Due to improved production capabilities among domestic fowl, the average lifespan of commercial laying hens has increased from 72 to 80 weeks (Liu et al., 2022). However, between 80 and 100 weeks of age, hens experience a decline in both egg production and quality. As a result, they are gradually removed from the flock and referred to as ‘spent hens’ and are classified as aged laying hens (Fan and Wu, 2022). The primary challenge faced by these aged laying hens is the decline in intestinal function (Gan et al., 2020; Abdollahi et al., 2021; Feng et al., 2021).

In recent years, there has been a significant surge of interest in extending the production period of hens. Intestinal health is closely associated with the overall well-being, defense mechanisms, and nutritional status of the host as they age (An et al., 2018). Maintaining gastrointestinal tract health is critically important in contemporary poultry farming practices aimed at enhancing production, especially in laying hens (Fu et al., 2022). The gastrointestinal is the primary pathway for nutrient absorption and the initiation of immune responses. However, this pathway is vulnerable to disruption from a variety of stressors, including environmental fluctuations, pathogens, and changes in feed. Furthermore, the gastrointestinal tract is essential for immune response, microbial defense, and hormonal regulation (Fu et al., 2022). Animal guts are host to trillions of microbes that perform vital functions, including the harvesting, storage, and expenditure of energy derived from dietary sources for the host, effectively acting as an additional organ (Becattini et al., 2016). Disruption of the balance among these microorganisms can adversely affect the health and productivity of hens. Consequently, significant efforts are being made to enhance the production performance of aged laying hens through nutritional interventions. These interventions include supplementing exogenous enzymes, prebiotics, probiotics, and minerals (Kim et al., 2012; Abdelqader et al., 2013b; Ghasemian and Jahanian, 2015). In recent years, interest in probiotics has surged, supported by increasing evidence of their efficacy as a promising approach to enhancing digestive health (Lee et al., 2018; Ji et al., 2019; Cai et al., 2020). Among these probiotics, PA has garnered considerable attention due to its potent physiological activity and resilience. PA represents a category of lactic acid bacteria notable for certain key traits, including its resistance to acidic environments, lack of porphyrin synthesis, and a strictly fermentative (homofermentative) metabolism that is facultatively anaerobic, primarily producing lactic acid as the main metabolic byproduct (Li et al., 2021). Furthermore, PA is recognized as a consistent producer of bacteriocins (Parada et al., 2007). Bacteriocins can colonize and persist in the human gut while having a beneficial effect on the host, including modulation of the composition of the gut microbiota, improvement of the host immune response, enhancement of the gut barrier function, and the modulation of immune system activities (Qiao et al., 2021). Collectively, these factors contribute to improved gut health, which subsequently enhances the productivity and quality of eggs produced by laying hens (Quarantelli et al., 2008; Mikulski et al., 2012; Mikulski et al., 2020; Shanmugam et al., 2024). Previous studies have indicated that PA can significantly improve both the production performance and egg quality of laying hens (Mikulski et al., 2012; Mikulski et al., 2020). However, the potential effects of PA on aged laying hens remain unclear. Therefore, the objective of this study is to elucidate the specific impacts of PA on the gut health of aged laying hens, ultimately aiming to improve laying performance and egg quality.

All experimental procedures were carried out in compliance with the guidelines established by the Animal Welfare Committee. Furthermore, the experimental protocol received approval from the Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University (No. 20181105).

The dietary probiotics have been shown to improve both egg production and egg quality by reducing pathogenic bacterial loads and preventing damage to the gut structure (Zhang et al., 2012; Forte et al., 2016; Upadhaya et al., 2016). The results of this study suggest that incorporating PA (CNCM I-4622) into the diet of laying hens can improve egg production rates and FCR, particularly at a dosage of 150 mg/kg. Additionally, our study found that while the inclusion of 100 mg/kg and 200 mg/kg of PA increased average egg weight, it also led to a decrease in the egg production rate. A recent study corroborated that the addition of 100 mg/kg of PA significantly improved the egg production rate of laying hens (Mikulski et al., 2020). In prior research, it was observed that a diet supplemented with 100 mg/kg of PA resulted in notable increases in egg mass and average egg weight, alongside a reduction in the feed conversion ratio (Mikulski et al., 2012). Previous reports have indicated that probiotics can improve nutrient absorption by enhancing the intestinal environment of animals, thereby contributing to increased egg weight (Nahashon et al., 1994). The observed increase in egg weight was primarily attributed to a higher proportion of eggshell and greater eggshell thickness, which subsequently reduced the rate of damaged eggs (Mikulski et al., 2012; Mikulski et al., 2020). Furthermore, Mikulski et al. (2020) noted that improvements in egg weight and shell quality may be linked to enhanced calcium absorption and retention associated with probiotic supplementation.

In our study, 150 mg/kg PA significantly decreased the color of egg yolk. Previous research has indicated that feed composition and conversion rates can influence egg yolk color, with carotenoid content in eggs also playing a crucial role (Zhao et al., 2019). Consequently, the impact of probiotics on egg yolk color necessitates further investigation. Our findings demonstrate that PA alters the levels of myristoleic acid, heptadecanoic acid, SFA, and n−3 PUFA in egg yolk. An earlier study revealed that the addition of the PA strain MA18/5 M (consistent with CNCM I-4622 composition) at 100 mg/kg led to a significant decrease in palmitoleic acid content in egg yolk, accompanied by notable increases in margaric, margaroleic, and linoleic acids, as well as a substantial rise in overall PUFA and n−6 PUFA content (Mikulski et al., 2012). Saleh et al. (2016) reported that the incorporation of lactic acid bacteria in feed significantly reduced palmitoleic acid levels in egg yolks. Conversely, this approach markedly elevated the concentrations of oleic acid, linoleic acid, palmitoleic acid, gamma-linolenic acid, arachidonic acid, and nervonic acid, resulting in an overall increase in polyunsaturated fatty acids and a decrease in saturated fatty acids. Research suggests that the mechanism by which probiotics elevate unsaturated fatty acids in the intestine may be linked to amino acids and the activity of lactic acid bacteria (Saleh et al., 2013). Similar studies by Haddadin et al. (1996) and Ramasamy et al. (2008) indicated that Lactobacillus had a minimal effect on the fatty acid composition of egg yolks, which may be attributed to the specific type of probiotic used and the dosage administered (Abdelqader et al., 2013a; Mikulski et al., 2020).

Serum biochemical indicators serve as reflections of alterations in tissue cell permeability and metabolic function within the body, making them sensitive markers of animal health status (Jia et al., 2019). This study found that the addition of 100 mg/kg significantly elevated serum TP, TG, and HDL-C levels. These findings align with previous research. Specifically, studies have demonstrated that probiotics enhance serum/plasma immunoglobulin levels and induce changes in immune cell populations and their phagocytic capabilities (Peralta-Sánchez et al., 2019). HDL-C serves as an indicator of excess cholesterol being excreted into the intestines (Wang W. W. et al., 2020). Consequently, this study indicates that incorporating 100 mg/kg of PA into the diet not only increased serum TG levels but also facilitated the excretion of surplus cholesterol into the intestine, thereby regulating blood lipid metabolism. Additionally, endotoxins, commonly referred to as lipopolysaccharides, are components of the cell wall of Gram-negative bacteria (Wang et al., 2021). D-LA, a byproduct of bacterial metabolism, has been proposed as a potentially valuable marker for assessing the extent of intestinal damage and gut barrier dysfunction (Murray et al., 1993). DAO, an enzyme present in elevated concentrations in the intestinal mucosa of humans and other mammals, serves as a marker for mucosal maturation and integrity (Lei et al., 2013). Our findings indicate that the addition of PA significantly reduced serum concentrations of DAO, consistent with the results reported by Lei et al. (2013). This suggests that intestinal permeability is largely dependent on the integrity of the intestinal barrier. Intracellular DAO, which acts as a biomarker for intestinal permeability, can be released into the circulatory system in substantial quantities. When there is a breach in the integrity of the intestinal barrier, the levels of DAO in the blood increase (Wu et al., 2022). Damage to the intestinal epithelial barrier is evidenced by the presence of abnormally tight junctions and increased epithelial permeability, leading to the influx of greater quantities of DAO, D-LA, and LPS into the peripheral circulation (Deng et al., 2023).

The morphology of the small intestine and the activity of digestive enzymes are critical indicators for assessing the digestive and absorptive functions in poultry. A reduction in VH and CD may lead to impaired nutrient absorption, increased secretion within the gastrointestinal tract, and reduced performance (Xu et al., 2003). In contrast, an increase in VH and the V/C is positively correlated with enhanced epithelial cell turnover. Previous studies have shown that probiotic supplementation can stimulate the proliferation of intestinal epithelial cells in chickens (Mappley et al., 2011). Yang et al. (2020) demonstrated that the dietary inclusion of Clostridium butyricum can significantly improve the VH and V/C ratios in the duodenum, jejunum, and ileum of laying hens. The results of our study indicated that the addition of PA significantly increased the height of ileal VH in laying hens while reducing the CD. Furthermore, PA was found to significantly increase the V/C. The observed improvement in the intestinal mucosal structure in the PA treatment group may lead to improved nutrient absorption and increased disease resistance, a finding supported by measurements of digestive enzyme activity. Previous research has demonstrated that probiotics can improve dietary digestibility by producing hydrolytic enzymes that facilitate nutrient absorption, including phytase, lipase, amylase, and protease (Wang and Gu, 2010; Flint et al., 2012). The existing literature on the effects of probiotics on intestinal digestive enzymes is extensive. For instance, a study found that the administration of Clostridium butyricum to the diet of broilers infected with Escherichia coli K88 resulted in enhanced activities of amylase, protease, and lipase (Zhang et al., 2016). This finding was also corroborated by Jiang et al. (2009). Our study’s results indicate that the addition of 150 mg/kg and 200 mg/kg PA significantly increased lipase activity and chymotrypsin activity in the ileal mucosa, respectively. Ogawa et al. (2015) demonstrated that Lactobacillus aerogenes SBT2055 significantly reduced lipase activity, increased the size of lipid emulsion droplets, and inhibited lipid absorption. This finding contrasts with the results of Gong et al. (2018), who showed that the addition of probiotics to basal diets can markedly enhance amylase and lipase activities. In their studies, Rodjan et al. (2018) and Wang et al. (2021) found that the inclusion of Bacillus subtilis did not significantly affect the lipase activity in broiler chickens. This discrepancy may be attributed to the varying efficacy of different probiotic strains in enzyme production and/or the stimulation of endogenous enzyme production in broilers (Wang and Gu, 2010). Additionally, differences in pH levels within the gastrointestinal tract may also contribute to these outcomes. It is well established that the pH of the gastrointestinal tract significantly influences nutrient absorption and the gut microbiome in birds (Liu et al., 2021). The activity of various digestive enzymes is contingent upon maintaining a specific pH range, which is essential for the enzymes to perform their digestive and absorptive functions (Wang et al., 2021).

Probiotics have been shown to protect the intestinal epithelium by preventing the entry of pathogens into the gut (Vicente et al., 2008), inducing the expression of intestinal protective factors (Lutgendorff et al., 2009), strengthening tight junctions (Mennigen et al., 2009), and reinforcing the cytoskeleton of epithelial cells (Sherman et al., 2005). SIgA is the primary antibody found in intestinal secretions and is the most prevalent immunoglobulin in the mucosa. Furthermore, SIgA plays a crucial role in the formation of SIgA-coated bacterial complexes, which are essential for maintaining intestinal immune homeostasis (Macpherson et al., 2008). In our study, the addition of PA reduced the concentration of SIgA in the jejunum and ileum, thereby maintaining intestinal immune balance. Additionally, we found that PA significantly decreased the expression of pro-inflammatory factors IL-8, INF-γ, and TNF-α in the jejunal mucosa. Furthermore, 150 mg/kg also significantly elevated the expression of Claudin. Several studies have demonstrated that the consumption of probiotics can suppress the expression of pro-inflammatory cytokines while enhancing the expression of anti-inflammatory cytokines (Hu et al., 2021; Wang et al., 2021). The production of inflammatory cytokines can bolster the immune response, providing protection against pathogen invasion. However, an excessive inflammatory response may lead to tissue damage. Collectively, these findings suggest that probiotics may stimulate the immune response to protect against pathogen intrusion and thus sustain intestinal equilibrium by maintaining a delicate balance between pro-inflammatory and anti-inflammatory reactions.

SCFAs are a class of saturated fatty acids characterized by having fewer than six carbon atoms. In addition to serving as the primary energy source for colonocytes, substantial evidence suggests that SCFAs play a pivotal role in maintaining health and modulating immune and inflammatory responses (van der Hee and Wells, 2021). A study conducted by Xu et al. (2021) demonstrated that the dietary inclusion of probiotics can elevate the levels of various SCFAs in cecal contents. Consistent with this research, our study indicated that the addition of PA resulted in a significant reduction in cecal chyme pH; this observation may be attributed to the increase in SCFA concentrations and alterations in gut microbial structure. The incorporation of PA into the diet of laying hens has been shown to enhance the concentrations of acetic acid, propionic acid, butyric acid, valeric acid, and isobutyric acid in the cecal chyme. Furthermore, research indicates that the observed increase in immunoglobulins and alterations in cytokine levels may be associated with enhanced SCFA production (Parada Venegas et al., 2019; Luu et al., 2020).

The significance of the commensal gut microbiota for the normal functioning of the gut’s immune system is paramount (Neish, 2009; Adeoye et al., 2016). Numerous studies have demonstrated that probiotics can positively influence the composition of the intestinal microbiota (Kristensen et al., 2016). Specifically, probiotic supplementation has been shown to significantly alter the overall structure of the fecal bacterial community, particularly in terms of β-diversity (Kristensen et al., 2016). It is well established that the intestinal microbiota has several physiological effects on the host, including the metabolization of dietary nutrients, the production of SCFAs from indigestible carbohydrates, the synthesis of amino acids and vitamins, and the regulation of metabolism (Kamada et al., 2012). The addition of PA resulted in the Simpson index, Chao1 index, observed species index, and ACE index in cecal chyme. At the phylum level, varying concentrations of PA altered the relative abundances of Bacteroidota, Fusobacteriota, Proteobacteria, and Euryarchaeota in the cecal digesta of laying hens. Similarly, at the genus level, different concentrations of PA affected the relative abundances of Butyricicoccus, Megamonas, Fusobacterium, and Lactobacillus. Notably, PA increased the relative abundances of Fusobacteriota and Euryarchaeota at the phylum level while significantly enhancing the relative abundances of Fusobacterium, Phascolarctobacterium, and Lactobacillus at the genus level. Research indicates that Fusobacteriota and Euryarchaeota metabolize carbohydrates into SCFAs, which confer several benefits to the host, including the provision of energy to gastrointestinal cells (Wang et al., 2022; Zhu et al., 2022a). Furthermore, Bacteroidetes play a crucial role in regulating lipid and bile acid metabolism, as well as maintaining energy balance in the host (Zhu et al., 2022c). Furthermore, research has demonstrated that incorporating probiotics into the diet can significantly enhance the concentration of lactic acid bacteria (Mountzouris et al., 2010) and improve the activity of the cecal microflora (Mountzouris et al., 2007). This indicates that probiotics selectively promote specific taxa (Schaub et al., 2022). In our study, an increase in PA concentration was accompanied by a corresponding rise in the relative abundance of Lactobacillus in cecal digesta, which may also contribute to the significant decrease in pH observed. These results align with findings from Lei et al. (2015) and Yang et al. (2012). Additionally, several studies have shown that lactic acid bacteria can inhibit the colonization of Campylobacter in the digestive tract of chickens (Kobierecka et al., 2017). Consequently, PA has the potential to significantly reduce the colonization of harmful bacteria within the intestinal tract, increase the concentration of beneficial bacteria, and optimize the intestinal microenvironment of laying hens, thereby enhancing production performance.

Recent investigations have demonstrated a close relationship between the host’s gut microbiota and metabolic pathways (Wang D. et al., 2020; Zhang J. et al., 2021; Zhang X. et al., 2021). Metabolomics, a novel analytical approach, enables the detection of changes in small endogenous metabolites that may be influenced by external factors or internal disruptions. This technique can be utilized to diagnose and predict the underlying mechanisms of these alterations (Vang et al., 2022; Von-Hafe et al., 2022). Analysis of the metabolomic data reveals that the differential metabolites following the addition of PA are primarily enriched in the arginine and proline metabolism pathways. These pathways have been proposed to be closely linked to inflammation (Wijnands et al., 2015). The study indicated that several metabolites, including spermine, D-proline, 3-methylhistidine, and 5-methylcytosine, were significantly upregulated upon the addition of PA. Notably, amino acid metabolism is a critical regulator of inflammation and atherosclerosis, with arginine and proline metabolism identified as the key pathways relevant to SII and hs-CRP (Zhu et al., 2022b). Furthermore, arginine is ultimately converted into putrescine, proline, and glutamine through the action of specific enzymes. Putrescine can be transformed into spermidine and spermine, while glutamine can enter the TCA cycle and be oxidized to provide energy, generating CO₂ (Zhang et al., 2022). Therefore, it is hypothesized that PA may alleviate intestinal inflammation in chickens by modulating the metabolic pathways of arginine and proline.

In conclusion, this study demonstrates that the inclusion of PA in the diet of the aged laying hen improves egg production by improving intestinal morphology, digestive enzyme activity, and intestinal permeability, as well as increasing SCFAs and the intestinal microbial structure in laying hens. These improvements may be linked to the metabolic pathways of arginine and proline. Ultimately, these findings contribute to a deeper understanding of the mechanisms through which dietary PA regulates both production performance and intestinal health in laying hens.