Antioxidant activity was analyzed using the DPPH IC₅₀ method(Kozmelj et al., 2024). A 0.01 mM DPPH solution and cardamom waste extract were prepared, mixed, and incubated for approximately 30 minutes at room temperature. The absorbance of the mixture was measured at a wavelength of 517 nm using a UV-Vis spectrophotometer. The antioxidant activity results are shown in .

Cardamom waste was extracted following the method of Gouda et al. (2021)(M. et al., 2021). The waste was dried in an oven at 50 °C and then ground into flour. The flour was dissolved in 96% ethanol (25 g/250 mL) and stirred until homogeneous. The solution was then sonicated at 37 °C with a wavelength of 50 Hz for 30 minutes, followed by incubation for 3 days. The filtrate was filtered and evaporated to remove ethanol. The drying process followed the method of Pham et al. (2015)(Pham et al., 2015), in which the evaporated material was dried in an incubator at 40 °C to produce a thick extract.

Encapsulation followed the method of Agusetyaningsih et al. (2022)(Agusetyaningsih et al., 2022). Maltodextrin was used as the coating material, dissolved in a ratio of 1:3. The dissolved maltodextrin was mixed with cardamom waste extract in a 5:1 ratio and dried using the freeze-drying method. Freeze-drying produced dry lumps of encapsulated material, which were then ground into particles approximately 20 µm.

Daily feed consumption and weight gain data were recorded from day 8 to day 35 of maintenance. Feed consumption was measured each morning by weighing the remaining feed (g) in each pen, using the following formula:

Feed intake (FI) = feed offered (g) - leftover feed (g)/number of birds

Body weight gain was calculated by weighing the chickens weekly in the morning, using the formula:

Average daily gain (ADG) = Final weight (g) - Initial weight (g)/Time (days)

On day 31, total excreta collection was conducted to measure crude protein digestibility. One chicken per replication, with body weight close to the group average (20 samples in total), was selected and placed in a battery cage coded according to the treatment. Excreta collection lasted 4 days, beginning with 24 hours of fasting, during which chickens had ad libitum access to water(Ruvini et al., 2021). The feed was supplemented with Fe₂O₃ as a digestibility marker. During collection, excreta were sprayed with 0.2 N HCl to bind nitrogen(Kikusato & Namai, 2025), then cleaned of feathers and other debris. The excreta were weighed, sun-dried under a net to prevent contamination, and reweighed before being ground. A 5 g sample of dry excreta was placed into a coded plastic container for laboratory analysis. Protein digestibility was calculated using the formula:

Protein digestibility (%) = (Protein consumption (g) - Protein in excreta(%))/Protein consumption (g) × 100

On day 35, chickens from each pen were randomly selected for hematology sampling. Blood was taken from the wing vein and collected in an ethylenediaminetetraacetic acid (EDTA) tube. The remaining blood was collected in a tube without an anticoagulant and allowed to clot for 2 hours at room temperature. After centrifugation at 2000 rpm for 15 minutes, serum was obtained and stored at -20°C until biochemical analysis. The chickens from which blood was taken were then weighed and slaughtered. The carcasses were separated into feathers, skin, head, feet, and internal organs before being weighed. Lymphoid organs, including the thymus, bursa of Fabricius, and spleen, were weighed using an analytical balance, and relative organ weight was calculated as follows:

Relative organ weight (%) = (Lymphoid organ weight (g ))/live weight (g) × 100

Digesta from the ileum and cecum were collected aseptically into coded tubes for microbiological analysis, and pH was measured using an electric pH meter. The number of microbes in the intestinal digesta was determined following the method of Yudiarti et al. (2024)(Yudiarti et al., 2024). Total bacteria were counted on potato dextrose agar (PDA) after aerobic incubation at 38 °C for 24 hours. Coliform bacteria were counted on MacConkey agar after aerobic incubation at 38 °C for 24 hours. Lactic acid bacteria were counted on de Man Rogosa Sharpe (MRS) agar after anaerobic incubation at 38 °C for 48 hours.

The number of leukocytes was determined using the Bürker chamber method(Ciborowska et al., 2024). Differential leukocyte counts were obtained by examining smear preparations under a light microscope with an immersion lens. The coverslip was used to prepare blood smears. The heterophil-to-lymphocyte (H/L) ratio was calculated by dividing the number of heterophils by the number of lymphocytes(Madej et al., 2024).

Data were analyzed using a completely randomized design (ANOVA) with SPSS Version 29. Significant differences among treatments were further tested using Duncan’s multiple range test.

The addition of 0.08% ECWE (T4) to the ration significantly (p<0.05) improved small intestine health. In this treatment, the LAB population increased to 7.34 log cfu/g. This increase was influenced by active compounds in cardamom extract, particularly phenolic compounds. According to Shehata et al. (2022)(Shehata et al., 2022), flavonoids are phenolic compounds that act as natural prebiotics metabolized by beneficial microbiota such as LAB. Flavonoids provide fermentation substrates for LAB and stimulate the expression of enzymes that accelerate the metabolism of simple carbohydrates into organic acids, including lactic and acetic acids.

Soundararajan et al. (2023)(Soundararajan et al., 2023) reported that flavonoid compounds in Moringa (Moringa oleifera) leaf meal act as modulators that support the growth of beneficial chicken gut microbiota and facilitate colonization. Lactic acid bacteria ferment glucose into lactic acid via the glycolysis pathway, lowering pH in the intestinal lumen. This acidic environment not only benefits LAB growth but also inhibits pathogenic bacteria such as E. coli and Salmonella. In this study, intestinal pH ranged from 6.03 to 6.45, lower than the 6.4–6.7 range reported by Sunu et al. (2019)(Sunu et al., 2019), in which prebiotic activity from garlic reduced pH.

Cardamom essential oils, including cineole, terpineol, and α/β-pinene, possess antibacterial properties but tend to be selective against pathogenic bacteria. These compounds damage gram-negative bacterial cell membranes by disrupting permeability and causing leakage of cell contents such as ions, ATP, and nucleic acids. This selectivity gives LAB a competitive advantage in dominating gut microbiota. Acidic pH changes in the intestine further promote the growth of Lactobacillus spp. and Bifidobacteria, suppressing pathogenic bacteria. Encapsulation technology protects flavonoids and essential oils from degradation during gastric digestion. The maltodextrin coating in the encapsulated particles allows gradual release in the small intestine, where LAB colonization occurs.

The H/L ratio is an indicator of stress levels in poultry; a higher ratio reflects a greater stress level. According to Jumadin et al. (2020)(Jumadin et al., 2020), factors influencing the H/L ratio include biological activity, environmental conditions, age, and feed. The normal H/L ratio is approximately 0.3–0.5. In this study, the ratio ranged from 0.73 to 0.83. The H/L ratio can also indicate the poultry immune system, with values of around 0.2 considered low, 0.5 normal, and 0.8 high.

The inclusion of ECWE in rations did not reduce stress levels in broiler chickens. Environmental conditions may have contributed to stress in the flock. According to Wang et al. (2023)(Wang et al., 2023), heterophils are part of the nonspecific immune response, functioning to limit pathogen spread before activation of the specific immune response, which involves lymphocytes.

The effects of encapsulated cardamom waste extract (ECWE) on the relative weights of lymphoid organs are presented in Table 6. The treatment significantly (p<0.05) increased the relative weights of the bursa of Fabricius, spleen, and thymus. The addition of ECWE to the ration resulted in a thymus relative weight of approximately 0.52%–0.55%, higher than the 0.33% average reported by Akbar et al. (2022)(Akbar et al., 2022). The thymus, located in the neck, plays a key role in the maturation of T lymphocytes. Factors affecting thymus size include lymphocyte production and protein intake. In this study, protein digestibility was high (75.66%–83.77%), indicating efficient amino acid absorption for immune cell formation.

Moniei et al. (2024)(Moniei et al., 2024) reported that the addition of turmeric flour to feed positively affected intestinal microflora, thereby improving thymus gland function. Cardamom’s main bioactive compounds—flavonoids, terpenoids, and essential oils—possess immunomodulatory activity. According to Gharechopogh et al. (2021)(Gharechopogh et al., 2021), antioxidants in medical plants and prebiotics can enhance thymus function in antibody production. However, flavonoids are heat- and oxidation-labile, making them susceptible to degradation before reaching the intestine. Encapsulation technology protects these compounds during digestion in the proventriculus and gizzard, allowing controlled release in the small intestine for optimal absorption.

Flavonoids can donate hydrogen atoms from hydroxyl groups (-OH) to neutralize free radicals, preventing lipid peroxidation of immune cell membranes in lymphoid organs. This antioxidant protection reduces lymphocyte apoptosis caused by oxidative stress, leading to an increased immune cell population.

The average relative weight of the spleen in this study ranged from 0.18 to 0.25%. Ampode and Federico et al. (2022)(Ampode & Federico, 2022) reported that adding 5% oregano powder (OP) significantly increased lymphoid organ weight by about 3%. The relative spleen weight observed here may be attributed to the activity of cardamom bioactive compounds and the protective effect of encapsulation, which maintains their availability in the digestive tract, thereby supporting lymphoid tissue function and development. Encapsulation preserves the integrity of the flavonoid chemical structure until it reaches the small intestine and ensures gradual release, allowing the concentration of absorbed active substances to remain stable and continuously stimulate the immune system.

The spleen is a lymphoid organ responsive to antigen stimulation and plays a role in lymphocyte formation for antibody production. According to Akbar et al. (2022)(Akbar et al., 2022), flavonoids in cardamom neutralize free radicals (ROS) by donating hydrogen atoms from hydroxyl groups, preventing lipid peroxidation in lymphocyte membranes. This process reduces lymphocyte apoptosis caused by oxidative stress, thereby increasing immune cell numbers in the lymph and contributing to greater organ mass.

Khukhodziinai et al. (2024)(Khukhodziinai et al., 2024) further noted that an increase in LAB in the intestine, promoted by flavonoids and essential oils, lowers intestinal pH, inhibits pathogenic bacteria, and improves amino acid absorption efficiency. Essential amino acids such as lysine and methionine are utilized in the synthesis of immunoglobulins and lymphocyte structural proteins, contributing to lymphocyte growth.

The bursa of Fabricius is a round-shaped lymphoid organ located between the cloaca and sacrum that plays a role in capturing antigens and forming antibodies(Wu et al., 2021). In this study, increasing ECWE supplementation raised the relative weight of the bursa of Fabricius to approximately 0.12%–0.23%. Morais et al. (2023)(Morais et al., 2023) also found that feeding garlic flour significantly increased bursa weight.

Cardamom essential oils, including cineole, terpineol, and α-pinene, are selectively antibacterial, inhibiting pathogenic bacteria without affecting beneficial bacteria by disrupting the phospholipid layer of pathogenic bacterial membranes. ECWE reduces intestinal infection burden and decreases the need for nonspecific immune system activation, allowing energy and nutrients to be directed toward the development of primary immune organs, including the bursa of Fabricius. Flavonoids further enhance the expression of immunostimulatory cytokines that stimulate B-cell proliferation and differentiation, leading to increased lymphoid follicles in the bursa and, consequently, greater organ weight and size.