Feed supplementation with the Ctx(Ile 21 )-Ha antimicrobial peptide associated with heat stress affects laying hens’ productive performance and physiology

Feed supplementation with the Ctx(Ile 21 )-Ha antimicrobial peptide associated with heat stress affects laying hens’ productive performance and physiology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Feed supplementation with the Ctx(Ile 21 )-Ha antimicrobial peptide associated with heat stress affects laying hens’ productive performance and physiology Lorenza Eivazian Brandão, Silvana Gomes Gonzalez, Diogo Sartori, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7993048/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study evaluated the effects of the supplementation of the antimicrobial peptide Ctx(Ile 21 )-Ha in laying hens under heat stress. A completely randomized design with a 2 × 3 factorial arrangement was used to evaluate two thermal environments (Comfort: 26°C and Heat stress: 35°C) and three peptide dosages (D1: 0.0mg/kg, D1: 2.5mg/kg and D2: 5.0mg/kg of feed). The peptide was synthesized by solid phase peptide synthesis, reaching 77.14% of efficiency, with purity degree > 95%. Microencapsulation via ionotropic gelation showed efficiencies of 79.84% (for 100 mg of peptide) and 58.47% (for 200 mg), with in vitro assays confirming controlled intestinal release. Eighteen 40-week-old hens were housed in climate-controlled chambers and supplemented with the antimicrobial peptide. The ingestion of microcapsules was 100% for D1 under both conditions and D2 under heat stress, but only 63.2% for D2 under comfort. Hens receiving 5.0 mg/kg of feed (D2) showed reduced feed intake, especially under heat stress, and slight weight loss, although egg production remained stable. Declines in eggshell quality were observed. Blood analyses showed no significant differences in glucose, hemoglobin, erythrocytes, proteins, or electrolytes for the different peptide doses tested. In conclusion, peptide did not significantly improve egg production or quality. Although no major physiological or productive changes were observed, this study contributes to the development of more sustainable poultry farming strategies. Animal welfare. Antimicrobial peptides. Blood count. Electrolytes. Shell quality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Antimicrobial peptides (AMPs) are naturally occurring molecules, intrinsic to the innate immune system of virtually all organisms [ 1 ]. AMPs belonging to the membranolytic class act by compromising the integrity of the plasma membrane of microorganisms, leading to their death and reducing the likelihood of microbial resistance [ 2 ]. This mechanism of action has sparked researchers' interest in investigating the effects of these peptides in laying hens as novel zootechnical additives to enhance their performance [ 3 ]. The use of AMPs in egg-laying poultry has proven effective in improving bird health, as they possess anti-inflammatory properties [ 4 ]. It can optimize intestinal mucosa while inhibiting/controlling the proliferation of pathogenic microorganisms, resulting in greater nutrient absorption and enhanced bird performance [ 5 ]. This class of peptides has shown promise as alternatives to conventional antibiotics in animal production systems, standing out for their antimicrobial and immunostimulatory capabilities. Studies indicate that AMPs can improve intestinal health, modulate the gastrointestinal microbiota, and enhance nutrient absorption and animal performance, representing a sustainable advancement in poultry production [ 6 ]. One of the major challenges faced by modern poultry farming is thermal stress caused by high temperatures, which not only compromises bird welfare but also negatively impacts their health and egg production [ 7 ]. Heat stress can impair the birds' immune system, leading to suppressed immune responses, atrophy of lymphoid organs such as the thymus and bursa of Fabricius, and alterations in immunity-related genes, making them more susceptible to diseases and parasites [ 5 , 8 , 9 , 10 ]. Prolonged exposure to high temperatures directly compromises productive performance, including feed intake, feed efficiency, and egg quality, significantly affecting the profitability of poultry systems [ 11 ]. Under heat stress, laying hens reduce their laying rate [ 12 ]. A reduction in eggshell weight and body weight of laying hens is observed in the first days of exposure to heat stress, but a reduction in Haugh unit values was observed after 2 to 4 weeks [ 13 ]. A consequence of elevated temperatures during the laying period is the increase in free radicals, which can lead to cell damage and inflammation in birds. Heat stress negatively affects the intestinal microbiota and lipid metabolism, suggesting that interventions modulating the microbiota may be beneficial to the animal [ 5 ]. For example, AMPs derived from lactic acid bacteria have been shown to combat a wide range of intestinal pathogens, promoting overall bird health [ 3 ]. Therefore, the use of AMPs in the diet of laying hens may be an effective strategy to counteract the adverse effects of heat stress by improving intestinal health, reducing inflammation, and enhancing nutrient absorption, thereby helping the organism overcome thermal stress conditions and improve egg quality. Specifically, the AMP Ctx(Ile 21 )-Ha, isolated from the skin secretion of the frog Boana albopunctata , a member of the Hylidae family [ 14 ], can destroy bacterial plasma membranes, leading to pathogen death [ 15 ]. The in vivo effect of microcapsules containing the AMP Ctx(Ile 21 )-Ha has been evaluated through dietary supplementation in laying hens previously infected with Salmonella enteritidis . A reduction in chick mortality during the first days of life was observed, along with a decrease in bacterial load, with a 69% reduction in mortality compared to the control group [ 8 ]. The same peptide reduced bacterial load in the liver of hens administered doses of 2.5 mg of Ctx(Ile 21 )-Ha/kg two days post-infection, indicating its efficacy against the infection [ 6 , 9 ]. Thus, considering the previously observed effects of the antimicrobial peptide (AMP) Ctx(Ile 21 )-Ha on bird health, the present study aimed to evaluate the use of this peptide as a protective agent in hens subjected to heat stress. The central hypothesis investigated is that the AMP Ctx(Ile 21 )-Ha, when properly formulated and supplemented in the feed of laying hens, can promote a protective response capable of mitigating the negative effects of thermal stress on the birds, resulting in improved zootechnical performance, egg quality, and blood parameters. Therefore, the objective of this study was to evaluate the effects of the antimicrobial peptide AMP Ctx(Ile 21 )-Ha on the body condition, egg production and quality, as well as the hematological parameters of laying hens exposed to heat stress. 2 Materials and Methods Synthesis, microencapsulation, and release assay of Ctx(Ile 21 )-Ha peptide Synthesis, purification and characterization of Ctx(Ile 21 )-Ha peptide The antimicrobial peptide Ctx(Ile 21 )-Ha was synthesized and manually obtained using the solid-phase peptide synthesis (SPPS) technique [ 16 ]. The polymer used as a support was Rink Amide resin. The synthesis protocol employed for the peptide was the Fmoc strategy, as shown in Fig. 1 . For the coupling of Fmoc-amino acids, the carboxyl groups were activated according to each method, as described below: 1) DIC/HOBT and 2) diisopropylethylamine DIEA/HBTU, for recoupling steps. The reaction time for each coupling method was two hours. The base-labile Fmoc group was removed by washing with 10% (w/v) piperazine in DMF/ethanol (9:1, v/v) for 20 minutes. To eliminate excess reagents and byproducts, washing cycles with DMF and DCM were performed. The cleavage between the peptide and the resin was carried out using TFA and a solution of ultrapure water and TIS. The filtrate obtained was lyophilized in a freeze-dryer (Liotop, model K108, Liobras, São Carlos, São Paulo, Brazil) to yield a white, flocculent material, representing the crude peptide. Purification was performed using high-performance liquid chromatography (HPLC) to separate synthesis impurities and analyze the purity of the peptide from the collected fractions. The peptide's purity level was determined in analytical mode using a C18 analytical column with a reversed phase (Shimadzu, model Prominence, Shimadzu Corporation, Kyoto, Japan), with solvents A (0.045% trifluoroacetic acid – TFA in ultrapure water) and B (0.036% TFA in acetonitrile – ACN), in runs with a gradient program from 5 to 95% solvent B over 30 minutes. The peptide was only used in subsequent steps when its purity level exceeded 95%. Peptide confirmation was achieved through mass spectrometry characterization using a triple quadrupole mass spectrometer (Shimadzu, model LCMS-8045, Shimadzu Corporation, Kyoto, Japan) with an ESI platform and direct injection, equipped with a Shimadzu UHPLC chromatograph, model Nexera X3 (Shimadzu Corporation, Kyoto, Japan) in scan mode. This tool allowed the determination of the molecular mass/charge ratio (MM/z) of the Ctx(Ile 21 )-Ha peptide, thereby ensuring its correct acquisition and characterization. Microencapsulation and coating of the Ctx(Ile 21 )-Ha peptide The microencapsulation processes were performed using the ionotropic gelation method [ 17 ]. A total of two microencapsulation processes were carried out: the first using 100 mg of the AMP Ctx(Ile 21 )-Ha and the second using 200 mg of the peptide, as shown in Fig. 2 . Briefly, the peptide was mixed with a solution of 10 g of sodium alginate and 500 mL of water, then homogenized using an Ultraturrax (Novatecnica, model NT 138, Novatecnica Laboratory Equipment, Piracicaba, São Paulo, Brazil) until a light brown broth was obtained. The crosslinking solution consisted of a 5% AlCl3 mixture placed on a magnetic stirrer to prevent aggregation. The peptide and alginate solution were dripped through a peristaltic pump coupled to a 200 µL pipette tip (Tecnopon, model LDP-101-3, MS Tecnopon Special Equipment Ltd., Piracicaba, São Paulo, Brazil) at a flow rate of 1 mL/min over the crosslinking solution. The microparticles were placed on a Büchner funnel connected to an Erlenmeyer flask and washed with 40 mL of ultrapure water, then subjected to drying in a continuous flow oven at 35°C for 24 hours. The coating of the microencapsulated particles was performed by immersing them in a solution of 10% hydroxypropyl methylcellulose phthalate (HPMCP), 25% ammonium hydroxide (NH4OH), 2.5% triethyl citrate, and 62.5% ultrapure water, in total of 250 mL of coating solution, followed by drying as described above. The coating was necessary to direct the microcapsules to the intestines of the hens. A Shimadzu spectrophotometer, model UV1800 (Shimadzu Corporation, Kyoto, Japan), was used to determine the microencapsulation efficiency by measuring the absorbance of the microcapsules in a 1:1 (w/v) solution of ultrapure water and ACN for 4 hours at a wavelength of 280 nanometers (nm). For this purpose, the Lambert-Beer law, represented in Eq. 1, was used as the basis for calculations: \(\:A=\:\epsilon\:lc\) Eq. (1) where: \(\:A\) is the absorbance; \(\:\epsilon\:\) represents the molar absorption coefficient, \(\:c\) is the molar concentration, and \(\:l\) corresponds to the optical path length (in cm) of the cuvette used. Peptide release assays in simulated solutions of avian intestinal and stomach fluids The simulated gastric fluid (SGF) was prepared with a volume of 10 mL of 0.1 mol/L HCl at pH 3.5. The pH was adjusted with 1 mol/L HCl. Simultaneously, the simulated intestinal fluid (SIF) was prepared using a solution of 10 mL of 0.1 mol/L HCl at pH 6, adjusted with 1 mol/L NaOH. A total mass of 50 mg of microcapsules were placed in contact with each simulated fluid. A mass of 50 mg of microcapsules were placed in contact with the SGF solution for 20 minutes at 42°C. The collection times for the solution were 1, 2, 5, 10, 15, and 20 minutes for SGF and 1, 2, 5, 10, 15, 20, 30, 45, and 60 minutes for SIF. The absorbance of the solutions was measured using a Shimadzu spectrophotometer, model UV1800 (Shimadzu Corporation, Kyoto, Japan) at 280 nm, which corresponds to the absorption wavelength of the tryptophan present in the primary sequence of the AMP Ctx(Ile 21 )-Ha [ 18 ] and used the Lambert-Beer equation (Eq. 1) to calculate the peptide concentration in the released solution. Experimental design This research was submitted and approved for the Animal Ethics Committee from the School of Sciences and Engineering at São Paulo State University (Unesp), Tupã Campus, under protocol number 01/2024. Two thermal treatments (Comfort and Heat Stress) and three different doses of peptides per treatment (Control, D1 and D2) were tested in this study. For each dosage, there were three experimental units (hens), totaling 18 Lohmann White laying hens aged 40 weeks, with beaks trimmed by the hot blade method, and weighing approximately 1.5 kg at the start of the experiment, sourced from a commercial farm. The experiment was conducted with a reduced number of animals, justified by low mass of the antimicrobial peptide Ctx(Ile 21 )-Ha produced, which is inherently associated with laboratory-scale peptide synthesis. Considering the absence of prior studies investigating the effects of this molecule on the health of laying hens, this experiment is characterized as a proof of concept. The hens were monitored for 35 consecutive days, with the first seven days allocated for acclimatization to the environmental chambers. The hens were divided into two groups of nine individuals each, with each group housed in a climatic chamber measuring 4.10 m x 3.50 m x 2.85 m (length, width, and height). Each hen was individually housed in a conventional cage with wire floor measuring 0.5 m × 0.5 m × 0.5 m (width × depth × height). During the acclimatization period, both groups were maintained at a constant air temperature of 26°C (measured value: 25.6 ± 0.94°C) from 4:00 AM to 9:30 PM and 24°C (measured value: 23.8 ± 0.85°C) from 9:30 PM to 4:00 AM. After the acclimatization period, one group remained under these environmental conditions, referred to as "Comfort." In the other climatic chamber, the air temperature was programmed to 35°C (measured value: 34.5 ± 0.72°C) from 4:00 AM to 9:30 PM and 26°C (measured value: 25.6 ± 0.94°C) from 9:30 PM to 4:00 AM, referred to as "Heat Stress." For temperatures of 24°C and 26°C, the mean and standard deviation of the relative humidity was 68 ± 5%, and for the temperature of 35°C, it was 56 ± 2%. Therefore, it was considered that in both treatments, relative humidity was controlled at around 60%. The heat stress conditions adopted in this study were based on the thermal comfort zone for laying hens, which typically ranges from 21°C to 28°C [ 11 ]. In both environmental treatments, the photoperiod consisted of 17.5 hours of artificial white light, activated at 4:00 AM and deactivated at 9:30 PM. Within each climatic chamber, the hens were randomly selected and distributed among the three peptide dosage treatments, as presented in Table 1 . Table 1 Treatments provided to hens according to peptide dosage and presence of thermal challenge. Dosages Description Thermal environment Presence of Ctx(Ile 21 )-Ha D0 Control Comfort No D1 2.5 mg/kg of feed Comfort Yes D2 5.0 mg/kg of feed Comfort Yes D0 Control Heat stress No D1 2.5 mg/kg of feed Heat stress Yes D2 5.0 mg/kg of feed Heat stress Yes The peptide dosage treatment was administered during the acclimatization period to allow the peptide to act within the hens' organisms, thereby creating a protective effect. The peptides were administered only once a week in a single dose, with each formulated peptide dose (D1 or D2) mixed into the feed. Individualized feeders enabled the weighing of leftover feed and peptides, and there was no competition for food. Management of hens and supplementation of microencapsulated Ctx(Ile 21 )-Ha peptide The hens were fed daily, with feed provided twice a day. The trough-type feeder was subdivided to match the width of the wire cages, allowing for individual monitoring of each hen's feed consumption throughout the experiment. A total of 110 g of feed per day per hen was provided, divided into two portions of 55 g at 8:30 AM and 4:00 PM. Water was offered ad libitum using nipple drinkers. The nutritional levels used (Table 2 ) were based on the Brazilian Tables for Poultry and Swine [ 19 ]. Table 2 Ingredients and nutritional composition of the experimental diets provided to laying hens. Ingredients Quantities (%) Corn 61.38 Soybean meal (46%) 24.00 Wheat bran 2.50 Pirius Emul Dry (plant-based lipid supplement) 1 0.30 Fine ground limestone (CaCO₃ source) 6.00 Coarse limestone gravel (CaCO₃ source) 4.50 Sodium chloride (common salt) 0.44 Toxin sequestrant (Calibrim Afla) 2 0.13 DL-methionine (99%) 0.05 L-Lysine (98%) 0.05 Pigment for egg yolk (PX1017 Pig laying) 3 0.25 Organic mineral and vitamin premix 4 0.10 Enzyme blend (phytase and carbohydrases) 5 0.10 Dicalcium phosphate (Free Phos) 0.20 Total 100.00 Nutritional levels Quantities (calculated) Metabolizable Energy (kcal/kg) 2790 Crude protein (%) 16.96 Total lysine (%) 0.91 Total methionine (%) 0.39 Total tryptophan (%) 0.21 Total threonine (%) 0.66 Calcium (%) 4.02 Available phosphorus (%) 0.44 Total phosphorus (%) 0.68 Ether extract (%) 2.90 Sodium (%) 0.18 Calcium/Potassium ratio 9.11 1 Prius Emul Dry: Commercial emulsified lipid supplement derived from plant-based ingredients, designed to enhance energy availability and nutrient absorption in poultry diets (Auster Nutri, Brazil; https://www.austernutri.com.br/prius/ ). 2 Calibrim Afla: Commercial aflatoxin sequestrant, typically based on bentonite or similar clay minerals. 3 PX1017 Pig laying: Commercial feed additive used for enhancing yolk pigmentation. 4 Organic mineral and vitamin premix: Provides essential vitamins (A, D₃, E, K₃, B₁, B₂, B₆, B₁₂, pantothenic acid, niacin, biotin, folic acid) and minerals (Zn, Mn, Fe, Cu, Se, I). 5 Enzyme blend: Includes phytase and carbohydrase enzymes to improve nutrient digestibility. The animals were weighed on the first day of housing in the climatic chambers using platform scales (B-160, Líder Co. Ltda, Araçatuba, São Paulo, Brazil). After the acclimatization period, the hens were weighed on days 1, 7, 14, 21, and 28 of the experiment. The leftover feed from each cage was monitored daily. Based on the daily feed consumption, the weekly Feed Conversion Ratio (FCR) was calculated for each dosage and temperature treatment (comfort or heat stress) using Eq. (2). \(\:FCR=\:\left(Total\:feed\:intake\:\right(g\left)\right)/\left(\:Total\:eggs\:weight\:\right(g\left)\right)\) Eq. (2) Analysis of egg production and egg quality Eggs were collected and counted daily. All eggs had their quality assessed daily. Specific gravity was determined following the methodology of Henriques et al. [ 20 ]. Egg weight, shell strength and thickness, albumen height, and Haugh unit were measured using a Digital Egg Tester (DET-6000, Nabel Co. Ltd., Kyoto, Japan®). Analysis of blood and physiological parameters Blood samples of 3 mL were collected on experimental days 1, 7, 14, 21, and 28, stored in EDTA K3 microtubes with anticoagulant, and 2.5 mL of serum clot activator was submitted to a complete blood count. Additionally, the levels of total proteins, pH, and electrolytes (Na+, K+, Cl-, and Ca 2+ ) were quantified. The rectal temperature of the hens was measured using a digital clinical thermometer from G-Tech (model TH1027, Joytech Healthcare Company, People’s Republic of China), and glucose levels were measured using 0.6 µL of fresh whole blood with an Accu-Check® Roche device (model Guide, Roche Diabetes Care Brazil, São Paulo, Brazil). Hemoglobin determination was performed by optical spectrophotometry at 540 nm using a colorimetric kit based on the cyanmethemoglobin (Drabkin) method and endpoint reaction (Labtest® Ref. 43 and Hemoglobin Standard Ref. 47). Replicates were performed using the Hemoglobin Standard Labtest®. After hemolysis, the samples were centrifuged in conical tubes to remove cellular nuclei before reading. Total protein determination was performed using manual refractometry with a veterinary protein and urine refractometer (Ion RHC-200®), with a measurement range of 0 to 12 g/dL and an accuracy of 0.2 g/dL. For cell counting, 10 µL of the EDTA-treated blood sample, previously homogenized, was diluted in 2 mL of the solution described by Natt-Herrick et al. [ 21 ]. Red blood cell and leukocyte counts were performed in a Neubauer chamber. Cells were diluted in 10 µL of blood to 2,000 µL of Natt-Herrick solution. The counting results were expressed as the number of cells using the correction factor described by Garcia-Navarro and Pachaly [ 22 ]. Leukocytes were counted in the four complete quadrants at the ends of the chamber. The results were expressed in mm³. Differential counting was performed by reading and differentiating one hundred blood leukocytes from microscopic examination of blood smears stained with Romanowsky-type dye. Electrolytes were measured using the selective electrode method, employing an IonPro® device, with linear ranges for Na+: 2.0 mmol/L, K+: 0.08 mmol/L, Ca2+: 0.04 mmol/L, Cl-: 2.0 mmol/L, and pH < 5.0%. Statistical analysis In the present study, a completely randomized design with a 2 × 3 factorial arrangement was used to evaluate two thermal environments (comfort: 26°C and 60% of relative humidity; and heat stress: 35°C and 60% of relative humidity) and three doses of Ctx(Ile 21 )-Ha antimicrobial peptide (D0: 0.0 mg/kg feed; D1: 2.5 mg/kg feed; and D2: 5.0 mg/kg feed), and their interaction effects. The main effects and their interactions were analyzed by ANOVA using the GLM procedure of Minitab 19 statistical software. Each cage was considered an experimental unit for egg production and quality performance. When significant differences (p ≤ 0.05) were found, the means were separated using Tukey's HSD test. The body condition of the laying hens (assessed weekly), and egg production (assessed daily) were analyzed and compared graphically. 3 Results Synthesis, purification and characterization of the Ctx(Ile 21 )-Ha peptide At the end of the process, an initial mass of peptidyl resin of 1,368 mg was obtained, and a final mass of 3,500 mg was achieved, resulting in a mass gain of 2,132 mg. After the coupling steps, the peptide cleavage occurred without complications, yielding a crude peptide mass of 2,700 mg, representing a synthesis efficiency of 77.14%. This efficiency is attributed to the inevitable loss of mass during solvent extractions due to the volatility of the products, errors related to the precision of supernatant extraction, and the temperature of the ether, which, despite being kept extremely cold, thaws very quickly, complicating the process. Following peptide purification, the chromatographic profile was generated using the Lab Solutions software coupled to the HPLC. Figures 3 and 4 show that the peptide exhibited a purity level greater than 95%, making it suitable for use in the experiment. Microencapsulation and coating of the antimicrobial peptide Ctx(Ile 21 )-Ha According to the calculations, it is evident that for the microparticles coated with HPMCP using an initial amount of 100 mg of the AMP Ctx(Ile 21 )-Ha, a total incorporation of 79.84 mg of this molecule was achieved, resulting in an encapsulation yield of 79.84% using the ionotropic gelation method. Similarly, for the microparticles coated with HPMCP using an initial amount of 200 mg of the peptide, a total incorporation of 116.94 mg of this molecule was obtained, resulting in an encapsulation yield of 58.47%. The calculations performed are detailed in the studies conducted by Roque-Borda et al. [ 8 ]. The lower incorporation efficiency occurred due to material loss during the dilution of the peptide with alginate in the microencapsulation solution preparation step. Additionally, the viscosity of the solution caused some of the material to adhere to the walls of the glass beaker, leading to partial loss of the solution. Release tests of microcapsules in stomach and intestinal solution The confirmation of the release in the simulated intestinal solution over time demonstrates that the peptide was released in the intestines of the hens for its complete action (Tables 3 and 4 ). This phenomenon was already reported by our research group in previous studies using the same microencapsulation process [ 8 , 9 ], demonstrating that the formulation is effective to release the peptide specifically in the intestine cavity. Although to a lesser extent, there was also a release of part of the microcapsule contents in the simulated gastric portion. The highest release occurred for dosage 2 at the 5-minute mark, with 9.74% of the peptide content being released. The highest release in the simulated intestinal portion of the hens occurred at the 5-minute mark for dosage 2. Table 3 Ctx(Ile 21 )-Ha peptide release profile in SGF from HPMCP coated microcapsules according to masses (%). 1’ 2’ 5’ 10’ 15’ 20’ 100 mg 3.02 4.48 3.53 5.46 0.00 0.00 200 mg 0.82 6.62 9.74 2.46 7.19 4.76 HPMCP 0.00 8.10 8.60 1.20 2.30 2.80 Table 4 Ctx(Ile 21 )-Ha peptide release profile in SIF from HPMCP coated microcapsules according to masses (%). 1’ 2’ 5’ 10’ 15’ 20’ 30’ 45’ 60’ 100 mg 20.67 14.11 93.28 12.10 13.86 12.10 12.86 10.59 23.95 200 mg 7.57 17.03 97.48 95.58 10.88 10.13 11.17 96.53 12.50 HPMCP 2.60 5.20 7.00 7.90 8.80 10.20 10.40 12.40 7.60 Analysis of feed intake, feed conversion and body condition No significant interaction (p > 0.05) between heat treatments and dosages was observed for total feed intake, total weight of eggs, and feed conversion (Table 5 ). Dosage levels did not affect these variables; however, under heat stress conditions, there was a reduction in feed intake and egg weight (p < 0.05). Table 5 Total feed intake, total weight of eggs, feed conversion, and rectal temperature of hens, for temperature conditions (Comfort: 26°C and 60% of relative humidity; Heat stress: 35°C and 60% of relative humidity) and experimental doses of peptides (D0: without peptide, D1: 2.5 mg/kg and D2: 5.0 mg/kg). Variables Experimental doses of peptides (P) Temperature conditions (T) P-value SEM 1 D0 D1 D2 Comfort Heat stress P T P*T Total feed intake (g) 3,243 3,084 3,091 3,4468 A 2,833 B 0.26 < 0.01 0.99 84.3 Total weight of eggs (g) 1,914 1,746 1,790 1,999 A 1,635 B 0.32 < 0.01 0.66 61.6 Feed conversion 1.69 1.80 1.73 1.73 1.76 0.51 0.69 0.35 0.03 Rectal temperature (°C) 41.1 41.1 41.1 41.0 B 41.2 A 0.84 < 0.01 0.93 0.04 1 Standard error of the mean. Means followed by different letters on the line differ statistically at 5% probability according to the F test. There was no difference in total feed intake, total egg weight, feed conversion, and rectal temperature in the groups that received different doses of peptide (Table 5 ). Among the groups under different temperature conditions, lower total feed intake (17.8% less) and lower total egg weight (18.3% less) were observed in the group subjected to heat stress. The rectal temperature of laying hens under heat stress was significantly higher than that of birds under comfort (p < 0.01). The body weight of the group under comfort conditions remained stable for all peptide doses, while the group under heat stress showed a reduction over time (Fig. 5 ). The standard error bars in the graph allow us to see that there was no difference between the peptide doses under the same environmental conditions throughout the experiment. In the stress treatment for D2, observing the intake data compared to the comfort treatment, heat stress affected the hens' feed intake as a consequence of the peptide supplementation. Egg production and egg quality In the comfort treatment, a decrease in production was observed for the group with a dose of 5 mg/kg of feed (0.92 ± 0.16 eggs/hen per day) compared to the groups with a dosage of 2.5 mg/kg of feed (D1) and 0 mg/kg of feed (D0 or control), both with 0.96 ± 0.11 eggs/hen per day (Fig. 6 ). In the heat stress treatment, the control group maintained a laying rate of 0.90 ± 0.15 eggs/hen per day, while the groups with a dosage of 5.0 mg/kg of feed (D2) and 2.5 mg/kg of feed (D1) presented laying rates of 0.85 ± 0.23 and 0.80 ± 0.23 eggs/hen per day, respectively. The loss in egg production of the heat-stressed group can be observed after the second administration of the peptide in the feed (downward arrows). This demonstrates a possible negative effect of this peptide on egg production under heat stress conditions. Significant differences were observed (p < 0.05) for the interaction of temperature treatments and peptide doses for the variables egg weight, shell thickness, shell resistance, and specific gravity) and no differences were observed for the variables Haugh unit and albumen height in the interaction or the treatments (Tabel 6). Table 6 Egg quality values for combined treatments of environmental condition (Comfort: 26°C and 60% of relative humidity; Heat stress: 35°C and 60% of relative humidity) and experimental doses of peptides (D0: control, without peptide; D1: 2.5 mg/kg of feed and D2: 5.0 mg/kg of feed). Variables Comfort Heat stress Comfort Heat stress P -value SEM 3 D0 D1 D2 D0 D1 D2 P 1 T 2 P*T Weight (g) 59.28 ab 60.64 a 58.65 b 54.95 c 53.97 c 54.25 c 59.48 A 54.47 B 0.14 < 0.01 0.01 0.20 Haugh Unit 87.19 86.62 88.24 89.37 88.44 85.60 87.34 88.01 0.44 0.62 0.06 0.44 Albumen height (mm) 7.73 7.62 7.87 7.85 7.83 7.46 7.74 7.73 0.77 0.87 0.19 0.07 Eggshell thickness (mm) 0.43 ab 0.41 bc 0.44 a 0.39 d 0.40 cd 0.37 e 0.43 A 0.39 B 0.24 < 0.01 < 0.01 0.01 Eggshell strength (kgf) 4.81 a 3.91 b 4.85 a 3.51 bc 4.05 b 3.31 c 4.58 A 3.60 B 0.25 < 0.01 < 0.01 0.05 Specific gravity (g/cm 3 ) 1081 a 1077 b 1081 a 1073 c 1077 b 1070 c 1080 A 1073 B 0.08 < 0.01 < 0.01 0.30 1 Experimental doses of peptides. 2 Temperature conditions. 3 Standard error of the mean. Means followed by different capital letters lowercase letters on the line differ statistically at 5% probability according to the F test. Means followed by different lowercase letters in the row differ statistically by 5% probability according to Tukey's test. With the exception of Haugh unit and albumen height, all other egg quality variables were worse in the heat-stressed group, when compared to the comfort group. Within the heat-stress treatment, no differences were observed between the D0 and D1 dosages, but the hens subjected to D2 had worse parameters of shell thickness and resistance. Under comfort conditions, the egg weight of the birds supplemented with D2 was lower, but the shell quality remained the same, when compared to the D0, with lower shell resistance and specific gravity being observed in the birds supplemented with D1. Blood parameters The mean blood parameters of the groups of hens subjected to the different doses of peptide administered for the comfort and heat stress treatments are shown in Table 7 . No differences (p > 0.05) were observed between the peptide dosages, nor interaction between temperature and peptide dosages. However, the variables sodium (Na+), chloride (Cl-), and calcium (nCa, iCa2+, and TCa) showed a reduction for the group under stress and without differentiation of peptide dosages. Table 7 Blood parameters for temperature conditions (Comfort: 26°C and 60% of relative humidity; Heat stress: 35°C and 60% of relative humidity) and experimental doses of peptides (D0: without peptide, D1: 2.5 mg/kg and D2: 5.0 mg/kg). Variable Experimental doses of peptides (P) Temperature conditions (T) P-value SEM 1 D0 D1 D2 Comfort Heat stress P T P*T Glucose (mg/dL) 194.79 199.83 193.69 196.20 195.75 0.23 0.82 0.76 1.49 Hemoglobin (g/dL) 8.68 8.49 8.96 8.80 8.61 0.26 0.49 0.21 0.11 Hematocrit (%) 28.85 27.44 28.75 28.50 28.30 0.11 0.81 0.76 0.30 Plasma proteins (g/dL) 7.92 8.35 7.71 8.19 7.79 0.09 0.09 0.99 0.12 Erythrocytes (10 6 /mm 3 ) 2.17 2.08 2.18 2.07 2.22 0.43 0.22 0.21 0.03 Leukocytes (10 3 /mm 3 ) 48.44 44.63 47.65 46.81 B 47.31 A 0.54 0.04 0.96 1.45 K + (mmol/L) 4.38 4.28 4.45 4.58 4.16 0.88 0.09 0.95 0.13 Na + (mmol/L) 150.76 150.65 151.38 152.72 A 149.10 B 0.92 0.02 0.83 0.74 Cl − (mmol/L) 119.71 120.03 120.06 120.84 A 119.01 B 0.61 < 0.01 0.06 0.20 nCa (mg/dL) 6.74 6.85 6.77 6.96 A 6.60 B 0.82 0.03 0.35 0.08 iCa 2+ (mg/dL) 6.08 6.20 6.14 6.29 A 5.98 B 0.75 0.04 0.35 0.07 TCa (mg/dL) 11.86 12.10 11.99 12.27 A 11.67 B 0.76 0.04 0.35 0.14 pH 7.25 7.26 7.26 7.25 A 7.27 B 0.94 0.43 0.97 0.01 1 Standard error of the mean. Means followed by different letters on the line differ statistically at 5% probability according to the F test. 4 Discussion This study investigated the effects of AMP supplementation on the production and quality of eggs from laying hens, with a focus on thermal stress conditions. The results indicated that, overall, AMP supplementation did not result in significant differences in egg production or quality. These findings highlight the need for further research on the use of AMPs in poultry farming, who emphasize the importance and relevance of innovative and sustainable practices for improving production systems [ 6 , 8 , 9 ]. No significant differences were observed in egg quality parameters, such as shell thickness and strength, even under thermal comfort conditions. Temperatures above this range negatively affected the hens' performance by impacting on their physiology, body temperature regulation, feed intake, and productivity. High temperatures reduce feed consumption, leading to lower weight gain and decreased egg production [ 11 ]. Additionally, heat causes metabolic and physiological changes, such as electrolyte imbalances and increased respiratory rate, further compromising productivity [ 11 , 12 , 23 ]. Although physiological indicators of stress, such as corticosterone levels, were not measured, the use of ambient temperature as a defining parameter is well supported in the literature to characterize heat stress in poultry [ 11 , 12 , 24 ]. The observed reduction in eggshell thickness in this study is associated with changes in calcium metabolism in birds, as shown in Table 7 . High temperatures disturb the acid-base balance and reduce the intake of essential nutrients, impairing the transport and absorption of calcium, essential for adequate shell formation, resulting in thinner and more fragile shells [ 11 , 25 ]. Heat stress is a critical factor that directly impacts the reproductive performance of laying hens and egg quality. Elevated environmental temperatures trigger a series of physiological and biochemical reactions in hens, significantly altering the quality of the eggs produced. This relationship is addressed by various authors who highlight how thermal stress affects eggshell mineralization and the composition of albumen and yolk [ 13 , 26 , 27 ]. These processes can impact calcium and phosphorus metabolism, which are essential for eggshell formation [ 28 ]. In this study, the AMP did not alter the measured electrolytes, and this impact was not observed in the experiment. AMP supplementation did not show significant results in mitigating the effects of thermal stress on body condition, egg production, or quality. Although some studies suggest that certain AMPs may benefit intestinal integrity and productivity in heat-stressed hens, these effects vary depending on composition, dosage, and experimental conditions [ 29 ]. Thus, the importance of effective thermal management strategies to mitigate the impacts of heat stress in poultry farming is emphasized. The results underscore the complexity of the effects of thermal stress on laying hens and the importance of integrated approaches that consider physiological, environmental, and nutritional aspects. In this context, the use of AMPs requires further investigation to elucidate their effects and potential applications, aligning with the demands of sustainable and efficient poultry farming [ 30 ]. The lack of specific analyses on the intestinal microbiota and inflammatory markers constitutes a limitation for the analysis of the effects of Ctx(Ile 21 )-Ha. The evaluation of these parameters would allow a more precise understanding of the mechanisms involved in immune responses and gastrointestinal integrity [ 31 , 32 ], so that future studies should consider the analysis of the microbiota profile and inflammatory indicators to deepen the understanding of the physiological effects of AMP. Additional investigations, such as tissue analyses, could provide insights into possible adverse effects on the digestive tract or other organs. A larger population study and investigation of the underlying mechanisms should be considered in future studies. In controlled experiments, statistical control of the effect of climate chambers should be added, alternating thermal environment treatments between chambers. 5 Conclusions The results demonstrated that, in the forms and concentrations tested, the AMP Ctx(Ile 21 )-Ha did not provide benefits for egg production or quality. There were no significant differences in blood parameters or egg quality with the use of Ctx(Ile 21 )-Ha. Future studies are essential to understand the mechanisms by which AMPs affect the metabolism and physiology of hens, including their impact on the intestinal microbiota and metabolic pathways related to nutrient consumption and absorption. Additionally, it will be important to investigate alternative administration methods and different concentrations of these compounds to maximize their potential benefits. This study expands the knowledge on the effects of AMP supplementation in laying hens under thermal stress conditions, aggregates more knowledge on antimicrobial peptides applied to poultry nutrition and shed light to the high potential of antimicrobial peptides as an effective alternative to conventional antibiotics, in line with current practices in sustainable and biosecurity poultry production. Abbreviations The following abbreviations are used in this manuscript: ACN – Acetonitrile AMPs – Antimicrobial peptides ANOVA – Analysis of variance D0 – Dosage, control, no antimicrobial peptide D1 – Dosage 1 (2.5 mg/kg of feed) D2 – Dosage 2 (5.0 mg/kg of feed) DCM – Dichloromethane DIC – Diisopropylcarbodiimide DIEA – Diisopropylethylamine hexafluorophosphate DMF – Dimethylformamide EDTA – Ethylenediaminetetraacetic acid ESI – Electrospray ionization FCR – Feed conversion ratio FMOC – Fluorenylmethyloxycarbonyl protecting group Hb – Hemoglobin HATU – 2-(1-H-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HBTU – 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOBT – N-hydroxybenzotriazole HPMCP – Hydroxypropyl methylcellulose phthalate iCa – Ionic calcium nCa – Normalized calcium NMM – N-methylmorpholine EDTA Ethylenediaminetetraacetic acid pH – Potential of hydrogen SGF – Simulated gastric fluid SIS – Simulated intestinal fluid SPFS – Solid-phase peptide synthesis TFA – Trifluoracetic acid TIS – Triisopropylsilane tCa – Total calcium UHPLC – Ultra-high performance liquid chromatography Declarations Ethics Statement The animal study protocol was approved by the Animal Use Ethics Committee of the São Paulo State University (protocol code 01/2024 and approval date 02/26/2024). Conflicts of Interest: The authors declare no conflicts of interest. Funding: Funding was provided by São Paulo Research Foundation – FAPESP (grants nº 2024/14650-1, 2020/14885-8, 2021/06706-9 and 2023/01834-4) and National Council for Scientific and Technological Development – CNPq (grant nº 304085/2021-9). Author Contribution L.E.V.N.B. contributed to the conceptualization, methodology, formal analysis, investigation, data curation, and both original draft preparation and review & editing of the manuscript. S.G.G. and W.Q.L. assisted with methodology and investigation and also contributed to the review and editing process. D.L.S aided in the conceptualization of the work and its subsequent review and editing. D.F.P., going further, contributed to the conceptualization, methodology, formal analysis, investigation, data curation, writing of the original draft, review & editing, visualization, and the acquisition of funding. 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Fermentation 10:540. 10.3390/ferment32ation10110540 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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11:50:13","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155766,"visible":true,"origin":"","legend":"","description":"","filename":"f393add848e14be0a2019259535ab2ef1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/3280d16202126f1b5f474661.xml"},{"id":96082351,"identity":"3664eb5e-1d90-4bf6-96d5-94e43665f910","added_by":"auto","created_at":"2025-11-17 11:50:13","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164599,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/7020ded5756ca79cf9e81897.html"},{"id":96082339,"identity":"d2504804-e1e0-45ec-bd51-d83d59fafa06","added_by":"auto","created_at":"2025-11-17 11:50:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":652263,"visible":true,"origin":"","legend":"\u003cp\u003eSolid-phase peptide synthesis (SPPS) technique flowchart employed in this work.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/346179412f9a22f21d092740.png"},{"id":96082335,"identity":"f56c6562-f6d3-4134-97da-3c821145a79b","added_by":"auto","created_at":"2025-11-17 11:50:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205614,"visible":true,"origin":"","legend":"\u003cp\u003eMicroencapsulation technique flowchart employed in this study.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/0c4254b3abb32f766e4daf88.png"},{"id":96247475,"identity":"b07d9088-0aca-4615-be97-e2f4655f3a4d","added_by":"auto","created_at":"2025-11-19 07:27:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57121,"visible":true,"origin":"","legend":"\u003cp\u003eChromatographic profile of the pure antimicrobial peptide Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/31c6f73993fca898a212edbb.png"},{"id":96248099,"identity":"34bc6640-eba9-4524-b8e0-b69577200740","added_by":"auto","created_at":"2025-11-19 07:28:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100066,"visible":true,"origin":"","legend":"\u003cp\u003eChromatographic profile of the pure antimicrobial peptide Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/d7c90db6e8eb90c9c2399f67.png"},{"id":96082342,"identity":"846ff9d2-d4d5-4590-96ee-3829500167f1","added_by":"auto","created_at":"2025-11-17 11:50:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":320168,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of the mean and standard error of the weight of laying hens during the experimental period, for each dosage tested (D0: 0.0 mg/kg of feed, D1: 2.5 mg/kg of feed, and D2: 5.0 mg/kg of feed) in each temperature treatment (Comfort: 26°C and 60% of relative humidity, and Heat stress: 35°C and 60% of relative humidity).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/50a6bdd3cdf1d4883059f552.png"},{"id":96249565,"identity":"fb46fd0d-e4f6-49eb-b548-97792362cd92","added_by":"auto","created_at":"2025-11-19 07:34:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":323906,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative production of eggs laid during the experimental period, for each dosage tested in each temperature treatment (Comfort: 26°C and 60% of relative humidity, and Heat stress: 35°C and 60% of relative humidity). The downward arrows indicate the days on which the peptide doses were applied (D0: 0.0 mg/kg of feed, D1: 2.5 mg/kg of feed, and D2: 5.0 mg/kg of feed).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/9192bff45244c24cd48f2c49.png"},{"id":100950986,"identity":"720a227c-2750-4e80-b57e-cd5515f638af","added_by":"auto","created_at":"2026-01-23 07:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3021683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7993048/v1/06c7e6ce-9370-44d0-8ae3-17a243cb351b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Feed supplementation with the Ctx(Ile 21 )-Ha antimicrobial peptide associated with heat stress affects laying hens’ productive performance and physiology","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAntimicrobial peptides (AMPs) are naturally occurring molecules, intrinsic to the innate immune system of virtually all organisms [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. AMPs belonging to the membranolytic class act by compromising the integrity of the plasma membrane of microorganisms, leading to their death and reducing the likelihood of microbial resistance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This mechanism of action has sparked researchers' interest in investigating the effects of these peptides in laying hens as novel zootechnical additives to enhance their performance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe use of AMPs in egg-laying poultry has proven effective in improving bird health, as they possess anti-inflammatory properties [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It can optimize intestinal mucosa while inhibiting/controlling the proliferation of pathogenic microorganisms, resulting in greater nutrient absorption and enhanced bird performance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This class of peptides has shown promise as alternatives to conventional antibiotics in animal production systems, standing out for their antimicrobial and immunostimulatory capabilities. Studies indicate that AMPs can improve intestinal health, modulate the gastrointestinal microbiota, and enhance nutrient absorption and animal performance, representing a sustainable advancement in poultry production [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne of the major challenges faced by modern poultry farming is thermal stress caused by high temperatures, which not only compromises bird welfare but also negatively impacts their health and egg production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Heat stress can impair the birds' immune system, leading to suppressed immune responses, atrophy of lymphoid organs such as the thymus and bursa of Fabricius, and alterations in immunity-related genes, making them more susceptible to diseases and parasites [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProlonged exposure to high temperatures directly compromises productive performance, including feed intake, feed efficiency, and egg quality, significantly affecting the profitability of poultry systems [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Under heat stress, laying hens reduce their laying rate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A reduction in eggshell weight and body weight of laying hens is observed in the first days of exposure to heat stress, but a reduction in Haugh unit values was observed after 2 to 4 weeks [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA consequence of elevated temperatures during the laying period is the increase in free radicals, which can lead to cell damage and inflammation in birds. Heat stress negatively affects the intestinal microbiota and lipid metabolism, suggesting that interventions modulating the microbiota may be beneficial to the animal [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For example, AMPs derived from lactic acid bacteria have been shown to combat a wide range of intestinal pathogens, promoting overall bird health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, the use of AMPs in the diet of laying hens may be an effective strategy to counteract the adverse effects of heat stress by improving intestinal health, reducing inflammation, and enhancing nutrient absorption, thereby helping the organism overcome thermal stress conditions and improve egg quality.\u003c/p\u003e\u003cp\u003eSpecifically, the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha, isolated from the skin secretion of the frog \u003cem\u003eBoana albopunctata\u003c/em\u003e, a member of the Hylidae family [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], can destroy bacterial plasma membranes, leading to pathogen death [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The in vivo effect of microcapsules containing the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha has been evaluated through dietary supplementation in laying hens previously infected with \u003cem\u003eSalmonella enteritidis\u003c/em\u003e. A reduction in chick mortality during the first days of life was observed, along with a decrease in bacterial load, with a 69% reduction in mortality compared to the control group [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The same peptide reduced bacterial load in the liver of hens administered doses of 2.5 mg of Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha/kg two days post-infection, indicating its efficacy against the infection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThus, considering the previously observed effects of the antimicrobial peptide (AMP) Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha on bird health, the present study aimed to evaluate the use of this peptide as a protective agent in hens subjected to heat stress. The central hypothesis investigated is that the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha, when properly formulated and supplemented in the feed of laying hens, can promote a protective response capable of mitigating the negative effects of thermal stress on the birds, resulting in improved zootechnical performance, egg quality, and blood parameters. Therefore, the objective of this study was to evaluate the effects of the antimicrobial peptide AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha on the body condition, egg production and quality, as well as the hematological parameters of laying hens exposed to heat stress.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003e\u003cb\u003eSynthesis, microencapsulation, and release assay of Ctx(Ile\u003c/b\u003e\u003csup\u003e\u003cb\u003e21\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)-Ha peptide\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eSynthesis, purification and characterization of Ctx(Ile\u003c/em\u003e\u003csup\u003e\u003cem\u003e21\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)-Ha peptide\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe antimicrobial peptide Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha was synthesized and manually obtained using the solid-phase peptide synthesis (SPPS) technique [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The polymer used as a support was Rink Amide resin. The synthesis protocol employed for the peptide was the Fmoc strategy, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For the coupling of Fmoc-amino acids, the carboxyl groups were activated according to each method, as described below: 1) DIC/HOBT and 2) diisopropylethylamine DIEA/HBTU, for recoupling steps. The reaction time for each coupling method was two hours. The base-labile Fmoc group was removed by washing with 10% (w/v) piperazine in DMF/ethanol (9:1, v/v) for 20 minutes. To eliminate excess reagents and byproducts, washing cycles with DMF and DCM were performed.\u003c/p\u003e\u003cp\u003eThe cleavage between the peptide and the resin was carried out using TFA and a solution of ultrapure water and TIS. The filtrate obtained was lyophilized in a freeze-dryer (Liotop, model K108, Liobras, S\u0026atilde;o Carlos, S\u0026atilde;o Paulo, Brazil) to yield a white, flocculent material, representing the crude peptide. Purification was performed using high-performance liquid chromatography (HPLC) to separate synthesis impurities and analyze the purity of the peptide from the collected fractions. The peptide's purity level was determined in analytical mode using a C18 analytical column with a reversed phase (Shimadzu, model Prominence, Shimadzu Corporation, Kyoto, Japan), with solvents A (0.045% trifluoroacetic acid \u0026ndash; TFA in ultrapure water) and B (0.036% TFA in acetonitrile \u0026ndash; ACN), in runs with a gradient program from 5 to 95% solvent B over 30 minutes. The peptide was only used in subsequent steps when its purity level exceeded 95%.\u003c/p\u003e\u003cp\u003ePeptide confirmation was achieved through mass spectrometry characterization using a triple quadrupole mass spectrometer (Shimadzu, model LCMS-8045, Shimadzu Corporation, Kyoto, Japan) with an ESI platform and direct injection, equipped with a Shimadzu UHPLC chromatograph, model Nexera X3 (Shimadzu Corporation, Kyoto, Japan) in scan mode. This tool allowed the determination of the molecular mass/charge ratio (MM/z) of the Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha peptide, thereby ensuring its correct acquisition and characterization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMicroencapsulation and coating of the Ctx(Ile\u003c/em\u003e\u003csup\u003e\u003cem\u003e21\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)-Ha peptide\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe microencapsulation processes were performed using the ionotropic gelation method [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A total of two microencapsulation processes were carried out: the first using 100 mg of the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha and the second using 200 mg of the peptide, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBriefly, the peptide was mixed with a solution of 10 g of sodium alginate and 500 mL of water, then homogenized using an Ultraturrax (Novatecnica, model NT 138, Novatecnica Laboratory Equipment, Piracicaba, S\u0026atilde;o Paulo, Brazil) until a light brown broth was obtained. The crosslinking solution consisted of a 5% AlCl3 mixture placed on a magnetic stirrer to prevent aggregation. The peptide and alginate solution were dripped through a peristaltic pump coupled to a 200 \u0026micro;L pipette tip (Tecnopon, model LDP-101-3, MS Tecnopon Special Equipment Ltd., Piracicaba, S\u0026atilde;o Paulo, Brazil) at a flow rate of 1 mL/min over the crosslinking solution. The microparticles were placed on a B\u0026uuml;chner funnel connected to an Erlenmeyer flask and washed with 40 mL of ultrapure water, then subjected to drying in a continuous flow oven at 35\u0026deg;C for 24 hours.\u003c/p\u003e\u003cp\u003eThe coating of the microencapsulated particles was performed by immersing them in a solution of 10% hydroxypropyl methylcellulose phthalate (HPMCP), 25% ammonium hydroxide (NH4OH), 2.5% triethyl citrate, and 62.5% ultrapure water, in total of 250 mL of coating solution, followed by drying as described above. The coating was necessary to direct the microcapsules to the intestines of the hens.\u003c/p\u003e\u003cp\u003eA Shimadzu spectrophotometer, model UV1800 (Shimadzu Corporation, Kyoto, Japan), was used to determine the microencapsulation efficiency by measuring the absorbance of the microcapsules in a 1:1 (w/v) solution of ultrapure water and ACN for 4 hours at a wavelength of 280 nanometers (nm). For this purpose, the Lambert-Beer law, represented in Eq.\u0026nbsp;1, was used as the basis for calculations:\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:A=\\:\\epsilon\\:lc\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEq.\u0026nbsp;(1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:A\\)\u003c/span\u003e\u003c/span\u003e is the absorbance; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\epsilon\\:\\)\u003c/span\u003e\u003c/span\u003e represents the molar absorption coefficient, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:c\\)\u003c/span\u003e\u003c/span\u003e is the molar concentration, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:l\\)\u003c/span\u003e\u003c/span\u003e corresponds to the optical path length (in cm) of the cuvette used.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePeptide release assays in simulated solutions of avian intestinal and stomach fluids\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe simulated gastric fluid (SGF) was prepared with a volume of 10 mL of 0.1 mol/L HCl at pH 3.5. The pH was adjusted with 1 mol/L HCl. Simultaneously, the simulated intestinal fluid (SIF) was prepared using a solution of 10 mL of 0.1 mol/L HCl at pH 6, adjusted with 1 mol/L NaOH. A total mass of 50 mg of microcapsules were placed in contact with each simulated fluid.\u003c/p\u003e\u003cp\u003eA mass of 50 mg of microcapsules were placed in contact with the SGF solution for 20 minutes at 42\u0026deg;C. The collection times for the solution were 1, 2, 5, 10, 15, and 20 minutes for SGF and 1, 2, 5, 10, 15, 20, 30, 45, and 60 minutes for SIF. The absorbance of the solutions was measured using a Shimadzu spectrophotometer, model UV1800 (Shimadzu Corporation, Kyoto, Japan) at 280 nm, which corresponds to the absorption wavelength of the tryptophan present in the primary sequence of the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and used the Lambert-Beer equation (Eq.\u0026nbsp;1) to calculate the peptide concentration in the released solution.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003e This research was submitted and approved for the Animal Ethics Committee from the School of Sciences and Engineering at S\u0026atilde;o Paulo State University (Unesp), Tup\u0026atilde; Campus, under protocol number 01/2024.\u003c/p\u003e\u003cp\u003eTwo thermal treatments (Comfort and Heat Stress) and three different doses of peptides per treatment (Control, D1 and D2) were tested in this study. For each dosage, there were three experimental units (hens), totaling 18 Lohmann White laying hens aged 40 weeks, with beaks trimmed by the hot blade method, and weighing approximately 1.5 kg at the start of the experiment, sourced from a commercial farm. The experiment was conducted with a reduced number of animals, justified by low mass of the antimicrobial peptide Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha produced, which is inherently associated with laboratory-scale peptide synthesis. Considering the absence of prior studies investigating the effects of this molecule on the health of laying hens, this experiment is characterized as a proof of concept.\u003c/p\u003e\u003cp\u003eThe hens were monitored for 35 consecutive days, with the first seven days allocated for acclimatization to the environmental chambers. The hens were divided into two groups of nine individuals each, with each group housed in a climatic chamber measuring 4.10 m x 3.50 m x 2.85 m (length, width, and height). Each hen was individually housed in a conventional cage with wire floor measuring 0.5 m \u0026times; 0.5 m \u0026times; 0.5 m (width \u0026times; depth \u0026times; height).\u003c/p\u003e\u003cp\u003eDuring the acclimatization period, both groups were maintained at a constant air temperature of 26\u0026deg;C (measured value: 25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u0026deg;C) from 4:00 AM to 9:30 PM and 24\u0026deg;C (measured value: 23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u0026deg;C) from 9:30 PM to 4:00 AM. After the acclimatization period, one group remained under these environmental conditions, referred to as \"Comfort.\" In the other climatic chamber, the air temperature was programmed to 35\u0026deg;C (measured value: 34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u0026deg;C) from 4:00 AM to 9:30 PM and 26\u0026deg;C (measured value: 25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u0026deg;C) from 9:30 PM to 4:00 AM, referred to as \"Heat Stress.\" For temperatures of 24\u0026deg;C and 26\u0026deg;C, the mean and standard deviation of the relative humidity was 68\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and for the temperature of 35\u0026deg;C, it was 56\u0026thinsp;\u0026plusmn;\u0026thinsp;2%. Therefore, it was considered that in both treatments, relative humidity was controlled at around 60%. The heat stress conditions adopted in this study were based on the thermal comfort zone for laying hens, which typically ranges from 21\u0026deg;C to 28\u0026deg;C [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In both environmental treatments, the photoperiod consisted of 17.5 hours of artificial white light, activated at 4:00 AM and deactivated at 9:30 PM.\u003c/p\u003e\u003cp\u003eWithin each climatic chamber, the hens were randomly selected and distributed among the three peptide dosage treatments, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTreatments provided to hens according to peptide dosage and presence of thermal challenge.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDosages\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThermal environment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePresence of \u003c/p\u003e\u003cp\u003eCtx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5 mg/kg of feed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.0 mg/kg of feed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHeat stress\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5 mg/kg of feed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHeat stress\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.0 mg/kg of feed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHeat stress\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe peptide dosage treatment was administered during the acclimatization period to allow the peptide to act within the hens' organisms, thereby creating a protective effect. The peptides were administered only once a week in a single dose, with each formulated peptide dose (D1 or D2) mixed into the feed. Individualized feeders enabled the weighing of leftover feed and peptides, and there was no competition for food.\u003c/p\u003e\u003cp\u003e\u003cb\u003eManagement of hens and supplementation of microencapsulated Ctx(Ile\u003c/b\u003e\u003csup\u003e\u003cb\u003e21\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)-Ha peptide\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe hens were fed daily, with feed provided twice a day. The trough-type feeder was subdivided to match the width of the wire cages, allowing for individual monitoring of each hen's feed consumption throughout the experiment. A total of 110 g of feed per day per hen was provided, divided into two portions of 55 g at 8:30 AM and 4:00 PM. Water was offered ad libitum using nipple drinkers. The nutritional levels used (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were based on the Brazilian Tables for Poultry and Swine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIngredients and nutritional composition of the experimental diets provided to laying hens.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eIngredients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eQuantities (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCorn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSoybean meal (46%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eWheat bran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePirius Emul Dry (plant-based lipid supplement)\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eFine ground limestone (CaCO₃ source)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCoarse limestone gravel (CaCO₃ source)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSodium chloride (common salt)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eToxin sequestrant (Calibrim Afla)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eDL-methionine (99%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eL-Lysine (98%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePigment for egg yolk (PX1017 Pig laying)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eOrganic mineral and vitamin premix\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eEnzyme blend (phytase and carbohydrases)\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eDicalcium phosphate (Free Phos)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e100.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNutritional levels\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eQuantities (calculated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMetabolizable Energy (kcal/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e2790\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e16.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal lysine (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal methionine (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal tryptophan (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal threonine (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e4.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAvailable phosphorus (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal phosphorus (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEther extract (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e2.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium/Potassium ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e9.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Prius Emul Dry: Commercial emulsified lipid supplement derived from plant-based ingredients, designed to enhance energy availability and nutrient absorption in poultry diets (Auster Nutri, Brazil; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.austernutri.com.br/prius/\u003c/span\u003e\u003cspan address=\"https://www.austernutri.com.br/prius/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Calibrim Afla: Commercial aflatoxin sequestrant, typically based on bentonite or similar clay minerals.\u003c/p\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e PX1017 Pig laying: Commercial feed additive used for enhancing yolk pigmentation.\u003c/p\u003e\u003cp\u003e\u003csup\u003e4\u003c/sup\u003e Organic mineral and vitamin premix: Provides essential vitamins (A, D₃, E, K₃, B₁, B₂, B₆, B₁₂, pantothenic acid, niacin, biotin, folic acid) and minerals (Zn, Mn, Fe, Cu, Se, I).\u003c/p\u003e\u003cp\u003e\u003csup\u003e5\u003c/sup\u003e Enzyme blend: Includes phytase and carbohydrase enzymes to improve nutrient digestibility.\u003c/p\u003e\u003cp\u003eThe animals were weighed on the first day of housing in the climatic chambers using platform scales (B-160, L\u0026iacute;der Co. Ltda, Ara\u0026ccedil;atuba, S\u0026atilde;o Paulo, Brazil). After the acclimatization period, the hens were weighed on days 1, 7, 14, 21, and 28 of the experiment. The leftover feed from each cage was monitored daily.\u003c/p\u003e\u003cp\u003eBased on the daily feed consumption, the weekly Feed Conversion Ratio (FCR) was calculated for each dosage and temperature treatment (comfort or heat stress) using Eq.\u0026nbsp;(2).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:FCR=\\:\\left(Total\\:feed\\:intake\\:\\right(g\\left)\\right)/\\left(\\:Total\\:eggs\\:weight\\:\\right(g\\left)\\right)\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEq.\u0026nbsp;(2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cb\u003eAnalysis of egg production and egg quality\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eEggs were collected and counted daily. All eggs had their quality assessed daily. Specific gravity was determined following the methodology of Henriques et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Egg weight, shell strength and thickness, albumen height, and Haugh unit were measured using a Digital Egg Tester (DET-6000, Nabel Co. Ltd., Kyoto, Japan\u0026reg;).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of blood and physiological parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBlood samples of 3 mL were collected on experimental days 1, 7, 14, 21, and 28, stored in EDTA K3 microtubes with anticoagulant, and 2.5 mL of serum clot activator was submitted to a complete blood count. Additionally, the levels of total proteins, pH, and electrolytes (Na+, K+, Cl-, and Ca\u003csup\u003e2+\u003c/sup\u003e) were quantified. The rectal temperature of the hens was measured using a digital clinical thermometer from G-Tech (model TH1027, Joytech Healthcare Company, People\u0026rsquo;s Republic of China), and glucose levels were measured using 0.6 \u0026micro;L of fresh whole blood with an Accu-Check\u0026reg; Roche device (model Guide, Roche Diabetes Care Brazil, S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e\u003cp\u003eHemoglobin determination was performed by optical spectrophotometry at 540 nm using a colorimetric kit based on the cyanmethemoglobin (Drabkin) method and endpoint reaction (Labtest\u0026reg; Ref. 43 and Hemoglobin Standard Ref. 47). Replicates were performed using the Hemoglobin Standard Labtest\u0026reg;. After hemolysis, the samples were centrifuged in conical tubes to remove cellular nuclei before reading.\u003c/p\u003e\u003cp\u003eTotal protein determination was performed using manual refractometry with a veterinary protein and urine refractometer (Ion RHC-200\u0026reg;), with a measurement range of 0 to 12 g/dL and an accuracy of 0.2 g/dL. For cell counting, 10 \u0026micro;L of the EDTA-treated blood sample, previously homogenized, was diluted in 2 mL of the solution described by Natt-Herrick et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRed blood cell and leukocyte counts were performed in a Neubauer chamber. Cells were diluted in 10 \u0026micro;L of blood to 2,000 \u0026micro;L of Natt-Herrick solution. The counting results were expressed as the number of cells using the correction factor described by Garcia-Navarro and Pachaly [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLeukocytes were counted in the four complete quadrants at the ends of the chamber. The results were expressed in mm\u0026sup3;. Differential counting was performed by reading and differentiating one hundred blood leukocytes from microscopic examination of blood smears stained with Romanowsky-type dye.\u003c/p\u003e\u003cp\u003eElectrolytes were measured using the selective electrode method, employing an IonPro\u0026reg; device, with linear ranges for Na+: 2.0 mmol/L, K+: 0.08 mmol/L, Ca2+: 0.04 mmol/L, Cl-: 2.0 mmol/L, and pH\u0026thinsp;\u0026lt;\u0026thinsp;5.0%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the present study, a completely randomized design with a 2 \u0026times; 3 factorial arrangement was used to evaluate two thermal environments (comfort: 26\u0026deg;C and 60% of relative humidity; and heat stress: 35\u0026deg;C and 60% of relative humidity) and three doses of Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha antimicrobial peptide (D0: 0.0 mg/kg feed; D1: 2.5 mg/kg feed; and D2: 5.0 mg/kg feed), and their interaction effects. The main effects and their interactions were analyzed by ANOVA using the GLM procedure of Minitab 19 statistical software. Each cage was considered an experimental unit for egg production and quality performance. When significant differences (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) were found, the means were separated using Tukey's HSD test. The body condition of the laying hens (assessed weekly), and egg production (assessed daily) were analyzed and compared graphically.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cb\u003eSynthesis, purification and characterization of the Ctx(Ile\u003c/b\u003e\u003csup\u003e\u003cb\u003e21\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)-Ha peptide\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the end of the process, an initial mass of peptidyl resin of 1,368 mg was obtained, and a final mass of 3,500 mg was achieved, resulting in a mass gain of 2,132 mg. After the coupling steps, the peptide cleavage occurred without complications, yielding a crude peptide mass of 2,700 mg, representing a synthesis efficiency of 77.14%. This efficiency is attributed to the inevitable loss of mass during solvent extractions due to the volatility of the products, errors related to the precision of supernatant extraction, and the temperature of the ether, which, despite being kept extremely cold, thaws very quickly, complicating the process.\u003c/p\u003e\u003cp\u003eFollowing peptide purification, the chromatographic profile was generated using the Lab Solutions software coupled to the HPLC. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show that the peptide exhibited a purity level greater than 95%, making it suitable for use in the experiment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroencapsulation and coating of the antimicrobial peptide Ctx(Ile\u003c/b\u003e\u003csup\u003e\u003cb\u003e21\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)-Ha\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAccording to the calculations, it is evident that for the microparticles coated with HPMCP using an initial amount of 100 mg of the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha, a total incorporation of 79.84 mg of this molecule was achieved, resulting in an encapsulation yield of 79.84% using the ionotropic gelation method. Similarly, for the microparticles coated with HPMCP using an initial amount of 200 mg of the peptide, a total incorporation of 116.94 mg of this molecule was obtained, resulting in an encapsulation yield of 58.47%. The calculations performed are detailed in the studies conducted by Roque-Borda et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The lower incorporation efficiency occurred due to material loss during the dilution of the peptide with alginate in the microencapsulation solution preparation step. Additionally, the viscosity of the solution caused some of the material to adhere to the walls of the glass beaker, leading to partial loss of the solution.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRelease tests of microcapsules in stomach and intestinal solution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe confirmation of the release in the simulated intestinal solution over time demonstrates that the peptide was released in the intestines of the hens for its complete action (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This phenomenon was already reported by our research group in previous studies using the same microencapsulation process [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], demonstrating that the formulation is effective to release the peptide specifically in the intestine cavity. Although to a lesser extent, there was also a release of part of the microcapsule contents in the simulated gastric portion. The highest release occurred for dosage 2 at the 5-minute mark, with 9.74% of the peptide content being released.\u003c/p\u003e\u003cp\u003eThe highest release in the simulated intestinal portion of the hens occurred at the 5-minute mark for dosage 2.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCtx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha peptide release profile in SGF from HPMCP coated microcapsules according to masses (%).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e100 mg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e200 mg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHPMCP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCtx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha peptide release profile in SIF from HPMCP coated microcapsules according to masses (%).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e30\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e45\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e60\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e100 mg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e12.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e23.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e200 mg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e97.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e96.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e12.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHPMCP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e12.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of feed intake, feed conversion and body condition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNo significant interaction (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between heat treatments and dosages was observed for total feed intake, total weight of eggs, and feed conversion (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Dosage levels did not affect these variables; however, under heat stress conditions, there was a reduction in feed intake and egg weight (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTotal feed intake, total weight of eggs, feed conversion, and rectal temperature of hens, for temperature conditions (Comfort: 26\u0026deg;C and 60% of relative humidity; Heat stress: 35\u0026deg;C and 60% of relative humidity) and experimental doses of peptides (D0: without peptide, D1: 2.5 mg/kg and D2: 5.0 mg/kg).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eExperimental\u0026nbsp;doses of\u0026nbsp;peptides\u0026nbsp;(P)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eTemperature\u0026nbsp;conditions\u0026nbsp;(T)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eD1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eD2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHeat\u0026nbsp;stress\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eP*T\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal feed intake\u0026nbsp;(g)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3,243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3,084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3,091\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3,4468 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2,833 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e84.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal weight of\u0026nbsp;eggs\u0026nbsp;(g)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,914\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,746\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,790\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1,999 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1,635 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e61.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFeed\u0026nbsp;conversion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRectal temperature\u0026nbsp;(\u0026deg;C)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e41.0 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e41.2 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Standard error of the mean. Means followed by different letters on the line differ statistically at 5% probability according to the F test.\u003c/p\u003e\u003cp\u003eThere was no difference in total feed intake, total egg weight, feed conversion, and rectal temperature in the groups that received different doses of peptide (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Among the groups under different temperature conditions, lower total feed intake (17.8% less) and lower total egg weight (18.3% less) were observed in the group subjected to heat stress. The rectal temperature of laying hens under heat stress was significantly higher than that of birds under comfort (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003eThe body weight of the group under comfort conditions remained stable for all peptide doses, while the group under heat stress showed a reduction over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The standard error bars in the graph allow us to see that there was no difference between the peptide doses under the same environmental conditions throughout the experiment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the stress treatment for D2, observing the intake data compared to the comfort treatment, heat stress affected the hens' feed intake as a consequence of the peptide supplementation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEgg production and egg quality\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the comfort treatment, a decrease in production was observed for the group with a dose of 5 mg/kg of feed (0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 eggs/hen per day) compared to the groups with a dosage of 2.5 mg/kg of feed (D1) and 0 mg/kg of feed (D0 or control), both with 0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 eggs/hen per day (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In the heat stress treatment, the control group maintained a laying rate of 0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 eggs/hen per day, while the groups with a dosage of 5.0 mg/kg of feed (D2) and 2.5 mg/kg of feed (D1) presented laying rates of 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 and 0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 eggs/hen per day, respectively. The loss in egg production of the heat-stressed group can be observed after the second administration of the peptide in the feed (downward arrows). This demonstrates a possible negative effect of this peptide on egg production under heat stress conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSignificant differences were observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for the interaction of temperature treatments and peptide doses for the variables egg weight, shell thickness, shell resistance, and specific gravity) and no differences were observed for the variables Haugh unit and albumen height in the interaction or the treatments (Tabel 6).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEgg quality values for combined treatments of environmental condition (Comfort: 26\u0026deg;C and 60% of relative humidity; Heat stress: 35\u0026deg;C and 60% of relative humidity) and experimental doses of peptides (D0: control, without peptide; D1: 2.5 mg/kg of feed and D2: 5.0 mg/kg of feed).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"16\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eHeat\u0026nbsp;stress\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHeat\u0026nbsp;stress\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c15\" namest=\"c13\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c16\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eD1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eD2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eD0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eD1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eD2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eP\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003eT\u0026nbsp;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c15\"\u003e\u003cp\u003eP*T\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWeight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59.28\u0026nbsp;\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60.64 \u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.65\u0026nbsp;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e54.95\u0026nbsp;\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e53.97\u0026nbsp;\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e54.25 \u0026nbsp;\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e59.48 \u0026nbsp;\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e54.47 \u0026nbsp;\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHaugh Unit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e87.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e86.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e88.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e89.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e88.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e85.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e87.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e88.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlbumen height (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEggshell thickness (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.43 \u0026nbsp;\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.41 \u0026nbsp;\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.44 \u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.39 \u0026nbsp;\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.40 \u0026nbsp;\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.37 \u0026nbsp;\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.43 \u0026nbsp;\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.39 \u0026nbsp;\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEggshell strength (kgf)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.81 \u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.91 \u0026nbsp;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.85 \u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.51 \u0026nbsp;\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.05 \u0026nbsp;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.31 \u0026nbsp;\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.58 \u0026nbsp;\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.60 \u0026nbsp;\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecific gravity (g/cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1081 \u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1077 \u0026nbsp;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1081 \u0026nbsp;\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1073 \u0026nbsp;\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1077 \u0026nbsp;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1070 \u0026nbsp;\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1080 \u0026nbsp;\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1073 \u0026nbsp;\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Experimental doses of peptides. \u003csup\u003e2\u003c/sup\u003e Temperature conditions. \u003csup\u003e3\u003c/sup\u003e Standard error of the mean. Means followed by different capital letters lowercase letters on the line differ statistically at 5% probability according to the F test. Means followed by different lowercase letters in the row differ statistically by 5% probability according to Tukey's test.\u003c/p\u003e\u003cp\u003eWith the exception of Haugh unit and albumen height, all other egg quality variables were worse in the heat-stressed group, when compared to the comfort group. Within the heat-stress treatment, no differences were observed between the D0 and D1 dosages, but the hens subjected to D2 had worse parameters of shell thickness and resistance. Under comfort conditions, the egg weight of the birds supplemented with D2 was lower, but the shell quality remained the same, when compared to the D0, with lower shell resistance and specific gravity being observed in the birds supplemented with D1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBlood parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mean blood parameters of the groups of hens subjected to the different doses of peptide administered for the comfort and heat stress treatments are shown in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. No differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were observed between the peptide dosages, nor interaction between temperature and peptide dosages. However, the variables sodium (Na+), chloride (Cl-), and calcium (nCa, iCa2+, and TCa) showed a reduction for the group under stress and without differentiation of peptide dosages.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBlood parameters for temperature conditions (Comfort: 26\u0026deg;C and 60% of relative humidity; Heat stress: 35\u0026deg;C and 60% of relative humidity) and experimental doses of peptides (D0: without peptide, D1: 2.5 mg/kg and D2: 5.0 mg/kg).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eExperimental doses of peptides (P)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eTemperature conditions (T)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eD1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eD2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eComfort\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHeat stress\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eP*T\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlucose (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e194.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e199.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e193.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e196.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e195.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHematocrit (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e28.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlasma proteins (g/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eErythrocytes (10\u003csup\u003e6\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeukocytes (10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e48.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e44.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46.81 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.31 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e (mmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e (mmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e150.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e150.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e151.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e152.72 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e149.10 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCl\u003csup\u003e\u0026minus;\u003c/sup\u003e (mmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e119.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e120.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e120.84 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e119.01 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003enCa (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.96 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.60 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eiCa\u003csup\u003e2+\u003c/sup\u003e (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.29 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.98 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTCa (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.27 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11.67 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.25 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.27 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Standard error of the mean. Means followed by different letters on the line differ statistically at 5% probability according to the F test.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study investigated the effects of AMP supplementation on the production and quality of eggs from laying hens, with a focus on thermal stress conditions. The results indicated that, overall, AMP supplementation did not result in significant differences in egg production or quality.\u003c/p\u003e\u003cp\u003eThese findings highlight the need for further research on the use of AMPs in poultry farming, who emphasize the importance and relevance of innovative and sustainable practices for improving production systems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. No significant differences were observed in egg quality parameters, such as shell thickness and strength, even under thermal comfort conditions.\u003c/p\u003e\u003cp\u003eTemperatures above this range negatively affected the hens' performance by impacting on their physiology, body temperature regulation, feed intake, and productivity. High temperatures reduce feed consumption, leading to lower weight gain and decreased egg production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, heat causes metabolic and physiological changes, such as electrolyte imbalances and increased respiratory rate, further compromising productivity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although physiological indicators of stress, such as corticosterone levels, were not measured, the use of ambient temperature as a defining parameter is well supported in the literature to characterize heat stress in poultry [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe observed reduction in eggshell thickness in this study is associated with changes in calcium metabolism in birds, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. High temperatures disturb the acid-base balance and reduce the intake of essential nutrients, impairing the transport and absorption of calcium, essential for adequate shell formation, resulting in thinner and more fragile shells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHeat stress is a critical factor that directly impacts the reproductive performance of laying hens and egg quality. Elevated environmental temperatures trigger a series of physiological and biochemical reactions in hens, significantly altering the quality of the eggs produced. This relationship is addressed by various authors who highlight how thermal stress affects eggshell mineralization and the composition of albumen and yolk [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These processes can impact calcium and phosphorus metabolism, which are essential for eggshell formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, the AMP did not alter the measured electrolytes, and this impact was not observed in the experiment.\u003c/p\u003e\u003cp\u003eAMP supplementation did not show significant results in mitigating the effects of thermal stress on body condition, egg production, or quality. Although some studies suggest that certain AMPs may benefit intestinal integrity and productivity in heat-stressed hens, these effects vary depending on composition, dosage, and experimental conditions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, the importance of effective thermal management strategies to mitigate the impacts of heat stress in poultry farming is emphasized.\u003c/p\u003e\u003cp\u003eThe results underscore the complexity of the effects of thermal stress on laying hens and the importance of integrated approaches that consider physiological, environmental, and nutritional aspects. In this context, the use of AMPs requires further investigation to elucidate their effects and potential applications, aligning with the demands of sustainable and efficient poultry farming [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe lack of specific analyses on the intestinal microbiota and inflammatory markers constitutes a limitation for the analysis of the effects of Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha. The evaluation of these parameters would allow a more precise understanding of the mechanisms involved in immune responses and gastrointestinal integrity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], so that future studies should consider the analysis of the microbiota profile and inflammatory indicators to deepen the understanding of the physiological effects of AMP.\u003c/p\u003e\u003cp\u003eAdditional investigations, such as tissue analyses, could provide insights into possible adverse effects on the digestive tract or other organs. A larger population study and investigation of the underlying mechanisms should be considered in future studies. In controlled experiments, statistical control of the effect of climate chambers should be added, alternating thermal environment treatments between chambers.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThe results demonstrated that, in the forms and concentrations tested, the AMP Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha did not provide benefits for egg production or quality. There were no significant differences in blood parameters or egg quality with the use of Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha.\u003c/p\u003e\u003cp\u003eFuture studies are essential to understand the mechanisms by which AMPs affect the metabolism and physiology of hens, including their impact on the intestinal microbiota and metabolic pathways related to nutrient consumption and absorption. Additionally, it will be important to investigate alternative administration methods and different concentrations of these compounds to maximize their potential benefits. This study expands the knowledge on the effects of AMP supplementation in laying hens under thermal stress conditions, aggregates more knowledge on antimicrobial peptides applied to poultry nutrition and shed light to the high potential of antimicrobial peptides as an effective alternative to conventional antibiotics, in line with current practices in sustainable and biosecurity poultry production.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\u003cp\u003eACN \u0026ndash; Acetonitrile\u003c/p\u003e\u003cp\u003eAMPs \u0026ndash; Antimicrobial peptides\u003c/p\u003e\u003cp\u003eANOVA \u0026ndash; Analysis of variance\u003c/p\u003e\u003cp\u003eD0 \u0026ndash; Dosage, control, no antimicrobial peptide\u003c/p\u003e\u003cp\u003eD1 \u0026ndash; Dosage 1 (2.5 mg/kg of feed)\u003c/p\u003e\u003cp\u003eD2 \u0026ndash; Dosage 2 (5.0 mg/kg of feed)\u003c/p\u003e\u003cp\u003eDCM \u0026ndash; Dichloromethane\u003c/p\u003e\u003cp\u003eDIC \u0026ndash; Diisopropylcarbodiimide\u003c/p\u003e\u003cp\u003eDIEA \u0026ndash; Diisopropylethylamine hexafluorophosphate\u003c/p\u003e\u003cp\u003eDMF \u0026ndash; Dimethylformamide\u003c/p\u003e\u003cp\u003eEDTA \u0026ndash; Ethylenediaminetetraacetic acid\u003c/p\u003e\u003cp\u003eESI \u0026ndash; Electrospray ionization\u003c/p\u003e\u003cp\u003eFCR \u0026ndash; Feed conversion ratio\u003c/p\u003e\u003cp\u003eFMOC \u0026ndash; Fluorenylmethyloxycarbonyl protecting group\u003c/p\u003e\u003cp\u003eHb \u0026ndash; Hemoglobin\u003c/p\u003e\u003cp\u003eHATU \u0026ndash; 2-(1-H-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate\u003c/p\u003e\u003cp\u003eHBTU \u0026ndash; 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate\u003c/p\u003e\u003cp\u003eHOBT \u0026ndash; N-hydroxybenzotriazole\u003c/p\u003e\u003cp\u003eHPMCP \u0026ndash; Hydroxypropyl methylcellulose phthalate\u003c/p\u003e\u003cp\u003eiCa \u0026ndash; Ionic calcium\u003c/p\u003e\u003cp\u003enCa \u0026ndash; Normalized calcium\u003c/p\u003e\u003cp\u003eNMM \u0026ndash; N-methylmorpholine EDTA Ethylenediaminetetraacetic acid\u003c/p\u003e\u003cp\u003epH \u0026ndash; Potential of hydrogen\u003c/p\u003e\u003cp\u003eSGF \u0026ndash; Simulated gastric fluid\u003c/p\u003e\u003cp\u003eSIS \u0026ndash; Simulated intestinal fluid\u003c/p\u003e\u003cp\u003eSPFS \u0026ndash; Solid-phase peptide synthesis\u003c/p\u003e\u003cp\u003eTFA \u0026ndash; Trifluoracetic acid\u003c/p\u003e\u003cp\u003eTIS \u0026ndash; Triisopropylsilane\u003c/p\u003e\u003cp\u003etCa \u0026ndash; Total calcium\u003c/p\u003e\u003cp\u003eUHPLC \u0026ndash; Ultra-high performance liquid chromatography\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics Statement\u003c/h2\u003e\u003cp\u003e The animal study protocol was approved by the Animal Use Ethics Committee of the S\u0026atilde;o Paulo State University (protocol code 01/2024 and approval date 02/26/2024).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eFunding was provided by S\u0026atilde;o Paulo Research Foundation \u0026ndash; FAPESP (grants n\u0026ordm; 2024/14650-1, 2020/14885-8, 2021/06706-9 and 2023/01834-4) and National Council for Scientific and Technological Development \u0026ndash; CNPq (grant n\u0026ordm; 304085/2021-9).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.E.V.N.B. contributed to the conceptualization, methodology, formal analysis, investigation, data curation, and both original draft preparation and review \u0026amp; editing of the manuscript. S.G.G. and W.Q.L. assisted with methodology and investigation and also contributed to the review and editing process. D.L.S aided in the conceptualization of the work and its subsequent review and editing. D.F.P., going further, contributed to the conceptualization, methodology, formal analysis, investigation, data curation, writing of the original draft, review \u0026amp; editing, visualization, and the acquisition of funding. E.F.V. contributed to the conceptualization, methodology, data curation, original draft writing, review \u0026amp; editing, visualization and, importantly, also secured funding.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\u003cp\u003eWe would like to thank the Research Group of the Laboratory of Precision Animal Science, the group \"PeSEAp\" - Peptides, synthesis, optimization and applied studies, both from the S\u0026atilde;o Paulo State University, Tup\u0026atilde; campus, as well as Yasmin Saegusa Tadayozzi and Wendell Queiroz Leite for kindly help with the experiments.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e\u003cp\u003eDocuments will be made available by the corresponding author([email protected]) upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDecker AP, Mechesso AF, Wang GS (2022) Expanding the landscape of amino acid-rich antimicrobial peptides: Definition, deployment in nature, implications for peptide design and therapeutic potential. 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Fermentation 10:540. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ferment32ation10110540\u003c/span\u003e\u003cspan address=\"10.3390/ferment32ation10110540\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Animal welfare. Antimicrobial peptides. Blood count. Electrolytes. Shell quality","lastPublishedDoi":"10.21203/rs.3.rs-7993048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7993048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the effects of the supplementation of the antimicrobial peptide Ctx(Ile\u003csup\u003e21\u003c/sup\u003e)-Ha in laying hens under heat stress. A completely randomized design with a 2 \u0026times; 3 factorial arrangement was used to evaluate two thermal environments (Comfort: 26\u0026deg;C and Heat stress: 35\u0026deg;C) and three peptide dosages (D1: 0.0mg/kg, D1: 2.5mg/kg and D2: 5.0mg/kg of feed). The peptide was synthesized by solid phase peptide synthesis, reaching 77.14% of efficiency, with purity degree\u0026thinsp;\u0026gt;\u0026thinsp;95%. Microencapsulation via ionotropic gelation showed efficiencies of 79.84% (for 100 mg of peptide) and 58.47% (for 200 mg), with \u003cem\u003ein vitro\u003c/em\u003e assays confirming controlled intestinal release. Eighteen 40-week-old hens were housed in climate-controlled chambers and supplemented with the antimicrobial peptide. The ingestion of microcapsules was 100% for D1 under both conditions and D2 under heat stress, but only 63.2% for D2 under comfort. Hens receiving 5.0 mg/kg of feed (D2) showed reduced feed intake, especially under heat stress, and slight weight loss, although egg production remained stable. Declines in eggshell quality were observed. Blood analyses showed no significant differences in glucose, hemoglobin, erythrocytes, proteins, or electrolytes for the different peptide doses tested. In conclusion, peptide did not significantly improve egg production or quality. Although no major physiological or productive changes were observed, this study contributes to the development of more sustainable poultry farming strategies.\u003c/p\u003e","manuscriptTitle":"Feed supplementation with the Ctx(Ile 21 )-Ha antimicrobial peptide associated with heat stress affects laying hens’ productive performance and physiology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 11:50:08","doi":"10.21203/rs.3.rs-7993048/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65f6204c-2b90-4fa2-aa9f-1099eae66b62","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-22T17:39:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 11:50:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7993048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7993048","identity":"rs-7993048","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}