Hen Egg Yolk Research Articles

Dietary stinging nettle (Urtica dioica) improves carotenoids content in laying hen egg yolk

ABSTRACT 1. This study assessed the addition of dried stinging nettle (SN) leaves at 0%, 2.5% and 5.0% in feed on egg production, egg quality, chemical composition and antioxidant content in eggs from laying hens. 2. Seventy-two Hy-Line Brown laying hens, housed in 36 enriched layer cages, were used in the study from 43 to 47 weeks of age. Feeding dry SN leaves did not affect (p > 0.05) egg production variables. 3. Dietary SN inclusion linearly increased (p < 0.001) carotenoid content more than six-fold, in addition to yellowness and redness of the yolks at the maximum inclusion. Providing eggs from hens fed carotenoid enriched diets, e.g. SN, may be used to increase carotenoids in human diets.

Unravelling the potential of yolkin for nutraceutical use: the origin, structure, and functional insights of a hen egg yolk polypeptide complex.

Nutraceuticals can reduce the risk of many diseases, such as cardiovascular disease, immune deficiencies, neurodegeneration, and others. Their delivery remains a challenge because it depends on many factors, most notably the stability of the bioactive compounds. Yolkin is a peptide complex isolated from hen egg yolk with immunomodulatory and neuroprotective potential. However, yolkin remains relatively poorly characterized. We aimed to determine the origin and glycosylation level of yolkin, its storage conditions, its thermal stability, and its aggregation ability and to assess its antioxidant, antihypertensive, and antidiabetic potential. The peptide composition of yolkin was shown to be homologous to that of vitellogenin II and vitellogenin I. These results indicate the stability of yolkin in a lyophilized form, preferably at 4 °C, with nonaggregation, antioxidant, and antidiabetic activities. As a result, yolkin can be considered to have significant therapeutic potential and represents a valuable tool for the development of novel nutraceuticals.

Passive Immunotherapy of Cynomolgus Monkeys with Anti-Rotavirus IgY.

Immunoglobulins Y (IgY) purified from egg yolks of hens represents an attractive, cost-effective alternative for the development of new diagnostic and therapeutic platforms. In this study, we evaluated the therapeutic efficacy of rotavirus-specific IgY in a cynomolgus monkey (Macaca fascicularis) model. Animals were experimentally infected with human rotavirus Group A (RVA), the most common cause of severe acute diarrhoea among young children worldwide. Animals were administered human RVA (3.1 × 107 FFU/mL) by oral gavage, challenged with 2.5 mg of anti-RVA IgY orally, and monitored for five days according to clinical, haematological and biochemical parameters; serum electrolyte levels; viral shedding; and histopathological changes. Immunotherapy with anti-RVA IgY had a protective effect against severe rotavirus-induced enteritis in four of the ten treated monkeys, as evidenced by histopathological findings. Although only one animal had diarrhoea, all but one exhibited virus shedding regardless of the treatment.

Salt induced slowdown of kinetics and dynamics during thermal gelation of egg-yolk.

We investigated the effect of the NaCl concentration (0.3-2M) on the structure and dynamics of hen egg yolk at room temperature and during thermal gelation at temperatures in the range of 66-90 °C utilizing low-dose x-ray photon correlation spectroscopy in ultra-small angle x-ray scattering geometry. With an increase in the salt concentration, we observe progressive structural and dynamic changes at room temperature, indicating the disruption of yolk components such as yolk-granules and yolk-plasma proteins. Temperature- and salt-dependent structural and dynamic investigations suggest a delay in the gel formation and aggregation of yolk low-density lipoproteins with increasing ionic strength. However, the time-temperature superposition relationship observed in all samples suggests an identical mechanism underlying protein aggregation-gelation with a temperature-dependent reaction rate. The sol-gel transition time extracted from kinetic and dynamic information follows Arrhenius's behavior, and the activation energy (460kJ/mol) is found to be independent of the salt concentration.

Reducing curing time via a shell‐less method: a comparative analysis of flavour characteristics in salted egg yolk

SummaryThe rapid shell‐less curing method significantly simplifies the processing cycle of salted egg yolks (SEYs). However, differences in flavour attributes between rapidly cured SEYs and traditionally cured SEYs have yet to be clearly established. Herein, a comprehensive evaluation of the volatile compounds in SEYs using HS‐SPME‐GC‐MS and HS‐GC‐IMS aims to delineate the flavour characteristics under varying preparation methods. Despite reduced oil exudation, the rapid salting process resulted in higher levels of free amino acids than the traditional approach. Hexanal, nonanal, 1‐octen‐3‐ol, 2‐pentyl‐furan, phenylacetaldehyde, benzaldehyde, 2‐octenal, and 2,5‐dimethyl‐pyrazine were identified as significant contributors to the SEY flavour profile. Conversely, salted hen egg yolks contained methional, decanal and 2‐methyl‐pyrimidine; whereas salted duck egg yolks presented with (E,E)‐2,4‐decadienal, 2,6‐diethyl‐pyrazine and nonanoic acid. Principal component analysis of the E‐nose revealed that the aroma profile of rapid curing (SHY‐R25) was more similar to that of traditional curing (SHY). The rapid preparation technology presented a viable alternative to traditional pickling, capable of partially replicating the flavour of traditional salted egg yolks within a single day. These findings contributed to a more scientific application and preparation of SEY flavouring bases, providing a theoretical basis for flavour enhancement and development.

Comparative Study of Gas and Liquid Chromatography Methods for the Determination of Underivatised Neutral and Acidic Cannabinoids and Cholesterol.

The aim of our study was to develop a gas chromatographic method coupled with mass spectrometry (GC-MS) for the determination of underivatised neutral (CBDs-N) and acidic (CBDs-A) cannabinoids (CBDs) and cholesterol (Chol). Emphasis was also placed on comparing our original GC-MS method with the currently developed C18-high-performance liquid chromatography with photodiode detection (C18-HPLC-DAD). A combination of a long GC column, shallow temperature column programme, and mass-spectrometry was employed to avoid issues arising from the overlap between CBDs and Chol and background fluctuations. The pre-column procedure for CBDs and Chol in egg yolks consisted of hexane extractions, whereas the pre-column procedure for CBDs in non-animal samples involved methanol and hexane extractions. CBDs-A underwent decarboxylation to CBDs during GC-MS analyses, and pre-column extraction of the processed sample with NaOH solution allowed for CBD-A removal. No losses of CBDs-N were observed in the samples extracted with NaOH solution. GC-MS analyses of the samples before and after extraction with NaOH solution enabled the quantification of CBDs-A and CBDs-N. CBDs-A did not undergo decarboxylation to CBDs-N during C18-HPLC-DAD runs. The use of the C18-HPLC-DAD method allowed simultaneous determination of CBDs-N and CBDs-A. In comparison to the C18-HPLC-DAD method, our GC-MS technique offered improved sensitivity, precision, specificity, and satisfactory separation of underivatised CBDs and Chol from biological materials of endogenous species, especially in hemp and hen egg yolk. The scientific novelty of the present study is the application of the GC-MS method for quantifying underivatised CBDs-A, CBDs-N, and Chol in the samples of interest.

Effect of salting and dehydration treatments on the physicochemical and gel properties of hen and duck egg yolks, plasma and granules.

Salted hen egg yolks are less oily and less flavorful than salted duck egg yolks. However, hen eggs have a more adequate market supply and have a broader application prospect than duck eggs. In the present study, egg yolks, plasma, and granules were dehydrated by adding 1% NaCl to simulate traditional curing process of salted egg yolk. The changes in the pickling process of hen egg yolks (HEY) and duck egg yolks (DEY) plasma and granules were compared to reveal the gelation mechanism and the underlying causes of quality differences in salted HEY and DEY. Salted HEY can be compared with the changes in DEY during the pickling process to provide a theoretical basis for the quality improvement of salted HEY to salted DEY. The results showed that both plasma and granules were involved in gel formation, but exhibited different aggregation behaviors. Based on the intermolecular forces, the HEY proteins achieved aggregation mainly through hydrophobic interactions and DEY proteins mainly through covalent binding. According to spin-spin relaxation time, HEY gels immobilized a large amount of lipid and interacted strongly with lipids. DEY gels showed much free lipid and had weak interaction with lipid. The microstructure showed that HEY proteins were easily unfolded to form a homogeneous three-dimensional gel network structure after salting, whereas heterogeneous aggregates were formed to hinder the gel development in DEY. Changes in protein secondary structure content showed that pickling can promote the transformation of the α-helices to β-sheets structure in HEY gels, whereas more α-helices structure was formed in DEY gels. The present study has demonstrated that different gelation behaviors of hen and duck egg yolk proteins (especially in plasma) through salting treatment led to the difference in the quality of salted HEY and DEY. © 2024 Society of Chemical Industry.

Differentiation of lipid profiles of hen eggs from different regions based on lipidomics combined with chemometrics

Hen egg yolk lipids from different regions were determined by ultra-high-performance liquid chromatography with high-resolution mass spectrometry. A total of 15 lipid classes and 346 lipid molecular species were tentatively identified. Principal component analysis showed that the egg lipid profiles varied a certain extent across different production regions. Hierarchical cluster analysis indicated that the egg lipid compositions from Jiangsu and Chungking were relatively similar, but significantly different to those from Inner Mongolia, Jiangxi, and Hainan. Differences in the content of some lipid molecules are obvious, for instance, the concentration of lysophosphatidylcholine (18:1/0:0) in Jiangsu egg and Chungking egg, and phosphatidylinositol (18:0/20:4) in Hainan egg, Changde egg and Jiangxi egg varied significantly from other origins. This study provides a foundation for comprehending the impact of diverse geographical environments on egg lipid profiles.

Diets comprising hen egg yolk and milk proteins as potential alternatives to shark egg-based diets for larvae of the Japanese eel Anguilla japonica

Diets comprising hen egg yolk and milk proteins as potential alternatives to shark egg-based diets for larvae of the Japanese eel Anguilla japonica

Yolkin, a Polypeptide Complex from Egg Yolk, Affects Cytokine Levels and Leukocyte Populations in Broiler Chicken Blood and Lymphoid Organs after In Ovo Administration.

Yolkin is a polypeptide complex isolated from hen egg yolk that exhibits immunomodulating properties. The aim of the present study was to determine whether in-ovo-delivered yolkin affects leukocyte populations and cytokine levels in broiler chickens. The experiment was carried out on eggs from Ross 308 broiler breeder birds. Yolkin was administered in ovo on the 18th day of incubation, once, at the following three doses: 1, 10, or 100 µg/egg. The immunological parameters were assessed in 1-, 7-, 14-, 21-, 28-, 35-, and 42-day-old birds kept under farming conditions and routinely vaccinated. The leukocyte populations were determined in the thymus, spleen, and blood. The cytokine (IL-1β, IL-2, IL-6, and IL-10) levels were determined in the plasma of the broiler chickens. Each experimental group included eight birds. The most pronounced effect of yolkin was an increase in the population of T cells, both CD4+ and CD8+, mainly in the blood. This effect on the lymphocyte subsets may be valuable regarding chicken immune responses, mainly against T-dependent antigens, during infection or after vaccination.

Clinical Features of Quail Egg Ingestion in Patients with Acquired Tolerance to Hen Eggs: A Case Series Study

Introduction: Patients with hen’s egg allergy are often instructed to avoid consuming other avian eggs, such as quail eggs. However, it is unclear whether patients with an acquired tolerance to hen eggs continue to avoid consuming quail eggs. This study aimed to evaluate the clinical features of quail egg ingestion. Methods: This prospective case series included children aged ≥1 year with hen’s egg allergy who were recruited between October 2019 and February 2021 in our hospital. We conducted an oral food challenge (OFC) with three boiled quail eggs to evaluate the clinical features of quail egg ingestion in patients with acquired tolerance to hen eggs. The primary outcome was a positive OFC after ingesting three quail eggs. Secondary outcomes were cross-antigenicity between hen and quail eggs observed through the skin prick test (SPT) and pattern of quail egg allergy, comprising the onset of reaction, and severity. The correlation between the diameters of the wheals with SPT in hen and quail eggs was evaluated using the Pearson product-moment correlation coefficient. Results: A total of 62 patients underwent the quail egg OFC. The median (interquartile range) age of the participants was 3 (2–5) years. Thirty-three (53%) patients had a history of anaphylaxis due to hen eggs. The median total immunoglobulin E (IgE) level in patients who underwent the OFC with half a heated whole hen’s egg was 271 (98–593) IU/mL. The median specific IgE level in egg white and ovomucoid was 9.7 (3.2–21.5) and 4.4 (1.3–6.9) UA/mL, respectively. The quail egg OFC results revealed that none of the 59 patients who ate the three quail eggs completely had an allergic reaction. The SPT-positive and SPT-negative rates in raw and boiled hen and quail egg whites were both correlated. The diameters of wheals with SPT in raw hen and quail egg whites and yolks were positively correlated. Conclusion: Patients with an acquired tolerance to hen eggs may not be required to avoid consuming quail eggs.

Lymphocyte stimulation test for diagnosing hen's egg yolk-induced enterocolitis syndrome.

There is currently little research into factors predicting the results of an initial diagnostic oral food challenge (OFC) test for food protein-induced enterocolitis syndrome (FPIES). The present study aimed to identify predictors of the diagnosis of hen's egg yolk-induced FPIES (HEY-FPIES). The present monocentric study was performed at Tokyo Metropolitan Children's Medical Center and included patients who underwent hen's egg yolk OFC (HEY-OFC) between March 2018 and March 2023 to assess for HEY-FPIES. The baseline characteristics of the groups and HEY-OFC positivity or negativity were then compared. Univariate analysis was conducted by using the Mann-Whitney U test or Fisher exact test. Receiver operator characteristic analysis was used to create probability curves. In total, 35 patients were analyzed; of these, 17 were HEY-OFC-positive. No significant difference was observed between the HEY-OFC-positive and HEY-OFC-negative groups in terms of background factors except for the HEY-LST value, which was significantly higher in the HEY-LST group (P= .027). Receiver operator characteristic analysis demonstrated that the area under the curve for HEY-OFC positivity using the HEY-LST value was 0.719 (95% CI= 0.541-0.897). The statistically optimal cutoff value for the HEY-LST was 610%, which had a clinical sensitivity and specificity of 64.7% and 83.3%, respectively. The present study demonstrated that the HEY-LST may be a useful predictor of the result of an initial OFC for HEY-FPIES.

Does dietary linseed or canola oil affect lipid metabolism, immunity, and n-3 polyunsaturated fatty acids content in quail eggs?

One of the most intriguing areas of research and innovation in the animal production and food sector recently has been designed-enriched products. These items are regarded as functional foods because they feature components that have advantageous physiological impacts on human health. In the production of poultry, designed eggs constitute a significant category of functional foods. The present study hypothesized that adding different kinds of oils to quail diets will help produce designer eggs rich in omega-3 and 6 fatty acids in addition to enhancing productive performance. So, this study examined how linseed (flaxseed) and canola oils with various levels can affect lipid metabolism, immune function, and the amount of n-3 polyunsaturated fatty acids (n-3 PUFA) in Japanese quail eggs. This work was conducted using 3 different vegetable oils (sunflower, linseed, and canola oils) and 3 different antioxidant supplements (0, 250 mg vitamin E/kg feed, and 1,000 mg ginger/kg feed) in a 3×3 factorial experiment. When linseed or canola oil was added to the diet, the number of fatty acids in the egg yolks of Japanese quail layers fell by (12.7 and 18.9%) and (41.4 and 24.6%), respectively. The amounts of saturated and monounsaturated fatty acids in total eggs fell by 21.9 and 14.6% and 24.5 and 15.8%, respectively, at 20 wk of age. However, when linseed and canola oil were added to the diet, the sum n-3 PUFA content in the egg yolk of Japanese quail-laying hens was noticeably raised at 15 and 20 wk of age. At 15 and 20 wk of age, the same groups' total n-6 PUFA content considerably increased compared to the group that did not receive flaxseed. In conclusion, during the laying period of Japanese quail, linseed oil, canola oil, vitamin E, or ginger positively affected productivity, blood hematology, constituents, resistance, lipid digestion system, and antioxidative properties in serum and egg yolk.

Effect of feed trace elements on eggs of five strains of laying hens and their health risk assessment.

Trace elements are essential for human nutrition, and their deficiencies or excesses are strongly associated with several diseases, such as cardiovascular ones. The current cross-sectional study investigated the concentration of essential trace elements (copper, non-metal selenium, iron, zinc, cobalt and manganese) in eggs and diets of five strains of laying hens. The yolk and albumen were analysed separately, and wet preparation was carried out following inductively coupled plasma-optical emission spectrometry detection. The target hazard quotients (THQs) for the non-carcinogenic disease were calculated by the United States environmental protection agency (USEPA) method. The highest concentrations of selenium, zinc and manganese were found in egg yolks of native hens (0.76, 44.22 and 6.52mg/kg, respectively). The highest amounts of copper and cobalt were recorded in the egg yolk of Lohman (2.07 and 0.023mg/kg, respectively). On the other hand, the egg yolk of Bovans contained the highest amount of iron (57.46mg/kg). Overall, the potential health risks were minimal, and the consumption of eggs was generally safe.

Correlation analysis between fertilized hen egg yolk extract and lipid metabolism

Correlation analysis between fertilized hen egg yolk extract and lipid metabolism

Effects of supplemental dried wild leek (Allium scorodoprasum L. subsp. rotundum) leaves on laying performance, egg quality characteristics, and oxidative stability in laying hens

This study aimed to examine the effect of dietary supplementation of dried wild leek (Allium scorodoprasum L. subsp. rotundum) leaves on laying performance, egg traits, antioxidant status, and oxidative stability in laying hens. For this purpose, a total of 96 Lohmann White laying hens aged 22 weeks allocated into four treatment groups each containing 24 hens. Hens were fed a diet supplemented with 0 (control), 1 (WL1), 2 (WL2), and 3 (WL3) g/kg dried wild leek (DWL) leaves. During the 10-week trial, egg weight was increased and feed efficiency was improved with 2 and 3 g/kg DWL leaves. No significant differences were observed among groups for egg production, feed intake, internal and external egg quality characteristics, and egg yolk cholesterol concentration. Levels of malondialdehyde and total antioxidant-oxidant status of egg yolk were not affected from DWL supplementation. However, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity was increased and oxidative stress index was decreased in egg yolk. Superoxide dismutase enzyme activity was increased in the group of WL3, and total antioxidant status levels were increased in the groups of WL2 and WL3 in serum. DWL supplementation reduced serum cholesterol concentration significantly. No marked changes were observed in other blood parameters. In conclusion, DWL is considered to be high antioxidant supplement due to having high antioxidant capacity and important bioactive compounds. Dietary supplementation of DWL leaves at 3 g/kg could be a viable and beneficial feed additive to improve egg weight and feed efficiency, increase DPPH radical scavenging activity in egg yolk and antioxidant status of hen. Therefore, the usage of DWL leaves in the laying hen diets will be beneficial for egg producers and poultry nutritionists to produce functional eggs having low cholesterol and high antioxidants.

Effect of Modified-Hen-Egg-Yolk-Lipid-Composition Diet on Plasma Lipids in Rats

Abstract This study aimed to investigate the effects of dietary supplementation with modified-hen-egg-yolk on plasma lipids and lipoprotein profiles in rats. During the four-week-experiment, 64 Wistar rats were divided into four groups of 16 (eight of both sexes), and fed commercial rat food (group C); food containing 70% commercial rat mixture and 30% freshly cooked egg yolk originating from laying hen eggs fed with 3% fish oil (group F); 3% palm olein (group P), or 3% lard (group L). The cooked egg yolk in the rat diet affected the concentrations of plasma total and LDL-cholesterol in males of the P and L groups. Cholesterol and total fat in the diet did not have a hypercholesterolemic effect on their own, but when in combination with fatty acid composition, they could contribute to an increase in plasma total and LDL cholesterol concentrations in rats. HDL-cholesterol was the most resilient plasma lipoprotein of rats to dietary treatments in our experiment. Compared to the control group, the addition of hen egg yolk to the rat diet regardless of its quality, adversely affected the values of HDL-C/TC and HDL-C/LDL-C in both males and females.

Low-density lipoprotein supplementation improves the quality of Holstein bulls' insemination doses

Despite the routine use of cryopreservation in dairy cattle, the subsequent deterioration in the quality of bull insemination doses (IDs) is an incentive to refine cryopreservation protocols, including the composition of the ID. The aim of this study was to examine, using a flow cytometry assay, whether the selected concentrations of low density lipoproteins (LDLs) extracted from hen-egg yolk may improve the post-thaw quality of the ID diluted with a plant-based extender. In total, 30 ejaculates from five sires (six trials per animal) were collected and processed at the Artificial Insemination Centre. The effect of the low density lipoprotein supplementation to the AndroMed® diluent at 4 and 8% (v/v) on the cryopreservation efficiency was tested. The effects of the modified extenders were determined on different sperm subpopulations by simultaneous quadruple staining for flow cytometry as the percentage of sperm with intact plasma membrane and acrosome (PMAI), PMAI sperm showing high mitochondrial membrane potential (HMMP), sperm with plasma membrane damage (PMD), and sperm with acrosome damage (AD). It was observed that the 8% LDL treatment was more effective (P < 0.05) in preserving all the analysed parameters than AndroMed® without any supplementation. The supplementation of the AndroMed® extender with 8% LDL resulted in the most optimal values of the PMAI (30.61 ± 1.13), HMMP (68.81 ± 1.25), PMD (68.69 ± 1.14), and AD (38.36 ± 1.13) compared to the control treatment (24.86 ± 1.13 for PMAI, 47.79 ± 1.25 for HMMP, 74.77 ± 1.14 for PMD, and 42.83 ± 1.13 for AD. The results of the study also demonstrated a synergistic positive effect of the LDL and soybean lecithin-based diluent on the spermatozoa post-thaw quality and resilience, based on 2 h long incubation. In conclusion, the soybean based semen extender treatment with LDL represents a beneficial tool to mitigate the detrimental effect of cryopreservation.

Mechanism of Molecular Activity of Yolkin—a Polypeptide Complex Derived from Hen Egg Yolk—in PC12 Cells and Immortalized Hippocampal Precursor Cells H19-7

Food-derived bioactive peptides able to regulate neuronal function have been intensively searched and studied for their potential therapeutic application. Our previous study showed that a polypeptide complex yolkin, isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), improved memory and cognitive functions in rats. However, the mechanism activated by the yolkin is not explained. The goal of the present study was to examine what molecular mechanism regulating brain-derived neurotrophic factor (BDNF) expression is activated by the yolkin complex, using in vitro models of PC12 cell line and fetal rat hippocampal cell line H19-7. It was shown that yolkin increased the proliferative activity of rat hippocampal precursor cells H19-7 cells and upregulated the expression/production of BDNF in a cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-dependent manner. Additionally the upregulation of carboxypeptidase E/neurotrophic factor–α1 (CPE/(NF-α1) expression was shown. It was also determined that upregulation of CREB phosphorylation by yolkin is dependent on cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) signaling pathway activation. Moreover, the impact of yolkin on the level of intracellular Ca2+, nitric oxide, and activation of extracellular signal-regulated kinases 1/2 (ERK 1/2 kinase) was excluded. These results emphasize that yolkin can act comprehensively and in many directions and may participate in the regulation of neurons’ survival and activity. Therefore, it seems that the yolkin specimen can be used in the future as a safe, bioavailable, natural nutraceutical helping to improve the cognition of older people.

Comparison of Anti-rabies Virus Nucleoprotein IgY Prepared by DNA Immunization and Protein Immunization.

Immunization of egg-laying hens with viral antigens efficiently produces large amounts of virus-specific IgY antibodies from egg yolks. A supply of practical and economical antibodies against the rabies virus is being desired worldwide. We immunized hens with the antigen gene DNA of the rabies virus, purified specific IgY antibodies from the egg yolk, and characterized the immuno-protein chemistry for use as a diagnosis. To prepare specific IgY antibodies against rabies virus nucleoprotein (RV-N) by DNA immunization, laying hens were pre-injected with λ-carrageenan or Freund's complete adjuvant to increase local immune activity (pre-immune stimulation), and then immunized with RV-N recombinant plasmid DNA. RV-N-specific IgY antibodies were prepared from egg yolks of immunized hens. For comparison, conventional protein antigen immunization was also used to induce the production of RV-N-specific IgY antibodies. Laying hens were immunized with an RV-N protein antigen and RV-N-specific IgY was purified from egg yolks. The binding activity against RV-N antigens was examined using IgY samples prepared by DNA (with pre-immune stimulation) and protein immunization. Immunohistochemical staining showed that IgY antibodies prepared by protein immunization strongly detected viral antigens in the brain sections of dogs infected with the virus, whereas IgY antibodies prepared by DNA immunization did not. Enzyme-linked immunosorbent assay was performed using a commercially available rabies vaccine (inactivated virus) treated with 10% formalin and heating (60°C, 30 min and 90°C, 5 min). IgY prepared by DNA immunization had weaker reactivity with denatured antigens and lower antigen concentrations than IgY prepared by protein immunization. These results suggest that it is necessary to develop a DNA immunization method for inducing IgY antibodies against the rabies virus that strongly bind to native and denatured antigens to prepare specific IgYs that can be used for antigen detection in clinical tests.