Biotechnological Applications of Eggshell: Recent Advances

The eggshell (ES) provides protection against pathogenic and physical insults while supplying essential metabolic and nutritional needs for the growing avian embryo. It is constituted mainly of calcium carbonate arranged as calcite crystals. The global chicken egg production in 2018 was over 76.7 million metric tons. In industrialized countries, about 30% of eggs are processed at breaker plants that produce liquid egg products and large quantities of solid ES waste. ES waste is utilized for a variety of low-value applications, or alternatively is disposed in landfill with associated economic and environmental burdens. The number of patents pertaining to ES applications has increased dramatically in recent years; of 673 patents granted in the last century, 536 (80%) were published in the last two decades. This review provides a snapshot of the most recent patents published between 2015 and 2020, with emphasis on different biotechnological applications of ES waste, and summarizes applications for biomedical, chemical, engineering, and environmental technologies. Biomedical technologies include the production of calcium lactate, calcium phosphate, and health-promoting products, while chemical technologies include plant growth promoters, food processing and production, and biodiesel oil catalysis along with active calcium, carbon, soluble proteins, organic calcium, and ultrafine calcium carbonate sources. Engineering technologies address material engineering and nanoparticle production, while environmental technologies pertain to production of biomass, solubilization of sludge as well as production of magnetic ES adsorbents and adsorption of heavy metals, organics, total nitrogen and fluoride, soil pollutants, and radioactive compounds. Although the number of ES-based patents has exponentially increased in the last decade, exploration of innovative top-down approaches and ES development as a physical platform are new endeavors that are expected to further increase the upscaling of ES waste exploitation.

Hypoxia Generated by Avian Embryo Growth Induces the HIF-α Response and Critical Vascularization

Cancer research has transformed our view on cellular mechanisms for oxygen sensing. It has been documented that these mechanisms are important for maintaining animal tissues and life in environments where oxygen (O2) concentrations fluctuate. In adult animals, oxygen sensing is governed by the Hypoxia Inducible Factors (HIFs) that are stabilized at low oxygen concentrations (hypoxia). However, the importance of hypoxia itself during development and for the onset of HIF-driven oxygen sensing remains poorly explored. Cellular responses to hypoxia associates with cell immaturity (stemness) and proper tissue and organ development. During mammalian development, the initial uterine environment is hypoxic. The oxygenation status during avian embryogenesis is more complex since O2 continuously equilibrates across the porous eggshell. Here, we investigate HIF dynamics and use microelectrodes to determine O2 concentrations within the egg and the embryo during the first four days of development. To determine the increased O2 consumption rates, we also obtain the O2 transport coefficient (DO2) of eggshell and associated inner and outer shell membranes, both directly (using microelectrodes in ovo for the first time) and indirectly (using water evaporation at 37.5°C for the first time). Our results demonstrate a distinct hypoxic phase (<5% O2) between day 1 and 2, concurring with the onset of HIF-α expression. This phase of hypoxia is demonstrably necessary for proper vascularization and survival. Our indirectly determined DO2 values are about 30% higher than those determined directly. A comparison with previously reported values indicates that this discrepancy may be real, reflecting that water vapor and O2 may be transported through the eggshell at different rates. Based on our obtained DO2 values, we demonstrate that increased O2 consumption of the growing embryo appears to generate the phase of hypoxia, which is also facilitated by the initially small gas cell and low membrane permeability. We infer that the phase of in ovo hypoxia facilitates correct avian development. These results support the view that hypoxic conditions, in which the animal clade evolved, remain functionally important during animal development. The study highlights that insights from the cancer field pertaining to the cellular capacities by which both somatic and cancer cells register and respond to fluctuations in O2 concentrations can broadly inform our exploration of animal development and success.

Modeling breast cancer by grafting patient tumor samples in the avian embryo: an in vivo platform for therapy evaluation coupled to large scale molecular analyses

Lack of preclinical patient-derived xenograft (PDX) cancer models in which to conduct large scale molecular studies seriously impairs the development of effective personalized therapies. We report here on an in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large scale RNAseq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple negative breast cancer (TNBC) and validated using TNBC standards of care (SOCs) and an investigational therapeutic agent.

Chorioallantoic Membrane Models of Various Avian Species: Differences and Applications

The chorioallantoic membrane model (CAM) of an avian embryo is used as an experimental model in various fields of research, including angiogenesis research and drug testing, xenografting and cancer research, and other scientific and commercial disciplines in microbiology, biochemistry, cosmetics, etc. It is a low-cost, low-maintenance, and well-available in vivo animal model that is non-sentient and can be used as an alternative for other mammal experimental models. It respects the principles of the “3R” rule (Replacement, Reduction, and Refinement)—conditions set out for scientific community providing an essential framework for conducting a more human animal research, which is also in line with constantly raising public awareness of welfare and the ethics related to the use of animal experimental models. In this review, we describe the chorioallantoic membrane of an avian embryo, focusing on its properties and development, its advantages and disadvantages as an experimental model, and the possibilities of its application in various fields of biological research. Since the most common chicken CAM model is already well known and described in many publications, we are particularly focusing on the advantages and application of less known avian species that are used for the CAM model—quail, turkey, and duck.

Mucosal and Systemic Immune Responses Modulated by in Ovo-Delivered Bioactive Compounds in Distinct Chicken Genotypes

BackgroundMucosal and systemic immune responses are different strategies to cope with environmental stimuli. In ovo delivery of prebiotic, probiotic, or synbiotic into the avian embryo allows for indigenous microbiota stimulation. Intestinal microbiota in animals is responsible for immune system maturation. The genetic component is critical in host-microbiota crosstalk and immune system development. The goal of this study was to compare mucosal and systemic immune responses in two distinct chicken genotypes stimulated in ovo. ResultsThe experiment was constructed in full-factorial design and was aimed to study the effects of two chicken genotypes (chicken broilers or native chickens), four in ovo-delivered compounds (GOS/galactooligosaccharides/prebiotic, Lactococcus lactis subsp. cremoris/probiotic, or GOS+L.lactis/synbiotic, vs. physiological saline) and three different stimuli (LPS or LTA vs. physiological saline) on a panel of cytokine genes (IL-1B, IL-2, IL-4, IL-6, IL-10, IL-12p40, and IL-17) expressed in caecal tonsils and spleen. We tested significance of the main effects and their interactions. Genotype had the most significant influence on all gene expression signatures in both tissues (P < 0.001 for all genes, except P < 0.05 for IL-10 in spleen). Immune challenge was the second most significant main effect, and influenced IL-1β, IL-6 IL-10 (P < 0.001), and IL-17 genes (P < 0.05) in caecal tonsils, and all genes in spleen (P < 0.001), except IL-4 (P > 0.05). In ovo stimulation influenced IL-2, IL-4, and IL-10 in caecal tonsils (P < 0.05), as well as IL-2 and IL-12p40 in spleen (P < 0.05). ConclusionsThe mucosal and systemic immune responses of chicken broilers and native chickens showed distinct patterns. Genotype influenced gene expression signatures of all immune-related genes, but chicken broilers developed stronger immune responses than native chickens. LPS triggered both mucosal (caecal tonsils) and systemic (spleen) immune responses in chicken broilers, but only systemic (spleen) in native chickens. In ovo stimulation with bioactive compounds (especially prebiotic) modulated innate immune responses to LPS. GOS delivered in ovo induced the most pronounced responses to LPS, which validated its further application as a potent immunomodulator for in ovo applications.

Angiogenesis in the Avian Embryo Chorioallantoic Membrane: A Perspective on Research Trends and a Case Study on Toxicant Vascular Effects

The chorioallantoic membrane (CAM) of the avian embryo is an intrinsically interesting gas exchange and osmoregulation organ. Beyond study by comparative biologists, however, the CAM vascular bed has been the focus of translational studies by cardiovascular life scientists interested in the CAM as a model for probing angiogenesis, heart development, and physiological functions. In this perspective article, we consider areas of cardiovascular research that have benefited from studies of the CAM, including the themes of investigation of the CAM’s hemodynamic influence on heart and central vessel development, use of the CAM as a model vascular bed for studying angiogenesis, and the CAM as an assay tool. A case study on CAM vascularization effects of very low doses of crude oil as a toxicant is also presented that embraces some of these themes, showing the induction of subtle changes in the pattern of the CAM vasculature growth that are not readily observed by standard vascular assessment methodologies. We conclude by raising several questions in the area of CAM research, including the following: (1) Do changes in patterns of CAM growth, as opposed to absolute CAM growth, have biological significance?; (2) How does the relative amount of CAM vascularization compared to the embryo per se change during development?; and (3) Is the CAM actually representative of the mammalian systemic vascular beds that it is presumed to model?