In this study, nine Jinfen White piglets and nine Mashen piglets were raised in the Datong Pig Breeding Farm (Shanxi Province, China). The piglets were weaned at the same age at 28 days and raised under the same nursery conditions. Both breeds of piglets were used for content sampling of the ileum at the newborn stage (1 day), weaning stage (28 days), and end of nursery stage (70 days). The feed formulation is provided in Supplementary Table 1. The piglets fasted for 12 h and were then allowed free access to fresh water prior to slaughter. The initial and final weight of each piglet are listed in Supplementary Table 2. The ileum samples were collected at three developmental stages, kept frozen in liquid nitrogen for transportation, and then stored at −80°C until use. The animal operating procedures involved in this study were approved by the Animal Care and Use Committee of Shanxi Agricultural University, and the methods were in accordance with the Guidelines for the Care and Use of Experimental Animals of the Ministry of Agriculture of China.

The ileum was fixed with 4% paraformaldehyde for 48 h and then soaked in paraffin. The paraffin-soaked tissue was cut into 4 μm sections and deparaffinized for a further 1 h at 62°C. Then, the sample was stained with hematoxylin–eosin, observed, and photographed under an optical microscope. For each ileum sample, a paraffin-embedded section was prepared and Image-Pro Plus software (version 6.0) was used to measure the villus length, crypt depth, and the ratio of villus length to crypt depth (V/C). Three slices were selected from each pig, and three non-overlapping fields were randomly observed in each slice.

Blood (5 mL) was collected from the vena cava before slaughter at different developmental stages of piglets, and serum was separated by centrifugation at 4000 r/min for 10 min at 4°C. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum antioxidant factors, including superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (T-AOC). All the above specific detection kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

Microbial genomic DNA was extracted from the ileum of different individuals using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Valencia, CA, United States) following the manufacturer’s instructions. The V3–V4 region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal bacterial 16S rRNA gene PCR amplicon primers (341F: 5′-ACTCCTACGGGRSGCGCAG-3′, 806R: 5′-GGACTACWGGGTATCTAATC-3′), and the PCR products were purified using a DNA Gel Extraction Kit (Tiangen, Beijing, China). All sample libraries were sequenced on the Illumina HiSeq 2500 platform.

The 16S rRNA sequencing data were processed using the Quantitative Insights into Microbial Ecology (QIIME1) (version 1.9.1) platform (Caporaso et al., 2010). The sequence reads were acquired after trimming out the barcodes and primers and were discarded with a >10% ambiguous base (N) or 20% low-quality base pairs (Q < 20). Then, the raw reads were filtered with a minimum overlap of 10 bp and a maximum mismatch ratio of 0.2, using FLASH (version 1.2.11) (Magoc and Salzberg, 2011). The reads were clustered into operational taxonomic units (OTUs) using de novo picking, with a 97% sequence identity threshold, and chimeras and singletons were removed using USEARCH software (version 7¹). Subsequently, OUTs were aligned to the SILVA rRNA specific database² to assign taxonomy, and a phylogenetic tree was generated within QIIME. The microbial alpha diversity indices of the samples were determined using Chao1, ACE, and Sobs (which measures richness based on rare OTUs), coverage, Shannon, and Simpson (which measure richness and evenness), and these indices were calculated using the Mothur program.³ Principal coordinate analysis (PCoA) plots, based on unweighted UniFrac distances, were visualized using EMPeror v0.9.3-dev. Subsequent downstream images were generated using the R package Phyloseq. Tax4Fun v1.0, which employs the 16S rRNA gene as a maker by using the Silva databases for taxonomy and OUT assignments, was used to predict the functionality of the ileum microbiota of the two pig breeds (Asshauer et al., 2015). The prediction of functions was inferred based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations for level 2 pathways. Pathways for which the relative abundance was <0.001% were dismissed.

The 16S rRNA gene sequence data used in this study were deposited in the NCBI SRA database under the accession number SRP321842.

The physiological features (villus height and crypt depth) and serum antioxidant factors are presented as mean ± SEM, and statistical analyses were performed using SPSS ver.26.0. Differences between the Jinfen White piglets and Mashen piglets were analyzed using Student’s t-test; ^∗∗ and ^∗ indicate that the differences between the samples are extremely significant (P < 0.01) or significant (P < 0.05), respectively. Differences at different time points were identified using ANOVA; values with the same or no letter superscripts mean no significant difference (P > 0.05), those with different small letter superscripts mean a significant difference with P < 0.05, and those with different capital letter superscripts mean a significant difference with P < 0.01.

To determine the physiological features of the ileum, we measured its villus length and crypt depth (Figure 1). We observed that the villus length of the ileum in the Jinfen White and Mashen piglets decreased from birth (1 day) to the end of nursery (70 days) stage, and there was a significant difference in the villus length between the two breeds on the 28 and 70 days (P < 0.05). Moreover, the villus length of the ileum in the Mashen piglets was higher than that in the Jinfen White piglets at all three stages, except at the end of the nursery stage (Figure 1A). In contrast, as depicted in Figure 1B, the crypt depth of the ileum in the Jinfen White and Mashen piglets at the end of the nursery stage were extremely significantly higher than in the other two stages (P < 0.01), and the crypt depth was significantly higher in the Mashen piglets than in the Jinfen White at the newborn (P < 0.01) and end of the nursery stages (P < 0.05). Furthermore, the villus/crypt ratio showed a downward trend, with no significant difference between the two breeds on the 1 and 70 days (Figure 1C).

Histological sections revealed that the villi of the ileum gradually became shorter with increasing age, but the transverse section of the villi became wider in the Jinfen White and Mashen piglets. However, intestinal folds were observed in the ileum of the Jinfen White piglets at birth, but not in the Mashen piglets. In addition, the ileum villi of the Jinfen White piglets were shorter than those of the Mashen piglets at birth, but in the Mashen piglets, the villi of the ileum were longer, and the surface area was larger than that in the Jinfen White piglets at the weaning stage and at the end of the nursery stage (Figure 1D).

The small intestine is the main organ for food digestion and nutrient absorption and is composed of the mucous membrane and muscle layer. Some indicators in the blood are often associated with growth, immune responses, and gut health. To determine the association between serum biochemical indexes (especially antioxidant factors) and intestinal microbiota, we measured the content of MDA, SOD, and T-AOC using the blood samples of piglets at three developmental stages (N = 3). In general, the levels of MDA, SOD, and T-AOC in the serum of the Jinfen White and Mashen piglets increased over time, except for the MDA and SOD content of the Mashen piglets at the weaning stage (Figures 2A,B), of which the SOD and T-AOC increased by an extremely significant amount in the Jinfen White piglets (P < 0.01). Additionally, as described in Figure 2, the levels of the three indexes in the serum of the Jinfen White piglets were extremely significantly higher than those in the Mashen piglets at the end of nursing stage (P < 0.01). We also found that the blood levels of SOD and T-AOC in the Jinfen White piglets were extremely significantly higher than those in the Mashen piglets at the weaning stage and end of nursery stage (P < 0.01) (Figures 2B,C). These results demonstrated that with development, the oxidative stress response of the Jinfen White piglets may be more significant than that of the Mashen piglets, especially on the 70th day of the growth period, which indicated the end of nursery stage and start of the feeding.

A total of 938,758 sequence reads were obtained from 18 ileum samples, with an average of 52,153 reads per sample (ranging from 32,195 to 71,847), covering 415,877,051 bp (Supplementary Table 3). After subsampling each sample to an equal sequencing depth (31,221 reads per sample) and clustering, 690 OTUs at 97% identity were obtained. From a taxonomic perspective, 21 phyla, 37 classes, 70 orders, 127 families, and 286 genera were identified in the ileum samples of 18 piglets. The alpha diversity indices are shown in Table 1 and Figure 3. Good’s coverage was at least 99% for all samples, suggesting that almost all bacteria in the ileum samples could be detected. For the Jinfen White piglets, the values of Chao1, ACE, and Shannon continuously increased and were significantly higher on the 70th day than on the 1st day and 28th day (P < 0.05), except for the Simpson indices (Figure 3D). However, the four indices did not change regularly in the Mashen piglets and did not reach the highest level at the endpoint (the 70th day). The changes in the four indices suggested that the richness and diversity increased during the Jinfen White piglet development, indicating that a subset of bacteria became dominant in the Jinfen White piglet’s ileum samples. In summary, the median values of the alpha diversity indices of the Mashen piglets were lower than those of the Jinfen White piglets (except on the 70th day of Simpson, and the 1st and 28th day of Shannon), revealing that the ileum of the Jinfen White piglets had a relatively richer microbial community diversity than the Mashen piglets.

Based on the relative abundance of the OUTs sequenced at the developmental stages of the two pig breeds, PCoA was used to analyze the differences in microbial community composition among the samples (Figure 4). As a result, the development of piglets was the first principal component, which explained 61.85% of the variation, indicating that development was the main factor affecting intestinal microbial changes. Interestingly, there were significant differences between the two breeds in the microbial variation trend. The microbial variation of the Jinfen White piglets was larger during the three developmental stages, while the microbial species of the Mashen piglets tended to be more stable during the entire development stage. Furthermore, microbial diversity was the second principal component, which explained 11.86% of the data variation, indicating that there were significant differences among individual piglets. The microorganisms of the Jinfen White piglets changed greatly before the weaning stage, and gradually stabilized at the nursery stage, while the microbes of the Mashen piglets were more similar from the newborn to the end of nursery stage. In summary, the intestinal microbiomes of different pig breeds were different, and the main factors affecting the stability of the intestinal microbiota of piglets were lactation and feed.

A total of 21 phyla were identified in the sequencing analysis, including Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria, Cyanobacteria, and others (Figure 5A). Firmicutes and Proteobacteria were the major phyla during the development of the Jinfen White and Mashen piglets, accounting for ∼90% of all OTUs. As the growth time increased, the proportion of Firmicutes increased from 12.32 to 83.21%, and the percentage of Proteobacteria decreased from 83.93 to 5.26% in the ileum of the Jinfen White piglets. For the Mashen piglets, the ratio of Firmicutes showed no significant difference between the 1st day and 28th day, while it increased from 80.97 to 99.75% at the end of the nursing stage, and the proportion of Proteobacteria first increased and then decreased during the Mashen piglet’s development stages (Table 2).

Bacterial community dynamics were also evaluated based on changes in the relative abundance at the genus level during the growth of the Jinfen White and Mashen piglets (Figure 5B). A total of 286 bacterial genera were identified using HTS. At the developmental stage, Pseudomonas was the dominant genus in the ileum of Jinfen White piglets (59.97, 26.85, and 3.83%), and Lactobacillus was the major genus in the ileum of the Mashen piglets (37.54, 29.46, and 86.91%). At the weaning stage, Lactobacillus was the dominant genus in both the Jinfen White and Mashen piglets (56.03 and 29.46%, respectively) (Table 3). Moreover, Streptococcus and Lactobacillus were the dominant bacteria at the end of nursery period in the Jinfen White and Mashen piglets, respectively. These results indicate that the microbial composition changes dynamically during the growth and development period of piglets. In this study, except for the primary stage of the Jinfen White piglets, Lactobacillus was the core bacteria in the ileum of all samples, which is frequently observed in the guts of pigs (Valeriano et al., 2017; Gao et al., 2019a).

As the abundance of metabolic pathways in microorganisms determines the status of the ileum, microbial metabolism can provide further insight into the development of pigs. As shown in Figure 6, carbohydrate metabolism and amino acid metabolism were the main metabolic pathways in all samples of the two pig breeds. For the Jinfen White piglets (Figure 6A), the abundance of genera associated with amino acid metabolism began to decline after 28 days, and carbohydrate metabolism began to increase. For the Mashen piglets (Figure 6B), on day 28, although the carbohydrate metabolism began to decrease and the amino acid metabolism began to increase, the two types of metabolisms showed an opposite tendency after 70 days.

Several predicted KEGG pathways (top 20) were differentially identified in the ileum microbiota of the Jinfen White and Mashen piglets (Figure 7). All the top 20 KEGG pathways had the lowest abundance in the MS_1d group and had a relatively high abundance on the 28th day in the Jinfen White and Mashen piglets. The pathways in the JFW_28d and MS_28d groups were similar, and these pathways were also similar in the JFW_1d samples. For instance, the “Carbon metabolism” pathway was predicted at a lower level in the microbiota of the JFW_70d group than in that of the JFW_1d and JFW_28d, but at a higher level in the microbiota of the MS_70d group than in that of MS_1d.

To investigate the possible correlations between the microbial community structure and environmental factors, we analyzed the Spearman correlation coefficient between the ileum microbiota composition of the genera (top 50) and several physiological features (villus length, crypt depth, SOD, MAD, and T-AOC) of the Jinfen White and Mashen pigs by hierarchical clustering (Figure 8). For the Jinfen White piglets (Figure 8A), 15 genera, including Ruminococcaceae_UCG-014, [Eubacterium]_coprostanoligenes_group, Blautia, Prevotellaceae_NK3B31_group, Kitasatospora, Christensenellaceae_R-7_group, Leuconostoc, Fructobacillus, Lactococcus, Holdemanella, unclassified_f__Lachnospiraceae, Subdoligranulum, Parabacteroides, Bacillus, and norank_f__Bacteroidales_S24-7_group showed significantly positive correlations with the contents of SOD, MDA, and T-AOC in the serum as well as with the crypt depth (P < 0.05), whereas some of them (Ruminococcaceae_UCG-014, [Eubacterium]_coprostanoligenes_group, Blautia, Kitasatospora, Christensenellaceae_R-7_group, Leuconostoc, Fructobacillus, Lactococcus, Holdemanella, and Subdoligranulum) had remarkably negative correlations with villus length (P < 0.05). Rothia demonstrated a significant negative correlation with villus length (P < 0.05). However, norank_c__Cyanobacteria was only significantly positively correlated with SOD content in the serum of Jinfen White piglets. These results revealed positive correlation between the ileum microbiota at the genus level and serum biochemical index of Jinfen White piglets, except for unclassified_k__norank and Herminiimonas. Unlike the correlation in Jinfen White piglets, most of the bacteria were significantly negatively correlated with the serum biochemical indexes and crypt depth in the ileum of Mashen piglets except Ruminococcaceae_UCG-005, Lactobacillus, Streptococcus, and unclassified_o__Lactobacillales, which had significantly positive correlations with the contents of T-AOC, MDA, or crypt depth (P < 0.05) (Figure 8B). The serum content of T-AOC was significantly negatively correlated with Clostridium_sensu_stricto_13 and Clostridium_sensu_stricto_2 (P < 0.05). Furthermore, villus length was significantly positively correlated with Turicibacter, Clostridium_sensu_stricto_1, unclassified_k__norank, Pseudomonas, unclassified_p__Proteobacteria, Aeromonas, Clostridium_sensu_stricto_2, Bacteroides, and Epulopiscium. Interestingly, some bacteria had different correlations with environmental factors, while others showed comparable correlations in the two pig breeds. For instance, Rothia had a different correlation with all the environmental factors in the two pig breeds, while Lactobacillus, Streptococcus, Fusobacterium, and Herminiimonas were positively correlated with villus length and negatively correlated with other factors. These results suggest that the environmental and host factors (villus length, crypt depth, SOD, MAD, and T-AOC) may affect the formation of microbial community structures in the Jinfen White and Mashen piglets.