Synbiotics for Broiler Chickens-In Vitro Design and Evaluation of the Influence on Host and Selected Microbiota Populations following In Ovo Delivery

Abstract

Synbiotics are synergistic combinations of prebiotics and probiotics. In chickens, synbiotics can be delivered in ovo to expedite colonization of the gut by beneficial bacteria. We therefore aimed to design synbiotics in vitro and validate them in broiler chickens upon in ovo delivery. The probiotic components of the synbiotics were Lactobacillus salivarius and Lactobacillus plantarum. Their growth was assessed in MRS medium supplemented with different prebiotics. Based on in vitro results (hatchability and growth curve), two synbiotics were designed: S1 -Lactobacillus salivarius with galactooligosaccarides (GOS) and S2 -Lactobacillus plantarum with raffinose family oligosaccharides (RFO). These synbiotics were delivered to Cobb broiler chicken embryos on day 12 of incubation at optimized doses (105 cfu egg-1 of probiotic, 2 mg egg-1 of prebiotic). Post hatching, 2,400 roosters were reared (600 individuals group-1 divided into eight replicate pens). Microbial communities were analyzed in ileal and cecal digesta on day 21 using FISH. Gene expression analysis (IL1β, IL4, IL6, IL8, IL12, IL18, IFNβ, and IFNγ) was performed on days 7, 14, 21, and 42 for the spleen and cecal tonsils with RT-qPCR. Body weight and feed intake of the roosters did not differ by the treatments. Microbial populations of Lactobacillus spp. and Enterococcus spp. in the ileum were higher in S1 and S2 than in the control. In the cecum, the control had the highest bacterial counts. S1 caused significant up-regulation of IL6, IL18, IL1β, IFNγ, and IFNβ in the spleen on day 21 and IL1β on day 7 (P < 0.05). In cecal tonsils, S1 caused significant down-regulation of IL12, IL8, and IL1β on day 42 and IFNβ on day 14 (P < 0.05). S2 did not elicit such patterns in any tissues investigated. Thus, we demonstrate that divergent effects of synbiotics in broiler chickens were reflected in in vitro tests.

Fig 1. Changes in optical density (OD) of culture media during incubation of Lactobacillus bacteria with prebiotics.

Incubation was performed with glucose as a control for (A) Lactobacillus salivarius and (B) Lactobacillus plantarum in presence of prebiotics RFO, Inulin, GOS, or beta-glucan. Growth of bacteria in the MRS medium supplemented with the prebiotics glucose, GOS, RFO, inulin, or beta-glucan was measured using the automated growth analyzer Bioscreen C (Oy Growth Curves Ab, Ltd.) for 72 h.

Fig 2

Chick hatchability in response to different doses of synbiotics delivered in ovo for (A) L. salivarius + GOS (S1) and (B) L. plantarum + RFO (S2). Six doses of two synbiotics were delivered at 12^th day of incubation. Combination of three doses of probiotics (10³, 10⁴, 10⁵ cfu egg^-1) with two doses of prebiotics (2 mg egg^-1 or 5 mg egg^-1) were tested. The highest dose that did not decrease hatchability (10⁵ cfu egg^-1 of prebiotic and 2 mg egg of probiotic) was selected for the field trial. One-way analysis of variance (ANOVA) was performed. Duncan’s post hoc test was applied to compare the mean values between pairs in the experimental groups (a, b: P < 0.05).

Fig 3. Selected bacterial counts (log cfu/ml digesta) in digesta determined by DAPI staining (total number of bacteria) and fluorescent in situ hybridization (FISH).

Hybridization was performed with oligonucleotides, whose sequence was based on the literature. A Carl Zeiss Microscope Axio Imager M2 (Carl Zeiss Jena GmbH, Jena, Germany) was used to determine bacterial count.

Fig 4

Changes in relative expression of the immune-related panel of genes in the spleen of broiler chickens injected in ovo with the synbiotics (A) Lactobacillus salivarius with GOS and (B) Lactobacillus plantarum with RFO. Analysis was performed using the ^ΔΔCt method to determine fold induction. Synbiotics were injected on day 12 of embryonic development. Sampling days were 7, 14, 21, and 42 days post hatching. Statistical analysis consisted of comparing the experimental groups with the control group by Student's t-test; * for P < 0.05. Each bar represents the mean (n = 5) ± standard error of the mean (SEM).

Fig 5

Changes in relative expression of immune-related panel of genes in the cecal tonsils of broiler chickens injected in ovo with synbiotics (A) Lactobacillus salivarius with GOS and (B) Lactobacillus plantarum with RFO. Analysis was performed using the ^ΔΔCt method to determine fold induction. Synbiotics were injected on day 12 of embryonic development. Sampling days were 7, 14, 21, and 42 days post hatching. Statistical analysis consisted of comparing the experimental groups with the control group by Student's t-test; * for P < 0.05. Each bar represents the mean (n = 5) ± standard error of the mean (SEM).