Activation of inflammatory immune gene cascades by lipopolysaccharide (LPS) in the porcine colonic tissue ex-vivo model.

The technique of challenging postmortem tissue explants with inflammation inducer such as lipopolysaccharide (LPS) followed by gene expression analysis is used widely for evaluating the immune-suppressing effect of bioactives. Using porcine colonic tissue as an ex-vivo model of mammalian intestinal gut, this study evaluated the effect of incubation time on the integrity of gene transcripts and activation of inflammatory immune gene cascade by LPS treatment. Post-slaughter colon was removed surgically and explants were incubated for 0, 3, 6 and 12 h and the abundance of mRNA transcripts of a panel of 92 immune genes were evaluated using quantitative polymerase chain reaction (qPCR) arrays. The mRNA transcripts were highly intact after 0 and 3 h of incubation; however, after 6 h the degradation was clearly evident. Following 3 h incubation, 98·8% and 100% mRNA transcripts were detectable in the colonic tissue harvested from weaned and mature pigs, respectively. In the explants of weaned piglets, LPS treatment activated inflammatory signalling pathways [high mobility group B1 (HMGB1), dendritic cell maturation, interleukin (IL)-6, IL-8, IL-17F], while these pathways were inhibited by dexamethasone treatment. Activations of inflammatory genes were also evident in the explants collected from the mature pigs subjected to ex-vivo incubation for 3 h in the absence or presence of LPS. It is concluded that the colonic explant remains physiologically viable and responsive to immunological challenge for up to 3 h ex-vivo.

The impact of vitamin D3 supplementation on the faecal and oral microbiome of dairy calves indoors or at pasture.

Vitamin D (VitD) is emerging as an immune regulator in addition to its established role in metabolism and mineral homeostasis. This study sought to determine if in vivo VitD modulated the oral and faecal microbiome in Holstein-Friesian dairy calves. The experimental model consisted of two control groups (Ctl-In, Ctl-Out) which were fed with a diet containing 6000 IU/Kg of VitD3 in milk replacer and 2000 IU/Kg in feed, and two treatment groups (VitD-In, VitD-Out) with 10,000 IU/Kg of VitD3 in milk replacer and 4000 IU/Kg in feed. One control and one treatment group were moved outdoors post-weaning at approximately 10 weeks of age. Saliva and faecal samples were collected after 7 months of supplementation and analysis of the microbiome was performed using 16S rRNA sequencing. Bray-Curtis dissimilarity analysis identified that both sampling site (oral vs. faecal) and housing (indoor vs. outdoor) had significant influences on the composition of the microbiome. The calves housed outdoors had greater microbial diversity in the faecal samples based on Observed, Chao1, Shannon, Simpson and Fisher measures in comparison to calves housed indoors (P < 0.05). A significant interaction between housing and treatment was observed for the genera Oscillospira, Ruminococcus, CF231 and Paludibacter in faecal samples. The genera Oscillospira and Dorea were increased while Clostridium and Blautia were decreased following VitD supplementation in the faecal samples (P < 0.05). An interaction between VitD supplementation and housing was detected in the abundance of the genera Actinobacillus and Streptococcus in the oral samples. VitD supplementation increased the genera Oscillospira, Helcococcus and reduced the genera Actinobacillus, Ruminococcus, Moraxella, Clostridium, Prevotella, Succinivibrio and Parvimonas. These preliminary data suggest that VitD supplementation alters both the oral and faecal microbiome. Further research will now be conducted to establish the significance of microbial alterations for animal health and performance.

Effects of dietary supplementation with a laminarin-rich extract on the growth performance and gastrointestinal health in broilers.

Restriction in antimicrobial use in broiler chicken production is driving the exploration of alternative feed additives that will support growth through the promotion of gastrointestinal health and development. The objective of this study was to determine the effects of dietary inclusion of laminarin on growth performance, the expression of nutrient transporters, markers of inflammation and intestinal integrity in the small intestine and composition of the caecal microbiota in broiler chickens. Two-hundred-and-forty day-old male Ross 308 broiler chicks (40.64 (3.43 SD) g) were randomly assigned to: (T1) basal diet (control); (T2) basal diet + 150 ppm laminarin; (T3) basal diet + 300 ppm laminarin (5 bird/pen; 16 pens/treatment). The basal diet was supplemented with a laminarin-rich Laminaria spp. extract (65% laminarin) to achieve the two laminarin inclusion levels (150 and 300 ppm). Chick weights and feed intake was recorded weekly. After 35 days of supplementation, one bird per pen from the control and best performing (300 ppm) laminarin groups were euthanized. Duodenal, jejunal and ileal tissues were collected for gene expression analysis. Caecal digesta was collected for microbiota analysis (high-throughput sequencing and QPCR). Dietary supplementation with 300 ppm laminarin increased both final body weight (2033 vs. 1906 ± 30.4, P < 0.05) and average daily gain (62.3 vs. 58.2 ± 0.95, P < 0.05) compared to the control group and average daily feed intake (114.1 vs. 106.0 and 104.5 ± 1.77, P < 0.05) compared to all other groups. Laminarin supplementation at 300 ppm increased the relative and absolute abundance of Bifidobacterium (P < 0.05) in the caecum. Laminarin supplementation increased the expression of interleukin 17A (IL17A) in the duodenum, claudin 1 (CLDN1) and toll-like receptor 2 (TLR2) in the jejunum and IL17A, CLDN1 and SLC15A1/peptide transporter 1 (SLC15A1/PepT1) in the ileum (P < 0.05). In conclusion, supplementation with laminarin is a promising dietary strategy to enhance growth performance and 300 ppm was the optimal inclusion level with which to promote a beneficial profile of the gastrointestinal microbiota in broiler chickens.

Colonic microbiome profiles for improved feed efficiency can be identified despite major effects of farm of origin and contemporary group in pigs.

While feed efficiency (FE) is a trait of great economic importance to the pig industry, the influence of the intestinal microbiome in determining FE is not well understood. The objective of this experiment was to determine the relative influence of FE and farm of birth on the pig colonic microbiome. Animals divergent in residual feed intake (RFI) were sourced from two geographically distinct locations (farms A + B) in Ireland. The 8 most efficient (low RFI (LRFI)) and 8 least efficient (high RFI, (HRFI)) pigs from farm A and 12 LRFI and 12 HRFI pigs from farm B were sacrificed. Colonic digesta was collected for microbial analysis using 16S ribosomal RNA gene sequencing and also for volatile fatty acid analysis. The α-diversity differed between the farms in this study, with pigs from farm A having greater diversity based on Shannon and InvSimpson measures compared to pigs from farm B (P < 0.05), with no difference identified in either Chao1 or observed measures of diversity (P > 0.05). In the analysis of ß-diversity, pigs clustered based on farm of birth rather than RFI. Variation in the management of piglets, weight of the piglets, season of the year, sanitary status and dam dietary influence could potentially be causative factors in this large variation between farms. However, despite significant variation in the microbial profile between farms, consistent taxonomic differences were identified between RFI groups. Within the phylum Bacteroidetes, the LRFI pigs had increased abundance of BS11 (P < 0.05) and a tendency toward increased Bacteroidaceae (P < 0.10) relative to the HRFI group. At genus level, the LRFI pigs had increased abundance of Colinsella (P < 0.05), a tendency toward increased Bacteroides and CF231 (P < 0.10). At species level, Ruminococcus flavefaciens had increased abundance in the LRFI compared to the HRFI animals. In conclusion, while farm of birth has a substantial influence on microbial diversity in the pig colon, a microbial signature indicative of FE status was apparent.

Pigs that are divergent in feed efficiency, differ in intestinal enzyme and nutrient transporter gene expression, nutrient digestibility and microbial activity.

Feed efficiency is an important trait in the future sustainability of pig production, however, the mechanisms involved are not fully elucidated. The objective of this study was to examine nutrient digestibility, organ weights, select bacterial populations, volatile fatty acids (VFA's), enzyme and intestinal nutrient transporter gene expression in a pig population divergent in feed efficiency. Male pigs (n=75; initial BW 22.4 kg SEM 2.03 kg) were fed a standard finishing diet for 43 days before slaughter to evaluate feed intake and growth for the purpose of calculating residual feed intake (RFI). Phenotypic RFI was calculated as the residuals from a regression model regressing average daily feed intake (ADFI) on average daily gain (ADG) and midtest BW0.60 (MBW). On day 115, 16 pigs (85 kg SEM 2.8 kg), designated as high RFI (HRFI) and low RFI (LRFI) were slaughtered and digesta was collected to calculate the coefficient of apparent ileal digestibility (CAID), total tract nutrient digestibility (CATTD), microbial populations and VFA's. Intestinal tissue was collected to examine intestinal nutrient transporter and enzyme gene expression. The LRFI pigs had lower ADFI (P<0.001), improved feed conversion ratio (P<0.001) and an improved RFI value relative to HRFI pigs (0.19 v. -0.14 SEM 0.08; P<0.001). The LRFI pigs had an increased CAID of gross energy (GE), and an improved CATTD of GE, nitrogen and dry matter compared to HRFI pigs (P<0.05). The LRFI pigs had higher relative gene expression levels of fatty acid binding transporter 2 (FABP2) (P<0.01), the sodium/glucose co-transporter 1 (SGLT1) (P<0.05), the glucose transporter GLUT2 (P<0.10), and the enzyme sucrase-isomaltase (SI) (P<0.05) in the jejunum. The LRFI pigs had increased populations of lactobacillus spp. in the caecum compared with HRFI pigs. In colonic digesta HRFI pigs had increased acetic acid concentrations (P<0.05). Differences in nutrient digestibility, intestinal microbial populations and gene expression levels of intestinal nutrient transporters could contribute to the biological processes responsible for feed efficiency in pigs.