Porcine bile acids improve performance by altering hepatic lipid metabolism and amino acid metabolism with different protein level diets in late laying hens

Abstract

As the extension of the egg-laying cycle, heightened energy and lipid metabolism cause excessive lipid accumulation, resulting in rapid decline in laying performance during the late laying period. Bile acids (BAs), synthesized from cholesterol in the liver, are potent metabolic and immune signaling molecules involved in lipid metabolism and the regulation of energy homeostasis. However, under different dietary protein levels, the role of BAs on hepatic lipid metabolism of laying hens at the late phase remains unclear. This experiment aimed to evaluate the effects of porcine BAs supplementation on performance, lipid metabolism, antioxidant status and amino acid metabolism in late-phase laying hens fed diets with different protein level. A total of 192 Hy-Line Brown laying hens (62 weeks of age) were randomly assigned to one of four treatment groups, in a 2 × 2 factorial design, with 8 replicates per treatment. The hens were fed diets with either normal protein (16.42 %) or low-protein (15.35 %) levels, with or without BAs supplementation (120 mg/kg for the first 56 days, followed by 200 mg/kg for the next 42 days). The results demonstrated that dietary BAs supplementation significantly enhanced egg production and feed intake (P < 0.05) although it has no notable effect on egg quality. Bile acids supplementation effectively reduced liver total cholesterol (TC), triglyceride (TG), as well as malondialdehyde (MDA) levels, while also ameliorating lipid deposition through the regulation of expression of lipid metabolism-related genes in late laying hens (P < 0.05). Additionally, the low-protein diets downregulated amino acid catabolism, thereby reducing serum uric acid content and enhancing protein utilization. Further analysis revealed that BAs also positively influenced trypsin activity and increased the expression of amino acid transporters, thereby improving amino acid availability (P < 0.05). In conclusion, this study demonstrated that dietary BAs supplementation could enhance the laying performance in late laying hens, primarily by improving hepatic lipid metabolism, antioxidant capacity, and amino acid availability.

Introduction

As the egg-laying cycle extends, the increased energy and lipid metabolism cause excessive lipid accumulation, resulting in rapid decline in laying performance at late laying period, which is a major concern for the global poultry industry. The liver, being central to both lipid and protein metabolism, also serves as the main organ for biotransformation and detoxification in laying hens (Trefts et al., 2017). However, as hens age, the physiological functions of the liver deteriorate, causing disruptions in lipid metabolism, a decline in protein utilization efficiency, and consequently, a reduction in overall production performance (He et al., 2021; Anene et al., 2023). Therefore, improving hepatic lipid metabolism and protein utilization efficiency in late-laying hens becomes essential to maintain optimal production levels.

Bile acids (BAs), the primary metabolic byproduct of hepatic cholesterol breakdown, play a crucial role in maintaining digestive health. These amphipathic molecules possess emulsifying properties that aid in the emulsification, digestion and intestinal absorption of dietary lipids and fat-soluble vitamins (Wahlström et al., 2016). Additionally, BAs are instrumental in regulating hepatic cholesterol homeostasis and maintaining the BAs pool through enterohepatic recirculation (Lefebvre et al., 2009; Kong et al., 2012). Importantly, in chickens, studies have shown that chenodeoxycholic acid (CDCA) improves hepatic lipid metabolism by downregulating genes involved in hepatic fat synthesis (Piekarski et al., 2016). Moreover, the body regulates endogenous BAs synthesis via farnesoid X receptor (FXR), a nuclear receptor for BAs, to mitigate disruptions caused by exogenous BAs on bile acid homeostasis (Kong et al., 2012; Sun et al., 2023). Furthermore, BAs also act as signaling molecules through FXR, influencing both protein synthesis and amino acid catabolism. For example, they stimulate protein synthesis while also participating in amino acid decomposition and ammonia detoxification processes (Kir et al., 2011; Massafra et al., 2017). Meanwhile, studies have demonstrated that fibroblast growth factor 19 (FGF19)-treated mice increased the synthesis of plasma albumin through FXR-FGF19 axis (Kir et al., 2011; Preidis et al., 2017). Conversely, FXR knockout mice increased blood ammonia levels, reduced detoxification function, and lower plasma urea concentration with a high-protein diet, indicating that FXR is directly involved in amino acid breakdown metabolism (Renga et al., 2011).

However, there is limited research on how exogenous BAs affect lipid and amino acid metabolism in laying hens, particularly in relation to varying crude protein levels in their diet. Therefore, this study aims to investigate the effects of supplementing BAs at different crude protein levels on laying performance, egg quality, BAs homeostasis, lipid metabolism, and amino acid metabolism in laying hens. This research could provide a novel approach for extending the laying period and alleviating the decline in production performance during the late laying period.

Materials and methods

Animal ethics statement

All animal management and experimental procedures were performed in accordance with the guidelines of the Animal Care Committee of Henan Agricultural University (Approval No. HENAU-2022-015).

Animals and experiment design

A total of 192 Hy-line brown (62 weeks of age) were randomly allocated to one of four experimental groups in a 2 × 2 factorial design: (1) a basal diet group (CON), (2) a basal diet supplemented with 120 mg/kg (62-69 weeks) and 200 mg/kg (70-75 weeks) BAs group (CON+BA), (3) a low-protein diet group (LP), and (4) a LP diet supplemented with 120 mg/kg (62-69 weeks) and 200 mg/kg (70-75 weeks) BAs group (LP+BA). Each group consisted of 8 replicates with 6 hens per replicate. The hens were housed in 3-layer vertical cages (3 birds/cage, 50 × 45 × 45 cm) located in a temperature-controlled room with an ambient temperature of 23-25°C and relative humidity of 30-50 %. A lighting schedule of 16 h light and 8 h dark was maintained throughout the trial. Water was freely available through nipple drinkers 35 cm above the floor of the cage. Feed was provided ad libitum by feeding twice a day (8:00, 1/2 (70 g); 14:00, 1/2 (70 g)).

The BAs products used in the trial were supplied by Henan 91 Chinese Medicine Research Institute Co., Ltd. (Zhengzhou, China) and were derived from the gallbladders of pigs, which are more readily available and inexpensive compared to alternative sources of BAs. The composition of the BAs was determined by liquid chromatography-tandem mass spectrometry (John et al., 2014), and consisted of 7.73 % hyocholic acid, 68.31 % hyodeoxycholic acid, and 18.96 % chenodeoxycholic acid. The dosage of BAs supplementation was adjusted based on previous studies (Sun et al., 2023; Goodwin et al., 2021; Yang et al., 2022b).

The trial lasted for 14 weeks, spanning the period from 62 to 75 weeks of age. The composition and nutrient levels of the experimental diets are shown in Table 1. Diets were formulated to meet the requirements recommended by the Hy-Line Brown Management Guide (Hy-line, 2018). Feed samples were dried in an oven (HB841-0, Hao Bei Co., Ltd., Suzhou, China) at 105 °C for 24 h, after which they were ground using a grinder (AF-04A, AO Li Co., Ltd., Wenling, China) to pass through a 1-mm sieve for subsequent chemical analysis. Crude ash content was determined using a muffle furnace (SX2-10T, Bo Xun Co., Ltd., Shanghai, China) following method 942.05, while crude protein content (N × 6.25) was analyzed using a Kjeldahl nitrogen apparatus (KDN-200, Jing Rui Co., Ltd., Jinan, China) according to method 984.13. Calcium and total phosphorus levels were assessed using ethylenediaminetetraacetic acid titration and ammonium metavanadate colorimetry, respectively, in accordance with method 985.01. All nutrient analyses were conducted following the AOAC guidelines (AOAC, 2005).

Table 1.

Experimental Diet Composition and Nutrients (as-fed basis, %).

Item	Normal protein	Low protein
Ingredients
Corn	65.75	67.30
Soybean meal	22.00	18.37
Bran	-	1.90
Liquid methionine	0.16	0.16
Stone powder	9.60	9.60
CaHPO4	0.80	0.80
Choline	0.14	0.14
Premix1	1.50	1.50
L-lysine·HCl, 78.6 %	-	0.10
DL-methionine, 99 %	0.05	0.08
L-threonine, 99 %	-	0.05
Total	100	100
Calculated nutrient content
Metabolizable energy, kcal/kg	2,650	2,650
Lys	0.77	0.74
Met	0.36	0.36
Thr	0.57	0.55
Assayed nutrient content
CP	16.42	15.35
Ca	3.59	4.01
TP2	0.44	0.40
Ash	14.00	15.08

¹Premix provided the following per kilogram of the diet: vitamin A, 4,500 IU; vitamin D, 5,500 IU; vitamin E, 16 IU; vitamin K, 0.5 mg; thiamine, 2.0 mg; riboflavin, 5.0 mg; pyridoxine, 4.5 mg; vitamin, B12, 24 mg; Cu (CuSO4·5H2O), 14 mg; Fe (FeSO4·7H2O), 85 mg; Zn (ZnSO4·7H2O), 75 mg; Mn (MnSO4·H2O), 78 mg; Se (NaSeO3), 0.7 mg; I (KI), 0.7 mg; calcium pantothenate, 15.0 mg; folate, 2.5 mg; biotin, 0.15 mg; nicotinic acid 42 mg.

²TP = Total phosphorus.

Sample collection

On the final day of the trial (Week 14), one hen per replicate was randomly selected for blood collection after overnight fasting. Blood samples were collected from the wing vein and subjected to centrifugation at 1,500 × g for 10 min at 4 °C using a JW-1060R centrifuge (Jia Wen Co., Ltd., Anhui, China) to isolate the serum. Following cervical dislocation, abdominal adipose tissue and liver was harvested. Tissues were divided into two parts: one part tissues were fixed in 4 % paraformaldehyde for morphological analysis, and the other part tissues were snap-frozen in liquid nitrogen. Additionally, samples from the jejunum and ileum were collected and immediately frozen in liquid nitrogen. All tissues were stored at −80 °C for subsequent analysis.

Laying performance

Egg production and egg weight were recorded daily throughout the trial. Feed intake was measured weekly, and the feed conversion ratio (FCR) was calculated accordingly. At the end of the trial, six eggs per replicate were collected for egg quality determination.

Egg quality

On the last day of 14-week experimental period, one egg per bird (n = 192) was collected for quality analysis. Eggshell thickness was measured at three locations using an eggshell thickness gauge (EFG-0503, ROBOTMATION, Japan). Egg shape index was calculated according to Cattaneo et al. (2025). Eggshell strength was measured by an eggshell strength tester (EFG-0503, ROBOTMATION, Japan). Albumen height and Haugh units were measured with a multifunctional egg tester (DET-6000, NABEL Co., Ltd. Japan). The percentages of yolk and shell were calculated as yolk weight and shell weight/egg weight, respectively, according to Hussein et al. (1992).

$$\text{Eggshell}\text{thickness},\text{mm}=\left(\text{large}\text{end}+\text{equatorial}\text{region}+\text{small}\text{end}\right)/3$$

$$\text{Egg}\text{shape}\text{index}=\text{long}\text{diameter}/\text{short}\text{diameter}$$

Serum biochemical parameters

Serum biochemical parameters, including albumin (ALB), globulin (GLB), total protein (TP), total BAs (TBA), blood urea nitrogen (BUN), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), were measured using an automatic biochemical analyzer (Hitachi 7,600, Hitachi High Technologies Corp., Tokyo, Japan) was used to determine serum. Hepatic TG, TC, HDL-C and LDL-C levels were determined by commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Antioxidant enzyme activity assay

Liver tissues (n = 8) were homogenized in phosphate-buffered saline (PBS) using a homogenizer (XU-YM-24, Xi Niu Co., Ltd., Shanghai, China) and subsequently centrifuged at 1,500 × g for 10 min with a centrifuge (ZD-ABDLXJ-01, Eppendorf Co., Ltd., Hamburg, Germany) to obtain the supernatant samples. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (T-AOC), the content malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), in liver were measured using assay kits (Jiancheng, Bioengineering Institute, Nanjing, China).

Hematoxylin and eosin (H&E) staining and Oil Red O staining

Abdominal adipose and liver tissues were fixed in 4 % paraformaldehyde and embedded in paraffin. Tissues were sectioned into 5-μm slices with hematoxylin and eosin (H&E) for histopathological analysis. Sections were dehydrated in alcohol, cleared with xylene and mounted for visualization using a light microscope (Olympus, Tokyo, Japan) through 200 x magnification. Then, digital images were obtained and quantitative analysis of H&E staining sections was performed using Image-Pro Plus 6.0 software. At least six slides per sample were evaluated by a blinded pathologist.

Liver samples were embedded and fixed immediately in optimal cutting temperature compound (OCT), sectioned, and subjected to Oil-Red O staining. Subsequently, liver sections were observed under 200 x magnification and digital images were captured. The area of lipid droplets was then analyzed using the ImageJ software according to Mehlem et al. (2013).

Quantitative real-time PCR

Total RNA was extracted from the liver using a total RNA extraction kit (TransGen Biotech Co.Ltd, Beijing, China) following the manufacturer's instructions. The quality and concentration of RNA were determined via agarose gel electrophoresis and spectrophotometry. The cDNA was synthesized using a reverse transcription kit (Takara, Dalian, China). Gene expression was quantified using SYBR Green qPCR Master mix (Takara, Dalian, China) with the 2^-ΔΔCt method. The β-actin was used as a housekeeping gene to normalize target gene expression. Primer sequences for the target genes are listed in Table S1.

Statistical analyses

Data were analyzed by ANOVA in a 2 × 2 factorial design using the GLM procedures of SPSS 23.0 software. The main effects of BA and LP, as well as their interactions, were evaluated. If significant differences (P < 0.05) were detected in the interaction of the main effects, Tukey's multiple range test was used to compare the means. The results were presented as means and their pooled standard errors (SEM). Differences with P < 0.05 were considered statistically significant.

Results

Laying performance

The effect of dietary protein level and BAs supplementation on laying performance are summarized in Table 2. Both LP diets and BAs treatments significantly decreased egg weight (P < 0.001). A significant interaction (P = 0.002) between BAs and dietary protein level was observed for daily egg production, with hens receiving normal protein diets supplemented with BAs achieving the highest egg production compared to other groups. An interaction (P = 0.011) was noted for feed intake, with the normal protein group supplemented with BAs showing the highest feed intake.

Table 2.

Production Performance¹.

Item			Egg production, %	Egg weight, g	ADFI, g	FCR
			62 to 75week	62 to 75week	62 to 75week	62 to 75week
	BA	LP
CON	-	-	86.73b	62.71a	114.37b	2.12
CON+BA	+	-	89.64a	62.32b	118.10a	2.12
LP	-	+	86.11b	62.31b	113.88b	2.14
LP+BA	+	+	85.43b	62.07c	114.34b	2.18
SEM			0.002	0.042	0.331	0.010
BA factor
-BA			86.42b	62.51a	114.13b	2.13
+BA			87.54a	62.19b	116.22a	2.15
LP factor
CON			88.18a	62.52a	116.24a	2.12
LP			85.77b	60.19b	114.11b	2.16
P-value
BA			0.028	<0.001	0.001	0.104
LP			<0.001	<0.001	0.001	0.112
Interaction			0.002	0.132	0.011	0.784

Abbreviations: ADFI, average daily feed intake; BA, bile acids; FCR, feed conversion ratio; SEM, standard errors; CON, basal diet; CON+BA, basal diet + 120 mg/kg (62-69 weeks) and 200 mg/kg (70-75 weeks) BA; LP, low-protein diet; LP+BA, LP diet +120 mg/kg (62-69 weeks) and 200 mg/kg (70-75 weeks) BA.

a,b Means with no common superscript within a column differ significantly (P < 0.05).

¹Each value represents the mean value of 8 replicates per treatment (n = 8).

Egg quality

The effects of the experimental diets on egg quality are presented in Table 3. At the end of Week 14, all egg quality indices measured, including egg shape index, eggshell strength, eggshell thickness, and proportions of albumen and yolk, were not remarkably affected by BAs supplementation or protein levels. However, BAs supplementation led to a reduction in egg weight (P = 0.015), and LP diets showed a tendency (P = 0.055) to decrease egg weight.

Table 3.

Egg Quality¹.

Item			Egg weight, g	Egg shape index	Eggshell Strength, N	Eggshell thickness, mm	Percentage of eggshell, %	Percentage of yolk, %	Yolk index	Albumen height, mm	Haugh units
	BA	LP
CON	-	-	62.97a	1.29	39.19	0.32	13.82	26.55	0.45	5.72	73.06
CON+BA	+	-	61.47ab	1.31	37.06	0.32	13.46	27.64	0.44	5.89	74.35
LP	-	+	61.78ab	1.29	36.49	0.32	13.54	27.70	0.44	5.82	73.92
LP+BA	+	+	60.30b	1.30	37.68	0.32	13.19	27.03	0.43	5.46	73.92
SEM			0.309	0.003	0.424	0.001	0.098	0.195	0.002	0.090	0.653
BA factor
-BA			62.38a	1.29	37.78	0.32	13.68	27.11	0.44	5.77	73.49
+BA			60.88b	1.30	37.38	0.32	13.33	27.34	0.43	5.67	72.54
LP factor
CON			62.22	1.30	38.15	0.32	13.64	27.11	0.44	5.80	73.70
LP			61.04	1.30	37.07	0.32	13.37	27.35	0.43	5.64	72.33
P-value
BA			0.015	0.118	0.898	0.132	0.071	0.513	0.094	0.596	0.468
LP			0.055	0.721	0.224	0.080	0.162	0.460	0.160	0.369	0.291
Interaction			0.986	0.505	0.966	0.442	0.972	0.115	0.555	0.144	0.087

Abbreviations: BA, bile acids; SEM, standard errors; CON, basal diet; CON+BA, basal diet + 120mg/kg (62-69 weeks) and 200mg/kg (70-75 weeks) BA; LP, low-protein diet; LP+BA, LP diet +120mg/kg (62-69 weeks) and 200 mg/kg (70-75 weeks) BA.

a,b Means with no common superscript within a column differ significantly (P < 0.05).

¹Each value represents the mean value of 8 replicates per treatment (6 eggs per replicate).

BAs homeostasis

Data on BAs synthesis, FXR target gene expression, liver function and H&E staining are displayed in Fig 1. The supplementation of BAs had no significantly effect on serum levels of liver enzymes (ALT, AST, ALP) (Fig 1A). Liver histology appeared normal, with no notable pathological change (Fig 1B). Neither serum nor liver TBA levels were affected by treatments (Fig 1C). The supplementation of BAs significantly increased hepatic FXR (P = 0.005) and SHP-1 (P = 0.002) mRNA expression, while decreasing expression of CYP7A1 (P = 0.018; Fig 1D). In the ileum, the supplementation of BAs increased FXR (P = 0.008) mRNA expression and decreased the expression of ASBT (P = 0.042; Fig 1E), with no significant interaction between two factors.

Fig. 1.

BAs Homeostasis.

Different superscripts in bars indicate a significance difference (P < 0.05). (A) Serum liver function indexes of laying hens (n = 6). (B) H&E staining of liver tissue sections (n = 6). (C) Serum and liver total bile acids concentration (n = 6). (D) mRNA levels of bile acids synthesis were detected by qRT-PCR in the liver (n = 8). (E) mRNA levels of bile acids transportation were detected by qRT-PCR in the ileum (n = 8). BAs = bile acids; LP = low protein; ALT= alanine aminotransferase; AST = aspartate aminotransferase; ALP = alkaline phosphatase; TBA = total bile acid; FXR = nuclear receptor subfamily 1 group H member 4; SHP-1 = nuclear receptor subfamily 0 group B member 2; LRH-1 = nuclear receptor subfamily 5 group A member 2; CYP7A1 = cytochrome P450 family 7 subfamily A member 1; CYP8B1 = cytochrome P450 family 8 subfamily B member 1;CYP7B1 = cytochrome P450 family 7 subfamily B member 1; CYP27A1 = cytochrome P450 family 27 subfamily A member 1; FGF19 = fibroblast growth factor 19; ASBT = solute carrier family 10 member 2; OSTβ = solute carrier family 51 beta subunit.

Serum biochemical parameters

The serum biochemical parameters are shown in Fig 2. Neither dietary protein level nor BAs supplementation affect serum concentrations of TG, TC, HDL-C and LDL-C (Fig 2A). The supplementation of BAs increased ALB concentration (P = 0.027; Fig 2B), and LP diets decreased serum UA content (P = 0.014; Fig 2C). A significant interaction (P = 0.005) was observed for BUN levels, with the LP diet supplemented with BAs resulting in the lowest BUN concentration. Low-protein diets also increased serum LEP (P = 0.016) and GC (P = 0.004), while BAs further elevated GC levels (P = 0.004; Fig 2D).

Fig. 2.

Serum Biochemical Parameters.

Each value represents the mean value of 6 replicates per treatment (n = 6). Different superscripts in bars indicate a significance difference (P < 0.05). (A) Serum lipid metabolism indexes of laying hens. (B) Plasma protein concentration in serum. (C) Serum urea nitrogen and uric acid content. (D) Serum hormones about lipid metabolism. BA = bile acids; LP = low protein; TC = total cholesterol; TG = triglyceride; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; ALB = albumin; GLB = globulin; TP = total protein; BUN = blood Urea Nitrogen; UA = blood uric acid; TH = thyroid hormone; LEP = leptin; GC = glucagon.

Lipid metabolism

The liver lipid metabolism is illustrated in Fig 3. The supplementation of BAs significantly decreased hepatic TG (P = 0.001), TC (P = 0.019) and lipid droplets content (P = 0.034; Fig 3A-C). Abdominal adipocyte size was reduced by BAs supplementation (P = 0.001; Fig 3D-E). Bile acids treatments decreased the expression of the genes related to lipid synthesis ACC (P = 0.025) and increased the expression of lipid oxidation gene PPARα (P = 0.010) in liver (Fig 3F-G). Low-protein diets upregulated the expression of lipid oxidation ACOX1(P = 0.021) and lipid transportation VTG2 (P = 0.011; Fig 3G-H).

Fig. 3.

Lipid Metabolism

Different superscripts in bars indicate a significance difference (P < 0.05). (A) Liver lipid metabolism indexes of laying hens (n = 6). (B) Oil Red O staining of liver tissue (n = 6). (C) Liver lipid droplet content (n = 6). (D) H&E staining of abdominal adipose tissues. (E) Abdominal adipocytes size (n = 6). (F-H) mRNA levels of lipid synthesis, lipid oxidation and lipid transportation were detected by qRT-PCR in the liver (n = 8).

BA = bile acids; LP = low protein; FASN = fatty acid synthase; ACC = acetyl-CoA carboxylase alpha; LXR = nuclear receptor subfamily 1 group H member 3; SREBP1c = sterol regulatory element binding transcription factor 1; PPARα = peroxisome proliferator activated receptor alpha; PPARγ = peroxisome proliferator-activated receptor gamma; ACOX1 = acyl-CoA oxidase 1; CPT1A = carnitine palmitoyltransferase 1A; VTG2 = vitellogenin 2; MTTP = microsomal triglyceride transfer protein; FABP4 = fatty acid binding protein 4; APOB = apolipoprotein B; VLDLR = very low-density lipoprotein receptor; APOV1 = apovitellenin 1.

Hepatic antioxidant enzyme activities

Hepatic antioxidant enzyme activities are shown in Fig 4. Low-protein diets decreased liver MDA content (P = 0.042), and supplementation of BAs further lowered MDA levels (P = 0.023). A significant interaction between BAs and dietary protein level was found for liver H₂O₂ content (P = 0.003), with LP diet plus BAs resulting in the lowest H₂O₂ content compared to the other groups.

Fig. 4.

Hepatic Antioxidant Enzyme Activity

Each value represents the mean value of 8 replicates per treatment (n = 8). Different superscripts in bars indicate a significance difference (P < 0.05). SOD = Superoxide Dismutase; GSH-Px = Glutathione Peroxidase; CAT = Catalase; T-AOC = Total antioxidant capacity; MDA = malondialdehyde; H₂O₂ = Hydrogen Peroxide.

Protein metabolism

The protein metabolism data are shown in Fig 5. A significant interaction between BAs and dietary protein level on trypsin activity (P = 0.049) was observed, with the normal protein diet receiving BAs supplementation exhibiting greater trypsin activity (Fig 5A). Chymotrypsin activity was unaffected by diets (Fig 5B). The supplementation of BAs significantly increased jejunal SLC7A5 expression (P = 0.030; Fig 5C), and LP diets elevated jejunal SLC6A19 expression (P = 0.005; Fig 5C). The genes related to protein synthesis (mTOR, ETF4EBP1 and RPS6KB1) were unaffected by diets (P > 0.05; Fig 5D). Low-protein diets decreased GS expression (P = 0.030), a gene related to protein degradation (Fig 5E). Interactions between BAs and protein levels were found for hepatic GA, GDH, and CPS1 expression, with the LP diet supplemented with BAs showing the lowest expression of these genes (P < 0.05; Fig 5E).

Fig. 5.

Protein Metabolism

Each value represents the mean value of 8 replicates per treatment (n = 8). Different superscripts in bars indicate a significance difference (P < 0.05). (A-B) Trypsin and Chymotrypsin activity in Jejunal digesta. (C) mRNA levels of amino acid and peptide transporters were detected by qRT-PCR in jejunal mucosa. (D-E) mRNA levels of protein synthesis and degradation were detected by qRT-PCR in liver.

SLC6A19 = solute carrier family 6 member 19; SLC7A7 = solute carrier family 7 member 7; SLC15A1 = solute carrier family 15 member 1; SLC7A5 = solute carrier family 7 member 5; SLC7A1 = solute carrier family 7 member 1; SLC1A1 = solute carrier family 1 member 1. mTOR = mechanistic target of rapamycin; EIF4EBP1 = eukaryotic translation initiation factor 4E bindingprotein 1; RPS6KB1 = ribosomal protein S6 kinase B1; GS = glutamate-ammonia ligase; GA = glutaminase 2; GDH = glutamate dehydrogenase 2; CPS1 = carbamoyl-phosphate synthase 1; SIRT4 = sirtuin 4; SIRT5 = sirtuin 5.

Discussion

This study investigated the effects of dietary BAs supplementation on production performance of laying hens under different crude protein levels. The results contribute to the growing body of research on BAs in poultry nutrition, particularly in relation to production efficiency. Consistent with previous research (Alagawany et al., 2020; Heo et al., 2023), the present study observed that LP diets were associated with a reduction in egg weight. Additionally, it is well known that dietary fat content is a key determinant of egg weight (Safaa et al., 2008). In this study, BAs supplementation also reduced egg weight, potentially due to the enhancement of lipid metabolism and fat absorption (Ge et al., 2019). This study confirmed that dietary treatments did not significantly affect the FCR, aligning with the findings of Sun et al. (2023). However, feed intake was notably increased in hens fed a normal-protein diet supplemented with BAs, suggesting that BAs improved lipid absorption and necessitated higher feed intake to maintain optimal production (Pérez-Bonilla et al., 2012; Barzegar et al., 2020). Interestingly, while BAs supplementation improved egg production rates in birds fed normal-protein diets, the effect was diminished under LP diets, likely due to the critical role of dietary protein in supporting maximum egg production (Hou et al., 2015). These findings imply that BAs may help sustain production, but their effectiveness is contingent upon adequate protein intake. Egg quality is one of the important indexes to evaluate the economic benefit of livestock and poultry products, which is affected by many factors (e.g., the diet and age of the hens) (Kowalska et al., 2021). In this study, both BAs and LP diets decreased egg weight without affecting other parameters as shell quality, consistent with earlier findings (Zhou et al., 2021; Yang et al., 2022a). Reduced egg weight in late-phase laying hens is advantageous, as larger eggs can reduce uniformity, cause dystocia, and increase feed consumption (Bain et al., 2016; Lu et al., 2021). Therefore, lowering egg weight may extend the laying period and improve overall efficiency.

Bile acids serve as effective emulsifiers in livestock and poultry production due to their amphipathic nature, which encompasses both hydrophilic and hydrophobic characteristics. These amphipathic molecules facilitate the emulsification, digestion, and intestinal absorption of dietary lipids and fat-soluble vitamins (Wahlström et al., 2016). However, the hydrophobic properties of BAs can also lead to cytotoxic effects, potentially resulting in gut dysbiosis and mucosal inflammation if their concentrations exceed safe thresholds (Allen et al., 2011; Li et al., 2017; Zhou et al., 2020). Consequently, it is imperative to administer BAs at appropriate levels in livestock and poultry production to mitigate these adverse effects. In this study, BAs supplementation did not result in any hepatic or systemic toxic effects, as evidenced by stable serum AST, ALT, and ALP levels. These findings are consistent with previous reports demonstrating the safety of BAs even at higher-than-recommended doses in poultry (Yang et al., 2022a, 2022b). Furthermore, BAs, as signaling molecules, can activate hepatic and intestinal FXR, which regulates BAs homeostasis through the enterohepatic circulation (Rajani and Jia, 2018; Tang et al., 2019). When BAs concentrations increase, FXR induces the binding of small heterodimer partner (SHP) to BAs response elements in hepatocytes, thereby inhibiting the transcription of the CYP7A1 gene (Kliewer, 2000; Lu et al., 2000; Li and Chiang, 2020). This study also confirmed that exogenous BAs activate hepatic FXR, inhibiting endogenous BAs synthesis and maintain BAs homeostasis. Additionally, previous studies have reported that the activation of intestinal FXR could inhibit apical sodium-dependent bile acid transporter (ASBT), thereby reducing BAs uptake and promoting their excretion (Lee et al., 2006). Similarly, our study observed that exogenous BAs can activate intestinal FXR, leading to the suppression of ASBT. Furthermore, the role of intestinal fibroblast growth factor 15/19 (FGF15/19) in mediating the feedback regulation of hepatic BAs synthesis has been well-documented (Zhang et al., 2013). However, in this study, the expression of intestinal FGF19 remained unchanged, implying that BAs synthesis may primarily depend on FXR-SHP regulation in chickens (Tarling et al., 2017). This finding highlights the need for further investigations to delineate the specific mechanisms involved in this regulatory process. Based on these findings, we hypothesize that BAs exert multiple effects through the activation of both hepatic and intestinal FXR, which not only regulate BAs homeostasis but also influence key metabolic pathways such as lipid and protein metabolism. Specifically, hepatic FXR activation inhibits endogenous BAs synthesis, which may contribute to improved lipid metabolism by reducing triglyceride and cholesterol levels, while intestinal FXR activation regulates BAs absorption, potentially enhancing nutrient utilization and reducing oxidative stress. These combined effects help explain the improved production performance observed in laying hens.

Lipid metabolism dysfunction is closely linked to oxidative stress, a major factor contributing to reduced egg production in hens (Yan et al., 2025). The reduction in abdominal fat cell size, hepatic triglyceride and cholesterol levels observed in this study suggests that BAs improve lipid metabolism, which is critical for maintaining metabolic health in late-laying hens. Additionally, since lipid dysregulation can exacerbate oxidative damage. In this study, by downregulating the expression of lipid synthesis genes like ACC and upregulating lipid oxidation gene PPARα, BAs help regulate lipid accumulation in the liver (Pineda Torra et al., 2003; Piekarski et al., 2016), which in turn alleviates hepatic oxidative stress by reducing hepatic MDA content, thus maintaining liver function and supporting sustained egg production. Moreover, as is well-known, lipid metabolism is regulated by various hormones, including insulin, leptin, and thyroid hormones, which inhibit lipid deposition and promote lipid breakdown (Li et al., 2019). In this study, LP diets increased serum insulin and thyroid hormones, which may be responsible for the upregulation of lipid oxidation gene ACOX1. Taken together, the beneficial effects of BAs on lipid metabolism and oxidative stress are interconnected and contribute to the overall enhancement of production performance in laying hens. By improving metabolic efficiency and reducing stress, BAs help optimize both egg production and egg quality, providing a valuable tool for improving poultry production under various dietary conditions.

The liver serves as both the primary site of lipid metabolism and the central hub for protein metabolism. The efficiency of protein utilization is influenced by dietary protein levels as well as the physiological functions of the liver (Trefts et al., 2017). Protein in feed is primarily digested and absorbed in the small intestine after initial digestion in the front end of the digestive tract. Previous studies have reported that dietary supplementation of BAs can increase intestinal pancreatic enzyme levels (Lammasak et al., 2019). In this study, there is a trend of BAs increasing pancreatic enzyme activity, maybe due to bile salts and bile enhancing autolytic activation of pancreatic protease (Lammasak et al., 2019). Low-protein diets improved the expression of amino acid transporters, consistent with earlier findings in weaned piglets (Morales et al., 2015). Additionally, BAs promoted intestinal amino acid transporter SLC7A5 expression, which may be linked to PPARα activation, enhancing protein digestion and absorption (Okamura et al., 2014). Meanwhile, BAs have been proved to regulate amino acid metabolism pathways and improve protein utilization efficiency, which may be similarly responsible for the BAs depressing the amino acid catabolism in LP diet and this was also supported by the decreased serum uric acid content in this study (Zhai et al., 2020; Yiyao et al., 2022). These findings imply that BAs supplementation may enhance protein utilization by not only promoting protein digestion and absorption but also by improving the overall metabolic environment in the liver. The regulation of lipid metabolism by BAs reduces fat accumulation and oxidative stress, which creates a more favorable physiological state for protein synthesis and utilization. This dual regulation of both lipid and protein metabolism underscores the potential of BAs to optimize overall metabolic efficiency, thus improving egg production and quality.

In conclusion, this study demonstrates that BAs supplementation provides multifaceted benefits to laying hens, particularly by optimizing lipid metabolism, reducing oxidative stress, and enhancing protein utilization. These effects are interconnected, with BAs improving both metabolic efficiency and physiological function, which ultimately supports sustained egg production. Importantly, BAs supplementation helped enhance the efficiency of low-protein diets by reducing fat accumulation, alleviating oxidative damage, and maintaining BAs homeostasis. This integrated metabolic regulation may extend the productive lifespan of laying hens while reducing feed costs without compromising performance. These findings offer a promising strategy for improving poultry production systems. Future research should focus on further elucidating the molecular mechanisms underlying the effects of BAs and how their interactions with dietary nutrients can be optimized to maximize production efficiency in poultry.

Declaration of competing interest

We declare that we have no competing interests.