Effects of heat stress on serum proteomics, laying performance and egg quality in aged laying hens

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Forty-eight 75-week-old Lohmann White hens were divided into two groups and subjected to either a comfort environment (26°C) or heat stress (35°C) for 28 days. Serum samples were collected on days 1 and 28 for proteomic analysis, while egg production and quality parameters were assessed daily throughout the experiment. Heat stress reduced egg production (11%), egg weight (16%), shell strength (30%), shell thickness (13.5%), and albumen height (12%). Proteomic analysis revealed the expression of proteins that bind to the heat shock proteins Hsp70 and Hsc70, exclusively in heat-stressed laying hens. Proteins related to coagulation rate were upregulated and when compared with field observations may indicate possible Disseminated Intravascular Coagulation (DIC). Apolipoprotein A and apovitelenin-1 were associated with decreased egg production and quality under heat stress. animal welfare avian hematology blood coagulation heat shock protein Shotgun proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Poultry farming faces significant challenges due to the increasing frequency and intensity of extreme weather events, such as heat waves, which directly impact animal production systems [ 1 – 2 ]. Heat stress negatively affects poultry physiology, leading to increased mortality in laying hens, alterations in metabolic rate and immune function, hormonal, endocrine, digestive, and reproductive imbalances, reduced laying performance, decreased feed intake, and compromised egg quality [ 3 – 4 ]. To restore homeostasis, birds activate compensatory mechanisms, including decreased feed consumption [ 5 ], increased water consumption [ 6 ], and peripheral vasodilation, often accompanied by wing elevation and feather ruffling to increase surface area for heat dissipation [ 7 ]. Furthermore, heat stress results in elevated body temperature [ 8 ] and an increased respiratory rate. This elevated respiratory rate leads to reduced blood carbon dioxide levels and an increase in blood pH, a condition known as respiratory alkalosis [ 9 ]. When ambient temperature and relative humidity are excessively high, birds are unable to effectively dissipate body heat, potentially reaching a critical physiological threshold and resulting in mortality [ 10 – 11 ]. Heat stress can also induce an increase in hepatic fat synthesis [ 9 , 12 – 13 ]. The liver then synthesizes excess triglycerides, which require transport to other tissues via very low-density lipoproteins (VLDL). However, heat stress can limit the availability of Apolipoprotein B (ApoB), which is essential for VLDL assembly and secretion, thus reducing VLDL transport and leading to an accumulation of triglycerides and total cholesterol in the liver, resulting in hepatic steatosis [ 12 ]. In response to heat stress, organisms induce the expression of specific proteins that serve as a protective mechanism to maintain cellular function and integrity [ 14 – 15 ]. These proteins participate in diverse metabolic and cellular processes, including the biogenesis of cellular components and the response to various stress stimuli [ 14 ]. Heat shock protein 70 (HSP70) serves as a prominent indicator of the response to heat stress [ 16 ] because, unlike most proteins whose synthesis is delayed, heat shock proteins (HSPs) are rapidly synthesized [ 17 ]. Under stress conditions, HSP70 functions to prevent the formation and aggregation of abnormal proteins and to restore the binding capacity of the progesterone receptor [ 18 ]. Khondowe et al. [ 19 ] observed variations in hepatic HSP70 protein expression among different local chicken breeds in Tanzania subjected to heat stress. Oloruntola et al. [ 20 ] reported an increase in serum HSP70 expression in chickens experiencing heat stress, and this elevation could be attenuated through dietary supplementation with vitamin C and mistletoe leaf powder. Several studies have correlated various proteins with egg formation and quality [ 21 – 22 ]. While samples for these studies are often extracted from different organs and from the egg itself [ 23 – 24 ], research investigating the expression of these proteins specifically in the blood of chickens remains limited. The aim of this study was to evaluate the effects of long-term heat stress on the abundance and expression of serum proteins in aged laying hens and to correlate these changes with global DNA methylation patterns, hematological parameters, production rates, and overall egg quality. 2 Materials and Methods The experiment was conducted in accordance with the ethical guidelines for animal experimentation established by the Animal Ethics and Welfare Committee of the Faculty of Science and Engineering, Tupã Campus, São Paulo State University Júlio de Mesquita Filho, Unesp/FCE. The study was approved by the Animal Use Ethics Committee (CEUA) under registration number 02/2022. 2.1 Description of the hens and housing conditions The experiment was conducted with 48 laying hens of the Lohmann White strain with 75 weeks old and with an average weight of 1.59 ± 0.16 kg. The hens were obtained from a commercial farm in the city of Tupã, State of São Paulo, Brazil (S 21° 56’ 36”, W 50° 27’ 45”). The birds were divided and housed in two climate chambers, in wire cages, and groups of four birds per cage, respecting the same housing density as the farm. The lighting was reproduced as that used on the commercial farm, with the lights turned on at 4h30 am and turned off at 10h00 pm, totaling a photo period of 17.5 hours of light. The feed provided to the birds had the same formulation as that used on the farm, with 2,785 Cal/kg of energy and 17% protein. The feed was offered in troughs twice a day in a total amount of 120 g/bird per day, and water was provided ad libitum through nipple drinkers. 2.2 Experimental treatments During the first seven days, the birds in the two climate chambers were subjected to an adaptation period, with a continuous ambient temperature of 26°C and 60% relative humidity. After adaptation, one of the chambers remained under these environmental conditions and the other had the air temperature raised to 35°C and 60% relative humidity, full time (continuous stress – 24 hours), totaling two temperature treatments. The first condition was called the Comfort (C) treatment, and the second condition was called Heat Stress (HS). The birds were subjected to the treatments 24 hours a day for 28 consecutive days. 2.3 Weighing of hens The hens were weighed individually every seven days using a platform scale (B-160, Líder Co. Ltda., Araçatuba, São Paulo, Brazil). The variation in hens' weight was compared between the comfort and heat stress groups. 2.4 Egg production and quality Egg production was recorded daily for each temperature treatment, and the laying rate was calculated by dividing the number of eggs by the total number of hens housed. All eggs produced in the last three days of the experiment were evaluated for specific gravity, egg weight, shell strength, shell thickness, albumen height and Haugh unit using the Digital Egg Tester (DET-6000, Nabel Co. Ltd., Kyoto, Japan®). 2.5 Collection and preparation of blood samples Blood samples were collected twice: on the first day of treatment and the last day of the experiment. Blood samples were collected from all hens, preferably by radial venipuncture, using a 25×7 mm needle and a 3 mL syringe. It was placed 0,5 mL in a microtube containing EDTA K3 as an anticoagulant and 2.5 mL in a tube with a coagulation accelerator and separating gel. The tubes with the coagulation accelerator and separating gel were kept at 24°C for 120 minutes, protected from light, to obtain complete coagulation and clot retraction. The serum was transferred to Eppendorf® microtubes, followed by freezing at -20°C, for use in proteomic analyses. 2.6 Complete blood count, plasma proteins and blood glucose The EDTA blood samples were homogenized for hematocrit analysis, red blood cell and leukocyte count, and determination of total plasma proteins and hemoglobin (Almosny et al., 2015, n.d.). Two capillary tubes for microhematocrit were filled, sealed and centrifuged at 12,500 rpm for 5 minutes in a Microcentrifuge (Mod. 211, Fanem® Ltda., Guarulhos, Brazil). After centrifugation, one tube was read in specific table for reading microhematocrit, provided by the manufacturer, while total plasma proteins were determined by manual refractometry in Refractometer (RHC-200-ATC), using the plasma obtained from the second capillary tube. The red blood cell and leukocyte count was performed in a Neubauer chamber after dilution in Nat-Herrick solution, with correction of values by specific factors [ 25 – 26 ]. Hemoglobin was quantified by spectrophotometry at 540 nm in semi-automatic biochemical analyzer Bio-2000® (Bioplus® Produtos para Laboratórios Ltda., Barueri, Brazil), using the hemoglobin cyanide method (Labtest® kit Ref. 43), using the Labtest® kit Ref. 47 as the Hemoglobin Standard. The glucose concentration was determined by an electrochemical method using a glucometer (Accu-Check®, Guide Roche®), with fresh blood, at the same time as sample collection. The values of the blood parameters were compared with the reference ranges compiled by Oliveira Boreli et al. [ 27 ]. 2.7 Global DNA methylation To quantify total genomic DNA, blood from two birds per cage was used on the first (D1) and last (D28) day of treatment. DNA was extracted using the Monarch kit (Biolab®, New England). DNA quantification was performed in a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific® Inc.). Methylation analysis was performed in duplicate using the Global DNA Methylation ELISA kit (Cell Biolabs® Inc.). The pretreated strips contained wells with a specific reagent for binding to methylated DNA, methylation-sensitive capture antibodies, and detection antibodies, which allowed photometric detection of the absolute amount of DNA methylation in each sample. The absorbance of the solution in the wells was measured with an ELISA spectrophotometer (Kasuaki-DR-200Bs-BI) at 450 nm. Methylation data were compared by Analysis of Variance (ANOVA) between collection days and temperature treatments. 2.8 Composition of samples for proteomic analysis Four samples were prepared in pool format for the proteomic analyses. Each pool consisted of 50 µL of blood serum from each of the hens (24 birds), from each blood collection and environmental treatment. The samples from the birds subjected to the Comfort treatment were identified by C1 and C28, and from the Heat Stress treatment by HS1 and HS28, with “1” and “28” representing the first and last day of the experiment. 2.9 Proteomic Analysis 2.9.1 Protein Quantification Considering the abundance of proteins present in blood serum, the crude samples were diluted in 50 mM Ammonium Bicarbonate solution, in the proportion (1:50). Protein quantification was performed by the Bradford reaction [ 28 ], prepared in technical triplicate, using the BioRad® Protein Assay kit, with commercial bovine albumin as the standard protein (Ref. 500-0006, Biorad®). To construct the calibration curve, four dilutions of the standard protein (1, 2, 4 and 6 µg/µL) were prepared in technical triplicate. The absorbance of the samples and the calibration curve were read at 595 nm in µQuant Bio-Tek® microplate spectrophotometer. 2.9.2 One-dimensional electrophoresis Electrophoretic analysis of the experimental samples under reducing and denaturing conditions (12% [m/v] SDS-PAGE) was performed to observe the quality and viability of the samples in relation to degradation aspects and the presence of proteins. This procedure was performed according to [ 29 ], using molecular markers present in the Calibration Low Molecular Weight kit (Ref. 17-0446-01, Cytiva®). 2.9.3 In-Solution Protein Digestion and Mass Spectrometry Analysis The experimental samples were subjected to enzymatic digestion according to (Cavecci-Mendonça et al., 2023), using standard surfactant RapiGest SF (Ref. 186001861, Waters®), and the enzyme trypsin at a concentration of 1:50 (enzyme: sample). After hydrolysis was interrupted, the samples were desalted using Sep-Pak Cartridges Sample Extraxt Products 1cc C18 columns (Ref. WAT054955, Waters). Peptide quantification was performed by chemiluminescence in a Qubit 2.0 Fluorometer using the Qubit Protein Assay Kit (Ref Q33212, Invitrogen). Shotgun LC-MS/MS mass spectrometry analyses were performed using an Ultimate 3000 LC liquid nanochromatography equipment (Dionex, Germering, Germany) coupled to a Q-Exactive mass spectrometry equipment (Thermo Fisher Scientific®, Bremen, Germany) according to the methodology proposed by Cavalcante et al., (2022). The spectrometry data were acquired using Thermo Xcalibur software (version 4.0.27.19, ThermoFisher Scientific® Inc.). 2.9.4 Data Analysis The raw mass spectrometry data in “. RAW” format was evaluated by RawVegetable® software [version 1.1.0.1] and then submitted to PatternLab® software [version 4.0.0.84] to obtain protein identification [ 30 ]. The main parameters used in this tool were: UNIPROT database (Taxonomy Gallus gallus), trypsin enzyme; permission of 2 missed cleavages; post-translational modification carbamidomethylating of cysteine residues; variable post-translational modification oxidation of methionine residues; MS and MS/MS tolerance errors of 0.0200 ppm. The maximum FDR (False Discovery Rate) rate ≤ 1%. A matrix compatible with the MetaboAnalyst® 6.0 program ( https://new.metaboanalyst.ca/ ) was constructed from the proteomic data using the spectral counts of each identified protein, which were normalized for each protein by the weighted average of the technical triplicates of each sample [ 31 ]. Partial Least Squares (PLS) analyses were used as the main method of multivariate analysis. Only signals present in 75% of the samples were considered for the generation of statistical models. The t-test (p ≤ 0.05) and fold change ≥ 2 were used to compare the mean protein abundances between each experimental group. Protein interactions were also investigated concerning their biological processes using the STRING® software ( http://string-db.org/ - version 10.5), using the basic parameters: cut-off score of 0.90, confidence as network edges, and PPI p-value of < 1.0e-16 [ 32 ]. 3 Results 3.1 Egg production and quality Figure 1 shows the daily egg production of each group, subjected to Comfort or Heat Stress. Figure 1 shows that from the third day of heat treatment onwards, the group under heat stress showed a significant reduction in the laying rate. In the Comfort group, the laying rate remained above 90%. Considering the entire experimental period, the group under Heat Stress produced 11% fewer eggs than the group in Comfort. Table 1 shows the result of the Fisher test at 5% for the egg quality variables between the Comfort and Heat Stress groups. The group under Heat Stress showed a significant reduction in egg quality. Egg weight was 16% lower, shell resistance and shell thickness were 30% and 13.5% lower, and albumen height was 12% lower. There was no statistical difference in the Haugh unit. Different letters indicate significant difference by Fisher's test (p < 0.05). 3.2 Weight of hens Figure 2 shows the weekly weight assessment of hens subjected to heat stress treatment, compared with the weight of hens raised in Comfort. Figure 2 shows that hens kept under Heat Stress lost weight in the first week. The impact of the 28-day Heat Stress treatment period resulted in a weight reduction of 13%, i.e., a loss of 200g on average per bird. 3.3 Complete blood count, plasma proteins and blood glucose During blood sampling, it was found that the blood from heat-stressed chickens clotted easily, many of them while still in the syringe and during extraction. EDTA samples from several animals had to be collected again due to loss of samples due to clotting. Figure 3 shows the box-plot graphs of the hematological variables for the first (graphs on the left) and last day of housing of the birds (graphs on the right). On the first day of housing, the birds selected for the two groups had similar blood parameters. After 28 days of experimentation, it was found that the birds in both treatments had a reduction in hemoglobin to levels below the limit established by the literature. It was also found that the hematocrit of the group under heat stress had an average at the lower limit established by the literature. Individual variations in total plasma proteins were observed, although the group average remained equivalent. 3.4 Global DNA methylation Figure 4 shows the global DNA methylation results for the comfort and heat stress groups on the first and last day of the experiment. Although there is a small visual difference in the graph of global DNA methylation of the samples between the groups, after 28 days of temperature treatment, the difference was not statistically significant. 3.5 Analysis of Protein Concentrations in Proteomic Samples The protein concentrations observed in each sample were as follows: 22.38 µg/µL (C1), 26.04 µg/µL (C28), 26.46 µg/µL (HS1), and 13.72 µg/µL (HS28). The protein profiles of the blood serums, when subjected to electrophoresis tests, exhibited high protein variability, with molecular masses ranging between 10 and 150 kDa and no signs of protein degradation. The electrophoretic image can be observed in Appendix I. Regarding the proteins present in the blood serum of the experimental groups, a comparison with theoretical data from the UNIPROT database for Gallus gallus taxonomy revealed the following: · Group C1: 125 total proteins identified, 90 proteins under analysis in maximum parsimony · Group C28: 122 total identifications, 83 in maximum parsimony · Group HS1: 108 total proteins identified, 85 proteins under analysis in maximum parsimony · Group HS28: 110 total proteins identified, 88 proteins under analysis in maximum parsimony The proteomic parameters for each analysis are detailed in Appendix II. Figure 5 presents the Venn diagrams for the groups of samples analyzed. Figure 5a shows 116 common proteins identified between the beginning and end of the Comfort treatment. Six proteins are exclusive to C1 and nine to C28. Figure 5b shows 98 common proteins present at the beginning and end of heat stress, ten of which are exclusive to HS1 and twelve to HS28. Figure 5c shows the interaction between all groups. The yellow-colored area of the Venn Diagram shows the distribution of proteins identified after 28 days of constant heat stress. 3.5.1 Protein differentiation between samples Eight proteins were exclusive to HS28, and among them, it is believed that they may stand out as potential candidates for heat stress biomarkers (Table 2). Regarding the proteins that presented differential abundance between the experimental groups, Figure 6 shows the Volcano plots obtained in the MetaboAnalyst software. Between groups HS1 and C1 (start of treatments) there were no significant differences in protein abundance. For the samples from the comfort group (Figure 6a), three proteins were more abundant and five decreased significantly. When comparing the HS28 vs. HS1 samples from birds subjected to heat stress, eleven proteins with differential abundance were noted (Figure 6b), with seven being higher and four being downregulated in HS28. When the groups were compared at the end of the experiment (Figure 6c), a more discrete variation was observed. Of the eleven proteins with differential abundance in the heat stress group, five of them were confirmed as differentials in comparison with the comfort group (HS28 vs. C28), and one protein that had not expressed divergences, Apolipoprotein A, appeared in this comparison. Table 3 and Table 4 shows the proteins with differences in abundance in each of the comparisons made between the samples. The tables were composed using information collected from the UniProt and String databases, and present the protein code, protein name, metabolic functions, and metabolic pathway and interactions with other proteins. The gene codes, when available, were important for later identification of the biological processes in which these proteins are involved. Table 3 presents seven proteins that had their expression increased in the HS28 samples relative to HS1 and C28: Fetuin B, Gelsolin, Histidine-rich glycoprotein, Prothrombin, Apolipoprotein A, Sushi domain protein, and Serpin G member 1. Most of these proteins (with the exception of Apolipoprotein A and Sushi domain protein) play a role in blood clotting. Appendix III provides detailed information on the variation in the amount of each protein. The proteins in Table 4 were found in smaller quantities in the HS28 sample. Apovitellin-1, for example, is essential for nutrient storage. It acts together with VTG1, VTG2 and VTG3, which are parts of lipovitellin-1, a precursor of the main yolk proteins. These proteins are vital for providing nutrients during the early development of oviparous animals. The expression of the π subunit of Hemoglobin was also reduced and is linked to the breakdown of hydrogen peroxide and the protection of cells against oxidizing substances. This subunit interacts with the beta subunit of Hemoglobin (HBBA), which is responsible for carrying oxygen from the lungs to the other tissues. 4 Discussion Quantitative analysis of serum proteins revealed eight unique proteins in samples from hens under heat stress after 28 days. These included Hsp70 and Hsc70, known biomarkers for heat stress [ 20 , 34 ]. HSC70 interacts with tumorigenicity suppressor proteins (ST14 and DNND2B) and the heat shock-related protein HSPA8, protecting the proteome. The expression of these proteins confirms heat stress and suggests attempted adaptation. Apolipoprotein A (up-regulated) and Apovitelenin-1 (down-regulated), interconnected proteins and VLDL components in laying hens, showed altered expression. APOV1, a potent lipoprotein lipase inhibitor, prevents triglyceride loss from VLDL during transport to oocytes [ 35 ]. Its inhibition promotes triglyceride accumulation in the liver [ 12 ], and consequently, liver fat accumulation, reducing feed conversion efficiency [ 36 ]. The laying rate decreased in the first week of heat stress, possibly related to increased abdominal fat. Li et al. [ 37 ] found that after five days of heat stress (35°C-37°C), laying hens exhibited a reduced laying rate due to fewer large, hierarchical yellow follicles. Fat synthesis in birds occurs mainly in the liver and impairment of this capacity implies hepatic steatosis [ 12 ]. Reducing the quantity or changing the quality of fats in the diet of laying hens under heat stress can mitigate liver damage and improve performance and adaptability to environmental conditions in hot areas [ 38 ]. No loss of Haugh unit was observed due to heat stress treatment. Narushin et al. [ 39 ] presents limitations in the formula for calculating the Haugh unit, mainly because it is restricted to the use of only two parameters, albumen height and egg weight, and questions its representativeness as an indicator of egg quality. Albumen height, and consequently the Haugh unit, tend to decrease with egg aging [ 40 ]. This study observed drops of approximately 16% in egg weight and 12% in albumen height in birds under heat stress. Based on these parameters, there is no doubt about the loss of internal egg quality due to heat stress treatment. These results are reinforced by the observed reduction in the Apovitellenin-1 protein, which consequently reduces interactions with Lipovitellenins, reduces VLDL in egg yolk, and compromises nutritional quality [ 41 ]. Shell quality was also compromised by heat stress. Shell thickness was reduced by approximately 13%, resulting in a loss of more than 30% in shell strength. The loss of shell rigidity is related to the loss of organic matter and phosphorus [ 23 , 42 ]. The increase in Gelsolin expression suggests modulations in actin polymerization, influenced by variations in free calcium concentrations [ 43 ]. The loss of internal and shell quality of eggs laid by hens under heat stress has also been reported in other studies [ 44 – 45 ], with reduced shell thickness being associated with reduced blood ionic calcium (iCa) in the first hours of heat stress [ 46 ] and increased potassium (K+) and sodium (Na+) [ 47 ]. In studies with layinh hens in the same age range (15–18 months of age), Lin et al. [ 48 ] found an increase in oxygen-reactive substances, concluding that enhanced enzymatic and non-enzymatic antioxidant systems acted together to alleviate heat stress-evoked oxidative damage in older hens. Attenuation of oxidative stress may provide protection against cardiomyopathies and inflammatory processes [ 49 – 50 ]. However, the potential increased susceptibility to pathologies in the medium term, resulting from reduced antioxidant capacity, is a concern. This complex adaptive response to prolonged heat stress suggests that immediate protection to the organism may weaken other biological functions in the long term. The absence of significant differences in blood counts between treatment groups suggests that maintaining blood homeostasis is a priority. These results coincide with the findings of Barrett et al. [ 46 ], who found that laying hens had increased blood pH between 4 and 6 hours of heat stress, with these values returning to pre-heat stress values after four weeks. The π subunit of hemoglobin, which was down-regulated in our study, is related to oxygen transport to tissues and the catabolic process of hydrogen peroxide, carried out by hepatic catalase. Lin et al. [ 51 ] state that the liver is more susceptible to oxidative stress than the heart, and that high body temperature can promote metabolic changes involved in the induction of oxidative stress, suggesting that the efficiency of thermoregulation may be related to protective mechanisms against oxidative stress. The negative regulation of the π subunit of hemoglobin suggests increased oxidative stress and hepatic inflammation [ 52 ]. Fibronectin, found among the exclusive proteins, is related to the blood coagulation process, as it binds to the actin of cells such as thrombocytes, and compounds such as fibrin and collagen. The expression of Fetuin B, Gelsolin, Prothrombin and Histidine-rich Glycoprotein are also strongly related to blood coagulation and fibrinolysis. During blood collections, an increase in the blood coagulation rate was observed in birds under heat stress. Birds naturally have a shorter coagulation time compared to other domestic species, and this increase in the coagulation rate and release of fibrinolysis products is compatible with Disseminated Intravascular Coagulation (DIC). DIC is a complex coagulation and hemorrhagic problem associated with liver dysfunction and occurs when the coagulation mechanism is overstimulated, with activation of the fibrinolytic mechanism [ 53 ]. Blood coagulation in chickens has been related to the availability of vitamin K [ 54 ]. Lipids, specifically phosphatides present in blood platelets, play an important role in activating blood coagulation. Activation of coagulation by phosphatide occurs at very low concentrations, with a sharp drop-in coagulation time at lower concentrations and a stabilization at higher concentrations [ 55 ]. The analysis of the metabolic pathways and interactions of the Ig-like domain-containing protein shows an interrelationship with Interleukin-6, which is a cytokine with a multifunctional role in the body, being essential in the regulation of the immune and inflammatory response. Characterizing the dynamics of interleukins during heat stress may be relevant in future studies. Due to the advanced age of the laying hens, changes in DNA methylation probably occurred before the experiment, resulting from environmental challenges while still on the farm. Oliveira Boreli et al. [ 27 ] did not observe changes in DNA methylation in blood samples from hens under heat stress. The effects of heat stress on chicks, evaluating liver proteomics and genomics, suggest that early exposure of individuals to high temperatures (40°C for 24 hours) contributes to the maintenance of homeostasis and inhibits biological damage in response to heat stress [ 56 ]. The analysis of grouped samples and the absence of physiological variables are the main limitations of this study. Blood coagulation was not a variable of interest, however, an increase in coagulation speed was observed during sample collection and the up-regulated expression of several proteins related to this process. There are few studies on blood coagulation associated with heat stress and this subject needs to be further investigated. Future studies should consider evaluating the blood profile in the first hours of exposure to heat stress and adding blood electrolyte analyses. It is suggested to focus on the analysis of responses related to oxidative resistance and homeostasis, to clarify the underlying mechanisms of adaptation and resistance to heat stress. The evaluation of additional biomarkers, such as malondialdehyde, IL-6 and IL-1, could further elucidate the dynamics of oxidative stress and its implications in birds subjected to adverse environmental conditions. 5 Conclusions Heat stress significantly affected the abundance of serum proteins in laying hens. Among the proteins expressed exclusively in the heat-stressed group, proteins that interact with the heat shock proteins Hsp70 and Hsc70 were found. There was an increase in the expression of proteins related to coagulation speed, indicating Disseminated Intravascular Coagulation (DIC). Egg production and quality decreased under heat stress and were associated with the proteins Apolipoprotein A (up-regulated) and Apovitelenin-1 (down-regulated). There was no association between the decrease in egg production and quality, and changes in the blood profile or DNA methylation in the blood after 28 days of heat stress for 75-week-old hens. Declarations Funding statement Funding was provided by National Council for Scientific and Technological Development – CNPq (Grant #304085/2021-9), and by São Paulo Research Foundation – FAPESP (Grants #2020/14885-8 and #2024/14650-1). Ethics statement All animal handling and care procedures followed the recommendations of the Animal Use Ethics Committee, under protocol no. 02/2022 of the Faculty of Science and Engineering of the São Paulo State University (UNESP). Author contributions statement The study conception and design were developed by SGG, FPdOB, FAS and DFP. Sample collection and other experimental data were conducted by SGG, FPdOB and ARG. Sample preparation and analysis by SGG, BCM, BCR and LDdS. Analisys of the data was performed by SGG, FPdOB, LDdS and DFP. The first draft of the manuscript was written by SGG, FPdOB, LDdS and DFP, and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript. 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21:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6710895/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6710895/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83732814,"identity":"61d6ac8d-ea9c-4772-b12e-26d30fa8f8f5","added_by":"auto","created_at":"2025-06-01 14:47:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30390,"visible":true,"origin":"","legend":"\u003cp\u003eDaily production of hens subjected to comfort and heat stress.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/608ecde2d646275f3cbc6bea.png"},{"id":83732818,"identity":"74fff5d7-5e74-4c7c-822b-3552f8156bf8","added_by":"auto","created_at":"2025-06-01 14:47:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13631,"visible":true,"origin":"","legend":"\u003cp\u003eWeekly weight assessment of hens raised in Comfort (blue line) and Heat Stress (red line), excluding the adaptation period.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/1d0b9f3e44d49a2e4d052afd.png"},{"id":83732816,"identity":"e4f8d22c-7895-43a3-a519-85b3851afd11","added_by":"auto","created_at":"2025-06-01 14:47:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56141,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot of hematological variables A) Hemoglobin, B) Erythrocytes, C) Hematocrit, D) Leukocytes, E) Glucose and F) Total plasma proteins, for the first (left) and last (right) day of housing of hens in comfort (blue box) and heat stress (red box). Reference ranges obtained from Oliveira Boreli et al. \u003cstrong\u003e[27]\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/019759afbd67c7e3246ed163.png"},{"id":83733173,"identity":"25edbe6b-a3b3-426f-8480-4a6ad7a02a3a","added_by":"auto","created_at":"2025-06-01 14:55:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14461,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal DNA methylation of blood samples from two birds per cage on the first and last day of the treatment, for the comfort (blue bar) and heat stress (red bar) groups.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/97cad786a67e3449b70086aa.png"},{"id":83732825,"identity":"b98dadb1-e252-40da-8af5-749d5a329e08","added_by":"auto","created_at":"2025-06-01 14:47:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87299,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagrams highlighting the common and exclusive proteins identified in the experimental groups a) C1 and C28, b) HS1 and HS28 and c) all groups. C1: Start of treatment in comfort at 26\u003csup\u003eo\u003c/sup\u003eC; C28: End of treatment in thermal comfort. \u0026nbsp;HS1: Start of treatment under heat stress at 35\u003csup\u003eo\u003c/sup\u003eC; HS28: End of treatment under heat stress.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/c072776f15511e732fc094f1.png"},{"id":83733680,"identity":"ae22941a-27ab-4c4e-be83-ce8e35aba7c4","added_by":"auto","created_at":"2025-06-01 15:11:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1046661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/8fe8a29f-4c98-4058-92ec-b9a8b944d67f.pdf"},{"id":83732820,"identity":"ac2fe607-c2e6-4a03-95c1-faf8bf2d78ff","added_by":"auto","created_at":"2025-06-01 14:47:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":140048,"visible":true,"origin":"","legend":"","description":"","filename":"Appendixs.docx","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/f78a6671d14b1aa3651507a6.docx"},{"id":83732822,"identity":"c19ebe5e-9e31-498a-bc30-6bfd4201e56d","added_by":"auto","created_at":"2025-06-01 14:47:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6784163,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6710895/v1/1fa8e896be98da063dfd9e18.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of heat stress on serum proteomics, laying performance and egg quality in aged laying hens","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePoultry farming faces significant challenges due to the increasing frequency and intensity of extreme weather events, such as heat waves, which directly impact animal production systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Heat stress negatively affects poultry physiology, leading to increased mortality in laying hens, alterations in metabolic rate and immune function, hormonal, endocrine, digestive, and reproductive imbalances, reduced laying performance, decreased feed intake, and compromised egg quality [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo restore homeostasis, birds activate compensatory mechanisms, including decreased feed consumption [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], increased water consumption [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and peripheral vasodilation, often accompanied by wing elevation and feather ruffling to increase surface area for heat dissipation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, heat stress results in elevated body temperature [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and an increased respiratory rate. This elevated respiratory rate leads to reduced blood carbon dioxide levels and an increase in blood pH, a condition known as respiratory alkalosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. When ambient temperature and relative humidity are excessively high, birds are unable to effectively dissipate body heat, potentially reaching a critical physiological threshold and resulting in mortality [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeat stress can also induce an increase in hepatic fat synthesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The liver then synthesizes excess triglycerides, which require transport to other tissues via very low-density lipoproteins (VLDL). However, heat stress can limit the availability of Apolipoprotein B (ApoB), which is essential for VLDL assembly and secretion, thus reducing VLDL transport and leading to an accumulation of triglycerides and total cholesterol in the liver, resulting in hepatic steatosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn response to heat stress, organisms induce the expression of specific proteins that serve as a protective mechanism to maintain cellular function and integrity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These proteins participate in diverse metabolic and cellular processes, including the biogenesis of cellular components and the response to various stress stimuli [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeat shock protein 70 (HSP70) serves as a prominent indicator of the response to heat stress [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] because, unlike most proteins whose synthesis is delayed, heat shock proteins (HSPs) are rapidly synthesized [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Under stress conditions, HSP70 functions to prevent the formation and aggregation of abnormal proteins and to restore the binding capacity of the progesterone receptor [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Khondowe et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] observed variations in hepatic HSP70 protein expression among different local chicken breeds in Tanzania subjected to heat stress. Oloruntola et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported an increase in serum HSP70 expression in chickens experiencing heat stress, and this elevation could be attenuated through dietary supplementation with vitamin C and mistletoe leaf powder.\u003c/p\u003e \u003cp\u003eSeveral studies have correlated various proteins with egg formation and quality [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While samples for these studies are often extracted from different organs and from the egg itself [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], research investigating the expression of these proteins specifically in the blood of chickens remains limited.\u003c/p\u003e \u003cp\u003eThe aim of this study was to evaluate the effects of long-term heat stress on the abundance and expression of serum proteins in aged laying hens and to correlate these changes with global DNA methylation patterns, hematological parameters, production rates, and overall egg quality.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003e The experiment was conducted in accordance with the ethical guidelines for animal experimentation established by the Animal Ethics and Welfare Committee of the Faculty of Science and Engineering, Tup\u0026atilde; Campus, S\u0026atilde;o Paulo State University J\u0026uacute;lio de Mesquita Filho, Unesp/FCE. The study was approved by the Animal Use Ethics Committee (CEUA) under registration number 02/2022.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Description of the hens and housing conditions\u003c/h2\u003e \u003cp\u003eThe experiment was conducted with 48 laying hens of the Lohmann White strain with 75 weeks old and with an average weight of 1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 kg. The hens were obtained from a commercial farm in the city of Tup\u0026atilde;, State of S\u0026atilde;o Paulo, Brazil (S 21\u0026deg; 56\u0026rsquo; 36\u0026rdquo;, W 50\u0026deg; 27\u0026rsquo; 45\u0026rdquo;).\u003c/p\u003e \u003cp\u003eThe birds were divided and housed in two climate chambers, in wire cages, and groups of four birds per cage, respecting the same housing density as the farm. The lighting was reproduced as that used on the commercial farm, with the lights turned on at 4h30 am and turned off at 10h00 pm, totaling a photo period of 17.5 hours of light. The feed provided to the birds had the same formulation as that used on the farm, with 2,785 Cal/kg of energy and 17% protein. The feed was offered in troughs twice a day in a total amount of 120 g/bird per day, and water was provided \u003cem\u003ead libitum\u003c/em\u003e through nipple drinkers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental treatments\u003c/h2\u003e \u003cp\u003eDuring the first seven days, the birds in the two climate chambers were subjected to an adaptation period, with a continuous ambient temperature of 26\u0026deg;C and 60% relative humidity. After adaptation, one of the chambers remained under these environmental conditions and the other had the air temperature raised to 35\u0026deg;C and 60% relative humidity, full time (continuous stress \u0026ndash; 24 hours), totaling two temperature treatments. The first condition was called the Comfort (C) treatment, and the second condition was called Heat Stress (HS). The birds were subjected to the treatments 24 hours a day for 28 consecutive days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Weighing of hens\u003c/h2\u003e \u003cp\u003eThe hens were weighed individually every seven days using a platform scale (B-160, L\u0026iacute;der Co. Ltda., Ara\u0026ccedil;atuba, S\u0026atilde;o Paulo, Brazil). The variation in hens' weight was compared between the comfort and heat stress groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Egg production and quality\u003c/h2\u003e \u003cp\u003eEgg production was recorded daily for each temperature treatment, and the laying rate was calculated by dividing the number of eggs by the total number of hens housed.\u003c/p\u003e \u003cp\u003eAll eggs produced in the last three days of the experiment were evaluated for specific gravity, egg weight, shell strength, shell thickness, albumen height and Haugh unit using the Digital Egg Tester (DET-6000, Nabel Co. Ltd., Kyoto, Japan\u0026reg;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Collection and preparation of blood samples\u003c/h2\u003e \u003cp\u003eBlood samples were collected twice: on the first day of treatment and the last day of the experiment. Blood samples were collected from all hens, preferably by radial venipuncture, using a 25\u0026times;7 mm needle and a 3 mL syringe. It was placed 0,5 mL in a microtube containing EDTA K3 as an anticoagulant and 2.5 mL in a tube with a coagulation accelerator and separating gel.\u003c/p\u003e \u003cp\u003eThe tubes with the coagulation accelerator and separating gel were kept at 24\u0026deg;C for 120 minutes, protected from light, to obtain complete coagulation and clot retraction. The serum was transferred to Eppendorf\u0026reg; microtubes, followed by freezing at -20\u0026deg;C, for use in proteomic analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Complete blood count, plasma proteins and blood glucose\u003c/h2\u003e \u003cp\u003eThe EDTA blood samples were homogenized for hematocrit analysis, red blood cell and leukocyte count, and determination of total plasma proteins and hemoglobin (Almosny et al., 2015, n.d.). Two capillary tubes for microhematocrit were filled, sealed and centrifuged at 12,500 rpm for 5 minutes in a Microcentrifuge (Mod. 211, Fanem\u0026reg; Ltda., Guarulhos, Brazil). After centrifugation, one tube was read in specific table for reading microhematocrit, provided by the manufacturer, while total plasma proteins were determined by manual refractometry in Refractometer (RHC-200-ATC), using the plasma obtained from the second capillary tube. The red blood cell and leukocyte count was performed in a Neubauer chamber after dilution in Nat-Herrick solution, with correction of values by specific factors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Hemoglobin was quantified by spectrophotometry at 540 nm in semi-automatic biochemical analyzer Bio-2000\u0026reg; (Bioplus\u0026reg; Produtos para Laborat\u0026oacute;rios Ltda., Barueri, Brazil), using the hemoglobin cyanide method (Labtest\u0026reg; kit Ref. 43), using the Labtest\u0026reg; kit Ref. 47 as the Hemoglobin Standard.\u003c/p\u003e \u003cp\u003eThe glucose concentration was determined by an electrochemical method using a glucometer (Accu-Check\u0026reg;, Guide Roche\u0026reg;), with fresh blood, at the same time as sample collection. The values of the blood parameters were compared with the reference ranges compiled by Oliveira Boreli et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Global DNA methylation\u003c/h2\u003e \u003cp\u003eTo quantify total genomic DNA, blood from two birds per cage was used on the first (D1) and last (D28) day of treatment. DNA was extracted using the Monarch kit (Biolab\u0026reg;, New England). DNA quantification was performed in a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific\u0026reg; Inc.). Methylation analysis was performed in duplicate using the Global DNA Methylation ELISA kit (Cell Biolabs\u0026reg; Inc.). The pretreated strips contained wells with a specific reagent for binding to methylated DNA, methylation-sensitive capture antibodies, and detection antibodies, which allowed photometric detection of the absolute amount of DNA methylation in each sample. The absorbance of the solution in the wells was measured with an ELISA spectrophotometer (Kasuaki-DR-200Bs-BI) at 450 nm. Methylation data were compared by Analysis of Variance (ANOVA) between collection days and temperature treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Composition of samples for proteomic analysis\u003c/h2\u003e \u003cp\u003eFour samples were prepared in pool format for the proteomic analyses. Each pool consisted of 50 \u0026micro;L of blood serum from each of the hens (24 birds), from each blood collection and environmental treatment. The samples from the birds subjected to the Comfort treatment were identified by C1 and C28, and from the Heat Stress treatment by HS1 and HS28, with \u0026ldquo;1\u0026rdquo; and \u0026ldquo;28\u0026rdquo; representing the first and last day of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Proteomic Analysis\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 Protein Quantification\u003c/h2\u003e \u003cp\u003eConsidering the abundance of proteins present in blood serum, the crude samples were diluted in 50 mM Ammonium Bicarbonate solution, in the proportion (1:50). Protein quantification was performed by the Bradford reaction [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], prepared in technical triplicate, using the BioRad\u0026reg; Protein Assay kit, with commercial bovine albumin as the standard protein (Ref. 500-0006, Biorad\u0026reg;). To construct the calibration curve, four dilutions of the standard protein (1, 2, 4 and 6 \u0026micro;g/\u0026micro;L) were prepared in technical triplicate. The absorbance of the samples and the calibration curve were read at 595 nm in \u0026micro;Quant Bio-Tek\u0026reg; microplate spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2 One-dimensional electrophoresis\u003c/h2\u003e \u003cp\u003eElectrophoretic analysis of the experimental samples under reducing and denaturing conditions (12% [m/v] SDS-PAGE) was performed to observe the quality and viability of the samples in relation to degradation aspects and the presence of proteins. This procedure was performed according to [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], using molecular markers present in the Calibration Low Molecular Weight kit (Ref. 17-0446-01, Cytiva\u0026reg;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3 In-Solution Protein Digestion and Mass Spectrometry Analysis\u003c/h2\u003e \u003cp\u003eThe experimental samples were subjected to enzymatic digestion according to (Cavecci-Mendon\u0026ccedil;a et al., 2023), using standard surfactant RapiGest SF (Ref. 186001861, Waters\u0026reg;), and the enzyme trypsin at a concentration of 1:50 (enzyme: sample). After hydrolysis was interrupted, the samples were desalted using Sep-Pak Cartridges Sample Extraxt Products 1cc C18 columns (Ref. WAT054955, Waters). Peptide quantification was performed by chemiluminescence in a Qubit 2.0 Fluorometer using the Qubit Protein Assay Kit (Ref Q33212, Invitrogen).\u003c/p\u003e \u003cp\u003eShotgun LC-MS/MS mass spectrometry analyses were performed using an Ultimate 3000 LC liquid nanochromatography equipment (Dionex, Germering, Germany) coupled to a Q-Exactive mass spectrometry equipment (Thermo Fisher Scientific\u0026reg;, Bremen, Germany) according to the methodology proposed by Cavalcante et al., (2022). The spectrometry data were acquired using Thermo Xcalibur software (version 4.0.27.19, ThermoFisher Scientific\u0026reg; Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.9.4 Data Analysis\u003c/h2\u003e \u003cp\u003eThe raw mass spectrometry data in \u0026ldquo;. RAW\u0026rdquo; format was evaluated by RawVegetable\u0026reg; software [version 1.1.0.1] and then submitted to PatternLab\u0026reg; software [version 4.0.0.84] to obtain protein identification [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The main parameters used in this tool were: UNIPROT database (Taxonomy Gallus gallus), trypsin enzyme; permission of 2 missed cleavages; post-translational modification carbamidomethylating of cysteine residues; variable post-translational modification oxidation of methionine residues; MS and MS/MS tolerance errors of 0.0200 ppm. The maximum FDR (False Discovery Rate) rate\u0026thinsp;\u0026le;\u0026thinsp;1%.\u003c/p\u003e \u003cp\u003eA matrix compatible with the MetaboAnalyst\u0026reg; 6.0 program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://new.metaboanalyst.ca/\u003c/span\u003e\u003cspan address=\"https://new.metaboanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was constructed from the proteomic data using the spectral counts of each identified protein, which were normalized for each protein by the weighted average of the technical triplicates of each sample [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePartial Least Squares (PLS) analyses were used as the main method of multivariate analysis. Only signals present in 75% of the samples were considered for the generation of statistical models. The t-test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) and fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 were used to compare the mean protein abundances between each experimental group. Protein interactions were also investigated concerning their biological processes using the STRING\u0026reg; software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://string-db.org/\u003c/span\u003e\u003cspan address=\"http://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e - version 10.5), using the basic parameters: cut-off score of 0.90, confidence as network edges, and PPI p-value of \u0026lt;\u0026thinsp;1.0e-16 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003ch2\u003e3.1 Egg production and quality\u003c/h2\u003e\n\u003cp\u003eFigure 1 shows the daily egg production of each group, subjected to Comfort or Heat Stress.\u003c/p\u003e\n\u003cp\u003eFigure 1 shows that from the third day of heat treatment onwards, the group under heat stress showed a significant reduction in the laying rate. In the Comfort group, the laying rate remained above 90%. Considering the entire experimental period, the group under Heat Stress produced 11% fewer eggs than the group in Comfort.\u003c/p\u003e\n\u003cp\u003eTable 1 shows the result of the Fisher test at 5% for the egg quality variables between the Comfort and Heat Stress groups. The group under Heat Stress showed a significant reduction in egg quality. Egg weight was 16% lower, shell resistance and shell thickness were 30% and 13.5% lower, and albumen height was 12% lower. There was no statistical difference in the Haugh unit.\u003c/p\u003e\n\u003cp\u003eDifferent letters indicate significant difference by Fisher\u0026apos;s test (p \u0026lt; 0.05).\u003c/p\u003e\n\u003ch2\u003e3.2 Weight of hens\u003c/h2\u003e\n\u003cp\u003eFigure 2 shows the weekly weight assessment of hens subjected to heat stress treatment, compared with the weight of hens raised in Comfort.\u003c/p\u003e\n\u003cp\u003eFigure 2 shows that hens kept under Heat Stress lost weight in the first week. The impact of the 28-day Heat Stress treatment period resulted in a weight reduction of 13%, i.e., a loss of 200g on average per bird.\u003c/p\u003e\n\u003ch2\u003e3.3 Complete blood count, plasma proteins and blood glucose\u003c/h2\u003e\n\u003cp\u003eDuring blood sampling, it was found that the blood from heat-stressed chickens clotted easily, many of them while still in the syringe and during extraction. EDTA samples from several animals had to be collected again due to loss of samples due to clotting.\u003c/p\u003e\n\u003cp\u003eFigure 3 shows the box-plot graphs of the hematological variables for the first (graphs on the left) and last day of housing of the birds (graphs on the right).\u003c/p\u003e\n\u003cp\u003eOn the first day of housing, the birds selected for the two groups had similar blood parameters. After 28 days of experimentation, it was found that the birds in both treatments had a reduction in hemoglobin to levels below the limit established by the literature. It was also found that the hematocrit of the group under heat stress had an average at the lower limit established by the literature. Individual variations in total plasma proteins were observed, although the group average remained equivalent.\u003c/p\u003e\n\u003ch2\u003e3.4 Global DNA methylation\u003c/h2\u003e\n\u003cp\u003eFigure 4 shows the global DNA methylation results for the comfort and heat stress groups on the first and last day of the experiment.\u003c/p\u003e\n\u003cp\u003eAlthough there is a small visual difference in the graph of global DNA methylation of the samples between the groups, after 28 days of temperature treatment, the difference was not statistically significant.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.5 Analysis of Protein Concentrations in Proteomic Samples\u003c/h2\u003e\n\u003cp\u003eThe protein concentrations observed in each sample were as follows: 22.38 \u0026micro;g/\u0026micro;L (C1), 26.04 \u0026micro;g/\u0026micro;L (C28), 26.46 \u0026micro;g/\u0026micro;L (HS1), and 13.72 \u0026micro;g/\u0026micro;L (HS28). The protein profiles of the blood serums, when subjected to electrophoresis tests, exhibited high protein variability, with molecular masses ranging between 10 and 150 kDa and no signs of protein degradation. The electrophoretic image can be observed in Appendix I.\u003c/p\u003e\n\u003cp\u003eRegarding the proteins present in the blood serum of the experimental groups, a comparison with theoretical data from the UNIPROT database for \u003cem\u003eGallus gallus\u003c/em\u003e taxonomy revealed the following:\u003c/p\u003e\n\u003cp\u003e\u0026middot; Group C1: 125 total proteins identified, 90 proteins under analysis in maximum parsimony\u003c/p\u003e\n\u003cp\u003e\u0026middot; Group C28: 122 total identifications, 83 in maximum parsimony\u003c/p\u003e\n\u003cp\u003e\u0026middot; Group HS1: 108 total proteins identified, 85 proteins under analysis in maximum parsimony\u003c/p\u003e\n\u003cp\u003e\u0026middot; Group HS28: 110 total proteins identified, 88 proteins under analysis in maximum parsimony\u003c/p\u003e\n\u003cp\u003eThe proteomic parameters for each analysis are detailed in Appendix II.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 5 presents the Venn diagrams for the groups of samples analyzed.\u003c/p\u003e\n\u003cp\u003eFigure 5a shows 116 common proteins identified between the beginning and end of the Comfort treatment. Six proteins are exclusive to C1 and nine to C28. Figure 5b shows 98 common proteins present at the beginning and end of heat stress, ten of which are exclusive to HS1 and twelve to HS28. Figure 5c shows the interaction between all groups. The yellow-colored area of the Venn Diagram shows the distribution of proteins identified after 28 days of constant heat stress.\u003c/p\u003e\n\u003ch3\u003e3.5.1 Protein differentiation between samples\u003c/h3\u003e\n\u003cp\u003eEight proteins were exclusive to HS28, and among them, it is believed that they may stand out as potential candidates for heat stress biomarkers (Table 2).\u003c/p\u003e\n\u003cp\u003eRegarding the proteins that presented differential abundance between the experimental groups, Figure 6 shows the Volcano plots obtained in the MetaboAnalyst software. Between groups HS1 and C1 (start of treatments) there were no significant differences in protein abundance.\u003c/p\u003e\n\u003cp\u003eFor the samples from the comfort group (Figure 6a), three proteins were more abundant and five decreased significantly. When comparing the HS28 vs. HS1 samples from birds subjected to heat stress, eleven proteins with differential abundance were noted (Figure 6b), with seven being higher and four being downregulated in HS28.\u003c/p\u003e\n\u003cp\u003eWhen the groups were compared at the end of the experiment (Figure 6c), a more discrete variation was observed. Of the eleven proteins with differential abundance in the heat stress group, five of them were confirmed as differentials in comparison with the comfort group (HS28 vs. C28), and one protein that had not expressed divergences, Apolipoprotein A, appeared in this comparison.\u003c/p\u003e\n\u003cp\u003eTable 3 and Table 4 shows the proteins with differences in abundance in each of the comparisons made between the samples. The tables were composed using information collected from the UniProt and String databases, and present the protein code, protein name, metabolic functions, and metabolic pathway and interactions with other proteins. The gene codes, when available, were important for later identification of the biological processes in which these proteins are involved.\u003c/p\u003e\n\u003cp\u003eTable 3 presents seven proteins that had their expression increased in the HS28 samples relative to HS1 and C28: Fetuin B, Gelsolin, Histidine-rich glycoprotein, Prothrombin, Apolipoprotein A, Sushi domain protein, and Serpin G member 1. Most of these proteins (with the exception of Apolipoprotein A and Sushi domain protein) play a role in blood clotting. Appendix III provides detailed information on the variation in the amount of each protein.\u003c/p\u003e\n\u003cp\u003eThe proteins in Table 4 were found in smaller quantities in the HS28 sample. Apovitellin-1, for example, is essential for nutrient storage. It acts together with VTG1, VTG2 and VTG3, which are parts of lipovitellin-1, a precursor of the main yolk proteins. These proteins are vital for providing nutrients during the early development of oviparous animals.\u003c/p\u003e\n\u003cp\u003eThe expression of the \u0026pi; subunit of Hemoglobin was also reduced and is linked to the breakdown of hydrogen peroxide and the protection of cells against oxidizing substances. This subunit interacts with the beta subunit of Hemoglobin (HBBA), which is responsible for carrying oxygen from the lungs to the other tissues.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eQuantitative analysis of serum proteins revealed eight unique proteins in samples from hens under heat stress after 28 days. These included Hsp70 and Hsc70, known biomarkers for heat stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. HSC70 interacts with tumorigenicity suppressor proteins (ST14 and DNND2B) and the heat shock-related protein HSPA8, protecting the proteome. The expression of these proteins confirms heat stress and suggests attempted adaptation.\u003c/p\u003e \u003cp\u003eApolipoprotein A (up-regulated) and Apovitelenin-1 (down-regulated), interconnected proteins and VLDL components in laying hens, showed altered expression. APOV1, a potent lipoprotein lipase inhibitor, prevents triglyceride loss from VLDL during transport to oocytes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Its inhibition promotes triglyceride accumulation in the liver [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and consequently, liver fat accumulation, reducing feed conversion efficiency [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The laying rate decreased in the first week of heat stress, possibly related to increased abdominal fat. Li et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] found that after five days of heat stress (35\u0026deg;C-37\u0026deg;C), laying hens exhibited a reduced laying rate due to fewer large, hierarchical yellow follicles.\u003c/p\u003e \u003cp\u003eFat synthesis in birds occurs mainly in the liver and impairment of this capacity implies hepatic steatosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Reducing the quantity or changing the quality of fats in the diet of laying hens under heat stress can mitigate liver damage and improve performance and adaptability to environmental conditions in hot areas [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNo loss of Haugh unit was observed due to heat stress treatment. Narushin et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] presents limitations in the formula for calculating the Haugh unit, mainly because it is restricted to the use of only two parameters, albumen height and egg weight, and questions its representativeness as an indicator of egg quality. Albumen height, and consequently the Haugh unit, tend to decrease with egg aging [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This study observed drops of approximately 16% in egg weight and 12% in albumen height in birds under heat stress. Based on these parameters, there is no doubt about the loss of internal egg quality due to heat stress treatment. These results are reinforced by the observed reduction in the Apovitellenin-1 protein, which consequently reduces interactions with Lipovitellenins, reduces VLDL in egg yolk, and compromises nutritional quality [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eShell quality was also compromised by heat stress. Shell thickness was reduced by approximately 13%, resulting in a loss of more than 30% in shell strength. The loss of shell rigidity is related to the loss of organic matter and phosphorus [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The increase in Gelsolin expression suggests modulations in actin polymerization, influenced by variations in free calcium concentrations [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe loss of internal and shell quality of eggs laid by hens under heat stress has also been reported in other studies [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], with reduced shell thickness being associated with reduced blood ionic calcium (iCa) in the first hours of heat stress [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and increased potassium (K+) and sodium (Na+) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn studies with layinh hens in the same age range (15\u0026ndash;18 months of age), Lin et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] found an increase in oxygen-reactive substances, concluding that enhanced enzymatic and non-enzymatic antioxidant systems acted together to alleviate heat stress-evoked oxidative damage in older hens. Attenuation of oxidative stress may provide protection against cardiomyopathies and inflammatory processes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, the potential increased susceptibility to pathologies in the medium term, resulting from reduced antioxidant capacity, is a concern. This complex adaptive response to prolonged heat stress suggests that immediate protection to the organism may weaken other biological functions in the long term.\u003c/p\u003e \u003cp\u003eThe absence of significant differences in blood counts between treatment groups suggests that maintaining blood homeostasis is a priority. These results coincide with the findings of Barrett et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], who found that laying hens had increased blood pH between 4 and 6 hours of heat stress, with these values returning to pre-heat stress values after four weeks.\u003c/p\u003e \u003cp\u003eThe π subunit of hemoglobin, which was down-regulated in our study, is related to oxygen transport to tissues and the catabolic process of hydrogen peroxide, carried out by hepatic catalase. Lin et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] state that the liver is more susceptible to oxidative stress than the heart, and that high body temperature can promote metabolic changes involved in the induction of oxidative stress, suggesting that the efficiency of thermoregulation may be related to protective mechanisms against oxidative stress. The negative regulation of the π subunit of hemoglobin suggests increased oxidative stress and hepatic inflammation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFibronectin, found among the exclusive proteins, is related to the blood coagulation process, as it binds to the actin of cells such as thrombocytes, and compounds such as fibrin and collagen. The expression of Fetuin B, Gelsolin, Prothrombin and Histidine-rich Glycoprotein are also strongly related to blood coagulation and fibrinolysis. During blood collections, an increase in the blood coagulation rate was observed in birds under heat stress. Birds naturally have a shorter coagulation time compared to other domestic species, and this increase in the coagulation rate and release of fibrinolysis products is compatible with Disseminated Intravascular Coagulation (DIC). DIC is a complex coagulation and hemorrhagic problem associated with liver dysfunction and occurs when the coagulation mechanism is overstimulated, with activation of the fibrinolytic mechanism [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Blood coagulation in chickens has been related to the availability of vitamin K [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Lipids, specifically phosphatides present in blood platelets, play an important role in activating blood coagulation. Activation of coagulation by phosphatide occurs at very low concentrations, with a sharp drop-in coagulation time at lower concentrations and a stabilization at higher concentrations [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe analysis of the metabolic pathways and interactions of the Ig-like domain-containing protein shows an interrelationship with Interleukin-6, which is a cytokine with a multifunctional role in the body, being essential in the regulation of the immune and inflammatory response. Characterizing the dynamics of interleukins during heat stress may be relevant in future studies.\u003c/p\u003e \u003cp\u003eDue to the advanced age of the laying hens, changes in DNA methylation probably occurred before the experiment, resulting from environmental challenges while still on the farm. Oliveira Boreli et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] did not observe changes in DNA methylation in blood samples from hens under heat stress. The effects of heat stress on chicks, evaluating liver proteomics and genomics, suggest that early exposure of individuals to high temperatures (40\u0026deg;C for 24 hours) contributes to the maintenance of homeostasis and inhibits biological damage in response to heat stress [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe analysis of grouped samples and the absence of physiological variables are the main limitations of this study. Blood coagulation was not a variable of interest, however, an increase in coagulation speed was observed during sample collection and the up-regulated expression of several proteins related to this process. There are few studies on blood coagulation associated with heat stress and this subject needs to be further investigated. Future studies should consider evaluating the blood profile in the first hours of exposure to heat stress and adding blood electrolyte analyses. It is suggested to focus on the analysis of responses related to oxidative resistance and homeostasis, to clarify the underlying mechanisms of adaptation and resistance to heat stress. The evaluation of additional biomarkers, such as malondialdehyde, IL-6 and IL-1, could further elucidate the dynamics of oxidative stress and its implications in birds subjected to adverse environmental conditions.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eHeat stress significantly affected the abundance of serum proteins in laying hens. Among the proteins expressed exclusively in the heat-stressed group, proteins that interact with the heat shock proteins Hsp70 and Hsc70 were found. There was an increase in the expression of proteins related to coagulation speed, indicating Disseminated Intravascular Coagulation (DIC).\u003c/p\u003e \u003cp\u003eEgg production and quality decreased under heat stress and were associated with the proteins Apolipoprotein A (up-regulated) and Apovitelenin-1 (down-regulated). There was no association between the decrease in egg production and quality, and changes in the blood profile or DNA methylation in the blood after 28 days of heat stress for 75-week-old hens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding was provided by National Council for Scientific and Technological Development \u0026ndash; CNPq (Grant #304085/2021-9), and by S\u0026atilde;o Paulo Research Foundation \u0026ndash; FAPESP (Grants #2020/14885-8 and #2024/14650-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal handling and care procedures followed the recommendations of the Animal Use Ethics Committee, under protocol no. 02/2022 of the Faculty of Science and Engineering of the S\u0026atilde;o Paulo State University (UNESP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study conception and design were developed by SGG, FPdOB, FAS and DFP. Sample collection and other experimental data were conducted by SGG, FPdOB and ARG. Sample preparation and analysis by SGG, BCM, BCR and LDdS. Analisys of the data was performed by SGG, FPdOB, LDdS and DFP. The first draft of the manuscript was written by SGG, FPdOB, LDdS and DFP, and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Available Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry data in this manuscript has been uploaded to the MassIVE Repository from the Computer Science and Engineering University of California, San Diego (https://massive.ucsd.edu/ProteoSAFe/) with the data set identifier MSV000095194.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNawaz, A.H., Amoah, K., Leng, Q.Y., Zheng, J.H., Zhang, W.L., Zhang, L., 2021. Poultry Response to Heat Stress: Its Physiological, Metabolic, and Genetic Implications on Meat Production and Quality Including Strategies to Improve Broiler Production in a Warming World. Front. Vet. 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Proteomic Analysis of the Protective Effect of Early Heat Exposure against Chronic Heat Stress in Broilers. Anim. Open Access J. MDPI 10, 2365. https://doi.org/10.3390/ani10122365 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-animals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Animals](https://link.springer.com/journal/44338)","snPcode":"44338","submissionUrl":"https://submission.springernature.com/new-submission/44338/3","title":"Discover Animals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"animal welfare, avian hematology, blood coagulation, heat shock protein, Shotgun proteomics","lastPublishedDoi":"10.21203/rs.3.rs-6710895/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6710895/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the impact of heat stress on serum proteomics, laying performance, and egg quality in aged laying hens. Forty-eight 75-week-old Lohmann White hens were divided into two groups and subjected to either a comfort environment (26\u0026deg;C) or heat stress (35\u0026deg;C) for 28 days. Serum samples were collected on days 1 and 28 for proteomic analysis, while egg production and quality parameters were assessed daily throughout the experiment. Heat stress reduced egg production (11%), egg weight (16%), shell strength (30%), shell thickness (13.5%), and albumen height (12%). Proteomic analysis revealed the expression of proteins that bind to the heat shock proteins Hsp70 and Hsc70, exclusively in heat-stressed laying hens. Proteins related to coagulation rate were upregulated and when compared with field observations may indicate possible Disseminated Intravascular Coagulation (DIC). Apolipoprotein A and apovitelenin-1 were associated with decreased egg production and quality under heat stress.\u003c/p\u003e","manuscriptTitle":"Effects of heat stress on serum proteomics, laying performance and egg quality in aged laying hens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-01 14:47:19","doi":"10.21203/rs.3.rs-6710895/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-07T14:07:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T15:17:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-21T20:38:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73998339954816827492922814136574661495","date":"2025-06-14T17:52:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159160881054313350377146834957278756829","date":"2025-06-13T20:12:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-03T09:32:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274750962832993767866007078578032921831","date":"2025-05-31T09:38:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10507921394111109165068528210073420090","date":"2025-05-31T07:33:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-28T17:05:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-21T13:17:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-21T11:42:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-21T11:38:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Animals","date":"2025-05-20T21:10:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-animals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Animals](https://link.springer.com/journal/44338)","snPcode":"44338","submissionUrl":"https://submission.springernature.com/new-submission/44338/3","title":"Discover Animals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65f6204c-2b90-4fa2-aa9f-1099eae66b62","owner":[],"postedDate":"June 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-24T07:23:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-01 14:47:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6710895","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6710895","identity":"rs-6710895","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}