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Early sex determination and genotyping of chicken embryos is crucial for ethical and resource-efficient animal research. We developed a reproducible workflow using whole genome amplification combined (WGA) with Kompetitive Allele Specific PCR (KASP) and standard endpoint PCR to perform in ovo sexing and genotyping from embryonic day 4 (ED4) to ED10. Across four study phases involving various chicken lines—including a genetically modified line, Araucana crossbreeds, and commercial layers (total n  = 819)—we evaluated methodological feasibility, accuracy, and hatchability. Both PCR methods demonstrated high success rates in yielding results (70–100%) and high identification accuracy (92–100%) throughout all developmental stages tested. While the overall efficiency improved with embryonic age, ED7 was identified as the optimal time point to balance reliable sampling with high hatchability. We conclude that this laboratory-scale, PCR-based protocol provides an accessible and precise method for research laboratories to identify sex and genotype simultaneously. This approach directly supports the 3R principles by preventing the hatching of surplus animals before the assumed onset of nociception (ED13).

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.

Humanity could descend into a pre-antibiotic world as a result of antimicrobial resistance (AMR). Phage defenses are considered “vintage heroes” in the battle against multidrug-resistant (MDR) bacteria. However, phage resistance continues to evolve, necessitating alternative strategies. A potential solution is combining phages with antimicrobials like silver nanoparticles (AgNPs) to enhance the phage therapy. This study focuses on a combination therapy using Bauhinia variegata L. -mediated AgNPs and Pseudomonas phage M12PA to target multidrug-resistant (MDR) Pseudomonas aeruginosa . The antibacterial properties of this combination were evaluated through comprehensive characterizations and assays. As a proof of concept, the AgNP-phage combination delayed phage resistance by 6–12 h, reduced mutation, and enhanced antibacterial efficiency. Anti-biofilm activity improved by ~ 40% compared to AgNPs alone at sub-inhibitory concentrations. AgNPs and phages have demonstrated excellent compatibility, and the phage titer did not drop noticeably over 30 days. Biocompatibility assessments, including cell viability and hemolysis assays, highlighted the safety of biosynthesized AgNPs. Wound healing efficacy was also demonstrated in mouse fibroblast cells and an in ovo angiogenesis model. This study underscores the efficiency of phage-AgNPs synergy to be a potential therapeutic approach for MDR bacterial wound infection, showing effective anti-biofilm activity, delayed phage resistance, and minimal toxicity.

Understanding when learning begins is critical for identifying the factors that shape both the developmental course and the function of information acquisition. Until recently, sufficient development of the neural substrates for any sort of vocal learning to begin in songbirds was thought to be reached well after hatching. New research shows that embryonic gene activation and the outcome of vocal learning can be modulated by sound exposure in ovo. We tested whether avian embryos across lineages differ in their auditory response strength and sound learning in ovo, which we studied in vocal learning (Maluridae, Geospizidae) and vocal non-learning (Phasianidae, Spheniscidae) taxa. While measuring heart rate in ovo, we exposed embryos to (i) conspecific or heterospecific vocalizations, to determine their response strength, and (ii) conspecific vocalizations repeatedly, to quantify cardiac habituation, a form of non-associative learning. Response strength towards conspecific vocalizations was greater in two species with vocal production learning compared to two species without. Response patterns consistent with non-associative auditory learning occurred in all species. Our results demonstrate a capacity to perceive and learn to recognize sounds in ovo, as evidenced by habituation, even in species that were previously assumed to have little, if any, vocal production learning. This article is part of the theme issue 'Vocal learning in animals and humans'.

In recent years, particular attention has been paid to the possible connections, similarities, and potential uses of animals, especially pets (dogs and cats), in research on the causes, characteristics, and treatment of cancers occurring in pets and humans. One of the most promising experimental research models used to explore these issues is the avian embryo chorioallantoic membrane (CAM). This review aims to highlight the problem of the occurrence of cancers in domestic animals, placing emphasis on types, incidence, and predispositions of dogs and cats. Methodology and applications in cancer studies of this unique model were presented in detail. Moreover, the advantages and disadvantages of this diagnostic tool, as well as potential and future perspectives, were also described. This review confirms that cancer research can be conducted without the use of animals. Furthermore, the CAM can provide a robust and reliable model for this type of research and provide translational potential as an ethical, cost‐effective model bridging laboratory and clinical research.

Recently, we found that in ovo feeding of l-leucine (l-Leu) afforded thermotolerance, stimulated lipid metabolism and modified amino acid metabolism in male broiler chicks. However, the effects of in ovo feeding of l-Leu on thermoregulation and growth performance until marketing age of broilers are still unknown. In this study, we investigated the effects of in ovo feeding of l-Leu on body weight (BW) gain under control thermoneutral temperature or chronic heat stress. We measured changes of body temperature and food intake, organ weight, as well as amino acid metabolism and plasma metabolites under acute and chronic heat stress in broilers. A total of 168 fertilized Chunky broiler eggs were randomly divided into 2 treatment groups in experiments. The eggs were in ovo fed with l-Leu (34.5 µmol/500 µl per egg) or sterile water (500 µl/egg) during incubation. After hatching, male broilers were selected and assigned seven to nine replicates (one bird/replicate) in each group for heat challenge experiments. Broilers (29- or 30-day-old) were exposed to acute heat stress (30 ± 1°C) for 120 min or a chronic heat cyclic and continued heat stress (over 30 ± 1°C; ages, 15 to 44 days). In ovo feeding of l-Leu caused a significant suppression of enhanced body temperature without affecting food intake, plasma triacylglycerol, non-esterified fatty acids, ketone bodies, glucose, lactic acid or thyroid hormones under acute heat stress. Daily body temperature was significantly increased by l-Leu in ovo feeding under chronic heat stress. Interestingly, in ovo feeding of l-Leu caused a significantly higher daily BW gain compared with that of the control group under chronic heat stress. Moreover, some essential amino acids, including Leu and isoleucine, were significantly increased in the liver and decreased in the plasma by l-Leu in ovo feeding under acute heat stress. These results suggested that l-Leu in ovo feeding afforded thermotolerance to broilers under acute heat stress mainly through changing amino acid metabolism until marketing age.

[This corrects the article DOI: 10.3389/fvets.2020.00105.].[This corrects the article DOI: 10.3389/fvets.2020.00105.].

Biosurfactants are increasingly recognized for their versatile applications across industries, including bioremediation and pharmaceuticals. Among these, lipopeptides, produced notably by Bacillus species, demonstrate promising surface activity and biocompatibility. This study investigates the toxicity of lipopeptides biosurfactants derived from the AF₃B strain of Bacillus velezensis. The research delves into the production of lipopeptides biosurfactants from AF₃B and their characterization through antibacterial bioassays, revealing a significant inhibition of various bacterial strains. In vitro assays demonstrate the ability of lipopeptides biosurfactants to suppress the growth of key mycotoxigenic fungi (Aspergillus niger and Fusarium oxysporum) maximum to 86%, confirming their potential as a biological control. In ovo toxicity is assessed by injecting fertile chicken embryos with varying doses (30, 15, 7.5, 3.75, and 1.87 mg/mL) of lipopeptides biosurfactants obtained from AF₃B. The embryos exhibit normal development and high survival rates, indicating negligible toxicity. Overall, this study highlights B. velezensis strain AF₃B as a valuable candidate against bacterial and fungal diseases and foodborne pathogens, and in healthcare sector, underscoring the potential of lipopeptides biosurfactants in diverse industrial applications. In ovo toxicity analysis of biosurfactant from Bacillus velezensis AF₃B.

While probiotic supplementation via feed or drinking water is well known to improve poultry gut health by modulating the microbiota and enhancing immune function, the effects of in ovo supplementation remain largely unexplored. In this study, we investigated the effects of administering a lactobacilli cocktail in ovo (embryonic day 18), post-hatch, and in combination on gut immunity and the succession of the cecal microbiota in broilers over 5 weeks. 16S rRNA gene-based sequencing of cecal contents revealed a steady increase in Shannon diversity during the first 2 weeks (PERMANOVA, p  < 0.005), with community structure stabilizing by week 3 across all groups. In ovo administration of lactobacilli improved early hatch rates and modulated microbial composition during early succession, including reductions in Klebsiella and Enterococcus , and enrichment of Lactobacillus , during the first two weeks (MaAsLin2, q  < 0.25). These microbiome shifts were accompanied by a reduction in the expression of the pro-inflammatory cytokines, including interferon gamma (IFN-γ), interleukin-1β (IL-1β), and IL-8 in the cecal tonsils. These findings highlight the transient yet critical role of early-life probiotic interventions in shaping gut microbial colonization and immune response in broiler chickens. More importantly, a single in ovo lactobacilli dose yielded effects comparable to weekly oral or combined administration.

This study was composed of two experiments which investigated the response of two strains (Arbor Acre and Cobb 500, respectively) of broiler chickens to in ovo injection of inorganic salts of zinc, copper and their combination. A total of 300 hatching eggs [only 148 (59.20 %) and 232 (90.27 %), respectively, were fertile] each of Arbor Acre and Cobb 500 strains of broiler chickens were used in both experiments. These eggs were distributed into four treatments: control, in ovo inorganic Zn (80 µg.egg−1), Cu (16 µg.egg−1) and combined Zn and Cu (80 µg.egg−1 Zn and 16 µg.egg−1 Cu). The data obtained in both experiments were subjected to Completely Randomized Design (CRD) at the 5 % probability level. The results showed increased hatchability (P < 0.05) in eggs injected with the combination of inorganic salts of Zn and Cu in Experiment I and daily intake was influenced in both experiments. The carcass traits, organ development and gut morphometry were not significantly influenced by the treatment groups. The total serum protein and albumin of the birds were significantly (P < 0.05) increased by in ovo injection of inorganic salts of Zn and Cu at day 49 in the Experiment I. The study concluded that in ovo injection of inorganic salts of Zn at 80 µg.egg−1 and/or Cu at 16 µg. egg−1 could be adopted to increase feed intake with: attendant enhanced growth, enhanced immune response, increased albumin and total protein contents of blood serum in the strains of broiler chickens used.