Constant chronic HS decreased the performance and affected normal physiological responses

Because one head of pig in the HS group dead on the 3rd day of experiment, the subsequent analysis of HS group was based on the data of the remaining 7 pigs. Effects of constant chronic HS on the performance and physiological responses of growing-finishing pigs were listed in Table 3. After 7-day constant chronic HS or feed limited, final body weight was declined (P < 0.05) compared with TN, and even caused body weight loss (0.70–0.96 kg). HS or PF treatment significantly decreased (P < 0.05) ADFI, about 61% reduction compared to TN, as well as water intake, but the ratio of water to feed was increased (P < 0.05). Compared with TN, HS significantly elevated (P < 0.05) the rectal temperature and body surface temperature (jugular, back, hind leg), while PF significantly decreased (P < 0.05) body temperature.

Table 3 Effects of constant chronic heat stress on the performance and physiological responses in growing-finishing pigs

Constant chronic HS decreased pH value in the small intestinal segments

As presented in Table 4, pH value of the digesta in the different small intestine sections (duodenum, jejunum, ileum) was measured. HS significantly decreased (P < 0.05) pH value of digesta in the jejunum and ileum compared with PF.

Table 4 Effects of constant chronic heat stress on pH value of small intestine segments in growing-finishing pigs

Constant chronic HS caused morphological injuries in the small intestine

As illustrated in Fig.1 and Fig.2, HS led to marked morphological injuries in the small intestine, such as villi tips desquamation and the lamina propria exposing. To quantify the extent of damage, villus height, microvillus height, and crypt depth were measured. As depicted in Tables 5 and 6, both HS and PF decreased villus and microvillus height (P < 0.05) in the duodenum, jejunum, and ileum compared with TN. In addition, HS also significantly decreased (P < 0.05) the ratio of villus height to crypt depth in different small intestine sections (duodenum, jejunum, and ileum).

Fig. 1
figure1

Effects of constant chronic heat stress on morphology of small intestine segments in growing-finishing pigs by H&E-staining (scale bar 1000 μm). TN, thermal neutral conditions (25 ± 1 °C); HS, heat stress conditions (35 ± 1 °C); PF, pair-fed with HS under TN conditions (25 ± 1 °C)

Fig. 2
figure2

Effects of constant chronic heat stress on the ultrastructure of small intestine segments in growing-finishing pigs by electron microscopy (scale bar 2 μm). The red arrows indicated the location of tight junction structure. TN, thermal neutral conditions (25 ± 1 °C); HS, heat stress conditions (35 ± 1 °C); PF, pair-fed with HS under TN conditions (25 ± 1 °C)

Table 5 Effects of constant chronic heat stress on villus height and crypt depth of small intestine segments in growing-finishing pigs
Table 6 Effects of constant chronic heat stress on microvillus height of small intestine segments in growing-finishing pigs, μm

Constant chronic HS elevated the expression of heat shock protein 70 and tight junction proteins

As shown in Fig.3, HS increased (P < 0.05) expression of HSP 70 in the duodenum, jejunum, and ileum, and induced up-regulation (P < 0.05) of the expression of tight junction protein ZO-1 in the duodenum and ileum, and Occludin in the ileum compared with TN and PF.

Fig. 3
figure3

Effects of constant chronic heat stress on the expression of heat shock protein and tight junction proteins of small intestine sections in growing-finishing pigs. A, B, C presented duodenum, jejunum, and ileum respectively. TN, thermal neutral conditions (25 ± 1 °C); HS, heat stress conditions (35 ± 1 °C); PF, pair-fed with HS under TN conditions (25 ± 1 °C). The bar graphs showed the protein band intensity. All data were expressed as the mean ± SEM (n = 8 for TN and PF, n = 7 for HS). Differences were determined by one-way ANOVA followed by LSD test. Groups without a common letter mean significant differences (P < 0.05). Abbreviations: HSP 70, heat shock protein 70, ZO-1, zonula occluden-1

Constant chronic HS activated the immune response in the ileum by NF-κB pathway

Immune-related gene expression of Toll-like receptor 2 (TLR-2), TLR-4, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-8 were found to be up-regulated (P < 0.05) by HS compared with TN (Fig.4A). Moreover, the protein expression of P65 was also increased by HS compared with TN (P < 0.05). Further, compared with TN, HS elevated (P < 0.05) gene expression of mucins (mucin-1, mucin-2) and antimicrobial peptide (protegrin 1–5 [PG1–5], porcine beta-defensin 2 [pBD-2]) (Fig.4B).

Fig. 4
figure4

Constant chronic heat stress provokes the immune response in the ileum by NF-κB pathway. Relative mRNA expression levels of TLR-2 (A), TLR-4 (A), TNF-α(A), IL-6 (A), IL-8 (A), mucin-1(B), mucin-2(B), PG1–5(B), pBD2(B) in the ileum of growing-finishing pigs were detected by Real Time-PCR. Protein expression of P65 (C) in the ileum of growing-finish pigs was detected by western blotting and the bar graphs showed the protein band intensity. All data were expressed as the mean ± SEM (n = 8 for TN and PF, n = 7 for HS). TN, thermal neutral conditions (25 ± 1 °C); HS, heat stress conditions (35 ± 1 °C); PF, pair-fed with HS under TN conditions (25 ± 1 °C). Differences were determined by one-way ANOVA followed by LSD test. Groups without a common letter mean significant differences (P < 0.05). Abbreviations: PG1–5, protegrin 1–5; pBD2, porcine beta-defensin 2; TLR-2, toll-like receptor 2; TLR-4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; IL-6, interleukin- 6; IL-8, interleukin- 8

Constant chronic HS influenced the intestinal microbial composition

After quality control, a total of 1,678,038 clean reads were procured from 23 fecal samples, and clustered into average 755 OTUs per sample, resulting in a total of 1770 OTUs for all samples, and the average sequence effective (the ratio of the number of clean reads to raw reads) was 94.11% per sample. The common and special OTUs proportion among groups was presented in Fig.5a.

Fig. 5
figure5

Effects of constant chronic heat stress on intestinal microbial communities. Venn diagram showed the proportion of common and special OTUs among groups (a). Alpha diversity index such as Chao1 (b) and Shannon (c) index indicated the diversity and evenness. Binary-Jaccard distance-based NMDS plot (d), OTUs-based PCA plot (e), unweighted unifrac distance-based PCoA plot (i), weighted unifrac distance-based PCoA plot (k) were used to display the distribution of the samples among groups. ANOSIM test was performed to detect differences in community structure between groups based on OTUs relative abundance (f, g, h). UPGMA clustering was conducted based on unweighted unifrac distance and weighted unifrac distance (j, i). Differences of α-diversity indices were determined by Wilcox rank-sum test, and differences of β-diversity indices were determined by ANOSIM test. * presented P < 0.05, *** presented P < 0.001, ns mean no significant difference. TN, thermal neutral conditions (25 ± 1 °C); HS, heat stress conditions (35 ± 1 °C); PF, pair-fed with HS under TN conditions (25 ± 1 °C). n = 8 for TN and PF, n = 7 for HS

There was no difference in Chao1 index among groups (Fig.5b). Differently, both HS and PF exhibited a lower Shannon index (P = 0.073, P = 0.095) compared with TN (Fig.5c). β diversity analysis was showed in OTUs-based PCA, and weighted and unweighted Unifrac distance-based PCoA plot, which indicated the distribution of the community of samples (Fig.5d, e, i, k), and shown divergence of the community structure among groups. Moreover, Anosim tests confirmed the dissimilarity (P < 0.05) community composition between each group (Fig.5f, g, h). LEfSe analysis identified 13 discriminative bacteria among the three groups (Fig.6A). Gammaproteobacteria (class), Bacilli (class), Pseudomonadales (order), Moraxellaceae (family), Lactobacillales (order), Bacillales (order), Planococcaceae (family), and Streptococcaceae (family) were more prevalent in HS. Ruminococcaceae (family), Lactobacillaceae (family), Bacteroidia (class), and Bacteroidales (order) were more abundant in TN. The biomarker microbe in PF was unidentified Clostridiales (family).

Fig. 6
figure6

Effects of constant chronic heat stress on the relative abundance of microbial species. The LEfSe analysis (LDA score ≥ 4) identified the biomarker species (A). The top 10 phylum, class, order, family, genus, and species and the significantly different microbial at each level were showed (B). TN, thermal neutral conditions (25 ± 1 °C); HS, heat stress conditions (35 ± 1 °C); PF, pair-fed with HS under TN conditions (25 ± 1 °C). Brackets indicated the affiliation between species. Differences were determined by Kruskal-Wallis test followed by Dunn test, and false discovery rate (FDR) values were estimated using the Bonferroni method to control for multiple testing (* presented FDR < 0.05, ns mean no significant difference). n = 8 for TN and PF, n = 7 for HS

The composition of top 10 phylum (Fig.6B), class (Fig.6C), order (Fig.6D), family (Fig.6E), genus (Fig.6F), and species (Fig.6G) were provided. Results indicated that Firmicutes (phylum, 54.65–65.49%), Bacteroidetes (phylum, 12.68–21.31%), and Proteobacteria (phylum, 10.00–30.65%) were major phylum (95.53–97.99%) of fecal microbiota in growing-finishing pigs. At genus level, the most prevalent microbes were Acinetobacter (5.45–21.65%), Kurthia (1.26–6.46%), and Lactobacillus (7.05–11.65%). Compared with TN, HS significantly increased (FDR < 0.05) the abundance of Proteobacteria (phylum), γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family) and Acinetobacter (genus), and Proteobacteria (phylum) became the second most enriched phylum instead of Bacteroidetes (phylum) in HS. The relative amount of Bacteroidetes (phylum, FDR < 0.05), Bacteroidia (class, FDR < 0.05), and Bacteroidales (order, FDR = 0.072) was decreased by HS. Belonging to Firmicutes (phylum), Clostridia (class), Clostridiales (order), Ruminococcaceae (family) was significantly decreased (FDR < 0.05) in HS when compared with TN, while unidentified_Clostridiales (family), and unidentified_Clostridium (genus) were increased (FDR < 0.05) in PF and HS compared with TN. However, also belonging to Firmicutes (phylum), Bacill (class) was decreased (FDR = 0.089, FDR < 0.05) in PF compared with TN or HS. Belonging to Bacill (class), the Bacillales (order), and Planococcaceae (family), and Kurthia (genus) presented different situations, and all was significant increased (FDR < 0.05) in HS independent of FI. Interestingly, HS didn’t affect (FDR > 0.05) the relative richness of Lactobacillales (order), Lactobacillaceae (family), Lactobacillus (genus), Lactobacillus reuteri (species), and Lactobacillus amylovorus (species) when compared with TN, but PF decreased (FDR < 0.05) the richness of the above microbes in comparison with HS, and decreased (FDR < 0.05) the richness of the above microbes except Lactobacillus amylovorus (species) in comparison with TN. Furthermore, HS significantly increased (FDR < 0.10) the abundance of Streptococcaceae (family) and Streptococcus (genus).

Associations of significantly differential intestinal microbes with the performance, intestinal morphological injuries indicators, and ileal immune response parameters under constant chronic HS

The Spearman correlation analysis between significantly differential intestinal microbes and the performance, morphological injuries indicators, and ileal immune response parameters was shown in Fig. 7.

Fig. 7
figure7

The Spearman correlation analysis between significantly differential microbes and the performance (A), small intestinal morphological injuries indicators (B), and ileal immune response parameters (C) under constant chronic HS. The heatmap of the correlation coefficient, the red represents positive correlation and the blue represents negative correlation, respectively (* presented P < 0.05, ** presented P < 0.01). Abbreviations: ADG, average daily gain; ADFI, average daily feed intake; V/C, the ratio of villus height to crypt depth; PG1–5, protegrin 1–5; pBD2, porcine beta-defensin 2; TLR-2, toll-like receptor 2; TLR-4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; IL-6, interleukin- 6; IL-8, interleukin- 8

As showed in Fig. 7A, final body weight and ADG, as well as ADFI, were negatively (P < 0.05) associated with Proteobacteria (phylum), γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family), Acinetobacter (genus), unidentified Clostridiales (family), and unidentified Clostridiales (genus). Final body weight was also correlated (negatively, P < 0.05) with Bacillales (order), Planococcaceae (family), and Kurthia (genus), but represented the opposite (P < 0.05) associations with Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order), and Ruminococcaceae (family). Moreover, ADG was positively (P < 0.05) associated with Bacteroidales (order), Ruminococcaceae (family), Lactobacillaceae (family), and Lactobacillus (genus), while negatively (P < 0.05) associated with Clostridium disporicum (species), Bacillales (order), Planococcaceae (family), and Kurthia (genus). Additionally, positively (P < 0.05) correlations between ADFI and Bacteroidales (order), Ruminococcaceae (family), Bacilli (class), Lactobacillales (order), Lactobacillaceae (family), Lactobacillus (genus), and Lactobacillus_reuteri (species) were observed, but represented the opposite (P < 0.05) correlation with Clostridium disporicum (species).

As displayed in Fig.7B, negative (P < 0.05) associations between Proteobacteria (phylum), and its aligned bacteria and jejunal villus height, ileal V/C, and ileal microvillus height were observed. Bacteroidetes (phylum) showed positively (P < 0.05) correlated with the duodenal villus height, jejunal and ileal microvillus height. Belong to Firmicutes (phylum), the unidentified Clostridiales (family), unidentified Clostridium (genus), Clostridium disporicum (species), Bacillales (order), Planococcaceae (family), and Kurthia (genus) shown positively (P<0.05) correlated with villus height, and microvillus height of small intestine sections. The value of V/C in different small intestine sections was all negatively (P < 0.05) correlated with Streptococcaceae (family) and Streptococcus (order). In addition, duodenal V/C was negatively (P < 0.05) associated with Bacillales (order), Planococcaceae (family), while jejunal V/C showed the same associations with Bacilli (class) and Lactobacillus amylovorus (species) as well as ileal V/C with Bacillales (order), Planococcaceae (family), and Kurthia (genus). Moreover, microvillus height was positively (P < 0.05) associated with Bacteroidetes (phylum), Bacteroidia (class), and Ruminococcaceae (family), but showed the opposite associations (P < 0.05) with γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family), Acinetobacter (genus), unidentified Clostridiales (family), unidentified Clostridium (genus), Clostridium disporicum (species), Bacillales (order), Planococcaceae (family), Kurthia (genus), Bacilli (class) and Lactobacillus amylovorus (species).

As exhibited in Fig.7C, antimicrobial peptide (PG1–5, pBD-2) and mucins (mucin 1) were associated (positively, P < 0.05) with Proteobacteria (phylum), γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family), Acinetobacter (genus), Streptococcaceae (family) and Streptococcus (genus), but showed the opposite correlated (P < 0.05) with Bacteroidetes (phylum), Bacteroidia (class). The gene expression of pro-inflammatory cytokine such as TNF-α and IL-6 was positively (P < 0.05) associated with Proteobacteria (phylum), γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family), Acinetobacter (genus), and negatively (P < 0.05) associated with Bacteroidetes (phylum), Bacteroidia (class). Positively (P < 0.05) associations between the protein expression of P65 with Pseudomonadales (order), Moraxellaceae (family), Bacillales (order), Planococcaceae (family), Kurthia (genus), Streptococcaceae (family) and Streptococcus (order) were found, but represented the opposite (P < 0.05) associations with Bacteroidetes (phylum) and Bacteroidia (class).

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