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.

Strategies to combat heat stress in poultry production-A review.

The effects of heat stress (HS) caused by high temperatures continue to be a global concern in poultry production. Poultry birds are homoeothermic, however, modern-day chickens are highly susceptible to HS due to their inefficiency in dissipating heat from their body due to the lack of sweat glands. During HS, the heat load is higher than the chickens' ability to regulate it. This can disturb normal physiological functioning, affect metabolism and cause behavioural changes, respiratory alkalosis and immune dysregulation in birds. These adverse effects cause gut dysbiosis and, therefore, reduce nutrient absorption and energy metabolism. This consequently reduces production performances and causes economic losses. Several strategies have been explored to combat the effects of HS. These include environmentally controlled houses, provision of clean cold water, low stocking density, supplementation of appropriate feed additives, dual and restricted feeding regimes, early heat conditioning and genetic selection of poultry lines to produce heat-resistant birds. Despite all these efforts, HS still remains a challenge in the poultry sector. Therefore, there is a need to explore effective strategies to address this long-lasting problem. The most recent strategy to ameliorate HS in poultry is early perinatal programming using the in ovo technology. Such an approach seems particularly justified in broilers because chick embryo development (21 days) equals half of the chickens' posthatch lifespan (42 days). As such, this strategy is expected to be more efficient and cost-effective to mitigate the effects of HS on poultry and improve the performance and health of birds. Therefore, this review discusses the impact of HS on poultry, the advantages and limitations of the different strategies. Finally recommend a promising strategy that could be efficient in ameliorating the adverse effects of HS in poultry.

Changes in the gene expression and methylation in chicken cecal tonsils after in ovo administration of bioactive substances.

Cecal tonsils are the main organs which generate an immune response and also the part of the GALT, thus they are in the close proximity of the intestinal microbiota and continuously exposed to microbe-associated molecular patterns. GALT developed regulatory and anti-inflammatory mechanisms which eliminate or tolerate microbiota. Bioactive substances in ovo administration ensures an early contact between the GALT and beneficial bacteria, which greatly promotes the development of tolerance. Our previous studies have shown that the administration of bioactive substances in ovo silences gene expression in the cecal tonsils. The research hypothesis assumes that negative silencing of expression is correlated with the level of methylation in the tonsils. Therefore the current study aimed to analyze the global and gene-specific DNA methylation profiles in the cecal tonsils of two distinct chicken genotypes administered in ovo with bioactive substances. Eggs of Ross 308 and Green-legged Partridgelike were stimulated on day 12 of incubation. The injected compounds were: probiotic-Lactococcus lactis subsp. cremoris, prebiotic-galactooligosaccharides, and synbiotic-combination of both. Chickens were sacrificed on d 42 post-hatching. Cecal tonsils was collected, RNA and DNA were isolated and intended to gene expression, gene methylation and global methylation analysis. Cecal tonsils changes were observed in the methylation of 6 genes: SYK, ANGPTL4, TNFRSF14, IKZF1, CYR61, SERPING. Analyzes showed that the suppression of gene expression is related to the level of methylation of individual genes. Based on the results obtained in the cecal tonsils, it can be concluded that the silencing of gene expression is of an epigenetic nature. This is another study aimed at analyzing the relationship between the host, its intestinal microbiota and the possibilities of its programming.

Can Myofascial Trigger Points Involve Nociplastic Pain? A Scoping Review on Animal Models.

Nociplastic pain is a non-specific, regional pain lasting more than three months, characterised by the onset of hypersensitivity, despite no clear evidence of tissue damage. It is a relatively new classified type of pain. As a result, there has not yet been much work describing its precise modelling. The mechanism of its formation needs to be clearly explained. Authors point out that the occurrence of myofascial trigger points (MTrPs) can lead to this type of pain as one possibility. This paper summarises the available literature on modelling nociplastic pain and MTrPs. It complies with studies describing animal model creation and presents the results of performed experiments. The literature search was conducted in December 2022 and included the following databases: PubMed, Scopus, and Web of Science. In this scoping review, six studies were included. Two described the creation of animal models of nociplastic pain, one adapted old models to nociplastic pain, and three described the modelling of MTrPs. This is the first paper pointing in the possible direction of detecting and studying the correlation between MTrPs and nociplastic pain in animal models. However, there is currently insufficient evidence to describe MTrPs as nociplastic, as few studies with animal models exist.

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.

Effect of prebiotics administered during embryo development on mitochondria in intestinal and immune tissues of adult broiler chickens.

Mitochondria are cellular organelles that are the place of many metabolic processes and thus have a significant impact on the proper functioning of the organism. These organelles respond easily to environmental stimuli and cellular energy demands. To ensure the proper functioning of mitochondria, a high supply of specific nutrients is needed. Literature reports suggest that a favorable profile of the intestinal microbiota may improve the functioning of the mitochondria. The gut microbiota transmits a signal to the mitochondria of the mucosa cells. This signaling alters mitochondrial metabolism, activates cells of the immune system, and alters intestinal epithelial barrier functions. The aim of the study is to determine the relative number of mtDNA copies and to analyze the mitochondrial expression of genes related to respiratory chain proteins and energy metabolism in the intestinal mucosa and cecal tonsils of broiler chickens injected on the d 12 of egg incubation with various prebiotics. 300 incubated eggs of Ross 308 broiler chicken on d 12 of incubation were injected with: control group with physiological saline, prebiotics: XOS3, XOS4, MOS3, and MOS4. On d 42 after hatching, 8 individuals from each group were sacrificed. Cecal mucosa and cecal tonsils were collected postmortem for DNA and RNA isolation. Relative mitochondrial DNA copy number analysis was performed by qPCR method using 2 calculation methods. Gene expression analysis of the cecal tonsils and cecal mucosa was performed by RT-qPCR for the gene panel selected based on literature data and gene functions related to mitochondria: CS, EPX (MPO), CYCS, TFAM, NRF1, ND2, MnSOD (SOD2). As the results showed the overall mt DNA copy number is stable in both tissues. The significant change in gene expression in cecal mucosa was induced by XOS4 and MOS3. Both prebiotics caused upregulation of gene expression. In cecal tonsils all prebiotics caused downregulation of entire set of genes under the analysis. Statistically significant results of gene expression were detected for CYCS, ND2, NRF, TFAM for all experimental groups.

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.

Effect of Zeolite Supplementation on Gene Expression in the Intestinal Mucosa in the Context of Immunosafety Support in Poultry.

Zeolite is an effective and non-toxic silicate mineral. Its properties are widely used in industry due to its sorption and ion exchange properties. Due to its excellent chemical properties, it has also great potential in poultry production as a food additive or supplement to bedding. This is of great importance for the biosafety and hygiene of production. The study aimed to analyse the effects of simultaneous application of zeolite to feed and bedding on production parameters and expression of genes related to intestinal tightness, organism defence, and immune response. Male Ross 308 broiler chickens were used in the experiment. In the experimental group, an external factor in the form of a powdery zeolite was used for feed and pelleted bedding. On the day of slaughter, the caecal mucosa was collected for gene expression analysis. We showed no significant changes in the tissue composition of the carcasses, but zeolite had a beneficial effect on the carcass yield. The analysis of the immune gene panel showed a significant increase in the expression of the interleukins and interferons genes. We have demonstrated the effect of zeolite on the improvement of the intestinal barrier and increasing the tightness of the intestines. There were no changes in gene expression related to the host's defence against infections; therefore, based on the obtained results, it was concluded that zeolite can be considered an immunomodulating factor of the immune system.

Avian Cell Culture Models to Study Immunomodulatory Properties of Bioactive Products.

Antimicrobial resistance is becoming a greater danger to both human and animal health, reducing the capacity to treat bacterial infections and increasing the risk of morbidity and mortality from resistant bacteria. Antimicrobial efficacy in the treatment of bacterial infections is still a major concern in both veterinary and human medicine. Antimicrobials can be replaced with bioactive products. Only a small number of plant species have been studied in respect to their bioactive compounds. More research is needed to characterize and evaluate the therapeutic properties of the plant extracts. Due to the more and more common phenomenon of antimicrobial resistance, poultry farming requires the use of natural alternatives to veterinary antibiotics that have an immunomodulatory effect. These include a variety of bioactive products, such as plant extracts, essential oils, probiotics, prebiotics, and synbiotics. This article presents several studies on bioactive products and their immunomodulatory effects tested in vitro and ex vivo using various avian cell culture models. Primary cell cultures that have been established to study the immune response in chickens include peripheral blood mononuclear cells (PBMCs), intestinal epithelial cells (IEC), and bone marrow-derived dendritic cells (BMDCs). Chicken lymphatic lines that can be used to study immune responses are mainly: chicken B cells infected with avian leukemia RAV-1 virus (DT40), macrophage-like cell line (HD11), and a spleen-derived macrophage cell line (MQ-NCSU). Ex vivo organ cultures combine in vitro and in vivo studies, as this model is based on fragments of organs or tissues grown in vitro. As such, it mimics the natural reactions of organisms, but under controlled conditions. Most ex vivo organ cultures of chickens are derived from the ileum and are used to model the interaction between the gastrointestinal tract and the microbiota. In conclusion, the use of in vitro and ex vivo models allows for numerous experimental replications in a short period, with little or no ethical constraints and limited confounding factors.

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.

Modulation of Intestinal Histology by Probiotics, Prebiotics and Synbiotics Delivered In Ovo in Distinct Chicken Genotypes.

The aim of the study was to determine the effect of probiotics, prebiotics and synbiotics administered in ovo on selected morphological parameters of the small intestine (duodenum, jejunum, ileum) in broiler chickens (Ross 308) and native chickens (Green-legged Partridge, GP). On the 12th day of embryonic development (the incubation period), an aqueous solution of a suitable bioactive substance was supplied in ovo to the egg's air cell: probiotic-Lactococcus lactis subsp. cremoris (PRO), prebiotic-GOS, galacto-oligosaccharides (PRE) or symbiotic-GOS + Lactococcus lactis subsp. cremoris (SYN). Sterile saline was injected into control (CON) eggs. After hatching, the chicks were placed in pens (8 birds/pen, 4 replicates/group) and housed for 42 days. On the last day of the experiment, all birds were individually weighed and slaughtered. Samples for histological analysis were taken directly after slaughter from three sections of the small intestine. In samples from the duodenum, jejunum and ileum, the height and width of the intestinal villi (VH) were measured and their area (VA) was calculated, the depth of the intestinal crypts (CD) was determined, the thickness of the muscularis was measured and the ratio of the villus height to the crypt depth (V/C) was calculated. On the basis of the obtained data, it can be concluded that the applied substances administered in ovo affect the production parameters and intestinal morphology in broiler chickens and GP. The experiment showed a beneficial effect of in ovo stimulation with a prebiotic on the final body weight of Ross 308 compared to CON, while the effect of the administered substances on the intestinal microstructure is not unequivocal. In GP, the best effect in terms of villi height and V/C ratio was found in the in ovo synbiotic group. Taking into account the obtained results, it can be concluded that chickens of different genotypes react differently to a given substance; therefore, the substances should be adapted to the genotype.

Proteome changes upon in ovo stimulation with Lactobacillus synbiotic in chicken liver.

The liver, as the main metabolic organ, plays a key role in many vital processes, including nutrient metabolism, fat digestion, blood protein synthesis, and endocrine management. As one of the immune organs, it has a remarkable ability to adequately activate the immune cells in response to metabolic signals. The anatomy of the liver ensures its close interaction with the gut so that nutrients and gut microbiota contribute to normal metabolism. In chickens, the intestinal microbiota plays an important role in supporting health and improving production parameters. The most effective method of stimulating the microbiota is to administer an appropriate bioactive compound during embryonic development. In ovo stimulation on d 12 of egg incubation involves the delivery of the substance into the air chamber. The aim of the study was to analyze the changes at the protein level after in ovo administration of the synbiotic on d 12 of egg incubation. Our study is the first to conduct a proteome analysis in liver after the administration of a Lactobacillus synbiotic in ovo. Eggs of broiler chickens were injected with a synbiotic-Lactobacillus plantarum with raffinose family oligosaccharides (RFO). On d 21 posthatching liver was collected. We performed analyses based on two-dimensional electrophoresis, matrix-assisted laser desorption/ionization (MALDI) time-of-flight, and MALDI Fourier-transform ion cyclotron resonance to obtain a global view of the hepatic proteome changes in response to in ovo injection. A representative pattern of significantly altered liver proteins was observed after stimulation with the synbiotic. A total of 16 protein spots were differentially expressed, with 5 downregulated and 11 upregulated spots. We conclude that the in ovo synbiotic treatment had the potential to accelerate the major energy-yielding metabolic pathways in the liver of adult broilers.

SNP prioritization in targeted sequencing data associated with humoral immune responses in chicken.

Our study aimed to identify single nucleotide polymorphisms (SNPs) with a significant impact on the innate immunity represented by antibody response against lipopolysaccharide (LPS) and lipoteichoid acid (LTA) and the adaptive immune response represented toward keyhole limpet hemocyanin (KLH) using the SNP prioritization method. Data set consisted of 288 F2 experimental individuals, created by crossing Green-legged Partridgelike and White Leghorn. The analyzed SNPs were located within 24 short genomic regions of GGA1, GGA2, GGA3, GGA4, GGA9, GGA10, GGA14, GGA18, and GGZ, pre-targeted based on literature references and database information. For the specific antibody response toward KLH at d 0 the most highly prioritized SNP for additive and dominance effects were located on GGA2 in the 3'UTR of MYD88. For the response at d 7, the most highly prioritized SNP pointed at the 3'UTR of MYD88, but potential causal additive variants were located within ADIPOQ and one in PROCR. The highest priority for additive and dominance effects in the antibody response toward lipoteichoic acid at d 0 was attributed to the same SNP, located on GGA2 in the 3'UTR region of MYD88. Two SNPs among the top-10 for additive effect were located in the exon of NOCT. SNPs selected for their additive effect on antibody response toward lipopolysaccharide at d 0 marked 3 genes - NOCT, MYD88, and SNX8, while SNPs selected for their dominance effect marked - NOCT, ADIPOQ, and MYD88. The top-10 variants identified in our study were located in different functional parts of the genome. In the context of causality three groups can be distinguished: variants located in exons of protein coding genes (ADIPOQ, NOCT, PROCR, SNX8), variants within exons of non-coding transcripts, and variants located in genes' UTR regions. Variants from the first group influence protein structure and variants from both latter groups' exhibit regulatory roles on DNA (UTR) or RNA (lncRNA).

Comparison of the Transcriptomic and Epigenetic Profiles of Gonadal Primordial Germ Cells of White Leghorn and Green-Legged Partridgelike Chicken Embryos.

The Green-legged Partridgelike fowl is a native, dual-purpose Polish chicken. The White Leghorn has been intensively selected for several decades to mainly improve reproductive traits. Primordial germ cells (PGCs) represent the germline stem cells in chickens and are the only cells that can transfer the information stored in the genetic material from generation to generation. The aim of the study was to carry out a transcriptomic and an epigenetic comparison of the White Leghorn and Green-legged Partridgelike gonadal PGCs (gPGCs) at three developmental stages: days 4.5, 8, and 12 of the embryonic development. RNA and DNA were isolated from collected gPGCs. The RNA was further subjected to microarray analysis. An epigenetic analysis was performed based on the global methylation analysis and qMSP method for the particular silenced genes demonstrated in transcriptomic analysis. Statistically significant differences between the gPGCs from both breeds were detected on the day 8 of embryonic development. Global methylation analysis showed significant changes at the methylation level in the White Leghorn gPGCs on day 8 of embryonic development. The results suggest faster development of Green-legged Partridgelike embryos as compared to White Leghorn embryos. Changes in the levels of gene expression during embryonic development are determined by genetic and environmental factors, and this variability is influenced by breed and gender.

The Impact of Hydrated Aluminosilicates Supplemented in Litter and Feed on Chicken Growth, Muscle Traits and Gene Expression in the Intestinal Mucosa.

The aim of the study was to compare the production, muscle traits and gene expression in the intestinal mucosa of chickens supplemented with aluminosilicates in feed and litter simultaneously. A total of 300 Ross 308 were maintained for 42 days. Group 1 was the control group. In group 2, 0.650 kg/m2 of halloysite was added to the litter and 0.5-2% to the feed (halloysite and zeolite in a 1:1 ratio); in group 3, we added zeolite (0.650 kg/m2) to the litter and 0.5-2% to the feed. The production parameters, the slaughter yield and analyses of muscle quality were analyzed. There was a higher body weight, body weight gain and feed conversion ratio on day 18 and 33 in group 3, and a higher feed intake on day 19-33 in groups 2 and 3 than in 1. A lower water-holding capacity was found in the breasts of group 2 and in the legs of group 3 compared to group 1. The expression of genes related to the immune response, host defense and intestinal barrier and nutrient sensing in the intestinal tissue was analyzed. The results show a beneficial effect on the immune status of the host without an adverse effect on the expression of genes related to intestinal tightness or nutritional processes. Due to the growth, meat characteristics and the positive impact of immunostimulant and regulating properties, aluminosilicates can be suggested as a litter and feed additive in the rearing of chickens.

miRNA Profiling in the Chicken Liver under the Influence of Early Microbiota Stimulation with Probiotic, Prebiotic, and Synbiotic.

Epigenetic regulation of gene expression is a form of interaction of the external environment on reading and transcription of genetic information encoded in nucleic acids. We provided evidence that early stimulation of the chicken microbiota with in ovo delivered synbiotics influenced gene expression and DNA methylation in the liver. Therefore, we hypothesize that the stimulation of microbiota by administering bioactive substances in ovo also affects the activity of miRNA in liver. For the analysis of miRNA activity, RNA was isolated from liver of adult broiler chicken and native chicken breed. The animals received a prebiotic, probiotic and synbiotic in ovo on day 12 of egg incubation. The analysis of miRNA expression was performed using the LNA method on a miRNA panel selected on the basis of previous microarray experiments. We have found increased miRNA expression activity after probiotic and synbiotic administration, especially in native chicken breed. Our results suggest that prebiotics reduce or do not affect miRNA activity. We have also shown that miRNA activity is regulated by the substance and genotype of the chicken. We can conclude that miRNAs constitute an important component of the molecular mechanism of host-probiotic interaction in liver.

TLR-Mediated Cytokine Gene Expression in Chicken Peripheral Blood Mononuclear Cells as a Measure to Characterize Immunobiotics.

Immunobiotics are probiotics that promote intestinal health by modulating immune responses. Immunobiotics are recognized by Toll-like receptors (TLRs) and activate cytokine gene expression. This study aimed to characterize cytokine gene expression in the chicken peripheral blood mononuclear cells (PBMC) stimulated with purified TLR ligands and live probiotics. PBMC were isolated from the whole blood. PBMC were stimulated with: lipopolysaccharide (LPS), CpG ODN, Pam3CSK4, Zymosan, galactooligosaccharides (GOS), Lactococcuslactis subsp. cremoris (L. lactis), and Saccharomyces cerevisiae at 42.5 °C and 5% CO2 for 3 h, 6 h, and 9 h. After each time-point, PBMC were harvested for RNA isolation. Relative gene expression was analyzed with RT-qPCR for cytokine genes (IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12p40, and IFN-É£) and reference genes (ACTB and G6PDH). Genes were clustered into pro-inflammatory genes, Th1/Th2 genes, and Th1-regulators. The gene expression differed between treatments in IL1-ß, IL-6, IL-8, IL-10, and IL-12p40 (p < 0.001). The genes IL-1ß, IL-6, and IL-8 had the highest fold change of mRNA expression at 3 h in response to TLR ligands. L. lactis up-regulated the pro-inflammatory genes at the 6 h time-point. L. lactis did not activate the anti-inflammatory IL-10 gene, but activated IL-12p40 at 6 h. Hereby, L. lactis was proven to exert immunostimulatory properties in PBMC.

Sensitivity analysis based on the random forest machine learning algorithm identifies candidate genes for regulation of innate and adaptive immune response of chicken.

Two categories of immune responses-innate and adaptive immunity-have both polygenic backgrounds and a significant environmental component. The goal of the reported study was to define candidate genes and mutations for the immune traits of interest in chickens using machine learning-based sensitivity analysis for single-nucleotide polymorphisms (SNPs) located in candidate genes defined in quantitative trait loci regions. Here the adaptive immunity is represented by the specific antibody response toward keyhole limpet hemocyanin (KLH), whereas the innate immunity was represented by natural antibodies toward lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The analysis consisted of 3 basic steps: an identification of candidate SNPs via feature selection, an optimisation of the feature set using recursive feature elimination, and finally a gene-level sensitivity analysis for final selection of models. The predictive model based on 5 genes (MAPK8IP3 CRLF3, UNC13D, ILR9, and PRCKB) explains 14.9% of variance for KLH adaptive response. The models obtained for LTA and LPS use more genes and have lower predictive power, explaining respectively 7.8 and 4.5% of total variance. In comparison, the linear models built on genes identified by a standard statistical analysis explain 1.5, 0.5, and 0.3% of variance for KLH, LTA, and LPS response, respectively. The present study shows that machine learning methods applied to systems with a complex interaction network can discover phenotype-genotype associations with much higher sensitivity than traditional statistical models. It adds contribution to evidence suggesting a role of MAPK8IP3 in the adaptive immune response. It also indicates that CRLF3 is involved in this process as well. Both findings need additional verification.