Ovary metabolome and cecal microbiota changes in aged laying hens supplemented with vitamin E

Abstract

This study was aimed to evaluate the effect of vitamin E (VE) on laying performance, VE deposition, antioxidant capacity, immunity, follicle development, estrogen secretion, ovary metabolome, and cecal microbiota of laying hens. One hundred and twenty XinYang Black-Feathered laying hens (70 wk old) were randomly assigned to 2 groups (6 replicates of 20 birds), and fed a basal diet (containing 20 mg/kg VE, control (CON) group) and a basal diet supplemented with 20 mg/kg VE (VE group). The experiment lasted for 10 wk. Results showed that VE supplementation increased laying performance, antioxidant capacity, and immunity, as evidenced by increased (P < 0.05) performance (laying rate), antioxidant (glutathione peroxidase, total superoxide dismutase, total antioxidant capacity, and catalase) and immune (immunoglobulins) parameters, and decreased (P < 0.05) feed/egg ratio and malondialdehyde. Meanwhile, VE group had higher (P < 0.05) pregrade follicles, ovary index and serum estrogen levels than CON group. 16S rRNA sequencing showed that VE supplementation altered the cecal microbiota composition by increasing Bacteroides, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001 and Megamonas abundances and reducing Christensenellaceae_R-7_group abundance (at genus level), which are mainly associated with the production of short-chain fatty acids. Metabolomic profiling of the ovary revealed that the major metabolites altered by VE supplementation were mainly related to follicle development, estrogen secretion, anti-inflammatory, antioxidant, phototransduction, bile acid synthesis, and nutrient transport. Furthermore, changes in cecal microbiota (at genus level) and ovary metabolites were highly correlated with laying performance, antioxidant, and immune parameters. In summary, VE contributed to the laying performance of aged laying hens by enhancing antioxidant, immune, and ovarian functions, promoting follicle development and estrogen secretion, and regulating gut microbiota and ovary metabolites. These findings will provide a new perspective on the mechanisms of egg production in aged poultry ovaries.

INTRODUCTION

Late in the laying period, oxidative stress is heightened in laying hens, and the antioxidant and immune capacities are reduced, resulting in a reduction in ovarian function (Xie et al., 2019). Decreased ovarian function, manifested by a reduction in the number of follicles, an increase in the rate of follicular atresia, and a decrease in reproductive hormone levels, results in a reduction in laying rate, which seriously hinders the improvement of poultry production efficiency (Hao et al., 2021; Qiang et al., 2023; Xu et al., 2023). Vitamin E (VE), also known as α-tocopherol, is a powerful antioxidant that sits on the cell membrane and can slow down the body's aging process by stopping the free radical chain reaction from oxidizing the cell membrane (Miyazawa et al., 2019). Previously, researchers have reported that VE can improve the production performance of aged breeder hens by increasing immunity and ovary antioxidant function, promoting estrogen secretion, and regulating gut microbiota (Amevor et al., 2021a, b; 2022a, b). However, there are few in-depth studies on the ovaries of poultry due to the supplementation of VE in aged laying hens.

Metabolomic is increasingly being used in nutritional studies to better understand how diet affects metabolic pathways and to develop biomarkers of dietary exposure (German et al., 2003; Odriozola and Corrales, 2015). Yuan et al. (2020) performed a metabolomic analysis of stearoyl-CoA desaturase during goose follicle development and identified cholesterol and pantothenic acid as potential biomarker metabolites of goose granulosa cells. Therefore, using metabolomic may provide a more comprehensive understanding of the underlying mechanisms of how VE regulates ovarian function.

Until now, no studies have focused on the effect of VE on ovary metabolome in laying hens in old age. Besides, the Xin Yang Black-Feathered laying hen is a Chinese breed with good meat quality and flavor, which is worthy of our in-depth study. On the other hand, in recent years, the age of commercial laying hens has been extended beyond 72 wk to 80 wk or even 100 wk to make better use of the poultry house facilities and to reduce production costs. Therefore, a feeding experiment was conducted using XinYang Black-Feathered laying hens (70 wk old) to identify the key regulatory metabolites in the ovary of chickens due to VE supplementation. In addition, laying performance, immune function, ovary antioxidant capacity, ovary index, follicle numbers, estrogen levels, and caecum microbiota were also determined to further validate the beneficial effects of VE. This research will provide new insights into the mechanisms of egg production in aged poultry ovaries, as well as a theoretical basis for extending the egg-laying cycle of XinYang Black-Feathered laying hens.

MATERIALS AND METHODS

Experimental Design, Birds, and Management

This study was conducted in strict accordance with the Regulations on the Administration of Laboratory Animals issued by the State Science and Technology Committee of the People's Republic of China. The Animal Care and Use Committee of Zhejiang A&F University approved all experimental procedures.

A total of 120 healthy XinYang Black-Feathered laying hens (70 wk old) with similar laying rate (71%) were randomly divided into control (CON) group and VE group (6 replicates/group, 20 hens/replicate). Prior to this, a 2-wk pretest was carried out to ensure that all hens used in this study had similar health status and laying rate. During the pretest period, the hens were fed a corn-soybean meal diet (Table 1, containing 20 mg/kg VE), which met the nutrient requirements of laying hens according to the NY/T-33-2004 feeding standard (China National Standard, 2004).

Table 1.

Composition and nutrient levels of the control diet (% dry matter).

Items	Content
Ingredients
Corn	60.30
Soybean meal	24.70
Soybean oil	1.00
Limestone	9.50
Dicalcium phosphate	2.00
Sodium chloride	0.30
DL-Methionine	0.20
Premix1	2.00
Total	100.00
Nutrient levels2
Metabolic energy (ME)/(MJ/kg)	10.88
Crude protein	15.70
Total phosphorus	0.66
Available phosphorus	0.48
Calcium	4.00
Methionine	0.42
Methionine + cysteine	0.72
Lysine	0.83

¹Provided per kg of diet: Vitamin A, 11,000 IU; Vitamin D₃, 3,000 IU; Vitamin E, 20 mg; Vitamin K₃, 3 mg; Vitamin B₁, 2.5 mg; Vitamin B₂, 8.5 mg; Vitamin B₆, 4 mg; Vitamin B₁₂, 0.025 mg; nicotinic acid, 40 mg; D-pantothenate calcium, 14 mg; folic acid, 0.8 mg; D-biotin, 0.125 mg; choline chloride, 500 mg; copper, 8 mg; manganese, 100 mg; iron, 80 mg; zinc, 85 mg; iodine, 0.8 mg; selenium, 0.3 mg.

²ME was a calculated value, while the others were measured values.

In another 4-wk pretest, the XinYang Black-Feathered laying hens (70 wk old) in 5 groups received a corn-soybean meal diet (containing 20 mg/kg VE) supplemented with 0, 20, 40, 80, and 160 mg/kg VE for 4 wk, respectively. The results showed that VE addition improved the laying rate and feed conversion rate of laying hens from 71 to 74 wk, and there was no significant difference in laying rate of laying hens from 71 to 74 wk between the 20, 40, 80, and 160 mg/kg VE groups. The feed/egg ratio of laying hens from 71 to 74 wk in the 20 mg/kg VE group was significantly lower than those in the 40, 80, and 160 mg/kg VE groups (Data not published). For the purpose of reducing breeding costs and increasing the utilization effectiveness, the VE group in this sudy received a corn-soybean meal diet (Table 1, containing 20 mg/kg VE) supplemented with 20 mg/kg VE (Shanghai Yuanye Bio-Technology Co. Ltd., Shanghai, China; purity ≥ 99%). The birds in CON group received the corn-soybean meal diet (Table 1, containing 20 mg/kg VE). Hens were housed in 5-tier ladder-type cages with 4 hens per cage and exposed to a photoperiod cycle of 16L: 8D. Cages were placed in a ventilated room with an average room temperature of 20℃ ± 3℃. Throughout the 10-wk experiment, all birds had ad libitum access to water via nipple drinkers and food troughs. Eggs were collected daily at 4 pm.

Laying Performance

Total egg number and egg weight per replicate were recorded daily. The initial and final feed weights of each replicate were determined on a weekly basis. According to the above records, laying performance parameters including laying rate, average egg weight, average daily feed intake (ADFI), and feed/egg ratio were calculated at week 5 and 10.

Sample Collection and Preparation

Twenty-four birds (2 hens per replicate) from the CON and VE groups were randomly selected and weighed at the end of the experiment after an 8 h fasting. Samples of blood taken from the wing vein were placed into coagulation-promoting vacuum tubes. Samples were left at room temperature for 4 h and centrifuged at 3,000 rpm for 15 min to collect serum. The birds were then slaughtered by exsanguination of the jugular vein postanesthesia. Follicles were sorted and counted. Follicles can be divided into pregrade follicles and preovulatory follicles (≥ 12 mm) according to their diameter (Hao et al., 2021). Among them, pregrade follicles can be divided into small white follicles (SWF) (1–2 mm), large white follicles (LWF) (2–6 mm), small yellow follicles (SYF) (6–8 mm), and large white follicles (LYF) (8–12 mm) (Sun et al., 2022). Ovary without follicles with a diameter of more than 1 mm were collected immediately and weighed. Ovary index (g/kg) was calculated (ovary weight (g)/bird body weight (kg)). Each bird's cecal contents were collected aseptically. The serum, ovary, and caecum samples were stored at –80°C for further analysis.

VE Content and Antioxidant Indexes

The levels of VE, glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), catalase (CAT), and malondialdehyde (MDA) in the serum and ovary were tested by using assay kits from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China) according to the instructions of the kits.

Immune Indexes

Serum concentrations of immunoglobulin M (IgM), immunoglobulin A (IgA), and immunoglobulin A (IgG) were measured using test reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) by specific enzyme-linked immunosorbent assay (ELISA) method following the manufacturer's instructions.

Hormonal Assays

Commercially ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) were used for measuring serum levels of estradiol (E₂), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) according to the protocols of the manufacturer.

16S rRNA Gene Sequencing of Cecal Microbiota

Genomic DNA from cecal microorganisms was extracted using the DNAiso Reagent (TaKaRa, Kyoto, Japan), according to the manufacturer's instructions. Gel electrophoresis was used to check DNA quality. Universal primer pairs (338F: 5ʹ-ACTCCTACGGAGGCACAG-3ʹ; 806R: 5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ) were used to amplify the V3-V4 hyper variable regions of bacterial 16S rRNA. Purified amplicons were pooled equimolarly and paired-end sequenced using an Illumina HiSeq2500 PE300 platform (Illumina, San Diego, CA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw paired-end reads were quality filtered using Trimmomatic software and then merged using FLASH 1.2.7 (https://ccb.jhu.edu/software/FLASH/index.shtml). UPARSE (version 7.1, https://drive5.com/uparse/) was used to cluster Amplicon Sequence Varian (ASV) with a 100% similarity requirement. The Majorbio Cloud Platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China) was used for data analysis. An RDP classifier algorithm was used to analyze the taxonomy of each 16S rRNA gene sequence with a 70% confidence threshold. Mothur-1.30.2 was used for the calculation of Ace, Chao, and Shannon indices. The principal coordinates analysis (PCoA) plot based on unweighted UniFrac was used to estimate pairwise distances among samples and to establish β-diversity. Linear discriminant analysis (LDA) and effect size measurements (LEfSe) were used to identify biomarkers of microbial taxa (based on LDA score > 2.0) between groups. Functional differences between CON and VE groups were calculated using a Wilcoxon rank-sum test.

Untargeted Metabolomics Analysis of Ovary

For metabonomic analysis, 12 ovary samples (20 ± 1 mg)were collected (6 from the CON group and 6 from the VE group, respectively). Methanol/water (70%, v/v) and 2-chlorophenylalanine (1 μg/mL) were used to extract metabolites from samples. After centrifugation (4℃, 12,000 rpm, 10 min), 200 μL supernatant was collected in a liquid chromatographic bottle for LC-MS/MS analysis. Mix equal amounts of extracts from all samples in the same group to prepare quality control samples. The UHPLC system (LC20, Shimadzu, Kyoto, Japan) was equipped with an ACQUITY UPLC HSS T3 C18 column (1.8 µm, 2.1 mm × 100 mm; Waters Corp., Milford, MA). Mobile phases A and B were ultra-pure water (0.1% formic acid) and acetonitrile (0.1% formic acid), respectively. The gradient program involved: 95A: 5B (v/v) at 0 min; 10A: 90B (v/v) at 11.0 min; 10A: 90B (v/v) at 12.0 min; 95A: 5B (v/v) at 12.1 min; 95A: 5B (v/v) at 14.0 min. The temperature of the column was set at 40°C, the injection volume was 2 µL and the flow rate was set at 0.4 mL/min. A Triple TOF-6600 mass spectrometer (AB SCIEX, Framingham, MA) was used for mass spectrometry detection in both positive and negative ion modes.

The self-built MWDB database (Metware Biotechnology Co., Ltd., Hubei, Wuhan, China) was used for data analysis. Metabolites with a variable important in projection (VIP) > 1 and fold change (FC) > 2 or FC < 0.05 were used as criteria for screening potential biomarkers. The most relevant pathways for birds from the CON and VE groups were enriched on the basis of the KEGG pathway analysis using the significantly regulated metabolites.

Statistical Analysis

Data (expect for gut microbes and ovary metabolites) were analyzed using Student's t-test. Results were expressed as “mean ± standard deviation”. P < 0.05 was used as the significance level. Figures were created using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). Relationships between gut microbes (at genus level), significant ovary metabolites, immunoglobulins, antioxidant parameters and performance parameters were analyzed based on Spearman correlation analysis.

RESULTS

Laying Performance

As illustrated in Table 2, compared with CON group, VE group significantly (P < 0.05) enhanced the laying rate (7.63, 13.41, 9.11%) but decreased the feed/egg ratio (7.51, 9.65, 8.8%) during weeks 71 to 75, 76 to 80 and 71 to 80. There were no differences (P > 0.05) in the average egg weight and ADFI during weeks 71 to 75, 76 to 80, and 71 to 78 between the CON and VE groups.

Table 2.

Effect of VE increment on laying performance of laying hens.

Items	CON	VE	P-value
71–75 W
Laying rate (%)	66.63 ± 2.07b	71.71 ± 2.36a	0.005
Average egg weight (g)	53.93 ± 0.88a	53.59 ± 1.34a	0.643
Feed/egg	2.85 ± 0.09a	2.64 ± 0.09b	0.003
ADFI (g)	101.73 ± 0.98a	100.73 ± 1.60a	0.258
76–80 W
Laying rate (%)	68.92 ± 2.39b	78.16 ± 2.44a	<0.001
Average egg weight (g)	54.29 ± 0.90a	53.75 ± 1.09a	0.408
Feed/egg	2.63 ± 0.07a	2.38 ± 0.07b	<0.001
ADFI (g)	94.83 ± 1.32a	96.91 ± 3.44a	0.237
71–80 W
Laying rate (%)	67.76 ± 0.02b	73.93 ± 2.13a	0.001
Average egg weight (g)	53.79 ± 0.93a	53.43 ± 0.99a	0.556
Feed/egg	2.79 ± 0.08a	2.54 ± 0.08b	<0.001
ADFI (g)	101.62 ± 1.51a	100.45 ± 0.92a	0.145

CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 6 per group).

The same superscript letters in the same row indicate no significant differences (P > 0.05), while different letters indicate that the difference is significant (P < 0.05).

VE Deposition

As shown in Figure 1, serum and ovary VE levels were increased (P < 0.05) by the VE group in comparison with CON group. The magnitudes of increase were (80.24%) and (158.97%) in serum and ovary, respectively.

Figure 1.

Effect of VE increment on VE content of laying hens. Note: CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 12 per group). Different superscript letters in the bar chart indicate significant differences (P < 0.05). (A) The content of VE in serum. (B) The content of VE in ovary.

Antioxidant Parameters

Based on the results in Figure 2, we found that VE group generally increased the antioxidant capacity of hens compared with CON group, which was exhibited as follows: increased (P < 0.05) GSH-Px activity (13.32%) in serum; increased (P < 0.05) T-SOD (9.55 and 10.6%),CAT (14.5 and 28.7%) and T-AOC (10.17 and 15.56%) levels in serum and ovary; decreased (P < 0.05) serum and ovary MDA concentrations (12.22 in serum and 10.5% in ovary).

Figure 2.

Effect of VE increment on antioxidant parameters of laying hens. Note: CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 12 per group). The same superscript letters in the bar chart for the same indicator indicate no significant differences (P > 0.05), while different letters indicate that the difference is significant (P < 0.05). (A) GSH-Px: glutathione peroxidase. (B) T-SOD: total superoxide dismutase. (C) T-AOC, total antioxidant capacity. (D) MDA: malondialdehyde. (E) CAT, catalase. The GSH-Px, T-SOD, T-AOC and CAT were expressed as specific activity (U/mgprot) in ovary and as (U/mL) in serum, respectively. The MDA was expressed as (nmol/mgprot) in ovary and as (nmol/mL) in serum, respectively.

Immune Parameters

According to Figure 3, we found that VE group increased (P < 0.05) IgG, IgM, and IgA levels in serum by 8.96, 9.21, and 7.91%, respectively, compared with CON group.

Figure 3.

Effect of VE increment on serum immune indexes of laying hens. Note: CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 12 per group). Different superscript letters in the bar chart indicate significant differences (P < 0.05). IgA, immunoglobulin A; IgM, immunoglobulin M; IgG, immunoglobulin G.

Ovary Index and Follicles Numbers

Ovary index and follicles numbers results are presented in Figure 4. Compared with CON group, VE group increased (P < 0.05) SWF (26.21%), LWY (18.89%), LYF (31.52%), and total follicles numbers (16.87%) as well as ovary index (14.17%) (Figures 4A–4C, Figure 4E, Figure 4G). However, the numbers of SYF and grade follicles were not affected (P > 0.05) by VE supplementation (Figure 4, Figure 4).

Figure 4.

Effect of VE increment on the number of follicles. Note: CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 12 per group). The same superscript letters in the bar chart indicate no significant differences (P > 0.05), while different letters indicate that the difference is significant (P < 0.05).

Serum Estrogen Levels

The results in Figure 5 illustrated that the addition of VE effectively (P < 0.05) increased serum FSH (21.86%) and LH (18.61%) levels (Figure 5, Figure 5). In comparison with CON group, VE group had a tendency to increase serum E₂ levels, but the difference was not significant (P > 0.05) (Figure 5A).

Figure 5.

Effect of VE increment on serum estrogen levels of laying hens. Note: CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 12 per group). The same superscript letters in the bar chart indicate no significant differences (P > 0.05), while different letters indicate that the difference is significant (P < 0.05). E₂: estradiol; FSH: follicle-stimulating hormone; LH: luteinizing hormone.

Cecal Microbiota

The Ace and Chao indices represent the community richness of samples, while the Shannon index reflects the community diversity of samples. The Ace and Chao indices (representing α-diversity) of cecal microbiota were lower (P < 0.05) in VE group than those in CON group (Figures 6A and 6B). No obvious changes (P > 0.05) were found in the Shannon index (also representing α-diversity) between the VE and CON groups (Figure 6C). In order to assess the similarity of microbial community structure (β-diversity) between the VE and CON groups, we also performed PCoA analysis (Figure 6D). The results of PCoA showed the trend of microbial community segregation between the VE and CON groups, and we found that the microbial communities were clearly clustered and could be clearly divided into 2 groups (Figure 6D). As demonstrated in Figure 6E, hens in VE and CON groups shared 618 OTUs of cecal microbiota in the Venn diagram. Birds from CON and VE groups had 632 and 317 particular OTUs, respectively (Figure 6E).

Figure 6.

Microbial diversity analysis of cecal contents between VE and CON groups. Note: The α-diversity between VE and CON groups was reflected by Ace index (A), Chao index (B) and Shannon index (C). (D) β-diversity between the VE and CON groups display as principal coordinates analysis analysis. (E) Venn diagram of 2 microbial communities based on ASV. CON, control group; VE, vitamin E group. Data were presented as mean ± standard deviation (n = 4 per group). *P < 0.05.

We analyzed the changes in microbiota composition at genus level (Table 3). The Bacteroides, Rikenellaceae_RC9_gut_group and Phascolarctobacterium accounted for the largest proportion of the microbiota. Compared with CON group, VE group increased (P < 0.05) Prevotellaceae_UCG-001 abundance but decreased UCG-005 and Norank_f__Oscillospiraceae abundances. Marked upregulation of Bacteroides, Rikenellaceae_RC9_gut_group, Phascolarctobacterium, Megamonas, Lactobacillus, Prevotellaceae_Ga6A1_group, Megasphaera and Fusobacterium abundances and downregulation of Ruminococcus_torques_group, Unclassified_f__Oscillospiraceae, Norank_f__norank_o__Clostridia_UCG-014, Christensenellaceae_R-7_group, Butyricicoccus, Parabacteroides and UCG-008 abundances were induced by VE supplementation, but the differences were not significant (P > 0.05).

Table 3.

Effect of VE increment on relative abundance of cecal microbiota species at genus level.

Items	CON	VE	P-value
Bacteroides	16.37 ± 6.31a	21.77 ± 3.58a	0.19
Rikenellaceae_RC9_gut_group	7.96 ± 2.75a	13.56 ± 7.20a	0.20
Phascolarctobacterium	7.10 ± 4.77a	9.59 ± 5.86a	0.54
Prevotellaceae_UCG-001	1.56 ± 0.67b	6.48 ± 3.79a	0.04
Ruminococcus_torques_group	4.57 ± 2.02a	2.72 ± 1.45a	0.19
Faecalibacterium	2.72 ± 1.63a	2.25 ± 0.38a	0.59
Unclassified_f__Oscillospiraceae	2.96 ± 1.26a	1.77 ± 0.45a	0.12
Megamonas	0.70 ± 0.59a	3.25 ± 2.42a	0.09
Norank_f__norank_o__Clostridia_UCG-014	2.34 ± 1.88a	1.02 ± 0.42a	0.22
Christensenellaceae_R-7_group	2.08 ± 1.52a	0.46 ± 0.34a	0.08
Butyricicoccus	1.67 ± 1.17a	0.87 ± 0.24a	0.23
Lactobacillus	0.83 ± 0.44a	1.27 ± 0.86a	0.39
Prevotellaceae_Ga6A1_group	0.10 ± 0.05a	1.67 ± 1.55a	0.09
Megasphaera	0.53 ± 0.81a	1.05 ± 0.33a	0.28
Fusobacterium	0.15 ± 0.17a	1.28 ± 1.78a	0.25
UCG-005	0.90 ± 0.38a	0.25 ± 0.06b	0.01
Parabacteroides	0.70 ± 0.40a	0.28 ± 0.04a	0.08
Norank_f__Oscillospiraceae	0.67 ± 0.42a	0.12 ± 0.04b	0.04
UCG-008	0.69 ± 0.63a	0.01 ± 0.02a	0.07

CON, control group; VE, vitamin E group.

Data were presented as mean ± standard deviation (n = 4 per group).

The same superscript letters in the same row indicate no significant differences (P > 0.05), while different letters indicate that the difference is significant (P < 0.05).

The results of LEfSe analysis showed that the taxonomic markers in the VE group were Bacteroidota (at phylum level), Bacteroidia (at class level), Bacteroidales (at order level), Prevotellaceae (at family level) and Prevotellaceae_UCG-001 (at genus level). Taxonomic markers in the CON group included the Clostridia (at class level), Oscillospirales (at order level) and Oscillospiraceae (at family level) (Figure 7).

Figure 7.

Effect size measurements analysis of cecal microbiota. Note: CON, control group; VE, vitamin E group. (A) Effect size measurements multi-level species hierarchy tree diagram. (B) Taxonomic biomarkers with linear discriminant analysis (LDA) score (log10) > 4. The LDA score is represented by the length of the histogram.

Ovary Metabolome

The metabolic profiles of CON group were well distinguished with those of VE group using the OPLS-DA and PCA analyses (Figures 8A and 8B), indicating that VE addition could cause marked changes in the ovary metabolites. In the scoring results of OPLS-DA, our results showed that the OPLS-DA model scores R²Y=1, Q² =0.942, indicating that the model has excellent stability (Figure 8C).

Figure 8.

Untargeted ovary metabolomics multivariate analysis. Note: CON, control group; VE, vitamin E group. (A) The scatter plot of principal component analysis (PCA) (n = 6 per group), the variance of metabolites between the CON and VE groups were shown in PCA plots. (B) The score chart of partial least squares discriminant analysis (PLS-DA) (n = 6 per group), PLS-DA plots display the discrimination of metabolites between the CON and VE groups. (C) The verification diagram of PLS-DA, the parameters R² and Q² were used as confirmation of the reliability of the model.

In addition, as demonstrated in Table 4, we identified 22 metabolites (up- regulated and down-regulated) as potential biomarkers after screening with P < 0.01 and FC ≧ 28 or FC ≦ 0.01. Compared with CON group, the contents of Leu-Enkephalin (L-ENK), cis-vaccenic acid, palmitoyl serotonin, buflomedil, fenaminosulf, 2-methoxyestradiol (2ME), lasalocid, ambroxol, vanillin and 3-phosphonoalanine in VE group were increased (P < 0.01); the contents of sphing-4-enine-1-phosphocholine, thujyl 19-trachylobanoate, polyporusterone G, morphine-3-glucuronide, 5-(Acetylamino)-2-hydroxybenzoic acid, 1-Arachidonoylglycerol, urobilin, triflusulfuron-methyl, 3-hydroxyechinenone, flumiclorac-pentyl and tetradecyl phosphonate in VE group were significantly decreased (P < 0.01).

Table 4.

Differential metabolites between the CON and VE groups.

Items	VIP	P-value	FC	Trend
Leu-Enkephalin	1.70	< 0.01	116.88	↑
Cis-vaccenic acid	1.72	< 0.01	91.88	↑
Palmitoyl serotonin	1.73	< 0.01	61.73	↑
Buflomedil	1.68	< 0.01	57.18	↑
Fenaminosulf	1.35	< 0.01	56.44	↑
Carnitine C18:3-OH	1.69	< 0.01	44.84	↑
2-Methoxyestradiol	1.69	< 0.01	33.43	↑
Lasalocid	1.69	< 0.01	33.28	↑
Ambroxol	1.51	< 0.01	32.23	↑
Vanillin	1.68	< 0.01	28.58	↑
3-Phosphonoalanine	1.60	< 0.01	28.15	↑
Sphing-4-enine-1-phosphocholine	1.68	< 0.01	0.01	↓
Thujyl 19-trachylobanoate	1.62	< 0.01	0.01	↓
Polyporusterone G	1.62	< 0.01	0.01	↓
Morphine-3-glucuronide	1.73	< 0.01	0.01	↓
5-(Acetylamino)-2-hydroxybenzoic acid	1.45	< 0.01	0.01	↓
1-Arachidonoylglycerol	1.70	< 0.01	0.01	↓
Urobilin	1.74	< 0.01	0.01	↓
Triflusulfuron-methyl	1.66	< 0.01	0.01	↓
3-Hydroxyechinenone	1.58	< 0.01	0.01	↓
Flumiclorac-pentyl	1.65	< 0.01	0.01	↓
Tetradecyl Phosphonate	1.65	< 0.01	0.01	↓

CON, control group; VE, vitamin E group. VIP was obtained on the basis of OPLS-DA.

The differences in metabolites between the CON and VE groups were assessed by the combination of the P-value of the test and the FC of the metabolites. Screening of differential metabolites with the following criteria: FC ≤ 0.02 or FC > 20, P < 0.01 and VIP > 1.

Arrow pointing up means that the metabolites were upregulated in the VE group compared to the CON group.

Arrow pointing down means that the metabolites were downregulated in the VE group compared to the CON group.

To assess potential metabolic pathways, KEGG enrichment analyses of the significantly different metabolites between the CON and VE groups were performed. As shown in Figure 9, our results showed that VE Supplementation mainly exhibited alterations in steroid hormone biosynthesis (P = 0.0008), followed by pyrimidine metabolism (P = 0.0049), phototransduction (P = 0.031), steroid biosynthesis (P = 0.036) and ABC transporters (P = 0.043).

Figure 9.

Analysis of KEGG pathways.

Spearman Correlation Analysis

We performed Spearman's correlation analysis to explore the potential relationships between the significantly selective different metabolites, selective gut microbes, production performance, antioxidant and immune parameters. As represented in Figure 10A, palmitoyl serotonin, ambroxol, cis-vaccenic acid, buflomedil and lasalocid were positively correlated with laying rate (P < 0.01), immunoglobulins (particularly IgM) and ovary antioxidant parameters (particularly T-SOD, T-AOC and CAT), and negatively correlated with the feed/egg ratio (P < 0.05). 2ME, LEK and vanillin were positively correlated with laying rate (P < 0.01) and CAT, and negatively correlated with the feed/egg ratio (P < 0.01) and MDA, whereas urobilin exhibited a reverse relationship. Fenaminosulf was positively correlated with laying rate (P < 0.01) and negatively correlated with the feed/egg ratio (P < 0.01). As shown in Figure 10B, Prevotelaceae_UCG-001 (at genus level) showed a significant positive correlation with laying rate (P < 0.01), IgG (P < 0.01) and serum GSH-Px (P < 0.05), and showed a significant negative correlation with the feed/egg ratio (P < 0.05). Megamonas (at genus level) was positively correlated with IgA (P < 0.05) and serum GSH-Px (P < 0.05), and negatively correlated with serum MDA (P < 0.05), whereas Christensenellaceae_R-7_group (at genus level) exhibited the opposite trend.

Figure 10.

Correlation analysis of ovary metabolites and gut microbes (at genus level) with immune, antioxidant and performance parameters. Note: positive and negative correlations are shown in red and blue panels (color intensity indicates the Spearman's r-value of the correlation in each panel). *P < 0.05, **P < 0.01. (A) The correlation of significantly changed ovary metabolites with immunoglobulins, ovary antioxidant parameters and performance parameters. (B) The correlation of gut microbes (at genus level) with immunoglobulins, serum antioxidant parameters and performance parameters.

DISCUSSION

Currently, our results showed that dietary addition of 20 mg/kg VE improved the laying rate and feed conversion rate of laying hens from 71 to 75 wk, 76 to 80 wk, and 71 to 80 wk, which were consistent with those of previous reports (Scheideler and Froning, 1996; Amevor et al., 2021b). The enhanced laying rate and reduced feed/egg ratio may be due to the increase in ovary and intestine antioxidant functions with VE supplementation, which promotes follicle development and improves intestinal digestion and absorption capacities.

It has been observed that the level of VE in the diet influences VE deposition in the serum and tissues of animals (Surai et al., 2016; Jiang, 2022). Shelton et al. (2014) found that sows fed diets supplemented with 44 mg/kg VE had higher plasma VE compared with sows fed a nonsupplemented diet. Otomaru et al. (2022) confirmed that the content of VE in serum increased after dietary supplementation with 300 mg/kg VE for 8 wks in 12-wk-old Japanese Black calves. In line with previous results, we found that dietary supplementation of VE (20 mg/kg) could increase serum and ovary VE levels in 80-wk-old XinYang Black-Feathered laying hens.

During the late phase of the laying cycle, fat storage increases in laying hens, which can easily lead to a lipid redox imbalance, resulting in the accumulation of reactive oxygen species (ROS), thus inducing oxidative stress (Nono et al., 2021; Miao et al., 2023). VE is an important component of cell membrane lipid bilayer and can bind oxygen radicals, thereby inhibiting the lipid peroxidation chain reaction and ultimately increasing the body's antioxidant capacity (Yilmaz et al., 2012). The study by Fu et al. (2022) found that dietary supplementation with 40 and 200 mg/kg VE increased liver T-AOC, SOD, and GSH-Px levels in 54 wk old male Jiangnan White breeding geese. Furthermore, the addition of 20 and 100 mg/kg VE to the diet of 50 wk old Lohmann laying hens for 12 wk significantly increased liver and serum GSH-Px activities (Ding et al., 2021). Supporting previous findings, the present study also found that dietary supplementation with 20 mg/kg VE improved CAT, T-AOC and T-SOD levels in the serum and ovary, and decreased serum and ovary MDA concentrations. Our results suggest that VE supplementation may improve ovary antioxidant status, indicating an increased ability of laying hens to scavenge ROS.

The improved levels of VE also contributed to the increased immunity. ROS production increases as the metabolism of activated immune cells increases (Forrester et al., 2018; Lin et al., 2021). Excessive ROS can damage immune active cells and reduce immunity (Nathan and Cunningham-Bussel, 2013). VE can effectively scavenge ROS, thereby increasing the function of lymphocytes, thus supporting the immune system and boosting the body's immune function (Niki, 2014, 2021; Saito, 2021). Liu et al. (2019) demonstrated that dietary addition of 30 mg/kg VE increased serum IgA, IgM and IgG concentrations of 44 wk old Brown-egg laying hens during Salmonella Enteritidis challenge. Consistent with previous study, the results of our experiment showed that dietary supplementation with 20 mg/kg VE improved the immunity of laying hens by increasing the secretion of IgA, IgM and IgG, indicating that the addition of VE had a pronounced influence on the immune function of laying hens during the late laying period.

The ovary is an important reproductive organ for follicle formation and development, and its normal function has a direct effect on the production performance of laying hens. The ovary index and the number of follicles are highly associated with the laying rate of hens (Xu et al., 2023). In our present study, VE supplementation at 20 mg/kg could increase ovary index and the number of SWF, LWF, LYF and total follicles. In hens, low follicular atresia rate is always associated with high egg production performance (Brady et al., 2021). However, oxidative stress in aged laying hens can lead to apoptosis of granulosa cells, resulting in increased follicular atresia and decreased egg production (Ma et al., 2021; Wang et al., 2023). In this study, VE addition enhanced the antioxidant and immune functions of hens, thereby alleviating the follicular atresia caused by oxidative stress and thus increasing the number of different follicular patterns (SWF, LWF and LYF). In addition, changes in reproductive hormone levels are thought to be responsible for the variation in follicle growth in aged laying hens (Lebedeva et al., 2010). At the end of the production cycle in laying hens, estrogen secretion decreases as ovary function declines, resulting in a decrease in the number of developing follicles and an increase in the number of atretic follicles (Colella et al., 2021). Our study demonstrated that supplementation with 20 mg/kg VE in the diet improved serum E₂, FSH and LH levels, which may be related to the fact that VE may improve the antioxidant function of ovary and slow down the process of ovary aging. Our results were consistent with reports by Das and Chowdhury (1999) and He et al. (2023). In this study, the ability of VE to increase estrogen levels may explain the improved number of different follicular patterns (SWF, LWF, and LYF) in the VE increment hens.

The gut microbiota plays a crucial role in regulating the health, nutrient absorption, and immune system of chickens, and can be easily influenced by various factors (Bindari and Gerber, 2022; Zhang et al., 2022). Specifically, differences in diet are a major factor in determining the composition and metabolism of gut microbiota (Mills et al., 2019). In the present study, dietary supplementation with 20 mg/kg VE reduced the α-diversity of microbial communities, as evidenced by the lower Ace and Chao indices. This may be due to VE's strong antioxidant function. During the late laying period of hens, oxidative stress leads to an imbalance of gut microbiota, resulting in an excessive proliferation of harmful microorganisms (Shandilya et al., 2022). VE supplementation can improve intestinal antioxidant capacity, thereby maintaining intestinal health and ultimately reducing the number of harmful microorganisms. However, the specific mechanisms will be the subjects of further investigation. Furthermore, our study showed that 20 mg/kg VE supplementation increased the abundance of Bacteroides, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001 and Megamonas and decreased the abundance of Christensenellaceae_R-7_group at the genus level. Bacteroides can ferment carbohydrates to produce short-chain fatty acids (SCFAs) (Lee and Hase, 2014; Nkosi et al., 2022). SCFAs can reduce intestinal pH and inhibit the growth of pathogens (Ali et al., 2022). Meanwhile, SCFAs are pivotal in restoring intestinal barrier function and ameliorating inflammatory response (Parada et al., 2019; Liu et al., 2021). In addition, SCFAs are an important source of energy for host utilization and can provide 70% of the energy required by colonic epithelial cells (Chambers et al., 2018; Banasiewicz et al., 2020). Rikenellaceae_RC9_gut_group may also degrade fiber to produce SCFAs (Lopetuso et al., 2013). Prevotellaceae _UCG-001 is also a SCFA-producing bacterium (Zhang et al., 2021; Meng et al., 2023). Megamonas has also been shown to utilize amino acids or carbohydrates to produce acetic and butyric acids, which are the most common representative of the SCFAs (Vargas et al., 2017; Han et al., 2021). A meta-analysis showed that the Christensenellaceae_R-7_group was more abundant in patients with intestinal diseases than in healthy controls (Mancabelli et al., 2017). Therefore, an increase in Bacteroides, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001, and Megamonas abundances and a decrease in Christensenellaceae_R-7_group abundance meant more protection for gut health in the VE increment group. Accordingly, the low α-diversity of microbial communities with VE increment could also be explained by the increased abundance of Bacteroides, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001 and Megamonas. In this study, Prevotellaceae_UCG-001 and Megamonas showed a positive correlation with serum antioxidant and immune parameters and laying rate. Besides, we also noticed a positive correlation of Bacteroides and Rikenellaceae_RC9_gut_group with serum antioxidant and immune parameters and laying rate, but the correlation was not significant. Therefore, we hypothesize that the microbiota altered by VE supplementation played an important role in enhancing antioxidant and immune functions and maintaining gut health, ultimately leading to an improvement in the laying rate and feed conversion rate of laying hens. Our findings were similar to the report by Niu et al. (2023) on New Pudong chickens. In addition, a previous study revealed that Rikenellaceae_RC9_gut_group was negatively associated with blood glucose (Song et al., 2021). Thus, our results suggested that VE might play a role in maintaining blood glucose homeostasis. Moreover, we also performed LEfSe analysis and found Bacteroidota (at phylum level), Bacteroidia (at class level), Bacteroidales (at order level), Prevotellaceae (at family level) and Prevotellaceae_UCG-001 (at genus level) dominated the VE increment group. The results of LEFSe analysis were in agreement with the results of gut microbial abundance.

In this study, analysis of the changes in ovary metabolites between the CON and VE groups using the OPLS-DA method showed that the L-ENK, cis-vaccenic acid, palmitoyl serotonin, buflomedil, fenaminosulf, 2ME, lasalocid, ambroxol and vanillin levels were higher in the VE group than those in the CON group. L-ENK has been shown to regulate female reproductive function through the hypothalamus-pituitary-ovarian axis (HPOA) (Ganeyan and Ganesh, 2023). After injection of L-ENK, the ovary index and oocyte diameter of prawn (Penaeus indicus) were significantly increased (Reddy, 2000). Another study by Vijayalaxmi and Ganesh (2017) found that L-ENK may stimulate oogonia proliferation in Oreochromis mossambicus through regulation of HPOA. Palmitoyl serotonin is a serotonin (5-hydroxytryptomine, 5-HT) receptor. Researches have shown that 5-HT may regulate the onset of estrus and the development of follicle via HPOA (Wada et al., 2006; Krsmanovic et al., 2010). Previous studies have shown that 5-HT can accumulate in the mouse ovary and stimulate follicle development (Nikishin et al., 2018; Alyoshina et al., 2022). In another study, Terranova et al. (1990) showed that 5-HT can stimulate E₂ secretion in hamster follicles in vitro. In the present study, L-ENK and palmitoyl serotonin had a high positive correlation with laying rate, which suggested that VE may regulate estrogen secretion and follicle development by increasing the ovary L-ENK and palmitoyl serotonin levels, thus improving the laying rate. Palmitoleic acid is the precursor of cis-vaccenic acid (Sec et al., 2015). Djoussé et al. (2014) confirmed that palmitoleic acid may increase the risk of heart failure via hypertension. The effects of high blood pressure and the risk of heart failure could be reduced by converting palmitoleic acid to cis-vaccenic acid. Buflomedil has the function of blood vessel dilation because it is a non‐selective competitive antagonist of α‐adrenoceptors on vascular smooth muscle (Wu et al., 2015). Our results indicated that VE might be involved in lowering blood pressure. Fenaminosulf is an active ingredient in a commercial fungicide (Li et al., 2022). Lasalocid is a feed additive that is widely used for the control of coccidiosis (Mahtal et al., 2020). In this study, lasalocid was highly positively correlated with immune and ovary antioxidant parameters and laying rate, while fenaminosulf had a slight positive correlation with immune and ovary antioxidant parameters. Thus, the upregulation of lasalocid and fenaminosulf in the VE increment group suggested that VE might have a similar function to that of antibiotics. Vanillin is the most widely used fragrance additive in the world for food products (Walton et al., 2003). Our results suggested that VE might be able to improve the aroma of egg yolk. 2ME is a potent inhibitor of inflammation. 2ME has been shown to inhibit the production of pro-inflammatory cytokines and tumor necrosis factor-α in the ischemia/reperfusion model of acute lung inflammation (Liao et al., 2021). In addition, 2ME has also been shown to have potent apoptotic activity against rapidly growing tumor cells (Schumacher and Neuhaus, 2006). Ambroxol has broad anti-inflammatory properties in vitro and in vivo (Beeh et al., 2008). In addition, vanillin can scavenge ROS via the oxidation product vanillic acid (Yalameha et al., 2023). Sefi et al. (2019) reported that vanillin significantly reduced MDA and hydrogen peroxide levels in the liver of adult male mice. In another previous study, vanillic acid has been shown to have anti-inflammatory effects in a variety of mouse models of inflammation (Calixto-Campos et al., 2015). Our results showed that 2ME, vanillin and ambroxol were positively correlated with ovary antioxidant parameters and laying rate. Thus, our results suggested that VE might improve the laying rate by enhancing anti-inflammatory functions and ovary antioxidant capacity. Urobilin is a catabolic product of conjugated bilirubin, and conjugated bilirubin is synthesized by the liver (Hamoud et al., 2018; Kipp et al., 2023). Urobilin was downregulated by VE supplementation in this study, suggesting that VE may reduce hepatic conjugated bilirubin levels. Elevated levels of conjugated bilirubin are positively correlated with liver and hepatobiliary diseases (Tamber et al., 2023). In this study, urobilin was negatively associated with laying rate, immunoglobulins and ovary antioxidant parameters. Our results indicated that VE might have a certain protective effect on the liver of laying hens, thus improving the laying rate.

The role of different metabolites in animal metabolism can be determined by metabolic pathway analysis. Based on KEGG enrichment analysis, we found that supplementation with 20 mg/kg VE significantly enriched ABC transporters, steroid biosynthesis, phototransduction, pyrimidine metabolism, and steroid hormone biosynthesis. The primary pathways included steroid hormone biosynthesis and pyrimidine metabolism, while the secondary pathways comprised phototransduction, steroid biosynthesis, and ABC transporters. There are 2 categories of steroid hormones: adrenocortical hormones and sex hormones. LH, FSH, E₂, testosterone, progesterone and prolactin are the main sex hormones. In this study, VE addition could enrich the biosynthesis of steroid hormones, suggesting that VE might play an important role in promoting the secretion of reproductive hormones, which was also consistent with the results of LH, FSH and E₂ in this experiment. Uridine and uracil are involved in pyrimidine metabolism and are converted by 5-nucleotidase. Increased levels of uracil and uridine have been observed in mature osteoblasts. Misra et al. (2021) suggested that both of these pyrimidines might be involved in the differentiation and maturation of osteoblasts. The increase in ROS levels induces osteoclastogenesis and stimulates osteoclast activity, leading to osteoporosis (Hu et al., 2023; Xi et al., 2023). A study by Wong et al. (2019) on mice suggested that dietary supplementation with VE could reduce ROS levels, thereby suppressing osteoclastogenesis, osteoclast activity and osteocyte apoptosis, and ultimately alleviate osteoporosis. Our study indicated that VE increment might stimulate bone growth and development, delay bone loss and then prevent osteoporosis. In animals, photoreception is a process of transmitting and converting signals. Animal photoreceptors are special nerve cells that are responsible for the conversion of light signals into electrical signals (Masek et al., 2023). It is well documented that the light cycle can regulate HPOA by affecting melatonin, gonadotropin-inhibiting hormone and dopamine (Surbhi and Kumar, 2015). A study by Liu et al. (2015) confirmed that compared to cold white light, blue and green light could increase total egg production, egg production rate, plasma estrogen levels, and SYF amount in 300-day-old Erlang Mountainous laying hens. Research has shown that ovary weight, SYF and LYF numbers, oviduct weight, oviduct length, and egg laying rate in 30-wk-old Beijing You chickens under continuous lighting were higher than those under intermittent lighting (Geng et al., 2022). Another study has shown that compared to 12 h of light exposure, light exposure ≥ 16 h could increase the number and weight of LWF and serum E₂, progesterone and FSH levels in 36 wk old Jinding laying ducks (Cui et al., 2021). In this experiment, the phototransduction pathway was enriched by VE supplementation, suggesting that VE may have beneficial effects on the production performance of laying hens by regulating light reception and transmission. Bile acid is a steroid that is converted from cholesterol in the liver and then excreted in the intestines. The main physiological functions of bile acid are to aid the digestion and absorption of fats and to regulate the metabolism of cholesterol (Zhang et al., 2023). Both steroid biosynthesis and primary bile acid biosynthesis were enriched by VE supplementation in the present study, indicating that VE may improve energy efficiency and reduce blood cholesterol levels. ABC transporters are a superfamily of membrane proteins that use the energy released by the hydrolysis of ATP to move bound substrates from one side of the cell membrane to the other (Liu, 2019). ABC transporters can be involved in the transport of ions, amino acids, nucleotides, polysaccharides, peptides and vitamins (Theodoulou and Kerr, 2015). In the present study, the enrichment of ABC transporters pathway in the VE increment group indicated that VE might enhance the normal transport function of cell membranes.

The findings of this study have to be seen in light of some limitations: First, only 6 ovary samples per group were selected when measuring the ovary metabolome. Due to the small sample size of the study, the results may not be an accurate reflection of the effects of dietary VE on ovary metabolites. This may limit the generalizability of this work. For this reason, a large sample size should be used in future studies. Second, granulosa cells have been shown to play a key role in the growth and development of follicles. Granulosa cells can regulate the metabolism and maturation of oocytes and the synthesis of estrogen. Moreover, granulosa cells can induce ovulation and regulate follicular atresia. However, the metabolome of granulosa cells was not measured in this study. This could be seen as a limitation of the study. Therefore, it would be very valuable to determine the metabolome of granular cells in future experiments. An additional limitation is that findings from animal models may not directly apply to humans. The effects of dietary VE on ovary metabolome in humans may differ from those observed in animal models.

CONCLUSIONS

In summary, this study showed that laying performance, immunity, antioxidant and ovary functions of aged laying hens were improved by dietary supplementation with 20 mg/kg VE. Furthermore, 16S rRNA sequencing of cecal microbiota showed that VE addition induced a change in microbiota composition by increasing Bacteroides, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001 and Megamonas abundances, which are involved in the production of SCFAs. In addition, metabolomics profiling of ovary revealed that the major metabolites altered by VE supplementation were mainly related to follicle development, estrogen secretion, anti-inflammatory, antioxidant, phototransduction, bile acid synthesis, and nutrient transport.