Early colonization of the intestinal microbiome of neonatal piglets is influenced by the maternal microbiome

doi:10.21203/rs.3.rs-2950703/v1

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Early colonization of the intestinal microbiome of neonatal piglets is influenced by the maternal microbiome

https://doi.org/10.21203/rs.3.rs-2950703/v1

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The intestinal microbiome plays a crucial role in animal health and growth by interacting with the host, inhibiting pathogenic microbial colonization and regulating immunity. This study investigated dynamic changes in the fecal microbial composition of piglets from birth to weaning and the relationship between piglet fecal microbiome and sows. Feces, skin, amniotic fluid, and vaginal samples were collected from eight sows and sixty-three piglets, and the 16S rRNA gene was sequenced. The results revealed that the Firmicutes, Bacteroidetes, and Proteobacteria dominated the piglets’ microbiome in their early stages, and Firmicutes and Bacteroidetes were crucial to maintaining a balance in the intestinal microbiome during nursing. Abundances of Christensenellaceae_R-7_group, Succinivibrio, and Prevotella increased in weaned piglets fed solid feed. Analysis of the microbiome from sows to piglets indicated a shift in the microbiome colonizing piglet intestines and became a significant constituent of the piglet intestinal microbiome. This study supports the theory that the neonatal intestinal microbiome is vertically transmitted from the mother. Further research is needed to integrate sows, piglets, and their environments to gain a better understanding of the early establishment of the piglet intestinal microbiome.

Biological sciences/Biotechnology

Biological sciences/Microbiology

Microbial complexes and communities, known as microbiota, coexist in the intestines of animals¹. The intestinal microbiome plays a crucial role in the health and growth of the host by interacting with it. It can inhibit pathogenic microbial colonization, produce short-chain fatty acids (SCFA), and regulate immunity^2,3. Scientists have recognized the importance of bacterial colonization of the gastrointestinal tract and its metabolic and protective function⁴.

The question of whether the fetal intestine and maternal uterus harbor a microbiome prior to delivery remains controversial⁵. However, several studies have reported that the colonization and establishment of the newborn intestinal microbiome likely begins prior to birth, as exposure to the microbiome can occur through the placenta, fetal membranes, and amniotic fluid^4,6. Exposure to colonizing bacteria continues at birth and during the first year of life, and profoundly influences lifelong health⁷.

Establishing a beneficial microbiome is critical during the weaning stage because early colonization by beneficial bacteria in the intestinal microbiome is essential for normal intestinal and immune system function^8,9. It is well-known that breastfeeding, and exposure to the external environment affect early intestinal microbial formation during the weaning period^6,10−13. As piglets are housed in the same space as their mothers until weaning, vertical transmission of the microbiome from their mothers plays an essential role in the development of the intestinal microbiome of newborns during the early growth stages^14,15. The abrupt change in diet from breast milk to more complex nutrient sources, such as formulated feed, is a major factor influencing shifts in the microbiome of piglets during weaning. The composition of the intestinal microbiome may change with shifts in the piglets’ diet.

A portion of the early intestinal microbiome derived from sows is retained despite changes and growth in various environmental factors such as changing hog barn, feed, water, stocking density, management and temperature. A balanced intestinal microbial environment is crucial for piglet growth and health⁹, which underscores the importance of the early microbial community derived from sows. Therefore, it is necessary to investigate the changes in the fecal microbial composition of piglets during the lactation period when piglets live with sows. This study investigated dynamic changes in the fecal microbial composition of piglets during the lactation period. Additionally, we examined the relationship between the fecal microbiome of piglets and sows and their environment from birth to weaning.

16S rRNA gene sequencing for species and strain-level microbiome analysis

We investigated the origin of the microbes obtained from the fecal samples of 63 healthy piglets born to 8 sows on a commercial pig farm. Samples were collected 1, 7, 14, 21, and 28 d after birth. Fecal (rectal swabs), vaginal (vaginal introitus swabs), skin (areolar skin swabs), amniotic fluid (newborn intraoral swabs), and milk samples were collected from each sow, and fecal (rectal swab) samples were collected from each piglet (Fig. 1a). Microbial DNA was extracted from 355 samples, and the V3-V4 region of the 16S rRNA gene was sequenced. After removing the chimeras and filtering for quality (Q-score > 25), 17, 874, 519 high-quality reads were obtained.

Microbial diversity in piglets from birth to weaning

To characterize the intestinal microbiome of piglets during the early growth period, we compared the relative abundances of major bacterial phyla. The dominant phyla observed in piglets during all growth periods were Firmicutes, Bacteroidetes, and Proteobacteria. The relative abundance of Firmicutes in the microbiome of piglets was high at approximately 79% immediately after birth (day 1) but decreased to approximately 57% by day 28. In contrast, Bacteroidetes had a low distribution of approximately 5% in the fecal samples, but gradually increased from day 7 onwards and reached approximately 25% by day 28. The relative abundance of Proteobacteria was approximately 9% immediately after birth, which increased to approximately 20% on day 7, and gradually decreased (Fig. 1c).

The core populations of bacteria present in all period groups of piglets included Clostridium sensu stricto 1, Turicibacterm, Romboutsia, Streptococcus, and Bacteroidetes. These were identified as significant constituents of the intestinal microbiome, with a relative abundance of over 1% (Supplementary Table S1, Fig. 1d). The relative abundance of Streptococcus, Lactobacillus, Escherichia-Shigella, and Bacteroidetes increased on day seven, whereas that of Succinivibrio, Christensenellaceae R-7 group, and Prevotella increased on day 21 (Fig. 1b).

The source and shared microbiome in piglets from birth to weaning

We compared the microbial abundance and diversity of the intestinal microbiome of piglets from birth to weaning. Alpha diversity was the highest on day 1 and lowest on day 7 in the feces, but gradually increased as the piglets grew aged. Notably, the piglets showed higher microbial diversity on days 1 and 28 than that of the fecal microbiome of the sows (p < 0.05, Fig. 2a). To examine the similarity of microbial communities between piglets and sows, we performed a principal coordinate analysis (PCoA) based on the Bray-Curtis distance. Our results showed that the microbial composition in the intestines of piglets changed over time, with the microbial community on day 1 showing an apparent difference from that in the other periods (7, 14, 21, and 28 d after birth). Furthermore, on day 1, the microbial community of the piglets was more similar to that of the sows compared to the other periods (Fig. 2b).

Transmission of the maternal microbiome from sows to piglets

This study aimed to investigate the effect of the vertical transmission of the maternal microbiome on the formation of the intestinal microbiome in piglets. The analysis was conducted on piglets at two stages: those born with a significant influence of the maternal microbiome on day 1, and those that were substantially affected by environmental factors on day 28. SourceTracker analysis was used to determine the proportion of microbial communities derived from maternal sources at each stage.

The results showed that on day 1, the piglets had a higher proportion of maternal microbiome (average 80%) than that on day 28 (average 37%). Specifically, colostrum (34%), skin (26%), and amniotic fluid (10%) contained in the maternal source significantly decreased to 10, 7, and 3% on day 28, respectively. However, maternal feces, which accounted for 6% of piglets’ microbiome on day 1, increased to 10% by day 28 (Fig. 3a). Furthermore, on day 28, piglets had a high proportion of unknown sources, suggesting that the external environment influenced the intestinal microbiome of piglets. To identify the microbiome transferred from sows, shared operational taxonomic units (OTUs) between piglets and their mothers were compared to identify the microbiome transferred from sows. On day 1, piglets shared 2,137 OTUs in the colostrum, 1,936 OTUs in the skin, 1,487 OTUs in the amniotic fluid, 1,289 OTUs in the vagina, and 818 OTUs in feces with their mothers. On day 28, piglets shared 920 OTUs in the colostrum, 887 OTUs in the skin, 748 OTUs in the amniotic fluid, 748 OTUs in the vagina, and 531 OTUs in feces with their mothers (Fig. 3b). Consistent with the SourceTracker analysis, maternal colostrum had the most significant impact on the piglet intestinal microbiome.

To investigate the microbiome transfer from sows to piglets, we compared the 16S rRNA sequences of sows and piglets. The findings revealed that the microbes that differed from that of the mother at birth mainly consisted of the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria. Although the transferred microbiome decreased as the piglets grew, it remained in the intestine even after separation from the sows. The transferred microbiome included significant piglet intestinal microbiota-forming microbes, such as Muribaculaceae, Bacteroidetes, Christensenellaceae_R-7_group, Prevotella, and Lactobacillus (relative abundance > 1%; Fig. 4, Supplementary Table S2).

The intestinal microbiome plays a crucial role in host health by breaking down indigestible fibers and producing crucial amino acids and vitamins^16–20. Several factors, including the mode of nutrient supply, birth environment, and maternal transmission of microorganisms, influence the initial composition of the intestinal microbiome^21,22. In newborns, the microbial community biomass is very low, and the external microbiome is introduced to perform various physiological functions and regulate the intestinal environment²². As the immune system of newborns is not fully developed, the intestinal microbiome supports and protects it from pathogenic infections and diseases ^23,24. Moreover, the intestinal microbiota enhances digestive function in newborns by improving nutrient absorption, inhibiting the colonization of pathogenic microorganisms, and stimulating the development of the intestinal immune system^25–27. Therefore, intestinal colonization by beneficial microbiomes is essential during the early stages of life. This study aimed to investigate the changes in the intestinal microbial community of piglets from birth to weaning and to elucidate the influence of the mother’s microbiome on the formation of the piglet’s intestinal microbiome.

Shortly after birth, the microbiome of piglets undergoes rapid changes owing to exposure to the external environment. In the present study, the microbiome of the piglets in the early stages was dominated by Firmicutes, Bacteroidetes, and Proteobacteria. Firmicutes play a vital role in maintaining the intestinal microbiome balance in newborns²⁸. Lactobacillus, which is mainly classified as a beneficial bacterium, has been reported to perform various physiological functions such as aiding digestion and supporting the immune system^29,30. Bacteroidetes are also dominant in the intestinal microbiome of newborns; Bacteroidetes use the lactose in breast milk as an energy source^31,32. The abundance of Bacteroidetes increased from days 1 to 7 in piglets fed only breast milk and dramatically decreased from days 14 to 28 during the weaning period. Additionally, the abundances of Streptococcus, Lactobacillus, Escherichia-Shigella, and Bacteroidetes increased on day 7, which is consistent with the findings of previous studies^33–35. Escherichia-Shigella is a microbe that causes diarrhea and indigestion in newborns, and colonizes the piglet intestine through the mucus in the birth canal^36–38. Previous studies have shown that breast milk can inhibit the colonization of Escherichia-Shigella in piglets by supplying immune cells, promoting the growth of beneficial bacteria, and inhibiting pathogen-intestine binding³⁹. Streptococcus and Lactobacillus are the dominant bacteria found in sow milk and are transferred to piglets via suckling^14,40. In piglets fed solid feed, on day 21, the abundance of Christensenellaceae_R-7_group, Succinivibrio, and Prevotella increased. These are towards the utilization of plant-based carbohydrates.

The findings of this study revealed that the piglets had a highly diverse intestinal microbiome from day one. Interestingly, these findings are in line with those of Collado⁴ and Aagaard⁶, who suggested that the colonization and establishment of the intestinal microbiome of newborns begins prior to birth. This is because the mother’s microbiome is transmitted to the fetus through the placenta, fetal membranes, and amniotic fluid.

The diversity of the intestinal microbiome in piglets decreased rapidly within the first week after birth, but gradually increased until 4 weeks. This suggests that various microbes were introduced into the intestines of neonatal piglets and only host-specific microbes were retained. As piglets live with sow before weaning, they are greatly influenced by the maternal microbiome. Thus, the microbial composition in piglet feces on day 1 was closely related to the microbiome of the mother’s milk, skin, and amniotic fluid, indicating that piglets are most influenced by the maternal microbiome during delivery (Fig. 2b). On day 1, the neonatal piglets consumed sow milk, suggesting that breast milk influenced the composition of the gut microbiome in piglets. The placenta produces amniotic fluid during fetal growth, which is vital to fetal health and development. The interaction between the microbiome, neonatal intestine, and amniotic fluid may affect the growth of the intestinal microbiome along with the nutrients provided to the fetus through the placenta^6,41. Our results also confirmed that the microbial compositions in the feces and amniotic fluid on day 1 were closely related. This finding is consistent with those of previous studies that reported that the microbiome detected in the feces of piglets on day 1 was the same as that in the amniotic fluid to which piglets were exposed in utero⁴².

The analysis of the influence of the sow-derived microbiome on the feces of piglets revealed that breast milk was the primary source of the microbiome in the intestines of piglets immediately after birth. This finding is consistent with the results obtained by comparing the microbiome composition of piglets and sows (Figs. 2b and3). Previous studies have also reported that breast milk plays the most crucial role in the formation of the piglet intestinal microbiome immediately after birth, with its contribution decreasing over time⁴⁴. We confirmed that the microbiome was transferred from sows to piglets and was retained until the post-weaning period. The microbiome transferred from sows to piglets consisted of significant components of the piglet intestinal microbiome. The microbial communities shared between sows and piglets are likely to interact and contribute to the health of both^45,46. Microbiomes transferred from the maternal intestine to the piglet intestine after birth likely establish early colonization and become significant constituents of the piglet intestinal microbiome. Our results support the theory that the neonatal intestinal microbiome is vertically transmitted from the maternal intestine. Although our study focused on piglets and their relationships with sows, it is essential to consider the influence of the pig farm environment. Additional studies that integrate sows, piglets, and their environments are necessary to gain a better understanding the early establishment of the piglet intestinal microbiome.

Our findings suggest that the initial intestinal microbiome is important because the bacteria derived from the sows that colonize the gastrointestinal tract of piglets maintain a constant ratio despite growth and changes in various environmental factors. This insight could further our understanding of the formation of the early microbial community in the intestines of piglets that receive microbes from sows and its impact on the health and robustness of piglets. Further research is needed to integrate sows, piglets, and their environments to gain a better understanding of the early establishment of the piglet intestinal microbiome as Identification of bacterial species.

Ethics statement

The experimental protocol used in this study was approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (permit number:2021-001). The experiment was conducted at a commercial pork production facility (Gimje-si, Republic of Korea). This study was carried out in compliance with the ARRIVE guidelines

Animal experiments

Eight sows (Landrace × Yorkshire) were selected for this study, and 63 infant piglets (Landrace × Yorkshire × Duroc; 1.5 ± 0.3 kg of average initial body weight) were cohoused with the sows by litter. Each piglet was ear notched for individual identification and dosed with a single intramuscular injection of Iron-Dextran and vaccine (circovirus and mycoplasma), following standard swine husbandry practices. The infant pigs were allowed to nurse freely until weaning after 21 d with creep feed.

Sample collection

Fecal, skin, amniotic fluid, and vaginal samples were collected rectally from each animal using a sterile NB bio microbial transport medium (NBG-2, Noble bio, Korea) wet with sterile phosphate-buffered saline (pH 7.0) 1, 7, 14, 21, and 28 d after birth. The samples were stored at − 80°C until DNA extraction.

Microbial DNA extraction and 16S rRNA gene sequencing

To analyze the microbial communities in sows and piglets, microbial DNA was extracted from the samples. The supernatant was removed by centrifugation at 10,000 × g for 10 min to obtain microbes in the transport medium. DNA was extracted from the collected microbes using a QIAGEN DNeasy PowerSoil Pro Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The DNA concentration and purity were determined using a SpectraMax Plus 384 spectrophotometer (Molecular Devices, USA). The extracted DNA was stored at − 80°C until subsequent analysis.

The extracted DNA was amplified using primers (341F/806R) specific to the hypervariable regions V3-V4 of the 16S rRNA gene. The amplification program consisted of 1 cycle at 95°C for 3 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final process at 72°C for 5 min. The PCR products were purified using AMPure XP beads (Beckman Coulter, Nyon, Switzerland) to remove free primers and primer dimers. A Nextera XT Index kit (Illumina, San Diego, CA, USA) was used to attach the index sequences to the purified PCR products and create libraries. DNA quality and product size were measured using a DNA 7500 chip and Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). The DNA libraries were sequenced using the Illumina MiSeq platform.

Microbial data analysis

The microbial community analysis was performed using the QIIME2 software (Quantitative Insights into Microbial Ecology, version 2020.8)⁴⁷. The DADA2 program was used to remove chimeric reads and low-quality sequences (quality score < 25) to obtain high-quality sequences, and amplicon sequence variants (ASVs) were classified using the dada2-denoise-paired method^48,49. ASVs were classified based on the Silva 138SSU reference database and a phylogenetic tree was constructed using a fast-tree algorithm⁵⁰. The Jvenn program (http://bioinfo.genotoul.fr/jvenn/example.html) was employed to visualize the number of communal and differential microbiomes between different growth periods of the piglets⁵¹. The alpha diversity of the microbial community was calculated using the Shannon diversity index and observed features to evaluate microbial diversity and richness. Principal coordinate analysis (PCoA) was conducted using the Bray-Curtis distance to measure the distances between the microbial compositions of the samples. The microbial diversity results were visualized using the phyloseq package in R software⁵².

Statistical analysis

The Wilcoxon test was used to calculate p-values. The Bayesian community-wide microbial source tracking algorithm SourceTracker was used to estimate the contribution of sow influence on piglet intestinal microbiome formation⁵³. The algorithm determines the proportion of each “source” (sow fecal, skin, amniotic fluid, and vaginal samples) that contributes to the “sink” (piglet fecal sample).

Acknowledgments

We would like to thank Editage (www.editage.co.kr) for English language editing.

Author contributions

J.I: Visualization, Writing-Original Draft, Investigation; J.C: Data Curation, Investigation, Formal analysis; N.K, Y.L: Resources, Software; M.S, S.J: Validation, Methodology; D.K ([email protected]) is responsible for the integrity of this work (Conceptualization, Visualization, Supervision, Writing-Review & Editing, Project administration).

Ethics approval and consent to participate

The experimental procedures were conducted in accordance with the regulations of National Institute of Animal Science (permit number: 2021-001). This study was carried out in compliance with the ARRIVE guidelines.

Data Availability Statement

The datasets used in this study are available from the corresponding author upon reasonable request.

Funding

This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ01581401), Rural Development Administration, Republic of Korea.

Competing Interests Statement

The authors declare no competing interests

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No competing interests reported.

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Early colonization of the intestinal microbiome of neonatal piglets is influenced by the maternal microbiome

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Abstract

Figures

Introduction

Results

16S rRNA gene sequencing for species and strain-level microbiome analysis

Microbial diversity in piglets from birth to weaning

The source and shared microbiome in piglets from birth to weaning

Transmission of the maternal microbiome from sows to piglets

Discussion

Methods

Ethics statement

Animal experiments

Sample collection

Microbial DNA extraction and 16S rRNA gene sequencing

Microbial data analysis

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

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