Investigation of Two Precipitation Methods for Extracting Immunoglobulin Y(IgY)from Egg Yolks

Two groups of hens(control and immunization group)were arranged in an experimental design with an immunization schedule of 3 injections of BSA antigen.IgY antibodies were extracted from egg yolks by two precipitation processes(chloroform and polyethylene glycol precipitates)and quantified using a standard curve of protein concentration.The purification of IgY was confirmed by SDS-PAGE.Total protein extracted from egg yoks were less contaminated with yellow pigments(lutein and zeaxanthin)nd by using chloroform precipitate.The 2 week post-immunization,IgY-1 concentration increased respectively to 3903±726μg.ml(chloroform-1 extraction process)and 2937±294μg.ml(PEG extraction process)(P<rd 0.01).After 3 immunization,IgY level obtaining from in immunization group extracted by chloroform process(6633±1166μg.ml-1)increased 2.7 times higher than that in control group(2482±414μg.ml-1).Whereas IgY concentrations obtained from PEG extraction process were not significantly different between the experimental group and control group.Chloroform and PEG precipitation methods had the same protein profile on the SDS-PAGE.IgY antibody was identified by the presence of bands corresponding with IgY heavy chain(67-70 kDa)and IgY light chain(25 kDa)for both precipitation processes.

Yolk free egg substitute improves the serum phospholipid profile of mice with metabolic syndrome based on lipidomic analysis

In this study,the impacts of egg consumption on mice model of metabolic syndrome(Met S)were comparatively investigated.Mice were divided into five groups(n=8):normal diet group(ND),high-fat diet group(HFD),HFD with whole egg group(WE),HFD with free-yolk egg substitute group(YFES),and HFD with lovastatin group(Lov).Main biochemical indexes and a non-targeted lipidomic analysis were employed to insight the lipid profile changes in serum.It was revealed that WE could significantly improve serum biochemical indexes by reducing body weight,low-density lipoprotein cholesterol(LDL-C)and total cholesterol(TC),while increasing high-density lipoprotein cholesterol.YFES exhibited remarkably better performance in increasing phosphatidylglycerol and phosphatidic acids,while decreasing phosphatidylinositol than WE.A total of 50 differential lipids biomarkers tightly related to glycerophospholipids metabolism were screened out.Carnitine C18:2 and C12:1,SM(d18:0/12:0),and SM(d18:1/14:1)were significantly upregulated in YFES compared to WE.YFES reduced expression of SREBP-1c and Cpt1a,while did not affect the expression of PPAR-α.Sphingomyelin biomarkers were positively related to the TC(|r|>0.6),while PPAR-αwas negatively correlated with triglyceride and LDL-C levels.To sum up,YFES attenuated HFD-induced Met S by improving the serum phospholipids,which account for its modulation of glycerophospholipid metabolism.

Comprehensive lipidomics and volatile compounds profiling reveals correlation of lipids and flavors in DHA-enriched egg yolk

DHA-enriched eggs have gained popularity due to their unique nutritional value,but their flavor can be challenging for some consumers to accept.The study analyzed the correlation of lipids and flavors in DHA-enriched egg yolks using comprehensive lipidomics and volatile compound analysis.The results showed that 411 lipids were detected in two egg yolk samples.Among them,148 lipid species,including 48 DHA-containing lipids,were significantly higher in DHA-enriched egg yolks than in Common ones(P<0.05).Furthermore,of the 24 volatile compounds detected,the contents of benzaldehyde,heptanal,hexanal,decanal and 2-nonanone in DHA-enriched egg yolks were significantly higher than in Common egg yolks(P<0.05).The“fishy”smell characteristic of DHAenriched egg yolks was mainly caused by volatile aldehydes,which may be produced through the hydrolysis of lipids in the egg yolk to free fatty acids and further oxidation.Analysis of the correlation network diagram revealed that phospholipids containing docosahexaenoic acid(DHA),linoleic acid,or oleic acid chains were the main contributors to the characteristic flavor of DHA-enriched egg yolks.Overall,this study explored the effect of different lipids on the flavor of DHA-enriched egg yolks and provided a theoretical basis for the production and improvement of DHA-enriched eggs.

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.

Chicken egg yolk antibodies (IgY) as non-antibiotic production enhancers for use in swine production： a review

In recent years, the use of in-feed antibiotics for growth and disease prevention in livestock production has been under severe scrutiny. The use and misuse of in-feed antibiotics has led to problems with drug residues in animal products and increased bacterial resistance. Chicken egg yolk antibodies （IgY） have attracted considerable attention as an alternative to antibiotics to maintain swine health and performance. Oral administration of IgY possesses many advantages over mammalian IgG such as cost-effectiveness, convenience and high yield. This review presents an overview of the potential to use IgY immunotherapy for the prevention and treatment of swine diarrhea diseases and speculates on the future of IgY technology. Included are a review of the potential applications of IgY in the control of enteric infections of either bacterial or viral origin such as enterotoxigenic Escherichia coil, Salmonella spp., rotavirus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus. Some potential obstacles to the adoption of IgY technology are also discussed.

The role of low-density lipoprotein （LDL） and high-density lipoprotein （HDL） in comparison with whole egg yolk for sperm cryopreservation in rhesus monkeys

Low-density lipoprotein （LDL） extracted from hen egg yolk has recently been considered to be superior to whole egg yolk in sperm cryopreservation of various animal species. Meanwhile, there was a notion that high-density lipoprotein （HDL） in egg yolk may have a negative effect on post-thaw survival. The role of LDL and HDL in sperm cryopreservation of rhesus monkeys has not been explored. The present study evaluates their effect in comparison with egg yolk with or without the addition of permeable cryoprotectant （glycerol） on sperm cryopreservation of rhesus macaques. In addition, various additives intended to change the lipid composition of LDL-sperm membrane complex have also been tested for their effectiveness in preserving post-thaw viability. Our findings indicated that LDL is the main component in egg yolk that is responsible for its protective role for sperm cryopreservation in rhesus monkeys. Regardless of the presence or absence of glycerol, the protective role of LDL is similar to that of egg yolk and we did not observe any superiority in post-thaw survival with LDL when compared to egg yolk. Modifying the lipid composition of LDL-sperm membrane complex with the addition of cholesterol, cholesterol loaded cyclodextrin and phosphatidylcholine also did not yield any improvements in pest-thaw survival; while addition of methyl-β-cyclodextrin reduced post-thaw motility. HDL plays a neutral role in sperm cryopreservation of rhesus monkeys. The present study suggests that egg yolk may still hold advantages when compared with LDL as effective components in extenders for sperm cryopreservation in rhesus monkeys.

The Effect of Different Levels of Sesame Oil on Productive Performance, Egg Yolk and Blood Serum Lipid Profile in Laying Hens

Addition of sesame oil into layer diets has been proved to enrich the proportion of polyunsatu-rated fatty acids in animal’s products. In this study, the effects of different levels of sesame oil in the diets on the performance, egg yolk and blood serum lipid profile of Isa Brown laying hens were investigated. A total of 96 layers were assigned into 4 groups to receive either 1 of 4 different diets contained 0.0%, 1.5%, 3.0% and 4.5% sesame oil, respectively. Sample of 12 eggs obtained from each groups were assessed for egg quality. The egg yolk fatty acid profile was determined with gas chromatography. Results revealed that the higher levels of sesame oil in the diet decreased egg production, egg weight, and egg yolk color except feed conversion ratio. In addition, supplementation of sesame oil increased the flow index of the eggs and the Haugh unite. The egg yolk lipid profile was not significantly different in the sesame oil fed groups, whereas, compare to control, it decreased the level of cholesterol. The blood serum lipid profile decreased in the sesame oil groups compare to control group. Meanwhile, monoacildigliserol also decreased in the sesame addition groups. In parallel with increasing levels of sesame oil, monounsaturated fatty acid (oleic acid) in the egg yolks significantly increased compared to the control (37.00%, 42.89%, 42.20% and 43.48%, respectively). It can be implied that sesame oil supplementation into the laying hens diet is necessary to produce monounsaturated fatty acid (MUFA) enriched eggs.

HPLC Analysis of Egg Yolk Phosphatidylcholine by Evaporative Light Scattering Detector

Egg yolk phosphatidylcholine(EYPC) is being widely used in food and pharmaceutical industries nowadays owing to its surface activity,pharmaceutical usefulness,and so on.Common determination methods of phospholipids were based on the American Oil Chemists' Society(AOCS) Official Method Ja7b-91,in which n-hexane/2-propanol/acetate buffer was used as the mobile phase.In order to achieve desired results,gradient elu-tion or buffer solution was used,which made the detection process more complicated.Moreover,water or buffer solution could affect the silica gel column both on its lifespan and the separation efficiency significantly.In this study,different mobile phase and detector were used to simplify EYPC analyzing process instead of using water within the mobile phase.The optimized HPLC operating conditions are as follows:pure methanol as a mobile phase,flow rate of 1.0 ml·min-1,silica gel column(250 mm×4.6 mm,5 μm,Inertsil GLTM),column temperature 30 ℃ and low temperature evaporative light scattering detector(40 ℃,0.35 MPa) as used.Under this optimal condition,the linear relative coefficient of the standard curve is 0.998 and the recovery was in the range of 96.83%-101.58% with a relative standard deviation of 1.79%(n=6).

Influence of Dietary Isoflavonoids on Stability of Egg Yolk Lipids

An experiment was conducted to investigate the influence of dietary tea polyphenol (TP) and daidzein (DA) on lipid stability of egg yolk. Two-hundred and seventy 27-week-old Hisex Brown laying hens were divided into nine groups, fed a diet supplemented with 0, 5, 10, 20, 40 mg kg-1 TP and 5, 10, 20, 40 mg kg-1 DA respectively for a period of 8 weeks ad libitum. The dietary addition of TP and DA both could reduce egg yolk LPO content significantly at the end of 4-week experiment (P < 0.05) and keep egg yolk lipid stable at the supplemental dose of 10 mg kg-1 diet respectively. A significant liner reduction in egg yolk LPO content was found with the increasing of dietary TP and DA. The egg yolk LPO content was decreased by 23.1 and 27.0% (P< 0.05) with the supplementation of TP and DA respectively at 40 mg kg-1 diet. The LPO contents in liver, plasma and egg yolk were decreased significantly with the dietary addition of TP or DA respectively (P < 0.05). There was no significant difference between the effects of TP and DA on improvement of layers' antioxidative capacity. The dietary TP and DA both could improve the bird's antioxidative level directly irrelevant to SOD and GSH-Px activities.

The Effect of Feeding Laying Hen Conventional and Omega-3 Enriched Diet on Fatty Acid Profiles in Egg Yolk Lipids

The aim of this research was to enrich table eggs with n-3 polyunsaturated fatty acids （n-3 PUFA）, especially with essential eicosapentaenoic acid （EPA） and docosahexaenoic acid （DHA）. After five weeks of feeding of laying hens with standard and experimental diets, there was analysis carried out to determine the fatty acid profile in egg yolks of both groups. Omega-3 eggs contain statistically significantly （P 〈 0.05） less sum of saturated fatty acids （∑SFA） and more∑n-3 PUFA, as well as narrower ratio of∑n-6/∑n-3 PUFA than conventional eggs. If referring to individual fatty acids of omega-3 eggs, there were increased portions （P 〈 0.05） of α-linolenic acid （α-LNA）, docosapentaenoic acid （DPA）, EPA and DHA determined in this research, which was in line with the research objective. Within the presented research, concentration of n-3 PUFA in egg yolk lipids of experimental group was two times higher than that in the control group. The ratio of ∑n-6 PUFA/∑n-3 PUFA was reduced from 12.04 to 6.17. Within their enzymatic system, laying hens can act as a bioreactor to naturally enrich their eggs with n-3 PUFA, especially with EPA and DHA. The results of this research proved that the fatty acid profile can be modified by designing of laying hens＇ diets with specific forages. Eggs were enriched with favorable fatty acids in the following order： α-LNA 〉 DHA 〉 DPA 〉 EPA.

Relations between the Elemental Composition Hens＇ Eggs and Feeds

Hens＇ eggs and corresponding feeds were sampled at commercial farms. The eggs were separated into egg yolk and egg white and freeze dried. After pressure digestion, total element contents of Ca, P, Mg, S, Na, Fe, Mn, Zn, Sr, Cu and Ba were detected by ICP-OES in the eggs, whereas, Pb, Ni, Sb, Sn, AI, Cd, Cr, Mo and V were all or largely below the detection limits. In addition, iodine was done by ICP-MS and Se after ashing with Mg-nitrate by hydride-generation-AAS. There were significant accumulations of P, S, Se and I in the egg yolk versus feed levels, as well as S and Na in the egg white. Just a few correlations between concentrations in feed and egg white resp. egg yolk were detected, indicating regulation mechanisms by homeostasis. Strontium showed the strongest trends between feeds and egg white and egg yolk, whereas, there was none for calcium. Among the main elements in the feeds, sulfur had the strongest effects on the egg compositions, e.g., on iodine and iron in the yolk and on sodium in the white, some of them might be non-linear.

A rapid and convenient dot-immunobinding assay for chicken egg-yolk antibodies

The dot-immunobinding assay was applied to investigate the characteristics of chicken egg yolk antibodies. This method of assay was proved to be a rapid and simple method to demonstrate and characterize the egg-yolk antibody IgY in comparison with the traditional ELISA assay. By using the BandScan software, the gray scale value of dots and the background could be determined. According to the intensity of dots (gray scale value) compared to the standard sample of 10μg, how much IgY remained can be determined in a shorter time.

Application of high-titred IgY antibodies in orthopox virus diagnostics

Layer chickens were immunized with three species of inactivated orthopox virus （vaccinia virus, calpox virus and cowpox virus）. Antibodies （IgY） were purified from egg yolks by improved polyethylene glycol precipitation. The development of IgY directed against orthopox virus antigens was followed by immunofluorescence assay, plaque reduction neutraliztion test and immunoelectron microscopy. Cross-reactivity of two IgY antibodies with cells infected by the different strains of the pox viruses was also investigated by different methods （immunofluorescence assay, plaque reduction neutraliztion test and Western blot）. Even in very high dilutions in immunofluorescence assay （titres up to 1：10^6 and 1：10^5, respectively） and persisted on a plateau over 10 months after four booster injections, it was showed that anti-vaccinia virus IgY and anti-calpox virus IgY were positive. Neutralizing activity and ultra-structural detection of antigen with gold-labelled antibodies were respectively observed in plaque reduction neutralization test and immunoelectron microscopy. Western blot analysis revealed specific binding of IgY to virus proteins. Thus, there was cross-reactivity between different orthopox viruses. Finally, orthopox virns-specific IgY antibodies bounded magnetic beads （Dynabead） were used to concentration of orthopox viruses. This study suggests that anti-pox virus IgY could serve as a useful tool for orthopox viruses diagnosis.

Alternatives to antibiotics as growth promoters for use in swine production:a review

In the past two decades, an intensive amount of research has been focused on the development of alternatives to antibiotics to maintain swine health and performance. The most widely researched alternatives include probiotics, prebiotics, acidiflers, plant extracts and neutraceuticals such as copper and zinc. Since these additives have been more than adequately covered in previous reviews, the focus of this review will be on less traditional alternatives. The potential of antimicrobial peptides, clay minerals, egg yolk antibodies, essential oils, eucalyptus oil-medium chain fatty acids, rare earth elements and recombinant enzymes are discussed. Based on a thorough review of the literature, it is evident that a long and growing list of compounds exist which have been tested for their ability to replace antibiotics as feed additives in diets fed to swine. Unfortunately, the vast majority of these compounds produce inconsistent results and rarely equal antibiotics in their effectiveness. Therefore, it would appear that research is still needed in this area and that the perfect alternative to antibiotics does not yet exist.

IgY technology: Methods for developing and evaluating avian immunoglobulins for the in vitro detection of biomolecules

The term“IgY technology”was introduced in the literature in the mid 1990s to describe a procedure involving immunization of avian species,mainly laying hens and consequent isolation of the polyclonal IgYs from the“immune”egg yolk(thus avoiding bleeding and animal stress).IgYs have been applied to various fields of medicine and biotechnology.The present article will deal with specific aspects of IgY technology,focusing on the currently reported methods for developing,isolating,evaluating and storing polyclonal IgYs.Other topics such as current information on isolation protocols or evaluation of IgYs from different avian species are also discussed.Specific advantages of IgY technology(e.g.,novel antibody specificities that may emerge via the avian immune system)will also be discussed.Recent in vitro applications of polyclonal egg yolk-derived IgYs to the field of disease diagnosis in human and veterinary medicine through in vitro immunodetection of target biomolecules will be presented.Moreover,ethical aspects associated with animal well-being as well as new promising approaches that are relevant to the original IgY technology(e.g.,development of monoclonal IgYs and IgY-like antibodies through the phage display technique or in transgenic chickens)and future prospects in the area will also be mentioned.

Olive Cake in Laying Hen Diets for Modification of Yolk Lipids

The possibility of improving egg yolk lipids of laying hens by olive cake （OC） feeding was investigated. Forty-two, 54-week-old, Lohman laying hens were fed for 12 weeks on 3 diets formulated to contain 0, 28.5 or 57 g OC/kg. Plasma cholesterol, triglycerides and high-density lipoproteins （HDL） were determined. Also total lipids, triglycerides, cholesterol, low-density lipoproteins （LDL）, Phospholipids and fatty acids were determined in egg yolk. Olive cake feeding at 28.5 or 57 g/kg diet decreased cholesterol and triglycerides of plasma, without affecting HDL. It also decreased total lipids, triglycerides, cholesterol, LDL and Phospholipids of yolk （P 〈 0.001）. Total （monounsaturated fatty acids, MUFA） was decreased with OC feeding at 28.5 g/kg diet, while it was increased with the 57 g/kg dietary OC. The two levels of OC decreased total （saturated fatty acids, SFA） but increased total （polyunsaturated fatty acids, PUFA）, n-6 PUFA, and n-3 PUFA of egg yolk, compared to the control. Thus, according to the results of the present experiment feeding hens 57 g OC/kg diet lowers cholesterol and total SFA and increases total MUFA, total PUFA, total n-3 PUFA and docosahexanoic acid in egg yolk.

Preparation and Determination of Immunological Activities of Anti-HBV Egg Yolk Extraction

To prepare an effective immune preparation to treat hepatitis B, hens were immunized with hepatitis B vaccines, and then anti-HBV egg yolk extraction （anti-HBV EYE） was refined from egg yolk by a dialyzable method. Its chemical characteristics were identified by ultraviolet spectrum, HPLC, Lowry analysis and pharmacopocia-raleted methods. The specific immunological activity was examined by leukocyte adherence inhibition （LAI） in vitro and delayed type hypersensitivity （DTH） in vivo. Anti-HBV EYE was a small dialyzable substance with molecular weight less than 12 kD containing 18 kinds of amino acids. The preparation could obviously inhibit LAI and DTH which was similar to hepatitis B virus-specific transfer factor of pig spleen. However, there were no similar effects observed in the nonspecific transfer factor （NTF） group, control egg yolk extraction （CEYE） group and hepatitis A virus （HAV） group. The results suggested that anti-HBV EYE contained hepatitis B virus-specific transfer factor （STF） and had the antigen-specific cell immune activity similar to PSHBV-TF. The STF obtained from egg yolk of the hens immunized with specific antigen, might be a potential candidate for immunoregulation in hepatitis B prevention and treatment.

Effects of drying methods on the structure and emulsifying capacity of egg yolk lecithin

The purpose of this study was to investigate the effects of different drying methods,including ultrasonic vacuum drying,vacuum drying,vacuum freeze-drying,hot-air drying and spray drying,on the structure and emulsifying capacity of egg yolk lecithin based on Raman spectra.The results showed that ultrasonic vacuum drying and spray drying can induce the vibration of C-N bonds in the polar O-C-C-N^(+)head skeleton of egg yolk lecithin.The shift of the peak attributed to the C-N bond from 717 cm^(−1)to 774 and 772 cm^(−1)indicated that the vibration of some C-N bonds in the O-C-C-N^(+)skeleton had transformed from gauche to trans.Ultrasonic vacuum drying exerted the most intense effect on the C-C skeleton of egg yolk lecithin,with the greatest vibration peaks at 1062 cm^(−1),1128 cm^(−1),and 1097 cm^(−1)in the Raman spectra of egg yolks.Specifically,it relieved gauche vibration and strengthened trans vibration in the C-C skeleton.Hence,the I_(gauche)/I_(trans)ratio of the egg yolk lecithin processed through ultrasonic vacuum drying decreased.Ultrasonic vacuum drying and spray drying decreased the I_(2850)/I_(2878)ratio of the vibration peak of C-H bonds in the lipid chains of egg yolk lecithin.The weakening of the symmetric stretching vibration of the C-H bond and the strengthening of antisymmetric stretching vibration indicated that orderliness among the molecular chains of lipid bilayer membranes had increased,whereas liquidity had decreased.The emulsifying capacities were highly significantly different among various egg yolk lecithin samples,in which the highest emulsifying capacity(49.58 m^(2)/g)was shown for the egg yolk lecithin prepared through vacuum freeze-drying,and ultrasonic vacuum drying produced the lowest emulsifying capacity(14.77 m^(2)/g).This study demonstrated that ultrasonic vacuum drying and spray drying drastically affected the structure of egg yolk lecithin.The appropriate drying method can be selected based on sample volume and production situation.

Sources,dynamics in vivo,and application of astaxanthin and lutein in laying hens:A review

Astaxanthin(Ax)and lutein are important fat-soluble pigments and essential nutrients for human and animal health.Haematococcus pluvialis microalga and Phaffia rhodozyma yeast are ideal species for commercial Ax production.Marigold flowers are a main source of commercial lutein.Dynamics of dietary Ax and lutein in the gastrointestinal tract are similar to lipids,but their activities are tremendously challenged by many physiological and dietary factors;few data are available about these in poultry.Dietary Ax and lutein have insignificant effects on egg production and egg physical properties,but have pronounced effects on yolk color,nutrition,and functionality.The two pigments can also enhance antioxidative capacity and immune function of laying hens.A few studies have shown that Ax and lutein can improve fertilization and hatchability of laying hens.Considering the pigmentation and health benefits of Ax and lutein from hen feed to human food,the commercial availability,chicken yolk improvement,and immune function of Ax and lutein are the focuses of this review.The potential roles of carotenoids in the cytokine storm and gut microbiota are also briefly presented.The bioavailability,metabolism,and deposition of Ax and lutein in laying hens are suggested for future research.

An Atomic Force Microscopy Study on Small Unilamellar Vesicle Structures on Mica

The vesicle structures of egg yolks phosphatidylcholine/didodecyldimethylammonium bromide （1 ： 1, mass ratio） deposited on mica were studied by atomic force microscopy （AFM） both in aqueous phase and air. In aqueous phase both bilayer and domelike vesicles with a mean diameter of 45 nm were observed, whereas in air the structure was more complicated depending on the initial concentration of vesicles. Vesicles with the original size could only be visualized at very low concentration with a mean diameter of 55 nm, which is a little larger than the result obtained in aqueous phase. At higher concentrations, fused large aggregates and multiple bilayer with a thickness ca. 4 nm of each bilayer were dominated. A plausible adsorption mechanism was proposed based on the experimental results.