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Background Purpose Methods Results Conclusions In ovo MR presents a promising and viable alternative to traditional in vivo small animal experiments. Sodium MRI complements proton MRI by providing potential access to tissue cellular metabolism. Despite its abundance, sodium MRI is challenged by lower MR sensitivity and faster relaxation times compared to proton MRI. Ensuring a high signal‐to‐noise ratio and effective B0 shimming is essential. Double‐tuned coils combining 23Na and 1H are frequently employed to achieve structural imaging and efficient shim adjustment.This study introduces a novel, highly optimized, double‐tuned coil design, specifically for MR scans of chick embryos.A tapered‐spiral, double‐tuned coil was designed and constructed following careful consideration of design parameters. The performance of the coil was rigorously assessed through bench tests, and final validation was conducted on a 7 T MRI scanner using a chick embryo.Bench tests demonstrated that the return losses for both 1H and 23Na coils were better than − 30 dB, and isolation factors were better than − 21 dB, indicating that the double‐tuned coil was well‐set, with negligible coupling between channels. MR images of chick embryos, obtained using the coil, validated the feasibility of utilizing the design concept for in ovo applications.The innovative design of the proposed double‐tuned coil, characterized by its unique arrangement, offers improved performance. This design has the potential to significantly enhance the quality of in ovo 1H and 23Na measurements. [ABSTRACT FROM AUTHOR]

Background: Patient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient – in vitro PDOs – in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns. Results: The main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient's liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient's sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking. Conclusions: We successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient's tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient's liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients. [ABSTRACT FROM AUTHOR]

Objectives: Patients with type 2 diabetes or prolonged diabetic condition are webbed into cardiac complications. This study aimed to ascertain the utility of chick embryo as an alternative to the mammalian model for type 2 diabetes-induced cardiac complications and chrysin as a protective agent. Methods: Diabetes was activated in ovo model (chick embryo) using glucose along with β-hydroxybutyric acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Alamar, and Kenacid blue assay were used to compare with chrysin-administered group. Blood glucose level, total cholesterol, triglyceride, and high-density lipoprotein were considered as endpoints. Diabetes was induced in Wistar albino rats by administering a high-fat diet and a subdued dose of streptozotocin (35 mg/kg, b.w). Percentage of glycated hemoglobin, creatinine kinase-MB, tumor necrosis factor-α, and C-reactive protein were evaluated and compared with chrysin administered group. Key findings: Chrysin treatment improved elevated blood glucose levels and dyslipidemia in a diabetic group of whole embryos. Condensed cellular growth and protein content as well as enhanced cytotoxicity in ovo were shielded by chrysin. Chrysin reduced cardiac and inflammatory markers in diabetic rats and provided cellular protection to damage the heart of diabetic rats. Conclusion: The protective action of chrysin in ovo model induced a secondary complication associated with diabetes, evidenced that the ovo model is an effective alternative in curtailing higher animal use in scientific research. [ABSTRACT FROM AUTHOR]

Extensive antibiotic use in the poultry industry has given rise to concerns of the proliferation and potential zoonotic transmission of antimicrobial-resistant pathogens. To mitigate the extent of antibiotic dosing, probiotics have been proposed as an alternate means of enhancing chick immune function by boosting host defense peptide (HDP) production. This study measured chick immune response to in ovo supplementation of a commercial poultry probiotic when subsequently exposed to a mimicked viral threat using polyinosinic-polycytidylic acid (poly(I:C)). To assess systemic immune responses to these conditions, real-time PCR was conducted to measure changes in expression of five immune-related genes in the spleen: TLR-3, AvBD-2, IFN-a, IFN-b, and IL-6. The chicks that received only the probiotic treatment saw a downregulation of TLR-3 (p50.0013) and an upregulation of AvBD-2 (p50.0105). The expression of IL-6 tended towards downregulation (p50.06) with a potential interaction between the probiotic and poly(I:C) treatments (p50.0794). Upregulation of AvBD-2 indicates a higher basal output of the chick's innate immune system upon early exposure to probiotics. These results suggest that enhanced systemic immune response in chicks can be achieved through early application of probiotics and is occurring through immunomodulation pathways that have not been fully elucidated. [ABSTRACT FROM AUTHOR]

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Abstract Background Patient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient – in vitro PDOs – in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns. Results The main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient’s liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient’s sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking. Conclusions We successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient’s tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient’s liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients. Graphical Abstract

Background and Aim: To combat enteric infections and antibiotic resistance in the poultry industry, researchers seek alternatives such as probiotics, prebiotics, and synbiotics as growth promoters. Synbiotics support probiotic growth through the supply of essential nutrients. The study’s objectives were to assess the most effective delivery methods for synbiotics and evaluate their growth, histomorphometric, and hematological impacts on Cobb-500 broilers. Materials and Methods: Two studies, independently conducted, employed a completely randomized design. One hundred and eighty viable eggs in the first trial were assigned to three groups: Control (T1), sterile water (T2), and synbiotic in sterile water (T3). On the 21st day of hatching, hatchability, day-old body weights, and ileum samples for histomorphometric analysis were recorded. In the second trial, out of 500 viable eggs, 200 eggs were fed in ovo with synbiotics (PoultryStar® sol, Biomin Singapore Pte Ltd, Singapore) on 17.5 days and 300 were set aside without in ovo injection. The treatments were control (T1), in water synbiotic (T2), in ovo synbiotic (T3), combination of in ovo synbiotic and synbiotic in feed (T4), and synbiotic in feed only (T5). On 21 and 42 days, blood, ileum, and visceral organ samples were collected for laboratory analysis. Data on weight gain, daily feed intake, and water consumption were recorded for 42 days. Results: The initial experiment’s results revealed a decrease in hatchability, slight weight increase, and significant intestinal morphological changes with the use of an in ovo synbiotic. Applying synbiotic through various methods in the second trial yielded better growth results, lower blood cholesterol, and significantly longer (p < 0.05) villi on 21 days. Conclusion: Using the in ovo method to administer synbiotics lowered hatchability. Use of synbiotics with any method or in combination enhances growth, ileum structure, dressing yield, feed efficiency, and cholesterol levels in blood. Synbiotics enhance gut health and overall performance in broilers when used through diverse approaches.

This study was undertaken to evaluate and compare the efficacy of different multi-antigen vaccines, including heat-inactivated, whole lysate, and subunit (outer membrane proteins [OMPs]) C. jejuni vaccines along with the immunostimulant CpG ODN in controlling Campylobacter colonization in chickens. In the first trial, 125 μg of C. jejuni OMPs and 50 μg of CpG ODN were administered individually or in combination, either in ovo to chick embryos or subcutaneously (SC) to one-day-old chicks. In the second trial, different concentrations of C. jejuni antigens (heat-killed, whole lysate, and OMPs) were administered SC to one-day-old chicks. The results of the first trial revealed that SC immunization with the combination of CpG ODN and C. jejuni OMPs elevated interferon (IFN)-γ, interleukin (IL)-1β, and IL-13 gene expression in the spleen, significantly increased serum IgM and IgY antibody levels, and reduced cecal C. jejuni counts by approximately 1.2 log10. In contrast, in ovo immunization did not elicit immune responses or confer protection against Campylobacter. The results of the second trial showed that SC immunization with C. jejuni whole lysate or 200 μg OMPs reduced C. jejuni counts by approximately 1.4 and 1.1 log10, respectively. In conclusion, C. jejuni lysate and OMPs are promising vaccine antigens for reducing Campylobacter colonization in chickens. [ABSTRACT FROM AUTHOR]

Summary: Various parts of Garcinia atroviridis have long been used to address various health issues and as a seasoning. Past research has highlighted the leaf extracts' diverse medicinal properties, including antioxidant, antihyperlipidaemic, antibacterial, antitumor, anti‐inflammatory, and antifungal effects. No research has explored the therapeutic effects of the ethanolic crude extract of G. atroviridis fruits (GAECE) for diabetes. This study aims to assess GAECE's antidiabetic potential using various methods, including in vitro and in ovo assays, cytotoxicity testing, phytochemical profiling, and in silico analysis to identify active compounds. The GAECE was prepared using the maceration method and evaluated for its inhibitory effect against DPP‐4, α‐glucosidase, and α‐amylase enzyme activities. The glucose uptake potential and the kinetic study of glucose uptake were performed to determine the time required to exert its glucose uptake potential. Moreover, the antihyperglycaemic effect was evaluated in diabetes induced in ovo model. Q‐TOF LCMS was performed to identify the phytochemical, which was then subjected to molecular docking and molecular dynamics (MD) simulation was conducted for 100 nanoseconds to validate the ligand–receptor complexes. GAECE exhibited promising antidiabetic potential, with half maximal inhibitory concentrations (IC50) of 0.129 ± 0.002 mg mL−1 for α‐amylase and 0.593 ± 0.01 mg mL−1 for α‐glucosidase; and showed an inhibition of 70.07 ± 0.96% for DPP‐4 enzymes at 0.5 mg mL−1. The 50% cell viability was found to be 0.87 ± 0.08 mg mL−1, 0.76 ± 0.04 mg mL−1, and 0.66 ± 0.03 mg mL−1 for 24, 48, and 72 h of treatment, respectively. Furthermore, GAECE showed potential for glucose uptake in differentiated L6 cells, resulting in a 136.7 ± 1.2% increase in glucose uptake. Kinetic analysis revealed peak potential at 10 min at the concentration of 0.4 mg mL−1. In in ovo diabetes induced model, GAECE reduced blood plasma glucose levels (152.4 ± 0.52 mg dL−1), which was similar to the effect of insulin. [ABSTRACT FROM AUTHOR]

Background and Aim: To combat enteric infections and antibiotic resistance in the poultry industry, researchers seek alternatives such as probiotics, prebiotics, and synbiotics as growth promoters. Synbiotics support probiotic growth through the supply of essential nutrients. The study's objectives were to assess the most effective delivery methods for synbiotics and evaluate their growth, histomorphometric, and hematological impacts on Cobb-500 broilers. Materials and Methods: Two studies, independently conducted, employed a completely randomized design. One hundred and eighty viable eggs in the first trial were assigned to three groups: Control (T1), sterile water (T2), and synbiotic in sterile water (T3). On the 21st day of hatching, hatchability, day-old body weights, and ileum samples for histomorphometric analysis were recorded. In the second trial, out of 500 viable eggs, 200 eggs were fed in ovo with synbiotics (PoultryStar® sol, Biomin Singapore Pte Ltd, Singapore) on 17.5 days and 300 were set aside without in ovo injection. The treatments were control (T1), in water synbiotic (T2), in ovo synbiotic (T3), combination of in ovo synbiotic and synbiotic in feed (T4), and synbiotic in feed only (T5). On 21 and 42 days, blood, ileum, and visceral organ samples were collected for laboratory analysis. Data on weight gain, daily feed intake, and water consumption were recorded for 42 days. Results: The initial experiment's results revealed a decrease in hatchability, slight weight increase, and significant intestinal morphological changes with the use of an in ovo synbiotic. Applying synbiotic through various methods in the second trial yielded better growth results, lower blood cholesterol, and significantly longer (p < 0.05) villi on 21 days. Conclusion: Using the in ovo method to administer synbiotics lowered hatchability. Use of synbiotics with any method or in combination enhances growth, ileum structure, dressing yield, feed efficiency, and cholesterol levels in blood. Synbiotics enhance gut health and overall performance in broilers when used through diverse approaches. [ABSTRACT FROM AUTHOR]

Abstract Background Dietary supplementation of xylooligosaccharides (XOS) has been found to influence gut health by manipulating cecal microbiota and producing microbe-origin metabolites. But no study investigated and compared the effect of in ovo feeding of xylobiose (XOS2) and xylotriose (XOS3) in chickens. This study investigated the effect of in ovo feeding of these XOS compounds on post-hatch gut health parameters in chickens. A total of 144 fertilized chicken eggs were divided into three groups: a) non-injected control (CON), b) XOS2, and c) XOS3. On the 17th embryonic day, the eggs of the XOS2 and XOS3 groups were injected with 3 mg of XOS2 and XOS3 diluted in 0.5 mL of 0.85% normal saline through the amniotic sac. After hatching, the chicks were raised for 21 d. Blood was collected on d 14 to measure plasma immunoglobulin. Cecal digesta were collected for measuring short-chain fatty acids (SCFA) on d 14 and 21, and for microbial ecology and microbial metabolic pathway analyses on d 7 and 21. Results The results were considered significantly different at P

ABSTRACT: Efficient poultry production can be accomplished using combined technologies. A combination of in ovo and ethno-veterinary technologies can deliver significant benefits, including reduced labour and production costs. This study evaluated the effect of in ovo administration of soursop leaf extract (SLE) on the hatching performance and physiology of Noiler chicks. A total of 550 eggs were incubated, and 460 fertile eggs were randomly distributed into 4 groups with 3 replicates on the 18th d of incubation. The eggs were then injected with 0.75 µg SLE, 1.5 µg SLE, and saline solution (negative control) at a rate of 0.2 ml in the air space. The noninjected group served as the positive control. At the end of the hatching period, the various groups were evaluated for embryo mortality, hatchability, hatch duration, organ weight, serum biochemistry, and chick quality. The results showed no significant differences (P > 0.05) in embryonic mortality, hatchability, organ weight, total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, and glucose among the treatment groups. However, chick weight, chick quality, and serum triglyceride levels were significantly (P < 0.05) higher in the extract-injected group. Additionally, incubation and hatch times were significantly lower (P < 0.05) in the SLE group compared to the other groups. In ovo administration of soursop leaf extract resulted in reduced incubation duration, hatch time, and embryo mortality. In conclusion, the in ovo injection of SLE improved hatch performance and chick quality.

Background: Dietary supplementation of xylooligosaccharides (XOS) has been found to influence gut health by manipulating cecal microbiota and producing microbe-origin metabolites. But no study investigated and compared the effect of in ovo feeding of xylobiose (XOS2) and xylotriose (XOS3) in chickens. This study investigated the effect of in ovo feeding of these XOS compounds on post-hatch gut health parameters in chickens. A total of 144 fertilized chicken eggs were divided into three groups: a) non-injected control (CON), b) XOS2, and c) XOS3. On the 17th embryonic day, the eggs of the XOS2 and XOS3 groups were injected with 3 mg of XOS2 and XOS3 diluted in 0.5 mL of 0.85% normal saline through the amniotic sac. After hatching, the chicks were raised for 21 d. Blood was collected on d 14 to measure plasma immunoglobulin. Cecal digesta were collected for measuring short-chain fatty acids (SCFA) on d 14 and 21, and for microbial ecology and microbial metabolic pathway analyses on d 7 and 21. Results: The results were considered significantly different at P < 0.05. ELISA quantified plasma IgA and IgG on d 14 chickens, revealing no differences among the treatments. Gas chromatography results showed no significant differences in the concentrations of cecal SCFAs on d 14 but significant differences on d 21. However, the SCFA concentrations were lower in the XOS3 than in the CON group on d 21. The cecal metagenomics data showed that the abundance of the family Clostridiaceae significantly decreased on d 7, and the abundance of the family Oscillospiraceae increased on d 21 in the XOS2 compared to the CON. There was a reduction in the relative abundance of genus Clostridium sensu stricto 1 in the XOS2 compared to the CON on d 7 and the genus Ruminococcus torques in both XOS2 and XOS3 groups compared to the CON on d 21. The XOS2 and XOS3 groups reduced the genes for chondroitin sulfate degradation I and L-histidine degradation I pathways, which contribute to improved gut health, respectively, in the microbiome on d 7. In contrast, on d 21, the XOS2 and XOS3 groups enriched the thiamin salvage II, L-isoleucine biosynthesis IV, and O-antigen building blocks biosynthesis (E. coli) pathways, which are indicative of improved gut health. Unlike the XOS3 and CON, the microbiome enriched the pathways associated with energy enhancement, including flavin biosynthesis I, sucrose degradation III, and Calvin-Benson-Bassham cycle pathways, in the XOS2 group on d 21. Conclusion: In ovo XOS2 and XOS3 feeding promoted beneficial bacterial growth and reduced harmful bacteria at the family and genus levels. The metagenomic-based microbial metabolic pathway profiling predicted a favorable change in the availability of cecal metabolites in the XOS2 and XOS3 groups. The modulation of microbiota and metabolic pathways suggests that in ovo XOS2 and XOS3 feeding improved gut health during the post-hatch period of broilers. [ABSTRACT FROM AUTHOR]

Glyphosate is an ingredient widely used in various commercial formulations, including Roundup®. This study focused on tight junctions and the expression of inflammatory genes in the small intestine of chicks. On the sixth day of embryonic development, the eggs were randomly assigned to three groups: the control group (CON, n = 60), the glyphosate group (GLYP, n = 60), which received 10 mg of active glyphosate/kg egg mass, and the Roundup®-based glyphosate group also received 10 mg of glyphosate. The results indicated that the chicks exposed to glyphosate or Roundup® exhibited signs of oxidative stress. Additionally, histopathological alterations in the small intestine tissues included villi fusion, complete fusion of some intestinal villi, a reduced number of goblet cells, and necrosis of some submucosal epithelial cells in chicks. Genes related to the small intestine (ZO-1, ZO-2, Claudin-1, Claudin-3, JAM2, and Occludin), as well as the levels of pro-inflammatory cytokines (IFNγ, IL-1β, and IL-6), exhibited significant changes in the groups exposed to glyphosate or Roundup® compared to the control group. In conclusion, the toxicity of pure glyphosate or Roundup® likely disrupts the small intestine of chicks by modulating the expression of genes associated with tight junctions in the small intestine. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021–2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry. [ABSTRACT FROM AUTHOR]

The viral diseases that occurred in recent years have increased the interest in non-toxic to healthy cells and naturally isolated agents to struggle with these diseases. The key intention of this research is to examine both antiviral potentials against the Infectious Bronchitis model virus (IBV) and cytotoxic activities on determined cell lines of different active ingredients and medical herbs extracts for developing new antiviral agents or drugs towards SARS-CoV-2. The antiviral potency of the samples against IBV was determined as in ovo virucidal antiviral activity in specific pathogen-free (SPF) embryonated chicken eggs (ECEs). To detect antiviral activity, the haemagglutination test was performed after 48 h of incubation for all samples. The cytotoxic activity of the samples was identified on HepG2, Caco-2, HeLa, HEK293, PANC-1, PC-3, A549, MDAMB-231, and CCD-34Lu cell lines by the MTT protocol. Hypericum perforatum extract was found to have a dominant role in cytotoxicity and antiviral activity. In addition, while nobiletin and Sambucus nigra do not exhibit cytotoxic activity on cells, they play a significant role in antiviral activity. As a consequence of our investigation, the cytotoxic and antiviral properties of Laurus nobilis, H. perforatum, and S. nigra extracts were found remarkable and the potential of these extracts was demonstrated. [ABSTRACT FROM AUTHOR]

Folate (vitamin B9) has been shown to reduce the prevalence of neural tube defects (NTDs). Many genes comprising Disabled‐1 (DAB1) and miRNAs have been shown to play important role in normal brain development. Reelin‐signalling has been shown to play key role in regulating of neuronal migration during brain development. The aim of this study was to evaluate the effects of in ovo administration of folic acid (FA) on DAB1 and gga‐miR‐182‐5p expression in the cerebral cortex of chick embryo. A total number of 30 hatching eggs were used in this study. The number of 10 eggs were injected into the yolk sac with FA (150 µg/egg), 10 eggs by normal saline (sham group) on embryonic day 11 and 10 eggs were left without injection as control. Then the cerebral cortices were collected on E19 and the expression of DAB1 and gga‐miR‐182‐5p was studied by Real‐Time PCR. The results showed that DAB1 expression in the cerebral cortex of FA‐treated, sham and control were 2.51 ± 0.13, 1.01 ± 0.04 and 1.03 ± 0.04 fold changes, respectively, and this amount for gga‐miR‐182‐5p were 0.54 ± 0.03, 1.09 ± 0.07 and 1.00 ± 0.06‐fold change respectively. Statistical analysis showed that there is a significant increase in DAB1 and a decrease in gga‐miR‐182‐5p expression in FA injected cerebral cortex as compared either with either SHAM or control (p < 0.0001). But, no significant change in DAB1 and gga‐miR‐182‐5p expression was observed between sham and the control group (p = 0.99 and p = 0.57 respectively). It is concluded that in ovo feeding of FA increases DAB1 and decreases gga‐miR‐182‐5p expression in the developing chick cerebral cortex. [ABSTRACT FROM AUTHOR]

This study was aimed to investigate the influence of in ovo injection (IO) into air cell (AC) or yolk sac (YS) of epigallocatechin-3 gallate (EG) and oleuropein (OL) as antioxidants on hatching, physiological and productive performance of heat-stressed chicks. 840 fertile eggs were chosen for IO on 12th day of incubation. Eggs were divided into 7 groups within 4 replications each as follows: negative control (NC) without IO and other groups involved IO of 100 μl distilled water and 5 mg each of EG and OL in both AC and YS sites of egg. After hatching, chicks were exposed to heat stress for 24 h and raised for 42 d. Compared with NC, the results revealed that main effect of IO with EG and OL increased (p≤0.05) hatchability and decreased deformed chicks and serum levels of glucose, cholesterol, aspartate aminotransferase, corticosterone and heat shock protein 70 with enhancing feed efficiency, superoxide dismutase, glutathione peroxidase and thyroxine in serum or liver. High chick length and relative chick weight with low serum protein carbonyl and feed intake were recorded (p≤0.05) by EG. Low rectal temperature and heterophil to lymphocyte ratio with high body weight were recordered (p≤0.05) by OL. No mainly altered effects between both IO sites on most variables measured. However, there were significant influences among interactive treatments which related to in ovo injected substance in an injection site-dependent manner. It is concluded that improved hatchability, physiological and productive characteristics of heat-stressed chicks were achieved by IO of EG and OL. [ABSTRACT FROM AUTHOR]

Background: In ovo application is the process of administering some nutrients or components into the egg. The main purpose of this application is to ensure that some nutrients are provided to chicks with a short incubation period. Few studies were conducted with taurine in fertile eggs; especially, no observation of hatchability and chick quality has been found. In addition, taurine has an anti‐stress impact that fights oxidative factors. Objective: To assess the hatchability and chick quality after in ovo taurine administration. To determine the stress that may occur as a result of in ovo application and whether taurine has a stress‐reducing effect. Methods: A total of 1200 fertile eggs from a 34‐week‐old broiler breeder (Ross 308) flock were categorized into 4 groups with 75 eggs per replicate: control (uninjected), taurine group (0.30 mL dissolved taurine in distilled water), sham control (sterile distilled water) and perforation (eggs perforated and then waxed). On day 14 of incubation, an in ovo injection was administered to the albumen. Data on hatching parameters and hepatic HSP70 levels were obtained using relevant formulas and western blotting, respectively. Results: Control chicks exhibited higher hatchability than other groups, with the taurine group showing the lowest hatchability. The HSP70 levels were the highest in the perforation group compared to the control group. An increase of 21.37% in the taurine group and 83.45% in the sham control group was observed compared to the control group. Conclusions: The findings suggest that in ovo application may induce increased stress, whereas taurine may have positive effects in mitigating the stress caused by in ovo application. [ABSTRACT FROM AUTHOR]

The final phase of embryonic development and the immediate period after hatching are pivotal moments in the life of broiler chickens. The lack of food for newly hatched chicks leads to decreased body weight. Under commercial conditions, the hatched eggs placed on the same hatching shelves are usually hatched within a period of 24-36 hours, which results in the early hatched chicks falling under harsh conditions of prolonged fasting period, and other periods are added to this period including determining sex, sorting, vaccination, and transporting to the farm, these factors directly contribute to the delay in growth. In addition, this period is very important, as the body weight increases during the first week 3-4 times, and the accompanying clear development in the digestive system, a negative impact on the immune ability of hatched chicks was observed as a consequence of the delay in obtaining food and thus become more sensitive to disease and thus the increase in mortality rates. To maximize the use of nutrients and the growth of chicks, early growth and development of the gastrointestinal tract (GIT) are crucial. It is possible to increase the rates of body growth and improve the gut development rate and thus increase the chicken's productive performance by conducting some strategies during the early stages of age, such as feeding the embryo by injecting eggs with nutrients or directly after the hatch, feeding newly hatched chicks immediately after pipping the eggs results in improved growth, increased the efficiency and development of digestive functions. This review discusses the early nutrition of various nutrients and their impacts on gut health, histomorphology, microbiology, and immunology. Additionally, it will shed light on the future vision of early nutrition as a strategy to modify gut health, promoting broiler chicks' health status and production. [ABSTRACT FROM AUTHOR]

ABSTRACT: In modern broilers, the period of embryonic development constitutes a greater proportion of a broiler's productive life. Hence, optimum embryonic development can exert a significant influence not only on chick hatchability and hatchling quality but also on overall broiler growth and performance. Further healthy and active hatchlings are correlated with improved posthatch performance. In this regard, probiotics are good candidates to mediate early-life programming. Therefore, we evaluated the effect of In ovo probiotic spray application on broiler hatchability and hatchling quality. The experiment was set out as a completely randomized study with 2 independent trials. In each trial, 540 eggs (Ross 308) were either sprayed with phosphate buffered saline (PBS; control) or probiotics [∼9 log CFU/egg of Lactobacillus rhamnosus NRRL B-442(LR) or Lactobacillus paracasei DUP 13076 (LP)] during incubation. On day 18, eggs were transferred to the hatcher and set up for hatching. Starting on day 19, eggs were observed for hatching to determine the spread of hatch and hatchability. Hatched chicks were then assessed for quality using the Tona and Pasgar score and morphometric measurements including hatchling weight, yolk-free-body-mass and hatchling length were measured. Further, chicks were reared in floor pens for 3 wk to assess posthatch growth. Overall, In ovo probiotic supplementation improved hatchability and hatchling quality. Specifically, the spray application of LP improved hatchability by ∼ 5% without affecting the spread of hatch. Further, both LR and LP significantly improved Pasgar and Tona score, indicating an improvement in hatchling quality. Also, LP and LR significantly improved hatchling weight, yolk-free-body-mass, and posthatch growth in chicks. LR significantly improved hatchling weight and hatchling length (P < 0.05). Moreover, this increase in posthatch growth was positively correlated with hatchling weight in the probiotic groups. Overall, our study demonstrates that In ovo probiotic application exerts a positive effect on hatchability, hatchling quality, and subsequent posthatch growth.

SUMMARY: The chicken gut microbiota plays important roles in host physiology and well-being. It can be impacted by several management and environmental factors like the use of antibiotic growth promoters (AGP) and heat stress (HS). Probiotics, essential oils (EO), and vitamins (such as folic acid) are bioactive substances that could potentially modulate the gut microbiota and promote its resilience to stressful environmental factors, especially when supplied as early as during embryonic development. This study evaluated the gut microbiota modulating potential of in ovo delivered probiotics, folic acid (FA), and in ovo + in-water delivered EO in broiler chickens, as compared to an in-feed AGP, under an HS challenge. Results from this study indicate that HS, AGP, and in ovo + in-water EO treatments modified chicken ceca bacterial populations and suggest that the microbiota-mediated role of AGP in growth promotion is related to improved biosynthesis of essential nutrients (amino acids and vitamins especially) and utilization of carbon sources derived from host diet and microbiome.

This study was aimed to investigate the influence of in ovo injection (IO) into air cell (AC) or yolk sac (YS) of epigallocatechin-3 gallate (EG) and oleuropein (OL) as antioxidants on hatching, physiological and productive performance of heat-stressed chicks. 840 fertile eggs were chosen for IO on 12th day of incubation. Eggs were divided into 7 groups within 4 replications each as follows: negative control (NC) without IO and other groups involved IO of 100 μl distilled water and 5 mg each of EG and OL in both AC and YS sites of egg. After hatching, chicks were exposed to heat stress for 24 h and raised for 42 d. Compared with NC, the results revealed that main effect of IO with EG and OL increased (p≤0.05) hatchability and decreased deformed chicks and serum levels of glucose, cholesterol, aspartate aminotransferase, corticosterone and heat shock protein 70 with enhancing feed efficiency, superoxide dismutase, glutathione peroxidase and thyroxine in serum or liver. High chick length and relative chick weight with low serum protein carbonyl and feed intake were recorded (p≤0.05) by EG. Low rectal temperature and heterophil to lymphocyte ratio with high body weight were recordered (p≤0.05) by OL. No mainly altered effects between both IO sites on most variables measured. However, there were significant influences among interactive treatments which related to in ovo injected substance in an injection site-dependent manner. It is concluded that improved hatchability, physiological and productive characteristics of heat-stressed chicks were achieved by IO of EG and OL.

The aim of the study was to determine differences in the proteome and peptidome and zinc concentrations in the serum and tissues of chickens supplemented with a multi-strain probiotic and/or zinc glycine chelate in ovo. A total of 1400 fertilized broiler eggs (Ross × Ross 708) were divided into four groups: a control and experimental groups injected with a multi-strain probiotic, with zinc glycine chelate, and with the multi-strain probiotic and zinc glycine chelate. The proteome and peptidome were analyzed using SDS-PAGE and MALDI—TOF MS, and the zinc concentration was determined by flame atomic absorption spectrometry. We showed that in ovo supplementation with zinc glycine chelate increased the Zn concentration in the serum and yolk sac at 12 h post-hatch. The results of SDS-PAGE and western blot confirmed the presence of Cu/Zn SOD in the liver and in the small and large intestines at 12 h and at 7 days after hatching in all groups. Analysis of the MALDI—TOF MS spectra of chicken tissues showed in all experimental groups the expression of proteins and peptides that regulate immune response, metabolic processes, growth, development, and reproduction.

Goal: This study presents a novel MRI coil design approach explicitly tailored for chick embryo measurements, with the primary objective of improving sensitivity and coverage. Methods: The limitations posed by conventional birdcage coils were addressed by introducing a curvature feature into a standard coil. The performance of the modified coil was assessed using EM simulations and experimental evaluations, which were subsequently validated using a 7 T MRI scanner. A comparative analysis was conducted against a standard quadrature low-pass birdcage coil to evaluate key factors. Results: The proposed coil demonstrated improved SNR and uniformity, particularly in the proximity of the end-rings. These results were consistent with the findings obtained from the simulations. Conclusions: The use of our innovative birdcage coil design holds promise and offers practical potential for in ovo studies.

The viral diseases that occurred in recent years have increased the interest in non-toxic to healthy cells and naturally isolated agents to struggle with these diseases. The key intention of this research is to examine both antiviral potentials against the Infectious Bronchitis model virus (IBV) and cytotoxic activities on determined cell lines of different active ingredients and medical herbs extracts for developing new antiviral agents or drugs towards SARS-CoV-2. The antiviral potency of the samples against IBV was determined as in ovo virucidal antiviral activity in specific pathogen-free (SPF) embryonated chicken eggs (ECEs). To detect antiviral activity, the haemagglutination test was performed after 48 h of incubation for all samples. The cytotoxic activity of the samples was identified on HepG2, Caco-2, HeLa, HEK293, PANC-1, PC-3, A549, MDAMB- 231, and CCD-34Lu cell lines by the MTT protocol. Hypericum perforatum extract was found to have a dominant role in cytotoxicity and antiviral activity. In addition, while nobiletin and Sambucus nigra do not exhibit cytotoxic activity on cells, they play a significant role in antiviral activity. As a consequence of our investigation, the cytotoxic and antiviral properties of Laurus nobilis, H. perforatum, and S. nigra extracts were found remarkable and the potential of these extracts was demonstrated.

With the rapidly increasing demand for poultry products and the current challenges facing the poultry industry, the application of biotechnology to enhance poultry production has gained growing significance. Biotechnology encompasses all forms of technology that can be harnessed to improve poultry health and production efficiency. Notably, biotechnology-based approaches have fueled rapid advances in biological research, including (a) genetic manipulation in poultry breeding to improve the growth and egg production traits and disease resistance, (b) rapid identification of infectious agents using DNA-based approaches, (c) inclusion of natural and synthetic feed additives to poultry diets to enhance their nutritional value and maximize feed utilization by birds, and (d) production of biological products such as vaccines and various types of immunostimulants to increase the defensive activity of the immune system against pathogenic infection. Indeed, managing both existing and newly emerging infectious diseases presents a challenge for poultry production. However, recent strides in vaccine technology are demonstrating significant promise for disease prevention and control. This review focuses on the evolving applications of biotechnology aimed at enhancing vaccine immunogenicity, efficacy, stability, and delivery. [ABSTRACT FROM AUTHOR]

The chorioallantoic membrane (CAM) model, generated during avian development, can be used in cancer research as an alternative in vivo model to perform tumorigenesis in ovo due to advantages such as simplicity, low cost, rapid growth, and being naturally immunodeficient. The aim of this systematic review has been to compile and analyze all studies that use the CAM assay as a tumor induction model. For that, a systematic search was carried out in four different databases: PubMed, Scopus, Cochrane, and WOS. After eliminating duplicates and following the established inclusion and exclusion criteria, a total of 74 articles were included. Of these, 62% use the in ovo technique, 13% use the ex ovo technique, 9% study the formation of metastasis, and 16% induce tumors from patient biopsies. Regarding the methodology followed, the main species used is chicken (95%), although some studies use quail eggs (4%), and one article uses ostrich eggs. Therefore, the CAM assay is a revolutionary technique that allows a simple and effective way to induce tumors, test the effectiveness of treatments, carry out metastasis studies, perform biopsy grafts of patients, and carry out personalized medicine. However, unification of the methodology used is necessary. [ABSTRACT FROM AUTHOR]

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The current study investigated the potentially harmful consequences of pure glyphosate or Roundup® on CYP family members and lipid metabolism in newly hatched chicks. On the sixth day, 225 fertilized eggs were randomly divided into three treatments: (1) the control group injected with deionized water, (2) the glyphosate group injected with 10 mg pure glyphosate/Kg egg mass and (3) the Roundup group injected 10 mg the active ingredient glyphosate in Roundup®/Kg egg. The results of the study revealed a reduction in hatchability in chicks treated with Roundup®. Moreover, change of Lipid concentration in serum and the liver-treated groups. Additionally, increased liver function enzymes and increased oxidative stress in the glyphosate and Roundup® groups. Furthermore, liver tissues showed histological changes and several lipid deposits in glyphosate-treated groups. Hepatic CYP1A2 and CYP1A4 expressions were significantly increased (p <.05) after glyphosate exposure, and suppression of CYP1C1 mRNA expression was significant (p <.05) after Roundup® exposure. The pro-inflammatory cytokines genes IFN-γ and IL-1β expression were significantly increased (p <.05) after Roundup® exposure. In addition, there were significant differences in the levels of expression genes which are related to lipid synthesis or catabolism in the liver. In conclusion, in ovo glyphosate exposure caused disruption of biotransformation, pro-inflammatory and lipid metabolism in chicks. [ABSTRACT FROM AUTHOR]

Simple Summary: In ovo feeding technology using eco-friendly products can play a significant role in poultry production. The current study aimed to evaluate the early intervention of green nanoparticles of silver and probiotics to enhance the embryonic development and, subsequently, the health status of hatched chicks by decreasing pathogenic bacteria populations. This study showed that in ovo green nanoparticles of silver, probiotics, and their combination improved the productivity, health status, and microbial counts of hatched one-day-old broilers. The aim of this study was to investigate the effects of the in ovo feeding of green nanoparticles of silver (Nano-Ag), probiotics, and their combination on hatchability, carcass criteria and internal organs, biochemical parameters, and cecal microbial populations in hatched one-day-old chicks. On day 18 of incubation, 250 live embryo eggs were weighed and randomly assigned to one of five treatment groups: a negative control group, a positive control group consisting of chicks injected with 0.2 mL physiological saline, a group consisting of chicks injected with 0.2 mL Nano-Ag, a group consisting of chicks injected with 0.2 mL probiotics (Bifidobacterium spp.), and a group consisting of chicks injected with 0.2 mL combination of Nano-Ag and probiotics (1:1). The results showed that the in ovo injection of Nano-Ag or probiotics, alone or in combination, had no effect on hatchability, live body weight, or internal organs but improved (p < 0.05) chick carcass yield compared to the control groups. Furthermore, in ovo feeding decreased (p < 0.05) serum levels of cholesterol, triglycerides, urea, creatinine, alanine aminotransferase, and aspartate aminotransferase, as well as cecal E. coli, but increased Bifidobacterium spp. when compared to the control groups. Based on these findings, in ovo injections of green Nano-Ag and probiotics, either alone or in combination, have the potential to improve chick health and balance the microbial populations in hatched one-day-old chicks. [ABSTRACT FROM AUTHOR]

In recent decades, neuraminidase/sialidase-based antivirals have been produced to suppress respiratory viral infections, including avian influenza, which relies on sialic acid as the entry point for viruses into cells. While neuraminidase has been extensively studied as an antiviral agent, numerous neuraminidases still have not been evaluated for their antiviral activities. Among these is NanB neuraminidase derived from Pasteurella multocida, which has received limited research attention. This study aimed to assess the potential of NanB neuraminidase in inhibiting H9N2 avian influenza virus infection in ovo. The research commenced with the molecular re-identification of the H9N2 A/Layer/Indonesia/WestJava-04/17 virus isolate, followed by determining the EID50 through Rapid HA test results. The toxicity of NanB neuraminidase was assessed by administering various doses to embryonated chicken eggs (ECE). The antiviral activity of NanB neuraminidase on ECE was evaluated through challenge tests, including treatment before, during, and after the challenge. The assessment involved monitoring the time of embryo death, virus titer through HA test, and viral copy number via RT-qPCR. The results indicated that the H9N2 virus titers capable of infecting 50% of ECE amounted to 108.83 EID50/mL. A dose of 0.258 U/mL of NanB neuraminidase was found to be toxic, leading to embryo mortality after 48 hours of incubation at 37 °C, while a nontoxic dose was determined to be 0.129 U/mL. The post-challenge treatment group exhibited the most significant reduction in virus titer in ECE. Notably, NanB neuraminidase derived from P. multocida demonstrated the ability to inhibit H9N2 avian influenza virus infection in the ovo model, with the optimal dosage of 0.129 U/mL. The observed decrease in virus titers in the hemagglutination assay and viral copy number assays suggests that NanB neuraminidase holds promise as a potential antiviral candidate for therapeutic approach. [ABSTRACT FROM AUTHOR]

Poultry vaccines are very important tools for disease prevention and may be administered collectively by drinking water or spray or individually by injection or oculonasal drop, whereas inactivated vaccines are administered by injection only. Poultry vaccines are increasingly delivered at the hatchery to day-old chicks or in ovo, because mass vaccination is much more efficiently implemented and controlled at the hatchery than on the farm. Mass administration on the farm by drinking water or spray requires strict compliance with guidelines regarding water quality, preparation of vaccines, and application, so as to cover the whole flock. Vaccination at the hatchery uses integrated machines to deliver vaccines to day-old chicks or, increasingly, in ovo at transfer from setters to hatchers. Regardless of the route and technology, a high quality of monitoring is critically important to maintain strict compliance and best practices from the vaccine vial to the bird, to guarantee efficient administration and intake of the vaccine by the whole flock and to secure the integrity of the vaccine itself. Major recent technical innovations in poultry vaccination covering both biology and technology open a very exciting era. Estudio recapitulativo- Tecnología de vacunación en la avicultura: principios de administración de vacunas. Las vacunas para la avicultura son herramientas muy importantes para la prevención de enfermedades y pueden administrarse colectivamente mediante agua de bebida y por aerosol, también individualmente mediante inyección o por gota oculonasal, mientras que las vacunas inactivadas se administran únicamente mediante inyección. Las vacunas avícolas se administran cada vez más en la planta de incubación a pollitos de un día o in ovo, porque la vacunación masiva se implementa y controla mucho más eficientemente en la planta de incubación que en las granjas. La administración masiva en granja mediante agua de bebida o aspersión requiere el cumplimiento estricto de las pautas relativas a la calidad del agua, preparación de vacunas y aplicación, de manera de cubrir a toda la parvada. La vacunación en la planta de incubación utiliza máquinas integradas para administrar vacunas a los pollitos de un día o, de manera más frecuente, in ovo en el momento del traslado de las incubadoras a las nacedoras. Independientemente de la ruta y la tecnología, una alta calidad del monitoreo es de vital importancia para mantener un cumplimiento estricto y las mejores prácticas desde el vial de la vacuna hasta el ave, para garantizar la administración y la captación eficiente de la vacuna por toda la parvada y para asegurar la integridad de la vacuna. Las importantes innovaciones técnicas recientes en la vacunación en avicultura que abarcan tanto la biología como la tecnología están iniciando una era fascinante. [ABSTRACT FROM AUTHOR]

Abstract Background In ovo application is the process of administering some nutrients or components into the egg. The main purpose of this application is to ensure that some nutrients are provided to chicks with a short incubation period. Few studies were conducted with taurine in fertile eggs; especially, no observation of hatchability and chick quality has been found. In addition, taurine has an anti‐stress impact that fights oxidative factors. Objective To assess the hatchability and chick quality after in ovo taurine administration. To determine the stress that may occur as a result of in ovo application and whether taurine has a stress‐reducing effect. Methods A total of 1200 fertile eggs from a 34‐week‐old broiler breeder (Ross 308) flock were categorized into 4 groups with 75 eggs per replicate: control (uninjected), taurine group (0.30 mL dissolved taurine in distilled water), sham control (sterile distilled water) and perforation (eggs perforated and then waxed). On day 14 of incubation, an in ovo injection was administered to the albumen. Data on hatching parameters and hepatic HSP70 levels were obtained using relevant formulas and western blotting, respectively. Results Control chicks exhibited higher hatchability than other groups, with the taurine group showing the lowest hatchability. The HSP70 levels were the highest in the perforation group compared to the control group. An increase of 21.37% in the taurine group and 83.45% in the sham control group was observed compared to the control group. Conclusions The findings suggest that in ovo application may induce increased stress, whereas taurine may have positive effects in mitigating the stress caused by in ovo application.

This study was undertaken to evaluate and compare the efficacy of different multi-antigen vaccines, including heat-inactivated, whole lysate, and subunit (outer membrane proteins [OMPs]) C. jejuni vaccines along with the immunostimulant CpG ODN in controlling Campylobacter colonization in chickens. In the first trial, 125 μg of C. jejuni OMPs and 50 μg of CpG ODN were administered individually or in combination, either in ovo to chick embryos or subcutaneously (SC) to one-day-old chicks. In the second trial, different concentrations of C. jejuni antigens (heat-killed, whole lysate, and OMPs) were administered SC to one-day-old chicks. The results of the first trial revealed that SC immunization with the combination of CpG ODN and C. jejuni OMPs elevated interferon (IFN)-γ, interleukin (IL)-1β, and IL-13 gene expression in the spleen, significantly increased serum IgM and IgY antibody levels, and reduced cecal C. jejuni counts by approximately 1.2 log10. In contrast, in ovo immunization did not elicit immune responses or confer protection against Campylobacter. The results of the second trial showed that SC immunization with C. jejuni whole lysate or 200 μg OMPs reduced C. jejuni counts by approximately 1.4 and 1.1 log10, respectively. In conclusion, C. jejuni lysate and OMPs are promising vaccine antigens for reducing Campylobacter colonization in chickens.

Metronidazole is an imidazole group bactericidal antibiotic used against anaerobic bacteria and some protozoa. There is no detailed information about the embryotoxic effect of Metronidazole. This study aims to determine the embryotoxic activity of Metronidazole using an in ovo method. A total of 210 fertile chicken eggs were placed in an incubator, divided into seven equal groups of 30. The first group was considered as the control group. On the seventh day of the study, Metronidazole was administered to the other six groups within 50 µL saline solution at doses of 250, 125, 62.5, 31.25, and 15.62 µg·egg-1 (5; 2.5; 1.25; 0.625; 0.312 mg·kg-1). At the end of the incubation period, the eggs hatched, and the number of live and dead embryos was determined. There were no significant differences in deaths between the groups (P>0.05 in all cases). No anomaly was detected in the macroscopic morphology of the embryos. As a result, it can be stated that Metronidazole may be safe for use during pregnancy. However, it is necessary to conduct molecular and histopathological studies to investigate the effects of this drug on organogenesis, especially in mammalian embryos.

In recent decades, neuraminidase/sialidase-based antivirals have been produced to suppress respiratory viral infections, including avian influenza, which relies on sialic acid as the entry point for viruses into cells. While neuraminidase has been extensively studied as an antiviral agent, numerous neuraminidases still have not been evaluated for their antiviral activities. Among these is NanB neuraminidase derived from Pasteurella multocida, which has received limited research attention. This study aimed to assess the potential of NanB neuraminidase in inhibiting H9N2 avian influenza virus infection in ovo. The research commenced with the molecular re-identification of the H9N2 A/Layer/Indonesia/WestJava-04/17 virus isolate, followed by determining the EID50 through Rapid HA test results. The toxicity of NanB neuraminidase was assessed by administering various doses to embryonated chicken eggs (ECE). The antiviral activity of NanB neuraminidase on ECE was evaluated through challenge tests, including treatment before, during, and after the challenge. The assessment involved monitoring the time of embryo death, virus titer through HA test, and viral copy number via RT-qPCR. The results indicated that the H9N2 virus titers capable of infecting 50% of ECE amounted to 108.83 EID50/mL. A dose of 0.258 U/mL of NanB neuraminidase was found to be toxic, leading to embryo mortality after 48 hours of incubation at 37 ℃, while a non-toxic dose was determined to be 0.129 U/mL. The post-challenge treatment group exhibited the most significant reduction in virus titer in ECE. Notably, NanB neuraminidase derived from P. multocida demonstrated the ability to inhibit H9N2 avian influenza virus infection in the ovo model, with the optimal dosage of 0.129 U/mL. The observed decrease in virus titers in the hemagglutination assay and viral copy number assays suggests that NanB neuraminidase holds promise as a potential antiviral candidate for therapeutic approach.

ABSTRACT: Early embryonic exogenous feeding of bioactive substances is a topic of interest in poultry production, potentially improving gastrointestinal tract (GIT) development, stimulating immunization, and maximizing the protection capability of newly hatched chicks. However, the biophysiological actions and effects of in ovo administered bioactive substances are inconsistent or not fully understood. Thus, this paper summarizes the functional effects of bioactive substances and their interaction merits to augment GIT development, the immune system, and microbial homeostasis in newly hatched chicks. Prebiotics, probiotics, and synbiotics are potential bioactive substances that have been administered in embryonic eggs. Their biological effects are enhanced by a variety of mechanisms, including the production of antimicrobial peptides and antibiotic responses, regulation of T lymphocyte numbers and immune-related genes in either up- or downregulation fashion, and enhancement of macrophage phagocytic capacity. These actions occur directly through the interaction with immune cell receptors, stimulation of endocytosis, and phagocytosis. The underlying mechanisms of bioactive substance activity are multifaceted, enhancing GIT development, and improving both the innate and adaptive immune systems. Thus summarizing these modes of action of prebiotics, probiotics and synbiotics can result in more informed decisions and also provides baseline for further research.

ABSTRACT: Probiotics are widely used as feed supplements in the poultry industry to promote growth and performance in chickens. Specifically, this supplementation starts around the time of lay and continues through the production cycle in laying hens. However, the embryonic period is critical to the growth and development of metabolically active organs thereby influencing subsequent health and productivity in adult birds. Therefore, the present study investigated the potential use of probiotics to promote embryonic growth in layers. Further, a pilot grow-out study was conducted to evaluate the effect of in ovo and in-feed probiotic application on pullet growth. For the study, fertile White Leghorn eggs were sprayed with phosphate buffered saline (control, CON) or probiotic cocktail (in ovo only, IO; Lactobacillus paracasei DUP 13076 and L. rhamnosus NRRL B 442) prior to and during incubation. The embryos were sacrificed on d 7, 10, 14, and 18 of incubation for embryo morphometry. On d 18, remaining eggs were set in the hatcher to assess hatchability and hatchling morphometry. For the pullet trial, hatchlings were raised on feed with or without probiotics until wk 5. Pullets were sacrificed weekly, and morphometric parameters were recorded. Results of our study demonstrate that in ovo probiotic application significantly improved relative embryo weight, crown-rump length, hatchability, and hatchling weight when compared to the control (P < 0.05). Further, this enhanced embryonic development was associated with a concomitant increase in posthatch growth. Specifically, pullets raised from probiotic-sprayed eggs had significantly improved crown-rump length, tibial length, tibial bone weight, and body weight when compared to the control (P < 0.05). Moreover, among the different treatment schemes employed in this study [CON (no probiotics), in-feed only (IF), IO only, and in ovo and in-feed probiotic supplementation (IOIF)], sustained probiotic supplementation (IOIF) was found to be the most effective in promoting growth. Therefore, in ovo and in-feed probiotic supplementation could be employed to promote embryo and pullet growth to support subsequent performance in layers.

This study aimed to evaluate the effects of in ovo injection of soy isoflavones (ISF) on hatchability, body weight, antioxidant status and intestinal development of newly hatched broiler chicks. One hundred and eighty fertile eggs were divided as follows: the control group, 3 mg/egg ISF (low dose) and 6 mg/egg ISF (high dose) on the 18th day of incubation. The results demonstrated that in ovo inclusion of 6 mg of ISF significantly increased hatchability and hatch weight. Both doses of ISF inclusion elevated the serum glutathione peroxidase and slightly decreased malondialdehyde compared to the control group. The high dose of ISF brings higher villus height and a higher villus/crypt ratio in chicks. Moreover, the mRNA levels of tumour necrosis factor‐ α and interferon‐gamma in the spleen were significantly decreased. The ISF treatments showed an improvement in intestinal enzyme expression levels of sucrose isomaltase and mucin 2 as well as tight junction protein (TJ) mRNA expression of claudin‐1 at high doses of ISF (p < 0.05) when compared with the other groups. Furthermore, the mRNA level of IGF‐1 was increased in the high doses of ISF compared to the control. Overall, these findings indicate that in ovo administration of ISF on the 18th day of incubation enhances hatchability, antioxidant status and intestinal morphometrics in hatched chicks and modulates the expression of proinflammatory cytokines, TJs and insulin‐like growth factor. In addition, the sustainability of antioxidants and other positive effects of ISF may increase chick viability and growth performance. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effect of in ovo co-supplementation of chicken embryos with a multi-strain probiotic containing effective microorganisms and zinc glycine chelate on total antioxidant capacity; concentrations of sulfhydryl groups, bityrosine bridges, formylkynurenines, hydroperoxides, proteins, corticosterone, pro- and anti-inflammatory cytokines and heat shock proteins; and the activity of catalase and superoxide dismutase in the serum, yolk sac and tissues of broiler chickens at 12 h and at 7 days after hatching. The results indicate high SOD activity in the small and large intestines of chicks at 12 h post-hatch in the groups receiving the multi-strain probiotic and in the small intestine and yolk sac of birds receiving the multi-strain probiotic and Zn-Gly chelate. High concentrations of TNF-α and IFN-γ in the yolk sac and serum after in ovo administration of Zn-Gly chelate were observed 12 h after hatching. The use of a probiotic and a probiotic with Zn-Gly chelate increased the total antioxidant capacity in the tissues of chickens. It can be concluded that in ovo administration of a multi-strain probiotic and Zn-Gly chelate can maintain the oxidant/antioxidant balance in chickens and increase the defense capacity against oxidative stress. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021–2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.

ABSTRACT: In the commercial egg industry, avian pathogenic Escherichia coli (APEC) can lead to significant economic loss. The Poulvac E. coli vaccine (PECV) is a commercially available attenuated live vaccine commonly applied via spray or drinking water to protect against losses associated with colibacillosis. The PECV has not been tested in layer hatching eggs using in ovo injection. Therefore, the purpose of this experiment was to determine the effects of injecting 50 μL of different doses of the PECV into Hy-Line W-36-layer hatching eggs on the hatchability and quality characteristics of hatchlings. At 18 d of incubation (DOI), treatments included 1 noninjected and 1 diluent-injected control. Furthermore, PECV treatments included a full dose (4.4 × 108 E. coli CFU) or serial dilutions of the full dose to produce 4.4 × 106, 4.4 × 104, or 4.4 × 102 CFU doses of E. coli. In ovo injections targeted the amnion. Percent hatchability of live embryonated eggs (HI), percent residue eggs, hatchling mortality, and female chick whole and yolk-free BW, relative yolk sac weight, and body length were among the variables examined. Treatment significantly (P < 0.0001) affected HI, with HI being highest in the control groups (97.3% in the noninjected and 94.2% in the diluent-injected), and with HI values being 89.0, 88.9, 84.4, and 71.2% in the 4.4 × 102, 4.4 × 104, 4.4 × 106, and 4.4 × 108 CFU E. coli dose treatments, respectively. The percentage of live embryos that did not complete hatch but that pipped internally (P = 0.024) or externally (P < 0.0001) were significantly affected by treatment, with percentages being highest in the 4.4 × 108 CFU treatment. Female chick body length was significantly (P < 0.0001) affected by treatment and was longer in both control groups and in the 1 × 102 CFU E. coli treatment in comparison to all other treatments. Yolk-free female chick BW was significantly (P = 0.034) affected by treatment and was lower in the 4.4 × 106 CFU and 4.4 × 108 CFU treatments when compared to the diluent-injected control group. An increase in the E. coli concentration administered in the amnion of embryonated layer hatching eggs at 18 DOI decreased hatch success and female chick yolk-free BW and body length.

ABSTRACT: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the main bacterial disease in poultry leading to significant economic losses worldwide. Antibiotic treatments favor the emergence of multidrug-resistant bacteria, and preventive measures are insufficient to control the disease. There is increasing interest in using the potential of bacteriophages, not only for phage therapy but also for prevention and biocontrol. This study aimed to evaluate the efficacy of a phage cocktail administered in ovo to prevent avian colibacillosis in chicks. When 4 different phages (REC, ESCO3, ESCO47, and ESCO58), stable under avian physiological conditions, were combined and inoculated at 17 embryogenic days (ED), they were transmitted to the newly hatched chicks. In a second trial, the 4-phage cocktail was inoculated into the allantoic fluid at ED16 and after hatch 1-day-old chicks were challenged with the O2 APEC strain BEN4358 inoculated subcutaneously. Two phages (REC and ESCO3) were still detected in the ceca of surviving chicks at the end of the experiment (7-days postinfection). Chicks that received the phages in ovo did not develop colibacillosis lesions and showed a significant decrease in intestinal BEN4358 load (8.00 × 107 CFU/g) compared to the challenged chicks (4.52 × 108 CFU/g). The majority of the reisolated bacteria from the ceca of surviving chicks had developed full resistance to ESCO3 phage, and only 3 were resistant to REC phage. The partially or complete resistance of REC phage induced a considerable cost to bacterial virulence. Here, we showed that phages inoculated in ovo can partially prevent colibacillosis in 1-wk-old chicks. The reduction in the APEC load in the gut and the decreased virulence of some resistant isolates could also contribute to control the disease.

Pharmacological and nutritional benefits of plant leaf extracts can be harnessed to address the problem of poor chick quality, performance and high mortality, which affect both hatchery and farm managers. This study sought to evaluate the in ovo effect of Soursop leaf extract (SLE) on the hatching and post-hatch performance of Noiler chicks. A total of 640 fertile eggs were randomly distributed into four groups of five replicates: (0.25 µg SLE), (0.5 µg SLE), (0.75 µg SLE) and a non-injected control. On day 18 of incubation, 0.2 ml of SLE was injected into the airspace of the eggs. At hatch, embryonic mortality, hatchability, and chick quality were evaluated. Hatched chicks were reared to assess feed intake, weight gain, feed conversion ratio (FCR) and mortality. Results showed no significant difference (p > 0.05) in embryonic mortality, hatchability and weight gain among the treatment groups. Chick weight and feed intake were significantly higher (p

Ascorbic acid, also known as vitamin C, is essential for the development of eggs and chicks due to its various important roles in biological processes such as collagen synthesis, antioxidant activity, iron absorption, immune system support, neurological development and enzyme cofactor. The injection of nutrients in ovo is a valuable technique used in poultry production due to the possibility of early nutrient delivery to chick embryos and improvement of this development. Front this, this study aimed to evaluate the effects of ascorbic acid use to in ovo feeding for poultry embryos. A total of 350 Rhode Island Red fertile eggs with viable embryos were randomly distributed in seven treatments with 50 replicates (eggs) each. The treatments were control (untreated eggs), eggs subjected to a saline solution (0.50% NaCl) and solutions containing increased levels of ascorbic acid (0.25, 0.50, 0.75, 1.00, and 1.25%). Evaluation of hatchability, embryo mortality, chicks' weight, and gastrointestinal tract development were performed. The increased addition of ascorbic acid in solutions to in ovo feeding caused a linear decrease (P=0.05) on hatchability, a linear increase (P=0.05) on intermediary embryo mortality. It was also observed a linear decrease (P=0.05) in late embryo mortality. In ovo injection of solutions with 0.50% of ascorbic acid provided heavier (P=0.05) chicks at birth with better (P=0.05) gizzard development. However, in ovo injection of ascorbic acid did not affect (P>0.05) the general development of the gastrointestinal tract. In conclusion, in ovo injection of ascorbic acid affected hatching characteristics. Increasing levels of ascorbic acid resulted in a linear decrease on hatchability and a sudden increase in intermediary embryo mortality. In ovo injection of solutions with 0.50% of ascorbic acid provided heavier chicks at birth with better gizzard development. However, it did not affect the development of the gastrointestinal tract of chicks with 1 day-old. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of in ovo feeding using albumin on hatchability, hatching weight, embryo mortality, as well as on organ development of Rhode Island Red chicks. A total of 245 fertile eggs with viable embryos were randomly distributed in seven treatments with 35 replicates (eggs) each. The treatments were untreated eggs and eggs treated with a buffer solution (0.50% saline) or solutions containing increased levels of albumin (0.5, 1.0, 1.5, 2.0, and 2.5%). On 17 days of incubation, eggs were injected with solutions containing albumin dissolved in 200 µL of 0.5% sterile saline. After the solutions were injected, the pinholes in the eggs were sealed with molten paraffin and moved to a hatcher machine. Evaluation of the chicks' weight, hatchability, and embryo mortality was made immediately post-hatch. Five viable chicks of each treatment were randomly selected and slaughtered by cervical dislocation to evaluate the heart, gastrointestinal organs and regional development. Data collected were subjected to polynomial regression (P=0.05) after a significant ANOVA result. In ovo feeding using albumin directly affected (P=0.05) the hatching characteristics of injected eggs. When eggs were injected with increased levels of albumin, there was a linear decrease (P=0.05) in hatchability and an increase in embryo mortality. The increased supplementation of albumin resulted in a linear growth (P=0.05) of gastrointestinal tract areas responsible for digestion (oropharynx plus esophagus, duodenal loop and cecum), and a linear decrease of heart, liver, pro-ventricle, gizzard and gastrointestinal tract areas responsible to absorption (jejunum plus ileum and colon plus rectum). [ABSTRACT FROM AUTHOR]

Melanoma, the tumor arising from the malignant transformation of pigment-producing cells—the melanocytes—represents one of the most severe cancer types. Despite their rarity compared to cutaneous melanoma, the extracutaneous subtypes such as uveal melanoma (UM), acral lentiginous melanoma (ALM), and mucosal melanoma (MM) stand out due to their increased aggressiveness and mortality rate, demanding continuous research to elucidate their specific pathological features and develop efficient therapies. Driven by the emerging progresses made in the preclinical modeling of melanoma, the current paper covers the most relevant in vitro, in vivo, and in ovo systems, providing a deeper understanding of these rare melanoma subtypes. However, the preclinical models for UM, ALM, and MM that were developed so far remain scarce, and none of them is able to completely simulate the complexity that is characteristic to these melanomas; thus, a continuous expansion of the existing library of experimental models is pivotal for driving advancements in this research field. An overview of the applicability of precision medicine in the management of rare melanoma subtypes is also provided. [ABSTRACT FROM AUTHOR]

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.