Dietary porcine bile acids alleviate lipid deposition and remodel ileal bile acid and microbiota profiles in laying hens fed a high-fat soybean meal-based diet

Abstract

This study was conducted to evaluate whether dietary porcine bile acids (PBA) alleviate lipid metabolic burden in laying hens fed a high-fat soybean meal-based diet. A total of 270 Hy-Line Brown laying hens at 60 wk of age were assigned to 3 dietary treatments for 12 wk: a corn-soybean meal basal diet (CON), a diet containing 20% high-fat soybean meal (HFSBM), and HFSBM supplemented with 60 mg/kg PBA (HFSBM+PBA). HFSBM reduced egg production during wk 9 to 12 and throughout the 12-wk feeding period (P = 0.001), whereas HFSBM+PBA maintained egg production at a level comparable with CON. Most conventional egg-quality traits, including egg shape index, albumen height, Haugh unit, yolk proportion, and eggshell strength, were not affected (P > 0.05). HFSBM increased serum triglyceride (TG), abdominal fat rate, hepatic TG, yolk total cholesterol (TC), and yolk TG, whereas PBA supplementation reduced serum TG (P = 0.005), prevented abdominal fat accumulation (P = 0.043), lowered hepatic TG (P < 0.001), restored yolk TC to a level comparable with CON (P = 0.041), and produced an intermediate response in yolk TG. Histological staining showed more apparent hepatocellular vacuolation and neutral lipid staining in HFSBM-fed hens, both of which appeared less prominent after PBA supplementation. Compared with HFSBM, HFSBM+PBA downregulated hepatic acetyl-CoA carboxylase, fatty acid synthase, and lipoprotein lipase expression (P < 0.05). Ileal bile acid profiling showed that PBA supplementation increased hyocholic acid, glycohyocholic acid, hyodeoxycholic acid, taurohyodeoxycholic acid, murideoxycholic acid, and taurodeoxycholic acid (P < 0.05), while tauroursodeoxycholic acid was reduced by HFSBM and partially restored by PBA. Ileal microbiota analysis showed increased Simpson diversity (P < 0.05), distinct community separation, enrichment of Ligilactobacillus, and reduced Enterococcus abundance in HFSBM+PBA hens. In conclusion, 60 mg/kg PBA prevented the decline in egg production observed with HFSBM and attenuated lipid deposition in laying hens, accompanied by downregulation of hepatic lipid metabolism-related genes and remodeling of ileal bile acid and microbial profiles.

Introduction

Dietary lipid supply is an important nutritional factor affecting laying performance and lipid distribution in laying hens. Moderate dietary fat supplementation can increase egg weight and alter egg component weight by increasing lipid availability for yolk formation (Whitehead et al., 1991; Grobas et al., 1999). However, responses to lipid-rich ingredients vary with lipid source, inclusion level, processing method, and stage of lay. In commercial formulation, lipid supply may also change when lipid-rich soybean ingredients replace part of a conventional corn–soybean meal diet. Full-fat or high-fat soybean products have been evaluated in laying-hen diets, with variable effects on laying performance, nutrient digestibility, egg quality, and egg lipid composition (Karimi et al., 2022; Maharjan et al., 2023; Toomer et al., 2024). Accordingly, high-fat soybean meal provides a practical ingredient-substitution setting in which changes in dietary composition and lipid supply occur together.

Excessive or prolonged lipid and energy input can impose a metabolic burden on laying hens, particularly during late lay, when laying performance remains high but metabolic resilience may decline (Liu et al., 2018; van Eck et al., 2023). Hepatic lipid accumulation is a major manifestation of this burden and is closely associated with impaired liver function, altered lipid transport, excessive adipose deposition, and reduced laying performance. In severe cases, hepatic steatosis may progress toward fatty liver hemorrhagic syndrome (FLHS), a multifactorial metabolic disorder influenced by nutritional, hormonal, environmental, and management factors (Rozenboim et al., 2016; Shini et al., 2019). Studies have shown that high-energy or imbalanced diets can predispose laying hens to hepatic lipid accumulation and FLHS-related pathological changes, although the magnitude of the response depends on energy source, protein level, age, and duration of exposure (Zhang et al., 2008; Zhuang et al., 2019; Du et al., 2024). These findings indicate that nutritional strategies capable of improving lipid utilization and reducing hepatic lipid deposition may be valuable for maintaining metabolic homeostasis and productive performance under lipid-enriched dietary conditions.

Nutritional strategies that regulate hepatic lipid synthesis or gut-liver metabolic communication have shown potential to reduce hepatic lipid accumulation in laying hens (Chen et al., 2023; Li et al., 2023, 2024; Hu et al., 2024b). Bile acids are amphipathic molecules with established roles in intestinal lipid digestion. They facilitate lipid emulsification, micelle formation, and lipase-mediated hydrolysis, thereby supporting the absorption of dietary lipids and fat-soluble nutrients (Krogdahl, 1985). Beyond this digestive function, bile acids also act as metabolic regulators through signaling pathways involved in lipid, cholesterol, glucose, and energy metabolism (Hylemon et al., 2009; Li and Chiang, 2015). In poultry nutrition, porcine bile acids (PBA) have attracted attention as a relatively defined and practical bile acid source. Previous studies in laying hens showed that PBA improved crude fat and energy utilization and enhanced fat-soluble vitamin absorption, supporting its role in lipid nutrient use (Yang et al., 2022a). In late-phase laying hens, dietary supplementation with 60 mg/kg PBA improved laying performance, modulated serum lipid metabolism, and altered cecal microbial composition (Yang et al., 2022b). In addition, exogenous bile salts have been reported to reduce hepatic lipid accumulation in aged laying hens, with suppressed lipogenic activity suggested as a contributing factor (Sun et al., 2023). These findings provide a nutritional and metabolic rationale for using PBA to alleviate lipid burden under diets with increased fat supply.

The metabolic effects of bile acids may also involve the intestinal bile acid pool and microbial ecology. Gut bacteria modify bile acids through deconjugation, dehydroxylation, dehydrogenation, and epimerization, whereas bile acids can selectively shape bacterial communities through antimicrobial pressure and host-mediated signaling (Ridlon et al., 2016; Guzior and Quinn, 2021; Collins et al., 2023; Larabi et al., 2023). Individual bile acids can differentially affect bacterial growth and microbial metabolism, indicating that changes in bile acid availability may be accompanied by selective microbial remodeling (Peng et al., 2024). Bile salt hydrolase activity represents a key microbial step in bile acid deconjugation and is common in many lactic acid bacteria and Lactobacillus-related taxa, providing a potential route for adaptation to bile-rich environments (Begley et al., 2006; Foley et al., 2019; O’Flaherty et al., 2018). These interactions are relevant to poultry because bile acid supplementation has been associated with altered gut microbiota, bile acid profiles, and hepatic lipid deposition under lipid-enriched dietary conditions (Wang et al., 2024b). Nevertheless, most available studies on PBA in laying hens have focused on conventional diets, production performance, serum biochemical indices, or cecal microbiota. Whether PBA can alleviate lipid metabolic disturbance induced by a high-fat soybean meal-based diet, and whether this response is accompanied by coordinated changes in hepatic lipid metabolism, ileal bile acid profiles, and ileal microbiota, remains insufficiently defined.

Therefore, the present study investigated the effects of dietary supplementation with 60 mg/kg PBA on production performance, systemic lipid deposition, and ileal bile acid and microbial profiles in laying hens fed a diet containing 20% high-fat soybean meal. We hypothesized that, relative to the unsupplemented high-fat soybean meal treatment, PBA supplementation would help maintain laying performance and attenuate lipid deposition, with these responses accompanied by changes in hepatic lipid metabolism-related gene expression and ileal bile acid and microbial profiles.

Materials and methods

Ethics statement

All experimental procedures involving laying hens were approved by the management of the Animal Care and Use Committee of Institute of Feed Research, Chinese Academy of Agricultural Sciences (approval No. AEC—CAAS-20240814).

Animals and experimental design

A total of 270 Hy-Line Brown laying hens at 60 wk of age were assigned to 3 dietary treatments in a completely randomized design, with 6 replicates per treatment and 15 hens per replicate. The dietary treatments consisted of a corn-soybean meal basal diet (CON), a diet containing 20% high-fat soybean meal (HFSBM), and the HFSBM supplemented with 60 mg/kg PBA (HFSBM+PBA). The HFSBM was produced by adding a lipid-rich soybean oil-processing by-product (soybean oil foots) back to commercially available conventional soybean meal, thereby increasing its crude fat content. The 20% inclusion level was selected to provide a substantial replacement of conventional soybean meal while remaining within the range evaluated for lipid-rich soybean ingredients in laying hens (Karimi et al., 2022; Maharjan et al., 2023; Toomer et al., 2024). At this inclusion level, the analyzed crude fat content of the diet increased from 1.86% in CON to 3.71% in HFSBM, whereas the calculated metabolizable energy remained similar between the two diets (11.25 and 11.14 MJ/kg, respectively). The PBA product, containing 60% total bile acids with hyodeoxycholic acid, chenodeoxycholic acid, and hyocholic acid as the primary components, was provided by Shandong Longchang Co., Ltd. (Dezhou, China). The nutrient composition of the high-fat soybean meal is presented in Supplementary Table S1, and the ingredient composition and nutrient levels of the experimental diets are shown in Table 1. Diets were formulated according to the Chinese Feeding Standard of Chickens (NY/T 33-2004; Ministry of Agriculture of the People’s Republic of China, 2004). The experiment lasted 13 wk, including a 1-wk adaptation period and a 12-wk feeding period.

Table 1.

Composition and nutrient levels of experimental diets (as-fed basis, %).

Itema	CON	HFSBM	HFSBM+PBA
Ingredientb
Corn	66.07	57.22	57.22
Soybean meal (44.8% CP)	23.25	8.10	8.10
High-fat soybean meal	—	20.00	20.00
Wheat bran	—	4.00	4.00
Dicalcium phosphate	1.00	1.00	1.00
Limestone	9.00	9.00	9.00
Sodium chloride	0.30	0.30	0.30
DL-methionine (99%)	0.12	0.12	0.12
Premix	0.13	0.13	0.13
Choline chloride	0.10	0.10	0.10
Phytase	0.03	0.03	0.03
Total	100.00	100.00	100.00
PBA product (mg/kg)	—	—	60
Nutrient levelc
Metabolizable energy (MJ/kg)	11.25	11.14	11.14
Crude protein	15.54	16.03	15.82
Crude fat	1.86	3.71	3.70
Calcium	3.57	3.58	3.50
Nonphytate phosphorus	0.35	0.36	0.36
Methionine	0.34	0.35	0.35
Methionine + cysteine	0.62	0.65	0.65
Lysine	0.73	0.82	0.82
Threonine	0.51	0.55	0.55
Tryptophan	0.16	0.18	0.18

^aCON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

^bNutritional composition of high-fat soybean meal and regular soybean meal is shown in Supplementary Table S1. Premix supplied per kilogram of diet: VA, 12,500 IU; VD₃, 4,125 IU; VE, 15 IU; VK, 2 mg; vitamin B₁, 1 mg; VB₂, 8.5 mg; VB₆, 8 mg; VB₁₂, 5 mg; Ca-pantothenate, 50 mg; niacin, 32.5 mg; biotin, 2 mg; folic acid, 5 mg; Mn, 65 mg; I, 1 mg; Fe, 60 mg; Cu, 8 mg; Zn, 66 mg.

^cCrude protein, crude fat and calcium are analyzed values. Others are calculated values.

Bird management

Hens were housed in three-tier battery cages (3 hens per cage; cage dimensions: 40 cm × 40 cm × 35 cm, providing approximately 533 cm² of floor space per hen) within a temperature-controlled room maintained at 22 to 26°C. The lighting regimen provided a 16-h light schedule daily with an intensity of approximately 20 lx. All birds were allowed ad libitum access to water and mash feed throughout the study.

Data and sample collection

At the end of the 12-wk trial, after a 12-h fast, two hens per replicate (12 hens per treatment) were selected for blood and tissue collection. For variables measured from two hens per replicate, values were averaged within replicate before statistical analysis, and the replicate mean was used as the experimental unit. Blood was drawn from the wing vein, allowed to clot in a 37°C water bath for 10 min, and centrifuged at 1,000 × g for 15 min to obtain serum, which was stored at −20°C until biochemical analysis. Birds were then slaughtered and dissected under aseptic conditions. The liver was excised, weighed, and photographed to record gross appearance; one portion was fixed in 4% paraformaldehyde for histomorphological examination, while a second portion was snap-frozen in liquid nitrogen and stored at −80°C for lipid concentration analysis and RT-qPCR. Abdominal fat pads were dissected and weighed. Ileal digesta were flushed from the distal ileum into sterile containers, snap-frozen in liquid nitrogen, and stored at −80°C for 16S rRNA sequencing and bile acid profiling. For 16S rRNA sequencing, ileal digesta from one of the two sampled hens in each replicate were used, yielding one microbiota sample per replicate.

Laying performance and egg quality

Mortality was recorded as it occurred throughout the trial. Daily egg number and total egg weight were recorded by replicate, and feed consumption was recorded biweekly. Egg mass was calculated as the total egg weight produced per replicate divided by the corresponding hen-days and expressed as g/hen per d. Feed conversion ratio (FCR) was calculated as total feed intake divided by total egg mass produced and expressed as g feed/g egg mass. Hen-day egg production, average egg weight, average daily feed intake, egg mass, and FCR were calculated for wk 1–4, 5–8, 9–12, and the overall 1–12 wk period.

At the end of wk 4, 8, and 12, six eggs per replicate with weight close to the replicate average were collected for egg quality determination. For each egg-quality trait, measurements obtained from the six eggs were averaged within replicate before statistical analysis. Egg shape index was calculated as egg length divided by egg width. Eggshell thickness (blunt end, tip, and equator) and eggshell strength were measured using an Eggshell Thickness Gauge and an Egg Force Reader (ORKA Technology Ltd., Ramat HaSharon, Israel), respectively. Albumen height, Haugh unit, and yolk color were determined using an Egg Analyzer (ORKA Food Technology Ltd., Ramat HaSharon, Israel). After removal of the chalazae, the yolk was separated and weighed. Thick and thin albumen were separated using a 40-mesh sieve and weighed as previously described (Zhou et al., 2025). Yolk proportion and thick albumen proportion were calculated relative to whole egg weight.

Liver index and abdominal fat rate

Liver index and abdominal fat rate were calculated as liver weight and abdominal fat pad weight, respectively, relative to live body weight (%).

Serum parameters

Serum total protein, albumin, globulin (calculated as total protein minus albumin), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and aspartate transaminase (AST) were determined using an automatic biochemical analyzer (Zhuoyue 300, Kehua Bio. Co., Ltd., Shanghai, China).

Hepatic and yolk lipid concentrations

Liver and freeze-dried yolk samples were homogenized in anhydrous ethanol (1:9 and 1:4, w/v, respectively) and centrifuged at 4,000 × g for 10 min at 4°C. The supernatant was used to determine TC and TG concentrations using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Hepatic TC and TG concentrations were expressed per gram of fresh liver tissue, whereas yolk TC and TG concentrations were converted to a fresh-weight basis and expressed per gram of fresh yolk (mg/g).

Histomorphology of liver

Liver tissue fixed in 4% paraformaldehyde was dehydrated, embedded in paraffin, and sectioned at 5-μm thickness, then stained with hematoxylin and eosin (H&E) for histological observation under light microscopy. For Oil Red O staining, fresh-frozen liver tissue was serially sectioned at 8-μm thickness using a Leica CM1900 cryostat (Shanghai, China) and stained with Oil Red O solution to visualize neutral lipid accumulation. Sections were photographed at × 400 magnification. Histological and Oil Red O staining observations were evaluated qualitatively.

RNA isolation and real-time quantitative PCR

Total RNA was extracted from liver tissue using the EasyPure RNA kit (TransGen Biotech Co., Ltd., Beijing, China), and RNA concentration was determined with an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., VT, USA). Reverse transcription was performed using the First-Strand cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd., Beijing, China). Quantitative PCR was conducted in triplicate on a CFX96 C1000^TM thermal cycler (Bio-Rad Laboratories, CA, USA). Relative mRNA expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL), and liver X receptor alpha (LXRα) was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001), normalized to β-actin; primer sequences are listed in Supplementary Table S2.

16S rRNA sequencing of ileal microbiota

Total DNA was extracted from ileal digesta using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA), and DNA quality was assessed by agarose gel electrophoresis. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplification consisted of initial denaturation at 95°C for 3 min, followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, with a final extension at 72°C for 10 min. PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and sequenced on the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). After demultiplexing, paired-end reads were quality-filtered using fastp (v0.19.6) and merged using FLASH (v1.2.11). Reads were trimmed when the mean quality score within a 50-bp sliding window was below 20, and reads shorter than 50 bp after trimming or containing ambiguous bases were discarded. Paired-end reads were merged with a minimum overlap of 10 bp and a maximum mismatch ratio of 0.2. The resulting high-quality sequences were denoised using the DADA2 plugin in QIIME2 (v2020.2) with recommended parameters to generate amplicon sequence variants (ASVs). ASVs assigned to chloroplasts or mitochondria were removed. Taxonomic assignment was performed using the Naive Bayes classifier implemented in QIIME2 against the SILVA 16S rRNA database (v138). Sequencing sufficiency was evaluated using rarefaction curves generated by sequential subsampling of the ASV table, which approached a plateau and indicated adequate sequencing coverage. The raw sequencing data have been deposited in the NCBI Sequence Read Archive under BioProject accession PRJNA1524582.

Ileal bile acid profiling

Ileal digesta samples were extracted with six volumes of ice-cold methanol. After homogenization and centrifugation, the supernatant was evaporated and reconstituted in 50% methanol. Bile acids were quantified using a Waters ACQUITY UPLC system coupled to an AB SCIEX QTRAP 6500+ mass spectrometer in negative electrospray-ionization MRM mode. Separation was performed on an ACQUITY UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm) at 50°C using water containing 0.025% formic acid and 10 mM ammonium acetate (A) and acetonitrile (9:1, v/v; B), at a flow rate of 0.4 mL/min and an injection volume of 5 μL. The gradient was programmed as follows: 5, 10, 20, and 28% B at 0, 0.5, 1, and 2 min, respectively; 28% at 8 min; 33, 45, 50, and 80% at 11, 20, 23, and 29 min; 100% from 29.01 to 31 min; and 5% from 31.01 to 33 min. CDCA-D4 and CA-D4 were used as internal standards. Pooled quality-control samples were periodically analyzed to monitor analytical reproducibility, with RSDs below 15%. MRM transitions and optimized potentials are provided in Supplementary Table S3. Peak integration and quantification were performed using MultiQuant software (v3.0.2).

Statistical analysis

Data other than microbiota were analyzed by one-way ANOVA using SAS 9.4 (SAS Institute Inc., Cary, NC, USA), with means separated by Duncan's Multiple Range Test. Production performance and egg quality data were analyzed separately for each reporting period and sampling week, respectively. The replicate was the experimental unit for all analyses; for bile acid profiling and RT-qPCR, values from the two hens within each replicate were averaged before analysis, whereas each microbiota sample represented one replicate (n = 6 per treatment). Data are expressed as means with pooled SEM. Differences were considered significant at P < 0.05.

Microbiota data were analyzed on the Majorbio Cloud Platform (Shanghai Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China) based on amplicon sequence variants. Alpha diversity was assessed using the Chao1 and Simpson indices, and beta diversity was evaluated by principal coordinate analysis based on Bray-Curtis distance. Differences in microbial community structure were tested using analysis of similarity (ANOSIM). Differentially abundant genera were identified using the Kruskal-Wallis test.

Results

Production performance

As shown in Table 2, dietary treatment did not affect egg production during wk 1 to 4 or wk 5 to 8. During wk 9 to 12 and throughout the 12-wk period, HFSBM reduced egg production compared with CON, whereas HFSBM+PBA maintained egg production at a level comparable with CON (P = 0.001). During wk 9 to 12, egg mass was lower in HFSBM than in CON and HFSBM+PBA (P < 0.05), whereas no significant differences were observed during the other periods (P > 0.05). HFSBM increased average egg weight during wk 5 to 8, wk 9 to 12, and the overall period, while HFSBM+PBA reduced average egg weight compared with HFSBM (P < 0.05). Average daily feed intake was lower in HFSBM+PBA than in CON and HFSBM during wk 5 to 8 and during the overall period (P < 0.05). Feed conversion ratio and mortality were not affected by dietary treatment (P > 0.05).

Table 2.

Effects of dietary porcine bile acid supplementation on production performance of laying hens fed a high-fat soybean meal-based diet¹.

Item2	Period	CON	HFSBM	HFSBM+PBA	SEM	P-value
EP, %	wk 1–4	84.70	83.60	83.58	0.79	0.568
	wk 5–8	85.48	80.52	81.31	1.71	0.221
	wk 9–12	84.01a	76.07b	83.46a	1.04	0.001
	wk 1–12	85.10a	79.87b	82.92a	1.63	0.001
EM, g/hen per d	wk 1–4	56.12	54.05	54.75	0.91	0.653
	wk 5–8	57.51	52.52	50.23	1.58	0.126
	wk 9–12	56.57a	51.42b	57.19a	0.98	0.001
	wk 1–12	56.74	52.67	54.05	1.06	0.090
AEW, g	wk 1–4	66.57	67.49	67.01	0.52	0.487
	wk 5–8	67.20b	68.11a	66.75b	0.20	0.008
	wk 9–12	66.88c	69.35a	67.78b	0.15	<0.001
	wk 1–12	66.91b	68.19a	67.40b	0.44	0.001
ADFI, g	wk 1–4	125.47	121.59	121.38	4.51	0.761
	wk 5–8	129.74a	123.79a	113.69b	2.45	0.006
	wk 9–12	125.70	123.62	121.92	2.65	0.576
	wk 1–12	126.99a	123.70a	118.93b	2.22	0.040
FCR	wk 1–4	2.20	2.15	2.11	0.10	0.851
	wk 5–8	2.26	2.27	2.25	0.10	0.739
	wk 9–12	2.25	2.38	2.20	0.09	0.276
	wk 1–12	2.24	2.34	2.27	0.10	0.509
Mortality, %	wk 1–4	0.00	0.00	2.22	0.74	0.391
	wk 5–8	0.00	0.00	1.11	0.39	0.391
	wk 9–12	1.11	1.11	0.00	0.53	0.539
	wk 1–12	1.11	1.11	3.33	0.91	0.559

¹n = 6 replicates per treatment (15 birds per replicate).

²EP, egg production; AEW, average egg weight; EM, egg mass; ADFI, average daily feed intake; FCR, feed conversion ratio (g feed/g egg mass); CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

^a–cWithin a row, means without a common superscript letter differ significantly (P < 0.05).

Egg quality

The effects of dietary porcine bile acid supplementation on egg quality are presented in Table 3. Dietary treatment had no significant effect on egg shape index, albumen height, Haugh unit, yolk proportion, or eggshell strength at any sampling week (P > 0.05). At wk 8, hens fed HFSBM and HFSBM+PBA had higher yolk color scores than those fed the CON diet (P = 0.002), whereas no difference was observed between HFSBM and HFSBM+PBA. Thick albumen proportion was higher in the HFSBM group than in the CON group at wk 4, with the HFSBM+PBA group showing an intermediate value (P = 0.019). Eggshell thickness was reduced by HFSBM and HFSBM+PBA compared with CON at wk 4 (P = 0.001), but no treatment effect was observed at wk 8 or wk 12.

Table 3.

Effects of dietary porcine bile acid supplementation on egg quality of laying hens fed a high-fat soybean meal-based diet¹.

Item2	Week	CON	HFSBM	HFSBM+PBA	SEM	P-value
Egg shape index	wk 4	1.31	1.30	1.31	0.01	0.637
	wk 8	1.31	1.33	1.31	0.01	0.083
	wk 12	1.32	1.30	1.31	0.01	0.448
Albumen height, mm	wk 4	5.59	6.05	5.92	0.38	0.433
	wk 8	7.73	7.62	7.69	0.35	0.962
	wk 12	5.21	5.91	5.44	0.38	0.224
Yolk color	wk 4	7.95	7.63	7.73	0.20	0.335
	wk 8	7.56b	8.27a	8.33a	0.20	0.002
	wk 12	7.77	7.68	7.33	0.23	0.233
Haugh unit	wk 4	73.20	78.73	77.86	2.95	0.112
	wk 8	85.48	83.77	85.26	2.07	0.794
	wk 12	66.89	71.41	69.10	2.82	0.440
Yolk proportion, %	wk 4	25.68	24.55	25.63	0.50	0.136
	wk 8	25.49	25.28	24.96	0.55	0.661
	wk 12	26.58	25.36	25.94	0.55	0.212
Thick albumen, %	wk 4	31.09b	34.01a	32.41ab	0.83	0.019
	wk 8	34.74	34.05	35.55	1.17	0.591
	wk 12	28.61	30.12	29.20	0.81	0.475
Eggshell thickness, mm	wk 4	0.46a	0.40b	0.40b	0.01	0.001
	wk 8	0.38	0.39	0.40	0.01	0.589
	wk 12	0.39	0.37	0.38	0.01	0.227
Eggshell strength, N	wk 4	35.21	31.90	31.70	2.02	0.247
	wk 8	30.48	29.97	32.43	2.23	0.609
	wk 12	25.53	27.78	28.32	1.81	0.307

¹n = 6 replicates per treatment (6 eggs per replicate).

²CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

^a,bWithin a row, means without a common superscript letter differ significantly (P < 0.05).

Serum biochemical parameters

The effects of dietary porcine bile acid supplementation on serum biochemical parameters are shown in Table 4. Hens fed the HFSBM had higher serum TG concentrations than those fed the CON diet, whereas HFSBM+PBA reduced serum triglyceride concentrations to a level comparable with CON (P = 0.005). Serum HDL-C was lower in the HFSBM+PBA group than in the CON group, with the HFSBM group showing an intermediate value (P = 0.015). No significant differences were observed among treatments in serum total protein, albumin, globulin, TC, or aspartate aminotransferase.

Table 4.

Effects of dietary porcine bile acid supplementation on serum biochemical parameters of laying hens fed a high-fat soybean meal-based diet¹.

Item2	CON	HFSBM	HFSBM+PBA	SEM	P-value
TP, g/L	59.41	64.27	56.05	2.76	0.092
ALB, g/L	21.96	22.24	21.18	0.81	0.627
GLB, g/L	38.32	42.43	34.73	2.39	0.095
TC, mmol/L	1.97	1.94	1.81	0.21	0.827
TG, mmol/L	28.58b	32.16a	29.72b	0.94	0.005
HDL-C, mg/dL	15.96a	14.24ab	12.06b	0.95	0.015
AST, U/L	245.80	262.25	248.25	9.47	0.231

¹n = 6 replicates per treatment (2 samples per replicate).

²TP, total protein; ALB, albumin; GLB, globulin; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; AST, aspartate aminotransferase; CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

^a,bWithin a row, means without a common superscript letter differ significantly (P < 0.05).

Liver and abdominal fat, hepatic and yolk lipid concentrations

The effects of dietary porcine bile acid supplementation on liver index, abdominal fat rate, and lipid concentrations in the liver and egg yolk are presented in Table 5, Table 6. Liver index was not affected by dietary treatment (P > 0.05). In contrast, HFSBM increased abdominal fat rate compared with CON, and this increase was prevented by PBA supplementation, with the HFSBM+PBA group showing a value similar to that of the CON group (P = 0.043). Consistent with the change in abdominal fat deposition, HFSBM markedly increased hepatic TG concentration, whereas PBA supplementation reduced hepatic TG to a level comparable with CON (P < 0.001). Hepatic TC concentration did not differ among treatments. In egg yolk, HFSBM increased TC concentration compared with CON, while PBA supplementation normalized yolk TC concentration (P = 0.041). Yolk TG concentration was also increased by HFSBM, with the HFSBM+PBA group showing an intermediate response between the CON and HFSBM groups (P = 0.014).

Table 5.

Effects of dietary porcine bile acid supplementation on liver index and abdominal fat rate of laying hens fed a high-fat soybean meal-based diet¹.

Item2	CON	HFSBM	HFSBM+PBA	SEM	P-value
Liver index, %	1.53	1.68	1.47	0.004	0.090
Abdominal fat rate, %	3.77b	5.28a	3.88b	0.284	0.043

¹n = 6 replicates per treatment (2 samples per replicate).

²CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

^a,bWithin a row, means without a common superscript letter differ significantly (P < 0.05).

Table 6.

Effects of dietary porcine bile acid supplementation on hepatic and yolk lipid concentrations of laying hens fed a high-fat soybean meal-based diet¹.

Item2	CON	HFSBM	HFSBM+PBA	SEM	P-value
Liver lipids
TC, mg/g	2.15	2.74	2.50	0.43	0.468
TG, mg/g	15.04b	37.51a	9.42b	4.50	<0.001
Yolk lipids
TC, mg/g	5.20b	9.80a	6.45b	1.37	0.041
TG, mg/g	69.65b	91.45a	79.65ab	4.85	0.014

¹n = 6 replicates per treatment (2 samples per replicate).

²TC, total cholesterol; TG, triglycerides; CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

^a,bWithin a row, means without a common superscript letter differ significantly (P < 0.05).

Liver appearance and histomorphology

As shown in Fig. 1, livers from the CON group displayed a dark-red appearance, whereas those from the HFSBM group appeared pale yellow-brown, consistent with increased hepatic lipid accumulation. This difference was less evident in the HFSBM+PBA group. Qualitative examination of representative sections showed more apparent hepatocellular vacuolation and Oil Red O-positive neutral lipid staining in the HFSBM group than in CON, while both features appeared less prominent in the HFSBM+PBA group.

Fig. 1.

Effects of dietary porcine bile acid supplementation on liver appearance and histological sections of laying hens fed a high-fat soybean meal-based diet. H&E staining and Oil Red O staining were performed at × 400 magnification. Scale bar = 200 μm. CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

Hepatic lipid metabolism-related gene expression

As shown in Fig. 2, dietary treatment significantly affected the hepatic mRNA expression of lipid metabolism-related genes. Compared with HFSBM, HFSBM+PBA downregulated the expression of ACC, FAS, and LPL (P < 0.05). The expression of FAS in the HFSBM+PBA group was reduced to a level comparable with CON, whereas LPL expression was lower in HFSBM+PBA than in both CON and HFSBM groups. For LXRα, both HFSBM and HFSBM+PBA showed lower expression than CON, with no difference between the two HFSBM-fed groups (P < 0.05).

Fig. 2.

Effects of dietary porcine bile acid supplementation on the mRNA expression of hepatic lipid metabolism-related genes in laying hens fed a high-fat soybean meal-based diet. FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; LPL, lipoprotein lipase; LXRα, liver X receptor alpha; CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids. Data are expressed as means ± SEM (n = 6). Bars with different letters (a, b) differ significantly (P < 0.05).

Ileal bile acid profile

Fig. 3 shows that dietary PBA supplementation significantly changed the ileal bile acid profile. The concentrations of hyocholic acid (HCA), glycohyocholic acid (GHCA), hyodeoxycholic acid (HDCA), taurohyodeoxycholic acid (THDCA), murideoxycholic acid (MDCA), and taurodeoxycholic acid (TDCA) were all higher in the HFSBM+PBA group than in the CON and HFSBM groups (P < 0.05), with no difference between CON and HFSBM. By contrast, tauroursodeoxycholic acid (TUDCA) was decreased in the HFSBM group compared with CON, whereas the HFSBM+PBA group showed an intermediate response (P = 0.038).

Fig. 3.

Effects of dietary porcine bile acid supplementation on ileal bile acid concentrations of laying hens fed a high-fat soybean meal-based diet. Data are expressed as Log₁₀ (concentration + 1) using box-and-whisker plots (n = 6 replicate means per treatment); concentration unit: ng/mg. Within each bile acid, groups without a common superscript letter differ significantly (P < 0.05). HCA, hyocholic acid; GHCA, glycohyocholic acid; HDCA, hyodeoxycholic acid; THDCA, taurohyodeoxycholic acid; MDCA, murideoxycholic acid; TUDCA, tauroursodeoxycholic acid; TDCA, taurodeoxycholic acid. CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

Ileal microbiota

As shown in Fig. 4A, Chao1 richness did not differ among treatments (P = 0.566), whereas the Simpson diversity index was higher in HFSBM+PBA than in both CON (P < 0.01) and HFSBM (P < 0.05). Beta diversity (PCoA) revealed clear separation among the three groups (Fig. 4B; ANOSIM, R = 0.425, P = 0.001). At the genus level, Ligilactobacillus was markedly more abundant in HFSBM+PBA than in CON and HFSBM (P = 0.005), whereas Enterococcus was more abundant in CON than in HFSBM and HFSBM+PBA (P = 0.003). Peptostreptococcus and Veillonella, both present at low relative abundance, also differed significantly among treatments (P = 0.006 and P = 0.017, respectively).

Fig. 4.

Effects of dietary porcine bile acid supplementation on ileal microbiota of laying hens fed a high-fat soybean meal-based diet. (A) Alpha diversity indices at ASV level: Chao1 richness index (left) and Simpson diversity index (right). (B) Beta diversity analysis: principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity at ASV level. Each dot represents one biological replicate (n = 6 per group). Group separation was assessed by ANOSIM. (C) Relative abundance of ileal microbiota at the genus level. Each bar represents the mean community composition of one treatment group. (D) Significantly different genera among groups identified by Kruskal-Wallis test. *P < 0.05, **P < 0.01. CON, control group fed a basal diet; HFSBM, group fed a diet containing 20% high-fat soybean meal; HFSBM+PBA, group fed the HFSBM diet supplemented with 60 mg/kg porcine bile acids.

Discussion

The HFSBM diet used in the present study provided a practical ingredient-substitution setting rather than a simple oil-supplementation model, with the responses to HFSBM reflecting the combined changes in fat content and other dietary components. In laying hens, hepatic lipid metabolism is closely coupled with follicular lipid deposition because the liver produces the lipoprotein precursors required for yolk formation. In particular, the onset of lay is accompanied by a hepatic shift from generic very-low-density lipoprotein (VLDL) production to yolk-targeted VLDL (VLDLy) particles, which are transported to growing follicles for yolk lipid deposition (Alvarenga et al., 2011; Walzem et al., 1999; Zaefarian et al., 2019; Ren et al., 2021). Consistent with this hepatic–follicular coupling, the higher average egg weight observed in HFSBM-fed hens is consistent with earlier studies showing that dietary fat can increase egg weight and egg component weight in laying hens (Whitehead et al., 1991; Grobas et al., 1999). This response did not constitute an overall improvement in laying performance, however, because egg production declined during wk 9–12 and over the entire feeding period. At the end of the trial, HFSBM-fed hens also had higher serum TG, abdominal fat rate, hepatic TG, and yolk TC and TG concentrations, indicating greater lipid deposition across multiple physiological compartments.

Despite consuming less feed during wk 5–8 and over the entire trial, hens fed HFSBM+PBA maintained egg production comparable with CON during wk 9–12 and over the 12-wk period, with unchanged FCR. This pattern is consistent with the digestive function of bile acids in promoting lipid emulsification, micelle formation, and lipase-mediated hydrolysis in the small intestine; in laying hens, porcine bile acids have been shown to improve crude fat and energy utilization and support late-phase laying performance (Krogdahl, 1985; Yang et al., 2022a,b). The higher thick albumen proportion in HFSBM-fed hens at wk 4 may partly reflect the greater lipid supply accompanying HFSBM substitution, as dietary fat has previously been associated with increased albumen deposition (Whitehead et al., 1991; Grobas et al., 1999). This early response was not accompanied by changes in albumen height or Haugh unit and was no longer evident at wk 8 or 12, indicating that the change in albumen partitioning was temporary. Although dietary calcium concentrations were similar among treatments, eggshell thickness was lower in both HFSBM-fed groups at wk 4. This difference was not accompanied by reduced eggshell strength and was absent at wk 8 and 12, indicating a transient change during the early feeding period; its nutritional basis remains to be determined. Apart from these transient changes, conventional egg-quality traits were largely unaffected, suggesting that the major response to PBA under HFSBM feeding was metabolic.

The most consistent response to PBA supplementation was the attenuation of lipid deposition across multiple physiologically connected lipid pools. In HFSBM-fed hens, elevated serum TG was accompanied by higher abdominal fat rate, hepatic TG concentration, yolk TC and TG concentrations, and more prominent hepatic lipid staining, indicating that the high-fat soybean meal-based diet affected lipid transport, storage, hepatic deposition, and reproductive lipid output simultaneously. This pattern is particularly meaningful in laying hens, because the liver is the major site of de novo fatty acid synthesis and also produces yolk precursors, including VLDL and vitellogenin, for follicular deposition (Alvarenga et al., 2011; Ren et al., 2021). Walzem et al. (1999) further described the shift toward yolk-targeted VLDLy production during lay, providing a physiological basis for linking hepatic lipid handling with yolk lipid allocation. Thus, the concurrent increases in hepatic TG and yolk lipids in HFSBM-fed hens suggest that the lipid-enriched diet disturbed not only hepatic fat storage but also the partitioning of lipids between metabolic and reproductive compartments. In the present study, 60 mg/kg PBA reduced serum TG, prevented abdominal fat accumulation, lowered hepatic TG, and restored yolk TC to a level comparable with CON, while yolk TG showed an intermediate response. Serum HDL-C was lower in HFSBM+PBA hens than in CON, whereas TC remained unchanged, showing that the serum lipid response was not uniform. In laying hens, circulating lipoprotein metabolism is closely coupled to hepatic lipid synthesis and yolk deposition (Walzem et al., 1999; Alvarenga et al., 2011); here, the decrease in HDL-C accompanied lower serum TG and reduced lipid deposition in the liver, abdominal fat, and egg yolk. The lower cumulative feed intake in HFSBM+PBA hens may also have contributed to these reductions, although ADFI did not differ among treatments during wk 9–12, indicating that the difference was not sustained throughout the trial.

These biochemical changes were supported by qualitative histological observations, in which hepatocellular vacuolation and Oil Red O-positive staining were visibly less prominent after PBA supplementation. The magnitude and distribution of this response are consistent with Yang et al., 2022b, who reported that the same dose of PBA reduced abdominal fat deposition and improved serum lipid metabolism in late-phase hens, and with Sun et al. (2023), who showed that exogenous bile salts alleviated hepatic lipid accumulation in aged laying hens. The higher hepatic TG concentration and greater Oil Red O-positive staining support hepatic lipid accumulation in HFSBM-fed hens. However, the unchanged mortality, serum AST activity, and liver index indicate that this accumulation was not accompanied by overt liver injury or FLHS. Collectively, these findings indicate that PBA moderated the HFSBM-induced lipid-deposition phenotype across circulating, storage, hepatic, and reproductive compartments, rather than affecting an isolated lipid index.

The expression of selected genes related to hepatic lipid synthesis and handling provides a molecular correlate for the reduced lipid deposition described above. ACC and FAS are key enzymes involved in de novo fatty acid synthesis and have been implicated in hepatic lipid accumulation in laying hens (Lv et al., 2018). In the present study, their expression was lower in HFSBM+PBA than in HFSBM, with FAS reduced to a level comparable with CON. This downregulation is consistent with the lower hepatic TG concentration and reduced Oil Red O staining observed after PBA supplementation, and parallels previous poultry studies in which bile acid supplementation reduced hepatic lipid accumulation together with suppressed lipogenic activity (Sun et al., 2023; Wang et al., 2024a). LPL showed a separate pattern, with the lowest expression observed in HFSBM+PBA hens, which may reflect reduced hepatic processing of TG-rich lipoprotein-derived fatty acids under the lipid-enriched diet (Jiang et al., 2020). Together, the changes in ACC, FAS, and LPL support the view that PBA attenuated hepatic lipid deposition by limiting both lipogenic drive and lipid-handling burden. Hepatic LXRα expression, however, remained lower in both HFSBM-fed groups than in CON and was not restored by PBA. This contrast with ACC, FAS, and LPL indicates that the transcriptional response to PBA was selective among the genes examined. This interpretation agrees with recent evidence in late-phase laying hens showing that dietary bile acids can reduce hepatic lipid deposition and modify lipid metabolic responses, although the underlying regulatory pathways may vary with bile acid source, dose, and dietary composition (Wang et al., 2024a).

The ileal bile acid profile complements the hepatic and tissue lipid responses to PBA. The measured ileal pool includes bile acids supplied with the PBA product, endogenous and reconjugated species returned to the intestine through biliary secretion, and metabolites potentially formed through intestinal microbial transformation. The porcine bile acid product used in this study was derived from swine bile and is characterized mainly by HDCA, CDCA, and HCA (Yang et al., 2022c). Accordingly, the marked increases in ileal HCA and HDCA in HFSBM+PBA hens are consistent with dietary delivery of these major PBA components, while the concurrent increases in GHCA and THDCA may reflect host conjugation and enterohepatic cycling of the supplemented bile acids. The increases in MDCA and TDCA further suggest secondary bile acid-related remodeling, which may involve intestinal microbial transformation and, in the case of TDCA, subsequent host reconjugation. This interpretation is consistent with the recognized role of gut microbiota in bile acid deconjugation and secondary bile acid formation, and with poultry studies linking bile acid supplementation to altered bile acid profiles and reduced hepatic lipid deposition (Guzior and Quinn, 2021; Wang et al., 2024b). TUDCA showed a different response, being reduced by HFSBM and partially restored by PBA, suggesting that the high-fat soybean meal-based diet also disturbed endogenous hydrophilic bile acid homeostasis. Given the reported effects of TUDCA in alleviating hepatic endoplasmic reticulum stress and steatosis in mice, this response may be relevant to the attenuation of hepatic lipid accumulation in HFSBM+PBA hens (Özcan et al., 2006). Because bile acids support intestinal lipid digestion and act as metabolic signals involved in lipid and cholesterol homeostasis, the remodeled ileal bile acid profile may have contributed to the lower hepatic and peripheral lipid accumulation observed in HFSBM+PBA hens (Hylemon et al., 2009; Li and Chiang, 2015).

PBA supplementation also reshaped the ileal microbial community. The unchanged Chao1 index together with the increased Simpson index and distinct PCoA separation suggests that PBA altered community evenness and composition rather than simply increasing bacterial richness. At the genus level, the enrichment of Ligilactobacillus in HFSBM+PBA hens was the most prominent response, whereas Enterococcus was more abundant in CON than in the two HFSBM-fed groups; Peptostreptococcus and Veillonella also differed among treatments but were present at low relative abundance. These changes are relevant to the remodeled bile acid profile described above, because bile acids can shape intestinal microbial communities through detergent-like antimicrobial activity and host-mediated signaling, while gut bacteria reciprocally modify bile acids through deconjugation and secondary bile acid formation (O’Flaherty et al., 2018; Collins et al., 2023; Larabi et al., 2023; Peng et al., 2024). The higher abundance of Ligilactobacillus may therefore reflect adaptation of bile-tolerant lactic acid bacteria to the expanded ileal bile acid pool. This interpretation is supported by evidence that Lactobacillus bile salt hydrolase substrate specificity affects bacterial fitness and colonization in bile-rich environments, although the genus-level 16S rRNA data obtained here do not resolve species identity or establish the bile acid-transforming capacity of the detected taxa (Foley et al., 2021). Consistent with this interpretation, porcine bile acid supplementation has been reported to alter gut microbial composition in late-phase laying hens, and bile acid supplementation under high-fat dietary conditions has been linked with concurrent changes in gut microbiota, bile acid profiles, and hepatic lipid deposition (Yang et al., 2022b; Hu et al., 2024a; Wang et al., 2024b). Moreover, bacterial bile acid modification through BSH activity has been shown to influence host lipid metabolism, supporting the biological plausibility of a microbiota-bile acid-lipid metabolism connection (Joyce et al., 2014).

Taken together, PBA supplementation alleviated HFSBM-induced lipid deposition in the liver, abdominal fat, and egg yolk, accompanied by lower hepatic ACC, FAS, and LPL expression and substantial remodeling of the ileal bile acid profile. The concurrent shift in ileal microbial composition further supports a coordinated hepatic–intestinal response to PBA supplementation.

Conclusion

In conclusion, feeding a high-fat soybean meal-based diet reduced egg production during the late phase of the trial and increased serum TG and lipid accumulation in the liver, abdominal adipose tissue, and egg yolk. Supplementation with 60 mg/kg porcine bile acids maintained egg production under this dietary condition and attenuated lipid deposition, as indicated by lower serum TG, abdominal fat rate, hepatic TG, and yolk TC, together with qualitatively less prominent hepatic lipid staining. These effects were accompanied by decreased hepatic ACC, FAS, and LPL expression, marked remodeling of the ileal bile acid profile, and shifts in ileal microbial community structure. Overall, porcine bile acids may represent a practical nutritional strategy to alleviate lipid metabolic burden in laying hens fed high-fat soybean meal-based diets.

Authorship contributions

Wenke Tao: Formal analysis, Data curation, Writing - original draft. Mengni Lei: Investigation, Methodology, Data curation. Uchechukwu Edna Obianwuna: Methodology, Writing - review & editing. Kai Qiu: Data curation, Validation. Jing Wang: Supervision, Formal analysis, Writing - review & editing. Haijun Zhang: Formal analysis, Investigation, Writing - review & editing. Guanghai Qi: Conceptualization, Methodology, Writing - review & editing. Shugeng Wu: Conceptualization, Supervision, Writing - review & editing. Jianmin Zhou: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Supervision, Project administration. All authors have read and agreed to the published version of the manuscript).

Disclosures

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