Maternal supplementation with Lactobacillus reuteri enhances colostrum sIgA secretion and immune function of offspring

Abstract

Specific maternal gut microbiota can activate immune responses in the gut-associated lymphoid tissues, driving the secretion of maternal immunoglobulins into the milk and facilitating the passive immunity of the offspring via the entero-mammary axis. This study aims to investigate the distinct gut microbiota profiles between large white (LW) sows and Ningxiang (NX) sows, and to explore probiotic for maternal and offspring intestinal health. Lactobacillus reuteri (LR) was significantly enriched in the feces of NX sows and positively correlated with colostral immunoglobulin (Ig) levels, specifically IgG (r = 0.568, P = 0.014), IgA (r = 0.573, P = 0.013), and IgM (r = 0.721, P = 0.0007). Maternal supplementation with LR isolated from NX pigs enhanced mammary IgA production and elevated secretory IgA (sIgA) levels in both colostrum and the ileum of mice. Importantly, maternal dietary LR supplementation improved immune functions of offspring mice challenged with enterotoxigenic escherichia coli (ETEC), as evidenced by reduced systemic inflammation (TNF-α, IFN-γ) and preserved intestinal barrier function. These results support the potential of NX pig-derived LR as a probiotic to improve the health of maternal and offspring via the gut-mammary axis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s42523-026-00550-z.

Background

The immune system of neonatal piglets is immature, making them highly susceptible to pathogen exposure from various environmental factors [9, 27]. Maternal milk provides macronutrients and bioactive molecules such as immunoglobulins that serve to protect the neonate against pathogens and to program the immune system of neonates [3, 35].

Emerging evidence has established a novel gut-mammary axis through which maternal gut microbiota orchestrates mammary immunity. During pregnancy or lactation period, immunoglobulins produced in the mammary gland rely on the migration of plasma cells into the mammary gland [25]. The presence of certain maternal microbiota in the gut is indispensable for the programming of immunoglobulin synthesis in colostrum [30]. Itestinal Peyer’s patches are activated by commensal bacteria, serving as the primary origin of plasma cells migrating to mammary tissue [8, 33]. In addition, the maternal microbiota can also stimulate antigen-presenting cells (dendritic cells) to induce the expression of inflammatory factors (IL-6 and TGF-β) which may facilitate the differentiation of B cells into IgA plasma cells resident in the mammary glands [4, 32].

A fiber-rich diet plays a crucial role in sustaining gut microbial homeostasis by enhancing microbial diversity and metabolic activity, including the production of short-chain fatty acids (SCFAs), which control gene expression to express molecules necessary for plasma B cell differentiation and support B cell responses in the steady state and during infection [16]. Ningxiang (NX) pigs, a Chinese indigenous breed with evolutionary adaptation to high-fiber diets, presents a unique model for investigating maternal microbial programming of offspring immunity. Compared to commercial breeds, NX piglets exhibit significantly enhanced disease resistance and reduced diarrheal incidence [19, 43]. We hypothesize that the distinctive maternal microbiota of NX pigs, particularly Lactobacillus reuteri populations, enhance passive immune transfer to offspring through the gut-mammary axis. Therefore, this study aimed to characterize breed-specific differences in colostral immunoglobulin profiles between NX and commercial sows and identify key differential microbiota associated with colostrum immunoglobulin production in sows. Furthermore, a pregnant mice model was used to verify the beneficial effects of Lactobacillus reuteri derived from NX pigs on gut homeostasis and immune capacity in both maternal and offspring.

Results

LR enrichment in NX sows correlated with colostral immunoglobulin profiles

The results of common alpha-diversity analysis (including observed species index, Chao index, Shannon index, and Simpson index) and beta diversity analysis (including bray curtis diversity distance and bray curtis cluster tree) are shown in Supplementary Fig. 1. The α-diversity analysis showed no significant differences between NX and LW sows. (Supplementary Fig. 1A, P > 0.05). Principal coordinates analysis (PCoA) revealed distinct clustering patterns between breeds (Fig. 1B) which was consistent with hierarchical clustering analysis (Supplementary Fig. 1B).

Fig. 1.

LR is a differential bacterium between NX and LW pigs and is positively correlated with immunoglobulin content in colostrum. (A) The concentrations of immunoglobulins (Ig) in the colostrum of two breeds of sows. (B) Principal Component Analysis (PCoA) of the bacterial community structure among NX and LW sows. (C) Linear discriminant analysis (LDA) scores derived from LEfSe analysis, showing biomarker taxa at the species level (LDA score) of > 3 and a significance of P < 0.05 determined by the Kruskal-Wallis rank sum test. (D) The spearman correlation between the gut discrepant microbiota with the levels of colostrum immunoglobulin. (E) Relative abundance of Lactobacillus reuteri and Lactobacillus crispatus in NX pigs. n = 9/group. *P < 0.05, **P < 0.01, and ***P < 0.001

Taxonomic profiling identified Firmicutes, Bacteroidetes, Spirochaetota, and Proteobacteria as dominant phyla (Supplementary Fig. 1C). At the genus levels, NX sows exhibited enrichment of Lachnospiraceae_unclassified, Ruminococcaceae_unclassified, Shigella, Clostridiales_unclassified, Clostridium, Ruminococcus, and Lactobacillus, while LW sows dominantly harbored Prevotella, Bacteroidales_unclassified and Streptococcus (Supplementary Fig. 1D). LEfSe analysis showed Lactobacillus_crispatus, Eubacterium_coprostanoligenes, Lactobacillus_reuteri, and Acinetobacter_haemolyticus were the discriminant bacterial species among the two breeds (Fig. 1C).

The colostral concentrations of IgG, IgM, and IgA in NX sows were significantly higher than those in LW sows (P < 0.05, Fig. 1A). Spearman’s correlation analysis revealed positive associations between immunoglobulin levels and the relative abundances of Clostridiales, Coprococcus_sp, Coriobacteriaceae, Eubacterium_coprostanoligenes, Lactobacillus_crispatus, Lactobacillus_reuteri, with showing high correlations with IgG, IgA and IgM concentrations (r = 0.5725; r = 0.5683; r = 0.7) (Fig. 1D). Furthermore, the relative abundance of Lactobacillus reuteri exhibited a trend toward being higher than that of Lactobacillus crispatus (P = 0.0606, Fig. 1E), and LR was therefore prioritized for subsequent isolation and functional validation.

Following isolation from NX feces, the LR demonstrated characteristic Gram-positive rod morphology (Fig. 2B and C) and reached stationary phase at 16 h with maximum titers 1.9 × 10⁹ CFU/mL (Fig. 2A).

Fig. 2.

Growth curve, colony characteristics and gram stain of Lactobacillus reuteri

LR increases colostral sIgA levels in postpartum mice

Building on the observed correlation between LR abundance and immunoglobulin levels in NX pigs, and considering the evolutionary conservation of key adaptive and mucosal immune mechanisms, including IgA production and the gut–mammary axis, across mammals, we utilized a gestating BALB/C mouse model to assess the impact of dietary LR supplementation (Fig. 3). LR supplementation significantly increased IgA concentrations in the colostrum and mammary gland, as well as sIgA concentrations in colostrum (P < 0.05, Fig. 4A). In addition, LR significantly increased mammary pIgR mRNA and protein levels (P < 0.05, Fig. 4B, C), concomitantly upregulating the mRNA expression of CCL28 and MAdCAM-1 (P < 0.05, Fig. 4D, E). Furthermore, the levels of IL-10 and TGF-β in the mammary gland were also significantly increased (P < 0.05, Fig. 4F).

Fig. 3.

Experimental design of mice

Fig. 4.

The effect of Lactobacillus reuteri on the content of sIgA in the ileum of postpartum mice. (A) Growth curve of Lactobacillus reuteri., (B) Colony characteristics of Lactobacillus reuteri., (C) Gram stain of Lactobacillus reuteri. (A) The content of IgA and sIgA in colostrum and mammary gland of postpartum mice. (B) Representative Western blot bands and corresponding quantitative analysis of relative pIgR protein expression in mammary gland of postpartum mice. (C-E) RT-PCR analysis of mammary gland mRNA expression of pIgR, CCL28, and MAdCAM-1 in postpartum mice. (F) RT-PCR analysis of mammary gland cytokine expression in postpartum mice. n = 8/group. *P < 0.05, ***P < 0.001

LR increases short-chain fatty acid levels in postpartum mice

To determine the colonization of LR in the intestine and its impact on the microbial community, qPCR was used to quantify the concentration of LR in the feces of postpartum mice and their offspring at weaning, while 16 S rDNA sequencing was employed to profile the ileal microbiota of the postpartum mice. LEfSe analysis identified significant differences in microbial composition between the groups in postpartum mice. Specifically, the LR group was enriched in Limosilactobacillus and Ruminococcus, whereas the CON group exhibited higher relative abundances of Lactobacillus and Bifidobacterium (Fig. 5A). Consistent with these findings, fecal LR abundance was significantly elevated in postpartum mice receiving LR supplementation (P < 0.05, Fig. 5B). However, no significant difference in fecal LR levels was observed in offspring at weaning (P > 0.05, Fig. 5C). Notably, LR supplementation was also associated with a marked increase in colonic butyrate concentration in postpartum mice (P < 0.05, Fig. 5G).

Fig. 5.

The effect of Lactobacillus reuteri on cytokine gene expression in the ileum and short-chain fatty acids in the colon of postpartum mice. (A) LEfSe analysis of the microbiota in the fresh ileal contents of maternal mice after delivery. (B) The content of Lactobacillus reuteri in the fresh feces of maternal mice during delivery. (C) The content of Lactobacillus reuteri in the fresh feces of offspring at weaning. (D-I) The content of short-chain fatty acid in the colon of postpartum mice. n = 8/group. *P < 0.05, **P < 0.01

LR increases intestinal TGF-β expression and sIgA secretion in postpartum mice

To investigate the status of maternal mucosal immunity, we examined the gene expression of ileal cytokines and the secretion of sIgA. The results showed that LR significantly increased the expression of TGF-β, pIgR, and CCL28 in the ileum (P < 0.05, Fig. 6D, E, F), and immunofluorescence analysis revealed an enhanced sIgA secretion (Fig. 6G).

Fig. 6.

The effect of Lactobacillus reuteri on colostrum sIgA and mammary gland cytokine gene expression in postpartum mice. (A-F) RT-PCR analysis of ileal cytokine expression in postpartum mice. (G) Immunostaining for sIgA in the ileum (Scale bar represents 100 μm). n = 8/group. *P < 0.05. n = 8/group. *P < 0.05

Maternal LR supplementation alleviates immune stress in offspring induced by ETEC challenge

To verify the protective effect of increased colostral sIgA on the offspring, we established an ETEC challenge model (Fig. 3). At weaning, offspring from NC dams were orally administered either PBS or ETEC, while offspring from LR-supplemented dams were administered ETEC. Within 24 h after ETEC infection, the body weight gain of offspring in the CON+ETEC group was significantly lower than that in the CON + PBS and LR+ETEC groups (P < 0.05, Fig. 7B). Meanwhile, the CON+ETEC group exhibited a marked increase in spleen index and serum LPS concentration, whereas the LR+ETEC group maintained levels comparable to the CON + PBS group (P < 0.05, Fig. 7A, C). Analysis of serum cytokines revealed that anti-inflammatory IL-4 levels were significantly decreased, while pro-inflammatory IFN-γ levels were significantly increased in the CON+ETEC group. In contrast, IL-4 and IFN-γ levels in the LR+ETEC group were similar to the CON + PBS group (P < 0.05, Fig. 7D, E). Furthermore, serum TNF-α levels in the LR+ETEC offspring were significantly lower than the CON+ETEC group (P < 0.05, Fig. 7F).

Fig. 7.

The offspring of dams fed Lactobacillus reuteri can alleviate immune stress induced by ETEC challenge. (A) Spleen index in the offspring mice. (B) Body weight changes in offspring mice within 24 h after ETEC challenge. (C) LPS levels in the serum of offspring mice. (D-I) Cytokine levels in the serum of offspring mice. n = 8/group. *P < 0.05, **P < 0.01, and ***P < 0.001

Maternal LR supplementation enhances intestinal immunity and barrier function in offspring

We examined cytokine expression and intestinal barrier function in the ileum of offspring. Compared with the CON+ETEC group, the LR+ETEC group exhibited a significant increase in the expression of the anti-inflammatory cytokine IL-10 and TGF-β, and a significant decrease in the pro-inflammatory cytokine IFN-γ (Fig. 8B, C, D). In addition, compared with the CON + PBS group, CON+ETEC offspring showed reduced expression of the E-cadherin and ZO-1, whereas LR+ETEC offspring exhibited significantly higher expression of E-cadherin and ZO-1 relative to the CON+ETEC group (Fig. 8E, F). Taken together, these findings support that LR improves offspring immunity and intestinal barrier function by increasing maternal colostral sIgA levels.

Fig. 8.

The effect of ETEC on cytokines and barrier function in offspring ileum. (A-D) RT-PCR analysis of ileal cytokine expression in offspring mice. (E-H) RT-PCR analysis of ileal barrier-related gene expression in offspring mice. n = 8/group. *P < 0.05, **P < 0.01, and ***P < 0.001

Discussion

NX pig exhibits robust resistance to roughage, and its high fiber diet shaped a distinctive intestinal microbial flora [41, 45]. Notably, we found that NX sows had higher colostral concentrations of IgA, IgG, and IgM compared with LW sows. This enhancement in maternal immunoglobulin production is likely influenced by the maternal intestinal microbiota through the Peyer’s patches–mammary gland pathway [33]. To identify the specific intestinal microbiota in NX sows associated with colostrum immunoglobulins, we employed 16 S rRNA microbial sequencing to analyze the differential microbiota and examined the correlation between these differential microbes and colostrum immunoglobulins. In this study, the abundance of phylum Firmicutes in fecal samples from NX sows accounted for 72.12%, which was significantly higher than that observed in LW sows (57.0%). The microbiota composition belonged predominantly to the Firmicutes phylum is susceptible to dietary fiber regimen [39], which is consistent to the dietary characteristics of NX pigs as a local pig breed with fiber-rich diet [15]. In view of gut microbiota composition on the genus level, NX sows had higher abundance of Clostridium and Ruminococcus, which were positively associated with the production of short-chain fatty acids that could modulate the immune response, repair intestinal barrier, and induce propagation of specific immune cell [10, 24]. Additionally, we found that NX sows had a higher abundance of LR and L. crispatus, which showed positive correlations with colostral immunoglobulin levels. Thus, LR and L. crispatus were identified from the porcine fecal microbiome as key differential bacterial species between NX and LW pigs. Howevers, the relative abundance of Lactobacillus reuteri exhibited a trend toward being higher than that of Lactobacillus crispatus, and LR was therefore prioritized for subsequent isolation and functional validation.

Indeed, recent studies have indicated that LR is a key component of the maternal microbiota that enhances IgA and sIgA production [22, 37]. Our findings similarly suggest that LR may serve as a keystone bacterial species associated with immunoglobulin secretion in the colostrum of NX pigs. To verify the association between LR and mammary immunoglobulin secretion, LR was isolated from the feces of NX pig and functionally validated in female mice. This cross-species validation strategy is supported by comparative immunological evidence indicating that the immune systems of humans, mice, pigs, and ruminants share a broadly conserved overall architecture, particularly with respect to fundamental adaptive and mucosal immune mechanisms [2]. Notably, LR derived from NX pigs significantly increased sIgA concentrations in colostrum and ileum, as well as IgA levels in the mammary glands of postpartum mice. IgA is produced by plasma cells in the intestinal lamina propria and plays essential roles in mucosal adaptive immunity by shaping and regulating the microbiota [13]. Importantly, maternal sIgA is transferred through breast milk to provide direct immune protection to neonates, enabling early-life defense against gut pathogens [34]. The production of IgA in the maternal mammary gland depends on intestinal humoral immune [25], where the gut microbiota plays a pivotal role in activating gut-associated lymphoid tissues to induce the differentiation of mucosal B cells into IgA-producing plasma cells [42]. In our study, LEfSe analysis revealed that LR supplementation not only consolidated its own dominance but also significantly enriched SCFA-producing taxa, specifically Ruminococcus and Lachnospira [14, 23]. The observed increase in intestinal butyrate levels may be associated with this microbial reconfiguration. Mechanistically, microbiota-derived SCFAs mediate anti-inflammatory responses, regulate immune cell functions, and foster mucosal tolerance primarily through the activation of G-protein-coupled receptors, notably GPR41, GPR43, and GPR109A [1]. Recent evidence highlights that microbiota-derived butyrate specifically engages GPR43 to promote the differentiation and function of T follicular helper cells within Peyer’s patches, thereby robustly enhancing IgA production [17]. Concurrently, our data demonstrated that LR supplementation significantly upregulated the expression of TGF-β. Interestingly, TGF-β serves as an indispensable synergistic enhancer for IL-21-induced IgA class switch recombination [7]. It not only dictates the direction of IgA switching and amplifies B cell proliferation but also crucially imprints the mucosal homing phenotype on these cells. In our study, LR supplementation concomitantly increased intestinal butyrate levels and upregulated TGF-β expression, supporting the existence of a coordinated microbiota–metabolite–immune axis that potentiates mucosal IgA responses and ultimately contributes to enhanced colostral sIgA output.

The mucosal chemokine CCL28 is a key regulator of the migration and accumulation of IgA-producing plasma cells in the mammary gland, thereby controlling maternal IgA transfer during lactation [38]. Our results showed that LR dramatically increased CCL28 expression in mammary gland and ileum. In the intestine, monomeric IgA associates with the small plasma cell–derived joining (J) chain to form IgA dimers that recognize the pIgR on the basolateral surface of mucosal intestinal epithelial cells, where pIgR mediates their transcytosis and ultimately releases sIgA onto the gut surface [11, 28]. Similarly, IgA produced by plasma cells in the mammary gland is transported across epithelial cells by the pIgR and subsequently released sIgA into the milk [21]. Our findings demonstrated that LR upregulated pIgR expression in the ileum and mammary gland of dams, accompanied by a significant increase in colostral and ileal sIgA concentrations. Similar to our findings, previous studies have also shown that breast milk contains higher sIgA levels correlates with both pIgR expression and IgA plasma cell accumulation in the lactating mammary gland of mice [5]. Therefore, these butyrate-mediated immune signals may activate the entero-mammary axis, driving the migration of IgA⁺ plasma cells from the intestine to the mammary gland, ultimately contributing to the enhanced secretion of colostral sIgA.

During delivery, infants are exposed to diverse and abundant microbial communities originating from the maternal vagina, feces, and skin [6]. After birth, extensive bacterial colonization of the neonatal gut further stimulates immune system development [18]. Microbiome-mediated immune responses play a crucial role in maintaining intestinal health [40]. Accordingly, in elucidating the protective mechanisms operating in our model, it is essential to distinguish whether the observed immune benefits in the offspring arise from passive transfer of immune factors through breast milk or from vertical microbial transmission from the mother. Our quantitative analyses confirmed that LR had successfully colonized the maternal gut at parturition. However, no corresponding enrichment was detected in the feces of the offspring prior to ETEC challenge. This finding suggests that maternal mice were unable to continuously transmit LR to their progeny after cessation of supplementation. Therefore, the enhanced resistance to ETEC observed in the offspring is more likely attributable to the benefits conferred by colostrum—specifically, elevated levels of sIgA—rather than to direct bacterial colonization of the neonatal intestine. Breast milk provides several immunological benefits, including protection against infection, mitigation of inflammation, and support for intestinal barrier development [36]. Colostrum-derived sIgA provides the first source of immune protection in the intestinal tract of suckling infants [21]. Meanwhile, sIgA promotes the development and maintenance of intestinal homeostasis during the transition from weaning to childhood and adulthood [26].

Diarrhea is a common disease in children and piglets, and ETEC is one of the major causative pathogens. ETEC releases adhesin and enterotoxin in intestine, which destroy intestinal barrier to cause diarrhea [44]. In addition, LPS is a major component of E. coli outer membrane, induces inflammatory responses [12, 20]. As reported by Zha et al., serum LPS concentrations are significantly elevated in ETEC-infected piglets [44]. Previous studies have also shown that weanling mice lacking passive sIgA from breast milk exhibit impaired epithelial barrier function, allowing pathogenic bacteria to colonize draining lymph nodes [26]. Furthermore, both plant-derived sIgA2 monoclonal antibodies and sIgA have been shown to protect against ETEC-induced diarrhea in murine infection [29, 31].

Our finding that higher concentrations of sIgA from breast milk reduced serum concentrations of LPS, IFN-γ, and TNF-α in ETEC-infected weaned mice, and alleviated ETEC-induced systemic inflammation and intestinal barrier damage. This is attributed to LR promoting the transport of endogenous sIgA in maternal and early exposure of offspring to passive sIgA in breast milk, which is beneficial to offspring intestinal development and immunological tolerance.

Consistent with these findings, our study suggested that higher concentrations of breast milk-derived sIgA significantly reduced serum LPS, IFN-γ, and TNF-α levels in ETEC-infected weaned mice and alleviated ETEC-induced systemic inflammation and intestinal barrier damage. These protective effects appear to be driven by LR -mediated enhancement of endogenous maternal sIgA transport and early-life exposure of offspring to passive sIgA, which support intestinal development and immunological tolerance in the offspring.

Conclusions

In summary, our results suggest that NX sows had higher colostrum immunoglobulins production, which might be associated with the maternal gut LR abundances. Subsequently, we isolated the LR from the feces of NX pigs and identified its beneficial effects on pregnant mice. Administration of LR derived from NX sows enhanced the sIgA production colostrum and the ileum and improved the ability of offspring to resist immune stress. While our findings demonstrate that LR supplementation promotes an intestinal immune-tolerant environment and enhances mammary gland homing signals, definitively identifying the specific immune cell subsets mediating this LR-induced activation and dissecting the underlying molecular mechanisms remain pivotal priorities for future research. Overall, our findings support the potential application of LR isolates from the Ningxiang pig gut as probiotics to improve intestinal health in maternal and offspring.

Methods

Sows and sample collection

All experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (China) and approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University (approval number: CACAHU 20240118). A total of nine NX sows and nine Large White (LW) sows of the same parity were maintained in standardized commercial farms specific to each breed, receiving breed-specific diets and farm-specific management, while all sows were confirmed to be healthy and matched for physiological stage. Fecal samples from rectum and colostrum samples from first teat were collected at onset of parturition and were immediately snap-frozen in liquid nitrogen and stored at -80 °C for further analysis.

Isolation and culture of the bacterial strain of LR from NX pig fecal samples

To increase the probability of successful strain isolation, fecal samples from multiple individual NX pigs were promptly combined into a single composite sample, thoroughly homogenized, and subjected to downstream isolation procedures. Briefly, 1 g fecal samples were suspended in normal saline solution and serially diluted to 10^− 6~10^− 8. And 100 µL diluted samples were plated anaerobically on MRS agar under anaerobic conditions at 37 °C for 24 h.

The absorbance of the bacterial solution at OD₆₀₀ was measured using a microplate reader (Infinite M PLEX, TECAN) at 2-hour intervals for 24 h. Bacterial solution (1 mL) was suspended in saline solution and serially diluted to 10^− 5~10^− 8. 200 µL diluted samples were plated anaerobically on MRS agar to count the number of LR. The plates were incubated at 37 °C for 24 h in an anaerobic condition.

Presumptive LR colonies (morphologically characterized as white, circular, convex, Gram-positive rods) were sub-cultured thrice on fresh MRS agar for purification. The 16 S rRNA gene of the single strain was amplified using two universal primers 27 F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGCTACCTTGTTACGACTT-3’) and sequenced by the Sanger method. The 16 S rRNA gene sequences were then aligned to the NCBI nucleotide sequence database to determine LR strains. The isolated strain was 99.86% sequence identity with Lactobacillus reuteri strain DSM 20,016.

Mice experiment design

As shown in Fig. 3, healthy 7-week-old BALB/C mice were purchased from Bo Rui Xin Biotechnology Co., Ltd. Female mice were naturally mated with males, and the presence of a vaginal plug was designated as gestational day 0.5 (GD 0.5). Due to the need to euthanize a subset of mice immediately after parturition while allowing the remaining mice to nurse their offspring until weaning, a total of 32 pregnant mice were randomly assigned to two groups(n = 16 per group) and fed either a standard chow diet (CON) or a diet supplemented with Lactobacillus reuteri (LR, 10¹⁰ CFU/kg). Dietary intervention for 8 pregnant mice per group began on GD 0.5 and continued throughout pregnancy. After parturition, the animals were anesthetized and euthanized by cervical dislocation. Ileal tissues were collected and fixed in 4% paraformaldehyde for subsequent immunofluorescence analysis. Colostrum, mammary gland tissue, ileal tissue, and colonic chyme were collected, snap-frozen in liquid nitrogen, and stored at − 80 °C for later analyses.

For the remaining 8 dams in each group, dietary intervention was administered from GD 0.5 until lactation day 7 (LD 7). From LD 7 to weaning (LD 21), all dams were fed a standard chow diet. To evaluate microbial transmission, fecal samples were collected from dams at parturition and from offspring at weaning. After weaning, healthy male offspring with comparable body weights were randomly selected. These mice were housed individually during the experiment, and the cages were randomly positioned under identical environmental conditions. From the CON group, two male offspring per litter (n = 16 total) were allocated into two subgroups (n = 8 each): CON + PBS group received 100 µL of PBS via oral gavage, and the CON+ETEC group received 10⁸ CFU/mouse of the enterotoxigenic Escherichia coli (ETEC, O149:K91, K88ac) suspension (100 µL, 10⁹ CFU/mL) via the same route. From the LR group, one male offspring per litter (n = 8 total) was assigned to the LR+ETEC group and administered 10⁸ CFU/mouse of the ETEC suspension (100 µL) via oral gavage. Serum and ileal tissues were collected 24 h post-infection for the assessment of endotoxin levels, cytokine profiles, and intestinal barrier function.

DNA extraction and 16 S rDNA gene sequencing

DNA extraction from the NX pig feces and the ileal contents of maternal mice was performed using the CTAB method according to the manufacturer’s instructions. The primers 341 F (5′- CCTACGGGNGGCWGCAG − 3′) and the reverse primer 805R (5′- GACTACHVGGGTATCTAATCC − 3′) were used to amplify the V3–V4 hypervariable region of the 16 S rRNA gene. The PCR products were purified (AMPure XT beads, Beckman Coulter Genomics, Danvers, MA, USA), quantified (Qubit, Invitrogen, USA) and sequenced on Illumina NovaSeq platform (LC-Bio). Paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH. Quality filtering on the raw reads were performed under specific filtering conditions to obtain the high-quality clean tags according to the fqtrim (v0.94). Chimeric sequences were filtered using Vsearch software (v2.3.4). After dereplication using DADA2 and obtained feature table and feature sequence.

Quantification of bacterial load by q-PCR

Genomic DNA from maternal and offspring mice feces was extracted using a Seno fecal genomic DNA extraction kit (China). The quantities of LR bacterial species present were analyzed via quantitative real-time PCR. A standard curve was created by measuring Cq values for dilutions of the template DNA concentration, ranging from 10⁹ to 10¹ copies/µL and the copy numbers of the target bacteria were calculated.

Quantitation of immunoglobulins

Colostrum was centrifuged for 10 min at 4 °C, and supernatants were collected. The mammary gland tissue was homogenated in PBS. Immunoglobulins A (IgA) and secretory immunoglobulin (sIgA) were measured using ELISA Quantitation Kits (Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer’s instruction.

Serum parameters assay

Serum lipopolysaccharide (LPS) was measured with the Endotoxin Test Kit (Kinetic Chromogenic Assay) (Xiamen Bioendo Technology co., Ltd., China) according to the manufacturer’s instructions.

Serum cytokines (IL-4, IFN-γ, TNF-α, IL-2, IL-6, IL-10) were measured using cytometric bead array with the Mouse Th1/Th2/Th17 CBA Kit (BD Biosciences, USA).

Western blotting

The mammary gland tissue was washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (P1048, Beyotime, China). Protein concentration was quantified using a bicinchoninic acid protein assay kit (P0012, Beyotime, China), and an equal amount of protein was subjected to SDS–polyacrylamide gel electrophoresis. Next, proteins were transferred onto polyvinylidene difluoride membranes, followed by incubation with primary and secondary antibodies (AF2800, HAF017, R&D Systems, America), and visualized using chemiluminescent reagent (P10300, NCM Biotech, China). The optical density of the signals on the film was quantified by ImageJ software.

Real-time PCR

Total RNA was extracted from mammary glands, ileal and colonic tissues using Trizol reagent (Takara, Japan). The concentration of each RNA sample was determined using Nanodrop One (Thermo Fisher Scientific, USA). cDNA was synthesized from 1000 ng total RNA using Premix Taq (Takara, Japan). RT-PCR was conducted using a commercial kit (TB Premix Ex Taq, Takara) by real-time PCR instrument (LightCycler480II, Roche, Germany) using the following temperature profiles: one cycle at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing at 60 °C for 60 s. Sample quantification cycle (Cq) values were normalized against that of the reference gene, β-actin. Fold changes compared with the control group were then calculated using the 2^−ΔΔCq method.

The primer sequences of interleukin-1β (IL-1β), interleukin-10 (IL-10), interleukin-13 (IL-13), tumor necrosis factor-alpha (TNF-α), transforming growth factor-β (TGF-β), Interferon-gamma (IFN-γ), polymeric immunoglobulin receptor (pIgR), C-C motif chemokine ligand 28 (CCL28), E-cadherin, occludin, zonula occludens-1 (ZO-1), claudin-1, and β-actin are listed in Supplementary Table 1.

Immunofluorescence analysis

The fixed, permeabilized, and sliced ileal samples were blocked with Quickblock blocking buffer for Immunol-staining (Beyotime), followed by incubation successively with primary and secondary antibodies. Stained tissues were viewed using a confocal fluorescence microscope (Zeiss).

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Normality was assessed using the Shapiro–Wilk test. For normally distributed data, comparisons were performed using two-tailed Student’s t-test (two groups) or one-way ANOVA followed by Tukey’s post hoc test (multiple groups). For data that did not meet normality assumptions, the Mann–Whitney test (two groups) or Kruskal–Wallis test followed by Dunn’s post hoc test (multiple groups) was applied. P < 0.01 indicated that the difference between groups was extremely significant, P < 0.05 indicated that the difference between groups was significant, and P > 0.05 indicated that the difference between groups was not significant. Correlation coefficients (Spearman’s p) were calculated, and statistical significance was determined using two-tailed p-values. The data were processed and analyzed by SPSS 23 software. Data statistical results were plotted by GraphPad Prism 8 software.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

LW pigs

Large white pigs

NX pigs

Ningxiang pigs

LR

Lactobacillus reuteri

ETEC

Enterotoxigenic Escherichia coli

LPS

Lipopolysaccharide

sIgA

Secretory immunoglobulin A

IgA

Immunoglobuli A

IL-1β

Interleukin-1β

IL-10

Interleukin-10

IL-13

Interleukin-13

TNF-α

Tumor necrosis factor-alpha

TGF-β

Transforming growth factor-β

IFN-γ

Interferon-gamma

pIgR

Polymeric immunoglobulin receptor

CCL28

C-C motif chemokine ligand 28

ZO-1

occludens-1

Author contributions

Qian Xie: Data curation, Visualization, Writing - Original Draft. Pengyi Tian: Writing - Review & Editing. Qing Duanmu: Methodology. Mei Yang: Validation. Chen Zhang: Supervision. Jing Wang: Supervision, Conceptualization. Bi E Tan: Conceptualization, Funding acquisition.

Funding

This work was supported by the Natural Science Foundation of Hunan Province (2024JJ1004) and Yuelushan Laboratory Seed Industry Special Project (YLS-2025-ZY02026).

Data availability

The sequences generated in this study are available in the NCBI Sequence Read Archive database (Accession Number: PRJNA1142906).

Declarations

Ethics approval and consent to participate

All experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (China) and approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University (approval number: CACAHU 20240118).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.