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An experiment was conducted to assess the response of chicks to in-ovo injection of Bacillus subtilis (probiotic), raffinose (prebiotic), and their combinations. The study used 1,500 embryonated eggs allotted to 10 groups/ 6 replicates (150 eggs/group). The experimental treatments were: 1) un-injected control (NC); 2) sham (sterile distilled water) (PC); 3) probiotic 4 × 105CFU/egg (LBS); 4) probiotic 4 × 106CFU/egg (HBS); 5) prebiotic 2 mg/egg (LR); (6 prebiotic 3 mg/egg (HR); 7) probiotic 4 × 105CFU + prebiotic 2 mg/egg (LBS+LR); 8) probiotic 4 × 105CFU + prebiotic 3 mg/egg (LBS+HR); 9) probiotic 4 × 106CFU + prebiotic 2 mg/egg (HBS+LR); and 10) probiotic 4 × 106CFU + prebiotic 3 mg/egg (HBS+HR). Results showed that in-ovo inclusion of Bacillus subtilis, prebiotic, and their combinations improved hatchability, yolk-free chick weight, and chick weight compared to the control group. Moreover, the in-ovo treatment reduced residual yolk weight on the day of hatch compared to the control group. Different levels of in-ovo B. subtilis alone or combined with raffinose significantly (P ≤ 0.001) reduced total bacterial count and total yeast and mold count compared to the negative control group. Total coliform and E. coli decreased significantly (P ≤ 0.001) in groups treated with probiotics, prebiotics, and synbiotics with different doses during incubation compared to those in the control. Clostridium spp. was not detected in the groups injected with B. subtilis alone or combined with raffinose. In-ovo probiotics and synbiotics (LBS+LR & LBS+HR) significantly (P ≤ 0.001) increased ileal villus length compared to other groups. In-ovo treatment increased mRNA expression of JAM-2 compared to the control group. The fold change significantly increased in group LBS+HR for genes MUC-2, OCLN, VEGF, SGLT-1, and EAAT-3 compared to the negative control. In conclusion, in-ovo injection of a low dose of B. subtilis plus a high or low dose of raffinose can positively affect hatching traits, cecal microbial populations, intestinal histomorphometry, nutrient transport- and intestinal function-related genes, and chick quality of newly hatched broiler chicks.

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This investigation was directed to examine the influence of copper oxide nanoparticles (CuO-NPs) on the hatchability traits, and chick quality of newly hatched broiler chicks. A total of 480 eggs were randomly divided into four treatment groups, each consisting of three duplicates. As a negative control (NC), the first group was not injected; the second group was injected with saline and served as a positive control (PC), the third and fourth groups were injected with 30 and 60 ppm of (CuO-NPs)/egg. Eggs were injected into the amniotic fluid on the eighteenth day of the incubation period. Results showed that the hatchability, chick yield %, yolk free-body mass (YFBM), chick length, shank length (SL), and relative weight of the heart, gizzard and intestine of day-old broiler chicks were all unaffected by the in ovo injection of CuO-NPs. The Pasgar Score was slightly improved compared to the NC and PC groups. Also, the in ovo administration of CuO-NPs (60 ppm/egg) significantly increased the intestine length. Both levels of CuO-NPs significantly increased the concentration of Cu ions in the hepatic tissue. Additionally, different levels of tissue damage were seen in the liver of the birds that were given low or high dosages of CuO-NPs. Conclusively, the in ovo injection of CuO-NPs has a good result on the appearance of the chicks (Pasgar score). However, negative effect of CuO-NPs on liver tissue may raise concerns about the potential risks of applying CuO-NPs in ovo administration.

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BACKGROUND: Selenium is an essential mineral for poultry. The conflicting reports about its in ovo injection are the justification for the more detailed investigation. OBJECTIVES: The aim of this study was to investigate the effects of in ovo injection of organic selenium on the hatching traits of broiler chickens and their performance. METHODS: Three hundred and twenty eggs of Ross 308 strain with an average weight of 65 g and 160 chicks were randomly divided into 4 treatment groups (each with 8 replicates of 10 eggs each for hatching parameters and 4 replicates of 10 chicks for broiler farming parameters): negative control (no injection), positive control (in ovo injection of 0.272 mL of normal saline solution) and 2 selenium treatments (in ovo injection of 2.72 or 5.44 µg of organic selenium). Injection was into the amniotic sac on the 10th day of incubation. Effects of in ovo injection on hatching and performance traits, blood parameters, immune responses, carcass characteristics, meat fatty acid profile, cecal microbial population and selenium consternation in the tibia were measured. RESULTS: Fewer chicks from the injected treatments hatched than from the negative control group (p < 0.01). However, the injection of selenium increased feed intake and the final weight of the birds (p < 0.01). Blood parameters were also affected. Glucose and cholesterol in experimental treatment chicks was lower than those of the controls (p < 0.01), whereas blood lipoproteins (VLDL, LDL and HDL) and the ratio of cholesterol to HDL was significantly increased in the treatments injected with selenium (p < 0.01). There was no significant difference in the immune response or microbial population between the experimental groups, but carcass components, such as thigh, breast, wing and abdominal fat weight, were significantly greater in the selenium treatments. CONCLUSIONS: Intra-egg injection of organic selenium produced favourable effects on performance of broiler chickens, although it had no effect on immune response or microbial population. However, the negative effect on hatching of chickens needs to be prevented to result in an acceptable economic return for the producer.

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Pathogens, such as Escherichia coli (E. coli), have been identified as significant causes of poultry mortality. Poultry can serve as potential sources of E. coli transmission, even when asymptomatic, posing a substantial threat to food safety and human health. The in ovo administration of antimicrobials is crucial for preventing and/or effectively combating acute and chronic infections caused by poultry pathogens. To achieve this goal, it is critical that antimicrobials are properly injected into embryonic fluids, such as the amnion, to reach target tissues and trigger robust antimicrobial responses. Several protocols based on antimicrobials were evaluated to meet these requirements. This review analyzed the impacts of antimicrobial substances injected in ovo on the control of E. coli in poultry. The reduction in infection rates, resulting from the implementation of in ovo antimicrobials, combined with efforts aimed at hygienic-sanitary action plans in poultry sheds, reinforces confidence that E. coli can be contained before causing large scale damage. For example, antimicrobial peptides and probiotics have shown potential to provide protection to poultry against infections caused by E. coli. Issues related to the toxicity and bacterial resistance of many synthetic chemical compounds represent challenges that need to be overcome before the commercial application of in ovo injection protocols focused on microbiological control.

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BACKGROUND: Early life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions. OBJECTIVES: In this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis. METHODS: A hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2'-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids. RESULTS: In comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch. CONCLUSIONS: Together our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells.

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1. Feathers are an important product from poultry, and the state of feather growth and development plays an important role in their economic value.2. In total, 120 eggs were selected for immunoblotting and immunolocalisation experiments of ERK and ß-catenin proteins in different developmental stages of goose embryos. The ERK protein was highly expressed in the early stage of goose embryo development, while ß-catenin protein was highly expressed in the middle stage of embryo development.3. The 120 eggs were divided into four treatment groups, including an uninjected group (BLANK), a group injected with 100 µl of cosolvent (CK), a group injected with 100 µl of AZD6244 containing cosolvent in a dose of 5 mg/kg AZD6244 containing cosolvent (AZD5) and a group injected with 100 µl of AZD6244 containing cosolvent in a dose of 15 mg/kg AZD6244 containing cosolvent (AZD15). The eggs were injected on the ninth day of embryonic development (E9). Samples were collected at E21.5 to observe feather width, feather follicle diameter, ERK and Wnt/ß-catenin pathway protein expression.4. The AZD5 and AZD15 doses were within the embryonic safety range compared to the BLANK and CK groups and had no significant effect on the survival rate and weight at the inflection point, but significantly reduced the feather width and feather follicle diameter (p < 0.05). The AZD6244 treatment inhibited ERK protein phosphorylation levels and blocked the Wnt/ß-catenin pathway, which in turn significantly down-regulated the expression levels of FZD4, ß-catenin, TCF4 and LEF1 (p < 0.05), with an inhibitory effect in the AZD15 group being more significant. The immunohistochemical results of ß-catenin and p-ERK were consistent with Western blot results.5. The small molecule inhibitor AZD6244 regulated the growth and development of feather follicles in goose embryos by the ERK and Wnt/ß-catenin pathways.

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Methionine (Met) is an essential and first limiting amino acid in the poultry diet that plays a significant role in chicken embryonic development and growth. The present study examined the effect of in ovo injection of DL-Met and L-Met sources and genotypes on chicken embryonic-intestinal development and health. Fertilized eggs of the two genotypes, TETRA-SL layer hybrid (TSL) - commercial layer hybrid and Hungarian Partridge colored hen breed (HPC) - a native genotype, were randomly distributed into four treatments for each genotype. The treatment groups include the following: 1) control non-injected eggs (NoIn); 2) saline-injected (SaIn); 3) DL-Met injected (DLM); and 4) L-Met injected (LM). The in ovo injection was carried out on 17.5 d of embryonic development; after hatching, eight chicks per group were sacrificed, and the jejunum was extracted for analysis. The results showed that both DLM and LM groups had enhanced intestinal development as evidenced by increased villus width, villus height, and villus area (P < 0.05) compared to the control. The DLM group had significantly reduced crypt depth, glutathione content (GSH), glutathione S-transferase 3 alpha (GST3), occludin (OCLN) gene expression and increased villus height to crypt depth ratio in the TSL genotype than the LM group (P < 0.05). The HPC genotype has overexpressed insulin-like growth factor 1 (IGF1) gene, tricellulin (MD2), occludin (OCLN), superoxide dismutase 1 (SOD1), and GST3 genes than the TSL genotype (P < 0.05). In conclusion, these findings showed that in ovo injection of Met enhanced intestinal development, and function, with genotypes responding differently under normal conditions. Genotypes also influenced the expression of intestinal antioxidants, tight junction, and growth-related genes.

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The objective of the present investigation was to ascertain the impact of in ovo administration of L-carnosine on physiological indicators in neonatal broiler chickens. A total of 280 viable broiler eggs were allocated to 7 distinct groups: control, Sham in ovo injection of sterile water on d 7 of incubation. Groups 3 and 4 were subjected to in ovo injections of L-carnosine (25 and 50 µg) on d 7 of incubation. Group 5, functioning as a sham in ovo, received an injection of sterile water on d 18 of incubation. Groups 6 and 7 were in ovo injected with L-carnosine (25 and 50 µg) on d 18 of incubation. All eggs were subjected to incubation, and the hatching rate and body weight were measured post-hatch. Subsequently, blood samples were collected, and the levels of biochemical constituents in the serum were determined. Based on the outcomes, the administration of L-carnosine (50 µg) on d 7 of incubation led to a significant increase in post-hatch body weight compared to the control group (P < 0.05). The in ovo injection of L-carnosine (25 and 50 µg) on d 7 and 18 of incubation resulted in a significant decrease in the levels of serum glucose, triglyceride (TG), low-density lipoprotein (LDL), phosphorus (P), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine transaminase (ALT) in the newly hatched chickens (P < 0.05). Furthermore, the in-ovo injection of L-carnosine (25 and 50 µg) on d 7 and 18 of incubation led to a significant increase in the levels of serum high-density lipoprotein (HDL), calcium, and total protein (TP) in the newly hatched chickens (P < 0.05). Nonetheless, L-carnosine did not have a significant effect on the levels of serum IgY and IgA in the newly hatched chickens (P > 0.05). These findings indicate that the in ovo administration of L-carnosine yielded favorable outcomes in neonatal broiler chickens.

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The aim of this study was to investigate the effects of in ovo testosterone injection into the yolk sac of embryos on physiology and development of broiler chicks during the early posthatching period. A total of 1,010 hatching eggs were obtained from the Ross genotype. Trial design was conducted with a noninjected group (control) and injection groups in which 100 µL sesame oil, or 100 µL sesame oil + 0.50 µmol testosterone were injected into the yolk sac of the embryo on d 6 or d 12 of incubation. Testosterone hormone level was measured in the egg yolk and albumen at onset of incubation, in the yolk sac on d 19 of incubation and in the residual yolk sac at hatching. Weights of chick, yolk sac and organ, morphological traits (body length, lengths of bilateral traits and beak length), asymmetrical development of bilateral morphological traits and body mass index were measured at hatching and on d 7 after hatching. Testosterone, corticosterone and growth hormone levels were determined in blood plasma obtained from male chicks at hatching and on d 7 of chick age. Chick weight was not affected, plasma testosterone level and brain weight decreased, while body mass index, plasma corticosterone and growth hormone levels increased by administering 0.50 µmol testosterone on d 12 of embryonic age. However, plasma testosterone and growth hormone levels did not change, chick weight increased, while plasma corticosterone level and the chick body length decreased by administering 0.50 µmol testosterone on d 6 of embryonic age. A significant interaction between chick age and in ovo testosterone administration resulted in an increase in lung weight of chicks. In conclusion, this study found that in ovo testosterone administered at different embryonic ages due to age-specific effects of testosterone in the yolk sac of embryo modulates development related to physiological parameters of male broiler chicks during early posthatching period.

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The effects of in ovo lactoferrin (Lf) injection on some physiological parameters and immune response of posthatch chicks were investigated. Live embryonated Fayoumi chicken eggs (n = 600) were randomly allocated into four groups. The first group as a control was noninjected eggs, the second group was only injected with 0.1 mL of NaCl 0.75% solution, and the third and fourth groups were injected with 50 and 100 µL Lf dissolved in 0.1 mL saline solution respectively. The eggs were injected on Day 15 of incubation in the amnion. The results illustrated that the hatchability of eggs in two Lf groups was significantly higher than in the control, NaCl groups. The residual yolk in chicks injected with Lf (100 µL/egg) was significantly lower than the control group (p < 0.05). In ovo Lf injection improved lipid profile, liver function, antioxidant indices, blood haematology, serum immunoglobulins and jejunum histomorphometry compared to the control group (p < 0.05). In ovo injection of Lf decreased significantly (p < 0.001) of pathogenic bacteria in residual yolk such as Salmonella, Shigella and Coliform compared to the control group. In conclusion, in ovo Lf injection can improve the hatchability, lipid profile, immune response and antioxidant indices and decline pathogens in the residual yolk, thus boosting the health status of newly hatched Fayoumi chicks.

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The combined effects of the in ovo injection of commercial Marek's disease vaccine (MDV) and various levels of 25-hydroxyvitamin D3 (25OHD3) on the hatch variables, immunological measurements, and gene expression of Ross 708 hatchling broilers were investigated. A total of 5 in ovo injection treatments that were applied at 18 d of incubation (doi) included: 1) noninjected (control); or a 50 µL solution volume of 2) MDV alone; or MDV combined with 3) 0.6 µg of 25OHD3; 4) 1.2 µg of 25OHD3; or 5) 2.4 µg of 25OHD3. At hatch, hatchability of set and live embryonated eggs, hatchling body weight, hatch residue analysis, serum IgY and alpha-1 acid glycoprotein (AGP) concentrations, and the expression of genes related to immunity (INFα, INFß, INFγ, TLR-3, and TLR-21) and vitamin D3 activity (1 α-hydroxylase, 24 hydroxylase, and vitamin D receptor) were determined. No significant treatment differences were observed for hatchability of set and live embryonated eggs, or for serum IgY and AGP concentrations. However, hatchling body weight was higher when MDV was combined with either 1.2 or 2.4 µg of 25OHD3 than when MDV was provided alone or in combination with 0.6 µg of 25OHD3. Also, in comparison to the noninjected treatment group, the expression of the genes for 1 α-hydroxylase and 24 hydroxylase was improved when MDV was combined with either 1.2 or 2.4 µg of 25OHD3. Lastly, expression of the genes linked to viral detection (TLR-3) and antibody production (INF-ß) was increased in those treatments that contained any level of 25OHD3. These results indicate that in comparison to controls, the effects of MDV were observed to be greater on hatchling BW and splenic gene expression when it was administered in combination with the 1.2 or 2.4 µg doses of 25OHD3. Further research is needed to determine the posthatch effects of the administration of various levels of 25OHD3 in combination with MDV.

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In ovo interventions are used to improve embryonic development and robustness of chicks. The objective of this study was to identify the optimal dose for in ovo delivery of oregano essential oil (OEO), and to investigate metabolic impacts. Broiler chickens Ross 308 fertile eggs were injected with 7 levels of OEO (0, 5, 10, 20, 30, 40, and 50 µL) into the amniotic fluid at embryonic d 17.5 (E17.5) (n = 48). Chick quality was measured by navel score (P < 0.05) and/or hatchability rates (P < 0.01) were significantly decreased at doses at or above 10 or 20 µL/egg, respectively, indicating potential toxicity. However, no effects were observed at the 5 µL/egg, suggesting that compensatory mechanisms were effective to maintain homeostasis in the developing embryo. To pursue a better understanding of these mechanisms, transcriptomic analyses of the jejunum were performed comparing the control injected with saline and the group injected with 5 µL of OEO. The transcriptomic analyses identified that 167 genes were upregulated and 90 were downregulated in the 5 µL OEO compared to the control group injected with saline (P < 0.01). Functional analyses of the differentially expressed genes (DEG) showed that metabolic pathways related to the epoxygenase cytochrome P450 pathway associated with xenobiotic catabolic processes were significantly upregulated (P < 0.05). In addition, long-chain fatty acid metabolism associated with ATP binding transporters was also upregulated in the OEO treated group (P < 0.05). The results indicated that low doses of OEO in ovo have the potential to increase lipid metabolism in late stages (E17.5) of embryonic development. In conclusion, in ovo delivery of 5 µL OEO did not show any negative impact on hatchability and chick quality. OEO elevated expression of key enzymes and receptors involved in detoxification pathways and lipid metabolism in the jejunum of hatchling broiler chicks.

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Intestinal morphology and regulation of nutrient transportation genes during the embryonic and early life of chicks influence their body weight and feed conversion ratio through the growing period. The intestine development can be monitored by measuring villus morphology and enzymatic activity and determining the expression of nutrient transporters genes. With the increasing importance of gut development and health in broiler production, considerable research has been directed towards factors affecting intestine development. Thus, this article reviews (1) intestinal development during embryogenesis, and (2) maternal factors, in ovo administration, and incubation conditions that influence intestinal development during embryogenesis. Conclusively, (1) chicks from heavier eggs may have a better-developed intestine than chicks from younger ones, (2) in ovo supplementation with amino acids, minerals, vitamins or a combination of several probiotics and prebiotics stimulates intestine development and increases the expression of intestine mucosal-related genes and (3) the long storage period, improper incubation temperature and imbalanced ventilation can negatively influence intestinal morphology and nutrient transporters gene expression. Finally, understanding the intestine development during embryonic life will enable us to enhance the productivity of broilers.

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This study aimed to evaluate the effectiveness of the embryonic injection of silver nanoparticles (SilNPs) on some productive traits and hepatic gene expression of lipopolysaccharide (LPS)-challenged broilers after a 42 d rearing period. 560 fertile eggs were randomly allocated to four groups and received either of the following treatments at d 7 of incubation, control (no injection), placebo (1 mL saline), SilNP20 (20 mg/kg silver nanoparticles), or SilNP40 (40 mg/kg silver nanoparticles). After the incubation, 320 hatchlings experienced a 42 d standard rearing period. Live body weight (LBW), feed intake (FI), and feed conversion ratio (FCR) were weekly recorded. At the end of the experiment, two birds from each replicate (n = 8 per treatment) were exposed to LPS intraperitoneal injection at 48, 24, and 12 h before slaughter time. They were also used for blood, intestinal, and microbial evaluations. The hepatic mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), transforming growth factor beta (TGF-ß), and insulin-like growth factor I (IGF-I) were assessed at d 1 and 42 of the experiment. Adminstration of SiLNPs improved LBW, FI, and FCR and also enhanced liver and spleen weights (P < 0.05). SilNP20 birds had significantly lower bursa of Fabricius weight (P < 0.05). SilNP20 had lower total cholesterol levels than others. There was a significant difference (P < 0.05) between SliNP40 and SilNP20 in the ratio of villus height to crypt width. Compared to control groups, chicks of SilNP20, but not SilNP40, showed a significant increase in the relative expression of TNF-α, IL-6, TGF-ß, and IFG-I genes at d 1. On d 42, however, both SilNP20 and SilNP40 had significantly higher TNF-α and TGF-ß levels than both controls. Silver nanoparticles did not significantly affect the microflora of the ileum and cecum in the current study. In summary, SilNPs administration to chick embryos showed a long-term positive effect on their productive performance.

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The total number of intestinal microbiotas is low, and the intestinal tract develops rapidly and imperfectly at the embryonic stage. Embryonic period as a particular physiological stage is an important time window to explore how to regulate organismal health by probiotics. Therefore, this experiment was conducted to investigate the effect of embryonic injection of Lactobacillus plantarum PA01 at embryonic d 14 (E14) on the microbiome of the contents of the gizzard, cecum at embryonic d 20 (E20) and cecum at d 1 posthatch (D1) by 16S rRNA sequencing. Results showed that PA01 had no significant effect on broiler body weight and yolk sac weight at E20 and D1 (P > 0.05). PA-01 altered the Shannon index and ß diversity of the gizzard at E20 (P < 0.05), increased the abundance of Firmicutes (P < 0.05), and decreased the relative abundance of Proteobacteria, Bacteroidota, and Actinobacteriota (P < 0.05). At the genus level of the microbiota, PA01 significantly increased the relative abundance of Lactiplantibacillus (P < 0.05). At 20 embryos, PA01 altered the α and ß diversity indices (P < 0.05) and decreased the relative abundance of Salmonella (P < 0.05) of the cecal microbiota. The biomarkers of PA01 group were Lactobacillales, Blautia, Lachnospiraceae, and Asinibacterium. Embryonic injection of PA01 altered the E20 intestinal microbes. PA01 altered the ß-diversity index of the 1-day-old cecum (P < 0.05), and there was no significant effect on microbial composition at the phylum and genus level (P > 0.05). LefSe analysis revealed that the biomarkers of the PA01 group were Lactobacillaceae, Lactiplantibacillus, Moraxellaceae, and Acinetobacter. Biomarkers in the Con group were Devosia, Bacillus, Nordella, Mesorhizobium, and Pseudolabrys. PA01 increased acetic acid in the gastrointestinal tract at E20 along with acetic and butyric acid in cecum of 1-day-old. In conclusion, embryo-injected L. plantarum PA01 altered the structure and metabolites of the microbial flora before and after hatching, in particular promoting the colonization of Lactobacillus.

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The current investigation was conducted to test the potential effects of in ovo feeding of DL-methionine (MET) on hatchability, embryonic mortality, hatching weight, blood biochemical parameters and development of heart and gastrointestinal (GIT) of breeder chick embryos. 224 Rhode Island Red fertile eggs were randomly distributed into seven experimental treatments: untreated egg (control), buffered saline (0.5% NaCl), and five solutions containing increased levels of MET (0.5, 1.0, 1.5, 2.0 and 2.5%) + 0.5% NaCl, being separated into four groups/replicates (each one with 8 eggs), totaling 32 eggs/treatment. All embryos submitted to in ovo injection with MET presented a decrease in the hatchability results and an increase in the results of intermediary embryonic mortality. Chicks hatched from eggs injected with until to 1.0% MET were heavier and presented better development of the heart and GIT, especially important organs and regions for digestion and nutrient absorption. Conclusively, the in ovo feeding using MET showed positive impacts on hatching weight and GIT development of breeder chicks. However, caused negative impacts on hatchability when used at high levels.

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In ovo administration as a possible alternative method of 6/85 MG vaccination was assessed. After 18 days of incubation (doi), the eggs were administered a particular dosage of a live attenuated 6/85 MG vaccine in either the air cell (AC) or amnion (AM). The treatments included non-injected eggs and eggs injected into the AC or AM with diluent alone as controls. Treatments also included eggs injected with diluent, which contained 1.73 × 102, or 1.73 × 104 CFU of 6/85 MG. Hatchability of viable injected eggs (HI) and residual embryonic mortality were determined at 22 doi. At hatch and at three weeks posthatch, one hatched chick per treatment replicate was bled and swabbed for the detection of 6/85 MG in the choanal cleft using PCR, serum plate agglutination (SPA), and ELISA methods. The results show that AC in ovo injection of 6/85 MG had no negative impacts on HI or on the live performance of pullets, but that it failed to provide adequate protection (p ≤ 0.0001) in hatchlings or three-week-old pullets. The 1.73 × 104 6/85 MG CFU dosage injected into the AM decreased the hatchability of injected eggs containing viable embryos (HI; p = 0.009) and was associated with a significant increase in late dead mortality (p = 0.001). Hatchling and three-week-old chick mortalities (p = 0.008) were significantly greater in the 1.73 × 104 CFU-AM treatment group in comparison with the other treatment groups. In addition, the 1.73 and 1.73 × 102 6/85 MG-AM treatments had no negative effects on the hatching process or on posthatch growth, and the 1.73 × 102 6/85 MG-AM treatment was more effective in the protection of pullets against MG (p ≤ 0.0001) as compared with the low dosage and non-injected treatment groups. Further research is needed to examine the influence of the 6/85 MG in ovo vaccine on layer immune competence.

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BACKGROUND: Under commercial conditions, chickens are fasted for the first 36 to 72 h post-hatch. Delayed access to feed and water causes delayed development of the intestinal tract and retarded performance. OBJECTIVES: This study focused on the chick's life as affected by in ovo feeding (IOF) and the animal's interaction with the feeding procedure. The birds in a factorial arrangement (2 × 3) were placed into six treatment groups in a completely randomised design. The treatment groups differed in feed procedure, 6 h [early feeding (EF)] or 36 h [common feeding (CF)] post-hatch, with or without IOF with beta-hydroxy-beta-methyl butyrate (HMB) or calcium gluconate (CG) in a saline solution, and were examined for hatchability and performance parameters until 24 h post-hatch. In addition, physical and histological characteristics of breast, jejunum and serum indices in 14 days post-hatch and performance criteria until 35 days of age were recorded. METHODS: On day 17 of the incubation period, eggs were subjected to the IOF procedure. One mL of sterile IOF solution including 0.1% HMB or 0.4% CG dissolved in 5% saline solution was injected into the eggs. RESULTS: Results showed that IOF groups had lower (p < 0.05) FCR than the control group. The highest mortality rate was noted in the control-CF group. The lowest myofibril density was related to the HMB-CF group. Myofibril periphery, area and diameter for the HMB-CF group were larger (p < 0.05) than those of the other groups. CONCLUSIONS: Results indicated that injection of HMB increased hatchability by almost 15%. The IOF of HMB improved the digestive tract and breast muscle development and improved FCR.

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In ovo immunization of chicken embryos with live vaccines is an effective strategy to protect chickens against various viral pathogens. The immunogenic efficacies of in ovo administration of lactic acid bacteria (LAB) in combination with live Newcastle disease (ND) vaccine were investigated in this study. Four hundred healthy 1-day-old fertilized specific pathogen-free (SPF) eggs of similar weights were randomly assigned to one of four treatments, with five replicates of each treatment and a total of 20 for each replicate. On day 18.5 of incubation, in ovo injections were given. The treatment groups are as follows: (I) no injection, (II) 0.9% physiological saline injection, (III) ND vaccine injection, and (IV) LAB as an adjuvant for ND vaccine injection. The ND vaccine adjuvanted with LAB significantly increased the daily weight gain, immune organ index, and small intestine histomorphological development in layer chicks while decreasing the feed conversion ratio (FCR). The results suggested that the LAB-adjuvant group significantly affected the relative expression of mucosal mucin protein (mucin-1) and zoccluding small circle protein-1 (ZO-1) (P < 0.05), whereas the relative expression of occludin mRNA was not significantly affected (P > 0.05) compared with the non-injected group. Meanwhile, we indicated that intra-amniotic synbiotic injection significantly maintained the balance of flora (P < 0.05). Compared with the non-injected group, the ND vaccine adjuvanted with the LAB group exhibited significant promotion of the HI and SIgA antibody titers in serum on day 21 (P < 0.05), induction of higher production of cytokines (IL-2, IL-4, IL-6, IFN-γ) in serum. In summary, in ovo injection of ND vaccine adjuvanted with LAB has a positive impact on the growth performance, immune function, and microbiome of growing chicks.

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The regulation of adipose deposition in broiler chickens is an important factor for production efficiency to poultry producers and health concerns to customers. Although vitamin A and its metabolite [all-trans retinoic acid (atRA)] have been used for studies on adipogenesis in mammals and avian, effects of embryonic atRA on adipose development in embryonic (E) and posthatch (D) ages in broiler chickens have not been studied yet. Different concentrations of atRA (0 M-2 µM) were injected in broiler eggs at E10, and adipose tissues were sampled at E16. Percentages of adipose tissues in chicken embryos were significantly increased in the group injected with 500 nM of atRA compared to the 0 M group (P < 0.05). In addition, the adipocyte cross-sectional area (CSA) was significantly greater by in ovo injection of 500 nM atRA compared to the injection of 0 M (P < 0.01). Moreover, in ovo atRA-injected embryos were hatched and BWs were measured at D0, D7, and D14. BWs were not different from those of the 0 M group. Percentages of adipose tissues and CSA of the in ovo atRA-injected group (500 nM) were not different from those of the 0 M group at D14. Taken together, the current study clearly showed that in ovo injection of atRA promoted adipose deposition with hypertrophy during embryonic development, but its effects were not maintained in early posthatch age in broiler chickens, implying that embryonic atRA has an important role in the regulation of adipose development in chicken embryos.

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