Hepatic Fatty Acid Profile and Gut Microbiota Changes in Response to Dietary Bile Acid Supplementation in Late-Laying Hens

Abstract

This research examined how dietary bile acids (BAs) influence hepatic fatty acid composition and cecal microbial community of late-laying hens. A total of 1080 Hy-Line Brown hens, aged 76 weeks, were randomly divided into four groups, each receiving a basal diet with 0 (CON), 200 (BA200), 300 (BA300), or 500 (BA500) mg/kg BAs for 35 days. Results showed that hepatic stearic acid (C18:0) concentrations were significantly reduced in all BAs-supplemented groups (p < 0.05). The BA300 group exhibited a significant increase in oleic acid (C18:1n9c), total monounsaturated fatty acids (MUFAs), and n-3 polyunsaturated fatty acids (PUFAs), alongside a decreased n-6/n-3 PUFA ratio (p < 0.05). Furthermore, the Chao1 index increased in all BAs-supplemented groups (p < 0.05), and the Simpson index was higher in BA300 (p < 0.05). LEfSe analysis indicated that the CON group had a higher abundance of opportunistic pathogens, while BA supplementation altered the cecal microbial composition. Notably, the BA300 group exhibited the most favorable microbial profile, characterized by enrichment of beneficial Enterococcus and improved microbial stability. In conclusion, supplementation with 300 mg/kg BAs most effectively improved hepatic fatty acid composition and cecal microbial diversity in late-laying hens, supporting its use as a nutritional strategy in the late laying phase.

1. Introduction

The egg industry represents a cornerstone of global sustainable food production, supplying essential nutrients vital for human health and nutrition [1,2]. With the increasing global population, eggs serve as an affordable and accessible source of high-quality protein sources and essential micronutrients [3]. However, a significant metabolic challenge arises during the late laying phase, when declining egg production leads to surplus energy intake exceeding the requirements for maintenance and egg synthesis, thereby promoting excessive lipid deposition in the liver. This metabolic imbalance leads to hepatic lipid accumulation, which precipitates a marked decline in laying performance and an increase in mortality, thereby constraining the economic sustainability of egg production [4,5,6]. Consequently, optimizing hepatic lipid metabolism in late-laying hens is a critical strategy for sustaining production efficiency.

Bile acids (BAs), synthesized from hepatic cholesterol, are essential hydroxysteroids involved in lipid metabolism [7]. During enterohepatic circulation, the molecular structure of BAs undergoes modifications through the action of hepatic enzymes and gut microbiota, thereby conferring the physicochemical properties that enable BAs to perform multiple physiological functions, including metabolic regulation, antimicrobial activity, and lipid solubilization during digestion [8]. Dietary supplementation with exogenous BAs has been shown to enhance growth performance, improve nutrient digestibility, and modulate hepatic lipid metabolism in broilers and laying hens [9,10,11]. BAs serve as signaling molecules influencing gut microbial community structure through their antimicrobial properties and activation of host receptors like farnesoid X receptor and Takeda G protein-coupled receptor 5, which regulate microbial BAs biotransformation [12,13]. The gut–liver axis represents a crucial mechanism by which dietary BAs can influence both liver metabolism and the balance of intestinal microbiota. Although prior studies have examined the impacts of BA supplementation on lipid metabolism and intestinal microbiota in poultry, most investigations have focused on broilers or hens during peak production, with limited data available specifically for the late laying phase [9,10,12,13]. A recent study by Fan et al. demonstrated that dietary bile acids supplementation decreased hepatic C20:0, C22:0, and C24:0 levels and reduced triglyceride accumulation, alongside downregulation of fatty acid synthase and stearoyl-CoA desaturase 1 mRNA expression in late-laying hens [14]. However, that study did not examine cecal microbiota responses, leaving the gut–liver axis dimension of BA supplementation unexplored. During this critical period, the concurrent decline in hepatic lipid clearance capacity and shifts in gut microbial composition may exacerbate metabolic dysfunction, yet the effects of exogenous BAs on hepatic fatty acid profiles and cecal microbiota in aged laying hens remain poorly understood. The present study addresses this gap by simultaneously evaluating hepatic fatty acid profiles and cecal microbiota composition in response to graded BAs doses, providing an integrated perspective on the gut–liver axis. Late-laying hens are particularly relevant for such investigation because aging is accompanied by declining hepatic metabolic capacity, hormonal changes, and progressive shifts in gut microbial composition, all of which may alter the response to exogenous BAs compared with younger birds.

This study evaluated the impact of dietary BAs supplementation at varying doses (200, 300, and 500 mg/kg) on hepatic total fatty acid composition and cecal microbial community structure in Hy-Line Brown laying hens aged 76 weeks. We hypothesized that dietary BAs supplementation would modulate hepatic fatty acid profiles and cecal microbiota composition in late-laying hens in a dose-dependent manner. The present study addresses this gap by simultaneously evaluating hepatic fatty acid profiles and cecal microbiota composition in response to graded BA doses, providing an integrated perspective on the gut–liver axis.

2. Materials and Methods

2.1. Animals and Treatments

The experiment took place at a commercial family farm in Kongjia Village, Ciyao Town, Ningyang County, Shandong Province, China. All animal experimental procedures and protocols were approved by the Animal Welfare and Use Committee of Shandong Agricultural University (SDAUA-2023-817). A total of 1080 Hy-Line Brown laying hens aged 76 weeks, maintaining consistent egg production and showing no clinical disease symptoms, were randomly allocated to four dietary treatment groups: a basal diet (CON) and the basal diet supplemented with 200 mg/kg (BA200), 300 mg/kg (BA300), or 500 mg/kg (BA500) of BAs. Each treatment comprised six replicates of 45 hens per replicate. The hens were housed in a three-tier cage system under a 16 h light/8 h dark photoperiod. The experiment consisted of a 7-day acclimation period followed by a 35-day experimental period. Animals were provided with ad libitum access to feed and water throughout the trial. The basal diet (

Table 1. Ingredients and nutrient levels of the basal diet (air-dry basis).

Ingredients, %	Content	Nutrient Levels 2, %	Analyzed Values
Corn	61.00	Metabolizable energy (MJ/kg)	11.30
Soybean meal	20.00	Crude protein	18.43
Wheat bran	6.00	Calcium	3.54
Soybean oil	2.00	Total phosphorus	0.80
Limestone	8.00	Non-phytate phosphorus	0.54
Sodium chloride	0.30	Lysine	0.71
DL-methionine	0.20	Methionine	0.46
Calcium monophosphate	2.00	Threonine	0.59
Premix 1	0.50	Tryptophan	0.18
Total	100.00

¹ The dietary premix per kilogram includes: 8000 IU of vitamin A, 1600 IU of vitamin D3, 12 IU of vitamin E, 0.5 mg of vitamin K, 2 mg of vitamin B1, 2.5 mg of vitamin B2, 3 mg of vitamin B6, 20 mg of nicotinamide, 15 mg of calcium D-pantothenate, 0.25 mg of folic acid, 8 mg of copper, 60 mg each of iron and manganese, 80 mg of zinc, 0.3 mg of selenium, and 0.35 mg of iodine. ² Nutrient levels were calculated values.

) was designed based on the Chinese Chicken Feeding Standard (NY/T 33-2004) to fulfill or surpass the nutrient needs for laying hens [15]. The BAs product, sourced from porcine origin and supplied by Shandong Zhongjing Biotechnology Co., Ltd. (Tai’an, China), contained 21.53% active components. These included 3.05% chenodeoxycholic acid, 6.59% phocaecholic acid, 0.24% hyocholic acid, 0.49% hyodeoxycholic acid, and 11.16% cholic acid.

2.2. Sample Collection

On day 35 of the experiment, two healthy laying hens per replicate, with body weights near the replicate mean, were selected after a 12 h fast. After euthanasia, cecal contents were placed in labeled cryogenic tubes and snap-frozen in liquid nitrogen and stored for downstream 16S rRNA gene sequencing. Liver tissue samples were collected, rapidly frozen in liquid nitrogen, and stored at −80 °C for analysis of hepatic total fatty acid composition. For hepatic fatty acid analysis, one liver sample per replicate was used (n = 6 per treatment group), while seven cecal content samples per treatment group (n = 7, 28 samples in total) were collected for 16S rRNA gene sequencing.

2.3. Determination of Hepatic Fatty Acid Composition

Hepatic fatty acid composition was analyzed using a fatty acid methyl ester (FAME) method in accordance with AOAC/AOCS guidelines, utilizing gas chromatography–mass spectrometry (GC-MS). Liver samples stored at −80 °C were thawed at 4 °C prior to analysis. About 50 mg of liver tissue was precisely weighed, homogenized, and combined with 3 mL of n-hexane. The mixture was vortexed and incubated at 50 °C for 30 min. Then, 3 mL of 0.4 mol/L KOH–methanol solution was added for derivatization, and the reaction was continued at 50 °C for another 30 min. After cooling to room temperature, 1 mL of distilled water was added, followed by thorough vortexing. After phase separation, 90 μL of the upper organic phase was mixed with 10 μL of a methyl nonadecanoate internal standard solution (100 μg/mL) for GC-MS analysis.

A DB-FastFAME capillary column (Agilent G3903-63011, Agilent Technologies, Santa Clara, CA, USA) was used for gas chromatographic separation. The injector was set at 250 °C with a split ratio of 10:1 and an injection volume of 1 μL. A constant flow rate of helium was used as the carrier gas. The oven temperature was initially set to 50 °C for 0.5 min, then increased to 194 °C at a rate of 35 °C/min and maintained for 3.5 min, followed by a rise to 240 °C at 9 °C/min, held for 1.0 min. Fatty acid composition was determined and measured using chromatographic retention times and mass spectrometric fragmentation patterns. Individual fatty acids were quantified by peak area and expressed as a percentage of total fatty acids.

2.4. 16S rRNA Gene Sequencing and Analysis

Composition of the cecal microbiota was characterized by 16S rRNA gene sequencing, following the method described by Wu et al. [16]. Genomic DNA was isolated from cecal chyme samples using the CTAB extraction method. DNA concentration and purity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and DNA integrity was verified by 1% agarose gel electrophoresis (100 V, 40 min). DNA samples were diluted to 1 ng/μL using sterile nuclease-free water for PCR amplification. The V4 region of the bacterial 16S rRNA gene was amplified using barcoded primers 515F/806R and Phusion^® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Ipswich, MA, USA). PCR products were analyzed via 2% agarose gel electrophoresis, purified with magnetic beads, quantified, and combined at equimolar concentrations. Target DNA fragments were extracted with a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Libraries were prepared with the TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA), and their quality was evaluated using Qubit fluorometric quantification and quantitative PCR (qPCR). Qualified libraries were sequenced on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Raw sequencing reads were assigned to samples according to unique barcode sequences and processed using the QIIME2 platform (version QIIME2-202202). Paired-end reads were processed by merging with FLASH (v1.2.11), quality filtering with fastp (v0.23.1), and chimera screening using the UCHIME algorithm. The DADA2 denoising pipeline was employed to generate amplicon sequence variants (ASVs). Taxonomic annotation utilized the SILVA SSU rRNA database (release 138). Subsequent analyses, including alpha diversity, beta diversity, principal coordinate analysis (PCoA), and linear discriminant analysis effect size (LEfSe), were performed on the obtained ASVs. Sequencing depth was normalized by rarefaction to approximately 64,687 sequences per sample for alpha and beta diversity analyses.

2.5. Statistical Analysis

Data normality was evaluated using IBM SPSS Statistics 27, with variables showing a Shapiro–Wilk test p-value above 0.05 deemed to satisfy the normality assumption. Data were analyzed using SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). A one-way ANOVA using the GLM procedure was conducted, followed by Tukey’s test for multiple comparisons of treatment means. In addition, linear and quadratic regression analyses were conducted using the REG procedure to evaluate dose–response effects of increasing dietary BAs levels. The experimental unit was defined as the replicate. For hepatic fatty acid analysis, one sample per replicate was analyzed (n = 6 per group), whereas for 16S rRNA sequencing, seven individual cecal samples per treatment group (n = 7) were used. Statistical significance was determined at p < 0.05. Pairwise comparisons of beta diversity were conducted using the Mann–Whitney U test based on UniFrac distance matrices, with 95% confidence intervals calculated. Alpha diversity indices were assessed using one-way ANOVA followed by Tukey’s multiple comparison test.

3. Results

3.1. Effects of Dietary BAs Supplementation on Hepatic Saturated Fatty Acid Composition

Table 2. Effects of dietary BAs supplementation on hepatic saturated fatty acid composition (% of total fatty acids).

Items	Treatments 1				SEM	p Value
	CON	BA200	BA300	BA500		ANOVA	Linear	Quadratic
C8:0	0.03	0.03	0.02	0.04	0.003	0.269	0.376	0.332
C12:0	0.01	0.01	0.02	0.01	0.001	0.135	0.204	0.144
C14:0	0.32	0.36	0.39	0.32	0.012	0.088	0.802	0.046
C15:0	0.04	0.05	0.05	0.04	0.002	0.275	0.845	0.147
C16:0	22.72	24.13	24.94	24.36	0.313	0.070	0.038	0.029
C17:0	0.07	0.08	0.07	0.07	0.002	0.361	0.445	0.231
C18:0	15.02 a	13.12 b	11.59 b	13.14 b	0.306	<0.001	0.007	<0.001
C20:0	0.10	0.10	0.08	0.11	0.004	0.139	0.671	0.166
C21:0	0.03	0.03	0.02	0.03	0.002	0.413	0.435	0.257
C22:0	0.05	0.05	0.04	0.06	0.003	0.193	0.610	0.227
C23:0	0.04	0.03	0.03	0.03	0.002	0.361	0.649	0.227
C24:0	0.02	0.02	0.02	0.03	0.001	0.147	0.767	0.089
Total SFA	38.45	38.01	37.27	38.23	0.227	0.292	0.505	0.248

¹ CON: basal diet; BA200: basal diet + 200 mg/kg bile acids; BA300: basal diet + 300 mg/kg bile acids; BA500: basal diet + 500 mg/kg bile acids. n = 6. SEM stands for standard error of the means. Means labeled with distinct lowercase letters indicate significant differences among the four treatments (p < 0.05).

illustrates the impact of dietary BAs supplementation on saturated fatty acid composition. Compared with the CON group, dietary BAs supplementation significantly reduced the proportion of C18:0 (p < 0.05), with the lowest value observed in the BA300 group. Furthermore, C18:0 and C16:0 both exhibited linear and quadratic effects in response to increasing dietary BA levels (linear and quadratic, p < 0.05), while C14:0 demonstrated a quadratic increase (quadratic, p < 0.05).

3.2. Effects of Dietary BAs Supplementation on Hepatic Monounsaturated Fatty Acid Composition

Table 3. Effects of dietary BAs supplementation on hepatic monounsaturated fatty acid composition (% of total fatty acids).

Items	Treatments 1				SEM	p Value
	CON	BA200	BA300	BA500		ANOVA	Linear	Quadratic
C16:1	2.95	3.29	2.70	2.62	0.116	0.165	0.125	0.209
C18:1n9c	33.51 b	33.66 ab	34.83 a	34.56 ab	0.185	0.017	0.007	0.022
C20:1	0.55	0.55	0.56	0.55	0.016	0.994	0.982	0.967
Total MUFA	37.00 b	37.50 ab	38.08 a	37.72 ab	0.119	0.007	0.008	0.004

¹ CON: basal diet; BA200: basal diet + 200 mg/kg bile acids; BA300: basal diet + 300 mg/kg bile acids; BA500: basal diet + 500 mg/kg bile acids. n = 6. SEM stands for standard error of the means. Means labeled with distinct lowercase letters indicate significant differences among the four treatments (p < 0.05).

illustrates the impact of dietary BAs supplementation on monounsaturated fatty acid composition. The BA300 group exhibited a significant increase in the proportion of C18:1n9c and total monounsaturated fatty acid (MUFA) content compared to the CON group (p < 0.05). Furthermore, both C18:1n9c and total MUFA exhibited linear and quadratic increases as dietary BAs levels rose (linear and quadratic, p < 0.05).

3.3. Effects of Dietary BAs Supplementation on Hepatic Polyunsaturated Fatty Acid Composition

Table 4. Effects of dietary BAs supplementation on hepatic polyunsaturated fatty acid composition (% of total fatty acids).

Items	Treatments 1				SEM	p Value
	CON	BA200	BA300	BA500		ANOVA	Linear	Quadratic
n-3 PUFA
C18:3n3	0.33	0.38	0.38	0.35	0.007	0.051	0.387	0.021
C20:5n3	0.25	0.25	0.29	0.25	0.009	0.183	0.775	0.460
C22:6n3	0.51	0.45	0.38	0.47	0.028	0.441	0.450	0.304
Total n-3 PUFA	0.58 b	0.63 ab	0.67 a	0.60 ab	0.011	0.024	0.430	0.018
n-6 PUFA
C18:2n6c	16.37	16.39	16.24	15.63	0.110	0.056	0.015	0.016
C18:3n6	0.09	0.09	0.10	0.09	0.004	0.825	0.454	0.693
C20:2n6	1.34	1.15	1.03	1.19	0.048	0.142	0.180	0.071
C20:3n6	1.37	1.36	1.22	1.57	0.060	0.247	0.415	0.246
C20:4n6	5.07	5.25	5.23	5.33	0.065	0.577	0.196	0.422
Total n-6 PUFA	22.90	23.09	22.79	22.63	0.110	0.516	0.262	0.391
Total PUFA	25.34	25.32	24.87	24.88	0.136	0.451	0.140	0.342
n-6 PUFA/n-3 PUFA	39.78 a	36.90 ab	34.42 b	38.17 ab	0.702	0.041	0.250	0.025

¹ CON: basal diet; BA200: basal diet + 200 mg/kg bile acids; BA300: basal diet + 300 mg/kg bile acids; BA500: basal diet + 500 mg/kg bile acids. n = 6. SEM stands for standard error of the means. Means labeled with distinct lowercase letters indicate significant differences among the four treatments (p < 0.05).

illustrates the impact of dietary BAs supplementation on the composition of polyunsaturated fatty acids. The BA300 group exhibited a significant increase in total n-3 polyunsaturated fatty acids (PUFAs) compared to the CON group (p < 0.05). In addition, C18:3n3 and total n-3 PUFA exhibited quadratic responses with increasing dietary BAs levels (quadratic, p < 0.05). Within the n-6 PUFA group, C18:2n6c exhibited both linear and quadratic reductions as dietary BAs levels (linear and quadratic, p < 0.05), while C20:2n6 demonstrated a tendency for a quadratic response (quadratic, p < 0.05). The BA300 group exhibited a significantly lower n-6 PUFA to n-3 PUFA ratio compared to the CON group (p < 0.05), with a notable quadratic response as dietary BAs levels increased (quadratic, p < 0.05).

3.4. Effects of Dietary BAs Supplementation on Hepatic Unsaturated Fatty Acid Composition

Table 5. Effects of dietary BAs supplementation on total unsaturated fatty acid composition (% of total fatty acids).

Items	Treatments 1				SEM	p Value
	CON	BA200	BA300	BA500		ANOVA	Linear	Quadratic
UFA	62.34	62.82	62.95	62.61	0.154	0.547	0.512	0.341
PUFA/ UFA	0.62	0.61	0.59	0.61	0.004	0.212	0.431	0.151

¹ CON: basal diet; BA200: basal diet + 200 mg/kg bile acids; BA300: basal diet + 300 mg/kg bile acids; BA500: basal diet + 500 mg/kg bile acids. n = 6. SEM stands for standard error of the means.

illustrates the impact of dietary BAs supplementation on the composition of unsaturated fatty acids (UFAs). There were no notable differences in total unsaturated fatty acid content or the PUFA/UFA ratio across the treatment groups (p > 0.05).

3.5. Effects of Dietary BAs Supplementation on Cecal Microbial Diversity

The species accumulation curve (Figure 1A) plateaued as sample size reached 28, suggesting sufficient sampling to capture most microbial species and accurately reflect community richness. Similarly, the rarefaction curves (Figure 1B) for all groups tended to level off at approximately 64,687 sequences, suggesting that the sequencing depth was sufficient and that the obtained data reliably represented microbial composition and diversity.

Effective sequences were processed into ASVs using the DADA2 pipeline. A total of 3806, 3446, 5149, and 4793 ASVs were identified in the CON, BA200, BA300, and BA500 groups, respectively. Among these, 2134, 1700, 3071, and 2706 ASVs were unique to each group, respectively, while 930 ASVs were shared among all groups (Figure 2).

The richness and evenness of the cecal microbial community were evaluated using the Chao1 index (Figure 3A), Shannon index (Figure 3B), Simpson index (Figure 3C), Dominance index (Figure 3D), Pielou_e index (Figure 3E), and observed ASV richness index (Figure 3F). In all groups supplemented with BAs, the Chao1 index showed a significant increase compared to the CON group (p < 0.05), with both linear and quadratic trends observed as dietary BAs levels increased (linear and quadratic, p < 0.05). Among all groups, the BA300 group exhibited the highest Chao1 value. The Simpson index significantly increased in the BA300 group (p < 0.05), with notable linear and quadratic increases corresponding to rising dietary BAs levels (linear and quadratic, p < 0.05).

Beta-diversity analysis of the cecal microbiota is presented in Figure 4. The Unweighted Unifrac distance heatmap (Figure 4) demonstrated variations in microbial community composition across samples, with pairwise dissimilarity values between 0.40 and 0.70. Samples within the same treatment group generally exhibited lower Unweighted Unifrac distances than those from different treatment groups, suggesting that dietary BAs supplementation influenced the overall cecal microbial community structure.

PCoA using unweighted UniFrac distances demonstrated that dietary BAs supplementation modified the cecal microbial community structure in late-laying hens (Figure 5A). The first principal coordinate accounted for 33.66% of the total variation, while the second explained 13.94%. A partial separation among treatment groups was observed along the ordination space. The BA300 and BA500 groups shifted positively along PC1, partially separating from the CON group, while the BA200 group exhibited a minor shift along PC2. Notably, the BA300 group occupied an intermediate position between the CON and BA500 groups along PC1, suggesting a gradual dose-dependent shift in microbial community structure with increasing BAs supplementation. The CON group showed a tight clustering near the center of the ordination space, indicating low inter-individual variation. In contrast, the BA500 group displayed a more dispersed distribution along PC1, suggesting increased heterogeneity in microbial composition with high-dose BAs supplementation. Despite these differences, partial overlap among groups remained, suggesting that the cecal microbiota retained a degree of compositional stability across treatments. Table S1 presents pairwise comparisons using unweighted UniFrac distances. A significant difference was observed between the CON and BA500 groups (p < 0.05), and between the BA200 and BA500 groups (p < 0.05). No significant differences were detected for the comparisons of CON vs. BA200, CON vs. BA300, BA200 vs. BA300, or BA300 vs. BA500 (p > 0.05).

Weighted UniFrac-based PCoA (Figure 5B) further indicated marked alterations in the cecal microbial community structure following dietary BA supplementation. The first principal coordinate accounted for 37.01% of the total variation, while the second explained 13.90%. The BA300 and BA500 groups exhibited a clearer distinction from the control, indicating that moderate-to-high BA levels more significantly influence the phylogenetically weighted microbial community composition. Nevertheless, partial overlap among groups indicated that the cecal microbiota maintained a certain degree of compositional stability across treatments. Weighted UniFrac-based pairwise comparisons (Table S2) revealed a significant difference only between the CON and BA300 groups (p < 0.05), with no significant differences found among the other group comparisons (p > 0.05).

3.6. Effects of Dietary BAs Supplementation on Cecal Microbial Relative Abundances

Figure 6 illustrates the impact of dietary BAs supplementation on the cecal microbial composition, highlighting the top 10 phyla. In all treatment groups, the predominant phyla were Bacteroidota and Firmicutes (Figure 6A). The heatmap analysis in Figure 6B indicated variations in microbial community composition across the treatment groups. The BA500 group exhibited a significant reduction in the relative abundance of Bacteroidota compared to the CON group (p < 0.05), with both linear and quadratic decreases correlating with higher dietary BAs levels (linear and quadratic, p < 0.05; Figure 6C). The BA500 group exhibited a significant increase in the relative abundance of Actinobacteriota compared to the CON group (p < 0.05). Additionally, both linear and quadratic trends were observed with rising dietary BAs levels (linear and quadratic, p < 0.05; Figure 6D).

Figure 7 illustrates the impact of dietary BAs supplementation on the cecal microbial composition, highlighting the top 10 genera. Bacteroides predominated in all treatment groups, with Rikenellaceae_RC9_gut_group and CHKCI001 following (Figure 7A). The heatmap analysis indicated variations in the relative abundance of dominant genera across the treatment groups (Figure 7B). The BA200 group exhibited a significant increase in the relative abundance of Rikenellaceae_RC9_gut_group compared to the CON group (p < 0.05; Figure 7C). The BA300 group exhibited a significant increase in the relative abundance of Clostridium_sensu_stricto_1 compared to the CON group (p < 0.05; Figure 7D).

LEfSe analysis identified bacterial taxa with significant differences among the treatment groups (Figure 8). The findings indicated a significant enrichment of Lysinibacillus, Prevotellaceae_UCG_001, Fournierella, Pseudomonas, Aeromonas, Parabacteroides, Flectobacillus, and Campylobacter in the CON group. The BA200 group was primarily distinguished by the abundance of Rikenellaceae_RC9_gut_group and F082. In contrast, Enterococcus and UCG_010 were identified as the predominant biomarkers in the BA300 and BA500 groups, respectively.

4. Discussion

In poultry, the liver serves as the central organ for fatty acid synthesis, oxidation, transport, and BAs biosynthesis. Consequently, alterations in hepatic fatty acid composition are considered important indicators of lipid metabolic status and hepatic health [17]. In this study, dietary BAs supplementation significantly altered the hepatic total fatty acid profile in late-laying hens, with the most pronounced responses observed in the BA300 group, including lower stearic acid (C18:0) and n-6/n-3 PUFA ratio, and higher oleic acid (C18:1n9c), total MUFA, and n-3 PUFA. In the liver, C18:0 is converted to C18:1n9c via the rate-limiting enzyme stearoyl-CoA desaturase 1 (SCD1) [18]. Enhanced SCD1 activity may contribute to increased cellular membrane fluidity and improved lipoprotein assembly efficiency, which may facilitate hepatic lipid export and reduce the risk of fatty liver syndrome in aging hens [19,20,21]. The concurrent decrease in C18:0 and increase in C18:1n9c observed in BA300 is consistent with potential benefits of BAs supplementation for hepatic health. Oleic acid is a major component of egg yolk lipids, and its increased hepatic availability may provide favorable substrates for yolk lipid synthesis; elevated oleic acid levels have been associated with enhanced yolk color intensity and improved egg production rates in laying hens [22,23]. Moreover, increasing n-3 PUFA levels and decreasing the n-6/n-3 PUFA ratio have important physiological effects. n-3 PUFAs exert well-established anti-inflammatory effects, regulate lipid metabolism, and maintain cell membrane integrity [24,25,26]. Their relative increase in the BA300 group may help mitigate the low-grade inflammatory state commonly observed in late-laying hens, thereby potentially supporting liver function [27,28]. The absence of significant changes in total saturated fatty acids or n-6 PUFA across groups suggested that BA supplementation may selectively influence specific fatty acid desaturation and elongation processes rather than broadly affecting overall fatty acid synthesis. The differences between the BA300 and BA500 groups were modest, suggesting that both doses exert beneficial effects on hepatic fatty acid composition, but the BA300 dose appeared to be the most effective overall. Although the percentage changes in individual fatty acids were relatively small, even modest shifts in hepatic fatty acid composition can be biologically meaningful, particularly when involving functionally important fatty acids such as oleic acid and n-3 PUFA. Notably, Fan et al. reported that BA supplementation decreased hepatic C20:0, C22:0, and C24:0 in late-laying hens, whereas the present study found no significant changes in these very-long-chain saturated fatty acids, possibly due to differences in basal diet composition; they also showed that BA200 increased C17:0 and C20:2, while BA300 reduced hepatic triglyceride content and downregulated FAS and SCD1 mRNA expression, providing complementary molecular evidence supporting the fatty acid profile changes observed herein [14].

The cecal microbiota is intricately connected to host metabolism through the gut–liver axis [29,30]. Alpha and beta diversity are widely utilized metrics in microbiome research [31]. This study found that dietary supplementation with BAs significantly impacted both alpha and beta diversity of the cecal microbiota. The Chao1 index was significantly increased in all BAs-supplemented groups, and the Simpson index was also significantly elevated in the BA300 group. The increases in the Chao1 and Simpson indices indicated that BAs supplementation enhanced both species richness and diversity [31]. This finding contrasts with the report by Yang et al., who observed that supplementation with 30, 60, or 90 mg/kg BAs had no significant effect on alpha diversity in the cecal microbiota of 69-week-old Hy-Line Brown laying hens [12]. This variation could be due to differing BAs concentrations and the age of the hens. PCoA using unweighted and weighted UniFrac distances demonstrated significant changes in microbial community structure across treatment groups, suggesting that dietary BAs supplementation may have modulated microbial diversity and community composition, although partial overlap among groups in PCoA plots suggests that these shifts were not complete. The discrepancy between unweighted and weighted UniFrac results suggests that BAs supplementation altered both the presence/absence of rare taxa (captured by unweighted UniFrac) and the relative abundance of dominant taxa (captured by weighted UniFrac), though the specific taxa driving these patterns differed between dose levels. Similar changes in beta diversity have been reported by Li et al. [32]. Because BAs possess antimicrobial activity and function as signaling molecules regulating both host metabolism and microbial ecology, dietary BAs supplementation may influence cecal microbial community assembly and composition [33,34]. Increased microbial diversity is generally associated with enhanced gut functional stability and improved resistance to environmental perturbations [35], which may underlie the changes in hepatic fatty acid composition observed in BAs-supplemented hens.

Bacteroidota and Firmicutes were the predominant phyla across all groups at the phylum level, aligning with the typical features of avian cecal microbiota [36]. Notably, the BA500 group exhibited a significantly reduced abundance of Bacteroidota and a markedly elevated abundance of Actinobacteriota. Members of Bacteroidota primarily degrade complex carbohydrates and produce short-chain fatty acids (SCFA) such as butyrate, which serves as an energy source for intestinal epithelial cells and helps maintain mucosal integrity [37,38], while also participating in BAs metabolism and facilitating the excretion of toxic compounds [39]. Certain genera within Actinobacteriota produce bioactive metabolites and contribute to intestinal homeostasis [40]. The shift in phylum-level composition at the highest BAs dose may therefore reflect a dose-dependent shift in the microbial metabolic landscape. At the genus level, BA200 showed a significant enrichment of Rikenellaceae_RC9_gut_group. This genus is associated with enhanced nutrient absorption and promotion of fatty acid metabolism, and its enrichment may be related to BAs-mediated improvements in gut health [41,42,43]. Clostridium sensu stricto 1 was significantly enriched in BA300; some species within this genus possess butyrate-producing capabilities [44], and butyrate serves as the primary energy source for intestinal epithelial cells while promoting barrier repair and anti-inflammatory responses [45]. This finding is mechanistically consistent with the favorable hepatic fatty acid profile observed in BA300, as butyrate-mediated improvements in intestinal barrier function may reduce the translocation of lipopolysaccharides into the portal circulation, thereby alleviating hepatic inflammatory burden. It should be noted, however, that intestinal barrier function and endotoxin translocation were not directly assessed in this study, and the proposed link between butyrate-producing taxa and hepatic inflammatory burden remains to be verified.

LEfSe analysis further demonstrated that dietary BAs supplementation significantly altered the composition of the cecal microbiota and promoted the enrichment of specific bacterial taxa at different supplementation levels. In this study, the CON group showed an enrichment of Pseudomonas, Aeromonas, Campylobacter, and Lysinibacillus. Several members of the genera Pseudomonas and Aeromonas are recognized as opportunistic pathogens, and their enrichment is often associated with microbial dysbiosis, intestinal inflammation, and potential gut barrier dysfunction, suggesting a less favorable intestinal environment [46]. Similarly, Lysinibacillus are generally maintained at low abundance in the healthy avian gut, and their overrepresentation is frequently associated with intestinal pathological conditions [47]. In addition, Campylobacter is a key colonizer of the poultry gut, linked to changes in microbial composition and host inflammation [48]. Therefore, the relatively high abundance of these taxa in the absence of BAs supplementation may reflect a less desirable microbial ecosystem in late-laying hens [49,50]. In contrast, BAs supplementation promoted the enrichment of bacterial taxa associated with improved gut function and host metabolism. The BA200 group was characterized by a higher abundance of Rikenellaceae_RC9_gut_group. Members of the family Rikenellaceae are considered important degraders of complex polysaccharides and have been positively associated with microbial fermentation and the production of SCFA, particularly acetate and propionate, which contribute to intestinal homeostasis and host energy homeostasis [51,52,53]. Previous studies have also reported positive associations between the abundance of Rikenellaceae_RC9_gut_group and improved gut metabolic function [54], suggesting that moderate BAs supplementation may enhance microbial fermentative capacity and nutrient utilization. The BA300 group was primarily characterized by the enrichment of Enterococcus. Although certain Enterococcus species may act as opportunistic pathogens, many commensal strains are regarded as beneficial members of the gut microbiota because of their ability to produce lactic acid, bacteriocins, and other antimicrobial compounds that inhibit pathogenic bacteria and contribute to microbial stability [55]. Therefore, the enrichment of Enterococcus in the BA300 group may indicate a more balanced microbial community with potentially enhanced colonization resistance against undesirable microorganisms [56,57]. In the BA500 group, UCG_010 was identified as the discriminative taxon. Although the ecological role of this taxon remains poorly characterized, previous microbiome studies have linked members of the Ruminococcaceae-related lineage to carbohydrate fermentation and gut metabolic activity [58,59]. The increased abundance of UCG_010 may therefore reflect a microbial response to higher dietary BAs supplementation and the accompanying alterations in cecal nutrient metabolism. Collectively, these findings suggested that dietary BAs supplementation modulated the cecal microbial community by reducing the relative abundance of potentially harmful bacteria while promoting taxa associated with carbohydrate fermentation, microbial stability, and metabolic health. Among the tested supplementation levels, 300 mg/kg BAs appeared to exert the pronounced effects on gut microbiota composition, which is consistent with its beneficial effects on microbial diversity and community structure observed in the present study.

5. Conclusions

In summary, under the conditions of this experiment, dietary supplementation with 300 mg/kg BAs improved hepatic lipid metabolism and gut microbiota composition in aged laying hens, and thus represents a practical nutritional strategy to mitigate metabolic decline during the late laying phase.