Rectal temperature and intestinal pH value
Piglets challenged with LPS in the CL, OL, AL, and OAL groups trended toward higher rectal temperatures than did piglets without LPS challenge in the C group (Fig. 1A). Piglets in the CL, OL, AL, and OAL groups had significantly higher stomach pH values than did piglets in the C group (P < 0.05)(Fig. 1B). Piglets in the AL and OAL groups had significantly lower duodenum, jejunum, and ileum pH values than did piglets in the C groups (P < 0.05). Inclusion of antibiotics or antibiotics plus olive extract in the diet significantly lowered the pH value in the jejunum and ileum compared with the CL groups (P < 0.05) (Fig. 1D, E). There was no significant difference in colon pH values in piglets among different treatment groups (P > 0.05)(Fig. 1F).
Serum Biochemical Characteristics
The LPS challenge significantly lowered serum glucose levels in the CL group compared with the C group (P < 0.05). Diet inclusion of olive extract significantly increased the serum glucose in OL piglets when compared with CL group piglets (P < 0.05). AL group piglets did not significantly differ from CL group piglets (P > 0.05). However, OAL group piglets, fed a diet with antibiotics plus olive extract, had significantly higher glucose than did the CL group piglets (P < 0.05)(Fig. 2A). The serum total protein content was significantly lower in CL, OL, and AL group piglets than in C group piglets (P < 0.05)(Fig. 2B). Serum total protein content was significantly lower in AL group piglets than in OAL group piglets (P < 0.05)(Fig. 2B). The piglets in OL groups had significantly lower total serum cholesterol than did piglets in the CL groups (P < 0.05)(Fig. 2C).
Serum Antioxidant Capacity
Compared with those in the C group, after i.p. injection of LPS, piglets in the CL group had significantly lower GSH-Px (P < 0.05) and SOD (P < 0.05) levels and higher MDA (P < 0.05) and NO (P < 0.05) levels in the serum (Fig. 3A-D). However, compared with the CL group, the piglets in OL, AL, and OAL groups had significantly higher GSH-Px (P < 0.05) and SOD (P < 0.05) levels and lower MDA (P < 0.05) and NO (P < 0.05) levels in the serum (Fig. 3A-D). There was no significant difference in these four parameters in serum of piglets from OL, AL, and OAL groups (P > 0.05)(Fig. 3A-D).
Compared with the C group, the piglets in the CL group had significantly higher levels of LDH (P < 0.05), ALT (P < 0.05), and AST (P < 0.05) and lower ALB levels (P < 0.05) in the serum (Fig. 3E-H). Compared with the CL group, the piglets in the OL, AL, and OAL groups had significantly lower serum LDH (P < 0.05), ALT (P < 0.05), and AST (P < 0.05) levels and higher ALB (P < 0.05) levels in the serum (Fig. 3E-H). The piglets in the OL and OAL groups were fed diets containing olive extract and had significantly lower serum LDH (P < 0.05) levels than did those in the AL groups (Fig. 3E).
Serum Inflammatory Factor And Spleen Index
LPS administration significantly increased the serum concentration of TNF-α (P < 0.05) and IL-6 (P < 0.05) in the CL group compared with the control group (Fig. 4A, B). Compared with those in the CL group, piglets in the OL, AL, and OAL groups had significantly lower serum TNF-α (P < 0.05) and IL-6 (P < 0.05) levels and significantly higher IL-10 (P < 0.05) levels (Fig. 4A-C). There were no significant differences in serum TNF-α, IL-6, and IL-10 levels in piglets from OL, AL, and OAL groups (P > 0.05)(Fig. 4A-C). Piglets challenged with LPS in the CL group had significantly higher spleen index than did C group piglets (P < 0.05, Fig. 4D). Spleen index values of piglets in OL, AL, and OAL groups did not significantly differ from those in CL group piglets (P > 0.05, Fig. 4D).
Intestinal Integrity And Histomorphology
The DAO and D-xylose was determined as indicators of the intestinal integrity. CL group piglets challenged with LPS had significantly higher serum DAO and D-xylose levels than did group C piglets (P < 0.05, Fig. 5A, B). The OL and OAL group piglets had significantly lower DAO and D-xylose concentrations than did CL group piglets (P < 0.05, Fig. 5A, B). The villus height and crypt depth, based on H&E staining, are shown in Fig. 6. The duodenum and ileum of piglets in control, OL, AL and OAL groups showed regular structures, while LPS stimulation destroyed the intestinal villus structure in the duodenum and ileum of piglets in the CL group (Fig. 6A, B). The duodenum and ileum of piglets in the CL group had significantly lower villus height (P < 0.05), higher crypt depth (P < 0.05) and lower villus height/crypt depth ratios (P < 0.05) than those of piglets in the control group (Fig. 6C, D). However, compared with those of the CL group, the duodenum and ileum of piglets in OL, AL, and OAL groups had significantly higher villi (P < 0.05), lower crypt depth (P < 0.05), and higher villus height/crypt depth ratios (P < 0.05) with the exception of ileum villus height in AL and OAL groups (P > 0.05) (Fig. 6C, D).
Piglet Fecal Bacterial Communities
The relative abundance of fecal microbiota at the family (Fig. 7A) and genus levels (Fig. 7B) were examined in pig fecal matter. There were 777, 694, 763, 680, and 733 OTUs obtained from the feces of piglets in C, CL, OL, AL, and OAL groups, respectively. Among these OTUs, 448 OTUs were shared and 197 OTUs were isolated (Fig. 7C). We performed LEfSe analysis to identify the significant ranking of abundant bacterial taxa among C, CL, OL, AL, and OAL groups. The cladogram highlighted 19 important bacterial taxa (Fig. 7D), and their significant effects were displayed using LDA scores (Fig. 7E). The threshold for the logarithmic LDA score for biomarkers was set at 2.0. The biomarker in C was p_251_o5 (c). The biomarkers in CL were Eubacterium_coprostanoligenes_group (g) and T34(p). The biomarkers in OL were Megasphaera (j), Veillonellaceae (k), and Selenomonadales (l). The biomarkers in AL were Lactobacillus (d), Lactobacillaceae (e), and Lactobacillales (f), which formed one branch, and Treponema_2 (q), Spirochaetaceae (r), and Spirochaetales (s) formed a second branch. Muribaculaceae (a), Tannerellaceae (b), Ruminococcaceae (h), Anaerovibrio (i), Succinivibrio (m), Succinivibrionaceae (n), and Aeromonadales (o) were biomarkers in OAL (Fig. 7E).
Chao 1 and Shannon and Simpson index values, which reflect species richness and evenness, did not differ among the five treatment groups (Fig. 8A-C). At the family level (Fig. 8D), there was no significant difference in relative abundance of Muribaculaceae, Tannerellaceae, Tannerellaceae, and Ruminococcaceae between the C and CL groups, while the CL group had a significantly higher relative abundance of Veillonellaceae and T34 (P < 0.05) and a lower relative abundance of Spirochaetaceae (P < 0.05) than did the C group. The relative abundance of Muribaculaceae was significantly lower in CL group piglets than in OAL group piglets (P < 0.05). The relative abundance of Tannerellaceae was significantly lower in CL group piglets than in OL, AL, and OAL group piglets (P < 0.05). The relative abundance of Lactobacillaceae was significantly lower in CL group piglets than in OL and AL group piglets (P < 0.05). The relative abundance of Ruminococcaceae was significantly lower in OL group piglets than in CL group piglets (P < 0.05). There was no significant difference in the relative abundance of Veillonellaceae in the intestines among the CL, OL, AL, and OAL group piglets (P > 0.05). The relative abundance of T34 was significantly lower in the OL, AL, and OAL group piglets than in the CL group piglets (P < 0.05). The relative abundance of Spirochaetaceae was significantly lower in CL group piglets than in OL and AL group piglets (P < 0.05).
At the genus level (Fig. 8E), LPS challenge induced a significantly higher relative abundance of Alloprevotella (P < 0.05) and a lower abundance of Clostridium_sensu_stricto_1, Lachnospiraceae_NK4A136_group, and Succinivibrio (P < 0.05) in CL group piglets. The relative abundance of Alloprevotella was significantly lower in OL, AL and OAL group piglets than in CL group piglets (P < 0.05). Dietary inclusion of olive extract in the OL group had significantly higher relative abundance of Clostridium_sensu_stricto_1 than did the CL group (P < 0.05). The relative abundance of Lactobacillus was significantly lower in CL group piglets than in OL and AL group piglets (P < 0.05). There was no significant difference in the relative abundance of Lachnospiraceae_NK4A136_group in the intestine of CL, OL, AL, and OAL group piglets (P > 0.05). The OL and OAL group piglets had significantly higher relative Succinivibrio abundance than did the CL group piglets (P < 0.05). There was no significant difference in relative Sutterella abundance between C and CL group piglets (P > 0.05). The relative abundance of Sutterella was significantly lower in CL group piglets than in the OL and OAL groups (P < 0.05). The relative Terrisporobacter abundance was significantly lower in the CL group piglets than in OL group piglets (P < 0.05). The OL and AL group piglets had significantly higher relative Treponema_2 abundance than did the CL groups (P < 0.05).