Integrated analyses of the intestinal microbiome and transcriptome in NingXiang piglets

doi:10.21203/rs.3.rs-3888036/v1

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Integrated analyses of the intestinal microbiome and transcriptome in NingXiang piglets

https://doi.org/10.21203/rs.3.rs-3888036/v1

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Background

NingXiang (NX) pig has been characterized as one of the best Chinese indigenous pig breeds, with high fertility and disease resistance.

However, its intestinal gene expression and gut microbial characteristics have not been studied.

Results

Here, we delineated and compared the profile of intestinal microbiome and transcriptome between NX and Duroc × Landrace × Large white (DLY) piglets by integrating metagenomic and transcriptomic analyses. We found that the abundance of several colonic Bacteroides species (B. fragilis, B. thetaiotaomicron and B. sp_PHL_2737), Prevotella species (P. intermedia, P. dentalis and P. enoeca), Clostridium species (C. scindens, C. butyricum and C. botulinum), Anaerrobutyricum and Lactobacillus_saerimneri are significantly increased in NX piglets than DLY piglets. Additionally, several archaea and fungal species were also dominantly enriched in the colon of NX piglets. Then, we identified some unique colonic bacterial species that may act as characteristics of these two pig breeds. Functional analyses of NX piglets-enriched microbes revealed their dominant function in methane, glycolysis and gluconeogenesis metabolism. Strikingly, the composition and function of the gut microbiota were significantly correlated with growth performance both in NX and DLY piglets. Finally, our mRNA-sequencing revealed the distinct intestinal gene expression pattern between NX and DLY piglets, and NX piglets exhibit improved intestinal barrier function and varied immune features than DLY piglets.

Conclusion

Together, our study revealed the characteristics of the gut microbiota and intestinal gene expression in NX piglets, providing the potential to explore its mechanisms in disease resistance and gut development.

NingXiang Pig

Intestinal transcriptome

Gut microbiome

Pig farming is influenced by both external and internal factors including breeds, nutrition levels, production systems and post-slaughter manufactures, among which the breeds act as a key factor affecting animal husbandry (Rosenvold and Andersen, 2003). Until now, the Duroc × Landrace × Large white (DLY), a typical lean-type European pig breed, has been well accepted as the most prevalent commercial pig breed for swine production, ascribed to its fast growth rate, high quality of meat and feed conversion ratios. However, the DLY piglets have also been recognized to be susceptible to environmental stimulus, increasing the diarrheal incidence and causing economic loss. Recent years, a variety of indigenous pig breeds have attracted researchers’ attentions, due to their dominant function in disease-resistance and high digestibility of dietary fibers. Chinese indigenous breeds, such as Chuanzang black pig, Tibetan pig, Liang-Shan pig, and Laiwu pig have been reported to exhibit improved environmental adaptability as compared to DLY pig (Ramayo-Caldas et al., 2012; Hua et al., 2014; Li et al., 2023; Liu et al., 2023). Therefore, it is of great importance to dissect their potential mechanisms in disease resistance and dietary functions.

The NingXiang (NX) pig is originated from Hunan province and have been approved by the Ministry of Agriculture as the geographical representation of Chinese agriculture products and acts as the important national livestock resource in China (Jiang et al., 2018; Gong et al., 2021). NX pigs exhibit excellent meat quality and flavors, with the high enrichment of unsaturated fatty acids and intramuscular fat in longissimus dorsi muscle when compared to commercial pig breeds (Feng Z.M. et al., 2012; Gong et al., 2021). The gut microbiota has been shown to act as the core effector to improve livestock production and increase economic benefits (Bergamaschi et al., 2020; Gardiner et al., 2020; Maltecca et al., 2020). A variety of small metabolites secreted by gut microorganisms regulate various biological process in the intestinal epithelial cells (Markowiak-Kopec and Slizewska, 2020). Conversely, the host genetic background also determines porcine intestinal function through modulating the composition and diversity of the gut microbiota (Yang et al., 2022). Thus, it is likely that both the gut microbiota and intestinal genetic features affect the gut health, which in turn largely affects the production values between commercial and indigenous pig breeds. The intestinal transcriptome and microbiome in DLY piglets have been well documented (Yang et al., 2017; Quan et al., 2019). While the characteristics of the gut microbiota and intestinal gene transcriptional pattern in NX pigs under normal feeding conditions are still a mystery.

Here, our study unraveled the detailed intestinal transcriptomic and metagenomic traits in NX piglets, and also compared these features with DLY piglets by integrating gut microbiome and transcriptome data. We found the distinct colonic microbial enrichment between NX and DLY piglets, with the increased abundance of several Bacteroides, Prevotella and Lactobacillus species in NX piglets, and the enrichment of some Streprococcus species and Megasphaera_elsdenii in DLY piglets. These distinct gut microbial species affected intestinal functions and displayed significant correlations with growth performance in these two breeds. Furthermore, RNA-seq and qPCR data revealed that NX piglets exhibit improved intestinal barrier function and varied immune reactions than DLY piglets, implying its dominant function in disease resistance and gut development.

ARRIVE guideline statement

The experimental details of animal research conducted in this study meets the requirements from the ARRIVE guidelines published in PLOS Biology doi:10.1371/journal.pbio.3000411 are described as follows:

Ethical statement

Ethics approval and consent to participate the animal study was reviewed and approved by Institution Animal Care and Use Committee of college of Animal Science and Technology, Hunan Agricultural University (No.43321809) (Changsha, China).

Animals, Experimental design, Diets, and Housing

The total of twenty 28-day-old male Duroc × Landrace × Large white (DLY) weaned piglets (n = 10), weighing 9.1 ± 0.9kg (mean ± SEM), and NingXiang (NX) weaned piglets (n = 10), weighing 7.0 ± 0.5kg (mean ± SEM), were obtained from the Yige Family Farm and Hunan ChuWeiXiang Agriculture and Animal Husbandry Farm (Changsha, China), respectively, and randomly divided into two groups: the NX group and the DLY group, 10 pens in each group, with one piglet per pen. Random numbers were generated using the standard = RAND() function in Microsoft Excel. Each piglet was individually kept in pens in a mechanically ventilated and temperature-controlled room at 22°C-24°C, humidity of 60%-65%, and all piglets had ad libitum access to drinking water and feed. The corn-soybean based diets were formulated to meet the recommended nutritional requirements from National Research Council [NRC,2012] (Table S1) and all piglets were fed to the same diet for 4 weeks after 7 days of adaptation. The experiment lasted for 4 weeks. 8 piglets from each group were slaughtered and then colonic samples were collected immediately after slaughter. Colonic mucosal samples were collected and stored immediately at -80°C until further transcriptomic and RT-qPCR analyses. Colonic chyme were collected and stored at -80°C until metagenomic analyses.

All piglets were fed twice per day and the average daily feed intake and average daily gain were recorded every day and calculated every week through the experimental trial. The visceral organs were collected after slaughter and evaluated by the organ index formula as follows: organ index (%) = (organ weight/body weight) × 100%.

Metagenomic DNA sequencing

The total of 8 colonic microbial DNA samples including bacteria, fungi and archaea from NX (n = 4) and DLY (n = 4) piglets were extracted according to the manufacturer’s instructions. All samples were sequenced on the Dr. Tom (BGI, Wuhan, China) platform. A paired-end library was constructed with 350-bp inserts for each sample. Low-quality reads and reads mapping to pig DNA were removed from the raw data. For taxonomic assignments, the high-quality reads from each sample were aligned against the integrated reference catalog of the Sus scrofa genome (NCBI_GCF_000003025.6_ Sscrofa11.1) and gut microbiome by bowtie 2 using the criterion of “identity > 90%,” genes from the existing reference gene catalog IGC inherited their original taxonomic annotation, and the relative abundance of a taxon was calculated from the relative abundance of its genes. To generate the annotation information, the protein sequences of genes were aligned against the functional database (BacMet, CARD, KEGG, EggNOG, COG, Swiss-Prot, CAZy, etc. ) using DIAMOND (Benjamin Buchfink et al., 2015) with an E value cutoff of 1e-5, based on the Kraken (Pal et al., 2014) LCA algorithm, the taxonomic annotation was assigned. The Nt (202011) database was selected to perform taxonomic annotation. To generate the taxonomic and functional abundance profiles, the Bracken(https://github.com/jenniferlu717/Bracken) software was used with the default settings. Based on the abundance profiles, the features (Genera, Phyla and KOs) with significantly differential abundances across groups were determined using Wilcoxon’s rank sum test (Kanehisa, 2000). Differentially enriched KEGG pathways were identified according to reporter scores (Huerta-Cepas et al., 2019). An absolute value of reporter score of 1.65 or higher was used as the detection threshold for significance. The alpha diversity was quantified by the Shannon index, Chao1 index, and Simpson index using the relative abundance profiles at gene, genus and KO levels with R package, respectively. The beta diversity was calculated using Bray-Curtis distance (Galperin et al., 2015) or Jensen-Shannon Divergence distance. Statistical analysis of Wilcoxon rank test and Kruskal-Wallis H test were calculated using R project.

RNA-seq analysis

The total of 8 colonic mucosal RNA from NX (n = 4) and DLY (n = 4) piglets were isolated and purified using TRIzol reagent (TaKaRa, Biomedical Technology Co., Ltd., Beijing, China) following the manufacturer’s procedure. The raw data was filtered with SOAPnuke (v1.5.2) (Chen et al., 2018) by 1) Removing reads containing adapters (adapter contamination); 2) Removing reads whose unknown base ('N' base) ratio was more than 10%; 3) Removing reads whose low-quality base ratio (Base quality less than or equal to 15) was more than 50%, afterwards clean reads were obtained and stored as FASTQ format. Then clean data were mapped to the reference genome by HISAT (v2.1.0) (Kim et al., 2015) and mapped to the assembled unique gene by Bowtie2 (v2.2.5). The expression level of genes was calculated by RSEM (v1.2.8) (Li and Dewey, 2011). Seven major functional databases (KEGG, GO, NR, NT, SwissProt, Pfam, and KOG) were selected to annotate assembled Unigene between NX and DLY samples. Within-group differential gene analysis was performed using DESeq (Wang et al., 2010) under the conditions of Fold Change ≥ 2 and Adjusted P value ≤ 0.001. Between-group differential gene analysis was performed using PossionDis under the conditions of Fold Change ≥ 2 and FDR ≤ 0.001.

RT-qPCR

Briefly, the total RNA was isolated and purified from colonic mucosa using TRIzol reagent (Takara Biomedical Technology Co., Ltd., Beijing, China) and gDNA Eraser Kit (Takara Biomedical Technology, Beijing, China). Then 1 ug of total RAN of each sample was used for 20ul of cDNA synthesis using the PrimeScript™ RT reagent Kit following the manufacturer’s protocol. Next, transcripts of interests were amplified using SYBR Green mix kit (Takara Biomedical Technology, Beijing, China) on the LightCyclerR480II platform (Roche). The relative mRNA expression of the selected genes were measured in at least three biological replicates and normalized to GAPDH or beta-actin. Primer sequences were listed in Supplementary Table S1.

Statistical analysis

Statistical significance was assessed using the unpaired Student’s t-tests in pairwise comparisons, as shown in related figures via using the SPSS statistical software. The mean and standard error of the mean (SEM) was used to denote error bars. Statistical significance was calculated based on the P-value (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001).

The growth performance does not statistically differ between DLY and NX piglets

To compare the growth performance between NX and DLY piglets, we measured the average daily feed intake (ADFI), average daily gain (ADG), feed conversion ratio (FCR) and organ index in the trial. As shown in Fig S1A, there was no significantly difference in ADFI between NX and DLY piglets, despite the DLY piglets exhibited the trend of higher ADFI than NX piglets. Regarding the ADG between DLY and NX piglets, DLY piglets showed a significantly increased ADG than NX piglets in the last week of the trial (Fig S1B). While the FCR and the organ index (organ weight/ body weight) between DLY and NX piglets did not show statistically significant p value (Fig S1C and S1D).

Pig breeds exhibit distinct colonic microbial characteristics

To compare the structure and function of gut microorganisms between NX and DLY piglets, we performed metagenomic analyses in the colon of these two breeds. Alpha-diversity analyses indicated that the DLY piglets consist of the higher total number of gut microorganisms in the colon when compared to NX piglets (Fig. 1A). The species diversity of gut microorganisms did not significantly differ between DLY and NX piglets which were measured by using Simpson and Shannon indices. While NX piglets manifested the trend of higher gut microbial species and richness than DLY piglets in the colon (Fig. 1A). There was a significantly difference between DLY and NX piglets in compositional similarity measured by Beta-diversity as shown in Fig. 1B.

To further uncover the distinct gut microbial composition between DLY and NX piglets, we analyzed the taxonomic composition of the colonic microbiota at different levels. DLY piglets had much more archaea distribution in the colon than NX piglets at the Kingdom level (Fig S2A and S3A). The phylum-level bacteria analyses found that the abundance of Bacteroidetes was greater in NX piglets than DLY piglets, while DLY piglets showed an increased abundance of Spirochaetes, Proteobacteria, Euryarchaeota and Fimicutes in the colon when compared to NX piglets (Fig S2B and S3B). At the Class level, NX piglets displayed a higher abundance of colonic Clostridia, Erysipelotrichia and Bacteroidia than DLY piglets (Fig S2C and S3C). Furthermore, the abundance of Clostridiales, Bacillales, Erysipelotrichales, Bacteroidales, Flavobacteriales and Enterobacterales were significantly increased in the NX piglets than DLY piglets at the order level (Fig S2D and S3D). Similarly, the increased abundance of Lactobacillaceae, Bacteroidaceae, Lachnospiraceae, Prevotellaceae, Ruminococcaceae, Clostridiaceae, Peptostreptococcaceae, Enterobacteriaceae, Odoribacteraceae, Rikenellaceae and Erysipelotrichaeae were observed in the colon of NX piglets at the Family level (Fig. S2E and S3E). Regarding the differences in the colonic microbiota between DLY and NX piglets at the Genus level, we found that the abundance of Bacteroides, Roseburia, Prevotella, Phocaeicola, Anaerrobutyricum, Lacobacillus were significantly enriched in NX piglets than that in DLY piglets (Fig. 1C). In a sharp contrast, the abundance of Megasphaera, Streptococcus and Treponema were significantly increased in DLY piglets than that in NX piglets.

NX and DLY piglets colonize different microbial species in the colon

Next, we discriminated the differentially colonized microbes (DCMs) at the species level between DLY and NX piglets, and identified 665 of gut microbial species that are specifically enriched in DLY piglets, and 371 of gut microbial species that are specifically enriched in NX piglets (Fig. 1D and Fig. 2A). Then we sought to compare the dissimilarity of colonic bacteria, archaea and fungi between these two breeds, respectively. Firstly, we evaluated the top 30 DCMs between DLY and NX piglets and found that the abundance of Clostridium species (C. butyricum, C. botulinum and C. perfringens), Bacteroides species (B. thetaiotaomicron and B. fragilis), Prevotella species (P. enoeca, P. intermedia and P. dentalis) and Lactobacillus_crispatus, are dominantly enriched in NX piglets. In contrast, DLY piglets showed the high level of colonic Streptococcus species (S. lutetiensis, S. infantarius, S. equninus, S. pasteurianus and S. lutetiensis) and Selenomonas_sputigena (Fig. 2B). Next, we also noticed the varied colonization of archaea between DLY and NX piglets. The abundance of Pyrodictium_delaneyi and Methanohalopilus_halophilus were significantly increased in NX piglet than DLY piglet, while the Halopenitus_persicus, Halolamina_sp_CBA1230, Halorubrum_sp.PV6 Halarchaeum_sp.CBA1220 and Methanosphaera_stadtmanae were preferentially colonized in the colon of DLY piglets (Fig. 2C). Lastly the abundance of some fungal species like Tetrapisispora_blattae and Zygosaccharomyces_rouxii were significantly increased in the colon of NX piglets than DLY piglets (Fig. 2D).

To further identify specific colonic bacterial genera that may act as the unique characteristics of these two breeds, LEFSe was used to evaluate the bacterial composition between DLY and NX piglets. Our analyses found that the species of Streptococcus (S. lutetiensis, S. pasteurianus, S. thermophilus, S. gallolyticus and S. infantarius) and Selenomonas_sputigens were specifically enriched in DLY piglets when compared with NX piglets. While the species of Bacteroides (B. fragilis, B. thetaiotaomicron, B. intestinalis and B. sp_PHL2737), Prevotella (P. dentalis, P. ruminicola, P. oris) were exclusively colonialized in NX piglets (Fig. 2E). Together, our analyses suggest that DLY and NX piglets display the vast differences in the colonization of gut microbial species.

NX piglets-enriched gut microbes modulate several metabolic functions

To further examine the function of the gut microbiome between these two breeds, we first assessed DEMs by conducting Kyoto Encyclopedia of Genes and Genomes (KEGG), Cazy, BacMet and Swissprot analyses. As shown in Fig. 3A-3D, all four analyses displayed statistically significant p value (p < 0.05) between NX and DLY piglets. KEGG data showed that the functional categories related to several metabolic pathways such as methane metabolism, fructose and mannose metabolism, glycerolipid metabolism, selenocompound metabolism, purines metabolism and D-amino acid metabolism were significantly enriched in the colon of NX piglets. While the function of DLY piglets-enriched gut microbes were dominant in phenylalanine metabolism, pentose and glucuronate interconversions, and oxidative phosphorylation pathway (Fig. 3E).

Then the Cazy analysis identified a subset of carbohydrate-active enzymes including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) that differ between DLY and NX piglets. There were 20 upregulated GHs and PLs in NX piglets when compared to DLY piglets, among which GH43, GH43_10, PL1, PL1_2 and carbohydrate esterases 12 (CE12) represented the most significantly upregulated ones. By contrast, GH25, GH13_31, GH13_26, GH13_3 and PL0 were slightly up-regulated in DLY piglets than NX piglets (Fig. 3F).

Next, we performed LDA analyses to further unravel the correlations between KEGG and DEMs. Our results showed that the Prevotella species (P.dentalls, P.oris, P.ruminicola) and Bacteroides species (B.fragilis, B.intestinalis, B.sp.PHL_2737 ) were positively correlated with several nutritional metabolic processes, such as amino acid metabolism, energy metabolism, carbohydrate and lipid metabolism. While the Streptococcus species and Acidaminocossus_fementans were positively correlated with xenobiotics biodegeneration and metabolism and nucleotide metabolism (Fig. 3G). Similarly, we also determined the correlations between carbohydrate-active enzymes and DEMs, and found that the Streptococcus species (S. equinus, S. infantanius, S. spuligena, S. pasteurianus) were positively correlated with GT112, GH4 and GH13_26 enzymes. And the activity of GH43, GH43_10, GH43_19, GH127, GH146, PL1 and CE0 were significantly correlated with the Prevotella species (P. dentails, P. ruminiccla) and Bacteroides species (B. thetaiotaomiccron, B. fragilis, B. intestinalis) (Fig. 3H).

Gut microbiota correlates with growth performance both in DLY and NX piglets

The gut microbiota has the ability to modulate nutrition level and physical state, affecting the growth performance of the host. Therefore, we sought to determine whether the gut microbiota correlates with ADG and ADFI in DLY and NX piglets by using Spearman correlation coefficient and Pearson correlation coefficient analyses. Our data found that the Bacteroides species (B.xylanisolvens, B.fragilis) and Clostridium species ( C.butyricum and C.botulinum) were negatively correlated with ADFI and ADG (Fig. 4A-4B), suggesting that the high abundance of Bacterides and Clostridium species may decrease the ADFI and ADG in DLY and NX piglets. Then, we further analyzed the correlations between the gut microbial function and ADFI and ADG. As shown in Fig. 4C-4F, despite the low correlations between the KEGG and ADFI and ADG, or the Cazy and ADFI and ADG were observed, there were still some small categories showing statistically significant p vulues. For instance, the small subunit ribosomal protein S12 (K02950), putative transposase (K07491), large subunit ribosomal protein L16 (K02878), ABC-2 type transport system permease protein (K01992) were negatively correlated with ADG and ADFI. While the ferrous iron transport protein A (K04758), transposase, IS30 family (K07482), large subunit ribosomal protein L33 (K02913), DNA-damage-inducible protein J (K07473) and small subunit ribosomal protein S16 (K02959) were positively correlated with ADG and ADFI (Fig. 4D). In addition, the PLs, such as PL1 and PL1_2, were negatively correlated with ADG, but not ADFI. The group of GH43_10, GH35, GH43, GT14 and GH127 showed negatively correlation with ADFI and ADG. But the PL0, GH29, GH20, GH29 and GH92 were positively correlated with ADG and ADFI, respectively (Fig. 4F). Lastly, the BacMet (a database for antibacterial biocide and metal resistance genes) and Card (the antibiotic resistance database) analyses showed that copper, hydrogen peroxide, penam and lincosamide antibiotic were positively correlated with ADFI, and the manganese was negatively correlated with ADG and ADFI, respectively (Fig. S4A-4D).

Intestinal genes expression pattern differs between DLY and NX piglets

To reveal the global mRNA gene expression pattern in these two breeds, we conducted mRNA sequencing in the colon of DLY and NX piglets. We identified 433 differentially expressed genes (DEGs), with 205 upregulated and 228 downregulated genes in NX piglets than DLY piglets (Fig. S5A). KEGG pathway analyses showed that DEGs are mainly involved in regulating cytokine-cytokine receptor interaction, intestinal immune activity, neuroactive ligand-receptor interaction, and chemokine signaling pathway (Fig S5B). Gene ontology analyses according to “Biological process “of significantly up- or downregulated genes revealed the enrichment of genes involved in carbohydrate binding activity, C-C chemokine activity and cytokine binding and transmembrane transporter activity (Fig S5C).

NX piglets exhibit improved intestinal barrier function than DLY piglets

Intestinal epithelium barrier not only contributes to the gut development but also acts as the core immune system to defend against microbial infections. Then, we compared the colonic barrier function between DLY and NX piglets. There were 30 DEGs related to intestinal barrier function that have been identified in our mRNA sequencing data, with 17 upregulated and 13 downregulated genes in NX piglets when compared to DLY piglets (Fig. 5A). As expected, qPCR experiment confirmed the significantly upregulation of a subset of intestinal barrier related genes like E-cadeherin, WAS (WASP actin nucleation promoting factor), TAD1 (tRNA-specific adenosine deaminase) and HCLS (hematopoietic cell-specific Lyn substrate 1) in the colon of NX piglets, in consistent with our mRNA sequencing data (Fig. 5A). These results indicate that NX piglets have better intestinal barrier function than DLY piglet.

Next, GO and KEGG analyses were further performed to examine the potential function of differentially expressed intestinal barrier genes, respectively. GO data showed that these upregulated genes are enriched in the binding activity of toll-like receptor and lipoteichoic acid receptor, oxidized low-density lipoprotein particle and short-chain fatty acid transmembrane transporter activity (Fig. 5B). While the group of downregulated genes showed dominant binding activity in cell adhesion molecule and protein domain specific factors (Fig. 5D). Moreover, KEGG analyses revealed the enrichment of upregulated genes in tight junction formation, fat digestion and absorption, PPAR (peroxisome proliferators-activated receptors) signaling pathway and cholesterol metabolism and lipid metabolism (Fig. 5C). Downregulated genes were involved in the formation of tight junction and defense against pathogenic Escherichia coli infection (Fig. 5E).

Varied intestinal immune reactions between NX and DLY piglets

Indigenous pig breeds have been shown to be associated with higher capacity of disease resistance. We first compared the expression of antimicrobial peptide genes (AMPs) in NX and DLY piglets. There were 22 differentially expressed AMPs, with 13 upregulated and 9 downregulated genes in NX piglets (Fig. 6A). Furthermore, RT-qPCR confirmed the significantly upregulated expression of regenerating islet-derived protein Ⅲ-γ (Reg-Ⅲ-γ) as well as two AMPs regulators Toll like receptor 1 (TLR1) and myeloid differentiation primary response gene 88 (Myd88)in NX piglets. While the mRNA expressions of beta-defensin 1 (PBD-1) and proline-arginine-rich (PR39) were drastically down-regulated in the colon of NX piglets when compared to DLY piglets (Fig. 6C). Our data suggest that these two breeds have different intestinal AMPs expression pattern and may exert distinct intestinal immune function. Additionally, we also examined the expression of inflammatory genes between NX and DLY piglets and identified 29 differentially expressed genes, with 20 upregulated and 9 downregulated inflammatory genes in the colon of NX piglets (Fig. 6B). Similarly, RT-qPCR validated the increased mRNA expressions of colonic interleukin-2 (IL-2), IL-17F, IL-18 and decreased mRNA expression of IL-8 and IL-22 in NX piglets when compared to DLY piglets (Fig. 6D). By conducting GO and KEGG analyses, we found that these differentially expressed immune responsive genes are mainly involved in modulating cytokine and chemokine activities, CCR chemokine receptor binding activity, and immune responsive signaling pathways including NF-kappaB inhibitor alpha (NF- κB) and TLRs signaling pathways (Fig. 6E-6H).

The gut microbiota has been reported to contribute to the maintenance of gut health by modulating various biological processes including the maturation of intestinal immune system and establishment of proper digestive and absorptive functions (Sender et al., 2016; Schirmer et al., 2019; González Olmo et al., 2021; Hu et al., 2022; Lynn et al., 2022). Distinct gut microbiota colonized in different pig breeds affect gut epithelium development and immune function, contributing to the host growth and defense against infections. In this study, we compared the diversity and composition of the intestinal microbiota and the pattern of intestinal gene transcription between NX and DLY piglets by integrating microbiome and transcriptome analyses. We found varied distribution of colonic bacteria, fungi, and archaea species in these two breeds and further functional analyses revealed that NX piglets-enriched microbes are mainly involved in nutrient and energy metabolism. Furthermore, this study established the correlation between the gut microbiota and growth performance in NX and DLY piglets, respectively. Additionally, by combining RNA-seq and RT-qPCR data, we found that NX and DLY piglets exhibit distinct colonic gene transcription patterns, with the increased expression of intestinal barrier related genes and varied immune responsive genes in NX piglets than DLY piglets. Taken together, this study comprehensively analyzed and compared the characteristics of intestinal microorganisms and gene expression in NX and DLY piglets (Fig. 7), supporting the notion that the gut microbial structures may represent the traits of different pig breeds, and the indigenous pig breeds are generally exhibiting dominant function in gut development and disease resistance.

Many studies have shown that the Bacteroides such as B. thetaioatomicron (Bth) VPI- 5482 strain and B. fragili play critical roles in sustaining the competitive microbial ecosystem and display high activities in digesting cellulose and polysaccharide (Coyne et al., 2008; Martens et al., 2008; Sonnenburg et al., 2010). Additionally, the function of a series of probiotics in regulating immune responses have also been documented, such as Lactobacillus (Zocco et al., 2006; Kuprys et al., 2020; Sato et al., 2020), Akkermansia (Sun et al., 2020; Ding et al., 2021; Gu et al., 2021), and Bifidobacterium (Sun et al., 2020). Dietary supplementation of B. fragilis activates the activity of natural killer T-cells in the intestine to alleviate the progression of colitis in murine model (Wieland Brown et al., 2013; An et al., 2014). In our study, the abundance of several probiotics like Bacteroides, Lachnoclostridium and Prevotrlla were significantly enriched in the colon of NX piglets. While the fraction of pathogenic bacteria, such as Streptococcus and Selenomonas, were drastically decreased in NX piglets. These results imply that NX piglet-enriched microbes may exhibit improved immune activity than DLY piglet. Moreover, functional analyses of DEMs showed that B. intestinalis, B. fragilis and L. crispatus were positively correlated with GH43, GH43_10 and GH127, all of which exhibit negatively correlations with ADG and ADFI both in NX and DLY piglets (Fig. 4), suggesting that these types of gut probiotics may have negative effects on ADG and ADFI. While some studies reported that dietary supplementation of Lactobacillus species, such as L. reuteri KT260178 (Yang et al., 2020b), L. reuteri KT260178 and L. salivarius MH 517354 (Yang et al., 2020a) improved ADG and ADFI in weaning piglets. Together, these data imply that distinct gut probiotics play different roles in regulating host behaviors to affect growth performance, probably by secreting different metabolites or biological compounds. The detailed mechanisms of how each microbial species modulate host growth performance awaits further investigation.

It has been shown that the high expression level of IL-17 is positively associated with intestinal barrier function (Shengjie Li et al., 2021; Leonardi et al., 2022; Shuying He et al., 2022). Probiotics like Bifidobacterium and Lactobacillus have the ability to enhance intestinal epithelial barrier function through promoting IL-17 production (Zareie et al., 2006; Anderson et al., 2010; Hu et al., 2018; Hu et al., 2023). In consistent with this notion, we found the high abundance of B. fragilis and L. crispatus as well as high expression level of IL-17 in the colon of NX piglets, which in turn exhibit improved intestinal epithelial barrier function than DLY piglets (Fig. 2 and Fig. 5). Therefore, our study confirmed the immune protective function of probiotics in the gut likely, but not exclusively, by improving gut epithelial barrier function.

Additionally, IL-17F has been shown to affect mucosal immune reactions by inducing the synthesis of IL-1β and IL-6 (Grivennikov et al., 2010; Chung et al., 2021). Bacteroides and Prevotella are capable of inducing IL-17B production through the TLR-Myd88 signaling pathway in the mice lung (Larsen, 2017; O’Dwyer et al., 2019; Yang et al., 2019). Interestingly, the expression of colonic TLR1, Myd88 and IL-17F as well as the abundance of Bacteroides and Prevotella species were significantly increased in NX piglet than DLY piglet (Fig. 6C-D). It is postulate that the Bacteroides and Prevotella activate the TLR-Myd88-IL-17F signaling pathway in the colon of NX piglets to initiate immune reactions. Moreover, IL-17F plays an important role in host defense against bacterial and fungal infection by recruiting neutrophils through the induction of chemokines (CXC) in infected sites (Ishigame et al., 2009; Li et al., 2019; Zhang et al., 2021). The compound of C-X-C Motif Chemokine Ligand 9 (CXCL9) and C-X-C Motif Chemokine Receptor 3 (CXCR3) promote the differentiation of CD4⁺T cells into Th17 cells, the main source of IL-17A production that are involved in anti-fungal infection (Zelante et al., 2009; Kawata et al., 2013; Chen et al., 2021; Sawada et al., 2021). Although the majority of colonic fungal distribution did not significantly differ between NX and DLY piglets, the remaining fungal species in NX piglet may be involved in the regulation of IL17F expression. Additionally, IL-17 also has been reported to defense against immune challenges by regulating antibacterial peptide genes (Kao et al., 2004; Kao et al., 2008; Songhet et al., 2010; Zheng et al., 2020). We found the varied expression of multiple AMPs in the colon of NX piglets. Whether these AMPs expression are regulated by IL17 in the colon of NX piglets and how these AMPs contribute to maintenance of immune homeostasis needs further investigation.

Lastly, we also identified some negative immune regulators that are drastically up-regulated in the colon of NX piglets, such as Suppressor Of Cytokine Signaling 3 (SOCS3) (Gao et al., 2018), SET Domain Containing 6, Protein Lysine Methyltransferase (SETD6) (Chen et al., 2016) and NLR Family Pyrin Domain Containing 12 (NLRP12) (Tuladhar and Kanneganti, 2020). NLRP12 inhibits IL-18 production and depletion of IL-18 enhanced the expression level of proinflammatory cytokines (Chen et al., 2017). Meanwhile, SOCS3 suppressed IL-10, IL-22 and IL-2 expression (Durbin et al., 1996; Park et al., 2000; Kisseleva T et al., 2002; Hunter, 2005). Thus, downregulation of serval other immune inflammatory genes like IL-22 and IL-8 in the colon of NX piglets may be result from the upregulation of these negative regulators. Notably, typical immune responsive pathways including the Toll-like receptor signaling pathway and NF- κB signaling pathway were both activated and inactivated in the colon of NX piglets. This indicated that NX piglets may have varied and versatile immune activities to establish intestinal homeostasis prior to and after immune challenges, likely rely on its colonization of the gut microbiota and the expression of core immune effectors.

In the present study, we have unraveled the characteristics of intestinal microbiome and transcriptome in NX piglets and compared these features with DLY piglets. We identified distinct colonic bacteria, fungi, and archaea species that may be acting as the characteristics of these two pig breeds, and further functional analyses revealed the dominant function of NX piglets-enriched microbes in nutrient and energy metabolism. Furthermore, this study also established the correlation between the gut microbiota and growth performance both in NX and DLY piglets. Finally, by combining the RNA-seq and RT-qPCR data, we found that NX and DLY piglets exhibit different colonic gene transcription patterns and NX piglets manifest improved intestinal barrier function and varied immune responsive genes expression than DLY piglets. Taken together, this study supports the notion that the gut microbial structures may represent the traits of different pig breeds, and the indigenous pig breeds are generally exhibiting dominant function in gut development and disease resistance.

Data Availability

The mRNA-seq and metagenomic DNA sequencing data are available at NCBI SRA under the accession number PRJNA1068200. This study did not generate any unique code.

Declaration of competing interests

The authors declare that they have no conflict of interest

Authors’ contributions

Zhenguo Hu, Xiongzhuo Tang, Yulong Yin: Conceptualization. Zhenguo Hu, Xiongzhuo Tang: Data analyses. Zhenguo Hu, Xiongzhuo Tang: Writing-Original draft. Zhenguo Hu, Yuezhou Yao, Feiyue Chen, Luya Feng, Zian Yuan, Junhao Deng, Lang Huang, Xiongzhuo Tang, Yulong Yin: Writing-Review & Editing. Zhenguo Hu, Xiongzhuo Tang: Revision.

Acknowledgements

This work was supported by the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-CXRC-038-01); Hunan Provincial Department of Education Scientific Research Project (23B0220). The figure illustration was created with BioRender.com.

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Integrated analyses of the intestinal microbiome and transcriptome in NingXiang piglets

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