Modulation of gene expression in immune-related organs by in ovo stimulation with probiotics and prophybiotics in broiler chickens.

In ovo stimulation has been studied intensively as an alternative to antibiotic use in poultry production. We investigated the potential use of a probiotic in combination with a phytobiotic as a prophybiotic for in ovo stimulation and reported its beneficial effects on the gut microbiome of broiler chickens. The current study further investigates the gene expression in the immune-related organs of these chickens to understand the tissue-specific immunomodulatory effects of the treatments. The selected prophybiotic (Leuconostoc mesenteroides with garlic aqueous extract) and its probiotic component alone were injected into ROSS308 chicken eggs on the 12th day of incubation, and gene expression in cecal tonsils, spleen, and liver at 35 days of age was determined using qPCR method. The relative expression of each treatment was compared to the positive control, chickens injected with physiological saline in ovo. The results displayed a downregulation of pro- and anti-inflammatory cytokines in the cecal tonsils of the probiotic group and the liver of the prophybiotic group. The spleen displayed upregulated AVBD1 in both groups and upregulated IL1-ß in the probiotic group. The probiotic group displayed increased expression of genes related to metabolism of energy (COX16), protein (mTOR), and lipids (CYP46A1) whereas the prophybiotic group displayed reduced expression of genes related to cholesterol synthesis (SREBP1) and glucose transportation (SLC2A2) in the liver. In conclusion, Leuconostoc mesenteroides differentially modulated gene expression in chickens when administered in ovo in combination with garlic aqueous extract. Further in ovo studies with different prophybiotic combinations are required to optimize the benefits in broiler chickens.

In-vitro selection of lactic acid bacteria to combat Salmonella enterica and Campylobacter jejuni in broiler chickens.

Campylobacter and Salmonella are the two most prominent foodborne zoonotic pathogens reported in the European Union. As poultry is one of the major sources of these pathogens, it is imperative to mitigate the colonization of these pathogens in poultry. Many strains of lactic acid bacteria (LAB) have demonstrated anti-Salmonella and anti-Campylobacter characteristics to varying degrees and spectrums which are attributed to the production of various metabolites. However, the production of these compounds and consequent antimicrobial properties are highly strain dependent. Therefore, the current study was performed to select a potent LAB and determine its causal attribute in inhibiting Salmonella enterica and Campylobacter jejuni, in-vitro. Six LAB (Lactiplantibacillus plantarum (LP), Lacticaseibacillus casei (LC), Limosilactobacillus reuteri (LR), Lacticaseibacillus rhamnosus (LRh), Leuconostoc mesenteroides (LM) and Pediococcus pentosaceus (PP)) and three serovars of Salmonella enterica (Typhimurium, Enterica and Braenderup) and Campylobacter jejuni were used in the current study. Spot overlays, well diffusion, co-culture and co-aggregation assays against Salmonella and well diffusion assays against Campylobacter jejuni were performed. Organic acid profiling of culture supernatants was performed using HPLC. The results indicated that LRh, LM and PP had the most significant anti-Salmonella effects while LP, LC, LM and PP displayed the most significant anti-Campylobacter effects. Lactic acid and formic acid detected in the culture supernatants seem the most likely source of the anti-Salmonella and anti-Campylobacter effects exhibited by these LAB. In conclusion, Leuconostoc mesenteroides displayed the most significant overall anti-pathogenic effects when compared to the other LAB strains studied, indicating its potential application in-vivo.

Prophybiotics for in-ovo stimulation; validation of effects on gut health and production of broiler chickens.

Probiotics and phytobiotics have demonstrated effective improvement of gut health in broiler chickens when individually administered in-ovo. However, their combined use in-ovo, has not been studied to date. We coined the term "prophybiotic" (probiotic + phytobiotic) for such a combination. The current study therefore, aimed to elucidate the effects of combined use of a selected probiotic and a phytobiotic in-ovo, on broiler gut health and production parameters, as opposed to use of probiotics alone. ROSS 308 hatching eggs were injected with either Leuconostoc mesenteroides (probiotic: PB) or L. mesenteroides with garlic aqueous extract (prophyiotic: PPB) on the 12th day of incubation. Relative abundances of bacteria in feces and cecal content (qPCR), immune related gene expression in cecal mucosa (qPCR) and histomorphology of cecal tissue (PAS staining) were analyzed along with production parameters (hatch quality, body weight, feed efficiency and slaughter and meat quality). PPB treatment increased the abundance of faecalibacteria and bifidobacteria in feces (d 7) and Akkermansia sp. in cecal content. Moreover, it decreased Escherichia coli abundance in both feces (d 34) and cecal content. PB treatment only increased the faecalibacteria in feces (d 7) and Akkermansia sp. in the cecal content. Moreover, PPB treatment resulted in up-regulation of immune related genes (Avian beta defensing 1, Free fatty acid receptor 2 and Mucin 6) and increased the crypt depth in ceca whereas PB treatment demonstrated a higher crypt depth and a tendency to increase Mucin 6 gene expression. Both treatments did not impair the production parameters studied. In conclusion, our results suggest that in-ovo PPB treatment may have enhanced potential in boosting the immune system without compromising broiler production and efficiency, as compared to the use of probiotic alone. Our study, highlights the potential of carefully selected PPB combinations for better results in improving gut health of broiler chickens.

In-vitro screening of compatible synbiotics and (introducing) "prophybiotics" as a tool to improve gut health.

Synbiotics have been intensively studied recently to improve gut health of humans and animals. The success of synergistic synbiotics depends on the compatibility of the prebiotic and probiotic components. Certain plant extracts possess both antimicrobial and prebiotic properties representing a potential use in combination with probiotics to improve the gut health. Here, we coined the term "prophybiotics" to describe this combined bioactivity. The current study aimed to select prebiotics that are preferred as an energy source and antimicrobial plant extracts which do not inhibit the growth, of six strains of lactic acid bacteria (LAB namely; Lactiplantibacillus plantarum, Lacticaseibacillus casei, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Leuconostoc mesenteroides, and Pediococcus pentosaceus) in-vitro to identify compatible combinations for potential synbiotic/prophybiotic use, respectively. Their growth kinetics were profiled in the presence of prebiotics: Inulin, Raffinose, and Saccharicterpenin with glucose, as the control, using carbohydrate free MRS broth media. Similarly, their growth kinetics in MRS broth supplemented with turmeric, green tea, and garlic extracts at varying concentrations were profiled. The results revealed the most compatible pairs of prebiotics and LAB. Turmeric and garlic had very little inhibitory effect on the growth of the LAB while green tea inhibited the growth of all LAB in a dose-dependent manner. Therefore, we conclude that turmeric and garlic have broad potential for use in prophybiotics, while the prebiotics studied here have limited use in synbiotics, with these LAB.

MicroRNA expression in immune tissues of adult chickens after embryo stimulation with bioactive substances.

The microbiota has a profound impact on the host organisms. The interaction between the host and its microbiota has an epigenetic mode of action. In poultry species, gastrointestinal microbiota might be stimulated before hatching. This stimulation with bioactive substances has a broad spectrum and long-term effects. This study aimed to examine the role of miRNA expression stimulated by host-microbiota interaction via administering a bioactive substance at the stage of embryonic development. This paper is a continuation of earlier research in the field of molecular analyzes in immune tissues after in ovo administration of bioactive substances. Eggs of Ross 308 broiler chicken and Polish native breed chicken (Green-legged Partridgelike) were incubated in the commercial hatchery. On day 12 of incubation, eggs were injected: the control group with saline (0.2 mM physiological saline), probiotic-Lactococcus lactis subsp. cremoris, prebiotic-galactooligosaccharides, and synbiotic-mentioned above prebiotic with probiotic. The birds were intended for rearing. miRNA expression analysis was performed using the miRCURY LNA miRNA PCR Assay in the spleen and tonsils of adult chickens. Six miRNAs differed significantly, at least between one pair of treatment groups. The most miRNA changes were observed in the cecal tonsils of Green-legged Partridgelike chickens. At the same time, only miR-1598 and miR-1652 showed significant differences between the treatment groups in the cecal tonsils and spleen of Ross broiler chickens. Only two miRNAs showed significant GeneOntology (GO)enrichment with the ClueGo plug-in. gga-miR-1652 target genes showed only 2 GOs significantly enriched: chondrocyte differentiation and early endosome. gga-miR-1612 target genes, the most significant GO was regulating the RNA metabolic process. The enriched functions were associated with gene expression or protein regulation, the nervous system, and the immune system. Results suggest that early microbiome stimulation in chicken might regulate the miRNA expression in different immune tissues in a genotype-dependent manner.

Pre-hatching and post-hatching environmental factors related to epigenetic mechanisms in poultry.

Epigenetic modifications are phenotypic changes unrelated to the modification of the DNA sequence. These modifications are essential for regulating cellular differentiation and organism development. In this case, epigenetics controls how the animal's genetic potential is used. The main epigenetic mechanisms are microRNA activity, DNA methylation, and histone modification. The literature has repeatedly shown that environmental modulation has a significant influence on the regulation of epigenetic mechanisms in poultry. The aim of this review is to give an overview of the current state of the knowledge in poultry epigenetics in terms of issues relevant to overall poultry production and the improvement of the health status in chickens and other poultry species. One of the main differences between birds and mammals is the stage of embryonic development. The bird's embryo develops outside its mother, so an optimal environment of egg incubation before hatching is crucial for development. It is also the moment when many factors influence the activation of epigenetic mechanisms, i.e., incubation temperature, humidity, light, as well as in ovo treatments. Epigenome of the adult birds might be modulated by nutrition, supplementation, and treatment, as well as modification of the intestinal microbiota. In addition, the activation of epigenetic mechanisms is influenced by pathogens (i.e., pathogenic bacteria, toxins, viruses, and fungi) as well as the maintenance conditions. Farm animal epigenetics is still a big challenge for scientists. This is a research area with many open questions. Modern methods of epigenetic analysis can serve both in the analysis of biological mechanisms and in the research and applied to production system, poultry health, and welfare.

Epigenetic modifications are phenotypic changes unrelated to the modification of the DNA sequence. In this case, epigenetics controls how the animal's genetic potential is used. The literature has shown that environmental modulation has a significant influence on the regulation of epigenetic mechanisms in poultry. The aim of this review is to give an overview of the current state of the knowledge in poultry epigenetics in terms of issues relevant to overall poultry production and the improvement of the health status in poultry. The bird's embryo develops outside its mother, so an optimal environment of egg incubation before hatching is crucial for development. It is also the moment when many factors influence the activation of epigenetic mechanisms, i.e., incubation temperature, humidity, light, as well as in ovo treatments. Epigenome of the adult birds might be modulated by nutrition, supplementation, and treatment, as well as modification of the intestinal microbiota. The activation of epigenetic mechanisms is influenced by pathogens as well as the maintenance conditions. Farm animal epigenetics is still a big challenge for scientists. Modern methods of epigenetic analysis can serve both in the analysis of biological mechanisms and in the research and applied to production system, poultry health, and welfare.