Comparison of the effect of different levels of hot red pepper or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens

Comparison of the effect of different levels of hot red pepper or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens | 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 Comparison of the effect of different levels of hot red pepper or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens Zahra Jahanbakhsh Rad, Sara Mirzaie Goudarzi, Abbas Farahavar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5745925/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 experiment compared the effect of different levels of hot red pepper (HRP) or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens from 41 to 49 weeks of age. Two hundred laying hens of Nick-Chick strain were allocated to five treatments, five replicates, and eight hens for each replicate in a completely randomized design. The treatments were: 1. control (basal diet without any HRP or synthetic pigment), 2, 3 and 4. control + 1, 2 or 3% HRP, respectively, and 5. control + synthetic pigment. The results showed that treatments had no significant effect on performance and egg quality traits ( P > 0.05), except for yolk colour and a* value (redness) that were significantly higher in the birds that were fed with HRP levels or synthetic pigment than control group ( P < 0.001). The higher immunoglobulin T and G was observed by HRP levels in comparison to the control ( P < 0.001). The inclusion of 3% HRP increased immunoglobulin M than other treatments ( P < 0.05). Plasma glucose and cholesterol showed a reduction in birds fed with HRP levels ( P < 0.001). However, plasma total protein and albumin were higher in the birds that were fed 2% and 3% HRP ( P < 0.001). In conclusion, HRP could be recommended as a safe and efficient alternative pigment to diet of laying hens due to its favorable effects on yolk pigmentation, immunity, and blood parameters. Egg production egg quality pigmentation yolk colour immunity layers Figures Figure 1 Introduction Heightened consumer apprehension regarding food safety has the potential to drive a transformation in animal feed production, leading to an increased utilization of phytobiotic feed additives. Herbs, categorized as phytobiotic feed supplements, include secondary metabolites or bioactive compounds such as flavonoids, alkaloids, carotenoids, and phenols (Mohanraj et al. 2018 ; Ivanišová et al. 2020 ). Such phytogenic characteristics are crucial for producing healthy and safe eggs, thereby enhancing consumer health and safety (Samantaray and Nayak 2022 ). Hot red pepper is a source of carotenoids called xanthophylls; so, it can be used as a natural yolk colourant that is safe and potentially healthier for egg consumers than synthetic or artificial colorants (Saleh et al. 2021 ). Egg yolk colour is an important characteristic that determines the choice of eggs by consumers (Spasevski et al. 2017 ). Associated with the geographical area, tradition, and culture, consumers prefer a particular colour of egg yolk (Marounek and Pebriansyah 2018 ). In industrial conditions in cages, many poultry farmers incorporate synthetic pigments into laying hens’ diets to achieve the desired orange colour in egg yolks, as corn grain lacks sufficient xanthophyll content (Hencken 1992 ). However, natural resources have been introduced to replace synthetic pigments because of rising public concern regarding using synthetic pigments (Spasevski et al. 2016 ; Sözcü 2019 ). The necessity for natural sources of pigmentation is a key consideration in poultry farming (Chaves et al. 2022 ). Conversely, the excessive use of certain synthetic pigments, like canthaxanthin, in poultry diets has been limited in certain regions due to potential harm to human vision caused by crystal formation in the retina. Hot red pepper is highly favored as a natural feed additive due to its natural, non-toxic, residue-free nature, and widespread availability (Munglang and Vidyarthi 2019 ). Hot red pepper contains capsaicin, a terpenoid compound with antibacterial properties (Singletary 2011 ). Capsaicin, an essential element of chili peppers, is involved in immune system modulation (Sri Sreshtaa et al. 2021 ). Capsinoids possess various biochemical and pharmacological attributes, such as anti-inflammatory, antioxidant, anticarcinogenic, and antiallergenic, effects, potentially lowering the risk of cancer (Munglang and Vidyarthi 2019 ). The humoral immune response plays a crucial role in immunity and can be evaluated by measuring antibody production and activity. Studies have been conducted regarding adding HRP to laying hen diets to assess its impact on productivity and egg quality. However, the results presented varied and inconclusive findings (Hanif et al. 2024 ). More studies should explore the various effects of HRP supplementation in poultry diets, particularly in terms of immunomodulatory activity (Abd El-Hack et al. 2022 ). Hence, we compared different levels of HRP or synthetic pigment concerning productive performance, egg quality, humoral and cellular immunity, and blood parameters in laying hens aged 41 to 49 weeks of age. Materials and methods Chemical analysis All ingredients were ground by a laboratory mill with a 1 mm screen (Retsch Model Z-I, Stuttgart, Germany). Dry matter, total ash, N, and ether extract contents were determined according to AOAC (2005). The amino acid contents were determined by NIRs (Evonik-Degussa, Hanau, Germany). Birds, Diets, and Management A total of 200 laying hens of Nick-Chick strain were allocated to five treatments, five replicates of eight hens each in a completely randomized design from 41 to 49 weeks of age. The following treatments were considered: 1: control (basal diet based on corn, wheat and soybean meal with no natural or synthetic pigment), 2, 3, and 4: control + 1, 2, or 3% HRP, respectively, and 5: control + synthetic pigment. Hot red pepper was purchased in dry form from the local market, then it was ground into a fine powder and mixed with experimental diets. Avixanthin 100 was used in the diet as a synthetic pigment, which contains 100 g of canthaxanthin/kg, and added 25 g/1000 kg in the experimental diets following the manufacturer’s guidelines (Lohmann company, China). Feed and water were available. Light was available for 16 hours per day. The temperature was 22 ± 2 ◦ C. Before the start of the experiment, all laying hens were treated (for ten days) on a basal diet to deplete the pigment reserves of birds, so that was the effect of the pigments on the colour of the egg yolk the same in all treatments. The basal diet was formulated according to Nick-Chick's laying hen nutritional recommendation guide (Nick Chick 2017). Table 1 shows the chemical analysis of the ingredients and Table 2 indicates the ingredient composition, calculated and determined chemical analysis of the diets. Productive performance Egg weight (EW) and egg production (EP) were noted once a day, whereas feed intake (FI) was determined weekly and they were applied to calculate the average daily feed intake (ADFI), egg mass (EM), and feed conversion ratio (FCR) both periodically and cumulatively. Egg quality Shape index, yolk index, yolk colour, Haugh unit, eggshell thickness, eggshell weight, and egg shell ratio was recorded every two weeks. Also, the evaluation of L*, a*, and b* (yellowness) values in the egg yolk was done by a Hunter Lab colorimeter (HP, China) device. Antibody Response Sheep red blood cells (SRBC) were employed as T-dependent antigens to assess the antibody response in treated hens. Each group comprised ten hens (two hens per replicate; n=5) that received intravenous injections of SRBC (3% suspension in PBS, 1ml per bird) at 47 weeks of age, followed by an SRBC suspension booster injection seven days later. Blood sampling was done at 7 and 14 days following the initial injection. Serum from each specimen was collected and heat-inactivated by incubating at 56 °C for 30 minutes. The serum samples were then assessed for IgT, IgM, and IgG anti-SRBC antibodies using the method outlined by Delhanty and Solomon (1966) and Qureshi and Havenstein (1994). To summarize, 50 µl of serum was mixed with an equal amount of PBS in the first column of a 96-well U-shaped plate and then incubated at 37 °C for 30 minutes. Following this, a serial dilution (1:2) was carried out, and 50 µl of 3% SRBC suspension was added to the wells. After 30 min incubation, IgT titers were read and several endpoints well with SRBC agglutination were recorded for each sample. For mercaptoethanol-sensitive (IgM) antibody measurement, 0.01 M mercaptoethanol (50 µl; Merck) in PBS was applied instead of PBS alone and continued according to the above-mentioned procedure. The antibody titers against SRBC were determined by expressing them as the base 2 logarithm of the reciprocal of the highest serum dilution causing complete agglutination. The differentiation between IgT and IgG levels was considered to be equal to the IgM antibody level. Toe web swelling index The toe web swelling index (cellular immune response) was was measured by basophilic skin sensitization reaction method (cutaneous basophilic hypersensitivity; CBH) or toe web swelling (TWS). At 49 weeks of age, two hens from every replicate (n=5) were randomly chosen and the thickness of the webs on the right and left foot (between 3rd and 4th toe web) was measured using a micrometer (INSIZE Company, China) as zero hour (Corrier and Deloach 1990). Then, 0.1 ml solution with phosphate saline was injected into the right foot membrane and 0.1 ml phytohemagglutinin (PHA) solution was injected into the left foot membrane in the same area. The thickness of the webbing at the injection sites was measured using a digital micrometer at 12, 24, and 72 hours post-injection. The swelling reaction was assessed by calculating the difference between the thickness of the toe web on the left foot after PHA injection and the thickness of the toe web on the right foot after PBS injection. The cellular immune response index was calculated utilizing the formula provided by Corrier (1990). In this equation, W represents the interdigital skin thickness index, RLP indicates the swelling of the left foot toe web after PHA injection, RL signifies the swelling of the left foot toe web before PHA injection, LLB denotes the swelling of the right foot toe web after PBS injection, and LL represents the swelling of the right foot toe web prior to PBS injection. Blood parameters When the hens reached 49 weeks of age, blood samples were obtained from two birds for each replicate (n=5) in heparinized tubes. These specimens underwent centrifugation for 15 minutes at 2000× g, followed by extracting and storing the plasma at –20 ºC until further biochemical assessment. Plasma levels of glucose, cholesterol, total protein and albumin, were quantified by commercial kits (Pars Azmun, Iran) by spectrophotometer (Varian Spectrophotometer AA220, Australia). Statistical analysis The study was done following a completely randomized design with five treatments and five replicates, each consisting of eight hens. The main effects were assessed through ANOVA utilizing the GLM procedure of the SAS software (2012). The Tukey’s test compared mean values ( P 0.05, Table 3). Egg quality Table 4 indicates the effect of treatments on egg quality traits. Shape index, yolk index, b* value, Haugh unit, eggshell thickness, shell weight, and eggshell ratio were not influenced by treatments ( P > 0.05). The use of 3% HRP significantly improved egg yolk colour compared to other treatments ( P < 0.001). Increased HRP levels or synthetic pigment in the diet decreased the L* value of the yolk colour in the control group ( P < 0.001). Also, a higher a* value of the yolk colour was observed by 3% HRP inclusion in the diet ( P 0.05). Figure 1 compares the egg yolk colour of the control group with various levels of HRP or synthetic pigment. Humoral immune response and toe web swelling index The lower total IgT, IgG, and IgM were observed in the controls compared to other treatments ( P < 0.001). In contrast, total IgT and IgG were higher at 2 and 3% HRP ( P < 0.001). Also, a higher IgM was observed at 3% HRP inclusion in the diet ( P 0.05). The highest index of toe web swelling was observed at 24 h after injection ( P < 0.001). No interaction was found between treatment and time for toe web swelling response to PHA (Table 6). Blood parameters Plasma glucose and cholesterol levels significantly decreased in hens fed with various concentrations of HRP ( P < 0.001). However, plasma total protein and albumin increased by using 2 and 3% of HRP inclusion in the diet ( P < 0.001; Table 7). Discussion The analytical values of the HRP used in the current study were: DM 93.21, total ash 7.83, crude protein 9.52, crude fat 8.50, and crude fiber 15.40%. Within the previously reported range of Niksiar (2018), the chemical compounds of HRP included: DM 94.60, total ash 8.68, crude protein 11.45, crude fat 7.00, and crude fiber 16.20%. In the current study, none of the performance parameters was affected by treatments. In agreement with our results, Aktaran Bala et al. (2020) declared that adding 4% of dried red pepper powder to the diet of laying hens compared to the control did not affect egg production rate, egg mass, and FCR. In another study, Tadeo and Gaffud (2024) reported that HRP inclusion in the diet did not affect the performance of native layers. Contrary to the results obtained in this experiment, Aikpitanyi and Imasuen (2023) reported that hen day production and egg mass were affected by including 1% red pepper, with significantly higher in this group compared to the control group. According to Zhao et al. (2023) dietary canthaxanthin supplementation significantly improved egg production and promoted ovulation of Huaixiang broiler breeders. Carotenoids, like ß-carotene, are able to promote the synthesis of egg yolk precursors. Hen age at trial start, pepper form, inclusion level, and supplementation duration can be the reasons among different studies. The impact of the experimental treatments on egg shape index and yolk index did not show statistical significance, aligning with the findings of Spasevski et al. (2017). As expected, the research demonstrated that the yolk pigment conversion efficiency from red pepper to yolk colour with increased levels of HRP supplementation, consistent with the findings of Abou-Elkhair et al. (2018) and Sozcu (2019). In the current study, laying hens that were fed with 1% HRP had the same yolk colour as the synthetic pigment group (10.30 vs. 10.83, respectively) (Figure 1). In another research, the improvement in egg colour scores was significant with red pepper powder. Nevertheless, other parameters related to egg quality unaffected (Aktaran Bala et al. 2020). It is suggested that phytochemicals’ capacity to readily accumulate in animal-derived products can potentially confer antioxidant, regenerative, and protective benefits from these substances (Akdemir et al. 2012). Aikpitanyi and Imasuen (2023) reported yolk colour improvements with red pepper inclusion in laying hens’ diets. Hanif et al. (2024) declared that diets treated with red pepper showed higher egg yolk colour than the control group. Carotenoid deposition considerably improved the colour of yolks. Xanthophylls play a crucial role in the pigmentation of bird egg yolks, accounting for 88 to 92% of the total carotenoids present in the yolk. The overall carotenoid concentration and ratio of the red carotenoids (astaxanthin, capsanthin, and canthaxanthin) to yellow carotenoids (zeaxanthin and lutein) affect the colour of the egg yolk and its components (yellowness or redness). Lokaewmanee et al. (2010) have shown a lower value of L* value for the paprika group which is in agreement with the current study. It has been reported that the lower yolk L* value was due to the accumulation of pigments in the yolk (Grashorn and Steinberg, 2002). Niksiar (2018) reported that the use of 1% HRP or synthetic pigment improved a* value compared to the control group in the egg yolk. Santos-Bocanegra et al. (2004) reported that capsaicin causes a dark red colour in the yolk. Lutein is a carotenoid that causes yellowing in egg yolks. Zeaxanthin is a carotenoid that causes a yellow colour and some of its derivatives such as zeaxanthin produce orange colour (Marusich et al. 1960). Despite this, the yolk yellowness value (b*) remained unaffected by the experimental treatments, consistent with the findings of Niksiar (2018). Consumer inclinations towards egg yolk colour can exhibit substantial diversity across countries and even within specific regions of a country. For instance, in the United States, the favored yolk colour typically ranges between 7 and 10 on the Roche yolk colour fan (RYCF), whereas certain European or Asian nations lean towards higher values, typically between 10 and 14 (Galobart et al. 2004). Eggshell thickness, shell weight, and shell ratio did not exhibit significant changes throughout the study period. Factors such as the calcium content and source in the diet, hen age, feed intake, and egg production percentage are crucial elements influencing eggshell quality (An et al. 2016). There have been limited animal studies exploring the potential chemo preventive properties of capsaicin (Abd El-Hack et al. 2022). The quantity of antibodies generated against sheep erythrocytes serves as an indicator of the status of the humoral immune system. Hot peppers are abundant in vitamin C, which plays a significant role in alleviating heat stress in birds (Abd El-Hack et al. 2019). Additionally, they are a plentiful source of carotenoids like vitamins E, C, and provitamin A (ß-carotene), famous for their antioxidant properties that combat the harmful impacts of free radicals, including cellular damage (oxidative stress) in broilers (Tawfeek et al. 2014). In agreement with the results outlined in this study, Sözcü (2010) noted a trend towards elevated IgG levels in layers that were provided with red pepper supplementation. This trend could be associated with the bioactive elements present in red pepper, notably capsaicin, as mixtures of active compounds in spicy red pepper have shown chemo preventive and chemotherapeutic benefits (Puvača et al. 2015). Chew and Park (2004) illustrated in human peripheral blood that the immune response modulation by dietary carotenoids operates through cytokine secretion. Cytokines possess the ability to exhibit adjuvant-like properties akin to vaccine stimulation, thereby triggering a certain immune reaction (Wigley and Kaiser 2003). Aktaran Bala et al. (2020) noted that cytokine levels, with the exception of transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and interleukin-4 (IL-4), remained constant following supplementation with 4% red pepper powder. The therapeutic attributes identified in the Capsicum genus were associated with their bioactive elements, notably capsaicin. Dihydro-capsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin represent some of the capsaicinoid compounds present in hot red pepper (Abou-Elkhair et al. 2018). The positive capsicum impacts on poultry nutrition could be linked to capsaicin, known for its bactericidal properties against intestinal pathogens like Clostridium spp, Salmonella spp, and Escherichia coli (Salem et al. 2021). The capsaicin content in red pepper releases neuropeptides such as substance P, neurokinin A, vasoactive intestinal peptide, and peptides associated with the calcitonin gene. These neuropeptides, upon reaching the intestine, stimulate smooth muscle activity, tissue regeneration, and immune processes, and safeguard against mucosal damage in the gastrointestinal tract (Sterner and Szallasi 1999). The decrease in heterophil percentage due to red pepper powder supplementation might be linked to the stress-reducing properties of pepper, which could have a positive impact on the hens’ immune system (Aktaran Bala et al. 2020). Inflammation of the foot of birds can indicate the ability of the cell-mediated immunity system. The PHA skin test can assess delayed-type cellular immune responses across different vertebrates and has been a standard tool in immunoecological investigations. Research indicated a notable presence of lymphocytes, neutrophils, eosinophils, basophils, and macrophages in tissues injected with PHA compared to control tissues injected with saline (Turmelleet al. 2010). The inflammation index reflects the stimulating impact on the generation of T lymphocyte clones specific to antigens. We assessed the immunomodulatory effects of HRP or synthetic pigment in measuring delayed-type cellular immune responses through the PHA skin test in laying hens. To our knowledge, limited research exists on the impact of HRP or synthetic pigment on cellular immunity in laying hens. The findings revealed that the web swelling unaffected by either natural or synthetic pigment, thus not confirming the hypothesis of our study. We hypothesized that the influence of HRP on cellular immunity is linked to its biologically active components, particularly capsaicin. Capsaicin has been recognized for its immune-modulating properties, capable of stimulating dendritic cells, a crucial cell type in the vertebrate immune system (Basu and Srivastava 2005). In opposition to our findings, Beltran et al. (2007) showed that capsaicin significantly improves both the quantity and activation status of skin-derived dendritic cells in draining lymph nodes. One plausible reason for the immune-modulating impact of capsaicin is its potential to regulate the cellular immune response by activating antigen-presenting cells, T-helper cells, and cytotoxic lymphocytes. However, there exists a distinct contrast between the outcomes from in vivo and in vitro assessments. For instance, previous in vitro studies indicated that capsaicin hindered IL-2 generation by inducing capacitative calcium entry into Jurkat cells, thereby diminishing T-cell functionality and cytokine generation (Park et al. 2004). Aktaran Bala et al. (2020) reported an elevation in serum TGF-β and TNF-α levels in the group administered with red pepper powder compared to the control group, suggesting a potential immune-enhancing effect of red pepper. TGF-β plays a crucial role in T cell development and exhibits significant anti-inflammatory properties, while TNF-α, generated by T cells, macrophages, and NK cells, serves as the primary regulatory cytokine in initiating an immune reaction during endothelial activation and acute inflammation (Zhang and Tracey 1998). The expected decline in response over time was due to the control of physiological processes related to the mobilization of cells that fight against external substances. The increased occurrence of basophils and eosinophils 24 hours after injection has been recognized as a cutaneous basophil hypersensitivity reaction (Stadecker 1977). Increased levels of HRP in the diet decreased blood glucose and cholesterol compared to the synthetic pigment or control group. In agreement with the data reported herein, Dougnon et al. (2014) revealed a decrease in the blood glucose levels of broilers receiving a diet treated with HRP in comparison to those receiving the control diet. Decreasing in plasma glucose due to increased insulin secretion by HRP. Capsaicin had a stimulant effect similar to epinephrine. Epinephrine has a strong impact on stimulating hepatic gluconeogenesis and causes the release of sugar in the blood, so this effect of epinephrine stimulates the release of insulin and subsequently decreases blood sugar (Kawada et al. 1986). Furthermore, Puvaca et al. (2015) validated its efficacy in lowering blood cholesterol levels. In agreement with the findings of the present study, Soliman and Al-Afifi (2020) observed a decrease in plasma cholesterol in broilers with 1% and 2% red pepper supplementation. The reduction in serum cholesterol can be attributed to active compounds like capsaicin and phenolic compounds present in red pepper, which inhibit the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) activity, thereby inhibiting cholesterol production (Lokaewmanee et al. 2010). Capsaicin found in hot pepper has been associated with decreased levels of LDL cholesterol and a lowered risk of heart-related mortality (Tadeo and Gaffud 2024). Moreover, the cholesterol-reducing impact of red pepper might be associated with its role to activate the hepatic cholesterol-7-hydroxylase enzyme, essential for converting cholesterol into bile acids, thereby lowering blood cholesterol levels (Adedoyin et al. 2019). This influence could be attributed to the inhibitory effects of the active components in hot red pepper on hepatic HMG-CoA activity, a pivotal enzyme in cholesterol synthesis, resulting in decreased cholesterol generation and absorption in the intestine. In the current study, diets supplemented with 2 or 3% HRP increased the total protein and albumin values. Our discovery aligns with results of Hassan et al. (2023) where supplementation with red pepper oil led to a notable rise in plasma albumin and total protein levels, credited to its bioactive components. Similarly, Husdfdggsein (2010) demonstrated an enhancement in serum total protein levels with 1.0% and 2.0% HRP in the diet compared to the control group. This effect could be attributed to improved food digestion by altering pH levels and potentially increasing acid production. Serum total proteins serve as crucial indicators of the body’s health status and productivity due to their various physiological functions. Consistent with our results, Abbas and Thamir Al-Jrrah (2022) who reported an increase in serum total protein levels when using red bell pepper fruit powder compared to the control group in Japanese quail layers. In general, adding different levels of HRP or synthetic pigment inclusion in the diet did not influence the performance of laying hens. Using a minimum of 1% HRP could be substituted with synthetic pigment to improve the egg yolk colour. One of the advantages of HRP compared to synthetic pigment was the improvement of the immune system and blood parameters which these effects were more pronounced with increasing levels of HRP. So, it is recommended to use HRP in the diet, which may be valuable for consumers. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Ethical approval All animal procedures were approved by the Ethics Committee of Bu-Ali Sina University, Faculty of Agriculture (approvement date: 17th of December 2018). Consent to participate The authors of this work have agreed to participate in present study. Consent for publication All authors agree with the content of this manuscript and gave their consent to submit the current manuscript to Tropical Animal Health and Production. Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by ZJR, SMG, AF, and FGK. Material preparation, data collection and analysis were performed by ZJR and SMG. The first draft of the manuscript was written by ZJR and SMG and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 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Potr S J F Sci 14:573–579. http://dx.doi.org/10.5219/1370 Kawada T, Hagihara K, Iwai K (1986) Effects of capsaicin on lipid metabolism in rats fed a high fat diet. J Nutr 116(7):1272–1278. https://doi.org/10.1093/jn/116.7.1272 Lokaewmanee K, Yamauchi KE, Komori T, Saito K (2010) Effects on egg yolk colour of paprika or paprika combined with marigold flower extracts. Ital J Anim Sci 9(4):e67.‏ https://doi.org/10.4081/ijas.2010.e67 Marounek M, Pebriansyah A (2018) Use of carotenoids in feed mixtures for poultry: a review. Agric trop subtrop 51(3):107–111. http://dx.doi.org/10.1515/ats-2018-0011 Marusich W, De Ritter E, Bauernfeind JC (1960) Evaluation of carotenoid pigments for coloring egg yolks. Poult Sci 39(6):1338–1345. https://doi.org/10.3382/ps.0391338 Mohanraj K, Karthikeyan BS, Vivek-Ananth RP, Bharath Chand RP, Aparna SR, Mangalapandi P, Samal A (2018)IMPPAT.A curated database of Indian medicinal plants, phytochemistry and therapeutics. Sci Rep 8(1):4329. https://doi.org/10.1038/s41598-018-22631-z Munglang NN, Vidyarthi VK (2019) Hot red pepper powder supplementation diet of broiler chicken- a review. Livest Res Int 7(3):159–167. NICK CHICK. Management Guide (2017). H&N international GmbH, Am Seedeich 9/27472, Cuxhaven/Germany. https://www.hn-int.com/eng/download/management-guides.php. Niksiar S (2018) Effect of insoluble fibre, fat source and natural or synthetic pigments on performance, egg quality and nutrient digestibility in laying hens. Thesis submitted for master of science in field of poultry nutrition. Bu-Ali Sina University, Hamedan, Iran. Park JY, Kawada T, Han IS, Kim BS, Goto T, Takahashi N, Fushiki T, Kurata T, Yu R (2004) Capsaicin inhibits the production of tumor necrosis factor alpha by LPS-stimulated murine macrophages, RAW 264.7: a PPARgamma ligand-like action as a novel mechanism. FEBS Lett 572(1-3):266–270. ‏ 10.1016/j.febslet.2004.06.084 Puvača N, Ljubojević D, Kostadinović LJ, Lević J, Nikolova N, Miscevic B, Könyves T, Lukač D, Popović S (2015) Species and herbs in broiler nutrition: hot red pepper ( Capsicum annuum L.) and its mode of action. World's Poult Sci 71(4):683–688. http://dx.doi.org/10.1017/S004393391500241X Qureshi MA, Havenstein GB (1994) A comparison of the immune performance of a 1991 commercial broiler with a 1957 randombred strain when fed “typical” 1957 and 1991 broiler diets. Poult Sci 73(12):1805–1812.‏ https://doi.org/10.3382/ps.0731805 Saleh AA, Gawish E, Mahmoud SF, Amber K, Awad W, Alzawqari MH, Shukry M, Abdel-Moneim AME (2021) Effect of natural and chemical colorant supplementation on performance, egg-quality characteristics, yolk fatty-acid profile, and blood constituents in laying hens. Sustainability 13(8):4503. https://doi.org/10.3390/su13084503 Salem HM, Ismael E, Shaalan M (2021) Evaluation of the effects of silver nanoparticles against experimentally induced necrotic enteritis in broiler chickens. Int J Nanomedicine 16:6783–6796. https://doi.org/10.2147/IJN.S319708 Samantaray L, Nayak Y (2022) The influences of black pepper, turmeric and fennel essential oils supplementation in feed on egg quality characteristics of layers. J Anim Health Prod 10(4):522–528. DOI http://dx.doi.org/10.17582/journal.jahp/2022/10.4.522.528 Santos-Bocanegra E, Ospina-Osorio X, Oviedo-Rondon EO (2004) Evaluation of xanthophylls extracted from tagetes erectus (marigold flower) and capsicum sp. (Red pepper paprika) as a pigment for egg-yolks compare with synthetic pigments. Int J Poult Sci 3(11):685–689. https://doi.org/10.3923/ijps.2004.685.689 SAS Institute (2012) SAS ® qualification tools user’s guide version 9.1.2. Cary, NC: SAS Institute Inc. Singletary K (2011) Red pepper: Overview of potential health benefits. Nutr Today 46 (1):33–47. https://doi.org/10.1097/NT.0B013E3182076FF2 Soliman NK, AlAfifi ShF (2020) The productive performance, intestinal bacteria and histomorphology of broiler chicks fed diets containing hot red pepper. Egypt Poult Sci J 40(1):345–357. Sözcü A ( 2019) Effects of supplementing layer hen diet with red pepper ( capsicum annuum L.) powder as natural yolk colourant on laying performance, pigmentation of yolk, egg quality and serum immunoglobulin levels. J Poult Res 16(2):80–85. https://doi.org/10.34233/jpr.600605 Spasevski N, Čolović D, Rakita S, Ikonić P, Đuragić O, Banjac V, Vukmirović Đ (2016) Fatty acid composition and β-carotene content in egg yolk of laying hens fed with linseed, paprika and marigold. Contemp Agric 65(1-2):15–22.‏ https://doi.org/10.1515/contagri-2016-0003 Spasevski N, Tasić T, Vukmirović D, Banjac V, Rakita S, Lević J, Ðuragić O (2017). Effect of different levels of marigold and paprika on egg production and yolk colour. Arch zootech 20(2):51–57.‏ Sri Sreshtaa V, Leslie Rani S, Brundha MP, Anjaneyulu K (2021) Dietary capsaicin and immune system. Ann Rom Soc Cell Biol 25(3):5809–5822. Stadecker MJ, Lukic M, Dvorak A, Leskowitz S (1977) The cutaneous basophil response to phytohemagglutinin in chickens. J Immunol 118(5):1564–1568. https://doi.org/10.4049/jimmunol.118.5.1564 Sterner O, Szallasi A (1999) Novel natural vanilloid receptor agonists: new therapeutic targets for drug development. Trends Pharmacol Sci 20(11):459–465. https://doi.org/10.1016/S0165-6147(99)01393-0 ‏Tadeo DB, Gaffud OM (2024) Laying performance of native chicken (Gallus gallus L.) Fed with natural egg enhancers. EJTAS 2(2):108–117. https://doi.org/10.59324/ejtas.2024.2(2).10 Tawfeek SS, Hassanin KMA, Youssef IMI (2014) The Effect of dietary supplementation of some antioxidants on performance, oxidative stress, and blood parameters in broilers under natural summer conditions. J World's Poult Sci 4(1):10–19. Turmelle AS, Ellison JA, Mendonça MT, McCracken GF (2010) Histological assessment of cellular immune response to the phytohemagglutinin skin test in brazilian free-tailed bats ( Tadarida brasiliensis ). J Comp Physiol B 180(8):1155–1164. https://doi.org/10.1007/s00360-010-0486-6 Wigley P, Kaiser P (2003) Avian cytokines in health and disease. Braz J Poult Sci 5(1):1–14.‏ https://doi.org/10.1590/S1516-635X2003000100001 Zhang M, Tracey KJ (1998) Tumor necrosis factor. In A.W. Thomson (editor). The Cytokine Handbook 3 rd edition. 517–547. San Diego, Academic Press. Zhao Z, Wu J, Liu Y, Zhuang Y, Yan H, Xiao M, Zhang L, An L (2023) Dietary canthaxanthin supplementation promotes the laying rate and follicular development of Huaixiang hens. Biol 12(11):1375. https://doi.org/10.3390/biology12111375 Tables Table 1 Determined chemical analysis of ingredients used in the experimental diets (g/kg; as fed) Corn Soybean Meal Wheat Wheat bran Dry matter 90.05 88.46 91.48 89.00 Total ash 1.20 6.80 1.60 6.34 Crude protein 7.68 44.30 9.84 14.2 Ether extract 3.95 2.30 2.10 3.8 Crude fiber 3.80 5.00 2.20 9.6 Amino acid profiles a Met 0.153 0.537 0.150 0.2 01 Met + Cys 0.309 1.035 0.372 0.502 Lys 0.216 2.415 0.281 0.600 Thr 0.243 1.411 0.269 0.480 Trp 0.050 0.538 0.107 0.298 Arg 0.325 3.010 0.445 1.073 Val 0.343 1.843 0.401 0.700 a Determined by Near Infrared Reflectance Spectroscopy (NIRs). Table 2 Ingredient composition, and calculated chemical analysis of (%; as is) experimental diets Levels of hot red pepper (%) Synthetic pigment d Ingredients Control 1 2 3 Corn 26.28 25.49 24.69 23.90 26.28 Wheat 35.00 35.00 35.00 35.00 35.00 Soybean meal, (44.30%) 23.48 23.92 24.36 24.80 23.48 Wheat bran a 3.00 2.00 1.00 0.00 3.00 Hot red pepper 0.00 1.00 2.00 3.00 0.00 Soy oil 1.42 1.75 2.09 2.43 1.42 Oyster shell 8.50 8.49 8.48 8.47 8.50 Dicalcium phosphate 1.36 1.38 1.40 1.42 1.36 Nacl 0.17 0.18 0.18 0.18 0.17 NaHCO3 0.15 0.15 0.15 0.15 0.15 DL-Methionine 0.14 0.14 0.15 0.15 0.14 Mineral premix b 0.25 0.25 0.25 0.25 0.25 Vitamin premix c 0.25 0.25 0.25 0.25 0.25 Calculated analysis AMEn (kcal/kg) 2700 2700 2700 2700 2700 Crude protein 16.78 16.78 16.78 16.78 16.78 Calcium 3.66 3.66 3.66 3.66 3.66 Available phosphorus 0.37 0.37 0.37 0.37 0.37 Sodium 0.15 0.15 0.15 0.15 0.15 Chloride 0.15 0.15 0.15 0.15 0.15 Digestible amino acids Lys 0.74 0.75 0.75 0.76 0.74 Met 0.36 0.36 0.36 0.36 0.36 Met + cys 0.59 0.59 0.59 0.59 0.59 Trp 0.19 0.19 0.18 0.18 0.19 Thr 0.50 0.50 0.50 0.50 0.50 DCAB (meq/kg) 210 208 207 205 210 a Hot red pepper is replaced by wheat bran in the diets. b Supplied per kilogram of diet: Manganese, 60 mg; Zinc 54 mg; Iron, 50 mg; Copper, 10 mg; Iodine, 0.7 mg; Selenium, 0.1 mg. c Supplied per kilogram of diet: trans-retinyl acetate, 3000 mg; cholecalciferol, 75 mg; tocopherol, 20 mg; menadione, 2.5 mg; thiamine, 1.5 mg; riboflavin, 4.4 mg; niacin, 20 mg; pantothenic acid, 8 mg; pyridoxine, 2.5 mg; biotin, 0.15 mg; folic acid, 1.1 mg; cyanocobalamin, 0.08 mg; choline chloride, 200 g. d Lohmann synthetic pigment was added 25 g/1000 kg as on top to diet. Table 3 Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on productive performance of laying hens from 41 to 49 weeks of age Levels of HRP (%) Control 1 2 3 Synthetic pigment SEM c P-value ADFI a (g/b/d) 100.81 101.41 99.14 102.39 101.34 1.933 0.5624 Egg weight (g) 60.09 60.77 60.05 61.13 59.82 0.845 0.5102 Egg production (%) 94.60 95.40 93.00 95.00 95.00 2.464 0.5477 Egg mass (g/d) 56.80 58.17 55.84 58.38 56.90 1.325 0.0815 FCR b 1.780 1.760 1.775 1.760 1.780 0.046 0.6973 a ADFI: Average daily feed intake. b Feed conversion ratio (ADFI/egg mass). c SEM: Standard error of the mean. Table 4 Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on egg quality traits from 41 to 49 weeks of age Control Levels of HRP (%) Synthetic pigment SEM c P-value 1 2 3 Shape index (%) 74.70 75.07 75.97 75.05 75.51 0.703 0.4383 Yolk index (%) 39.04 39.51 39.89 38.72 38.23 0.623 0.0861 Yolk colour a 4.86 d 10.30 c 12.27 b 13.93 a 10.83 c 0.262 0.0001 L* (lightness) b 55.88 a 54.00 b 52.36 c 50.52 d 52.99 bc 0.547 0.0001 a* (redness) b -3.40 c 0.66 b 3.46 ab 5.27 a 2.06 bc 0.676 0.0001 b* (yellowness) b 22.60 22.55 23.65 23.36 22.78 0.749 0.5052 Haugh unit 84.49 85.35 84.86 82.24 75.85 5.377 0.3820 Eggshell thickness (mm) 0.675 0.672 0.673 0.670 0.665 0.007 0.7672 Eggshell weight (g) 5.80 5.83 5.93 5.95 5.89 0.130 0.7303 Eggshell ratio (%) 9.53 9.44 9.65 9.59 9.71 0.149 0.4310 a Determined by Roche Yolk Colour Fan. b Measured by Hunter Lab colorimeter. c SEM: Standard error of mean. Table 5 Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on the humoral immunity (log 2 ) in laying hens Control Levels of HRP (%) Synthetic pigment SEM d P-value 1 2 3 Humoral immunity IgT a 0.62 c 1.65 b 2.91 a 2.46 a 1.42 b 0.426 0.0001 IgG b 0.20 c 0.82 bc 1.90 a 1.06 b 0.40 bc 0.269 0.0001 IgM c 0.42 b 0.84 ab 1.01 ab 1.40 a 1.02 ab 0.294 0.0471 a IgT: total antibodies against SRBC, b IgG: 2-mercaptoethanol-resistant antibodies, c IgM: 2-mercaptoethanol-sensitive antibodies. d SEM: Standard error of the mean. Table 6 Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on the toe web swelling index (mm) in laying hens Hours after PHA b injection Control Levels of HRP (%) Synthetic pigment SEM a P-value 1 2 3 Treat Time Treat ×Time 24 1.12 1.26 1.00 0.87 1.09 0.131 0.2891 48 0.78 0.78 0.74 0.67 0.68 0.121 0.9514 72 0.65 0.68 0.54 0.42 0.55 0.102 0.4324 Treatment average 0.85 0.90 0.76 0.65 0.77 0.069 0.1218 0.0001 0.9851 a SEM: Standard error of mean. b phytohemagglutinin. Table 7 Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on blood parameters in laying hens at 49 weeks of age Control Levels of HRP (%) Synthetic pigment SEM a P-value 1 2 3 Glucose (mg/dl) 216.05 a 195.34 b 174.46 c 149.09 d 218.18 a 7.857 0.0001 Cholesterol (mg/dl) 109.57 a 66.94 b 78.83 b 67.92 b 111.25 a 8.755 0.0001 Total protein (g/dl) 3.41 b 3.47 b 4.80 a 4.91 a 3.45 b 0.555 0.0010 Albumin (g/dl) 3.91 b 3.69 b 4.58 a 4.55 a 3.32 b 0.296 0.0010 a SEM: Standard error of the mean. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5745925","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433461045,"identity":"ffea5278-d084-49b2-b464-a98583a0fbb8","order_by":0,"name":"Zahra Jahanbakhsh Rad","email":"","orcid":"","institution":"Bu Ali Sina University Faculty of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"Jahanbakhsh","lastName":"Rad","suffix":""},{"id":433461046,"identity":"aaa475dd-468b-4f2d-a7cc-e30dd8a3fbb0","order_by":1,"name":"Sara Mirzaie Goudarzi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFACxgYgIcHALwETYCZWi+QM4rVAgcENYlXKTzvc9vFHjYWc8e3mx595GOzkGdh5H+A3/HZi8wyJYxLGZneOmUnzMCQbNjCzG+DXIp3YzGDAJpG47UaCGTMPA3MCAzMbAYfNBmpJ+CdRv3lG+megw+oJa2EAOozhYJtEgoFEjgHQYYcJawH5hbGxT8Jwxp0zZZJzDI4bthF2WPpjxh/f6uT5Z7dv/vCmolqen/8YAYehWcrAQMgno2AUjIJRMAqIAABiSzkA9D4HYAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8118-9922","institution":"Bu Ali Sina University Faculty of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Sara","middleName":"Mirzaie","lastName":"Goudarzi","suffix":""},{"id":433461047,"identity":"245c10f9-746e-42b3-8f9e-2a60ba28a68a","order_by":2,"name":"Abbas Farahavar","email":"","orcid":"","institution":"Bu Ali Sina University Faculty of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Abbas","middleName":"","lastName":"Farahavar","suffix":""},{"id":433461048,"identity":"46d02467-a16c-484a-af69-ad1fc99cd469","order_by":3,"name":"Farhad Ghafouri Kesbi","email":"","orcid":"","institution":"Bu Ali Sina University Faculty of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Farhad","middleName":"Ghafouri","lastName":"Kesbi","suffix":""}],"badges":[],"createdAt":"2025-01-01 11:28:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5745925/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5745925/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79274501,"identity":"8a6d03de-bd22-45d6-b954-56a307222914","added_by":"auto","created_at":"2025-03-26 11:57:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":540598,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of egg yolk colour of control group with different\u003c/p\u003e\n\u003cp\u003elevels of hot red pepper or synthetic pigment, using Roche yolk colour fan.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5745925/v1/efa3d8fc24ca152eec08317f.png"},{"id":84548887,"identity":"ad223491-fac2-41c3-a9cd-0ba6e449e739","added_by":"auto","created_at":"2025-06-13 09:50:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1586448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5745925/v1/3dbe5afa-6419-404d-a7a9-8e9e9d7be66e.pdf"}],"financialInterests":"","formattedTitle":"Comparison of the effect of different levels of hot red pepper or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeightened consumer apprehension regarding food safety has the potential to drive a transformation in animal feed production, leading to an increased utilization of phytobiotic feed additives. Herbs, categorized as phytobiotic feed supplements, include secondary metabolites or bioactive compounds such as flavonoids, alkaloids, carotenoids, and phenols (Mohanraj et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ivanišov\u0026aacute; et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Such phytogenic characteristics are crucial for producing healthy and safe eggs, thereby enhancing consumer health and safety (Samantaray and Nayak \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hot red pepper is a source of carotenoids called xanthophylls; so, it can be used as a natural yolk colourant that is safe and potentially healthier for egg consumers than synthetic or artificial colorants (Saleh et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Egg yolk colour is an important characteristic that determines the choice of eggs by consumers (Spasevski et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Associated with the geographical area, tradition, and culture, consumers prefer a particular colour of egg yolk (Marounek and Pebriansyah \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In industrial conditions in cages, many poultry farmers incorporate synthetic pigments into laying hens\u0026rsquo; diets to achieve the desired orange colour in egg yolks, as corn grain lacks sufficient xanthophyll content (Hencken \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). However, natural resources have been introduced to replace synthetic pigments because of rising public concern regarding using synthetic pigments (Spasevski et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; S\u0026ouml;zc\u0026uuml; \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The necessity for natural sources of pigmentation is a key consideration in poultry farming (Chaves et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Conversely, the excessive use of certain synthetic pigments, like canthaxanthin, in poultry diets has been limited in certain regions due to potential harm to human vision caused by crystal formation in the retina. Hot red pepper is highly favored as a natural feed additive due to its natural, non-toxic, residue-free nature, and widespread availability (Munglang and Vidyarthi \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hot red pepper contains capsaicin, a terpenoid compound with antibacterial properties (Singletary \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Capsaicin, an essential element of chili peppers, is involved in immune system modulation (Sri Sreshtaa et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Capsinoids possess various biochemical and pharmacological attributes, such as anti-inflammatory, antioxidant, anticarcinogenic, and antiallergenic, effects, potentially lowering the risk of cancer (Munglang and Vidyarthi \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The humoral immune response plays a crucial role in immunity and can be evaluated by measuring antibody production and activity. Studies have been conducted regarding adding HRP to laying hen diets to assess its impact on productivity and egg quality. However, the results presented varied and inconclusive findings (Hanif et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). More studies should explore the various effects of HRP supplementation in poultry diets, particularly in terms of immunomodulatory activity (Abd El-Hack et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hence, we compared different levels of HRP or synthetic pigment concerning productive performance, egg quality, humoral and cellular immunity, and blood parameters in laying hens aged 41 to 49 weeks of age.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eChemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll ingredients were ground by a laboratory mill with a 1 mm screen (Retsch Model Z-I, Stuttgart, Germany). Dry matter, total ash, N, and ether extract contents were determined according to\u0026nbsp;AOAC (2005). The amino acid contents were determined by NIRs (Evonik-Degussa, Hanau, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBirds, Diets, and Management\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 200 laying hens of Nick-Chick strain were allocated to five treatments, five replicates of eight hens each in a completely randomized design from 41 to 49 weeks of age. The following treatments were considered: 1: control (basal diet based on corn, wheat and soybean meal with no natural or synthetic pigment),\u003cspan dir=\"RTL\"\u003e\u0026nbsp;2, 3, and 4: control +\u003c/span\u003e 1, 2, or 3% HRP, respectively, and 5: control + synthetic pigment.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eHot red pepper was purchased in dry form\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003efrom the local market, then it was ground into a fine powder and mixed with experimental diets. Avixanthin 100 was used in the diet as a synthetic pigment, which contains 100 g of canthaxanthin/kg, and added 25 g/1000 kg in the experimental diets following the manufacturer\u0026rsquo;s guidelines (Lohmann company, China). Feed and water were available. Light was available for 16 hours per day. The temperature was 22 \u0026plusmn; 2 \u003csup\u003e◦\u003c/sup\u003eC. Before the start of the experiment, all laying hens were treated (for ten days) on a basal diet to deplete the pigment reserves of birds, so that was the effect of the pigments on the colour of the egg yolk the same in all treatments. The basal diet was formulated according to Nick-Chick\u0026apos;s laying hen nutritional recommendation guide (Nick Chick 2017). Table 1 shows the chemical analysis of the ingredients and Table 2 indicates the ingredient composition, calculated and determined chemical analysis of the diets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProductive performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEgg weight (EW) and egg production (EP) were noted once a day, whereas feed intake (FI) was determined weekly and they were applied to calculate the average daily feed intake (ADFI), egg mass (EM), and feed conversion ratio (FCR) both periodically and cumulatively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEgg quality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShape index, yolk index, yolk colour, Haugh\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eunit, eggshell thickness, eggshell weight, and egg shell ratio was recorded every two weeks. Also, the evaluation of L*, a*, and b* (yellowness) values in the egg yolk was done by a\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eHunter\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eLab colorimeter (HP, China) device.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody Response\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSheep red blood cells (SRBC) were employed as T-dependent antigens to assess the antibody response in treated hens. Each group comprised ten hens (two hens per replicate; n=5) that received intravenous injections of SRBC (3% suspension in PBS, 1ml per bird) at 47 weeks of age, followed by an SRBC suspension booster injection seven days later. Blood sampling was done at 7 and 14 days following the initial injection. Serum from each specimen was collected and heat-inactivated by incubating at 56 \u0026deg;C for 30 minutes. The serum samples were then assessed for IgT, IgM, and IgG anti-SRBC antibodies using the method outlined by Delhanty and Solomon (1966) and Qureshi and Havenstein (1994). To summarize, 50 \u0026micro;l of serum was mixed with an equal amount of PBS in the first column of a 96-well U-shaped plate and then incubated at 37 \u0026deg;C for 30 minutes. Following this, a serial dilution (1:2) was carried out, and 50 \u0026micro;l of 3% SRBC suspension was added to the wells. After 30 min incubation, IgT titers were read and several endpoints well with SRBC agglutination were recorded for each sample. For mercaptoethanol-sensitive (IgM) antibody measurement, 0.01 M mercaptoethanol (50 \u0026micro;l; Merck) in PBS was applied instead of PBS alone and continued according to the above-mentioned procedure. The antibody titers against SRBC were determined by expressing them as the base 2 logarithm of the reciprocal of the highest serum dilution causing complete agglutination. The differentiation between IgT and IgG levels was considered to be equal to the IgM antibody level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eToe web swelling index\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe toe web swelling index (cellular immune response) was was measured by basophilic skin sensitization reaction method (cutaneous basophilic hypersensitivity; CBH) or toe web swelling (TWS).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAt 49 weeks of age, two hens from every replicate (n=5) were randomly chosen and the thickness of the webs on the right and left foot (between 3rd and 4th toe web) was measured using a micrometer (INSIZE Company, China) as zero hour (Corrier and Deloach 1990).\u0026nbsp;Then, 0.1 ml solution with phosphate saline was injected into the right foot membrane and 0.1 ml phytohemagglutinin (PHA)\u0026nbsp;solution was injected into the left foot membrane in the same area.\u0026nbsp;The thickness of the webbing at the injection sites was measured using a digital micrometer at 12, 24, and 72 hours post-injection. The swelling reaction was assessed by calculating the difference between the thickness of the toe web on the left foot after PHA injection and the thickness of the toe web on the right foot after PBS injection. The cellular immune response index was calculated utilizing the formula provided by Corrier (1990).\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAVQAAAAvCAYAAAC8ADIDAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAAkBSURBVHhe7ZxLjw1PGMZ7/h8Agw8gxsKERCJucUtIxoiFnbASCSHEhoXLxEaCGMHCQlhI7BASK+KSkBgkrhlBLFziAwzCF5j/eWrqpaamqruqu073YZ5f0lPnVF9OvVXv+7zVdfpM12iLjBBCSGX+0yUhhJCKUFAJISQRFFRCCEkEBZUQQhJBQSWEkERQUAkhJBEUVEIISQQFlRBCEkFBJYSQRFBQCSEkERRUQghJBAWVEEISQUElhJBETCpB/fLlS7Znz55sy5Ytuoak4uLFi6pfHz9+rGsImXxMGkG9evVqtm7dumzBggXqNUnLrl27sr1792b79u1TSasT+PbtW3bq1Kls8eLF6jWpxt+UNNHWN2/e6HfF4PgkduH/oRaxefNm/M/U39vw8LDeM0Z/f7+qNxkaGhp3Do5pCmkLShfSfnvr6ekZHRwcHB0ZGdFHjhFjG843j5Wtu7t7dPfu3ROuXQex9gL7WB84F9cZGBjQNc0AH5V22PbYY4L3PszjsPmwfcLcFi1alPsZ7cQea18MhPo0joM98N1OBGMNvbI1CqAO7fbZduHChdErV67od+UIElQAAUBH2w01B8IGDUQ9BqAJ4QAS4HkOgGPQRrRV2okSwYg6lzig40Ntw2fj2Fu3bumaP32TJ8btooy9nz9/VvvgBy5nNRGf8AVvu4EtaGeeiMmYFAlDjN3iE2b/4Xz5rCZE1RzrIrEI9Wns64Sk6QJiasaZgDrx77yYw74qohosqPggNMYG9SK2NmgY9sGpmkKcpKgNcBA4kg3OhQ02GCDsgzAWIQFlO6k4ehPE2ou2Yx8cNgRcu4lkAdBG2JeHBFeRSMbYLT5hB7Rco6n+wFi4xtQmxqebTpouEOsunzYpGgfoRBXNqrSGijWH58+fZy3B0DXjOXLkSHbo0KFs9uzZuqZ+zpw5k7U6ObcNWF9rdaBaY3Uxffp0/eoPT548UeWyZctUmceLFy9UG2bMmKFrxvjx44d+VS9l7H327Jkq16xZo8oiNm3alN29e7f2tUt88Xjt2rXs2LFjusbNvXv3slbgqDX1PGLsFp+YO3euKm2mTZumX9UH+v/ly5fesTaJ8emVK1cqny7q5zrB2j38rgrQiVYyzk6fPq1r4qgkqOhMCObUqVN1zR+wyAu2b9+uyqaAM+FLiTwkaFasWKFKQb682rlzpypNQgNSHNoeaCQjiFpr5qNr6qOMvTHBBpYvX65K+ay6uHTpkir7+vpU6aJdIgOfQDDayVvatHXrVlXWSUxCCPVpoamk6QK+iwmK+F0VoBe4Xhm7SguqzE5dgomGHD58ODt37tyEWZkPXK+rqyt4W79+vT7Tj3xrN2vWLFX6kKBZunSpKjHLQUJAxoPgHThwQNWbhAakOLQMNPoGg7Vx40blvCdOnFD1dVLG3levXkUFm/Du3Tv9qh7QTohant99+PBBlSEiE2M3fAKzNgH9iqcMDh48mA0ODmYbNmzQe+pD+j8kIYT6tNBU0nTx6NEjVfb29qqyCtALiHMZu4IF1b5dkdmpy3HPnj2bLVmyJMqBcAsxOramG7TduXNHn1kdZGYwc+ZMJdYIyJs3b2bnz5//PWszEaGOuQ1ctWqVujY+A0shWCb5+PFjI8shsfYCzERigq0p0M45c+bod26ePn2qyhCRCbVbfALLDZL00a8nT57MhoeHnUmqDh48eBCUEGJ82qbupOkCyQuETuDymD9/virL2BUsqAsXLtSvxjrfNzuFYZh1ITj/BuT2DwIHoR4ZGVGzDNT7bhslIOfNm6fKPCBeuJ6ZDD59+pQdP348aPAxE5cADdkkMHyUsVeuafpAKlLbFwJEBoSKTIjd4hNDQ0O/xxlCClHdsWOH6t8i2tEXSAiY3BQR49OdCOxMxZQpU/SreErd8ufNTnHbODAw0OgXUTHItH716tWqhE0IAIjO/fv3VZ2NBCRm1XmIeBWt4eaBmbgEaMhW1KYy9kqwpVifskltXwgIvv7+fv3OT4zdLp+AYB89elT17Y0bN3Stn9R9IYK7du1aVeYR6tMkn2hBxSD5Zqeyb//+/bomHJzrysK+LWQNVZzj69evqnQht+RmFpcs/fbtW1Xa+AISyUS+jAO2eHUCZez1BRuWB3w/4/3165cq5fapU8gTGSwVmL+uibHb5xPSDz9//lRlnfgSAu4i7Sc5Qn26UwlJkHUQLah5s9Nt27apW/2QW1kbOK0rC/u20DVU3M7isSUfsp5ozqglgGSfiQScHZBwUgSauS4n4pVioTwVsfYCJEmXw+KRNN+am9jue4SoXaCdWFLx8f79e1XaInP79u3s+/fv45YBQu32+QS4fPmyKpsI+NevX6vSTgh46sBcF47xaZNOSpriz7KWWgVJRKXsaolTEPiVBw7v7u6e8IC67GuJl67pHPIe7JeHk3t6erwP3dvnia3mA804BsejbwRcD+9xbNHD43VRxl45x/yVD86VHyv4bMNntEREv6sPeWDftk9Am7DfBDZirMyH92PsdvkE9uN6qMc5dSP+Z4+B/ELPfHg/1KdtpK9dsVU3EuemDTYYExwDu3z+AYp8KI9gQZWBMB1McA1Ip4BOQXDbTg0nQJtlwzEm0qmol+DBL0lEJF2bGZA4z9zXtNOVsRelCK1r8wWbiFET/iB2IsBsxE99m4hMjN0+n0A/wh+a6AP4vIi5byvj0zawsYmk6QN2uPQJSCI1N9/YYOzLJsFgQf2bkQCH85D2IgkMAt0UEAE7YZC0NJk0fYT89LQIJGQIc9kJ0KQQVIDORkeF/E6ZlAPBBSFr4hbXBKKOsW5S1P9lOiFp+kAyrTJxgu+67m5CmTSCCpB10GFNB/y/CBJVU7e4LnBLK+IOASBp6JSk6UOWO2RJIwYIaRUxBV3405q6E/LPgeeA8Y329evXc5/0IGHg8amHDx+qfyRuPznQaaCteDoh5CfDAE954IH+qnZRUAkhJBGlfilFCCFkIhRUQghJBAWVEEISQUElhJBEUFAJISQRFFRCCEkEBZUQQhJBQSWEkERQUAkhJBEUVEIISQQFlRBCEkFBJYSQRFBQCSEkCVn2P/cpI2LaoksPAAAAAElFTkSuQmCC\" width=\"340\" height=\"47\"\u003e\u003c/p\u003e\n\u003cp\u003eIn this equation, W represents the interdigital skin thickness index, RLP indicates the swelling of the left foot toe web after PHA injection, RL signifies the swelling of the left foot toe web before PHA injection, LLB denotes the swelling of the right foot toe web after PBS injection, and LL represents the swelling of the right foot toe web prior to PBS injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the hens reached 49 weeks of age, blood samples were obtained from two birds for each replicate (n=5) in heparinized tubes. These specimens underwent centrifugation for 15 minutes at 2000\u0026times; g, followed by extracting and storing the plasma at\u0026nbsp;\u0026ndash;20 \u0026ordm;C until further biochemical assessment. Plasma levels of glucose, cholesterol, total protein and albumin, were quantified by commercial kits (Pars Azmun, Iran) by spectrophotometer (Varian Spectrophotometer\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eAA220, Australia).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was done following a completely randomized design with five treatments and five replicates, each consisting of eight hens. The main effects were assessed through ANOVA utilizing the GLM procedure of the SAS software (2012). The Tukey\u0026rsquo;s test compared mean values (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Immunity data were evaluated as repeated measurements.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eProductive performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the whole experimental period, productive performance was not affected by treatments (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05, Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEgg quality\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 4 indicates the effect of treatments on egg quality traits. Shape index, yolk index, b* value, Haugh unit, eggshell thickness, shell weight, and eggshell ratio were not influenced by treatments (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). The use of 3% HRP significantly improved egg yolk colour compared to other treatments (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). Increased HRP levels or synthetic pigment in the diet decreased the L* value of the yolk colour in the control group (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). Also, a higher a* value of the yolk colour was observed by 3% HRP inclusion in the diet (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). However, treatments did not affect the b* value of the yolk colour (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). Figure 1 compares the egg yolk colour of the control group with various levels of HRP or synthetic pigment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHumoral immune response and toe web swelling index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lower total IgT, IgG, and IgM were observed in the controls compared to other treatments (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). In contrast, total IgT and IgG were higher at 2 and 3%\u0026nbsp;HRP\u0026nbsp;(\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). Also, a higher IgM was observed at 3%\u0026nbsp;HRP\u0026nbsp;inclusion in the diet (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Table 5). However, toe web swelling index was not affected by treatments (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). The highest index of toe web swelling was observed at 24 h after injection (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001).\u0026nbsp;No\u0026nbsp;interaction was found between treatment and time for toe web swelling response to PHA (Table 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma glucose and cholesterol levels significantly decreased in hens fed with\u0026nbsp;various concentrations of\u0026nbsp;HRP\u0026nbsp;(\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). However, plasma total protein and albumin increased by using 2 and 3% of\u0026nbsp;HRP\u0026nbsp;inclusion in the diet (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001; Table 7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe analytical values of the\u0026nbsp;HRP used in the current study were: DM 93.21, total ash 7.83, crude protein 9.52, crude fat 8.50, and crude fiber 15.40%. Within the previously reported range of Niksiar (2018), the chemical compounds of HRP included: DM 94.60, total ash 8.68, crude protein 11.45, crude fat 7.00, and crude fiber 16.20%.\u003c/p\u003e\n\u003cp\u003eIn the current study, none of the performance parameters was affected by treatments. In agreement with our results, Aktaran Bala et al. (2020) declared that adding 4% of dried red pepper powder to the diet of laying hens compared to the control did not affect egg production rate, egg mass, and FCR. In another study, Tadeo and Gaffud (2024) reported that HRP inclusion in the diet did not affect the performance of native layers. Contrary to the results obtained in this experiment, Aikpitanyi and Imasuen (2023) reported that hen day production and egg mass were affected by including 1% red pepper, with significantly higher in this group compared to the control group. According to Zhao et al. (2023) dietary canthaxanthin supplementation significantly improved egg production and promoted ovulation of Huaixiang broiler breeders. Carotenoids, like \u0026szlig;-carotene, are able to promote the synthesis of egg yolk precursors. Hen age at trial start, pepper form, inclusion level, and supplementation duration can be the reasons among different studies.\u003c/p\u003e\n\u003cp\u003eThe impact of the experimental treatments on egg shape index and yolk index did not show statistical significance, aligning with the findings of Spasevski et al. (2017). As expected, the research demonstrated that the yolk pigment conversion efficiency from red pepper to yolk colour with increased levels of HRP supplementation, consistent with the findings of Abou-Elkhair et al. (2018) and Sozcu (2019). In the current study, laying hens that were fed with 1% HRP had the same yolk colour as the synthetic pigment group (10.30 vs. 10.83, respectively) (Figure 1). In another research, the improvement in egg colour scores was significant with red pepper powder. Nevertheless, other parameters related to egg quality unaffected (Aktaran Bala et al. 2020). It is suggested that phytochemicals\u0026rsquo; capacity to readily accumulate in animal-derived products can potentially confer antioxidant, regenerative, and protective benefits from these substances (Akdemir et al. 2012). Aikpitanyi and Imasuen (2023) reported yolk\u0026nbsp;colour\u0026nbsp;improvements with red pepper inclusion in laying hens\u0026rsquo; diets. Hanif et al. (2024) declared that diets treated with red pepper showed higher egg yolk\u0026nbsp;colour\u0026nbsp;than the control group. Carotenoid deposition considerably improved the colour of yolks. Xanthophylls play a crucial role in the pigmentation of bird egg yolks, accounting for 88 to 92% of the total carotenoids present in the yolk. The overall carotenoid concentration and ratio of the red carotenoids (astaxanthin, capsanthin, and canthaxanthin) to yellow carotenoids (zeaxanthin and lutein) affect the colour of the egg yolk and its components (yellowness or redness). Lokaewmanee et al. (2010) have shown a lower value of L* value for the paprika group which is in agreement with the current study. It has been reported that the lower yolk L* value was due to the accumulation of pigments in the yolk (Grashorn and Steinberg, 2002).\u0026nbsp;Niksiar (2018) reported that the use of 1% HRP or synthetic pigment improved a* value compared to the control group in the egg yolk.\u0026nbsp;Santos-Bocanegra\u0026nbsp;et al. (2004) reported that capsaicin causes a dark red\u0026nbsp;colour\u0026nbsp;in the yolk. Lutein is a carotenoid that causes yellowing in egg yolks. Zeaxanthin is a carotenoid that causes a yellow colour and some of its derivatives such as zeaxanthin produce orange colour (Marusich et al. 1960). Despite this, the yolk yellowness value (b*) remained unaffected by the experimental treatments, consistent with the findings of Niksiar (2018). Consumer inclinations towards egg yolk colour can exhibit substantial diversity across countries and even within specific regions of a country. For instance, in the United States, the favored yolk colour typically ranges between 7 and 10 on the Roche yolk colour fan (RYCF), whereas certain European or Asian nations lean towards higher values, typically between 10 and 14 (Galobart et al. 2004).\u0026nbsp;Eggshell thickness, shell weight, and shell ratio did not exhibit significant changes throughout the study period. Factors such as the calcium content and source in the diet, hen age, feed intake, and egg production percentage are crucial elements influencing eggshell quality (An et al. 2016).\u003c/p\u003e\n\u003cp\u003eThere have been limited animal studies exploring the potential chemo preventive properties of capsaicin (Abd El-Hack et al. 2022).\u0026nbsp;The quantity of antibodies generated against sheep erythrocytes serves as an indicator of the status of the humoral immune system.\u0026nbsp;Hot peppers are abundant in vitamin C, which plays a significant role in alleviating heat stress in birds (Abd El-Hack et al. 2019). Additionally, they are a plentiful source of carotenoids like vitamins E, C, and provitamin A (\u0026szlig;-carotene), famous for their antioxidant properties that combat the harmful impacts of free radicals, including cellular damage (oxidative stress) in broilers (Tawfeek et al. 2014).\u0026nbsp;In agreement with the results outlined in this study, S\u0026ouml;zc\u0026uuml; (2010) noted a trend towards elevated IgG levels in layers that were provided with red pepper supplementation. This trend could be associated with the bioactive elements present in red pepper, notably capsaicin, as mixtures of active compounds in spicy red pepper have shown chemo preventive and chemotherapeutic benefits (Puvača et al. 2015). Chew and Park (2004) illustrated in human peripheral blood that the immune response modulation by dietary carotenoids operates through cytokine secretion. Cytokines possess the ability to exhibit adjuvant-like properties akin to vaccine stimulation, thereby triggering a certain immune reaction (Wigley and Kaiser 2003). Aktaran Bala et al. (2020)\u0026nbsp;noted that cytokine levels, with the exception of transforming growth factor-beta (TGF-\u0026beta;), tumor necrosis factor-alpha (TNF-\u0026alpha;), and interleukin-4 (IL-4), remained constant following supplementation with 4% red pepper powder. The therapeutic attributes identified in the Capsicum genus were associated with their bioactive elements, notably capsaicin. Dihydro-capsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin represent some of the capsaicinoid compounds present in hot red pepper (Abou-Elkhair et al. 2018). The positive capsicum impacts on poultry nutrition could be linked to capsaicin, known for its bactericidal properties against intestinal pathogens like Clostridium spp, Salmonella spp, and Escherichia coli (Salem et al. 2021).\u0026nbsp;The capsaicin content in red pepper releases neuropeptides such as substance P, neurokinin A, vasoactive intestinal peptide, and peptides associated with the calcitonin gene. These neuropeptides, upon reaching the intestine, stimulate smooth muscle activity, tissue regeneration, and immune processes, and safeguard against mucosal damage in the gastrointestinal tract (Sterner and Szallasi 1999). The decrease in heterophil percentage due to red pepper powder supplementation might be linked to the stress-reducing properties of pepper, which could have a positive impact on the hens\u0026rsquo; immune system (Aktaran Bala et al. 2020).\u003c/p\u003e\n\u003cp\u003eInflammation of the foot of birds can indicate the ability of the cell-mediated immunity system.\u0026nbsp;The PHA skin test can assess delayed-type cellular immune responses across different vertebrates and has been a standard tool in immunoecological investigations. Research indicated a notable presence of lymphocytes, neutrophils, eosinophils, basophils, and macrophages in tissues injected with PHA compared to control tissues injected with saline (Turmelleet al. 2010).\u0026nbsp;The inflammation index reflects the stimulating impact on the generation of T lymphocyte clones specific to antigens. We assessed the immunomodulatory effects of HRP or synthetic pigment in measuring delayed-type cellular immune responses through the PHA skin test in laying hens.\u0026nbsp;To our knowledge, limited research exists on the impact of HRP or synthetic pigment on cellular immunity in laying hens. The findings revealed that the web swelling unaffected by either natural or synthetic pigment, thus not confirming the hypothesis of our study. We hypothesized that the influence of HRP on cellular immunity is linked to its biologically active components, particularly capsaicin. Capsaicin has been recognized for its immune-modulating properties, capable of stimulating dendritic cells, a crucial cell type in the vertebrate immune system (Basu and Srivastava 2005).\u0026nbsp;In opposition to our findings, Beltran et al. (2007) showed that capsaicin significantly improves both the quantity and activation status of skin-derived dendritic cells in draining lymph nodes.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eOne plausible reason for the immune-modulating impact of capsaicin is its potential to regulate the cellular immune response by activating antigen-presenting cells, T-helper cells, and cytotoxic lymphocytes. However, there exists a distinct contrast between the outcomes from in vivo and in vitro assessments. For instance, previous in vitro studies indicated that capsaicin hindered IL-2 generation by inducing capacitative calcium entry into Jurkat cells, thereby diminishing T-cell functionality and cytokine generation (Park et al. 2004).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAktaran Bala et al. (2020) reported an elevation in serum TGF-\u0026beta; and TNF-\u0026alpha; levels in the group administered with red pepper powder compared to the control group, suggesting a potential immune-enhancing effect of red pepper. TGF-\u0026beta; plays a crucial role in T cell development and exhibits significant anti-inflammatory properties, while TNF-\u0026alpha;, generated by T cells, macrophages, and NK cells, serves as the primary regulatory cytokine in initiating an immune reaction during endothelial activation and acute inflammation (Zhang and Tracey 1998).\u0026nbsp;The expected decline in response over time was due to the control of physiological processes related to the mobilization of cells that fight against external substances. The increased occurrence of basophils and eosinophils 24 hours after injection has been recognized as a cutaneous basophil hypersensitivity reaction (Stadecker 1977).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIncreased levels of HRP in the diet decreased blood glucose and cholesterol compared to the synthetic pigment or control group.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eIn agreement with the data reported herein, Dougnon et al. (2014) revealed a decrease in the blood glucose levels of broilers receiving a diet treated with HRP in comparison to those receiving the control diet.\u0026nbsp;Decreasing in plasma glucose due to increased insulin secretion by HRP. Capsaicin had a stimulant effect similar to epinephrine. Epinephrine has a strong impact on stimulating hepatic gluconeogenesis and causes the release of sugar in the blood, so this effect of epinephrine stimulates the release of insulin and subsequently decreases blood sugar (Kawada et al. 1986).\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eFurthermore, Puvaca et al. (2015) validated its efficacy in lowering blood cholesterol levels. In agreement with the findings of the present study, Soliman and Al-Afifi (2020) observed a decrease in plasma cholesterol in broilers with 1% and 2% red pepper supplementation. The reduction in serum cholesterol can be attributed to active compounds like capsaicin and phenolic compounds present in red pepper, which inhibit the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) activity, thereby inhibiting cholesterol production (Lokaewmanee et al. 2010).\u0026nbsp;Capsaicin found in hot pepper has been associated with decreased levels of LDL cholesterol and a lowered risk of heart-related mortality (Tadeo and Gaffud 2024). Moreover, the cholesterol-reducing impact of red pepper might be associated with its role to activate the hepatic cholesterol-7-hydroxylase enzyme, essential for converting cholesterol into bile acids, thereby lowering blood cholesterol levels (Adedoyin et al. 2019). This influence could be attributed to the inhibitory effects of the active components in hot red pepper on hepatic HMG-CoA activity, a pivotal enzyme in cholesterol synthesis, resulting in decreased cholesterol generation and absorption in the intestine.\u003c/p\u003e\n\u003cp\u003eIn the current study, diets supplemented with 2 or 3% HRP increased the total protein and albumin values. Our discovery aligns with results of Hassan et al. (2023) where supplementation with red pepper oil led to a notable rise in plasma albumin and total protein levels, credited to its bioactive components. Similarly,\u0026nbsp;Husdfdggsein\u0026nbsp;(2010) demonstrated an enhancement in serum total protein levels with 1.0% and 2.0% HRP in the diet compared to the control group. This effect could be attributed to improved food digestion by altering pH levels and potentially increasing acid production. Serum total proteins serve as crucial indicators of the body\u0026rsquo;s health status and productivity due to their various physiological functions. Consistent with our results, Abbas and Thamir Al-Jrrah (2022)\u0026nbsp;who reported\u0026nbsp;an increase in serum total protein levels when using red bell pepper fruit powder compared to the control group in Japanese quail layers.\u003c/p\u003e\n\u003cp\u003eIn general, adding different levels of HRP or synthetic pigment inclusion in the diet did not influence the performance of laying hens. Using a minimum of 1% HRP could be substituted with synthetic pigment to improve the egg yolk colour. One of the advantages of HRP compared to synthetic pigment was the improvement of the immune system and blood parameters which these effects were more pronounced with increasing levels of HRP. So, it is recommended to\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003euse HRP in the diet, which may be valuable for consumers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eAll animal procedures were approved by the Ethics Committee of Bu-Ali Sina University, Faculty of Agriculture (approvement date: 17th of December 2018).\u003c/p\u003e\n\u003ch2\u003eConsent to participate\u003c/h2\u003e\n\u003cp\u003eThe authors of this work have agreed to participate in present study.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors agree with the content of this manuscript and gave their consent to submit the current manuscript to Tropical Animal Health and Production.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by ZJR, SMG, AF, and FGK. Material preparation, data collection and analysis were performed by ZJR and SMG. The first draft of the manuscript was written by ZJR and SMG and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\n\u003cp\u003eThe authors thank the personnel of the Poultry Research Unit of Bu-Ali Sina University for the care of birds.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement:\u003c/h2\u003e\n\u003cp\u003eAll available data incorporated into the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbbas RJ, Thamir Al-Jrrah IA (2022) Influence of feeding natural and synthetic lycopene on performance, egg quality and serum parameters of Japanese quail layers. 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San Diego, Academic Press.\u003c/li\u003e\n \u003cli\u003eZhao Z, Wu J, Liu Y, Zhuang Y, Yan H, Xiao M, Zhang L, An L (2023) Dietary canthaxanthin supplementation promotes the laying rate and follicular development of Huaixiang hens. Biol 12(11):1375. https://doi.org/10.3390/biology12111375\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTable 1\u003c/strong\u003e Determined chemical analysis of ingredients used in the experimental diets (g/kg; as fed)\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"564\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eCorn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003eSoybean Meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eWheat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eWheat bran\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eDry matter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e90.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e88.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e91.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e89.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eTotal ash\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e6.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e6.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e7.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e44.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e9.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e14.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eEther extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e3.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eCrude fiber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e3.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eAmino acid profiles \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eMet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.2 01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eMet + Cys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.502\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eLys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2.415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.600\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eThr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.243\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1.411\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.480\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eTrp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.298\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eArg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1.073\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003eVal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1.843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.700\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e\u0026nbsp; \u0026nbsp;a\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eDetermined by Near Infrared Reflectance Spectroscopy (NIRs).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Ingredient composition, and calculated chemical analysis of (%; as is) experimental diets\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"606\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 197px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eLevels of hot red pepper (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eSynthetic pigment \u003csup\u003ed\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003eIngredients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 81px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eCorn\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e26.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e25.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e24.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e23.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e26.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eWheat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e35.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e35.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e35.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e35.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e35.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eSoybean meal, (44.30%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e23.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e23.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e24.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e24.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e23.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eWheat bran \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHot red pepper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eSoy oil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eOyster shell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e8.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e8.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e8.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e8.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e8.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eDicalcium phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eNacl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eNaHCO3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eDL-Methionine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eMineral premix \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eVitamin premix \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eCalculated analysis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eAMEn (kcal/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e2700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2700\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e16.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e16.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e16.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e16.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e16.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eCalcium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eAvailable phosphorus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eSodium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eChloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eDigestible amino acids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eLys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eMet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eMet + cys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eTrp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eThr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eDCAB (meq/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Hot red pepper is replaced by wheat bran in the diets.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Supplied per kilogram of diet: Manganese, 60 mg; Zinc 54 mg; Iron, 50 mg; Copper, 10 mg; Iodine, 0.7 mg; Selenium, 0.1 mg.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e Supplied per kilogram of diet: trans-retinyl acetate, 3000 mg; cholecalciferol, 75 mg; tocopherol, 20 mg; menadione, 2.5 mg; thiamine, 1.5 mg; riboflavin, 4.4 mg; niacin, 20 mg; pantothenic acid, 8 mg; pyridoxine, 2.5 mg; biotin, 0.15 mg; folic acid, 1.1 mg; cyanocobalamin, 0.08 mg; choline chloride, 200 g.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003e Lohmann synthetic pigment was added 25 g/1000 kg as on top to diet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on productive performance of laying hens from 41 to 49 weeks of age\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"678\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eLevels of HRP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eSynthetic pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eSEM\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eADFI \u003csup\u003ea\u003c/sup\u003e (g/b/d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e100.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e101.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e99.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e102.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e101.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.5624\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eEgg weight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e60.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e60.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e60.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e61.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e59.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.5102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eEgg production (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e94.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e95.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e93.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e95.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e95.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.5477\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eEgg mass (g/d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e56.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e58.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e55.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e58.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e56.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.0815\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eFCR \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.775\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e1.780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.6973\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eADFI: Average daily feed intake.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eFeed conversion ratio (ADFI/egg mass).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e SEM: Standard error of the mean.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on egg quality traits from 41 to 49 weeks of age\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"659\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003eLevels of HRP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSynthetic pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSEM \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eShape index (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e74.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e75.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e75.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e75.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e75.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eYolk index (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e39.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e39.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e39.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e38.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e38.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0861\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eYolk colour \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.86 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e10.30 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e12.27 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10.83 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eL* (lightness) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e55.88 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e54.00 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e52.36\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e50.52 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e52.99 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.547\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003ea* (redness) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e-3.40 \u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.66 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3.46 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5.27 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.06 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eb* (yellowness) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e22.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e22.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e23.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e23.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e22.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.749\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5052\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eHaugh unit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e84.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e85.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e84.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e82.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e75.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5.377\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.3820\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eEggshell thickness (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.675\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.672\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.673\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.670\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.665\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.7672\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eEggshell weight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e5.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e5.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.7303\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eEggshell ratio (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e9.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e9.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4310\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Determined by Roche Yolk Colour Fan.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eMeasured by Hunter Lab colorimeter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e SEM: Standard error of mean.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on the humoral immunity (log\u003csub\u003e2\u003c/sub\u003e) in laying hens\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"647\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eControl\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003eLevels of HRP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSynthetic pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSEM \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eHumoral immunity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eIgT \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.62 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.65 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.46 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.42 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eIgG \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.20 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.82 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.90 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.06 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.40 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eIgM \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.42 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.84 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.01 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.40 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.02 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.0471\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e IgT: total antibodies against SRBC, \u003csup\u003eb\u003c/sup\u003e IgG: 2-mercaptoethanol-resistant antibodies, \u003csup\u003ec\u003c/sup\u003e IgM: 2-mercaptoethanol-sensitive antibodies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003e SEM: Standard error of the mean. \u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"705\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"bottom\" style=\"width: 705px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 6\u0026nbsp;\u003c/strong\u003eComparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on the toe web swelling index (mm) in laying hens\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eHours after\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;PHA\u003csup\u003eb\u003c/sup\u003e injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eLevels of HRP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSynthetic pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSEM \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTreat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTime\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTreat \u0026times;Time\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.2891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.9514\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.4324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTreatment average\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.1218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.9851\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"top\" style=\"width: 705px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e SEM: Standard error of mean.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e phytohemagglutinin.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7\u003c/strong\u003e Comparison of the effect of different levels of hot red pepper (HRP) or synthetic pigment on blood parameters in laying hens at 49 weeks of age\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"654\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 217px;\"\u003e\n \u003cp\u003eLevels of HRP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSynthetic pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSEM \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eGlucose (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e216.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e195.34 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e174.46\u003csup\u003ec\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e149.09 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e218.18 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e7.857\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eCholesterol (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e109.57 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e66.94 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e78.83 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e67.92 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e111.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e8.755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eTotal protein (g/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3.41 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3.47 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4.80\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.91\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.45 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eAlbumin (g/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3.91 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3.69\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4.58 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.55 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.32 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e SEM: Standard error of the mean.\u003c/p\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":"Egg production, egg quality, pigmentation, yolk colour, immunity, layers","lastPublishedDoi":"10.21203/rs.3.rs-5745925/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5745925/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis experiment compared the effect of different levels of hot red pepper (HRP) or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens from 41 to 49 weeks of age. Two hundred laying hens of Nick-Chick strain were allocated to five treatments, five replicates, and eight hens for each replicate in a completely randomized design. The treatments were: 1. control (basal diet without any HRP or synthetic pigment), 2, 3 and 4. control\u0026thinsp;+\u0026thinsp;1, 2 or 3% HRP, respectively, and 5. control\u0026thinsp;+\u0026thinsp;synthetic pigment. The results showed that treatments had no significant effect on performance and egg quality traits (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), except for yolk colour and a* value (redness) that were significantly higher in the birds that were fed with HRP levels or synthetic pigment than control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The higher immunoglobulin T and G was observed by HRP levels in comparison to the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The inclusion of 3% HRP increased immunoglobulin M than other treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Plasma glucose and cholesterol showed a reduction in birds fed with HRP levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, plasma total protein and albumin were higher in the birds that were fed 2% and 3% HRP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In conclusion, HRP could be recommended as a safe and efficient alternative pigment to diet of laying hens due to its favorable effects on yolk pigmentation, immunity, and blood parameters.\u003c/p\u003e","manuscriptTitle":"Comparison of the effect of different levels of hot red pepper or synthetic pigment on performance, egg quality, immune response, and blood parameters in laying hens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 11:57:08","doi":"10.21203/rs.3.rs-5745925/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":"89ed9c79-ef0e-47c9-944f-bd946429b264","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-13T09:42:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-26 11:57:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5745925","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5745925","identity":"rs-5745925","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}