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Epigenetic mechanisms play a pivotal role in hereditary processes, shaping phenotypic outcomes across generations. This study investigates the transgenerational impacts of in ovo injection of bioactive substances on gene expression and DNA methylation in the male gonads using the Green-legged Partridgelike chickens as a model organism. Synbiotic PoultryStar ® (Biomin; PS) and choline were injected in ovo on the 12th day of egg incubation. In the F1 generation, three groups were established: (1) control (C, 0.9% physiological saline); (2) PS synbiotic (SYN, 2 mg/embryo); and (3) PS synbiotic combined with choline (SYNCH, 2 mg/embryo of synbiotic and 0.25 mg/embryo of choline). In subsequent F2 and F3 generations, groups SYN and SYNCH were further divided into two subgroups each: (A) only injected in F1 embryos (SYNs and SYNCHs); and (B) repeatedly injected in every generation (SYNr and SYNCHr). At 21 weeks post-hatching, gonadal tissues were sampled from F2 and F3 male chickens for transcriptomic and reduced representation bisulfite sequencing (RRBS). Synbiotic alone produced minimal and diminishing changes in gene expression across generations. In contrast, the single co-administration of synbiotic and choline in F1 embryos (SYNCHs) led to 1,897 differentially expressed genes (DEGs) and 786 differentially methylated regions (DMRs) in F3. Repeated administration across generations (SYNCHr) resulted in an even greater number of DEGs (2,804) and DMRs (2,880) in F3, reflecting a cumulative exposure effect. DEGs in SYNCH groups were mainly enriched in pathways related to cytoskeletal organization and extracellular matrix. In SYNCHs, methylation changes were associated with TGF-beta signaling, whereas SYNCHr showed additional enrichment in Wnt signaling, focal adhesion, and adipocytokine signaling pathways. Integrative analysis revealed coordinated changes in gene expression and DNA methylation, particularly in the F3 generation, identifying 37 genes (47 DMRs) in SYNCHs and 194 genes (306 DMRs) in SYNCHr. This study highlights the potential of prenatal epigenetic interventions to induce gene expression and DNA methylation changes across generations in reproductive tissues.

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.

In ovo delivery of prebiotics and synbiotics in chickens allows for the development of intestinal microflora prior to hatching, which boosts their robustness. The goal of this study was to determine the transcriptomic profile of the spleen (S), cecal tonsils (CT), and large intestine (LI) of adult chickens injected with prebiotics and synbiotics in ovo. On day 12 of embryo development, incubating eggs were injected with prebiotics: inulin alone (P1) or in combination with Lactococcus lactis subsp. lactis IBB2955 (S1), galactooligosaccharides (GOS) alone (P2) or in combination with Lactococcus lactis subsp. cremoris IBB477 (S2); control group (C) was mock injected with physiological saline. Gene expression analysis was conducted using an Affymetrix Chicken Gene 1.1 ST Array Strip. Most of the differentially expressed genes (DEG) were detected in the cecal tonsils of P2 (378 DEG), and were assigned to gene ontology categories: lymphocyte proliferation, activation and differentiation, and cytokine production. Ingenuity pathway analysis of the DEG (CT of P2) indicated the inhibition of humoral and cellular immune responses, e.g., role of NFAT in regulation of immune responses, phagocytosis, production of nitric oxide, NF-κB, IL-8, and CXCR4 signaling. The DEG with the highest up-regulation from S1 and P2 were involved in gene expression (PAPOLA, RPL27A, RPLP1, and RPS29) from P1 and P2 in transport (BEST4, SLC9A3, and SLC13A2), metabolism (OGT, ALPP, CA4, and CA7), signaling (FGG, G3BP2, UBB, G3BP2, CACNA1G, and ATP6V0A4), and immune responses (MSMB, LGALS3, CABIN1, CXCR5, PAX5, and TNFRSF14). Two DEG influencing the complement system (SERPING1 and MIR1674) were down-regulated in P2 and S1. In conclusion, GOS injected in ovo provided the most potent stimulation of the host transcriptome. This is likely due to its strong bifidogenic effect, which triggers proliferation of indigenous embryonic microflora in ovo, and indirectly influences gene expression regulation in host tissues, especially cecal tonsils.

Epigenetic modifications can influence the phenotypes of subsequent generations through intergenerational and transgenerational effects. The aim of the research was to assess the impact of epigenetic factors acting during embryonic development on the structure and cellular composition of lymphoid organs in three generations of chickens. Two groups of eggs were injected once (in the F1 generation) with a synbiotic (SYNs) or a synbiotic + choline (SYNCHs), while two other groups were injected in each successive generation (F2, F3—SYNr, SYNCHr). Synbiotic administration resulted in an increased cortex/medulla ratio of the thymus in the F1 but not in subsequent generations. In the spleen, an intergenerational effect (from F1 to F2) was found in the choline-supplemented (SYNCHs) groups but not in the SYNs groups. Not all changes observed in the F1 were evident in the F2 generation. No intergenerational effect was found in the cecal tonsil, and no transgenerational effects were observed in any of the tested organs. In ovo administration of synbiotics with choline may induce intergenerational phenotypic effects on specific immune organs. However, these effects either persisted through the first two generations or appeared solely in the F1 or F2 generations. Changes were evident in young birds but not in mature ones.

Objective Regulation of gene expression during embryo development on the basis of migration of primordial germ cells (PGCs) in vivo has been rarely studied due to limited cell number and the necessity to isolate PGCs from a large number of embryos. Moreover, little is known about the comprehensive dynamics of the transcriptome in chicken PGCs during early developmental stages. The current study investigated transcriptome dynamics of chicken PGCs at key developmental stages: 4.5, 8 and 12 days of embryo incubation. PGCs were collected, and RNA was isolated using a commercial kit for single cells. The isolated RNA was subjected to microarray analysis (Agilent Technologies). Results Between 8 and 12 days of incubation, the highest number of genes was regulated. These data indicate that the most intense biological activity occurs between 8 and 12 days of embryo development. Heat map showed a significant decrease in gene expression on day 8, while it increased on day 12. The development of a precise method to isolate bird PGCs as well as the method to isolate RNA from single cells isolated from one embryo allows for early molecular analysis and detection of transcriptome changes during embryonic development.

The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo’s feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.

The aim was to investigate the impact of an automatic in ovo injection of the raffinose family oligosaccharides (RFO) extracted from the seeds of Lupinus luteus L, on the chicken performance and resistance in a production environment. At day 12 of incubation, a total of 57,900 eggs (Ross 308) were divided into two groups: 1/ Control, injected with 0.9% NaCl and 2/ RFO group, injected with 1.9 mg/egg of the lupin seed extract, dissolved in 0.2 mL NaCl. The performance parameters, biochemical indices (lipid profile, hepatic parameters), gut histomorphology and duodenum structure, oxidative stability of the meat and microbiological counts of the major commensal microbiota species were analyzed. Mortality, body weight, and feed conversion ratio (FCR) were not affected. By day 42, several health indices were improved with RFO and were reflected in a beneficial lipid blood profile, increased villi surface and better combating opportunistic pathogens through reduction of Clostridia and decreased coccidia counts. The RFO increased meat oxidation, but only at the beginning of the storage. The RFO sourced from local legumes can be considered a promising prebiotic for broiler chickens. In ovo delivery of prebiotics and/or synbiotics should be further optimized as an important strategy for the earliest possible modulation of chicken resistance.

Background A previous study showed that prebiotics and synbiotics administered in ovo into the egg air cell on the 12th day of incubation enhance the growth and development of chickens. However, the influence of this procedure on the development and efficiency of the innate immune system of broiler chickens is unclear. Therefore, the aim of this study was to evaluate whether the early (on the 12th day of embryo development) in ovo administration of selected prebiotics (inulin − Pre1 and Bi 2 tos − Pre2) and synbiotics (inulin + Lactococcus lactis subsp. lactis IBB SL1 − Syn1 and Bi 2 tos +  L. lactis subsp. cremoris IBB SC1 − Syn2) influences the innate immune system. Results Chickens (broiler, Ross 308) that were treated with Pre1 exhibited a decreased H/L ratio on D7, but an increased H/L ratio was observed on D21 and D35. In the remaining experimental groups, an increase in the H/L ratio was observed on D21 and D35. The oxidative potential of leukocytes measured using the NBT test increased on D21 in Pre2 and Syn1 groups. The rate of the phagocytic ability of leukocytes increased in Pre1 and Syn1 groups on D21. The phagocytic index decreased in Pre1 and Syn2 groups on D21 and D35. Concurrently, the count of WBC in circulating blood decreased on D21 in Pre1, Pre2, and Syn1 groups. The hematocrit value was increased in Syn1 chickens on D21, in Pre1 chickens on D35, and in Syn2 chickens on both time points. Conclusions Early in ovo treatment of chicken embryos with prebiotics and synbiotics may temporarily modulate not only the production/maturation of leukocytes but also their reactivity.

The present study aimed to test the synbiotic PoultryStar® solUS delivered in ovo to evaluate its effect on hatchability, productive performance and meat quality, compared to its post-hatch administration in water. On the twelfth day of embryonic incubation, 1200 fertile eggs were divided into synbiotic groups injected with 2 mg/embryo (T1) and 3 mg/embryo (T2), a saline group injected with physiological saline and an uninjected control group (C). After hatching, 120 male chicks/group were reared and chicks from the saline group were supplemented with the synbiotic via drinking water (T3). Hatchability was low in both T1 and T2 groups. Growth performance was not affected by the treatments. However, in the second rearing phase (15–36 days), birds from the C and T3 groups were heavier than T1 birds, due to a higher feed intake and daily weight gain. Neither route of synbiotic administration influenced final body weight (at 56 days), weight and yield of the carcass or commercial cuts. Physico-chemical properties, total lipid, cholesterol and fatty acid composition of breast muscle were not affected by the treatments. Considering its exploratory nature, this study has raised many questions that need further investigation, such as the bioactive combination and the effect on embryonic development.

The effect of the in ovo application of selected prebiotics and synbiotics on the humoral immune response against T-dependent (SRBC) and T-independent (dextran) antigens and delayed-type hypersensitivity (DTH) to phytohemagglutinin was studied. On the 12th day of incubation, 800 eggs (Ross 308) were divided into five groups and injected into the egg air chamber with prebiotic inulin (Pre1), Bi2tos (Pre2), a synbiotic composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1), a synbiotic composed of Bi2tos and L. lactis subsp. cremoris IBB SC1 (Syn2), and physiological saline (control group; C). The chickens were immunized twice at the 7th and 21st day of life with SRBC and dextran. A DTH test was performed on the 7th, 21st, and 35th day. The application of prebiotics and synbiotics had no significant effect on the humoral immune response. SRBC-immunized in ovo Pre1- and Pre2-treated chickens showed significantly higher serum IgG levels than the control. A significant effect on the DTH reaction was detected on the 7th (Pre1 < C) and 21st (Pre2 > Syn2) day. However; Bi2tos may transiently stimulate the cellular immune response on the 21st day. It may be concluded that the application of inulin in an egg air chamber on the 12th day of incubation may stimulate the secondary immune response. The inulin-treated group exhibited a lower mortality rate than the control group.