Dynamics of the magnum microbiome and predicted metabolic pathways across the egg-laying cycle in commercial laying hens

Abstract

The microbiome of a chicken's reproductive tract is essential for egg production and safety. It helps regulate the immune system, preventing the transmission of pathogens like Salmonella and Staphylococcus that can contaminate eggs and pose health risks. Older hens may experience reduced immune function, which can disrupt their microbiome and increase the likelihood of harmful bacterial growth. We hypothesize that age-related shifts in the oviduct microbiome influence egg production and safety. This study aims to identify microbial communities and predicted pathways in the magnum of laying hens across egg production phases and to detect any changes that may affect reproductive health and egg quality. In this study, magnum mucosa samples were aseptically collected from the hens at the peak production phase (37 weeks of age), the mid-decline phase (67 weeks of age), and the declined production phase (87 weeks of age). After DNA extraction, 16S rRNA gene sequencing was performed on an Illumina platform, and microbial diversity was analyzed using CLC bioinformatics tools. The microbial metabolic pathways were compared between groups. Raw data were analyzed using QIIME2, PICRUSt2, and STAMP v2. The level of significance was considered at P < 0.05. The magnum samples showed significant differences in alpha and beta diversity across ages. While all age groups displayed the same core phyla, there were significant changes in relative abundance in Brevibacillus, Lactobacillus, and Bacteroides. The relative abundance of the species Phocaeicola barnesiae, associated with increased egg production, significantly decreased with age. In addition, metabolic microbiome profiling showed differences in microbial biosynthesis of essential amino acids, such as l-methionine and l-lysine, between age groups. Predicted enzyme profiles revealed a functional shift in the magnum microbiota from predominantly aerobic metabolism in younger hens to enhanced anaerobic and fermentative pathways in middle and older hens, suggesting age-associated microbial remodeling. This study revealed key differences in microbial community diversity and their predicted metabolic and enzymatic pathways in the magnum across varying age and egg production levels, providing insight into age-associated functional shifts that may inform strategies to optimize reproductive health and sustained productivity in laying hens.

Introduction

The poultry industry is one of the fastest-growing sectors in agriculture, motivated by consumer demand for both chicken meat and eggs. In 2022, the U.S. poultry sector generated $76.9 billion in sales, a 67% increase from 2021. Sales of broilers jumped by 60%, turkey sales by 21%, and egg sales soared by 122%, reflecting the increasing reliance on poultry products (Grossen, 2023). To meet this growing demand, poultry producers have focused on improving production efficiency, with hens capable of producing 500 eggs over 100 weeks (Bain et al., 2016). Central to this is understanding the hen’s reproductive system, including its microbial communities, as the functioning of these organs affects both internal and external egg quality, as well as foodborne illness.

The reproductive system of the hen consists of the ovary, responsible for housing yolk as it matures, and the oviduct, a complex, multisegmented organ responsible for egg development. The oviduct itself is composed of the infundibulum (picks yolk from the ovary), magnum (site of albumen biosynthesis), isthmus (site of shell membrane synthesis), uterus (site of eggshell mineralization), and vagina (final positioning of shelled egg for oviposition) segments. The reproductive system is hormonally controlled, and each segment of the oviduct has a unique microbial community within its lumen. The cloaca serves as the exit point for the reproductive, urinary, and digestive systems, and is therefore a potential reservoir for fecal and environmental bacteria, posing a risk of contamination to the freshly laid egg and oviductal microbiome (Shterzer et al., 2020).

Bacterial contamination of the yolk, albumen, eggshell membranes, or eggshells can occur before the egg is laid due to pathogenic bacterial colonization of the oviductal microbiome. Research has shown that Salmonella enterica can colonize the oviduct and survive within the albumen after incorporation into the egg (Humphrey et al., 1991). Egg-associated foodborne illnesses are a serious public health concern, with 316 outbreaks reported to the CDC from 2000 to 2020 commonly associated with Listeria, Staphylococcus, and Salmonella internal contamination (Bunning and Tsevdos, 2023). The chicken oviductal microbiome plays a vital role in maintaining reproductive health, forming a key component of the mucosal barrier that protects against pathogenic bacteria. Much research on the human reproductive microbiome, particularly in the vaginal tract, has shown that it is typically dominated by Lactobacillus species (Muhleisen and Herbst-Kralovetz, 2016). Hens experience significant negative changes to egg production and overall reproductive health as they age. Older hens tend to exhibit lower egg production rates, increased occurrences of cracked and thin eggshells, and decreased albumen height (Joyner et al., 1987). Thin eggshell cuticles exacerbate health and safety risks by allowing bacteria to infiltrate the egg more easily. Additionally, aging hens undergo immunosenescence, a weakening of the immune system like that observed in humans (Lavoie, 2005). Reproductive disorders such as ovarian cancer and bacteria-caused oviductal egg impaction also increase with age, pointing to a weakened reproductive tract that is more susceptible to bacterial infection (Johnson and Giles, 2013; Gingerich and Shaw, 2021). Prior studies focusing on the impact of age primarily characterized the overall oviduct microbiome across anatomical segments or investigated microbial transmission between the reproductive and intestinal tracts (Wen et al., 2021; Lee et al., 2019; Ellwood et al., 2025). However, our study specifically focuses on age-associated changes in the magnum microbiome across distinct egg production stages and over an extended timeline. To our knowledge, magnum-specific microbiome alterations across reproductive aging have not been comprehensively characterized. Our goal is to document the age-related changes to the microbial communities of the different segments of the hen’s reproductive tract, as the functioning of these organs impacts both internal and external egg qualities.

In this study, we investigated specifically the magnum microbiome as a potential site of broad age-related changes for several key reasons. Namely, the magnum is the primary site of albumen synthesis and remains in direct contact with the developing egg for approximately 2–3 hours, during which time the magnum, under estrogen regulation, secretes the protein-rich albumen (egg white). Notably, the albumen has been identified as the most frequent site of internal Salmonella contamination among oviductal segments (Humphrey et al., 1991). We expect this issue to be exacerbated with age, as evidenced by several age-related changes in the hen. First, estrogen levels have been shown to decrease with age in hens, as they do in mammals, due to a decrease in the number of ovarian follicles (Liu et al., 2018). Second, albumen height has been shown to decrease with hen age, highlighting a decline in important egg quality metrics (Munro, 1971). We hypothesize that the magnum will be a vital area for microbiota diversity and pathway analysis, with key differences evident across age groups. Understanding these differences can lead to safer egg production, especially in long-life layers.

Materials and methods

Experimental design

All animal experiments were carried out in accordance with the approved protocol from the University of Hawaii Institutional Animal Care and Use Committee (IACUC) (Approval No. 17-2605). Hy-Line-W36 hens (n = 45) were sourced from a local commercial layer farm on Oahu Island, Hawaii, USA. All hens selected for this study were kept under identical housing conditions and management practices, following the guidelines for Hy-Line W-36 hens, and provided with a production-stage-specific layer diet without experimental probiotic supplementation. We collected samples at three production phases: peak egg production (37 weeks of age), mid-decline production (67 weeks of age), and declined production (87 weeks of age), with 15 hens per group.

Sample collection

All birds were euthanized via carbon dioxide asphyxiation. To examine the bacterial communities in the magnum of the hen’s oviduct, we first aseptically scraped small pieces of the magnum luminal surface with a scalpel to access the bacteria found in the mucosa layer. These samples were snap-frozen and stored at −80°C until DNA extraction. Of the 15 hens per group initially collected, 8 were used for further analysis, based on visual confirmation of the egg in the oviduct, to increase the likelihood of detecting biologically meaningful age-associated patterns despite the limited sample size.

DNA extraction and 16 s rRNA sequencing

The DNA was then extracted from the tissue samples using the Qiagen DNeasy Blood and Tissue Kits. NanoDrop One (Thermo Fisher Scientific, Madison, WI, USA) was used to assess bacterial DNA concentration. The DNA samples (n = 8/group) were sequenced at the University of Hawaii at Manoa's Advanced Studies in Genomics, Proteomics, and Bioinformatics core facility. The amplification of the V3-V4 hypervariable regions of the 16S rRNA gene was performed following the Illumina 16S Metagenomic Sequencing Library guidelines.

DNA sequence analysis

Microbial bioinformatics analysis was conducted using CLC Genomics Workbench version 12.0.1, including the CLC Microbial Genomics Module. Sequencing analyses were performed in accordance with the procedures outlined in Qiagen’s OTU Clustering Step-by-Step Tutorial (Qiagen, Hilden, Germany) and as previously described (Al Amaz et al., 2025). In short, raw reads were quality-filtered in the CLC Microbial Genomics Module as part of the OTU clustering workflow. Adapter and primer trimming were performed with a quality score limit of 0.05, allowing a maximum of two ambiguous nucleotides, and short reads (<5 bp) were discarded. Samples with low sequencing depth were filtered using the “Filter Samples Based on Number of Reads” tool with a minimum threshold of 100 reads and 50% of the median read count. OTUs with low abundance were removed using a minimum combined abundance threshold of 10 prior to downstream diversity analyses. Chimeric sequences were identified and excluded during OTU clustering. OTU clustering was performed against the Greengenes 97% OTU reference database. For alpha and beta diversity analyses, a maximum-likelihood approach based on multiple sequence alignment of OTU sequences was used to construct a phylogenetic tree using MUSCLE in the CLC workbench. Alpha diversity was quantified using Simpson’s index and Shannon entropy, with results visualized using boxplots. Beta diversity was assessed using unweighted and weighted UniFrac distances, and the results were visualized using principal coordinate analysis (PCoA). To evaluate the statistical significance of differences in beta diversity, a permutational multivariate analysis of variance (PERMANOVA) was performed. To identify differentially abundant taxa (at the order, family, and genus levels), a one-way ANOVA was applied to the OTU table following the removal of OTUs with a relative abundance of less than 10. Additionally, differences in microbial metabolic pathways between groups were evaluated. Functional prediction and pathway comparison were performed using QIIME2, PICRUSt2, and STAMP v2. Statistical significance was set at P < 0.05.

Results

Alpha and beta diversity of magnum microbiota

In the diversity analyses, the microbial communities of hens in the magnum at 37, 67, and 87 weeks of age were evaluated.

Alpha diversity was assessed to determine how within-sample microbial richness and evenness varied among the age groups. This metric reflects both the number of microbial species present and their relative abundance within each hen, providing a measure of the overall complexity of the oviductal microbiome at different stages of the laying cycle. In this study, Shannon entropy and Simpson’s index were used to assess alpha diversity (Fig. 1). Shannon entropy captures the uncertainty in predicting the species identity of a randomly selected individual, thereby showing species richness. In contrast, Simpson’s index calculates the likelihood that two individuals randomly drawn from the same sample belong to a different species. Results from both Simpson’s index (Fig. 1a) and Shannon entropy (Fig. 1b) revealed a statistically significant difference (P < 0.05) in microbial diversity between the magnums of 37-week and 87-week hens.

Fig. 1.

Effects of age on Alpha Diversity. A) Simpson’s index B) Shannon entropy where X axis represent the age of the hens and Y axis represent diversity index values. Pairwise comparisons showed that, for both indices, the 37-week hens differed significantly from both the 67-week hens and the 87-week hens (P < 0.05 for both comparisons).

Beta diversity, on the other hand, compares the differences in microbial community composition across age groups. In this study, Unweighted and Weighted UniFrac distances were used to assess beta diversity. Weighted Unifrac incorporates microbial abundance, whereas Unweighted Unifrac considers only presence or absence. Beta diversity analysis using PERMANOVA provided quantitative support for the PCoA clustering pattern. For unweighted UniFrac distances, microbial community composition differed significantly among age groups (pseudo-F = 5.40758, P = 0.00053). Pairwise PERMANOVA showed significant differences between 37- and 67-week groups (pseudo-F = 5.58818, Bonferroni-adjusted P = 0.01911) and between 37- and 87-week groups (pseudo-F = 10.02760, Bonferroni-adjusted P = 0.00140), while the difference between 67- and 87-week groups was not significant (Bonferroni-adjusted P = 0.21632). Similar patterns were observed using weighted UniFrac distances, with significant overall differences among age groups (pseudo-F = 2.78825, P = 0.00498). Pairwise comparisons showed significant differences between 37- and 67-week groups (pseudo-F = 3.84983, Bonferroni-adjusted P = 0.02471) and between 37- and 87-week groups (pseudo-F = 4.02010, Bonferroni-adjusted P = 0.01585), whereas 67- and 87-week groups did not differ significantly (Bonferroni-adjusted P = 1.00000). Both analyses (Fig. 2) demonstrated that the microbial communities of 37-week and 87-week hens were significantly distinct.

Fig. 2.

Effects of age on microbial Beta Diversity. A) Unweighted Unifrac B) Weighted Unifrac. The X- (PCo1), Y- (PCo2), and Z- (PCo3) axes indicate the first, second, and third principal coordinates, respectively, and the values in parentheses represent the percentages of community variation explained by each coordinate. For both indices, significant differences were observed between the 37-week and 67-week hens, as well as between the 37-week and 87-week hens (P < 0.05)..

Magnum microbiota profile

This study examined the microbial communities in the magnum region of the hen’s oviduct, focusing on both the phylum and genus levels. After filtering out low-abundance OTUs, the phylum-level composition of microbiota across all three age groups was analyzed (Fig. 3a). Firmicutes and Proteobacteria were the most dominant phyla in all age groups. Firmicutes were detected at 52%, 54%, and 56% in 37-week-, 67-week-, and 87-week hens, respectively. Proteobacteria were found at 32%, 18%, and 24% for 37-week, 67-week, and 87-week hens, respectively.

Fig. 3.

Effects of age on the average relative abundance of the microbiota at the A) Phylum level, B) Family level, and C) Genus level.

At the family level (Fig. 3b), the microbial profile was predominantly composed of Ruminococcaceae, Paenibacillaceae, and Lachnospiraceae. Relative abundance of Ruminococcaceae was found to be 5%, 12%, and 15% for 37-week, 67-week, and 87-week hens, respectively. Paenibacillacea’s relative abundance was found at levels 24%, 4%, and 2% for 37-week, 67-week, and 87-week hens, respectively. Following the same format, Lachnospiraceae was found at relative abundance levels of 4%, 6%, and 8%.

At the genus level (Fig. 3c), the microbial profile was enriched in the unknown genus from the Ruminococcaceae family, Brevibacillus, and Lactobacillus. Their relative abundances were (in order of 37-week hens, 67-week hens, and 87-week hens): Unknown Genus (Family Ruminococcaceae): 4%, 8%, 10%, Brevibacillus: 16%, 4%, and 2%, and Lactobacillus: 4%, 6%, and 8% respectively.

Specific bacterial abundance in magnum microbiota

More detailed analysis of order, family, genus, and species-level abundance is shown in (Fig. 4). At the order level (Fig. 4a), both Bacteriodales and Clostriodiales were shown to significantly increase (P < 0.05) in abundance from 37-week to 87-week hens. At the family level (Fig. 4b), Ruminococcaceae, Lachnospiraceae, Comamonadaceae, and Clostridiaceae had a significant increase in abundance with age from 37-week to 87-week hens(P < 0.05). Family Coriobacteriaceae also significantly increased in abundance but from 37-week hens to 67-week hens (P < 0.05). Conversely, family Paenibacillaceae and Sphingomonadaceae significantly decreased in abundance with increased age from 37-week to 87-week hens. At the genus level (Fig. 4c), the abundance of Brevibacillus, Paenibacillus, Sphingomonas, and Coprococcus significantly decreased (P < 0.05) from 37-week hens to 87-week hens. However, Lactobacillus, Bacteroides, Ruminococcus, Blautia, Prevotella, and Sutterella significantly increased (P < 0.05) with age from 37-week hens to 87-week hens. At the species level (Fig. 4d), Faecalibacterium prausnitzii significantly increased with age, while Anoxybacillus kestanbolensis and Phocaeicola barnesiae abundance significantly decreased between 37- week hens and 87-week hens.

Fig. 4.

Effects of age on significantly abundant microbiota at the A) Order level, B) Family level, C) Genus level, and D) Species level. Data shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Metabolic pathways of the magnum microbiota

PICRUST2 predicted analysis of the functional capacity of the magnum microbiota in 87-week hens versus 37-week hens (Fig. 5a) revealed differences in biosynthesis pathways of essential amino acids. We found that four biosynthesis pathways of l-methionine were significantly enriched in 37-week hens as compared to 87-week hens. These are “L-methionine biosynthesis I”, “Superpathway of l-methionine biosynthesis (by sulfate)”, “L-methionine biosynthesis III”, and “Superpathway of l-methionine biosynthesis (transulfation)”. Additionally, “L-lysine biosynthesis II” and “L-arginine biosynthesis III (via N-acetyl-l-citrulline” were significantly raised in 37-week hens. However, “L-lysine biosynthesis I”, “L-lysine biosynthesis III”, and “L-lysine biosynthesis VI” were significantly raised in 87-week hens compared to 37-week hens. 87-week hens also saw enrichment in “L-isoleucine biosynthesis IV” and “Superpathway of l-threonine biosynthesis”.

Fig. 5.

Effects of age on predicted microbial metabolic pathways of amino acid biosynthesis for A) 87-week vs 37-week hens and B) 67-week vs 37-week hens. P < 0.05 at 95% confidence intervals.

We also looked at predicted differences in biosynthesis pathways of essential amino acids for 67-week hens versus 37-week hens (Fig. 5b). We found that three l-methionine biosynthesis pathways were significantly higher in 37-week hens versus 67-week hens: “L-methionine biosynthesis I”, “Superpathway of l-methionine biosynthesis (by sulfate)”, and “L-methionine biosynthesis III”. 37-week hens also had enriched “L-arginine biosynthesis III (via N-acetyl-l-citrulline)”. However, “L-lysine biosynthesis I”, “L-lysine biosynthesis III”, and “Superpathway of l-threonine biosynthesis” were significantly raised in 67-week hens. Similarly, four l-isoleucine biosynthesis pathways were significantly higher in 67-week hens: “Superpathway of l-isoleucine biosynthesis I”, “L-isoleucine biosynthesis II”, “L-isoleucine biosynthesis III”, and “L-isoleucine biosynthesis IV”.

No significant differences were observed between 87-week hens versus 67-week hens in terms of essential amino acid biosynthesis pathways.

Enzyme classification of magnum microbiota

This study also considered predicted enzymes of the magnum microbiota using PICRUSt2. We found that for 87-week hens versus 37-week hens (Fig. 6a) 87-week old hens had a microbiota significantly enriched in both “[formate-C-acetyltransferase]-activating enzyme” and "oxaloacetate decacarboxylase”. While, 37-week hens were significantly enriched in “cytochrome-c oxidase” and “pyruvate dehydrogenase (acetyl-transferring)” enzymes as compared to 87-week hens.

Fig. 6.

Effects of age on predicted microbial enzyme classification A) 87-week vs 37-week hens, B) 67-week vs 37-week hens, and C) 87-week vs 67-week hens. P < 0.05 at 95% confidence intervals.

Whereas for 67-week hens versus 37-week hens (Fig. 6b) “cytochrome-c oxidase” and “pyruvate dehydrogenase (acetyl-transferring)” enzymes were significantly raised in 37-week hens versus 67-week hens. While 67-week hens were significantly enriched in “[formate-C-acetyltransferase]-activating enzyme” and "oxaloacetate decarboxylase”.

Enzyme classifications for 87-week hens versus 67-week hens were also explored. 87-week hens showed significantly elevated levels of “adenine deaminase” and “carboxypeptidase Taq” when compared to 67-week hens.

Discussion

Age-related changes in microbial composition and functioning have long been documented in the vaginal microbiome of human females (Park et al., 2023). However, there is limited research on how the hen's reproductive microbiome may change with age, and, to our knowledge, no studies have focused specifically on the magnum segment of the oviduct (Wen et al., 2021; Lee et al., 2019; Ellwood et al., 2025). This study found a significant increase in alpha diversity of the hens’ magnum microbiome with increasing age and therefore a greater number of OTU’s in the 87-week hens. This change could be explained similarly to the increased diversity among women of advanced maternal age, in which it has been theorized that there is an increase in the number of non-core bacterial species and a decrease in the abundance of core bacteria (Huang et al., 2022). A recent study investigating changes in the gut microbiome across peak, mid, and declining egg production similarly found increased alpha diversity in older hens compared with younger hens. The authors concluded that age-related microbial shifts help to maintain physiological and immune homeostasis, ultimately influencing egg production across different stages of the laying cycle (Shahid et al., 2026a). Beta diversity analysis here showed that the magnum microbiome of 37-week hens differed significantly from those of 67-week and 87-week hens, consistent with other recent findings (Ellwood et al., 2025). However, 67-week hens and 87-week hens did not differ significantly. Therefore, peak-production hens have a distinct magnum microbiome that varies in both abundance and composition from mid-declined production-age hens and declined production-age hens.

The microbial profile of the magnum was shown to consist of the same core phyla across all three age groups. In order of abundance, these were Firmicutes, Proteobacteria, and Bacteroidetes. These results are consistent with several other studies and appear to be the core bacterial species of laying hens (Wen et al., 2021; Shterzer et al., 2020). The core known genera of the magnum were: Brevibacillus, Lactobacillus, and Bacteroides. Bacteroides and Lactobacillus have previously been reported as part of the core genera of mature laying hens (Lee et al., 2019).

Order Clostridiales was shown to increase significantly in relative abundance across ages. Within this order, the families: Ruminococcacae (and Genus Ruminococcus), Lachnospiraceae, and Clostridiaceae significantly increased with age. All three families are obligate anaerobic, gram-positive bacteria. They are considered commensal gut bacteria and are important producers of butyrate (Rychlik, 2020). In the gut microbiome of chickens, butyrate has been shown to inhibit colonization of Salmonella enterica and Clostridium perfringens (Liu et al., 2021). It is possible they play a similar role in the reproductive microbiome, however the overall physiological environments of the gut and oviduct differ considerably and bacteria could in-turn behave differently. In addition, butyrate has been reported to reduce the expression of proinflammatory cytokines (Onrust et al., 2015). Together, this could demonstrate that an increased abundance of butyrate-producing bacteria is compensating for an increased inflammatory state in the magnum microbiome or for an increased risk of pathogenic bacterial colonization with age. Similarly, our research showed species Faecalibacterium prausnitzii, a key species of Order Clostridiales, significantly increased in abundance with age. Faecalibacterium prausnitzii is a commensal gut bacteria. Its increased abundance may indicate increased bacterial transmission between the intestinal tract, the external environment, and the upper oviduct (magnum) with age.

Within the family Lachnospiraceae, the genera Blautia and Coprococcus differed significantly by age. Genus Coprococcus is a known bacteria of the hen’s gut microbiome and is associated with gut barrier function (Wang et al., 2024). Here, Coprococcus abundance in the magnum significantly increased between 37-week and 67-week hens but decreased between 37-week and 87-week hens, indicating elaborate changes to intestinal health or bacterial oviduct transmission with age. Genus Blautia significantly increased in abundance from 37-week hens to 87-week hens in the magnum microbiome. Blautia coccoides, used as a representative of the genus Blauita, has been shown to activate B cells in the hen to produce immunoglobulin A (Xie et al., 2024). Immunoglobulin A (IgA) at mucosal surfaces interacts with commensal bacteria to support their adhesion to intestinal epithelial cells while simultaneously tagging pathogenic microbes for removal, thereby helping maintain microbial homeostasis. A similar mechanism may operate in aged hens, where IgA-mediated regulation of the microbiome could help compensate for other age-associated declines in immune function (Lavoie, 2005).

Family Paenibacillaceae was shown to significantly decrease in abundance in the magnum from 37-week hens to 87-week hens. The genus Paenibacillus also significantly decreased with age. Paenibacillus species are gram-positive, spore-forming bacteria that have often been shown to exhibit probiotic traits when fed in the diet (Moon et al., 2022). For instance, P. polymyxa has also been used as a probiotic feed additive, shown to improve gut microbiota composition (Zhou et al., 2024). Feeding chickens purified bacteriocin from P. polymyxa has been used to control Campylobacter jejuji, a common cause of foodborne illness (Stern et al., 2005). P. xylanexedens, when used as a food additive, was linked to decreased abundance of E. coli and increased Lactobacillus in the gut microbiome (Dablool et al., 2024). However, Paenibacillus strains can have significant variability in function, and probiotic traits cannot be assumed for all. Genus Brevibacillus, within Family Paenibacillacae, also significantly decreased in abundance from 37-weeks to 87-weeks. Although research on Brevibacillus is limited, the closely related genus Bacillus has been shown to have numerous impacts on the microbiome and egg production. Dietary supplementation of B. velezensis has been linked to improved egg production and egg quality, in terms of increased albumen height, in laying hens (Ye et al., 2020). We expect Paenibacillaceae to play a similar protective and microbiome-stabilizing role in the reproductive tract of 37-week hens, particularly given the functional parallels observed in related genera. Members of this family are known to produce antimicrobial compounds, such as bacteriocins, that inhibit pathogenic bacteria and help maintain a balanced microbial environment (Grady et al., 2016). Their higher abundance in younger hens suggests they may contribute to the early-life stability of the oviductal microbiome. Likewise, the abundance of Anoxybacillus kestanbolensis, a species within the order Bacillales, was significantly reduced with advancing age in the magnum. This reduction may reflect age-related shifts in the oviductal environment, such as hormonal changes or altered mucosal immunity that make it less supportive of bacteria commonly associated with a healthy, functional reproductive tract in younger hens. Together, these microbial trends highlight the dynamic nature of the oviductal microbiome and its potential linkage to reproductive aging.

Our research found that the order Bacteriodales increased significantly with age, along with two of its key genera: Prevotella and Bacteroides. Recently, certain Prevotella species have been linked to improved laying performance (Liu et al., 2024). An increased presence of the genus Bacteroides, along with Genus Lactobacillus (order Lactobacillales), in the gut and reproductive microbiomes of laying hens has been linked to increased egg production (Su et al., 2021). Lactobacillus is among the most frequently used probiotics in the poultry feed industry (Halder et al., 2024). Lactobacillus is thought to inhibit pathogenic bacteria by producing lactic acid, which lowers the pH of their environment. Similarly, Bacteroides are known for their roles in nutrient metabolism and the production of short-chain fatty acids (SCFAs) (Shin et al., 2024). SCFAs can impact host physiology by supporting epithelial health, modulating local immune responses, and enhancing energy availability, all of which are essential for maintaining optimal reproductive performance (Shahid et al., 2026b; Liu et al., 2021). The increased abundance of Prevotella, Bacteroides, and Lactobacillus with age could compensate for the known declines in immune function and egg production in aged hens. However, Lactobacillus and Bacteroides contain significant diversity between strains, and their functions in the microbiome cannot be assumed universally.

Our research also found a significant decrease in abundance between 37-week and 87-week hens in the species Phocaeicola barnesiae. An increased abundance of Phocaeicola barnesiae, previously classified as Bacteroides barnesiae, in the reproductive tract has been positively associated with higher egg production, a relationship that becomes particularly meaningful when considered alongside the well-established decline in egg production as hens age (Su et al., 2021). Several Phocaeicola species have been identified as SCFA producers, which are known to provide energy and overall benefit the host (Da Silva Morais et al., 2024). In younger hens, where P. barnesiae abundance tends to be higher in the magnum, these microbial functions may help maintain a more favorable oviductal environment for albumen synthesis. As hens age and P. barnesiae levels decline, the reduction in these beneficial metabolic and immunomodulatory effects may contribute, at least in part, to the known decline in egg quality. This suggests that the presence of P. barnesiae could act as a microbial indicator or even a microbial contributor to sustain reproductive efficiency in laying hens.

This study also investigated the predicted functional capacity of the magnum microbial community in 37-week (peak production) hens, 67-week (mid-declined production) hens, and 87-week (declined production) hens. We examined biosynthesis pathways of essential amino acids. Interestingly, we found a significant predicted enrichment of three l-methionine biosynthesis pathways in the magnum microbiome of 37-week hens as compared to 67-week hens and four l-methionine biosynthesis pathways as compared to 87-week hens. Methionine is an essential amino acid in chickens and the first limiting amino acid in the diet. It has important roles in growth performance and immune function (Alagawany et al., 2020). Research has shown that adding dietary methionine to broiler diets positively influenced antibody production and certain immune responses (Tsiagbe et al., 1987). Potentially predicted decreased methionine biosynthesis in 67-week and 87-week hens may be coupled with their immunosenescence, exerting a strong impact on avian immunity in older age. Added dietary methionine has also been shown to affect egg production, increasing albumen crude protein (Shafer et al., 1998). Since albumen is synthesized in the magnum, it is reasonable to think that this predicted increased methionine biosynthesis by the microbiota could readily affect the developing egg. 67-week hens and 87-week hens also had a significant predicted decrease in the l-lysine biosynthesis II pathway compared to 37-week hens. Lysine is another essential amino acid and plays major roles in the body composition and growth of chickens (Belloir et al., 2019). Increased dietary lysine has been shown to significantly affect albumen weight (Prochaska et al., 1996). Hence, it would be reasonable to assume that changes in lysine biosynthesis pathways with age could be linked to the known decline in albumen quality. 37-week hens also uniquely saw predicted increased biosynthesis of essential l-arginine from the magnum microbiota as compared to both 67-week hens and 87-week hens. Research has shown that increased dietary Arginine can positively influence egg production and egg weight (Silva et al., 2012). Arginine produced by the magnum microbiota could be more readily taken up by the developing egg, yielding similar results. However, these PICRUSt2 predictions are based solely on 16S rRNA composition and are largely inferential, requiring experimental validation using approaches such as metabolomics, transcriptomics, qPCR, or enzymatic assays.

This study also identified significant differences in predicted enzyme levels across age groups, particularly for enzymes that act under aerobic or anaerobic conditions. 37-week hens were predicted to have significantly elevated levels of “cytochrome-c oxidase” and “pyruvate dehydrogenase (acetyl-transferring)” enzymes when compared to both 67-week hens and 87-week hens. Cytochrome-c oxidase is a key enzyme in cellular respiration in aerobic bacteria (Pitcher and Watmough, 2004; Hederstedt, 2022). Similarly, pyruvate dehydrogenase (acetyl-transferring) is necessary for aerobic metabolism (Nuzzo et al., 2015). Predicted elevated levels of both could indicate a microbiota that favors aerobic conditions in young peak-producing hens. Conversely, the microbiota of 67-week and 87-week hens showed significantly elevated levels of [formate-C-acetyltransferase]-activating enzyme and oxaloacetate decacarboxylase as compared to 37-week hens. Elevated levels of both oxaloacetate decacarboxylase, which has roles in anaerobic citrate fermentation, and the activating enzyme for formate-C-acetyltransferase, important for anaerobic glucose metabolism, could likewise denote an aged magnum microbiota consisting more of anaerobic or facultative anaerobic bacteria (Balsera et al., 2011; Knappe and Sawers, 1990). Past research has shown that the cecum of layer hens is heavily characterized by its anaerobic fermentative microbial populations (Ricke et al., 2022). Therefore, the aged hen’s shift towards enzymes important for anaerobic metabolism and fermentation could further denote increased bacterial transmission and influence between the cecum and upper oviduct (magnum) with age.

A limitation of the present study is the relatively modest sample size (n = 8/group), and the findings should therefore be interpreted as exploratory. Additionally, the avian oviduct comprises anatomically and functionally distinct regions that may support region-specific microbial communities. The age-associated microbial alterations observed in the magnum may reflect both localized physiological changes and broader systemic effects of aging. Future studies incorporating larger cohorts and additional reproductive tract regions will be important for further validating and expanding upon these findings. Although ASV-based methods (e.g., DADA2 and Deblur) are increasingly preferred for higher taxonomic resolution and reproducibility, this study used a 97% OTU clustering approach via the CLC Genomics Workbench pipeline to maintain consistency across samples and evaluate broader microbial community patterns. Furthermore, integrating microbiome analysis with host physiological parameters in future investigations could potentially establish functional relationships between reproductive tract microbiota and hen productivity. For instance, hormonal profiling could help elucidate a stronger connection between age-associated estrogen decline and microbial shifts, which, in our current study, is only inferred from prior literature. A further limitation of the present study is that extraction blanks and sequencing negative controls were not included during sample processing and sequencing. While contamination control is an important consideration in low-biomass microbiome studies, all samples were collected aseptically and processed under consistent laboratory conditions to minimize potential sources of contamination. However, low-abundance taxa should be interpreted with some caution, as environmental or reagent-associated contaminants cannot be completely excluded.

Conclusion

This study demonstrates that the magnum microbiome of laying hens undergoes marked age-related changes in both composition and functional capacity. Alpha diversity significantly increased with age, indicating that 87-week hens had a greater number of OTUs relative to peak-production 37-week hens. In contrast, beta diversity analyses revealed clear and significant compositional differences between 37-week and 87-week birds, confirming that a distinct shift in oviductal microbial community structure accompanies aging. Several known butyrate-producing taxa, including members of Ruminococcaceae, Lachnospiraceae, and Clostridiaceae increased in abundance with age, suggesting increased SCFA production that may compensate for the reduced immune function and increased inflammatory state associated with aging. Despite these increases, aging hens exhibited notable declines in taxa associated with reproductive performance. Most prominently, P. barnesiae, a species positively associated with egg production, significantly decreased between 37-week and 87-week hens. This decline is consistent with the well-documented age-related reduction in egg production and suggests that P. barnesiae may serve as a microbial indicator of reproductive efficiency. Functional metabolomic predictions indicated age-associated declines in microbial contributions to albumen-related amino acid biosynthesis, though these findings are based on 16S rRNA composition and would require further experimental verification. The magnum microbiome of 87-week hens showed predicted significantly reduced biosynthetic capacity for key amino acids, including l-methionine, l-lysine, and l-arginine, all of which are critical for albumen protein synthesis. These declines parallel the decreased albumen quality and egg production observed in older hens. Further, PICRUSt2 enzyme predictions showed an age-related shift from aerobic metabolism in 37-week hens to increased anaerobic and fermentative activity in older hens. Together, these findings highlight that aging reshapes both the microbial composition and metabolic and enzymatic capabilities of the hen’s magnum microbiome. The combined loss of beneficial species such as P. barnesiae and the diminished microbial capacity to supply essential amino acids likely contribute to the deterioration of reproductive performance with advancing age. This work underscores the importance of the reproductive microbiome as a potential target for microbial or nutritional interventions to sustain egg production and oviductal health in aging laying hens.

Funding

B.M. received funding for the research from a USDA Multistate (2059R) grant provided by the CTAHR University of Hawaii at Manoa. The author(s) declare that financial support was received for the research, authorship, and/or publication of this article.

CRediT authorship contribution statement

Ava Vankempen: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Md Ahosanul Haque Shahid: Writing – review & editing, Validation, Methodology, Formal analysis, Data curation. Birendra Mishra: Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Disclosures

All authors declare no conflicts of interest regarding this article.

Data availability

The metagenomics sequence data used in this study have been submitted to the NCBI database (accession no: PRJNA1299480).