Biological Evaluation, Molecular Docking, and in Ovo Hatchability Assessment of Selected Mannich-Type...

1,2,4-Triazole derivatives are widely investigated as bioactive heterocyclic compounds with diverse biological properties; however, their potential effects on avian embryonic development remain insufficiently characterized. This study aimed to synthesize selected Mannich-type 1,2,4-triazol-5-one derivatives and evaluate their in vitro biological activities, predicted enzyme interactions, and in ovo hatchability outcomes in broiler embryos. Three derivatives (3a-3c) were synthesized and structurally characterized using Fourier-transform infrared (FT-IR), proton nuclear magnetic resonance (¹H-NMR), and carbon-13 nuclear magnetic resonance (¹³C-NMR) spectroscopy. Their reducing capacity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, metal-chelating capacity, and antimicrobial activity were evaluated. Molecular docking analyses were performed against carbonic anhydrase II and catalase, using acetazolamide and 3-amino-1,2,4-triazole as reference inhibitors, respectively. For the in ovo assessment, fertilized Ross 308 eggs were allocated to non-injected control, vehicle control, and compound-treated groups. Compounds 3a, 3b, and 3c were administered on incubation day 17 at 20, 10, and 5 mg/egg, respectively, as literature-supported, compound-specific exploratory exposure levels. The compounds exhibited weak reducing capacity, negligible DPPH radical-scavenging activity, pronounced metal-chelating capacity, and selective antimicrobial activity against several bacterial strains, whereas no inhibition was observed against Escherichia coli. Docking analysis predicted favorable interactions with both enzyme targets, with compound 3c showing the most favorable binding energies. Baseline egg weights were comparable among groups, indicating that initial egg weight was unlikely to influence hatchability outcomes. Complete hatch failure occurred in all compound-treated groups, whereas hatchability was observed in the control groups. These findings suggest that the tested derivatives may adversely affect embryonic development under the applied in ovo conditions. Their pronounced metal-chelating capacity, together with the predicted interactions with carbonic anhydrase II and catalase, provides a plausible mechanistic framework for interpreting the observed embryonic outcomes. The molecular docking results complement the experimental findings and provide supportive in silico evidence for these potential interactions. These findings contribute to the limited literature on the in ovo evaluation of synthetic Mannich-type 1,2,4-triazol-5-one derivatives and provide preliminary evidence regarding their embryotoxic potential. Further dose-dependent, mechanistic, histopathological, and enzyme-based studies are required to clarify the biological basis of the observed effects and to guide future evaluation of these compounds in in ovo, biomedical, and veterinary applications.