Open Veterinary Journal, (2026), Vol. 16(4): 2299-2312

Research Article

10.5455/OVJ.2026.v16.i4.30

Ethanolic chicory leaf extract enhances hematological profile, gut microbiota, and intestinal morphology in rabbits

Maryam Hassan, Ayesha Saddiqa, Razia Iqbal, Rimsha Yaseen^* and Kiran Aftab

Department of Zoology, Faculty of Life Sciences, University of Gujrat, Punjab, Pakistan

*Corresponding Author: Rimsha Yaseen. Department of Zoology, Faculty of Life Sciences, Hafiz Hayat Campus, University of Gujrat, Punjab, Pakistan. Email: 22011714-017 [at] uog.edu.pk; rimshayaseen7818 [at] gmail.com

Submitted: 06/11/2025 Revised: 22/02/2026 Accepted: 02/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Prebiotics contribute substantially to livestock physiology by promoting health equilibrium. Chicory leaves have been a subject of research as a natural prebiotic and exhibit various phytoconstituents that act as nutraceutical agents. However, the in vivo efficacy of ethanolic leaf extract remains under-researched.

Aim: This study aimed to examine the impact of ethanolic chicory leaf extract (CLE) on the blood profile, intestinal microbial species, and histology of male rabbits.

Methods: To prepare the leaf extract, ethanol was used as the solvent and was extracted using a rotatory evaporator. Forty-eight rabbits were divided into 4 groups (12/group). G₀ was kept as the control, while the other groups G₁, G₂, and G₃ were given doses of 1, 2, and 3 ml/kg Body weight of CLE, respectively, on a daily basis for 45 days. On 15th, 30th, and 45th day samples (n=4 per group) were collected to assess hematological parameters via complete blood count, Escherichia coli and Lactobacillus colonies through plate count agar, and intestinal tissue histology by microtomy.

Results: One-way Analysis of variance indicated a dose-dependent impact of CLE on the research parameters as the greatest improvements were observed in the highest-dose group (G₃), followed by the medium (G₂) and low (G₁) dose groups compared to the control group. There was improvement in all blood parameters (p ≤ 0.05), whereas there was a reduction of E. coli and an increment of Lactobacillus population (p ≤ 0.05) in the ileum and cecum. Histological examination showed an increase in villus length and crypt depth, along with a decrease in villus width (p ˂ 0.05).

Conclusion: Administration of CLE to rabbits acts as a microbiota-modulatory agent that boosts gut health and structure while maintaining the blood profile. Hence, it can be used as a natural tonic for livestock to promote bio-functionalities and can be a potential alternative to synthetic prebiotics.

Keywords: Chicory leaf extract, E. coli, Hematological indices, Histology, Lactobacillus.

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Introduction

In the last two decades, many countries have shifted toward natural and safe feed additives to increase livestock production due to the side effects of traditional antibiotics (Juśkiewicz et al., 2011; Saeed et al., 2017). An emerging global trend involves incorporating phytogenic compounds found in medicinal plants into animal feed to lower the prevalence of gastrointestinal diseases. Moreover, they promote health through immunomodulation and antioxidant effects and increase nutrient digestion while providing antibiotic alternatives at a lower cost (Alagbe, 2019; Kuralkar and Kuralkar, 2021).

Cichorium intybus L. serves as a natural prebiotic because it contains abundant fructans and inulin. The leaves of the plant contain multiple essential minerals, such as potassium, calcium, phosphorus, and sodium, as well as phenols and vitamins A and C (Al-Snafi, 2016; Skoufos et al., 2020). The medicinal properties of chicory leaves have been employed in traditional practices because their flavonoids and sesquiterpene lactones offer a natural solution for multiple health conditions, such as their ethanolic extract, which is used for treating chronic periodontitis (Babaei et al., 2018). They have a high content of protein (14.70%), ash (10.91%), crude ether extract (3.68%), and crude fiber (16.78%) with relatively less carbohydrate content (68.50%), making them a nutritionally beneficial and safe addition to salads and vegetable dishes (Abbas et al., 2015).

Many studies have highlighted the substantial influence of prebiotics on the composition and metabolic activity of gut microbiota, which significantly affects the health of the host. Dietary modifications account for almost 57% of changes in the gut microbiota, whereas the genetics of the host play a limited role (12%) in gut microbiome modification (Zhang et al., 2010). The production of metabolites by the intestinal microbiome is directly linked to food consumption, which has a significant influence on physiological functions, such as vitamin production, nutrient absorption, modulation of the host’s organ-specific immune response, and maintenance of gut integrity (Petropoulos et al., 2017). Dietary fibers in chicory regulate the configuration, diversity, and functions of the gut microbiota and the composition of their metabolites, such as short-chain fatty acids (SCFAs), resulting in a healthy intestine, improved body metabolism, and improved animal behavior (Li et al., 2008; Lockyer and Stanner, 2019). Chicory extract is the main ingredient in Cichorium intybus L. formula (CILF) (Janda et al., 2021). A gut microbiota analysis was carried out in CILF-treated rats suffering from hyperuricemia nephropathy, and the results indicated a decrease in the abundance of bacteroides, whereas an increase in beneficial bacteria, such as Lactobacillaceae, Ruminococcaceae, and Bifidobacterium, was observed (Amatjan et al., 2023). Research evidence from various animal models, including broilers, rabbits, and pigs, shows that chicory leaf consumption enhances fermentation in the gut microbiota, as reflected by positive changes in cecal biochemical indicators (Liu et al., 2012; Khoobani et al., 2019).

Although the potential benefits of phytogenic feed additives in rabbit nutrition are well-documented, the specific effects of chicory leaf extract on rabbit health remain underexplored. This study employed ethanolic leaf extract of chicory because of its safety profile, capacity to solubilize a diverse range of both polar and non-polar phytochemical constituents, stability in preserving bioactive compounds, and extended shelf life. These attributes improve the bioavailability of pharmacologically significant compounds and facilitate their proper absorption. We hypothesized that the cumulative biological effects of the active components present in ethanolic CLE would elicit beneficial alterations in hematological parameters and prompt shifts in the microbial populations of Lactobacillus and Escherichia coli due to their antimicrobial properties. Moreover, we speculated that the prebiotic properties of chicory would improve intestinal histology, thereby enhancing the tissues’ overall health and functionality. To achieve this objective, we formulated various doses of the extract and administered it orally to an animal model to explore its biological activity by assessing its systemic effects under standard dietary conditions. This research seeks to contribute to the development of innovative, plant-based solutions for promoting animal health and reducing reliance on antibiotics by evaluating the efficacy of this natural extract. As a rich source of bioactive compounds, chicory leaf extract holds promise as a sustainable and natural health modulator, aligning with the growing demand for eco-friendly livestock production systems.

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Materials and Methods

Animals, facilities, and experimental design

A total of 48 male New Zealand White rabbits with 1,200 g (±200 g) of initial weight were housed in cages (5ft × 3ft). The animals were subjected to standard experimental conditions of a 12-hour photoperiod, 65% humidity, and a temperature of 16°C–20°C (Volek and Marounek, 2011). All rabbits were separated into four groups: G₀, G₁, G₂, and G₃, whereas G₀ was designated as the reference group. Each group comprised 12 specimens, and four replicates were chosen at each trial using a fully Completely randomized design (CRD). A standard diet was developed based on the nutrient requirements mentioned in the Merck Sharp & Dohme veterinary manual, with the amendments to meet the experimental conditions as mentioned in Table 1 (Mayer, 2021). The doses of ethanolic CLE were designed based on the therapeutic range established by El Debs et al. (2011) and were optimized according to our experimental settings. The experimental groups were orally administered doses of 1, 2, and 3 ml/kg Body weight (BW) as drops for 45 days, guaranteeing constant individual dosing with actual extract concentrations of 75, 150, and 225 mg/kg, respectively (Fig. 1). At each experimental interval (days 15, 30, and 45) blood of 4 rabbits from all groups was collected, induced with isoflurane, and humanely euthanized. Blinding was maintained throughout the study as all hematological, microbial, and histological samples were labeled with coded identifiers to minimize bias. The animals’ carcasses were disposed of through incineration in accordance with the institutional biosafety rules and the instructions given by AVMA guidelines for animal euthanasia (Underwood and Anthony, 2020).

Table 1. Ingredients and nutritional composition of the experimental diets (as-fed basis).

Fig. 1. Illustration of the experimental design.

Preparation of the leaf extract

Intact chicory leaves, free from any damaged parts, were collected at the mature vegetative stage from the University of Gujrat botanical garden. The leaves were then washed and dried under shade at 25^oC–32^oC for 7–10 days to prevent thermal degradation of the heat-sensitive phytoconstituents of chicory leaves. Once the leaves were completely dried, they were ground into a fine powder with a yield of 350 g. This powder was then soaked in 3,500 ml of 96% ethanol (1:10) (w/v) for 72 hours. Filtration with Whatman No. 1 filter paper was performed, and the filtrate was transferred into a flask. The solvent was evaporated through a rotatory evaporator at 45^oC leaving 22.75 g of a semi-solid paste. Finally, 6.5% (w/w) of the product was stored in sterile, dark, and sealed containers at room temperature. For the experimentation, the dry extract was freshly reconstituted with distilled water to attain a stock concentration of 75.83 mg/ml, and the final product was 300 ml of liquid extract, which was placed in a flask covered with aluminum foil. This standardized extract composition was employed for all subsequent doses to guarantee consistency (Saggu et al., 2014; Sakeran et al., 2014; Sapiun et al., 2020).

Sample collection and analysis

Hematological studies

To study the hematological attributes, blood samples were collected from the jugular vein using a 3 ml syringe with a 25-gauge (G) needle. Before blood collection, the animals were fasted for 8 hours night; however, water access was maintained. Blood was collected the following morning to avoid the occurrence of circadian variations. These samples were preserved in 3 ml K3EDTA tubes and categorized into groups such as G₀, G₁, G₂, and G₃ (Parasuraman et al., 2010). Blood samples were analyzed for hemoglobin (Hb), red blood cells (RBCs), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, white blood cells (WBCs), and differential leukocyte count (neutrophils, lymphocytes, monocytes, and eosinophils) using an automated hematology analyzer (model HKTE0112 Guangzhoun Hekang) (Oikonomidis et al., 2021).

Examination of microbial species

The digestive system was dissected, and the alimentary canal was removed to quantify bacterial colonies of 4 rabbits from each group. The ileal and cecal contents (1 g) were collected aseptically from the distal ileum and cecal base, respectively. Samples were collected into 50 ml Falcon tubes, which were labeled accordingly. These samples were mixed with saline and glycerin in a ratio of 9:1 and then placed on ice to prevent the growth of microflora. Subsequently, serial dilution of these samples was performed with normal saline solution (1:10), and their weight was documented. Lastly, 0.1 ml of the sample from each dilution was plated on agar plates in technical triplicate, having specific media for bacterial growth (Garrido et al., 2004; Hashemi et al., 2012). Gram-negative E. coli was quantified on MacConkey agar incubated aerobically at 37^oC for 24 hours. They were identified as pink to brick red, non-mucoid colonies and were confirmed via the Indole test. Lactobacillus was measured on MRS agar, incubated anaerobically at 37^oC for 48 hours, and appeared as white, dew drops on the medium. A confirmatory Gram-positive, catalase-negative rod test was performed to confirm the presence of Lactobacilli (Engberg et al., 2000; Giuliano et al., 2019). The identification process was replicated three times using independent subcultures (n=3) to ensure validity. Lastly, the bacterial colonies flourished in the respective media, and the results were reported as the logarithm of colony-forming units per gram (CFU/g) of the ileal and cecal contents (Hashemi et al., 2012) using the following formula:

Log CFU/g=log [(No. of bacterial colonies counted on plate/vol. of culture plate) × dilution factor].

Histological analysis

After euthanasia, the digestive system was separated, and the small intestine was cut into small sections of almost 2–3 mm thickness. These sections were cleaned with distilled water and dipped into 0.085% saline solution to remove debris. Tissues were fixed with Bouin’s fluid, washed with 70% alcohol, and dehydrated with increasing concentrations of alcohol (70%, 80%, 95%, and absolute alcohol). Tissues were cleared with Xylene and incubated in paraffin wax at 60^oC for 12 hours, and sections of 4–5 mm thickness were cut with a microtome. Tissues were rehydrated in an ascending series of alcohol before staining with hematoxylin and eosin (Nwachukwu et al., 2021). Histological examination was performed using a LABOMED binocular compound microscope (Lx 400). Quantitative morphometric analysis of villus length (VL), villus width (VW), and crypt depth (CD) was conducted using ImageJ software. The 200 µm scale bar was used to calibrate digital images, captured at 100x, and we quantified 10 villi per section were quantified. Statistical analysis was conducted, and results were given as Mean ± SD.

Statistical analysis

Data were collected and arranged using Microsoft Excel 365. All statistical analyses were conducted using IBM SPSS Statistic 23.0 software. The normality of all variables and homogeneity of variance were assessed using Shapiro–Wilk and Levene tests, respectively. When the data were not normal, they were converted to a logarithmic scale and analyzed by one-way Analysis of variance, where all treatment groups were kept as fixed factors. Tukey’s post hoc test was applied to evaluate the significant dose-response relationship. The intergroup comparisons were evaluated through Mean ± SD per experimental unit (individual animal), and p-value ˂ 0.05 was considered statistically significant.

Ethical approval

This study was conducted in the Animal Hematology and Immunology Laboratory, Department of Zoology, Hafiz Hayat Campus, University of Gujrat, Punjab, Pakistan. The experimental protocol adhered to the ARRIVE guidelines and was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Gujrat (Approval No. 2/2024).

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Results

Hematological indices

The administration of ethanolic CLE to rabbits for 45 days improved the hematological parameters compared with the control group (Table 2). The levels of hemoglobin, red blood cells, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, platelet count, WBC, and differential leukocytes (neutrophils, lymphocytes, monocytes, and eosinophils) were increased (p-value ≤ 0.05) in all dosage groups, with the highest values recorded in group G3 (3 ml of ethanolic CLE), followed by groups G2 (2 ml) and G1 (1 ml), as shown in Figure 2.

Table 2. Effect of ethanolic CLE concentrations on rabbit blood profile.

Fig. 2. Effect of different ethanolic CLE doses on rabbit blood profile Bars represent the mean values of hemoglobin (Hb), red blood cells (RBCs), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, white blood cells (WBC), and differential leukocyte count (neutrophils, lymphocytes, monocytes, and eosinophils) in all groups, displaying significant modulations in all parameters (p < 0.05).

Microbial shift

A prominent shift in the population of two key microbial species was observed after oral ingestion of ethanolic CLE for 45 days. It significantly enhanced Lactobacillus abundance in the ileum and cecum (p-value ≤ 0.05). The abundance of E. coli declined throughout the trial (p-value ≤ 0.05) compared with the control group (Fig. 3). Tukey’s test showed the highest number of Lactobacillus colonies in group G3 (3 ml of ethanolic CLE) at the end of the trial (Table 3).

Table 3. Effect of ethanolic CLE concentrations on E. coli and Lactobacillus colony count in rabbit ileum and cecum.

Fig. 3. Effect of different ethanolic CLE doses on the intestinal microflora population in rabbits Bars represent the mean bacterial count in all groups, displaying a significant increase in Lactobacillus and a concurrent decrease in E. coli count (p < 0.05).

Histology of the intestinal

Table 4 shows the intestinal development of rabbits fed ethanolic CLE at concentrations of 1 ml, 2 ml, and 3 ml for 45 days. Histomorphometry results revealed higher VL and increased CD in all treated groups than in the control group. Conversely, VW was decreased in all ethanolic CLE-treated groups compared with the control group (p ˂ 0.05). The maximum VL, CD, and minimum VW were prominent in the highest dose group G3, which was supplemented with 3 ml/kg BW of ethanolic CLE on the 15th, 30th, and 45th day of the experiment, as shown in Figure 4a–c.

Table 4. Effect of ethanolic CLE concentrations on rabbit intestinal development.

Fig. 4. Photomicrographs of intestinal tissue sections of rabbits illustrating slight variations in all treatment groups on day 15 (a), followed by a noticeable change in G2 and G3 on day 30 (b) and prominent changes in all dosage groups on 45th (c) day. Morphological variations displayed an increase in crypt depth and villus length and a decrease in villus width (p ˂ 0.05) (H&E stain; X100).

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Discussion

The quest for alternatives to antimicrobial treatments in livestock has led to the exploration of functional feed ingredients that promote disease resistance and enhance vitality (Peng and Biao, 2021). The rising need for natural substances has led to increased interest in phytogenic compounds, which offer a residue-free natural solution to boost animal health (Placha et al., 2022). Raw chicory leaves contain numerous beneficial bioactive constituents, such as phenolics and flavonoids; however, these compounds are often confined within phytochemical reservoirs, limiting their absorption by the body. The aqueous extract of chicory leaves contains a higher concentration of inulin but lower total flavonoid content, which reduces its antioxidant potential (Malik et al., 2017; Kandil et al., 2019). On the contrary, our findings suggest that the ethanolic extraction method facilitates the efficient release and concentration of phenolic compounds, thereby enhancing the potential and bioavailability of the extract (Khalil et al., 2019; Al-Haliem et al., 2025). Thus, the phytogenic compounds present in chicory leaf extract are renowned as potential substitutes for synthetic antibiotics (Janda et al., 2021). This context provides a basis for evaluating the potential benefits of different doses of ethanolic chicory leaf extract as a functional food ingredient on various physiological parameters in rabbits. Nevertheless, this screening study aimed to determine the biological potency of CLE by assessing the synergistic impact of the entire leaf, rather than the isolated chemicals it contains.

Hematological profiles can be used to evaluate health status and optimal nutrition, which reveal vital information about how external factors impact immune function, physiological balance, and overall well-being of animals (Beigh et al., 2018). Different doses of ethanolic chicory leaf extract in rabbits led to a progressive rise in different hematological indices, including RBC-related parameters, WBC-related parameters, and differential leukocyte count throughout the 45-day study. The enhancement of hematological parameters within the normal range indicates that ethanolic CLE can maintain a healthy blood profile. Similar results were obtained in a study where the administration of 3 g/kg chicory leaf extract in the broiler diet enhanced various hematological parameters, such as white blood cell count, phagocytic index, phagocytic activity, eosinophil %, lymphocytes %, and monocytes % (Abo Ghanima et al., 2023). The inulin fractions of chicory leaf produce SCFAs, which lower intraluminal gut pH and increase mineral solubility, particularly iron, resulting in enhanced erythropoiesis. In the end, this event improves the blood profile (Scholz-Ahrens and Schrezenmeir, 2007; Lobo et al., 2009; Samanta et al., 2013). Additionally, the chemical profile of chicory leaves contains large quantities of chicoric acid, which helps in cytoprotection by keeping the RBC membrane intact, increasing their lifespan, and the number of RBCs circulating in blood surges (Liu et al., 2017). Another investigation revealed the improvement in blood profile following an increase in RBC count, WBC count, hemoglobin concentration, and hematocrit% by the addition of 0.75 g dried chicory leaves in rabbit’s diet (Mahmoud, 2018). Zanzer and Theis (2024) reported that SCFA fermentation in the colon enhances the expression of antimicrobial peptides and improves innate immunity by activating myeloid cells. These findings suggest that chicory leaf extract refines hematopoiesis and modulates differential leukocytes, as demonstrated in our experiment. A further explanation by Paço et al. (2022) highlighted that sesquiterpene lactones present in chicory play a major role in inhibiting the formation of proinflammatory cytokines, such as Interleukin-1, Interleukin-6, and Tumor necrosis factor-α, which reduces oxidative stress and enhances the bioavailability of iron and smoothens erythropoiesis, leading to a healthy blood profile. Meanwhile, improvements in platelet count are associated with the presence of caffeic acid in chicory leaves, which was found to weaken in vivo antiplatelet activity (Schumacher et al., 2011). Furthermore, chicory was reported to boost immunity by suppressing harmful microorganisms in the gastrointestinal tract (Mahmood et al., 2015). It was evident in a 12-week study where people suffering from non-alcoholic fatty liver disease were given brewed chicory leaves, and they showed significant improvements. The chicory-treated group displayed a notable increase in the number of RBCs, platelets, Hb, total cholesterol, fasting blood sugar, and low-density lipoproteins compared with the reference group (Faraji et al., 2022).

Regarding the antimicrobial activity of ethanolic CLE, a significant shift in microbial dynamics was observed, characterized by a marked decrease in pathogenic bacterial strains (E. coli) and a concomitant increase in beneficial bacteria (Lactobacillus). These specific species were focused on to assess the eubiotic index because they serve as representative markers of intestinal health in nutritional studies. The shift in these sentinel species is considered an indicator of the beneficial-to-pathogenic ratio that reflects the effect of CLE on gut hemostasis (Mohamed et al., 2025). Though, this study lacks mechanistic insight into the quantification of intermediate metabolites such as SCFAs and inflammatory markers. However, similar studies on chicory reported that SCFAs, which are the end products of inulin and fructooligosaccharides present in chicory, improve gut microbial communities and their histology, causing a prominent effect on the intestinal microbial biochemistry and its activity (Kulkarni et al., 2024; Reimer et al., 2024). The concentration of butyrate, which is produced as a by-product during the manufacturing of SCFAs, increases with the supplementation of inulin (Underlings et al., 2019; Wang et al., 2020), which maintains the intestinal barrier and possesses antioxidant and anti-inflammatory properties that result in the flourishment of Lactobacilli along with other beneficial strains (Le Bastard et al., 2020). Moreover, the remarkable quantity of flavonoids and phenolic acid in chicory leaves stops the oxidation of nutrients and antimicrobial components in the gut and effectively disrupts the membrane functions of pathogenic microbes and alters the permeability of cellular membranes, thereby suppressing their growth (Papadimitriou et al., 2015; Nwafor et al., 2017). The presence of such functional elements in the chicory extract remarkably controls the proliferation of microbiota in the gut lumen, resulting in an acidic environment in the intestine that restricts the colonization of pathogenic microorganism (Gurram et al., 2021). Comparable effects on gut microbiota were documented in a study where the inclusion of 0.10%, 0.15%, and 2.0% chicory extract in broilers’ diet inhibited the growth of harmful bacteria E. coli, whereas the growth of beneficial bacteria Lactobacillus was enhanced, which improved their growth performance (p-value 0.0001) (Khoobani et al., 2019). The experiment by Verma et al. (2013) also demonstrated a prominent antimicrobial effect of CLE in various solvents possessing inhibitory activity for E. coli strain. Additionally, experimental diets of 0.5%, 1.0%, and 1.5% chicory powder in broilers significantly (p < 0.05) reduced the E. coli and Salmonella counts and improved the Lactobacillus count compared with the control group (Gurram et al., 2021). It was reported that when young rabbits were fed fresh chicory leaves, their cecal biochemistry improved, which was evident through dropped values of NH₃ and pH and higher volatile fatty acid content, indicating a balanced microflora and increased fermentation in the gut (Castellini et al., 2007). However, in an alternative study by Juśkiewicz et al. (2011) found that the polyphenols of chicory leaves, such as chlorogenic acid and polyphenolic glucosides, are comparatively less fermentable; hence, they raise the pH of the gut and reshape the microbial balance. On the contrary, our research reports an increase in Lactobacillus colonies, which indicates an acidic gut environment. Lactobacillus colonization and proliferation yield lactic acid, which serves as an antimicrobial that suppresses the growth of harmful bacteria and supports the development of beneficial bacteria. Additionally, Lactobacillus can adhere to the intestinal wall, forming a compact flora membrane that prevents pathogenic bacteria from reproducing on it (Goyal et al., 2013; Papadimitriou et al., 2015). Thus, Lactobacillus enhancement also plays a role in boosting growth performance, strengthening immune and defense mechanisms, and promoting a more abundant and diverse gut microbiota, particularly in the cecum of rabbits (Wang et al., 2017).

The intestinal mucosa is the hub of macrophages, which are key factors in managing inflammatory responses and maintaining microbial equilibrium (Elshahed et al., 2021). Hence, the absorptive and digestive ability of the intestine depends on the mechanisms occurring in the mucosal wall, which can be calibrated by using prebiotics and dietary supplements, which can improve the activity of certain microorganisms, influencing the performance and intestinal proficiency in animals (Leone and Ferrante, 2023). The present study reveals the morphological alterations in the intestine due to the use of ethanolic CLE, providing a new foundation for using chicory leaf extracts as a prebiotic. Increased villus length is a key factor in the smooth transportation and processing of amino acids, fatty acids, and glucose. The phytochemicals and inulin present in the CLE enhanced VL, facilitating the extraction of more nutrients from the available diet (Fatima et al., 2025; Wang et al., 2025). In contrast, phenolic acid, flavonoids, and caffeic acid derivatives play a prominent role in protecting the intestine from inflammation, leading to a healthy mucosal environment that fosters a shorter diffusion path for nutrient transport and absorption. Thus, the reduced villus width in the chicory-treated groups points to the streamlined mucosal structure of the intestine, reflecting the anti-inflammatory properties of the CLE (Xia et al., 2026). Similar morphological changes were observed in a study that showed that rats fed a 5% chicory diet exhibited significant increases in jejunal villus height and goblet cell count. The chicory-fed group had increased cecum weight relative to the control group. These alterations in gut morphology, attributed to the presence of chicory, are believed to change the metabolism of nutrients, specifically lipids and cholesterol (Kim, 2002). Deeper crypts play an essential role in maintaining the structural integrity and absorptive surface area of the intestine by supplying mature enterocytes to the villus tip. Furthermore, an increased VL and CD ratio is linked with the presence of dynamic mitosis in the intestinal mucosa (Samanya and Yamauchi, 2002; Chichlowski et al., 2007). It is evident from a study where the inclusion of 1% and 1.5% chicory in broilers feed resulted in the improved villus length and crypt depth, which accelerated the absorption of the nutrition and digestive secretory function (Gurram et al., 2021). Other research in broilers fed with chicory extract showed that the antimicrobial activity of chicory leaves suppresses E. coli in the small intestine of broilers. Consequently, it caused an increase in villus height, villus surface area, and crypt depth, which supported the healthier intestine in the experimental birds (Ayssiwede et al., 2011). Chicory extract displays morphological stabilization and functionality in animals by amplifying the number, length, and surface area of the intestinal villi (Abo Ghanima et al., 2023). The antimicrobial properties of chicory extract showcase the dynamic internal environment of the gut, which induces structural augmentations in the villi, resulting in the efficient absorption of the available nutrients, making it a potential prebiotic for livestock (Izadi et al., 2013). 

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Conclusion

Our findings provide compelling evidence that ethanolic CLE is a potential nutraceutical agent for improving various physiological parameters in rabbits. The results demonstrated that CLE exerts beneficial effects on hematological indices and intestinal morphology, concomitant with a favorable modulation in the pathogenic and useful bacteria characterized by a decrease in E. coli and an increase in Lactobacillus colony count. These outcomes have significant implications for our understanding of the therapeutic profile of chicory as a natural feed additive to enhance its nutritional value and animal health. To extend this conclusion to commercial livestock production, future studies should characterize chicory leaf extract in various polar protic solvents while examining the molecular mechanisms and biochemical pathways in other animal species with a larger sample size.

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Acknowledgments

None.

Conflict of interest

The authors declare no conflicts of interest.

Funding

This study received no external funding.

Authors' contribution

Maryam Hassan conducted the experiments, animal handling, sample collection, laboratory analysis, tissue processing, data collection, and results interpretation. Ayesha Saddiqa and Rimsha Yaseen helped with the methodology and participated in the preparation of the manuscript. Razia Iqbal supervised the study. Kiran Aftab edited and revised the manuscript. All authors have read and approved the final version of the manuscript.

Data availability

Data will be available from the corresponding author upon reasonable request.

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