Effects of Leuconostoc lactis on the antioxidant ability and indole-3-acetaldehyde metabolism via regulating the gut microbiota-liver axis in aged laying hens

doi:10.21203/rs.3.rs-7378456/v1

Lactobacillus has antioxidant properties that may benefit poultry production. However, there is no systematic research on antioxidant of Lactobacillus strain and its effects on regulating nutrient metabolism in aged laying hens. This study investigated the influence of Leuconostoc lactis on production and antioxidant capacity in aged laying hens and explored the key biomarkers associated with tryptophan-skatole metabolism and its effects on the intestinal microbiota-liver axis. Hens supplemented with L. lactis showed a higher laying rate, reduced hepatic MDA levels, and increased T-AOC in comparison with the control group (CG). Indole-3-acetaldehyde (IAld) levels were elevated in both feces and yolk, and skatole decreased in feces by the L. lactis group compared to CG. The total polyunsaturated fatty acids (PUFAs), C18:3n3, and C18:2n6c in yolk were raised in the L. lactis group relative to CG. In the liver, mRNA levels of AhR, CYP2D6, and CPT-1 were markedly upregulated in the L. lactis group relative to CG. The L. lactis-treated group also exhibited higher alpha diversity in fecal samples at 30 days and in ileal samples at 60 days. Further, we conducted the hepatocyte validation experiment and found that MDA levels were significantly reduced, and T-AOC was increased in both the L. lactis and IAld-treated groups compared with the CG. IAld treatment significantly affected p38, and NF-κB, and Nrf2 cytokine expression in hepatocytes. The findings provide a reference for the use of L. lactis in improving production and intestinal nutrition in aged laying hens.

L. lactis

Antioxidant ability

Indole-3-acetaldehyde

Tryptophan-skatole metabolism

Aged laying hens

The egg production (EP) of aged laying hens directly affects the economic profitability of breeding farms. High-intensity EP in laying hens can disrupt the gut microbiota (GM), cause immune imbalance, and impair intestinal function, all of which contribute to the decrease in EP during the late laying period. This period is generally characterized by an egg-laying rate (ELR) of less than 80% after 48 weeks of age ^[1–4].

With advancing age, laying hens experience reduced activity and levels of antioxidant enzymes, alongside diminished free radical-scavenging capacity. This decline leads to the accumulation of excess free radicals and peroxides generated through cellular metabolism ^[5–6]. Consequently, the antioxidant potential of internal organs weakens, upsetting the balance between pro-oxidant and antioxidant processes and ultimately resulting in oxidative stress ^[7]. Thus, boosting antioxidant capacity in aged laying hens is critical for sustaining high production performance.

Nutritional interventions may slow aging by alleviating oxidative stress ^[8–9]. Lactobacillus is classified as a probiotic microorganism that, when administered in sufficient amounts, provides health benefits to the host by regulating GM composition. A growing body of research has shown that Lactobacillus not only help maintain intestinal microbial balance and preserve gastrointestinal barrier integrity, but also exert multiple functions including antioxidant, anti-inflammatory, and antiviral effects ^[10–12]. Lactobacillus has antioxidant properties and is important in maintaining animal intestinal homeostasis. It had been said that Lactobacillus can eliminate reactive oxygen radicals based on its antioxidant enzyme system ^[13], a previous study has proven that Lactobacillus plantarum 4 − 2 could alleviate oxidative stress via Keap1-Nrf2 pathway ^[14], L. reuteri, a species of Lactobacillus, has anti-oxidative stress effects, which could alleviateoxidative stress and inflammatory response in animals ^[15–16]. However, few studies on the ability of Lactobacillus strain to alleviate antioxidant stress and its influences on growth performance and nutrition metabolism in aged laying hens has been reported.

Here, we screened a new probiotic strain, Leuconostoc lactis ZLL028, which was isolated from the cecum of healthy laying hens in China. Our previous data had shown L. lactis ZLL028 had in vitro antioxidant activities. However, its application in the aged laying hens remains relatively limited. Based on these findings, this study evaluated the influence of this strain on EP and antioxidant capacity in aged laying hens. Moreover, it also analyses the GM composition and serum metabolism to identify biomarkers of tryptophan metabolism affected by L. lactis ZLL028 through the GM-liver axis, further validating the regulatory mechanisms of these biomarkers through in vitro cell experiments.

2.1 Bacterial strain

L. lactis ZLL028, originally obtained from the healthy laying hen feces, was deposited at the China General Microbiological Culture Collection Center under preservation number CGMCC 26853). Cultivation was carried out on de Man, Rogosa, and Sharpe (MRS) agar. After harvesting cells by centrifugation, they were resuspended in protective agents. The prepared suspension underwent freeze-drying using an Epsilon 2–60 lyophilizer (Martin Christ GmbH, Germany), resulting in a lyophilized powder with a final concentration of 10⁹ CFU/g.

2.2 Animal experiments

A total of 320 Jingfen 1 laying hens (550 days of age), comparable in body weight and ELR were selected. The hens were randomly assigned to two treatment groups, each containing eight replicates of 20 hens. All hens were housed at 26 ± 2°C with 50 ± 10% relative humidity and a 16-hour light/8-hour dark cycle, with darkness from 22:00 to 06:00. Food and water were unrestricted, with the dietary formulation following the guidelines of the Chinese Feeding Standard of Chickens (NY/T 33-2004) (Table 1). The control group (CG) had a basal diet, whereas the experimental group was given the same diet supplemented with 1% L. lactis ZLL028. A one-week pre-feeding period was followed by a 60-day experimental period. Feeding management was conducted according to conventional standards.

Table 1

Test ration composition and nutrient levels (air-dried basis)
Ingredients, %	Contents
Corn	64.38
Soybean	24.00
Soybean oil	0.12
Limestone	9.06
CaHPO₄	0.89
DL-Met	0.14
Lysine	0.01
NaCl	0.30
Choline chloride	0.10
Premix ¹⁾	1.00
Total	100.00
Nutrient levels ²⁾
ME, MJ/kg	11.15
CP, %	15.63
Ca, %	3.55
TP, %	0.47
Lys, %	0.80
Met, %	0.38
¹⁾ The premix provided the following per kg of diets: VA 4 100 IU, VE 5 IU, VD₃ 500 IU, VK₃ 0.5 mg, VB₁ 0.8 mg, VB₂ 2.2 mg, VB₅ 2.2 mg, VB₆ 3 mg, VB₁₂ 0.4 mg, folic acid 0.25 mg, nicotinic acid 10 mg, biotin 0.1 mg, Fe 50 mg, Cu 6 mg, Zn 50 mg, Mn 30 mg, I2 0.3 mg, Se 0.1 mg.
²⁾ CP, Ca and TP are measured values, M E and AA contents are calculated values.

Table 2

Number of metabolites among different treatments in aged laying hens
Pathway name	R vs. C	regulate
Indoline	pos	down
Indole-3-Carboxaldehyde	pos	down
3-Methylindole	pos	down
Indole	pos	down
Isoindoline	pos	down
indole-3-acetaldehyde	pos	up
Serotonin	pos	up
Quinolinic Acid	neg	up
Indole-3-Acetic Acid	pos	down
3-Indoleacrylic Acid	pos	up
Indole-3-Carboxaldehyde	pos	down
R vs. C, Lactobacillus-treated vs. control pigs.

2.3 Growth performance

During the experimental period, eggs were collected daily at 10 AM and 3 PM. The total number of eggs laid per replicate and their weights were noted to calculate the average egg weight (AEW) and EP rate. The intake of feed was assessed weekly and the average daily feed intake (ADFI) and feed-to-egg ratio (FER) were calculated.

2.4 Collection of serum and fecal samples

Feces (10 samples/group) were collected on days 0 and 30, placed immediately in liquid nitrogen, and kept at -80°C until analysis. After the 60-day experiment, ten hens from each group were euthanized, and ileal and cecal contents were collected. Blood samples were obtained on day 60 via precaval venipuncture before morning feeding and werecentrifuged at 4°C for 10 min at 1, 238 × g, and sera were kept at -80°C until analysis of antioxidant indices.

2.5 Antioxidant indices, and mRNA determination of CYP450 and AhR in the liver

At the end of experiment, the hens (n = 8) were selected and sacrificed by cervical dislocation. The liver samples were collected, and liver antioxidant indices were assessed using kits for determining total antioxidant capacity (T-AOC; cat. no. A10015-2-1) and malondialdehyde (MDA; (cat. no. A003-1-2) from the Nanjing Jiancheng Institute of Biological Engineering, as directed.

Cytochrome P450 (CYP) and AhR levels were determined by qRT-PCR. Total RNA was obtained from liver tissue (n = 15 per group) using a universal mRNA purification kit (TaKaRa, Japan). Reverse transcription was performed with a kit containing gDNA Eraser (TaKaRa), and qRT-PCR was conducted using SYBR Fast qPCR Mix (TaKaRa) on a StepOne Plus real-time PCR system (Life Technologies, USA). Amplification involved denaturation at 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s. Target gene expression was normalized to 18S rRNA (for the liver) using the 2^−△△Ct method.

2.6 Analysis of the yolk long chain fatty acids (LCFAs) composition

The yolk LCFAs constituents were determined using gas chromatography (GC, 6890 series, Agilent Technologies) following a slightly modified version of the Sukhija and Palmquist (1998) method ^[17]. A total of 20 LCFAs standards and an internal standard (C11:0) (Sigma-Aldrich, USA; HPLC grade, > 99%) were included. Serum samples (1 mL) were mixed with 4 mL of ethyl chloride and methanol, after which 1 mL of hexane solution was added. The mixture was maintained for 2 h at 80°C, allowed to cool to room temperature (RT), and added to 5 mL of a KCl solution (100 g/L). A 1 µL aliquot was injected into a capillary column (60 m × 250 µm × 0.25 µm, DB-23, Agilent Technologies), with a stationary phase of cyanopropyl methyl silicone. The column operated at a 1:20 split ratio, with detector and injector temperatures set at 270°C and 260°C, respectively. Nitrogen at a flow rate of 2.0 mL/min served as the carrier gas.

2.7 Measurement of Trp metabolites

The concentrations of indole derivatives and skatole in feces were assessed using high-performance liquid chromatography (HPLC) with minor modifications based on a previously described method. Briefly, cecal and colonic digesta samples (0.2 g) were weighed into centrifuge tubes, and 1.5 mL of acetonitrile was added. The samples were vortexed for 10 min and kept for 30 min at -20°C. After centrifugation at 5000 × g for 10 min at 4°C, the resulting supernatant was filtered with a 0.22-µm membrane before being processed using a Waters Alliance HPLC system (e2695 separation module; Waters Corp., USA). Moreover, ultra-performance liquid chromatography coupled with an Orbitrap spectrometer (UPLC-Orbitrap-MS/MS) was used to quantify indole-3-pyruvic acid (IPYA), indole-3-acetaldehyde (IAld), and indole-3-acetic acid (IAA) in fecal samples. The extraction and identification procedures followed a previously described method.

2.8 Metagenome sequencing analysis of cecum samples

DNA was collected from fecal samples (n = 10) using the E.Z.N.A. Stool DNA Kit (Omega Biotek, USA). Following quality assessment, the DNA was fragmented and used for paired-end library preparation. Sequencing was carried out on aNovaSeq 6000 platform (Illumina, USA) at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

The raw reads underwent quality control using fast-. Gene prediction was conducted with Prodigal, and alignment of the clean reads was performed via SOAPaligner. Functional annotation was achieved by mapping the non-redundant gene catalog to the NCBI NR and KEGG databases using Diamond. This process enabled taxonomic and functional analysis of the sequencing data.

2.9 LC-MS analysis of serum samples

LC-MS analysis was performed on 12 serum samples (n = 6 per group). Serum samples (50 mg) were homogenized in a water-methanol solution (1:4, v/v), ground for 30 min at 60 Hz, kept at -20°C for 30 min, and centrifuged for 15 min at 13,000 rpm (4°C). The resulting supernatant was analyzed using a Thermo UHPLC-Q Exactive HF-X system equipped with an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm; Waters, USA) maintained at 40°C. The mobile phase included 0.1% formic acid in acetonitrile:water (5:95, v/v) (phase A) and 0.1% formic acid in isopropanol:acetonitrile:water (47.5:47.5:5, v/v) (phase B). Samples (2 µL) were injected at 400 µL/min, with quality control samples analyzed every five runs. Data processing was performed on the Majorbio Cloud Platform, with metabolite identification using the HMDB and KEGG databases. PCA and OPLS-DA were applied to assess metabolic differences, and significant metabolites were identified based on VIP ≥ 1 (OPLS-DA) and P < 0.05 (t-tests).

2.10 Analysis of antioxidant ability for L. lactis and IAld in hepatocyte hepatocytes injury model

Another experiment was performed to investigate the effects of bacterial strain on hepatocytes. Hepatocytes were kindly provided by Dr. Liao from the Institute of Animal Sciences, CAAS (Beijing, China) and were routinely grown in DMEM with fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C with 5% CO₂. Hepatocytes were incubated in a fresh medium containing either the basal medium (control), L. lactis (10⁸ CFU/mL), or IAld (20 mg/kg IAld) for 2 h. Following incubation and medium removal, the cells were rinsed with cold PBS and those from the first three wells of the replicates were harvested into cold saline and sonicated at 4°C for 2 min. Cells from the second three-well set were collected in TRIzol, pooled, and frozen for analysis of metallothionein (MT) mRNA. T-AOC and MDA levels were measured using kits, and MT concentrations were determined with an ELISA kit (Mlbio, Shanghai, China).

Total RNA was isolated using TRIzol and DNA was removed with DNase I (TaKaRa). Quality assessment was performed using a 2100 Bioanalyzer (Agilent Technologies) and quantification with an ND-2000 spectrophotometer (Thermo Fisher). High-quality RNA (OD_260/280 = 1.8–2.2, OD_260/230 ≥ 2.0, RIN ≥ 8.0, 28S:18S ≥ 1.0, and > 10 µg total RNA) was used for construction of libraries. For sequencing, 5 µg of total RNA was processed using the TruSeq™ RNA Sample Preparation Kit (Illumina). mRNA was isolated via poly-A selection with oligo (dT) beads, fragmented (100–400 bp), and transcribed to cDNA using a SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) with random hexamer primers (Illumina). The cDNA underwent end-repair, phosphorylation, and adenylation, followed by size selection (200–300 bp) on 2% low-range ultra agarose. PCR amplification (15 cycles) was performed using Phusion DNA polymerase (NEB, USA). Library quantification was performed with a TBS-380 fluorometer (Turner BioSystems, USA) with sequencing on an Illumina HiSeq 4000 platform (2 × 150 bp).

Raw paired-end reads were processed using Sickle and SeqPrep for quality control and trimming. Clean reads were aligned to the reference genome using Bowtie²⁴, allowing up to two mismatches per read without deletions or insertions. Operon identification was conducted by expanding gene regions based on site depth. Novel transcribed regions were defined within overlapping 15-kbp windows (5-kbp overlap), requiring at least two mapped reads per window in the same orientation. DEGs were identified using Cuffdiff, with expression level quantification by the FPKM method. DEGs were defined by log₂ fold change > 2.0 and FDR < 0.05. Functional enrichment of DEGs was evaluated using KEGG pathway and Gene Ontology (GO) analyses via KOBAS and Goatools, with enrichment considered significant at a Bonferroni-corrected P < 0.05.

2.11 Statistical analyses

The general linear model program in SAS software (SAS Institute, USA) was used to analyze growth performance, alpha-diversity indices, antioxidant indices, Trp metabolites. Tukey's univariate test was applied for statistical analysis. evaluation. P < 0.05 represented statistical significance.

3.1 Growth performance

Dietary supplementation with L. lactis ZLL028 significantly increased the ELR of hens by 7.29% compared with the CG. However, the FER, ADFI, and AEW did not differ significantly between the groups (P > 0.05) (Fig. 1).

3.2 Antioxidant indices and mRNA expression of CYP450 and AhR in the liver

Liver MDA levels were significantly lower, while T-AOC was elevated in the L. lactis- treated hens (LG) relative to CG. AhR (P = 0.001) and CYP2D6 (P = 0.001) mRNA levels were higher in the LG, whereas CYP1A2 expression showed no marked difference (P = 0.056). Moreover, mRNA levels of CPT-1 (P = 0.001) was markedly raised in the LG (Fig. 2).

3.3 Yolk LCFAs composition

The LCFAs profiles in the yolk, including polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs), and saturated fatty acids (SFAs), were determined. The total MUFAs (P = 0.013) and C22:1n9 (P = 0.009) were msrkedly decreased, while the total PUFAs (P = 0.018), and C18:3n3 (P = 0.015), C18:2n6c (P = 0.020) were increased in the LG relative to the CG (Fig. 3).

3.4 Trp metabolites analysis

The LG showed markedly lower concentrations of IAA (505.78 μg/kg vs. 651.70 μg/kg, P = 0.005) and skatole (905.80 μg/kg vs. 1514.50 μg/kg, P = 0.002) but higher levels of IAld (330.71 μg/kg vs. 235.69 μg/kg, P = 0.001) compared with the CG (Fig. 4A).

In yolk samples, IAA levels were significantly decreased in the LG in comparison with the CG (17.61 μg/kg vs. 75.20 μg/kg, P = 0.002), whereas IAld concentrations were significantly higher (1099.47 μg/kg vs. 983.93 μg/kg, P = 0.004). Skatole was not detected in either group (Fig. 4B).

3.5 The GM community affected by L. lactis

The GM composition of the feces at day 0 and 30, and GM composition of ileum and cecum at day 60 were analyzed at the end of the experiment. Similarities and differences in GM composition between treatment groups were assessed at the operational taxonomic unit (OTU) level using PLS-DA (Fig. 5A-C). The LG was significantly different from the CG in fecal samples at day 30, as well as in ileum and cecum samples at the end of the experiment. The Venn diagram indicated that 1347 OTUs were common to all samples, while the LG exhibited a higher number of unique OTUs in fecal samples at day 30 (1894 vs. 344), as well as in ileum (36 vs. 7) and cecum (155 vs. 116) samples at the end of the experiment (Fig. 5D). Alpha diversity, measured using the Chao index (Fig. 3B), was higher in fecal samples at day 30 and in ileum samples in the LG compared with the CG (Fig. 5E).

At both day 30 and day 60, Bacillota, Bacteroidota, Actinomycetota, and Pseudomonadota were the abundant phyla in fecal and cecal samples, whereas Pseudomonadota, Actinomycetota, Chlamydiota, and Chordata were predominant in ileum samples. At day 30, the Bacillota abundance was significantly lower in the LG in comparison with the CG (51.64% vs. 59.59%, P = 0.008), whereas Actinomycetota abundance was higher (18.49% vs. 8.29%, P = 0.028) in fecal samples. At the end of the experiment, Chordata abundance in ileum samples was significantly lower in the LG (58.79% vs. 66.63%, P = 0.047), whereas Actinomycetota abundance was higher (5.43% vs. 0.18%, P = 0.033). In cecal samples, Bacillota abundance was significantly higher (30.50% vs. 27.56%, P = 0.008), while Bacteroidota abundance was lower (55.59% vs. 58.45%, P = 0.028) in the LG compared with the CG. At the genus level, in fecal samples at day 30, the abundance of Alistipes (0.59% vs. 0.46%, P = 0.038) and Acinetobacter (0.35% vs. 0.08%, P = 0.018) was higher, whereas Escherichia abundance was lower (1.71% vs. 2.63%, P = 0.013) in the LG in comparison with the CG. At the end of the experiment, Lactobacillus (0.57% vs. 0.31%, P = 0.037) and Alistipes (4.62% vs. 3.92%, P = 0.024) were markedly more abundant in ileum samples in the LG (Fig. 6A-D).

3.6 Metabolomics analysis

Metabolomic analysis was performed using a significance threshold of P < 0.05, an FC >1.2 or <0.8, and a VIP score >1.0. A total of 809 metabolites, including 475 positive ions and 334 negative ions, were detected. The KEGG database was used to identify pathways associated with the detected metabolites. The enriched compounds were primarily classified as phospholipids, amino acids, and other metabolites (Fig. 7A). KEGG pathway enrichment analysis indicated significant involvement in lipid metabolism, amino acid metabolism, and digestive system, among other pathways (Fig. 7B). Further classification using the HMDB revealed the presence of 11 indoles and derivatives, accounting for 1.74% of the total metabolites (Fig. 7C). Identified positive ion metabolites included indoline, indole-3-carboxaldehyde, 3-methylindole, indole, isoindoline, IAld, serotonin, indole-3-acetic acid, 3-indoleacrylic acid, and indole-3-carboxaldehyde, while quinolinic acid was identified as a negative ion metabolite (Supplementary Table 1).

3.7 Hepatocytes proliferation, antioxidant analysis in vitro by L. lactis and IAld

Hepatocyte viability and proliferation were significantly increased following treatment with L. lactis, but no marked variations were found between L. lactis and IAld in proliferation rate (147.1% vs. 149.1%) (Fig. 8A). Furthermore, MDA levels in hepatocytes significantly decreased (P = 0.020), while T-AOC significantly increased (P = 0.020) in the LG and IAld group (IG) in comparison with the CG. However, the T-AOC index was higher in the IG compared with the LG (5.71 U/mL vs. 4.40 U/mL), whereas the MDA level was seen lower in the IG than the LG group (1.01 nmol/mL vs. 0.81 nmol/mL) (Fig. 8B).

3.7.2 RNA-seq analysis

DEGs were defined as having a threshold of P ≤ 0.05 and absolute |logFC| ≥ 1. Gene expression comparisons revealed a total of 30, 108 detected genes across the CG, LG, and IG. Overall, 810 DEGs were detected in the LG vs. CG, including 491 upregulated and 319 downregulated genes. In the IG vs. CG, 281 DEGs were detected, with 66 upregulated and 215 downregulated genes. The IG and LG comparison identified 1, 994 DEGs, of which 628 were upregulated and 1, 366 downregulated.

GO annotation classified DEGs into the categories of cellular component, molecular function, and biological process. The LG vs. CG had 19, 2, and 19 GO terms in these categories, respectively, while the IG vs. CG contained 19, 2, and 20 GO terms. The IG vs. LG had 17, 2, and 10 GO terms. GO clustering analysis revealed that GO terms ‘cellular process’, ‘binding’, and ‘cellular anatomical entity’ were significantly upregulated in both the LG and IG compared with the CG (Fig. 9A-C).

Pathway analysis was performed based on the KEGG pathway database to predict the significantly enriched metabolic. It identified 304, 159, and 335 enriched KEGG pathways in the LG vs. CG, IG vs. CG, and IG vs. LG, respectively. Signal transduction pathways, particularly the signal transduction, and signaling molecules and interaction were markedly enriched in the LG vs. CG, IG vs. CG, and IG vs. LG (Fig. 9D-F).

3.7.3 Cytokine detection of p38/NF-κB axis in hepatocytes by L. lactis and IAld

Then, we analyzed the expression of key genes in the p38, NF-κB and Nrf2 signal pathway, and the results showed that mRNA levels of p38, NF-κB were significantly decreased and Nrf2 was significantly increased in hepatocytes in the LG and IG compared to the CG (P = 0.001). No significant variation was seen between IG vs. LG regarding p38 mRNA expression (Fig. 10 A-C).

Aging is a systemic process characterized by the gradual accumulation of tissue and cellular damage, leading to impaired homeostasis. In laying hens, the late phase of the laying cycle is associated with a significant decline in egg quality and production, abnormal hormone secretion, and immunosuppression, all of which contribute to reduced production performance. Previous research has shown that adding Lactobacillus can improve nutrient digestion and absorption, improving growth performance and feed conversion efficiency ^[18–19]. Lokapirnasari et al. (2019) ^[20] reported that supplementation with probiotics such as Bifidobacterium spp. and L. casei improved growth performance and EP in organic laying hens. In this study, L. lactis ZLL028 supplementation significantly increased the ELR of aging hens by 7.29%. Lactobacillus strains produce organic acids, hydrogen peroxide, lactoferrin, and bacteriocins, which exhibit bacteriostatic or bactericidal properties ^[21]. The organic acids reduce the digestive tract pH, thus increasing the activity of key digestive enzymes ^[22]. Therefore, investigating the metabolic processes of Lactobacillus in aged laying hens is important for understanding its role in the production of beneficial compounds.

Production performance declines with age due to ovarian senescence and hormonal changes associated with oxidative stress (OS) in laying hens ^[23]. Continuous EP further exposes hens to OS, leading to physiological imbalances that negatively affect growth performance. Lactobacillus, as a probiotic, has demonstrated significant antioxidant properties and potential applications in OS mitigation. Studies suggest that Lactobacillus exerts antioxidant effects by producing compounds that regulate OS, altering GM homeostasis and affecting OS-associated pathways in the host ^[24–25]. In our present study, to assess the antioxidant capacity in aged laying hens, T-AOC and MDA levels were analyzed in both serum and liver, the results showed a marked increase in T-AOC and decrease in MDA in the LG in comparison with the CG. T-AOC serves as a comprehensive indicator of antioxidant function ^[26], while MDA is a lipid peroxidation byproduct that reflects the extent of oxidative injury ^[27]. Previous studies have demonstrated that Lactobacillus rhamnosus can counteract deoxynivalenol-induced reductions in hepatic antioxidant enzyme activity ^[28]. This effect may be due to the ability of L. rhamnosus to stimulate antioxidant enzyme systems, particularly under OS conditions ^[29–30]. Moreover, yolk LCFAs composition analysis revealed a decrease in total MUFAs and C22:1n9, along with an increase in total PUFAs, C18:3n3, and C18:2n6c in the LG compared with the CG. These results indicate that the antioxidant influence of Lactobacillus may be linked with the desaturation of PUFAs.

Trp-derived metabolites affect multiple physiological mechanisms and contribute to systemic and intestinal homeostasis in both disease and health ^[31]. Trp serves as a precursor for several indoles, including skatole and indole, the primary end products of bacterial metabolism in the intestine, which are excreted in feces or absorbed into the bloodstream and processed in the liver ^[32]. The pathways involved in indole production and the key enzymes regulating this process have been identified, but the factors affecting skatole formation remain unclear ^[33]. In pigs, skatole deposition is influenced not only by microbial production in the gut but also by its metabolism in vivo ^[34]. The liver is the primary site of skatole degradation, with CYP enzymes playing a key catalytic role ^[34–35]. Specific CYP isoforms, including CYP2E1, CYP2A, and CYP1A, have been implicated in skatole degradation ^[35]. Sun et al. (2024) ^[36] reported that mulberry leaf treatment upregulated CYP1A1 expression, reducing skatole deposition in backfat but not in serum, these findings suggest that CYP1A1 activation affects skatole deposition and that mulberry leaves may enhance the degradation of skatole by inducing hepatic CYP1A1 expression in finishing pigs. AhR is a ligand-activated transcription factor, CYP1As are controlled by the AhR pathway, and tryptophan and its metabolites clould activate AhR and initiate CYP1A transcription. In this study, L. lactis decreased skatole content in feces, and hepatic AhR and CYP2D6 mRNA levels were markedly higher in the LG relative to the CG. The LG also exhibited lower levels of IAA in both yolk and feces, suggesting that L. lactis may regulate skatole metabolism by modulating hepatic CYP450 activity. This study demonstrated that CPT1 mRNA level was markedly raised in the LG relative to the CG. These findings suggest a potential association among AhR, and CYP450 indicating that L. lactis may affect Trp metabolism and its regulatory pathways.

Multiple studies suggest that poultry age is a major factor affecting GM composition ^[37–40]. At the phylum level, Bacteroidetes, Firmicutes, and Proteobacteria exhibited the highest relative abundance in all groups, while Verrucomicrobia was detected in the cecum of cage-free laying hens ^[41]. Commensal microbiota plays a key role in food digestion, nutrient metabolism, and synthesis ^[42]. The production stage significantly affects GM development ^[43], and imbalances in GM composition may contribute to decreased egg quality and production ^[44]. Wang et al. (2023) ^[45] reported that bacterial diversity was generally higher in the early laying period than at peak production, with Fusobacteriota abundance increasing during the peak period and Cyanobacteria being more prevalent in the early phase. Higher GM diversity and richness are linked to superior health outcomes ^[46]. However, longitudinal studies indicate that GM composition can fluctuate daily, affecting host physiology and health, particularly in laying hens with long production cycles. As hens age, changes occur in both the structure and abundance of GM ^[47]. Here, L. lactis enhanced Actinomycetota abundance in feces on day 30 and in the ileum on day 60 compared with the CG. Actinomycetota have gained increasing research attention due to their ability to produce bioactive secondary metabolites relevant to pharmacological and biotechnological applications ^[48]. However, at the end of the experiment, Bacillota abundance increased while Bacteroidota abundance decreased in the cecum of the LG. At the genus level, Alistipes numbers were markedly greater in feces on day 30 and in the ileum on day 60 in the LG. Moreover, Lactobacillus was elevated significantly in ileum samples on completion of the experiment in the LG relative to the CG.

The GM plays a key role in Trp metabolism, affecting amino acid metabolic pathways. Around 5% of dietary Trp bypasses absorption in the small intestine and reaches the large intestine, where intestinal bacteria contribute to its metabolism. Both Gram-positive and Gram-negative bacteria, including Bacteroides spp., Clostridium spp., and Escherichia coli, produce tryptophanase, an enzyme that converts Trp into indole. Moreover, bacteria such as Bifidobacterium and Lactobacillus possess enzymes that facilitate the synthesis of indole acid derivatives, including indole-lactic acid (ILA), which is further transformed into IPA by Peptostreptococcus and Clostridium spp. Various bacterial species also contribute to the enzymatic conversion of Trp into IAA, and the concentration of indole in the mouse gastrointestinal tract has been linked to indole-producing Bacteroides ^[49]. Furthermore, Ruminococcus plays a key role in modulating Trp metabolism in the neonatal porcine colon. Skatole concentrations in animals are affected by age, sex, breed, and diet, primarily due to variations in microbial communities involved in the production of skatole ^[50–51]. Trp is either derived from the upper intestinal mucosa or obtained from the diet and can be degraded into indole or converted into IAA, which GM then metabolizes into skatole. Altering dietary composition may serve as a strategy to minimize odor emissions at the source ^[52–53]. Moreover, acidic conditions promote indole production, while alkaline environments favor IAA synthesis ^[54–55]. Lactobacillus can produce organic acids to reduce pH value of intestinal tract, and Lactobacillus strains can improve the microbiota composition, however, our current understanding of skatole production affected by Lactobacillus remains limited, but reducing the production of fecal skatole at its source through regulating microbiota composition using Lactobacillus strains may be a viable strategy.

Analysis of indole derivatives in feces, yolk, and serum metabolomics revealed that IAld levels were increased in both yolk and feces compared with the CG, with IAld also showing enrichment in serum. These results suggest the potential of IAld as an L. lactis biomarker in affecting Trp metabolism and reducing skatole production. To further investigate this mechanism, cell proliferation, antioxidant, and transcriptomic analyses were conducted. IAld has been shown to induce IFN-1 production and prevent local inflammation via AhR activation. Mackenzie et al. (2023) ⁵⁶ reported that Lactobacillus uses Trp to generate IAld, which activates AhR receptors to mediate antitumor immunity. Similarly, Hou et al. (2018) ^[57] reported that IAld, produced through Lactobacillus-mediated Trp metabolism, activates the AhR-IL-22-Stat3 signaling pathway to promote cell proliferation and intestinal mucosal repair.

In the present study, MDA levels in hepatocytes significantly decreased, whereas T-AOC increased following IAld treatment. Kurata et al. (2019) ^[58] reported that skatole induces apoptosis of intestinal epithelial cells via the p38-MAPK pathway but does not affect the ERK-MAPK and JNK-MAPK pathways. Zhuang et al. (2022) ^[59] also showed that IAld could alleviates chondrocytes inflammation through the AhR-NF-κB signalling pathway. Consistent with these findings, it was consistent with the above results, our present study showed that L. lactis and IAld affected the signal pathway, especially for the MAPK, NF-kappa B and Nrf2 signaling pathway, these results suggest that L. lactis may regulate Trp metabolism by modulating metabolic signaling pathways.

In conclusion, dietary supplementation with L. lactis enhanced antioxidant ability in aged laying hens, improved laying performance, and modulated GM. Moreover, L. lactis affected Trp metabolism and reduced skatole production, with IAld identified as a key biomarker regulating the tryptophan-skatole pathway. Although some unanswered questions need better and more intensive investigations, this study presents a foundation for developing specialized probiotics that improve animal health and performance while potentially reducing environmental pollution in poultry production.

CRediT authorship contribution statement

ZCX: carried out the hens experiment and writing-original draft; LH and CMX: formal analysis and data curation; ZZF: carried out the hens experiment; ZDY: rewriting-original and data curation. All the authors read and approved the final manuscript.

Ethics statement

This study was performed according to the relevant animal welfare guidelines (IHVM11-2302-8) of the Animal Care and Use Committee of the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, China.

Funding

This study was funded by the Innovation Capacity Building Project of the Beijing Academy of Agriculture and Forestry Sciences (KJCX20251102), the Public institution project of Institute of Animal Husbandry and Veterinary Medicine (XMS202516), Innovation Capacity Building Project of the Beijing Academy of Agriculture and Forestry Sciences (KJCX20240507).

Declaration of Competing Interest

The authors declare that there is no conflict of interest.

Availability of data and materials

The sequences of metagenomes were submitted to GenBank under BioProject PRJNA1252277 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1252277). The datasets of Metabolomic sequence presented during the current study are available under https://www.ebi.ac.uk/metabolights/MTBLS12964.

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No competing interests reported.

SupplementedTable1.docx

Effects of Leuconostoc lactis on the antioxidant ability and indole-3-acetaldehyde metabolism via regulating the gut microbiota-liver axis in aged laying hens

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Bacterial strain

2.2 Animal experiments

2.3 Growth performance

2.4 Collection of serum and fecal samples

2.5 Antioxidant indices, and mRNA determination of CYP450 and AhR in the liver

2.6 Analysis of the yolk long chain fatty acids (LCFAs) composition

2.7 Measurement of Trp metabolites

2.8 Metagenome sequencing analysis of cecum samples

2.9 LC-MS analysis of serum samples

2.10 Analysis of antioxidant ability for L. lactis and IAld in hepatocyte hepatocytes injury model

2.11 Statistical analyses

3. Results

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1