Dietary supplementation of Ferula badrakema and sodium saccharin: Effects on the growth performance, blood metabolites, immune response, antioxidant status, and gut histomorphology in broiler chicks

Dietary supplementation of Ferula badrakema and sodium saccharin: Effects on the growth performance, blood metabolites, immune response, antioxidant status, and gut histomorphology in broiler chicks | 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 Article Dietary supplementation of Ferula badrakema and sodium saccharin: Effects on the growth performance, blood metabolites, immune response, antioxidant status, and gut histomorphology in broiler chicks Hamidreza Khajavi, Ahmad Hassanabadi, Mahdi Askari Badouei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7871471/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract This study investigated the effects of dietary supplementation with Ferula badrakema (FB) root powder and sodium saccharin(SAC) on broiler chicks. A total of 468 one-day-old male Ross 308 chicks were allocated in a completely randomized design with a 3 × 2 factorial arrangement, consisting of three levels of FB (0%, 0.75%, and 1.5%) and two levels of SAC (0% and 0.15%), with six replicates per treatment. SAC supplementation significantly increased feed intake (FI) during days 1–10 and over the entire experimental period (days 1–42) (P < 0.05). The addition of FB further increased FI in diets containing SAC but decreased it in diets without SAC. SAC also improved body weight gain (WG) (P < 0.05) and significantly elevated blood uric acid levels (P < 0.05). Orthogonal contrasts showed that FB supplementation significantly increased blood uric acid, albumin, and phosphorus concentrations (P < 0.05), while reducing cholesterol and triglyceride levels (P < 0.05). Additionally, FB enhanced total antibody titers and IgG concentrations on day 35, as well as IgG and IgM levels on day 42. FB supplementation reduced malondialdehyde (MDA) concentrations and increased superoxide dismutase (SOD) activity (P < 0.05), indicating an improved antioxidant status. Both SAC and FB increased jejunal villus height (P < 0.05), and their interaction significantly reduced cecal Escherichia coli counts (P < 0.05). In conclusion, SAC mitigated the FB-induced reduction in feed intake, whereas FB improved immune function, intestinal morphology, and blood lipid profiles in broiler chicks. Biological sciences/Biochemistry Health sciences/Diseases Health sciences/Health care Biological sciences/Immunology Health sciences/Medical research Biological sciences/Physiology Biological sciences/Zoology Broiler chickens Ferula badrakema immune response performance sodium saccharin Figures Figure 1 Introduction Antibacterial compounds play a crucial role in maintaining microbial balance and promoting a healthy gut microflora in poultry. However, the overlap between antibiotics used in poultry production and those applied in human medicine has raised significant concerns about the potential transfer of antibiotic-resistant bacterial strains to humans through poultry products. In response to these concerns particularly regarding the use of antibiotics as growth promoters, extensive research has focused on identifying effective and safe alternatives. Many of these alternatives aim to enhance poultry performance by modulating the intestinal microbial population. Among the promising alternatives investigated in recent years are medicinal plants and their extracts [ 1 , 2 ] . Numerous studies have demonstrated beneficial effects of medicinal plants on poultry growth performance [ 3 , 4 ] , with many species exhibiting notable antimicrobial activity. In addition to their antimicrobial effects, medicinal plants can improve digestive function and nutrient absorption, stimulate appetite, and reduce serum lipid concentrations [ 5 ] . The genus Ferula , belonging to the family Peucedaneae and subfamily Apioideae, comprises approximately 133 species distributed across the Mediterranean region. Iran is particularly rich in Ferula species, with more than 70 having been chemically characterized. Species of this genus display considerable biological diversity and exhibit antibacterial, antifungal, anticancer, and antioxidant activities [ 6 , 7 ] . Ferula badrakema is a resinous and aromatic plant native to Iran, predominantly found in Tandoureh National Park, Dargaz County, North Khorasan Razavi Province. Its antibacterial and antifungal properties are attributed mainly to its high content of α-pinene and β-pinene. Similar to other Ferula species, FB is a rich source of sesquiterpene coumarins. Although its volatile compounds have not yet been studied, investigations of other Ferula species in various regions of the world have yielded promising pharmacological results [8–9−10] . Saccharin (1,2-benzisothiazol-3(2H)-one-1,1-dioxide) is a sulfonamide derivative originally synthesized from coal tar compounds [ 11 ] . It is approximately 300–500 times sweeter than sucrose. Currently, high-intensity sweeteners are widely used in the diets of pigs and ruminants [12–13− 14] . Due to their intense sweetness and negligible caloric value, such compounds are employed to enhance feed palatability, as the perception of sweetness has been linked to increased FI [ 15 ] . Previous studies have reported that dietary supplementation with sweeteners can increase FI and consequently improve growth performance in livestock [16–17−18] . For example, Japanese quails have been shown to prefer sucrose solutions over plain water due to enhanced palatability. However, limited research has explored the physiological effects of sweeteners on the gastrointestinal tract of broilers [ 15 ] . Given that many medicinal plants, including FB, possess a bitter taste that may reduce FI and hinder chick growth, this study aimed to evaluate the combined effects of FB root powder and saccharin supplementation. Specifically, this research sought to determine whether saccharin could mitigate the bitter taste of FB and to assess its effects on growth performance, immune response, blood serum metabolites, antioxidant status, cecal Escherichia coli counts, and carcass traits in broiler chicks. Materials and Methods Ethical approval This experiment was conducted in accordance with the comprehensive animal welfare guidelines approved by the Animal Care and Use Committee of Ferdowsi University of Mashhad, Mashhad, Iran (Approval Reference Number: 48465). The study was performed in compliance with the ARRIVE 2.0 guidelines regarding study design, the number and use of experimental animals, randomization procedures, statistical analyses, and other relevant aspects. Birds, Diets , and Housing A total of 468 one-day-old male Ross 308 broiler chicks were obtained from a commercial hatchery. The chicks were individually weighed and randomly allocated to six treatments with six floor-pen replicates per treatment, each containing 13 birds, following a completely randomized design arranged in a 3 × 2 factorial layout. The treatments consisted of three levels of FB root powder (0%, 0.75%, and 1.5%) and two levels of saccharin (SAC; 0% and 0.15%) supplemented in a basal diet. All birds received standard basal diets during the starter (1–10 days), grower (11–24 days), and finisher (25–42 days) phases, formulated according to Ross 308 broiler nutrition specifications (Aviagen, 2019) (Table 1). Feed and water were provided ad libitum throughout the experimental period. A lighting program of 18 hours of light and 6 hours of darkness was maintained daily. The initial room temperature was set at 32 °C and gradually reduced by 0.5 °C per day until it reached 21 °C at 21 days of age. Average daily feed intake, WG, and feed conversion ratio (FCR) were measured at the end of each rearing phase. Mortality was recorded daily and used to adjust FI data. At 42 days of age (maximum weight 2,700 g), one bird per replicate representative of the average pen weight, was selected, humanely euthanized by cervical dislocation, and dissected for carcass, organ, and intestinal weight determinations. All birds were anesthetized with CO 2 inhalation prior to the cervical dislocation to ensure minimal distress or pain to the animals. Blood Collection and Analysis At 42 days of age, one bird from each replicate was selected for blood sampling. Blood samples were collected from the wing vein using syringes without anticoagulant. After centrifugation (3000 × g for 10 min at 4 °C), serum samples were stored at –20 °C until analysis. Serum concentrations of total protein, glucose, cholesterol, albumin, high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), uric acid, triglycerides, calcium, and phosphorus were determined using a commercial auto-analyzer (Alcyon model) and diagnostic kits (ParsAzmoon Co. Tehran, Iran) at the Laboratory of the Applied Pharmaceutical Research Center, Tabriz, Iran. Humoral immune response The humoral immune response was evaluated by injecting 0.1 mL of a 25% SRBC suspension into the breast muscle of one bird per replicate on days 28 and 35. Blood samples were collected from the wing vein on days 35 and 42 to measure primary and secondary antibody responses. Two milliliters of blood were collected 7 days after each SRBC injection. After clotting, serum was separated by centrifugation (3000 × g for 10 min at 4 °C) and incubated for 30 min at 56 °C to inactivate complement proteins. Total anti-SRBC antibody titers, immunoglobulin G (IgG), and immunoglobulin M (IgM) levels were determined. Antibody titers were expressed as the log₂ of the highest serum dilution that agglutinated 0.05 mL of a 2.5% SRBC suspension in phosphate-buffered saline [19] . Lipid Peroxidation (MDA) and Antioxidant Enzyme Activity Liver samples were stored in a potassium chloride solution (1.15% w/v; pH 4.7) and homogenized at 4 °C. The homogenate was centrifuged at 5000 × g for 15 min, and the supernatant was used to determine total SOD and glutathione peroxidase (GPx) activities as well as total protein concentration. Absorbance was measured using an auto-analyzer (Alcyon 300, Abbott, USA). TSOD activity was determined colorimetrically using a Ransod kit [ 20 ] , and GPx activity was measured enzymatically using a Ransel kit [ 20 ] . Serum MDA concentration was determined by reaction with thiobarbituric acid followed by extraction with normal butanol and spectrophotometric measurement at 532 nm against a standard curve. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the IFCC method with commercial kits (ParsAzmoon Co., Tehran, Iran). For serum MDA quantification, 500 µL of serum was mixed with 3 mL of 1% phosphoric acid, vortexed, and reacted with 1 mL of 0.675% thiobarbituric acid. The mixture was incubated in a water bath for 45 min, cooled, extracted with 3 mL of normal butanol, vortexed for 2 min, and centrifuged (3000 × g for 10 min). The absorbance of the supernatant was read at 532 nm, and MDA concentration was calculated using a standard curve. Total antioxidant capacity (TAC) was determined by mixing 20 µL of serum with 1 mL of chromogen solution. A blank (distilled water + chromogen) and standard (standard solution + chromogen) were prepared similarly. Absorbance was measured at 600 nm at 37 °C using the Alcyon 300 device, before and after adding 200 µL of substrate. TAC values were obtained using a commercial Randox kit at the Applied Pharmaceutical Research Center, Tabriz, Iran. Jejunal Morphology At 42 days of age, one bird per replicate was humanely euthanized, and a 1 cm segment was collected from the midpoint of the jejunum. Morphometric parameters were evaluated according to the method described by Brudnicki et al., 2017 [21] . Cecal Escherichia coli Population count At 42 days of age, one bird per replicate was randomly selected and slaughtered. Cecal contents were aseptically collected into sterile containers and transported on ice to the microbiology laboratory for analysis. Because one of the study objectives was to assess the reduction of Escherichia coli , quantification of cecal E. coli populations was performed using the culture method described by Koju et al. (2022) [ 22 ] . Statistical Analysis Data were tested for normality using the UNIVARIATE procedure in SAS 9.4 (SAS Institute, 2012) [23 ] . Non-normally distributed data were arcsine-transformed prior to analysis. Data were analyzed using the GLM procedure of SAS in a completely randomized design with a 3 × 2 factorial arrangement (three FB levels and two SAC levels). Mean comparisons were conducted using Tukey’s test at P < 0.05, and orthogonal contrasts were performed between diets containing FB and those without FB. Results Essential Oil Composition Gas chromatography–mass spectrometry (GC–MS) analysis was performed to determine the chemical constituents of the essential oil extracted from the root of FB. The results showed that the oil contained considerable amounts of phenolic compounds (35.34 μg/mL) and flavonoids (21.37 μg/mL). These bioactive compounds are well known for their antioxidant and anti-inflammatory properties and play important roles in maintaining general health, particularly by supporting digestive and immune functions. The relatively high concentrations of phenols and flavonoids suggest that FB root may serve as a valuable natural source for use in poultry nutrition, as well as in the pharmaceutical and herbal supplement industries. Growth Performance The effects of dietary supplementation with FB root powder and SAC on the growth performance of broiler chicks are presented in Table 2. The main effect of SAC on FI showed that supplementation significantly increased FI during both the 1–10 d and 1–42 d periods compared with the control group (P < 0.05). In contrast, FB supplementation alone did not significantly affect FI during any growth phase. Notably, birds receiving 0.15% SAC consumed significantly more feed than those without SAC supplementation. A significant interaction between FB root powder and SAC was observed for FI during the 25–42 d and 1–42 d periods (P < 0.05). Birds fed 1.5% FB with 0.15% SAC consumed significantly more feed than those fed 1.5% FB without SAC. Supplementation with 0.15% SAC significantly improved WG during the 1–10 d period (P < 0.05). Similarly, BWG during the 25–42 d phase was higher in birds receiving 0.15% SAC compared with un-supplemented birds (P < 0.05). Over the entire rearing period (1–42 d), the inclusion of 0.15% SAC resulted in significantly greater BWG compared with the control (P < 0.05). Carcass Traits As shown in Table 3, dietary treatments did not significantly affect the relative weights of major body organs, including the heart, liver, small intestine, abdominal fat, thighs, breast, and whole carcass (P > 0.05). Likewise, the interaction between FB and SAC levels had no significant influence on internal organ weights. Orthogonal comparisons between FB-supplemented and control groups revealed no significant differences in carcass characteristics. Blood Serum Metabolites The effects of dietary FB root powder, with and without SAC, on blood serum biochemical parameters are presented in Table 4. The interaction between FB and SAC significantly influenced serum uric acid concentration at 42 d of age (P < 0.05). Increasing dietary FB levels resulted in elevated serum albumin concentrations (P < 0.05), while SAC supplementation had no effect on albumin levels. FB supplementation significantly decreased serum cholesterol concentration (P < 0.05), whereas SAC inclusion did not affect cholesterol levels. The FB × SAC interaction was not significant for serum cholesterol. A significant interaction between SAC and FB was detected for triglyceride levels (P < 0.05); the lowest triglyceride concentration was recorded in birds fed 0.75% FB without SAC, whereas the highest occurred in the group without both FB and SAC. The effect of FB on serum phosphorus concentration was also significant (P < 0.05), while SAC had no effect. The FB × SAC interaction was not significant for phosphorus levels. Orthogonal comparisons between control and FB-supplemented groups revealed that FB significantly affected serum uric acid, cholesterol, triglyceride, and phosphorus concentrations (P < 0.05), but not total protein, HDL-C, LDL-C, glucose, or calcium. Humoral Immune Response The effects of FB root powder, with and without SAC, on antibody titers (IgM, IgG, and total immunoglobulin (IgT)) against SRBC at 35 and 42 d of age are shown in Table 5. SAC supplementation did not significantly influence antibody titers at 35 d; however, the 1.5 g/kg FB diet significantly increased IgT secretion in response to the SRBC antigen at 42 d (P < 0.05). FB supplementation significantly affected antibody production, with the 15 g/kg diet resulting in higher IgT and IgG titers at 35 d, and higher IgG and IgM titers at 42 d, compared with the control. No significant differences were observed for IgM titers at 35 d or IgT at 42 d. The FB × SAC interaction did not significantly affect antibody responses at either age. Lipid Peroxidation and Antioxidant Enzyme Activities As shown in Table 6, FB supplementation significantly reduced serum MDA concentrations at 42 d of age (P < 0.05). However, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and TAC in serum were not significantly affected by FB or SAC, either alone or in combination. According to Table 7, SOD activity in liver tissue increased significantly with rising FB levels (P < 0.05), whereas GPx activity did not differ among treatments. As presented in Table 8, increasing dietary FB levels significantly reduced MDA concentrations and enhanced TAC in liver tissue (P < 0.05). Jejunal Morphology The effects of FB root powder, with and without SAC, on jejunal morphology at 42 d of age are summarized in Table 8. The inclusion of SAC and FB root powder significantly increased jejunal villus height (P < 0.05). The villus height-to-width ratio was significantly increased by dietary inclusion of 0.15% SAC (P < 0.05). Furthermore, supplementation with 1.5% FB root powder increased the villus height-to-crypt depth ratio and the absorptive surface area of the jejunum (P < 0.05). However, FB and SAC supplementation, alone or in combination, had no significant effects on villus width, crypt depth, or their interactions. The FB × SAC interaction was not significant for any jejunal morphometric parameters. Cecal Escherichia coli Population As illustrated in Figure 1, the interaction between FB root powder and SAC significantly reduced the cecal population of Escherichia coli in broiler chicks at 42 d of age (P < 0.05). Discussion Different levels of FB root powder had no significant effect on the FCR of broiler chicks during any rearing period. Although the effects of medicinal plants on broiler performance are inconsistent, several studies suggest that such plants and their extracts do not significantly affect average daily FI [1] . For example, Abdullahi et al. (2013) reported that dietary supplementation with Ferula gummosa root powder at levels up to 3% did not significantly influence the growth performance of broiler chicks, likely due to the absence of a sweetener in their diets [26] . The positive effects of dietary supplementation with essential oils from medicinal plants on WG and FCR in broilers have been previously documented [24] , consistent with the findings of the present study. The interaction between FB root powder and SAC increased FI, suggesting that while the bitter taste of FB may reduce palatability, SAC effectively neutralized this bitterness and enhanced feed consumption in the SAC-supplemented groups. Previous research has indicated that essential oils from medicinal plants such as rosemary and oregano, when included at concentrations of 0.1%, can improve BWG, FI, and FCR [2] . Overall, improvements in growth performance associated with medicinal plants are likely due to their bioactive compounds, including flavonoids and phenolic compounds, which possess anti-inflammatory and antioxidant properties. These compounds can positively influence digestive activity, improve nutrient utilization efficiency, and inhibit harmful microorganisms in the gut [25] . The lack of significant effects of FB and SAC supplementation on carcass traits in the current study is consistent with previous findings [26] , which showed no significant effects on organ weights or intestinal length following supplementation with Ferula gummosa root powder. It has been suggested that essential oils from medicinal plants may reduce abdominal fat deposition by lowering serum lipid levels, thereby improving carcass quality and promoting consumer health [27] . Conversely, Viana et al. (2019) [28] reported no significant differences in organ weights in mice treated with essential oils, which aligns with the results of the present study. In this study, dietary supplementation with FB root powder effectively reduced blood cholesterol concentrations. The active compounds present in medicinal plants have been reported to lower blood lipid levels [29] , reduce cholesterol, and even offer protective effects against cancer [30] . Similar cholesterol-lowering effects of medicinal plant essential oils have been observed in broiler chicks [31] . A significant reduction in blood glucose levels in saccharin-treated rats compared with controls has also been reported, supporting findings from other animal studies [11] . Oral administration of SAC may influence carbohydrate metabolism indirectly, possibly due to its effects on liver function. Studies have shown that triglyceride and total cholesterol levels decrease following SAC administration in rats [11] , consistent with the results of the present study. This reduction may be attributed to the direct or indirect influence of SAC on lipid metabolism and lipid peroxidation. Reduced blood triglyceride levels may result from decreased hepatic synthesis of very low-density lipoproteins (VLDL) [32], while reductions in total cholesterol may be associated with decreases in triglyceride-rich lipoproteins. The intestinal microbiota plays a crucial role in cholesterol metabolism by converting bile acids derived from hepatic cholesterol. Therefore, the use of antimicrobial compounds—including antibiotics and medicinal plants—can reduce blood cholesterol levels [32] . The observed increase in blood uric acid levels in this study is consistent with findings from other animal studies [11] , which reported elevated blood urea levels in SAC-treated mice. The significant increase in serum phosphorus concentration in broiler chicks supplemented with FB root powder suggests that this medicinal plant may enhance phosphorus metabolism through physiological and nutritional mechanisms. Bioactive compounds in FB could stimulate intestinal phosphorus absorption, increase digestive enzyme activity, and promote the release of phosphorus from phytic acid by modifying the intestinal microflora. Additionally, the combination of FB with SAC may improve FI by masking FB’s bitterness, resulting in greater phosphorus consumption. FB may also influence phosphorus-regulating hormones such as vitamin D and parathyroid hormone, enhancing absorption and reducing renal excretion. Collectively, these effects suggest that FB can improve serum phosphorus levels, particularly in well-formulated diets. Supplementation with FB root powder enhanced antibody production in broiler chicks, consistent with previous reports [33] demonstrating that essential oils from medicinal plants improve both humoral and cellular immune responses. The presence of phenolic and flavonoid compounds in FB likely contributes to improved immunity through their antioxidant and antibacterial properties [34] . Alkaloids, another class of bioactive plant compounds, can bind to serum albumin and act as antigens, thereby stimulating immune responses and antibody production [35] . The phenolic and flavonoid constituents of FB, together with its antioxidant properties, appear to strengthen the immune system. The observed increases in total antibody, IgG, and IgM levels in broilers may be attributed to these compounds. Flavonoids, known for their diverse biological functions—including immune enhancement, cholesterol regulation, blood pressure modulation, and disease prevention—likely contributed to the improved immune response. Previous studies have shown that thymol and carvacrol, active compounds in several medicinal plants, enhance immune function through antibacterial, antiviral, and antioxidant effects [36] , in agreement with the current findings. Rapid growth in broiler chickens often leads to increased free radical production and fat accumulation, elevating oxidative stress and predisposing birds to diseases, particularly cardiovascular disorders. The inclusion of antioxidant-rich feed ingredients can strengthen the endogenous antioxidant defense system and alleviate oxidative stress. Although aspartate aminotransferase (AST) activity is often associated with cellular damage, experimental treatments in this study did not significantly affect AST or alanine aminotransferase (ALT) levels, indicating no liver impairment. These enzymes typically occur in low concentrations in the blood, and marked elevations are generally linked to hepatic obstruction. Therefore, the lack of significant changes suggests that FB and SAC did not adversely affect liver function. In the current experiment, SOD activity increased with higher FB supplementation, implying that the phenolic and flavonoid constituents of FB may enhance the antioxidant defense system. This response indicates a protective mechanism against oxidative stress and suggests that FB could aid in scavenging free radicals from tissues. FB supplementation also reduced serum MDA concentrations, reflecting lower lipid peroxidation. TAC, an indicator of overall antioxidant status, was not significantly affected by FB or SAC, suggesting that antioxidant homeostasis was maintained across treatments [37] . The activity of antioxidant enzymes, including SOD and GPx, was also evaluated. SOD neutralizes superoxide radicals, preventing their interaction with biological membranes and the formation of more reactive species. In conjunction with catalase and GPx, SOD facilitates the conversion of hydrogen peroxide into water and molecular oxygen [38] . GPx detoxifies lipid hydroperoxides formed during membrane lipid peroxidation. Under stress, chickens typically show increased SOD activity as an adaptive response to oxidative pressure [39] . Reactive oxygen species, such as superoxide radicals, can damage the intestinal mucosa and impair nutrient absorption. Given the antioxidant potential of FB, its inclusion in diets may enhance SOD and GPx activity [40] , thereby protecting intestinal integrity and improving nutrient utilization. In contrast to previous reports showing decreased villus height following SAC supplementation [15] , the present study found an increase in villus height under similar conditions. Increased villus height and crypt depth expand the absorptive surface area of the intestine, improving nutrient uptake. A thinner intestinal epithelium further facilitates rapid absorption [41] . Longer villi and shallower crypts are associated with improved nutrient absorption efficiency and reduced FCR. The intestinal villi contain absorptive enterocytes, goblet cells, and enterochromaffin cells, with enterocytes—located at the villus tips—playing the primary role in nutrient absorption. Therefore, villus height serves as a reliable indicator of intestinal health and absorptive capacity [42] . The combined inclusion of FB and SAC likely increased the number of enterocytes in the jejunum, enhancing nutrient absorption. Although this finding contrasts with earlier studies [43] , it agrees with others reporting that herbal medicines from the Ferula genus, including FB and Ferula gummosa , reduce Escherichia coli populations in the digestive tract [44] . Ferula gummosa exhibits strong antibacterial properties against both Gram-positive and Gram-negative bacteria, including E. coli , Salmonella typhimurium , and Pseudomonas aeruginosa [45] . The major components of FB and Ferula gummosa essential oils—α- and β-pinene—are known for their antimicrobial and insecticidal properties [26] . Hence, modulation of gut microflora by these compounds can enhance performance, suggesting that such natural additives could serve as effective alternatives to antibiotic growth promoters [46] . Conclusion The results of this experiment demonstrate that supplementation with 0.15% sodium saccharin mitigates the reduction in feed intake caused by the bitterness of Ferula badrakema root powder in broiler diets. Additionally, the inclusion of 0.75% FB root powder enhances immune responses and improves blood biochemical parameters by reducing cholesterol and triglyceride concentrations. Furthermore, FB supplementation strengthens antioxidant status, increases jejunal villus height and absorptive surface area, and decreases Escherichia coli populations in the cecum of broiler chicks. Declarations Funding This work was financially supported by the Faculty of Agriculture, Ferdowsi University of Mashhad, Iran, under Grant Number 3/48465. Author Contribution HR.K.: Writing first draft; Investigation; Data curation; Software. A. H.: Project administration; Supervision, Software; Methodology; Conceptualization; Funding acquisition; Writing – review and editing. M.AB: Advising; Review and editing. Acknowledgements The authors express their gratitude to the Vice President for Research at the Ferdowsi University of Mashhad, Iran. Data Availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Rafeeq, M. et al. The use of some herbal plants as effective alternatives to antibiotic growth enhancers in poultry nutrition. World's Poult . Sci . J . 78(4), 1067-1085. (2022). https://doi.org/10.1080/00439339.2022.2108362 Cross, D. E., McDevitt, R. M., Hillman, K., & Acamovic, T. The effect of herbs and their associated essential oils on performance, dietary digestibility and gut microflora in chickens from 7 to 28 days of age. Br. Poult. 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(2017). https://www.thejaps.org.pk/docs/v-27-04/03.pdf Koju, P. et al. Antimicrobial resistance in E. coli isolated from chicken cecum samples and factors contributing to antimicrobial resistance in Nepal. Trop. Med. Infect. 7(9), 249-262. (2022). https://doi.org/10.3390/23. SAS. SAS User's Guide: Statistics, Version 9.4. (SAS Institute Inc., 2012). El-Sayed, Y., Khalil, W., Fayez, N., & Mohamed Abdel-Fattah, A. F. Enhancing effect of oregano essential oil and Bacillus subtilis on broiler immune function, intestinal morphology and growth performance. BMC Veterinary Research. 20(1), 112 (2024). https://doi.org/10.1186/s12917-024-03960-w Zhang, F. et al. Dietary oregano aqueous extract improves growth performance and intestinal health of broilers through modulating gut microbial compositions. J. Anim. Sci. Biotec. 14, 77-91. (2023). https://doi.org/10.1186/s40104-023-00857-w Abdollahi, Z., Hassanabadi, A., & Golian, A. Effects of Ferula gummosa Boiss. root on performance, microbial population and nutrient digestibility in broiler chicks. Iran. J. Anim. Sci. Res . 5, 112-118. (2013). (In Persian). https://doi.org/10.22067/ijasr.v5i2.28288 Elbaz, A. M. et al. Effects of garlic and lemon essential oils on performance, digestibility, plasma metabolite, and intestinal health in broilers under environmental heat stress. BMC vet Res. 18, 430 (2022). https://doi.org/10.1186/s12917-022-03530-y Viana, A. F. S. et al. (−)-Myrtenol accelerates healing of acetic acid-induced gastric ulcers in rats and in human gastric adenocarcinoma cells. Eur. J. Pharmacol. 854, 139-148. (2019). https://doi.org/10.1016/j.ejphar.2019.04.025 Al-Khalaifah, H. et al. Effect of ginger powder on production performance, antioxidant status, hematological parameters, digestibility, and plasma cholesterol content in broiler chickens. Animals , 12(7), 901. (2022). https://doi.org/10.3390/ani12070901 Reboredo-Rodríguez, P., & Varela-López, A. Essential Oils from Aromatic Plants in Cancer Prevention and Treatment. In Nutraceuticals and Cancer Signaling: Clinical Aspects and Mode of Action. 61-81. (Springer International Publishing, Cham 2021). https://link.springer.com/chapter/10.1007/978-3-030-74035-1_4#citeas Molinero, N., Ruiz, L., Sánchez, B., Margolles, A., & Delgado, S. Intestinal bacteria interplay with bile and cholesterol metabolism: implications on host physiology. Frontiers. physiol. 10, 185. (2019). https://doi.org/10.3389/fphys.2019.00185 Roberfroid, M. B. Prebiotics: concept, definition, criteria, methodologies, and products. CRC Press: Boca Raton, FL, USA, pp. 39-69. (2008). https://www.taylorfrancis.com/chapters/edit/10.1201/9780849381829-8 Lotfollahian, H., Rasi, H. M., & Najmabadi, A. Investigating the effect of fennel root powder on productive performance, immune system and meat quality of broiler chickens. J Anim Prod. 25, 445-459. (2023). https://doi.org/%E2%80%8E%E2%80%8E%E2%80%8E10.22059/jap.2023.362688.623755 Satorov, S., Mavlonazarova, S., Yusufi, S., & Dushenkov, V. Total Polyphenol Content, Antioxidant Potential, Antibacterial and Antifungal Properties of Ferula L . Species Growing in Tajikistan. J. Drug Alcohol Res. 13, 1-9. (2024). https://doi.org/10.4303/JDAR/236424 Divsalar, K., Saravani, R., Meymandi, M., Taei, M., & Sheykh, A. A. Electrophoretic profile of albumin, α1, α2, β and γ globulin in sera of opioid dependants and non-dependants. Yafte 9: 13-19 (2008). (In Persian). https://www.sid.ir/paper/80026/en Mączka, W., Twardawska, M., Grabarczyk, M., & Wińska, K. Carvacrol—A natural phenolic compound with antimicrobial properties. Antibiotics , 12(5), 824. (2023). https://doi.org/10.3390/antibiotics12050824 Al-Surrayai, T., & Al-Khalaifah, H. Dietary supplementation of fructooligosaccharides enhanced antioxidant activity and cellular immune response in broiler chickens. Front. vet. sci. 9, 857294 (2022). https://doi.org/10.3389/fvets.2022.857294 Finkel, T., & Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature. 408: 239-247 (2000). https://www.nature.com/articles/35041687#citeas Azad, M., Kikusato, M., Maekawa, T., Shirakawa, H., & Toyomizu, M. Metabolic characteristics and oxidative damage to skeletal muscle in broiler chickens exposed to chronic heat stress. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 155 : 401-406 (2010). https://doi.org/10.1016/j.cbpa.2009.12.011 Mishra, B., & Jha, R. Oxidative stress in the poultry gut: potential challenges and interventions. Front. vet. sci. 6, 60 (2019). https://doi.org/10.3389/fvets.2019.00060 Nguyen, T. N. D., Le, H. N., Eva, P., Alberto, F., & Le, T. H. Relationship between the ratio of villous height: crypt depth and gut bacteria counts as well production parameters in broiler chickens. J. Agric. Dev. 20 1-10 (2021). https://doi.org/10.52997/jad.1.03.2021 Bonis, V., Rossell, C., & Gehart, H. The intestinal epithelium–fluid fate and rigid structure from crypt bottom to villus tip. Front. cell dev. biol. 9, 661931 (2021). https://doi.org/10.3389/fcell.2021.661931 Asili, J., Sahebkar, A., Bazzaz, B. S. F., Sharifi, S., & Iranshahi, M. Identification of essential oil components of Ferula badrakema fruits by GC-MS and 13C-NMR methods and evaluation of its antimicrobial activity. J. Essent. Oil-Bear. Plants. 12, 7-15 (2009). https://doi.org/10.1080/0972060X.2009.10643685 Alimoradi Tamrin, Z., Darmani Kohi, H., & Gavi Hosseinzadeh, N. Effect of xylanase and galbanum essential oil ‎( Ferula gummosis Boiss ) supplementation of wheat-based diets on performance and intestinal microflora of‎ broiler chickens. Anim. Prod. 20, 1-14 (2018). https://doi.org/10.22059/jap.2018.231919.623178 Ghasemi, Y., Faridi, P., Mehregan, I., & Mohagheghzadeh, A. Ferula gummosa fruits: an aromatic antimicrobial agent. Chem. Nat. Compd. 41, 311-314 (2005). https://link.springer.com/article/10.1007/s10600-005-0138-3 Ayalew, H., Zhang, H., Wang, J., Wu, S., Qiu, K., Qi, G., Tekeste, A., Wassie, T., & Chanie, D. Potential feed additives as antibiotic alternatives in broiler production. Front. Vet. Sci. 9, 916473 (2022). https://doi.org/10.3389/fvets.2022.916473 Tables Tables 1 to 8 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":28736,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of dietary supplementation of \u003cem\u003eFerula badrakema\u003c/em\u003e root powder with and without sodium saccharin sweetener on the Escherichia coli (Log\u003csub\u003e10\u003c/sub\u003e CFU/g) at the age of 42 d.\u003c/p\u003e\n\u003cp\u003eSAC, Sodium saccharin; FB, \u003cem\u003eFerula badrakema\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e Means with different letters within a column are significantly different (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eTreatments: 1: SAC0*FB0 2: SAC0*FB0.75 3: SAC0*FB1.5 4: SAC0.15*FB0 5: SAC0.15*FB0.75 6: SAC0.15*FB1.5\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7871471/v1/d169e38fab220f37a7c91982.png"},{"id":98444771,"identity":"6b967212-5736-45e9-987f-669a5b512069","added_by":"auto","created_at":"2025-12-17 17:17:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":717482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7871471/v1/fc1dbae1-b14b-4983-be8a-c2aa18b30792.pdf"},{"id":98432130,"identity":"d473cd96-a9e1-4dcd-9740-2934fa7b8d9f","added_by":"auto","created_at":"2025-12-17 16:49:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":70781,"visible":true,"origin":"","legend":"","description":"","filename":"Tables9.docx","url":"https://assets-eu.researchsquare.com/files/rs-7871471/v1/d27eb98475a9dade0b2924ba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary supplementation of Ferula badrakema and sodium saccharin: Effects on the growth performance, blood metabolites, immune response, antioxidant status, and gut histomorphology in broiler chicks","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibacterial compounds play a crucial role in maintaining microbial balance and promoting a healthy gut microflora in poultry. However, the overlap between antibiotics used in poultry production and those applied in human medicine has raised significant concerns about the potential transfer of antibiotic-resistant bacterial strains to humans through poultry products. In response to these concerns particularly regarding the use of antibiotics as growth promoters, extensive research has focused on identifying effective and safe alternatives. Many of these alternatives aim to enhance poultry performance by modulating the intestinal microbial population.\u003c/p\u003e \u003cp\u003eAmong the promising alternatives investigated in recent years are medicinal plants and their extracts \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have demonstrated beneficial effects of medicinal plants on poultry growth performance \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, with many species exhibiting notable antimicrobial activity. In addition to their antimicrobial effects, medicinal plants can improve digestive function and nutrient absorption, stimulate appetite, and reduce serum lipid concentrations \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eFerula\u003c/em\u003e, belonging to the family Peucedaneae and subfamily Apioideae, comprises approximately 133 species distributed across the Mediterranean region. Iran is particularly rich in \u003cem\u003eFerula\u003c/em\u003e species, with more than 70 having been chemically characterized. Species of this genus display considerable biological diversity and exhibit antibacterial, antifungal, anticancer, and antioxidant activities \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFerula badrakema\u003c/em\u003e is a resinous and aromatic plant native to Iran, predominantly found in Tandoureh National Park, Dargaz County, North Khorasan Razavi Province. Its antibacterial and antifungal properties are attributed mainly to its high content of α-pinene and β-pinene. Similar to other \u003cem\u003eFerula\u003c/em\u003e species, FB is a rich source of sesquiterpene coumarins. Although its volatile compounds have not yet been studied, investigations of other \u003cem\u003eFerula\u003c/em\u003e species in various regions of the world have yielded promising pharmacological results \u003csup\u003e[8\u0026ndash;9\u0026minus;10]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSaccharin (1,2-benzisothiazol-3(2H)-one-1,1-dioxide) is a sulfonamide derivative originally synthesized from coal tar compounds \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. It is approximately 300\u0026ndash;500 times sweeter than sucrose. Currently, high-intensity sweeteners are widely used in the diets of pigs and ruminants \u003csup\u003e[12\u0026ndash;13\u0026minus; 14]\u003c/sup\u003e. Due to their intense sweetness and negligible caloric value, such compounds are employed to enhance feed palatability, as the perception of sweetness has been linked to increased FI \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Previous studies have reported that dietary supplementation with sweeteners can increase FI and consequently improve growth performance in livestock \u003csup\u003e[16\u0026ndash;17\u0026minus;18]\u003c/sup\u003e. For example, Japanese quails have been shown to prefer sucrose solutions over plain water due to enhanced palatability. However, limited research has explored the physiological effects of sweeteners on the gastrointestinal tract of broilers \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven that many medicinal plants, including FB, possess a bitter taste that may reduce FI and hinder chick growth, this study aimed to evaluate the combined effects of FB root powder and saccharin supplementation. Specifically, this research sought to determine whether saccharin could mitigate the bitter taste of FB and to assess its effects on growth performance, immune response, blood serum metabolites, antioxidant status, cecal Escherichia coli counts, and carcass traits in broiler chicks.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was conducted in accordance with the comprehensive animal welfare guidelines approved by the Animal Care and Use Committee of Ferdowsi University of Mashhad, Mashhad, Iran (Approval Reference Number: 48465). The study was performed in compliance with the ARRIVE 2.0 guidelines regarding study design, the number and use of experimental animals, randomization procedures, statistical analyses, and other relevant aspects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBirds, Diets\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e and Housing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 468 one-day-old male Ross 308 broiler chicks were obtained from a commercial hatchery. The chicks were individually weighed and randomly allocated to six treatments with six floor-pen replicates per treatment, each containing 13 birds, following a completely randomized design arranged in a 3 × 2 factorial layout. The treatments consisted of three levels of FB root powder (0%, 0.75%, and 1.5%) and two levels of saccharin (SAC; 0% and 0.15%) supplemented in a basal diet.\u003c/p\u003e\n\u003cp\u003eAll birds received standard basal diets during the starter (1–10 days), grower (11–24 days), and finisher (25–42 days) phases, formulated according to Ross 308 broiler nutrition specifications (Aviagen, 2019) (Table 1). Feed and water were provided \u003cstrong\u003e\u003cem\u003ead libitum\u003c/em\u003e\u003c/strong\u003e throughout the experimental period. A lighting program of 18 hours of light and 6 hours of darkness was maintained daily. The initial room temperature was set at 32 °C and gradually reduced by 0.5 °C per day until it reached 21 °C at 21 days of age.\u003c/p\u003e\n\u003cp\u003eAverage daily feed intake, WG, and feed conversion ratio (FCR) were measured at the end of each rearing phase. Mortality was recorded daily and used to adjust FI data. At 42 days of age (maximum weight 2,700 g), one bird per replicate representative of the average pen weight, was selected, humanely euthanized by cervical dislocation, and dissected for carcass, organ, and intestinal weight determinations. All birds were anesthetized with CO\u003csub\u003e2\u003c/sub\u003e inhalation prior to the cervical dislocation to ensure minimal distress or pain to the animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood \u003c/strong\u003e\u003cstrong\u003eCollection\u003c/strong\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cstrong\u003eAnalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 42 days of age, one bird from each replicate was selected for blood sampling. Blood samples were collected from the wing vein using syringes without anticoagulant. After centrifugation (3000 × g for 10 min at 4 °C), serum samples were stored at –20 °C until analysis.\u003c/p\u003e\n\u003cp\u003eSerum concentrations of total protein, glucose, cholesterol, albumin, high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), uric acid, triglycerides, calcium, and phosphorus were determined using a commercial auto-analyzer (Alcyon model) and diagnostic kits (ParsAzmoon Co. Tehran, Iran) at the Laboratory of the Applied Pharmaceutical Research Center, Tabriz, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHumoral immune response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe humoral immune response was evaluated by injecting 0.1 mL of a 25% SRBC suspension into the breast muscle of one bird per replicate on days 28 and 35. Blood samples were collected from the wing vein on days 35 and 42 to measure primary and secondary antibody responses.\u003c/p\u003e\n\u003cp\u003eTwo milliliters of blood were collected 7 days after each SRBC injection. After clotting, serum was separated by centrifugation (3000 × g for 10 min at 4 °C) and incubated for 30 min at 56 °C to inactivate complement proteins. Total anti-SRBC antibody titers, immunoglobulin G (IgG), and immunoglobulin M (IgM) levels were determined. Antibody titers were expressed as the log₂ of the highest serum dilution that agglutinated 0.05 mL of a 2.5% SRBC suspension in phosphate-buffered saline \u003csup\u003e[19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid \u003c/strong\u003e\u003cstrong\u003ePeroxidation\u003c/strong\u003e\u003cstrong\u003e (MDA) and \u003c/strong\u003e\u003cstrong\u003eAntioxidant Enzyme Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver samples were stored in a potassium chloride solution (1.15% w/v; pH 4.7) and homogenized at 4 °C. The homogenate was centrifuged at 5000 × g for 15 min, and the supernatant was used to determine total SOD and glutathione peroxidase (GPx) activities as well as total protein concentration. Absorbance was measured using an auto-analyzer (Alcyon 300, Abbott, USA).\u003c/p\u003e\n\u003cp\u003eTSOD activity was determined colorimetrically using a Ransod kit \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e, and GPx activity was measured enzymatically using a Ransel kit \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. Serum MDA concentration was determined by reaction with thiobarbituric acid followed by extraction with normal butanol and spectrophotometric measurement at 532 nm against a standard curve. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the IFCC method with commercial kits (ParsAzmoon Co., Tehran, Iran).\u003c/p\u003e\n\u003cp\u003eFor serum MDA quantification, 500 µL of serum was mixed with 3 mL of 1% phosphoric acid, vortexed, and reacted with 1 mL of 0.675% thiobarbituric acid. The mixture was incubated in a water bath for 45 min, cooled, extracted with 3 mL of normal butanol, vortexed for 2 min, and centrifuged (3000 × g for 10 min). The absorbance of the supernatant was read at 532 nm, and MDA concentration was calculated using a standard curve.\u003c/p\u003e\n\u003cp\u003eTotal antioxidant capacity (TAC) was determined by mixing 20 µL of serum with 1 mL of chromogen solution. A blank (distilled water + chromogen) and standard (standard solution + chromogen) were prepared similarly. Absorbance was measured at 600 nm at 37 °C using the Alcyon 300 device, before and after adding 200 µL of substrate. TAC values were obtained using a commercial Randox kit at the Applied Pharmaceutical Research Center, Tabriz, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJejunal \u003c/strong\u003e\u003cstrong\u003eMorphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 42 days of age, one bird per replicate was humanely euthanized, and a 1 cm segment was collected from the midpoint of the jejunum. Morphometric parameters were evaluated according to the method described by Brudnicki et al., 2017 \u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCecal \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePopulation count\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 42 days of age, one bird per replicate was randomly selected and slaughtered. Cecal contents were aseptically collected into sterile containers and transported on ice to the microbiology laboratory for analysis. Because one of the study objectives was to assess the reduction of \u003cem\u003eEscherichia\u003c/em\u003e\u003cem\u003e coli\u003c/em\u003e, quantification of cecal \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e coli\u003c/em\u003e populations was performed using the culture method described by Koju et al. (2022) \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e22\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were tested for normality using the UNIVARIATE procedure in SAS 9.4 (SAS Institute, 2012) \u003csup\u003e[23\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. Non-normally distributed data were arcsine-transformed prior to analysis. Data were analyzed using the GLM procedure of SAS in a completely randomized design with a 3 × 2 factorial arrangement (three FB levels and two SAC levels). Mean comparisons were conducted using Tukey’s test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and orthogonal contrasts were performed between diets containing FB and those without FB.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEssential Oil Composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGas chromatography–mass spectrometry (GC–MS) analysis was performed to determine the chemical constituents of the essential oil extracted from the root of FB. The results showed that the oil contained considerable amounts of phenolic compounds (35.34 μg/mL) and flavonoids (21.37 μg/mL). These bioactive compounds are well known for their antioxidant and anti-inflammatory properties and play important roles in maintaining general health, particularly by supporting digestive and immune functions. The relatively high concentrations of phenols and flavonoids suggest that FB root may serve as a valuable natural source for use in poultry nutrition, as well as in the pharmaceutical and herbal supplement industries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePerformance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of dietary supplementation with FB root powder and SAC on the growth performance of broiler chicks are presented in Table 2.\u003c/p\u003e\n\u003cp\u003eThe main effect of SAC on FI showed that supplementation significantly increased FI during both the 1–10 d and 1–42 d periods compared with the control group (P \u0026lt; 0.05). In contrast, FB supplementation alone did not significantly affect FI during any growth phase. Notably, birds receiving 0.15% SAC consumed significantly more feed than those without SAC supplementation.\u003c/p\u003e\n\u003cp\u003eA significant interaction between FB root powder and SAC was observed for FI during the 25–42 d and 1–42 d periods (P \u0026lt; 0.05). Birds fed 1.5% FB with 0.15% SAC consumed significantly more feed than those fed 1.5% FB without SAC.\u003c/p\u003e\n\u003cp\u003eSupplementation with 0.15% SAC significantly improved WG during the 1–10 d period (P \u0026lt; 0.05). Similarly, BWG during the 25–42 d phase was higher in birds receiving 0.15% SAC compared with un-supplemented birds (P \u0026lt; 0.05). Over the entire rearing period (1–42 d), the inclusion of 0.15% SAC resulted in significantly greater BWG compared with the control (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCarcass\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTraits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 3, dietary treatments did not significantly affect the relative weights of major body organs, including the heart, liver, small intestine, abdominal fat, thighs, breast, and whole carcass (P \u0026gt; 0.05). Likewise, the interaction between FB and SAC levels had no significant influence on internal organ weights. Orthogonal comparisons between FB-supplemented and control groups revealed no significant differences in carcass characteristics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSerum Metabolites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of dietary FB root powder, with and without SAC, on blood serum biochemical parameters are presented in Table 4. The interaction between FB and SAC significantly influenced serum uric acid concentration at 42 d of age (P \u0026lt; 0.05). Increasing dietary FB levels resulted in elevated serum albumin concentrations (P \u0026lt; 0.05), while SAC supplementation had no effect on albumin levels.\u003c/p\u003e\n\u003cp\u003eFB supplementation significantly decreased serum cholesterol concentration (P \u0026lt; 0.05), whereas SAC inclusion did not affect cholesterol levels. The FB × SAC interaction was not significant for serum cholesterol. A significant interaction between SAC and FB was detected for triglyceride levels (P \u0026lt; 0.05); the lowest triglyceride concentration was recorded in birds fed 0.75% FB without SAC, whereas the highest occurred in the group without both FB and SAC.\u003c/p\u003e\n\u003cp\u003eThe effect of FB on serum phosphorus concentration was also significant (P \u0026lt; 0.05), while SAC had no effect. The FB × SAC interaction was not significant for phosphorus levels. Orthogonal comparisons between control and FB-supplemented groups revealed that FB significantly affected serum uric acid, cholesterol, triglyceride, and phosphorus concentrations (P \u0026lt; 0.05), but not total protein, HDL-C, LDL-C, glucose, or calcium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHumoral Immune Response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of FB root powder, with and without SAC, on antibody titers (IgM, IgG, and total immunoglobulin (IgT)) against SRBC at 35 and 42 d of age are shown in Table 5. SAC supplementation did not significantly influence antibody titers at 35 d; however, the 1.5 g/kg FB diet significantly increased IgT secretion in response to the SRBC antigen at 42 d (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eFB supplementation significantly affected antibody production, with the 15 g/kg diet resulting in higher IgT and IgG titers at 35 d, and higher IgG and IgM titers at 42 d, compared with the control. No significant differences were observed for IgM titers at 35 d or IgT at 42 d. The FB × SAC interaction did not significantly affect antibody responses at either age.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid Peroxidation and Antioxidant Enzyme Activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 6, FB supplementation significantly reduced serum MDA concentrations at 42 d of age (P \u0026lt; 0.05). However, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and TAC in serum were not significantly affected by FB or SAC, either alone or in combination.\u003c/p\u003e\n\u003cp\u003eAccording to Table 7, SOD activity in liver tissue increased significantly with rising FB levels (P \u0026lt; 0.05), whereas GPx activity did not differ among treatments. As presented in Table 8, increasing dietary FB levels significantly reduced MDA concentrations and enhanced TAC in liver tissue (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJejunal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMorphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of FB root powder, with and without SAC, on jejunal morphology at 42 d of age are summarized in Table 8. The inclusion of SAC and FB root powder significantly increased jejunal villus height (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eThe villus height-to-width ratio was significantly increased by dietary inclusion of 0.15% SAC (P \u0026lt; 0.05). Furthermore, supplementation with 1.5% FB root powder increased the villus height-to-crypt depth ratio and the absorptive surface area of the jejunum (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eHowever, FB and SAC supplementation, alone or in combination, had no significant effects on villus width, crypt depth, or their interactions. The FB × SAC interaction was not significant for any jejunal morphometric parameters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCecal\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 1, the interaction between FB root powder and SAC significantly reduced the cecal population of\u0026nbsp;\u003cem\u003eEscherichia coli\u003c/em\u003e in broiler chicks at 42 d of age (P \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDifferent levels of FB root powder had no significant effect on the FCR of broiler chicks during any rearing period. Although the effects of medicinal plants on broiler performance are inconsistent, several studies suggest that such plants and their extracts do not significantly affect average daily FI \u003csup\u003e[1]\u003c/sup\u003e. For example, Abdullahi et al. (2013) reported that dietary supplementation with \u003cem\u003eFerula gummosa\u003c/em\u003e root powder at levels up to 3% did not significantly influence the growth performance of broiler chicks, likely due to the absence of a sweetener in their diets \u003csup\u003e[26]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe positive effects of dietary supplementation with essential oils from medicinal plants on WG and FCR in broilers have been previously documented \u003csup\u003e[24]\u003c/sup\u003e, consistent with the findings of the present study. The interaction between FB root powder and SAC increased FI, suggesting that while the bitter taste of FB may reduce palatability, SAC effectively neutralized this bitterness and enhanced feed consumption in the SAC-supplemented groups. Previous research has indicated that essential oils from medicinal plants such as rosemary and oregano, when included at concentrations of 0.1%, can improve BWG, FI, and FCR \u003csup\u003e[2]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOverall, improvements in growth performance associated with medicinal plants are likely due to their bioactive compounds, including flavonoids and phenolic compounds, which possess anti-inflammatory and antioxidant properties. These compounds can positively influence digestive activity, improve nutrient utilization efficiency, and inhibit harmful microorganisms in the gut \u003csup\u003e[25]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe lack of significant effects of FB and SAC supplementation on carcass traits in the current study is consistent with previous findings \u003csup\u003e[26]\u003c/sup\u003e, which showed no significant effects on organ weights or intestinal length following supplementation with \u003cem\u003eFerula gummosa\u003c/em\u003e root powder. It has been suggested that essential oils from medicinal plants may reduce abdominal fat deposition by lowering serum lipid levels, thereby improving carcass quality and promoting consumer health \u003csup\u003e[27]\u003c/sup\u003e. Conversely, Viana et al. (2019) \u003csup\u003e[28]\u003c/sup\u003e reported no significant differences in organ weights in mice treated with essential oils, which aligns with the results of the present study.\u003c/p\u003e\n\u003cp\u003eIn this study, dietary supplementation with FB root powder effectively reduced blood cholesterol concentrations. The active compounds present in medicinal plants have been reported to lower blood lipid levels \u003csup\u003e[29]\u003c/sup\u003e, reduce cholesterol, and even offer protective effects against cancer \u003csup\u003e[30]\u003c/sup\u003e. Similar cholesterol-lowering effects of medicinal plant essential oils have been observed in broiler chicks \u003csup\u003e[31]\u003c/sup\u003e. A significant reduction in blood glucose levels in saccharin-treated rats compared with controls has also been reported, supporting findings from other animal studies \u003csup\u003e[11]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOral administration of SAC may influence carbohydrate metabolism indirectly, possibly due to its effects on liver function. Studies have shown that triglyceride and total cholesterol levels decrease following SAC administration in rats \u003csup\u003e[11]\u003c/sup\u003e, consistent with the results of the present study. This reduction may be attributed to the direct or indirect influence of SAC on lipid metabolism and lipid peroxidation. Reduced blood triglyceride levels may result from decreased hepatic synthesis of very low-density lipoproteins (VLDL) [32], while reductions in total cholesterol may be associated with decreases in triglyceride-rich lipoproteins.\u003c/p\u003e\n\u003cp\u003eThe intestinal microbiota plays a crucial role in cholesterol metabolism by converting bile acids derived from hepatic cholesterol. Therefore, the use of antimicrobial compounds\u0026mdash;including antibiotics and medicinal plants\u0026mdash;can reduce blood cholesterol levels \u003csup\u003e[32]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe observed increase in blood uric acid levels in this study is consistent with findings from other animal studies \u003csup\u003e[11]\u003c/sup\u003e, which reported elevated blood urea levels in SAC-treated mice. The significant increase in serum phosphorus concentration in broiler chicks supplemented with FB root powder suggests that this medicinal plant may enhance phosphorus metabolism through physiological and nutritional mechanisms. Bioactive compounds in FB could stimulate intestinal phosphorus absorption, increase digestive enzyme activity, and promote the release of phosphorus from phytic acid by modifying the intestinal microflora. Additionally, the combination of FB with SAC may improve FI by masking FB\u0026rsquo;s bitterness, resulting in greater phosphorus consumption. FB may also influence phosphorus-regulating hormones such as vitamin D and parathyroid hormone, enhancing absorption and reducing renal excretion. Collectively, these effects suggest that FB can improve serum phosphorus levels, particularly in well-formulated diets.\u003c/p\u003e\n\u003cp\u003eSupplementation with FB root powder enhanced antibody production in broiler chicks, consistent with previous reports \u003csup\u003e[33]\u003c/sup\u003e demonstrating that essential oils from medicinal plants improve both humoral and cellular immune responses. The presence of phenolic and flavonoid compounds in FB likely contributes to improved immunity through their antioxidant and antibacterial properties \u003csup\u003e[34]\u003c/sup\u003e. Alkaloids, another class of bioactive plant compounds, can bind to serum albumin and act as antigens, thereby stimulating immune responses and antibody production \u003csup\u003e[35]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe phenolic and flavonoid constituents of FB, together with its antioxidant properties, appear to strengthen the immune system. The observed increases in total antibody, IgG, and IgM levels in broilers may be attributed to these compounds. Flavonoids, known for their diverse biological functions\u0026mdash;including immune enhancement, cholesterol regulation, blood pressure modulation, and disease prevention\u0026mdash;likely contributed to the improved immune response. Previous studies have shown that thymol and carvacrol, active compounds in several medicinal plants, enhance immune function through antibacterial, antiviral, and antioxidant effects \u003csup\u003e[36]\u003c/sup\u003e, in agreement with the current findings.\u003c/p\u003e\n\u003cp\u003eRapid growth in broiler chickens often leads to increased free radical production and fat accumulation, elevating oxidative stress and predisposing birds to diseases, particularly cardiovascular disorders. The inclusion of antioxidant-rich feed ingredients can strengthen the endogenous antioxidant defense system and alleviate oxidative stress.\u003c/p\u003e\n\u003cp\u003eAlthough aspartate aminotransferase (AST) activity is often associated with cellular damage, experimental treatments in this study did not significantly affect AST or alanine aminotransferase (ALT) levels, indicating no liver impairment. These enzymes typically occur in low concentrations in the blood, and marked elevations are generally linked to hepatic obstruction. Therefore, the lack of significant changes suggests that FB and SAC did not adversely affect liver function.\u003c/p\u003e\n\u003cp\u003eIn the current experiment, SOD activity increased with higher FB supplementation, implying that the phenolic and flavonoid constituents of FB may enhance the antioxidant defense system. This response indicates a protective mechanism against oxidative stress and suggests that FB could aid in scavenging free radicals from tissues.\u003c/p\u003e\n\u003cp\u003eFB supplementation also reduced serum MDA concentrations, reflecting lower lipid peroxidation. TAC, an indicator of overall antioxidant status, was not significantly affected by FB or SAC, suggesting that antioxidant homeostasis was maintained across treatments \u003csup\u003e[37]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe activity of antioxidant enzymes, including SOD and GPx, was also evaluated. SOD neutralizes superoxide radicals, preventing their interaction with biological membranes and the formation of more reactive species. In conjunction with catalase and GPx, SOD facilitates the conversion of hydrogen peroxide into water and molecular oxygen \u003csup\u003e[38]\u003c/sup\u003e. GPx detoxifies lipid hydroperoxides formed during membrane lipid peroxidation. Under stress, chickens typically show increased SOD activity as an adaptive response to oxidative pressure \u003csup\u003e[39]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eReactive oxygen species, such as superoxide radicals, can damage the intestinal mucosa and impair nutrient absorption. Given the antioxidant potential of FB, its inclusion in diets may enhance SOD and GPx activity \u003csup\u003e[40]\u003c/sup\u003e, thereby protecting intestinal integrity and improving nutrient utilization.\u003c/p\u003e\n\u003cp\u003eIn contrast to previous reports showing decreased villus height following SAC supplementation \u003csup\u003e[15]\u003c/sup\u003e, the present study found an increase in villus height under similar conditions. Increased villus height and crypt depth expand the absorptive surface area of the intestine, improving nutrient uptake. A thinner intestinal epithelium further facilitates rapid absorption \u003csup\u003e[41]\u003c/sup\u003e. Longer villi and shallower crypts are associated with improved nutrient absorption efficiency and reduced FCR. The intestinal villi contain absorptive enterocytes, goblet cells, and enterochromaffin cells, with enterocytes\u0026mdash;located at the villus tips\u0026mdash;playing the primary role in nutrient absorption. Therefore, villus height serves as a reliable indicator of intestinal health and absorptive capacity \u003csup\u003e[42]\u003c/sup\u003e. The combined inclusion of FB and SAC likely increased the number of enterocytes in the jejunum, enhancing nutrient absorption.\u003c/p\u003e\n\u003cp\u003eAlthough this finding contrasts with earlier studies \u003csup\u003e[43]\u003c/sup\u003e, it agrees with others reporting that herbal medicines from the \u003cem\u003eFerula\u003c/em\u003e genus, including FB and \u003cem\u003eFerula gummosa\u003c/em\u003e, reduce \u003cem\u003eEscherichia\u003c/em\u003e\u003cem\u003e coli\u003c/em\u003e populations in the digestive tract \u003csup\u003e[44]\u003c/sup\u003e. \u003cem\u003eFerula gummosa\u003c/em\u003e exhibits strong antibacterial properties against both Gram-positive and Gram-negative bacteria, including \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e \u003csup\u003e[45]\u003c/sup\u003e. The major components of FB and \u003cem\u003eFerula gummosa\u003c/em\u003e essential oils\u0026mdash;\u0026alpha;- and \u0026beta;-pinene\u0026mdash;are known for their antimicrobial and insecticidal properties \u003csup\u003e[26]\u003c/sup\u003e. Hence, modulation of gut microflora by these compounds can enhance performance, suggesting that such natural additives could serve as effective alternatives to antibiotic growth promoters \u003csup\u003e[46]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this experiment demonstrate that supplementation with 0.15% sodium saccharin mitigates the reduction in feed intake caused by the bitterness of \u003cem\u003eFerula badrakema\u003c/em\u003e root powder in broiler diets. Additionally, the inclusion of 0.75% FB root powder enhances immune responses and improves blood biochemical parameters by reducing cholesterol and triglyceride concentrations. Furthermore, FB supplementation strengthens antioxidant status, increases jejunal villus height and absorptive surface area, and decreases\u0026nbsp;\u003cem\u003eEscherichia coli\u003c/em\u003e populations in the cecum of broiler chicks.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the Faculty of Agriculture, Ferdowsi University of Mashhad, Iran, under Grant Number 3/48465.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHR.K.: Writing first draft; Investigation; Data curation; Software. A. H.: Project administration; Supervision, Software; Methodology; Conceptualization; Funding acquisition; Writing \u0026ndash; review and editing. M.AB: Advising; Review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors express their gratitude to the Vice President for Research at the Ferdowsi University of Mashhad, Iran.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRafeeq, M. et al. The use of some herbal plants as effective alternatives to antibiotic growth enhancers in poultry nutrition. \u003cem\u003eWorld\u0026apos;s Poult\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e Sci\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e J\u003c/em\u003e\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e 78(4), 1067-1085. (2022). https://doi.org/10.1080/00439339.2022.2108362\u003c/li\u003e\n\u003cli\u003eCross, D. E., McDevitt, R. M., Hillman, K., \u0026amp; Acamovic, T. 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Prod. \u003c/em\u003e20, 1-14 (2018). https://doi.org/10.22059/jap.2018.231919.623178\u003c/li\u003e\n\u003cli\u003eGhasemi, Y., Faridi, P., Mehregan, I., \u0026amp; Mohagheghzadeh, A. Ferula gummosa fruits: an aromatic antimicrobial agent. \u003cem\u003eChem. Nat. Compd. \u003c/em\u003e41, 311-314 (2005). https://link.springer.com/article/10.1007/s10600-005-0138-3\u003c/li\u003e\n\u003cli\u003eAyalew, H., Zhang, H., Wang, J., Wu, S., Qiu, K., Qi, G., Tekeste, A., Wassie, T., \u0026amp; Chanie, D. Potential feed additives as antibiotic alternatives in broiler production. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e 9, 916473 (2022). https://doi.org/10.3389/fvets.2022.916473\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 8 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Broiler chickens, Ferula badrakema, immune response, performance, sodium saccharin","lastPublishedDoi":"10.21203/rs.3.rs-7871471/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7871471/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the effects of dietary supplementation with \u003cem\u003eFerula badrakema \u003c/em\u003e(FB) root powder and sodium saccharin(SAC) on broiler chicks. A total of 468 one-day-old male Ross 308 chicks were allocated in a completely randomized design with a 3 × 2 factorial arrangement, consisting of three levels of FB (0%, 0.75%, and 1.5%) and two levels of SAC (0% and 0.15%), with six replicates per treatment. SAC supplementation significantly increased feed intake (FI) during days 1–10 and over the entire experimental period (days 1–42) (P \u0026lt; 0.05). The addition of FB further increased FI in diets containing SAC but decreased it in diets without SAC. SAC also improved body weight gain (WG) (P \u0026lt; 0.05) and significantly elevated blood uric acid levels (P \u0026lt; 0.05). Orthogonal contrasts showed that FB supplementation significantly increased blood uric acid, albumin, and phosphorus concentrations (P \u0026lt; 0.05), while reducing cholesterol and triglyceride levels (P \u0026lt; 0.05). Additionally, FB enhanced total antibody titers and IgG concentrations on day 35, as well as IgG and IgM levels on day 42. FB supplementation reduced malondialdehyde (MDA) concentrations and increased superoxide dismutase (SOD) activity (P \u0026lt; 0.05), indicating an improved antioxidant status. Both SAC and FB increased jejunal villus height (P \u0026lt; 0.05), and their interaction significantly reduced cecal \u003cem\u003eEscherichia coli\u003c/em\u003e counts (P \u0026lt; 0.05). In conclusion, SAC mitigated the FB-induced reduction in feed intake, whereas FB improved immune function, intestinal morphology, and blood lipid profiles in broiler chicks.\u003c/p\u003e","manuscriptTitle":"Dietary supplementation of Ferula badrakema and sodium saccharin: Effects on the growth performance, blood metabolites, immune response, antioxidant status, and gut histomorphology in broiler chicks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 12:17:40","doi":"10.21203/rs.3.rs-7871471/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-12-28T04:47:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18183052795098061952531469875994559367","date":"2025-12-15T18:19:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-10T17:22:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T08:52:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-24T02:16:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T15:31:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-22T15:00:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c5cee42-51ea-4b50-a864-c01ecf62c579","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59522389,"name":"Biological sciences/Biochemistry"},{"id":59522390,"name":"Health sciences/Diseases"},{"id":59522391,"name":"Health sciences/Health care"},{"id":59522392,"name":"Biological sciences/Immunology"},{"id":59522393,"name":"Health sciences/Medical research"},{"id":59522394,"name":"Biological sciences/Physiology"},{"id":59522395,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-12-15T12:17:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 12:17:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7871471","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7871471","identity":"rs-7871471","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}