene treatment group (LYC), the lycopene complex treatment group (CLYC), and the β-carotene complex treatment group (CCBA), respectively. After one week acclimation period, the hens in the CON group were fed a basal diet, the LYC group hens were supplemented with 120 mg/kg lycopene, the CLYC group hens were supplemented with 60 mg/kg lycopene, 250 mg/kg curcumin, 250 mg/kg allicin, and 500 mg/kg sodium butyrate, the CCBA group hens supplemented with 60 mg/kg β-carotene, 250 mg/kg curcumin, 250 mg/kg allicin, and 500 mg/kg sodium butyrate for 6 weeks. Daily feed intake and egg production were recorded for each group throughout the trial period. Egg quality parameters, including egg weight, yolk weight, yolk score, yolk weight, Protein weight, and yolk score were measured weekly (n\u0026thinsp;=\u0026thinsp;5 for each group per time point). The experiment was conducted for 42 days. On day 21, blood samples were collected via venipuncture, and fresh fecal samples were obtained for subsequent analysis (n\u0026thinsp;=\u0026thinsp;10 per group). At the end of the experiment, blood was collected. Body weights, liver, spleen, and intestinal weight were recorded. The following instruments were used for measurements: MA2 protein height analyzer (Beijing Heng Odd Instrument Co., Ltd., China), Roche colorimetric (Nanjing Mingao Instrument Equipment Co., Ltd., China), and a precision electronic balance (accuracy\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001g; Mettler-Toledo International Inc). The compositions and nutrition levels of the basal diets are shown in Supplementary Table\u0026nbsp;1. All laying hens are fasted for 4 h to facilitate anesthetic absorption. They are then deeply anesthetized via intramuscular injection of ketamine and xylazine. Once the absence of pedal reflex was confirmed, euthanasia was performed by an intravenous injection of an overdose of sodium pentobarbital into the wing vein. Death was confirmed by the absence of corneal reflex and cessation of breathing.\u003c/p\u003e\n\u003ch3\u003eGrowth performance and egg nutrition test\u003c/h3\u003e\n\u003cp\u003eThe daily feed intake (ADFI) and egg production (ADE) were recorded for each group. The feed conversion ratio (FCR) was calculated (FCR\u0026thinsp;=\u0026thinsp;ADFI/ADE). Laying rate was determined by dividing (FCR\u0026thinsp;=\u0026thinsp;ADE/Total hen number) \u0026times;100%. Organ indices were computed as (organ weight / body weight) \u0026times; 100%. Chemical analysis was conducted by Huace Testing (China) using validated methods: Vitamin A was quantified via HPLC-UV (325nm) following saponification and purification; β-carotene was analyzed by HLPC-UV/Vis (450nm) after organic solvent extraction; and polyunsaturated fatty acids (PUFAs) were determined as fatty acid methyl esters (FAMEs) by DC-FID following lipid extraction and methylation, with all analyses employing external standard quantification. Specially, vitamin A analysis utilized a C18 column (4.6tamin \u0026times;250 mm, 5 \u0026micro;m) separation after solid phase extraction, β-carotene was separated on a C30 column (4.6 \u0026times;250 mm, 5 \u0026micro;m) following silica gel purification, and PUFA analysis employing an HP-88 capillary column (100 m\u0026times; 0.25 mm \u0026times;0.2 \u0026micro;m) with reference to a 37-component FAME mix.\u003c/p\u003e\n\u003ch3\u003eQuantification of Antioxidant Compounds in Egg Yolk and Serum\u003c/h3\u003e\n\u003cp\u003eBlood samples (n\u0026thinsp;=\u0026thinsp;10 for each group) were collected and maintained at 4\u0026deg;C for 30 min prior to serum separation by centrifugation (3500g, 15 min). For egg white analysis, 0.5 mL aliquots were homogenized with 1mL of Dulbecco\u0026rsquo;s phosphate-buffered saline (D-PBS), supplemented with 52.52 mg polyethylene glycol 8000 (PEG8000), and centrifuged (14,000g, 10 min) to collect the supernatant fractions. Egg yolk samples (0.5 mL) were processed similarly using D-PBS homogenization followed by chloroform extraction (1.5 mL) and centrifugation (1000g, 30 min). Analyte concentrations in both matrices were normalized to sample volume (\u0026micro;g/mL). Oxidative stress markers, including Malondialdehyde (MDA), Catalase (CAT), Superoxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC), and Glutathione Peroxidase (GSH-Px) were analyzed using standardized assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., China) following manufacturer specifications. For egg yolk antioxidant assessments, reducing power was determined by physiological saline extraction, while 2,2-diphenyl-1-picrydrazyl (DPPH) radical scavenging activity was measured using ethanol extraction methodology [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Serum levels of High-density lipoprotein (HDL), Low-density lipoprotein (LDL), Total Cholesterol (TC), and Triglyceride (TG) were quantified using standardized assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., China) following manufacturer specifications, with triplicate measurements per sample.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistological observation of the intestine and the number of goblet cells\u003c/h2\u003e \u003cp\u003eIleal, hepatic, and splenic tissues (n\u0026thinsp;=\u0026thinsp;10 for each group) from laying hens were fixed in 4% paraformaldehyde solution, dehydrated through an ethanol series, embedded in paraffin, and sectioned at 5 \u0026micro;m thickness using a rotary microtome. Tissue sections were stained with hematoxylin and eosin (Soleberg Biotechnology, Beijing, China) for histological examination. Morphometric analysis was performed using an Olympus BX53 light microscope (Olympus corporation, Japan), with villus height and crypt depth measurements using ImageJ software (National Institutes of Health, USA). Goblet cells were quantified following Periodic Acid Schiff (PAS) staining (Leagene Biotechnology Co., Ltd., China) using standard histochemical protocols.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemical measurement\u003c/h3\u003e\n\u003cp\u003eSplenic, hepatic, and ileal sections (5 \u0026micro;m) on slides were treated with xylene, different concentration gradients of alcohol, and washed in PBS (PH\u0026thinsp;=\u0026thinsp;7.4 for 3 min). Antigen retrieval was performed using boiling sodium citrate antigen buffer for 20 min. After cooling to room temperature, the slides were incubated with the pre-oxygenase blocking solution (Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) at 37\u0026deg;C for 10 min. After washing in PBS, the slides were incubated overnight at 4\u0026deg;C with mouse anti-chicken immunoglobulin A (IgA) primary antibodies (1:100, Monoclonal, Southern Biotech, Cat. No.8330-01, U.S.A.). The slides treated with PBS instead of the primary antibody served as the negative control. After being exposed to biotinylated secondary antibody goat anti-mouse IgG (Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) for 20 min at 37\u0026deg;C, slices were incubated with streptavidin (Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) for 10 min at 37\u0026deg;C. Under dark conditions. Under dark conditions, the slices were immersed in diaminobenozidine hydrochloride (DAB, Fuzhou Meixin Biotechnology Development Co., Ltd., Fujian, China) to develop the immunoreaction. This reaction was quenched by immersion in distilled water after the brown staining developed. Slices were counter-stained with H\u0026amp;E followed by hydrochloric acid alcohol color separation. They were finally washed with tap water, dehydrated in ethanol, and cleared using xylene.\u003c/p\u003e \u003cp\u003eSections on the slide were viewed under the light microscope (Olympus, Tokyo, Japan), and any IgA-positive areas in the five different microscope fields for each tissue were measured with Image J (National Institutes of Health, Bethesda, MD U.S.A) and the average calculated.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and real-time fluorescence quantitative analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from the ileum, liver, and spleen (n\u0026thinsp;=\u0026thinsp;10 for each group) using Trizol reagent, with concentration and purity assessed by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). Following reverse transcription using a High-Capacity cDNA kit, qPCR analysis was performed in triplicate on a CFX Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR Green chemistry. Tissue-specific reference genes were employed: \u003cem\u003eβ-actin\u003c/em\u003e for liver (\u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eACO\u003c/em\u003e, \u003cem\u003eLXR-α\u003c/em\u003e, \u003cem\u003ePPAR-α\u003c/em\u003e, and \u003cem\u003eSCD\u003c/em\u003e), and spleen (\u003cem\u003eTNF-α\u003c/em\u003e, \u003cem\u003eIFN-γ\u003c/em\u003e, \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-2\u003c/em\u003e, and \u003cem\u003eIL-10\u003c/em\u003e). Relative gene expression was calculated using 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e, with all primers (Sangong Biotechnology, Shanghai, China) demonstrating 90\u0026ndash;110% amplification efficiency (sequences in Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSequencing analysis of bacterial colonies and statistical analysis\u003c/h2\u003e \u003cp\u003eOn day 21 of the experiment, total microbial RNA was extracted from hen faces (n\u0026thinsp;=\u0026thinsp;5 for each group). Sequencing analysis of bacterial colonies was performed by analyzing the KEGG database and the MetaCyc database. Finally, the metagenomeSeq method was combined with calling the fitFeatureModel function to use a zero-included log-normal model to fit the distribution of each pathway/group, and the results of this model were used to determine the significance of the difference. Group distributions use the results of this model to discriminate the significance of the difference.\u003c/p\u003e \u003cp\u003eData were analyzed for significance using SPSS, and the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error was plotted using Prism software. \u003cem\u003eP\u003c/em\u003e-values less than 0.05 were considered statistically significant, and \u003cem\u003eP\u003c/em\u003e-values less than 0.01 were considered highly statistically significant. * In the figure, lower case letters in the table indicate a \u003cem\u003eP\u003c/em\u003e-value less than 0.05, and ** in the figure and uppercase letters indicate a P-value less than 0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of lycopene on growth performance\u003c/h2\u003e \u003cp\u003eAs shown in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e, no significant changes in ADFI, ADE, and FCR were observed in the four groups during the first two weeks of the experiment. At days 21, 35, and 42, ADE was significantly increased in the three treatment groups compared with CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). At days 28 and 42, ADFI was remarkedly higher in CLYC group compared with CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). And ADFI was significantly increased in the CCBA group compared with CON group, at day 42 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Mokhtar et al [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] found that supplementing broiler diets with 100 mg/kg lycopene significantly improved growth performance. Sarker et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] reported that dietary supplementation of lycopene improved the average daily weight gain in broilers. Although we supplemented lycopene to the laying hen diet, our findings same with those reported in previous studies.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of lycopene on growth performance.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eGrowth performance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADFI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eADE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFCR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e7 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e112.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e14 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e119.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e118.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e119.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e119.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e21 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e129.47\u0026thinsp;\u0026plusmn;\u0026thinsp;4.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e129.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e133.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e28 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e123.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e127.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e130.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e127.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e35 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e126.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e42 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e129.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e134.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e130.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of lycopene on basic physiological parameters and egg quantity of eggs on 21 d and 42 d\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, egg weight has significantly increased on days 21, 28, 35, and 42 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), the yolk weight has significantly increased on days 21, 35 and 42 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and yolk score has significantly increased on days 14, 21, 28, 35, and 42 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, organ indices analysis revealed significantly increased liver and ovary indices in the LYC and CCBA groups than in the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), while spleen and tubal indices remained unchanged (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the content of β-carotene in the serum was shown on days 42, LYC, CLYC, and CCBA groups β-carotene content has significantly increased compared with CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the β-carotene content in the serum was shown on day 42. Compared to the CON group, the LYC, CCBA, and CLYC groups showed a significant improvement (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The β-carotene, UFA, and vitamin A content in the LYC and CCBA groups significantly increased than those in the CON and CLYC groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and the α-linolenic acid, Linolenic acid, and Arachidonic acid content in the LYC group remarkedly higher than those in the CLYC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the nutritional compounds content in the eggs was shown on day 42. The content of β-carotene and UFA in the LYC and CCBA groups significantly increased compared to the CON and CLYC groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), the content of vitamin A in the LYC, CLY, and CCBA groups remarkedly higher than that in the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and CLYC group also significantly increased than in the LYC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). It is plausible that this nutritional improvement contributes to the documented protective effects of UFAs on the intestinal epithelium, which are underpinned by the reinforcement of tight junctions and the inhibition of necroptosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, the observed significant increase in the vitamin content of these eggs suggests that their consumption could contribute to improved human health due to this nutritional enhancement. The research by An et al [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] demonstrated that dietary supplementation with either 20 mg/kg lycopene or 1.7% ketchup for 28 days significantly increased egg weight and egg production in Hy-line Brown laying hens. Similarly, Ma et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] reported that supplementation with 10, 20, and 30 mg/kg lycopene enhanced the ADG of broilers. Based on the experimental data, egg weight in the CLYC group was significantly higher than that in the CON group at days 21, 28, and 42. Additionally, the yolk color score in the LYC group was significantly increased compared to the CON group after day 14. Olson et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] demonstrated that dietary lycopene supplementation enhanced yolk color and immune function, which is consistent with our experimental findings. These results indicate that dietary lycopene supplementation can significantly increase egg quality and improve growth performance in laying hens.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of lycopene on intestinal microbiota on day 21\u003c/h2\u003e \u003cp\u003eIn the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, microbiota composition was analyzed. The fecal microbiota composition was analyzed across four experimental groups. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, at the phylum level, \u003cem\u003eFirmicutes\u003c/em\u003e represented the dominant phylum in all groups, followed by \u003cem\u003eFusobacteria\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e. At the genus level, \u003cem\u003eLactobacillus\u003c/em\u003e was the most abundant genus, with \u003cem\u003eStreptococcus\u003c/em\u003e and \u003cem\u003eClostridiaceae_Clostridium\u003c/em\u003e showing secondary dominance. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Veen diagram analysis demonstrated both unique and shared microbial species among groups. The CON group contains 733 unique species, while the LYC, CLYC, and CCBA groups contain 296, 513, and 372 unique species, respectively. Intersection analysis revealed that CON shared 253, 238, and 248 species with LYC, CLYC, and CCBA groups, respectively. The LYC group shared 181 and 188 species with the CLYC and CCBA groups, respectively. While CLYC and CCBA groups shared 206 species. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, alpha diversity showed no significant difference among groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). However, NMDS analysis revealed distinct clustering patterns, with CLYC and CCBA groups demonstrating particularly similar microbiota profiles (Stress\u0026thinsp;=\u0026thinsp;0.0704). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, the heat map of species variability among groups. Six phyla (\u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003ePhascolarctobacterium\u003c/em\u003e, \u003cem\u003eFaecalibacterium\u003c/em\u003e, \u003cem\u003eDesulfovibrio\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, and \u003cem\u003eOscillospira\u003c/em\u003e) were significantly enriched in the CON group, four phyla (\u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eGallibacterium\u003c/em\u003e, \u003cem\u003eCampylobacter\u003c/em\u003e, and \u003cem\u003eVeillonella\u003c/em\u003e) were significantly enriched in the LYC group, two phyla (\u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e) alone were significantly enriched in the CLYC group, and one phyla (\u003cem\u003eEnbacterium\u003c/em\u003e) was significantly enriched in the CCBA group. Two phyla (\u003cem\u003eEnterichia\u003c/em\u003e and \u003cem\u003eSMB53\u003c/em\u003e) were significantly enriched in the LYC and CCBA groups, four phyla (\u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eMegamonas\u003c/em\u003e, \u003cem\u003eCandidatus Arthromitus\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e) were significantly enriched in the CON and CLYC groups, two phyla (\u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eMegamonas\u003c/em\u003e) were significantly enriched in the CON and CCBA groups, and two phyla (\u003cem\u003eMegamonas\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e) were significantly enriched in the CLYC and CCBA groups. The metabolic pathway statistics, differential analysis of metabolic pathways is shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, improvements were observed in the following pathways: \u003cem\u003eamino acid biosynthesis, cofactor, prosthetic, group, electron, carroer, and vitamin biosynthesis, fatty acid and lipid biosynthesis, nucleoside and nucleotide biosythesis, carbothydrate degradation, carboxylate degration, fermentation and glycolysis\u003c/em\u003e. Compared with the CON group, the LYC group presented highly significant differences in the following pathway: \u003cem\u003eP101-PWY\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and \u003cem\u003eCRNFORCAT-PWY\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, the relative abundance of microbiota in feces showed that there was no significant difference among the four groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). The intestinal microbiota plays a pivotal role in regulating host physiological homeostasis, including promoting intestinal tissue development, enhancing immune responsiveness, and facilitating nutrient metabolism and absorption [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, analysis revealed that the LYC group exhibited decreased \u003cem\u003eFirmicutes\u003c/em\u003e abundance but no significant change in the \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e (F/B) ratio compared to the CON group. Previous studies have established that deviations in the gut F/B ratio, whether an increase or decrease, can be indicative of health risks [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, as previous studies associate a reduced F/B ratio with inhibited short-chain fatty acids (SCFAs) production, altered cholesterol metabolism, and elevated cardiovascular risk [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In present study, however, lycopene supplementation did not significantly alter this ratio. As one of the predominant genus in the laying hens' gut microbiota, \u003cem\u003eLactobacillus\u003c/em\u003e produces acetic acid, which has a well-documented beneficial effect on gut health [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Heatmap analysis of the gut microbiota revealed a significant enrichment of \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eVeillonella\u003c/em\u003e in the LYC group. Notably, both are short-chain fatty acid producing genera, with \u003cem\u003eTuricibacter\u003c/em\u003e being a known producer of butyrate and \u003cem\u003eVeillonella\u003c/em\u003e of acetate. As both taxa generate SCFAs maintain intestinal barrier integrity, suppress inflammation, and promote microbial homeostasis, these enrichments may functionally contribute to lycopene\u0026rsquo;s beneficial effects [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, metabolism pathway analysis revealed significant enrichment of the 101-PWY and CRNFORCAT-PWY pathways in the LYC group. Consistent with established literature, 101-PWY known as \u0026lsquo;Pyruvate Fermentation to Butanoate I,\u0026rsquo; is a canonical microbial metabolic pathway responsible for the biosynthesis of butyrate, a primary metabolite from pyruvate. CRNFORCAT-PWY facilitates the bacterial degradation of creatine, wherein creatine is dismantled to sarcosine and urea. Sarcosine is further metabolized, ultimately generating glycine and acetate, while urea is hydrolyzed to release ammonia, providing a nitrogen source. Previous studies have demonstrated that butyrate specifically upregulates MUC gene expression in intestinal goblet cells, notably MUC3, via histone deacetylase (HDAC) inhibition [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Acetic acid, which participated in cellular metabolic pathways, maintains intestinal integrity, and regulates lipid and carbohydrate metabolism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, we are fully convinced that the addition of lycopene to the diet not only promotes hepatic lipid metabolism but also promotes intestinal health.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of lycopene on antioxidant and metabolism factors in serum and eggs on day 42\u003c/h2\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the content of CAT has a significant increase in the CLYC and CCBA groups than that in the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Compared to the CON group, the content of GSH-Px and SOD was remarkedly higher than that in the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), and the content of SOD in the CCBA group also significantly increased than that in the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The correlation analysis is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. Piselman-related analysis of antioxidant substances in serum and eggs shows that the CAT level in the serum was strongly correlated with SOD and RP in eggs, the GSH-Px level was strongly correlated with DPPH, T-AOC, and SOD in eggs, and the SOD level in serum was strongly correlated with DPPH, T-AOC, and SOD in eggs. TG level in the CON group remarkedly higher than LYC, CLYC, and CCBA groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. HDL levels were higher in both the LYC and CCBA groups compared to the CON group. Furthermore, LDL levels were lower in all three treatment groups relative to the CON group, although this decrease did not reach statistical significance. Additionally, TG content was significantly lower in all three treatment groups compared to the CON group. Previous studies have shown that lycopene reduces the production of pro-inflammatory cytokines and decreases inflammatory damage in both hypercholesterolemic and high-fat diet-induced obese rats [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, previous studies demonstrated that dietary lycopene supplementation significantly reduced serum and brain total TC, TG, and LDL-C levels in high-fat diet-fed rats, findings consistent with the results of the present study [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects of lycopene on the goblet cell number and IgA-positive expression of the ileum\u003c/h2\u003e \u003cp\u003eThe number of goblet cells of the ileum among the four groups was shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. In the CCBA group, the number of goblet cells significantly increased compared with the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the immunohistochemical results of ileal IgA were shown. Compared with the CON group, IgA-positive expression was significantly increased in the LYC, CLYC, and CCBA groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Based on observed increases in ileal goblet cell numbers and IgA-positive expression in hens, dietary lycopene supplementation appears to enhance ileal physiological function. These results are in line with our previous research which also demonstrated that[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of lycopene on hepatic PCNA expression and lipid metabolism\u003c/h2\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the effects of lycopene on liver and the relative expression of lipid metabolic genes were shown. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the PCNA staining in the liver of the LYC group significantly increased compared with the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the relative expression of lipid metabolic genes was shown. The relative expression of \u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eLXR-α\u003c/em\u003e, and \u003cem\u003ePPAR-α\u003c/em\u003e significantly increased in the CLYC and CCBA groups compared with CON group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The relative expression of \u003cem\u003eLXR-α\u003c/em\u003e in the LYC group was remarkedly higher compared with CON group \u003cem\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e In the LYC group, ACC and LXR-α expression were significantly upregulated compared to the CON group. Although other lipid metabolic genes demonstrated non-significant upward trends in both LYC and CLYC groups, these changes did not reach statistical significance. Collectively, these findings indicate that dietary lycopene supplementation modulates hepatic lipid metabolic pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of lycopene on splenic PCNA expression and inflammatory genes\u003c/h2\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the effects of lycopene on spleen and the relative expression of inflammatory genes were shown. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the PCNA staining in the spleen showed no significant difference among the four groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). In the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, the relative expression of inflammatory genes was shown. The relative expression of TNF-α, IL-1β, and IL-2 significantly increased in the CON group compared with LYC, CLYC, and CCBA groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The relative expression of IFN-γ and IL-10 remarkedly higher in the CCBA group compared with CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and the relative expression of IL-10 also significantly increased in the CLYC group compared with CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). As the largest peripheral immune organ, the spleen plays an indispensable role in immune cell growth and differentiation. Research investigating LYC effects on splenic immune function has gained increasing attention. Recent studies demonstrated that LYC counteracts aflatoxin-induced immunosuppression in mice by inhibiting splenic oxidative stress and apoptosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consequently, we assessed the expression of inflammation factors in splenic tissue. The results demonstrated significantly reduced expression of these factors in all treatment groups compared to the CON group, consistent with previous findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, based on the present study, we demonstrated that the replacement of β-carotene by lycopene in the additive package can improve the antioxidant, lipid metabolism function, and egg quality of laying hens. The immune function was also examined, which modulates gut microbiota and enhances intestinal barrier function, which is associated with increasing intestinal IgA levels. This makes a strong case for promoting the use of lycopene in poultry production, and offers a reference basis for delivering egg products of higher quality, greater nutritional value, enhanced safety, and improved health benefits.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors would like to thank Yuan Gao, Huali Shi, Zhongbo Sun, Qi Lin, Qiyue Wu, and Shuai Zhang from Jilin Agriculture University for their assistance with the administration of the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions:\u003c/strong\u003e Conceptualization, M.C., Y.D., J.P., J.L., Y.S., X.Y., M.W. and X.Z.; methodology, M.C., J.P., Y.S., J.L., Y.D., and X.Z.; formal analysis, M.C., Y.S. and X.Z.; resource, M.C., Y.S.; data curation, M.C., and J.P.; writing\u0026mdash;\u0026mdash;original draft preparation, M.C.; Y.S.; writing \u0026mdash;\u0026mdash;review and editing, M.C., J.P., Y.S., and X.Z.; project administration, M.C., J.P., Y.S. X.Y. Y.L., W.S. and X.Z.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was funded by the National Natural Science Foundation of China (No. 32472995).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003eThis study was approved by the Experimental Animal Ethical Committee of Jilin agriculture university.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The raw sequences from 16S rRNA sequencing are available at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the BioProject accession numbers PRJNA1285354 (http://www.ncbi.nlm.nih.gov/bioproject/1285354).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKaulmann A, Bohn T (2014) Carotenoids, inflammation, and oxidative stress--implications of cellular signaling pathways and relation to chronic disease prevention. 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Food Funct 10(7):3868-3879.http://10.1039/c8fo02300j\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lycopene, Egg quantity, Growth performance, Intestinal barrier, Lipid metabolism, Microbiota","lastPublishedDoi":"10.21203/rs.3.rs-8414745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8414745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe strategic focus on improving the quality of eggs, a foundational and nutritious food, offers a straightforward method to elevate the nutritional value of diets. However, studies on strategies to improve egg quality remain limited. Lycopene, a lipophilic carotenoid derived from red fruits and vegetables, enhances growth performance, lowers blood lipid levels, and improves antioxidant capacity. Therefore, in this study, a total of 360 laying hens were randomly divided into four groups. The CON group was fed a basic diet, the LYC group supplemented with 120 mg/kg lycopene, whereas the CCBA group supplemented with 60 mg/kg β-carotene, 250 mg/kg curcumin, 250 mg/kg allicin, and 500 mg/kg sodium butyrate, the CLYC group replaced β-carotene with lycopene, while other ingredients remained the same. For the yolk score, the LYC group significantly increased at days 14, 21, 28, 35, and 42 compared to the CON group (\u003cem\u003eP\u0026lt; 0.05\u003c/em\u003e). The UFA and Vitamin content are significantly elevated in the LYC group compared to the CON group (\u003cem\u003eP\u0026lt;0.05\u003c/em\u003e). The result of lipid metabolism genes was shown, the relative expression of \u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eLXR-α\u003c/em\u003e, and \u003cem\u003ePPAR-α\u003c/em\u003e significantly increased in the CLYC and CCBA groups compared with the CON group (\u003cem\u003eP\u0026lt; 0.05\u003c/em\u003e), the relative expression of \u003cem\u003eLXR-α\u003c/em\u003e in the LYC group remarkedly higher than the CON group \u003cem\u003e(P\u0026lt; 0.05). \u003c/em\u003eAdditionally, the heatmap analysis showed that \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eVeillonella\u003c/em\u003e were enriched in the LYC group. The results of this study indicated that lycopene supplementation significantly improved egg quality, hepatic lipid metabolism, and systemic immunity in laying hens. Furthermore, analysis of gut revealed that lycopene increased ileal goblet cell amount and IgA production, while enriching beneficial microbiota.\u003c/p\u003e","manuscriptTitle":"Dietary supplementation with lycopene can effectively enhance egg quality and antioxidant function in laying hens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-24 09:07:37","doi":"10.21203/rs.3.rs-8414745/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54e0c376-8f53-4e3d-9c8b-54d12d929e9a","owner":[],"postedDate":"December 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-26T08:54:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-24 09:07:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8414745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8414745","identity":"rs-8414745","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}