Storing eggs at low temperature prior to incubation is common practice in the broiler hatchery industry; however, prolonged storage (beyond 7 d) is known to increase early embryonic mortality and reduce chick quality and performance. To better understand the basis of this mortality, we previously published milestone criteria to evaluate morphological and cellular properties of the freshly laid embryo. Using these criteria, in the present study we checked the effects of storage at 18°C and 12°C for up to 28 d on hatchability and chick quality. Furthermore, using a 3D high-resolution episcopic microscopy (HREM) imaging system combined with standard and confocal microscopy and cell viability markers, we analyzed the effects of the different storage conditions on embryonic developmental stage, cytoarchitectural properties, mitotic index and cell survival. A total of 1,483 eggs from a young flock were divided in 2 groups, 18°C and 12°C, and stored for 7, 14, 21, and 28 d. Following storage, randomly selected 1,222 eggs were incubated, and the hatched chicks were evaluated for chick quality parameters. Nonhatched eggs were also analyzed to determine the stage of embryonic mortality. The remaining 261 eggs were isolated and analyzed for developmental stage, cytoarchitecture, mitotic index, and cell death following storage. Hatchability rates beyond 7 d of storage at 12°C were significantly improved compared to 18°C, and chick quality remained high. Similar results were obtained for an old flock's eggs (n = 1,350). Analyzing the embryos, at each time point, we found that at 12°C, the developmental progression during storage slows significantly, mitotic index-which at this temperature may indicate mitotic arrest-increases and the rate of early apoptosis is half than at 18°C. Moreover, the HREM system and histological sections showed that embryos stored at 18°C for prolonged times undergo dramatic cytoarchitectural changes that may be maladaptive to resuming normal development after diapause. We thus demonstrate the usefulness of the milestone criteria for predicting and studying the storage conditions that will allow for better performance in hatchery practice.

Introduction Antimicrobial resistance is (AMR) an emerging global public health problem. Rationale use of antibiotic can prevent the rise of antimicrobial resistance. The objective of this study was to understand the knowledge, attitude and practice on antibiotic usage and AMR among the veterinarians and para-veterinarians in Bhutan. Method A cross-sectional questionnaire survey among the veterinarians and para-veterinarians was conducted from June to July 2020. A score of one to the correct answers and zero for the wrong answers was allotted to each respondent answers. The total score was added and those who scored above the mean was categorized as having good knowledge and favourable attitude. Result A total of 219 animal health workers participated in this study. The mean knowledge score was 12.05 ±1.74 with 38.8% of the respondents having good knowledge on antibiotic use and AMR. Similarly, the mean scores for the attitude level were 8.32±1.61 with 51% them having favorable attitude towards antibiotic usage and AMR. The mean practice score was 3.83±1.06 with 77% of them having good practices on antibiotic use. The respondents who read national plan on AMR were found to have good knowledge on antibiotics and AMR (AOR: 2.39; 95% CI: 1.19–4.82). The female respondents (AOR: 2.16; 95% CI: 1.01–4.61), respondents from the eastern region (AOR: 2.53; 95% CI: 1.18–5.44), west central (AOR: 3; 95% CI: 1.30–6.92), animal health supervisors (AOR: 9.77; 95% CI: 1.98–48.29), and livestock production supervisors (AOR: 2.77; 95% CI: 1.21–6.35) have favorable attitude towards antibiotics and AMR. Conclusions Our study identified that most animal health workers in Bhutan had poor knowledge on antibiotics usage and AMR. Therefore, regular awareness education on antibiotics and AMR in the form of refresher course/training must be provided to the animal health workers in the country to avoid inappropriate use of antibiotics.

The pioneering study of Eyal-Giladi and Kochav (EG&K; Eyal-Giladi and Kochav, 1976) on the early developmental stages-from fertilization, through oviposition, to the gastrulation process-set the standard for characterizing chicken embryos, and has been used in numerous studies over the years. During uterine development, the chicken embryo undergoes dramatic changes, extremely rapid cell cycles, massive cell death, and axial determination processes. However, once the egg is laid, the temperature drops and the embryo enters into a diapause-like state. This phenomenon is utilized to store fertile eggs prior to incubation. The ability to resume development to hatching, following storage, relies on several factors, including the number of living cells and the embryonic developmental stage. These factors are highly influenced by the storage conditions-mainly duration and temperature. Thus, to study the effects of storage conditions on embryonic viability, a comprehensive characterization of the starting point-shortly after oviposition-is needed. In this study, we characterized freshly laid broiler eggs from Ross 308 flocks for embryonic developmental stage, total cell count, and cell viability. Using the novel high-resolution episcopic microscopy (HREM) system, we show, for the first time, high-resolution 3D morphological models of blastoderms which allow for highly accurate embryonic staging. Staging was also done under a dissecting microscope thus allowing for a direct side-by-side comparison of the two methods. Analysis of freshly laid blastomeres showed that the total nucleus count increases with developmental stage from ∼60,000 at stage X EG&K to ∼130,000 at stage XIII EG&K, whereas the proportion of mitotic index and dying cells at oviposition are ∼2% and ∼5%, respectively. Moreover, staging embryos from young and old flocks revealed that the blastoderms of the old flocks are more developed. Specifically, the predominant embryonic stages were XI and XII EG&K in young and old flocks, respectively. Collectively, we characterized parameters that can serve to analyze the maladaptive effects of prolonged storage under various conditions on embryo survival.