In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

Predator-induced maternal effects determine adaptive antipredator behaviors via egg composition.

In high-risk environments with frequent predator encounters, efficient antipredator behavior is key to survival. Parental effects are a powerful mechanism to prepare offspring for coping with such environments, yet clear evidence for adaptive parental effects on offspring antipredator behaviors is missing. Rapid escape reflexes, or "C-start reflexes," are a key adaptation in fish and amphibians to escape predator strikes. We hypothesized that mothers living in high-risk environments might induce faster C-start reflexes in offspring by modifying egg composition. Here, we show that offspring of the cichlid fish Neolamprologus pulcher developed faster C-start reflexes and were more risk averse if their parents had been exposed to cues of their most dangerous natural predator during egg production. This effect was mediated by differences in egg composition. Eggs of predator-exposed mothers were heavier with higher net protein content, and the resulting offspring were heavier and had lower igf-1 gene expression than control offspring shortly after hatching. Thus, changes in egg composition can relay multiple putative pathways by which mothers can influence adaptive antipredator behaviors such as faster escape reflexes.

Deoxynivalenol and T-2 toxin cause liver damage and egg quality degradation through endoplasmic reticulum stress in summer laying hens.

The present study aimed to find whether low doses of mixed mycotoxins would affect egg quality in laying hens, and to explore the oxidative stress induced liver damage through endoplasmic reticulum during summer stress. A total of 96 Jinghong laying hens, 36 wks of age, were divided into four treatments, with eight repetitions per treatment and three hens per repetition. All the hens were raised in summer (average temperature: 31.3 ± 0.5℃; average humidity: 85.5 ± 0.2%) for 28d. One treatment was fed a basal diet as control (CON), and the other three treatments were fed the same diets containing 3.0 mg/kg deoxynivalenol (DON), 0.5 mg/kg T-2 toxin (T-2), and 1.5 mg/kg DON + 0.25 mg/kg T-2 toxin (Mix). Albumen height and Haugh unit were decreased (P < 0.05) in the Mix group on day 14 and 28. The activity of total antioxidant capacity, glutathione peroxidase, catalase, and superoxide dismutase were decreased (P < 0.05) in the DON, T-2, and Mix groups. The alkaline phosphatase level in DON, T-2, and Mix groups was significantly increased (P < 0.05). The level of interleukin-1ß, interferon-γ, and tumor necrosis factor-α in the Mix group were higher (P < 0.05) than CON, DON, and T-2 groups. Mix group upregulated the mRNA expressions of protein kinase RNA-like ER kinase, activating transcription factor4, IL-1ß, nuclear factor-κ-gene binding, and nuclear respiratory factor 2 in the liver (P < 0.05). The results showed that low doses of DON and T-2 toxin could cause oxidative stress in the liver, but DON and T-2 toxin have a cumulative effect on virulence, which can reduce egg quality and cause endoplasmic reticulum stress in the liver.

The Effects of Different Doses of Canthaxanthin in the Diet of Laying Hens on Egg Quality, Physical Characteristics, Metabolic Mechanism, and Offspring Health.

Currently, there is a dearth of in-depth analysis and research on the impact of canthaxanthin on the production performance, egg quality, physical characteristics, and offspring health of laying hens. Furthermore, the metabolic mechanism of cantharidin in the body remains unclear. Therefore, to solve the above issues in detail, our study was conducted with a control group (C group), a low-dose canthaxanthin group (L group), and a high-dose canthaxanthin group (H group), each fed for a period of 40 days. Production performance was monitored during the experiment, in which L and H groups showed a significant increase in ADFI. Eggs were collected for quality analysis, revealing no significant differences in qualities except for yolk color (YC). The YC of the C group almost did not change, ranging from 6.08 to 6.20; however, the trend in YC change in other groups showed an initial intense increase, followed by a decrease, and eventually reached dynamic equilibrium. By detecting the content of canthaxanthin in the yolk, the YC change trend was found to be correlated with canthaxanthin levels in the yolk. The content of unsaturated fatty acid increased slightly in L and H groups. Following the incubation period, the physical characteristics and blood biochemical indices of chicks were evaluated. It was observed that the shank color of chicks in the L and H groups was significantly higher than that in the C group at birth. However, by the 35th day, there were no significant differences in shank color among the three groups. Further investigation into the metabolic mechanism involving canthaxanthin revealed that the substance underwent incomplete metabolism upon entering the body, resulting in its accumulation as well as metabolic by-product accumulation in the yolk. In summary, this study highlighted the importance of understanding canthaxanthin's role in production performance, egg quality, and offspring health, providing valuable insights for breeders to optimize feeding strategies.

Changes of Potent Odorants in Salted Duck Egg Yolk before and after Roasting.

As the second most widely consumed eggs, duck eggs are made into preserved eggs, salted duck eggs, and roasted duck eggs to extend their shelf-life. To investigate the differences in potent odorants (POs) between salted duck egg yolk (SDEY) and roasted duck egg yolk (RDEY), the volatiles in SDEY and RDEY were extracted through solvent extraction coupled with solvent-assisted flavor evaporation and were assayed with gas chromatography-mass spectrometry-olfactometry. A total of 45 volatiles were identified in two samples, 24 odor-active compounds (OACs) were screened, and more OACs were in RDEY. The flavor-dilution (FD) factors of OACs were obtained by aroma extract dilution analysis and ranged from 3 to 6561. Twenty-two OACs with FD factors ≥ 9 were quantitated, and the results indicated the concentrations of OACs in yolk increased greatly after salted duck eggs were roasted. Based on the concentrations and thresholds, odor activity values (OAVs) were determined; 17 odorants with OAVs ≥ 1 were determined as POs. Acetoin was the most PO in SDEY; there were more POs in RDEY, including 2-ethyl-3,6-dimethylpyrazine, acetoin, 2-acetyl-3-methylthiophene, dihydro-4-hydroxy-2(3H)-furanone, etc. The outcomes obtained have reference values for making better use of duck eggs in the food industry.

A Cost-Effective and Sensitive Method for the Determination of Lincomycin in Foods of Animal Origin Using High-Performance Liquid Chromatography.

BACKGROUND: Lincomycin (LIN) is extensively used for treating diseases in livestock and promoting growth in food animal farming, and it is frequently found in both the environment and in food products. Currently, most of the methods for detecting lincomycin either lack sensitivity and precision or require the use of costly equipment such as mass spectrometers. RESULT: In this study, we developed a reliable high-performance liquid chromatography-ultraviolet detection (HPLC-UVD) method and used it to detect LIN residue in 11 types of matrices (pig liver and muscle; chicken kidney and liver; cow fat, liver and milk; goat muscle, liver and milk; and eggs) for the first time. The tissue homogenates and liquid samples were extracted via liquid-liquid extraction, and subsequently purified and enriched via sorbent and solid phase extraction (SPE). After nitrogen drying, the products were derivatized with p-toluene sulfonyl isocyanic acid (PTSI) (100 µL) for 30 min at room temperature. Finally, the derivatized products were analyzed by HPLC at 227 nm. Under the optimized conditions, the method displayed impressive performance and demonstrated its reliability and practicability, with a limit of detection (LOD) and quantification (LOQ) of LIN in each matrix of 25-40 µg/kg and 40-60 µg/kg, respectively. The recovery ranged from 71.11% to 98.30%. CONCLUSIONS: The results showed that this method had great selectivity, high sensitivity, satisfactory recovery and cost-effectiveness-fulfilling the criteria in drug residue and actual detection requirements-and proved to have broad applicability in the field of detecting LIN in animal-derived foods.

Modification of the Protein Amino Acid Content in Hen Eggs as a Consequence of Different Concentrations of Lupine and Soy in Feed.

The effect of the diet modification (soybean and lupine addition) on the content of protein and amino acids (AA) in eggs was studied. Both the sampling day and the diet influenced the total protein content. In albumen, the lowest protein content (10.6%) was noted after administering a diet containing 25% lupine; in the same egg the yolk contained the most proteins (16.7%). In the content of nonessential AA (NAA) in egg yolks, differences were noted only for cysteine, with its the highest content in the yolks of the control group. The stable content of essential yolk amino acids (EAA) was observed only for isoleucine, leucine, tryptophan and phenylalanine. The highest contents of EAA and NAA were recorded in the yolks of the control group (~47 and ~53 g/100 g of protein, respectively) and in the group with 25% additions of lupine (~42 and ~51 g/100 g of protein, respectively). AA with constant content in the tested albumens were methionine, tryptophan and alanine. The highest content of EAA (>~42 g/100 g of protein) and NAA (>~62 g/100 g of protein) were determined in albumen of eggs determined in the group with at least 20% additions of lupine. The highest content of EAA for humans delivered eggs from groups 4-6 (with the addition of soy into the diet ≤5%). The protein sources used in the hen diet significantly influenced the content of protein and individual AA in the produced eggs.

Determination of Plasmalogen Molecular Species in Hen Eggs.

(1) Background: Plasmalogens are vinyl ether-type glycerophospholipids that are characteristically distributed in neural tissues and are significantly reduced in the brains of individuals with dementia compared to those in healthy subjects, suggesting a link between plasmalogen deficiency and cognitive decline. Hen eggs are expected to be a potential source of dietary plasmalogens, but the details remain unclear. (2) Methods: We evaluated the fresh weight, dry weight, total lipid, neutral lipids, glycolipids, and phospholipids in the egg yolk and egg white of hen egg. Then, the molecular species of plasmalogens were quantified using HPLC-ESI-MS/MS. (3) Results: In egg yolk, the total plasmalogen content was 1292.1 µg/100 g fresh weight and predominantly ethanolamine plasmalogens (PE-Pls), specifically 18:0/22:6-PE-Pls, which made up 75.6 wt% of the total plasmalogen. In egg white, the plasmalogen content was 31.4 µg/100 g fresh weight and predominantly PE-Pls, specifically 18:0/20:4-PE-Pls, which made up 49.6 wt% of the total plasmalogen. (4) Conclusions: Plasmalogens were found to be more enriched in egg yolk than in egg white. It was found that humans are likely to ingest almost 0.3 mg of total plasmalogens from one hen egg. These findings highlight the importance of plasmalogens in the daily diet, and it is recommended to explore the impact of long-term dietary plasmalogen intake to assess its effect on human health. This provides a viewpoint for the development of new food products.

Moisture sorption isotherm and thermodynamic analysis of egg powders dried by foam-mat-assisted refractance window drying.

BACKGROUND: In the designed study, the moisture sorption isotherm and thermodynamic properties of refractance-window-dried egg white powder (EWP), egg yolk powder (EYP) and whole egg powder (WEP) were determined. RESULTS: The adsorption isotherm of egg powders was classified as type II, giving a sigmoidal shape. Peleg showed the highest goodness of fitting for egg powder. The monolayer moisture content of egg powders varied between 0.02 and 0.09 g g-1. According to the thermodynamic analysis, an increase in moisture content led to a decrease in net isosteric heat of sorption (qst), and the highest decrease was observed in EWP. The qst of EWP was calculated as 18 637 kJ mol-1, while it decreased to 3104 kJ mol-1 at 0.2 g g-1 moisture content. Like qst, sorption entropy decreased from 52.07 to 7.14 J mol-1 K-1 with increasing water activity (aw) for EWP. Increasing temperature caused a decrease in spreading pressure and was higher in EWP samples. Increasing moisture content and temperature also decreased Gibbs free energy level. CONCLUSION: Moisture sorption isotherm (MSI) is used to predict the amount of bound water in a material at a specific relative humidity and temperature. To inhibit chemical and microbiological spoilage, the drying process must continue until the monolayer moisture content is reached. In this study, the monolayer moisture content of powdered eggs for safe storage was demonstrated by MSI. Additionally, by determining the thermodynamic functions of egg powder, valuable information was obtained related to predicting the energy requirement in drying processes. © 2024 Society of Chemical Industry.

The impact of dietary oregano essential oil supplementation on fatty acid composition and lipid stability in eggs stored at room temperature.

*1. In many countries, eggs are not refrigerated and must be stored at room temperature. The objective of this study was to explore the effects of dietary oregano oil (275 mg/kg; ORE) versus an unsupplemented control diet (CON) on laying hens on the shelf life and fatty acid profile of eggs.2. Treatments were randomly distributed into 10 pens containing 27 birds each. A total of 200 eggs were collected from both groups on the same day and were stored for either 0, 10, 21 and 35 d. At each storage time, egg yolks were analysed for fatty acid profile and lipid peroxidation.3. The main indicator of lipid peroxidation, malondialdehyde (MDA), was significantly lower in ORE eggs compared to CON eggs (p = 0.001). Storage time had a significant impact on MDA concentrations (p = 0.023), with the highest found after 35 d. Significant differences were found for individual fatty acids, saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA). Palmitic acid, stearic acid, oleic acid, linoleic acid and arachidonic acid were significantly lower in ORE eggs compared to CON eggs (p < 0.05). Palmitoleic acid (p = 0.002), linolenic acid (p = 0.001) and docosahexaenoic acid (DHA, p = 0.001) were significantly higher in ORE eggs.4. Storage only affected oleic, linolenic, linoleic, arachidonic and docosahexaenoic acids (p < 0.05). Total SFA, MUFA, n-6 and ratio of n-3 to n-6 (n-3:n-6) PUFA were significantly higher in CON eggs (p < 0.05). The ratio of SFA to PUFA (SFA:PUFA, p = 0.005) and total n-3 PUFA (p = 0.001) were significantly higher in ORE eggs.5. The n-3:n-6 ratio was significantly impacted by treatment (p = 0.021) and storage (p = 0.031) with no significant interaction. This ratio is important for human health indication and could lead to the development of designer eggs.

Effects of dietary local hemp seed meal as soybean meal alternative on productive performance, egg quality and yolk fatty acid composition of laying hens.

Supply problems and rising soybean meal prices have an impact on increasing feed costs. Hemp seed meal (HSM) with high protein content has the potential to be used as an alternative to soybean meal. This study evaluated the impact of dietary HSM of Narli Sarayi variety as a substitute for soybean meal on productive performances, egg quality and yolk fatty acid composition. A total of 120 Lohmann Brown laying hens aged 50 weeks were allocated into 4 groups and 10 repetitions. Birds received treatment without HSM (control group), or soybean meal substituted with 4%, 8% and 12% HSM. Dietary 4% significantly increased (p < 0.05) egg production and decreased FCR compared with 8% and 12% HSM group but did not differ from the control group in an overall period of 6 weeks. The inclusion of the 12% HSM group significantly decreased (p < 0.05) egg production. Meanwhile, there was no influence of hemp seed meal (p > 0.05) on feed intake, egg weight, body weight change, egg shape index, albumen index, albumen weight, Haugh unit, yolk weight, yolk index and eggshell thickness. Dietary 8% and 12% HSM significantly increased (p < 0.05) eggshell weight and yolk colour compared with control and 4% HSM groups. There was a significant increase (p < 0.05) in omega-3 fatty acid concentration and a decrease in yolk omega-6 to omega-3 fatty acids ratio with an increase in dietary HSM. It was concluded that dietary up to 12% HSM of the Narli Sarayi variety decreased egg production and increased FCR. Increasing dietary levels of HSM increased eggshell weight, yolk colour and omega-3 fatty acids content and decreased the omega-6 to omega-3 fatty acids ratio.

Heavy metal contamination in eggs on poultry farms and ecological risk assessment around a gold mine area in northern Thailand.

The aim of this study was to analyze and compare the Hg, Pb, Cd, and Mn levels in egg feed, soil, and water among laying hens, laying ducks, and free-grazing duck farms in contaminated and uncontaminated areas. This study revealed that the Hg concentration in the eggs of free-grazing ducks was significantly greater than that in the eggs of laying hens and ducks in both contaminated and uncontaminated areas. However, the Pb and Mn levels in the eggs of laying ducks and free-grazing ducks were significantly greater than those in the eggs of laying hens in the contaminated area. Unfortunately, the Hg, Pb, Cd, and Mn concentrations in the feed, soil, and water from these three farms in both areas were not significantly different (P > 0.05). Hg and Cd were confirmed to be enriched in the egg albumin fraction, while Pb and Mn were found mainly in the egg yolk. However, egg consumption from free-grazing duck farms was the riskiest to Hg, Pb, and Mn contamination in the contaminated area. Additionally, the ecological risk factor (ER) in the soil revealed that all the farms were at considerable to high environmental risk for Cd except for Hg and Pb. Although the potential ecological risk index (RI) indicated a moderate risk for all farms in both contaminated and uncontaminated areas, these results were not consistent with our hypothesis. Therefore, the information gained in this study could be useful for setting up mitigation strategies and making decisions about public health concerns related to health hazards, especially for ecological risk assessments of heavy metal contamination.

Impact of different range areas on behavior, welfare, and performance of Naked Neck chickens.

This study aimed to evaluate the effect of different environmental enrichment tools on behavior, welfare, productive performance, and egg quality traits of Naked Neck chickens. A total of 1080 Naked Neck female chickens (30 weeks old) were used in the present study. A completely randomized experimental design, with 3 treatments having 4 replicates of 90 birds each, was applied. Treatments consisted of 3 different range areas A (121.9 m2), B (152.4 m2), and C (190.5 m2). Behavior (walking, feeding/foraging, sitting, aggressiveness wing flapping, perching, dust bathing, and range use), welfare (foot pad dermatitis, lameness, gait score, and feather pecking), productive performance (egg weight, egg production %, egg mass, and feed intake), egg quality traits (morphometry, egg weight, shell, albumen, and yolk characteristics) were evaluated. There was no influence (P > 0.05) of different range area on bird's behavior, and egg quality traits except walking and rang usage. The range area C promoted productive performance including egg weight, egg production %, egg mass and feed intake, but it did not influence welfare traits. It might be concluded that Naked Neck chicken when reared in free range with different range enrichments did not reveal any difference regarding behavioral parameters, egg morphometry, and egg quality traits. The range area within minimum length and effectively used environmental enrichments (perching and dust bathing area) should be used for backyard poultry to obtain ideal performance.

Hemoglobin polymorphism and its association with morphometric and egg production traits in Ethiopian indigenous and Sasso chicken breeds.

The present study investigated the biochemical polymorphism of hemoglobin (Hb) and its relationship with performance traits of Ethiopian indigenous and Sasso chicken breeds. A total of 284 chickens reared in three agro-ecologies were examined for genetic diversity and associations with productive traits at Hb locus using agarose gel electrophoresis. The results showed that the HbA allele was dominant in both breeds, and a higher proportion of male chickens were HbAA genotypes, while females were predominantly HbBB types. In the highland agro-ecology, chickens with the HbAA genotype were the most dominant, whereas in mid- and low-land agro-ecologies, chickens with HbBB and HbAB genotypes were found to be more frequent. A moderate level of expected heterozygosity was obtained with 0.47 and 0.445 for indigenous and Sasso chickens, respectively, with an average effective number of alleles per locus of 1.89 and 1.80. Moreover, chickens with HbAA genotypes showed significantly (p ≤ 0.05) higher body weight and linear body measurements than those of HbAB and HbBB genotypes. However, for appendage body structures (comb and wattle dimensions), chickens with the HbAB and HbBB genotypes had higher mean values. Additionally, clutch size (14.2 ± 0.4), clutch length (21.8 ± 0.7), and eight-month egg production (84.1 ± 1.2) were significantly (p ≤ 0.05) higher for hens with HbBB genotypes, followed by those with HbAB-types. Therefore, the considerable hemoglobin variability and significant associations of Hb variants with the performance traits can be sought as guiding information for further genetic improvement interventions in the chicken breeds under investigation. Further microsatellite marker-based genotyping is recommended to validate the higher morphometric values for HbAA genotypes and the better egg production for HbBB and HbAB genotypes.

Effects of substitution of crude palm oil with palm-pressed fibre oil on the laying hen's performance, egg production and egg quality traits.

This study aimed to evaluate the effects of substituting a crude palm oil (CPO) diet with palm-pressed fibre oil (PPFO) on laying hens' performance, egg production, carcass characteristic and egg quality. A total of 150 Hisex Brown laying hens were randomly assigned to five treatments: a basal diet containing 4% CPO (T1-control) and basal diets in which CPO was substituted by 25% (T2), 50% (T3), 75% (T4) or 100% (T5) PPFO. These diets were fed to laying hens ad libitum for 16 weeks. Compared to the T1 diet, dietary treatments T2, T3 and T4 had no significant effect (P > 0.05) on the feed intake, body weight gain, feed conversion ratio, egg number, egg production, egg weight and egg mass of the laying hens during the entire experimental period. Nonetheless, a significant (P < 0.05) reduction in feed intake, egg number, egg production and egg mass were observed in hens fed T5 compared to those fed the T1 (control) diet. Except for heart and spleen weights, other carcass characteristics were not affected by treatment (P < 0.05). Skin yellowness and breast meat redness increased significantly (linearly, P < 0.05) with increasing proportions of PPFO. Likewise, yolk colour scores increased in proportion to the dietary level of PPFO (linearly, P < 0.05). These findings suggest that PPFO can be used as a novel, cost-effective lipid source at a level of 3% in layer diets as a substitute for CPO to improve eggs' quality parameters without any harmful effects on laying performance.

Effect of Perilla seeds inclusion on the performance, egg quality characteristics, biochemical parameters and egg yolk fatty acid composition of laying hens.

This study investigates the effect of supplementation of Perilla seeds (PS) on the performance, egg quality, blood biochemical parameters, and egg yolk fatty acids composition in the diet of egg-laying chicken. A total of 1600 Lohmann laying hens were randomly assigned to four different groups with 4 replicates each (100 chickens/replicate) and were subjected to varying PS concentrations (PS0, PS6, PS12, and PS18; 0%, 6%, 12%, and 18%, respectively) for four weeks, including an acclimation period of one week. The results showed no significant differences among the groups for average egg weight (P > 0.005). The laying rate (%), feed conversion ratio (FCR) and average feed intake (AFI) decreased significantly for birds fed on 18% PS as compared to the other treatments (P < 0.005). Haugh unit, albumin height, egg-shape index and eggshell thickness among hens fed PS diets were greater averaging 80.53, 7.00, 1.29, 0.34 compared to 76.84, 6.86, 1.25 and 0.32 from Control hen eggs (P < 0.05). Serum analysis showed a trend towards elevated levels of glucose (Glu), total protein (TP) and aspartate aminotransferase (AST) among treatments. Total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) decreased for the birds fed on 6% PS. The fatty acid composition of egg yolk showed a substantial reduction for α-linolenic acid and docosahexaenoic acid increased significantly by the incorporating PS in the diet (P < 0.001). PS incorporation in diets resulted in significant improvements in both performance indicators and greater amounts of α-linolenic acid and DHA in egg yolks. These findings indicate that PS at 6% inclusion has the potential to improve fatty acid profiles of egg yolk without any adverse effect on performance of egg quality.

Transcriptome and histological analyses on the uterus of freckle egg laying hens.

BACKGROUND: In this study, we explored the characteristics and causes of freckle formation. We collected 15 normal and freckled eggs each for eggshell index testing and hypothesized that the structure and function of the uterus would have a direct effect on freckled egg production given that eggshells are formed in the uterus. To test this hypothesis, we collected uterine tissue from laying hens (418 days of age) that laid normal (Group C, n = 13) and freckled (Group T, n = 16) eggs for 7 consecutive days. RESULTS: When we examined the eggshell quality, we found that the L value was significantly lower (P < 0.05) in the freckled site group of freckled eggs compared to the normal egg group during the detection of blunt pole, equator, and sharp pole of the eggshell color. The a-values of the three positions were significantly higher (P < 0.05) in the freckled site group of freckled eggs, and the a-values of the blunt pole were significantly lower (P < 0.05) in the background site group of freckled eggs, compared to the normal egg group. The b-values were significantly higher (P < 0.05) at three locations in the freckled site group of freckled eggs compared to the normal egg group. During the detection of eggshell thickness, the blunt pole was significantly higher (P < 0.05) in the freckled egg site group of freckled eggs compared to the normal egg group, and there was no significant difference between the other groups (P > 0.05). There was no significant difference (P > 0.05) between the transverse and longitudinal diameters of the eggs in each group.We then performed histopathology and transcriptome analyses on the collected tissue. When compared with group C, uterine junctional epithelial cells in group T showed significant defects and cilia loss, and epithelial tissue was poorly intact. From transcriptomics, genes that met (|log2FC|) ≥ 1 and P < 0.05 criteria were screened as differentially expressed genes (DEGs). We identified a total of 136 DEGs, with 101 up- and 35 down-regulated genes from our RNA-seq data. DEGs identified by enrichment analyses, which were potentially associated with freckled egg production were: IFI6, CCL19, AvBD10, AvBD11, S100A12, POMC, and UCN3. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that pathways were associated with immunoreaction and stress stimulation, e.g., complement activation, interleukin-1 cell reactions, viral responses, cell reactions stimulated by corticotropin releasing hormone, steroid hormone mediated signaling pathways, staphylococcal infections, B cell receptor signaling pathways, and natural killer cell mediated cytotoxicity. CONCLUSIONS: From these data, freckled areas deepen freckled eggshell color, but background areas are not affected. At the same time,we reasoned that freckle eggs may result from abnormal immune responses and impaired uterine functions induced by stress. Therefore, the uterus of laying hens in a state of stress and abnormal immune function can cause the appearance of freckled eggs.

Development, validation, and use of a monitoring method for fipronil and its metabolites in chicken eggs by QuEChERS with online-SPE-LC-Q/Orbitrap analysis.

RATIONALE: The residues of fipronil and its metabolites in chicken eggs pose a threat to human health, so regular monitoring is necessary. However, the pretreatments of the existing detection methods are complex and time-consuming. A simple and streamlined pretreatment method is needed to improve the detection efficiency. METHOD: A rapid, efficient, and facile approach employing the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method with online solid-phase extraction liquid chromatography tandem Q Exactive Orbitrap high-resolution mass spectrometry (online-SPE-LC-HRMS) was established and evaluated for the determination of fipronil, fipronil-desulfinyl, fipronil-sulfone, and fipronil-sulfide in chicken eggs. An improved sample preparation technique combining QuEChERS and online-SPE was developed. Negative targeted ion fragmentation scanning and targeted-selected ion monitoring of HRMS were adopted to identify and quantify the target analytes. RESULTS: The proposed pretreatment method took a few steps in <13 min to achieve excellent recoveries and negligible interference. High selectivity was acquired with the adoption of Q/Orbitrap HRMS. The limit of quantification (LOQ) of the analytes was 2.5 µg kg-1 , meeting the detection requirements of the maximum residue level enacted by the Codex Alimentarius Commission, Japan, and the United States for the sum of fipronil and its metabolites. Extraction recoveries at three spiked concentration levels were within 84.56% to 93.84%, with relative standard deviation ≤5.87%. CONCLUSION: The established method is efficient and easy to operate and displays satisfactory LOQs, recoveries, accuracy, and precision. This approach serves as a reference method for monitoring eggs while providing potential solutions for fipronil determination in more complicated matrices.

Total mercury in different egg tissues provides insights to mercury metabolisms in bird bodies.

Mercury (Hg) pollution in birds has been widely reported, but the metabolism of Hg in bird bodies remains unclear. Measurement of Hg concentrations in bird tissues (muscles and organs) could provide insights into the metabolism of Hg in bird bodies, however, this approach is often invasive. To avoid invasive procedures, we conducted feeding experiments using chickens and used eggs as a proxy for understanding Hg metabolism in chicken bodies. For the control group, various THg concentrations were observed in egg whites, egg yolks, and eggshells, but the THg concentration trends for different egg tissues were not statistically different (P > 0.05). For the Hg feeding group (0.3 mg/kg body weight, feeding once), Hg peaks were observed in egg yolks and egg whites at different time periods, suggesting different response time to dietary Hg in chicken body tissues. Mercury in egg yolks peaked at Day 6, suggesting their quick response to dietary Hg. Egg whites reached Hg peak at Day 20, exhibiting a slower response to dietary Hg. Eggshells did not show a Hg peak, perhaps due to their predominant inorganic components that do not trigger Hg bioaccumulation. We measured THgyolk/THgwhite ratios in various chicken eggs purchased from three areas in Guizhou, SW China. The THgyolk/THgwhite ratios for Huaxi, Hezhang and Wuchuan were 1.33 (0.57-2.41), 7.89 (4.27-19.47) and 2.64 (1.68-4.22), respectively, to suggest different exposure history for chickens. This study provides new insights into the metabolism and lifetime of Hg in bird bodies.

Fluorescent probe of quantum dots and zinc oxide in a highly selective polymer simultaneously determined florfenicol and sparfloxacin.

A nanocomposite fluorescent probe was fabricated for the simultaneous determination of florfenicol and sparfloxacin based on fluorescence quenching. The probe was synthesized by integrating nitrogen-doped graphene quantum dots (N-GQDs), cadmium telluride quantum dots (CdTe QDs) and zinc oxide nanoparticles (ZnO) into a molecularly imprinted polymer (MIP). The determination was based on the quenching of fluorescence emissions from N-GQDs by florfenicol, detected at 410 nm, and the quenching of fluorescence emissions from CdTe QDs by sparfloxacin, detected at 550 nm. The fluorescent probe was highly sensitive and specific with good linear relationships for florfenicol and sparfloxacin in the range  0.10 to 100.0 µg L-1. The limits of detection for florfenicol and sparfloxacin were 0.06 and 0.10 µg L-1, respectively. The fluorescent probe was used to determine florfenicol and sparfloxacin in food samples and the results agreed well with the results of chromatographic determination. Recoveries of spiked milk, egg and chicken samples reached 93.3-103.4% with good precision (RSD < 6%). The advantages of the nano-optosensor include high sensitivity and selectivity, simplicity, rapidity, convenience, good accuracy and precision.