D. pentandra leaves were collected from mango host trees located in Semarang, Central Java, Indonesia, specifically within the campus area of Universitas Diponegoro under natural field conditions. Leaves were dried at room temperature without exposure to direct sunlight. The dried leaves were ground using a grinder mixer until they became a fine powder and were extracted with 70% ethanol at room temperature for 72 h at a ratio of 1:5 (b/v). The resulting extracts were filtered using filter paper to obtain the filtrate. The filtrate was evaporated using a vacuum rotary evaporator to obtain a paste-shaped extract of D. pentandra. After the evaporation process, the extract was encapsulated with maltodextrin as a coating agent using the freeze-drying method (Jamdar et al., 2021). The coating material consisted of maltodextrin dissolved in distilled water at a ratio of 1:3. The maltodextrin solution was then mixed with the D. pentandra leaf extract filtrate at a ratio of 5:1 and subsequently dried using the freeze-drying method to obtain encapsulated D. pentandra leaf extract powder. Antioxidant activity of the leaf material and encapsulated extract was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, following the spectrophotometric principle described by (Yismairai et al., 2019). Briefly, the sample solution was mixed with DPPH reagent, incubated in the dark, and absorbance was measured against a reagent blank. Radical-scavenging activity was calculated as inhibition (%) = [(A control – A sample)/A control] × 100. The same sample concentration, reaction time, solvent, wavelength, and analytical batch were used for both materials. The reported values represent percentage inhibition under these assay conditions. The antioxidant activity of D. pentandra leaves and encapsulated D. pentandra leaf extract is shown in Table 1.

The research procedures were approved by the Animal Research Ethics Committee of the Faculty of Animal and Agricultural Sciences, Universitas Diponegoro (62-03/A-08/KEP-FPP).

A total of 300 unsexed Cobb CP 707 broiler chicks with an average body weight of 46.7 ± 2.2 g were randomly assigned to four treatments with five replicates following standard experimental designs in poultry studies: T0 (density of 9 birds/m²), T1 (density of 17 birds/m²), T2 (density of 17 birds/m² + 0.5 g encapsulated D. pentandra leaf extract), and T3 (density of 17 birds/m² + 1 g encapsulated D. pentandra leaf extract). The stocking density treatments (9 and 17 birds/m²) were selected based on Agusetyaningsih et al. (2025), who reported broiler studies using comparable low- and high-density conditions (9-10 vs 14-16 birds/m²) to induce production stress and evaluate physiological responses. Similarly, the inclusion levels of encapsulated D. pentandra leaf extract (0.5 and 1.0 g/kg feed) were based on prior studies reporting effective phytogenic supplementation ranges of 0.5-1.5 g/kg feed in broilers, particularly under high stocking density conditions (Agusetyaningsih et al., 2025). Broiler chicks were reared in an open-house system using litter (rice husks). Chickens were fed commercial feed from 0 to 7 days of age. At 8-35 days of age, chickens were fed a pre-formulated feed (Table 2) with different doses of encapsulated D. pentandra leaf extract.

The sample size was determined based on previous studies to ensure adequate statistical power (Sugiharto et al., 2019); (Li et al., 2019). Broilers were reared in an open-sided house with natural ventilation using rice husk litter (8-10 cm depth), which is commonly applied in tropical poultry production systems(Soliman & Hassan, 2020). The ambient temperature ranged from 28 to 32 °C with relative humidity between 60%-75%, and a continuous lighting program (23 h light:1 h dark) was applied (Osti et al., 2017). Temperature and humidity were recorded twice daily.

All chickens were vaccinated with an active ND vaccine (Medivac ND Clone 45®, PT. Medion Farma Jaya) administered at the age of 22 days through drinking water. Body weight and feed consumption were recorded weekly from the age of 8 to 35 days. At 35 days of age, one chicken per replicate was randomly selected for blood collection via the brachial vein. The collected blood was transferred into vacutainer tubes containing an anticoagulant (EDTA) for complete blood count analysis, or into tubes without anticoagulant for serum separation. Serum was obtained by centrifuging the blood samples at 3,000 rpm for 15 min and subsequently stored at freezing temperature until further analysis. At the end of the 35-day experimental period, the sampled chickens were slaughtered, and their lymphoid organs, including the bursa of Fabricius, spleen, and thymus, were collected.

Performance of broilers. Broiler performance was evaluated based on daily body weight gain, daily feed intake, final body weight, and feed conversion ratio (FCR). Daily body weight gain was calculated following (Agusetyaningsih et al., 2022) by recording the body weight of birds on day 35 to obtain the final weight, subtracting the initial weight measured at the end of day 7, and dividing the difference by the experimental duration (28 days). Daily feed intake was determined by dividing the total feed consumed during the trial by the length of the experimental period. The feed conversion ratio (FCR) was calculated as the ratio between feed intake and body weight gain during the treatment period. In this study, the final body weight and FCR are presented as mean ± standard deviation.

Complete blood counts. To assess the hematological parameters, one bird was randomly selected from each replicate, and blood samples were collected via the brachial vein. Blood was drawn using a 3 mL syringe fitted with a 25G needle and then transferred into EDTA tubes (0.5 mL). The samples were subsequently subjected to hematological analysis in the laboratory. The parameters measured included Red blood cells (RBC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Complete blood counts were determined using the dilution flask method as described by (Almuthaybiri et al., 2025). Hemoglobin levels were measured using the Sahli method. Erythrocyte and leukocyte counts were determined based on the dilution method, and a Bürker chamber was used for cell counting. Hematocrit values were determined using the microhaematocrit technique, while differential leukocyte counts were assessed using a light microscope.

Serum protein profiles of broilers. During the fifth week of the experiment, one bird was randomly selected from each replicate pen for blood sampling. Approximately 2 mL of blood was collected from the brachial vein, resulting in five independent serum samples per treatment. Blood sampling was performed using a 3 mL syringe fitted with a 25 G needle. The collected blood was transferred into plain tubes and allowed to stand for approximately 15 minutes to facilitate serum separation. The serum was then separated from the remaining cellular components by aspirating it with a pipette and subsequently used for biochemical analysis. The analyses followed the procedures described by (Sugiharto et al., 2016) with minor modifications. The albumin levels were determined by the bromocresol green (BCG) method. SGOT and SGPT levels were determined using the International Federation of Clinical Chemistry (IFCC) method.

Relative immune organ weights of broilers. The relative weights of immune organs (bursa of Fabricius, spleen, and thymus) were calculated following the method described by (Choi et al., 2021)by dividing the organ weight by live body weight and multiplying the result by 100%.

Antioxidant activity. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were determined as described by Agusetyaningsih et al. (2022). SOD activity was evaluated based on the ability of the sample to inhibit pyrogallol auto-oxidation. The reaction mixture consisted of 50 mM Tris-HCl buffer (pH 8.2), 1 mM diethylenetriaminepentaacetic acid, and the sample. The reaction was initiated by adding pyrogallol to achieve a final concentration of 0.2 mM, followed by kinetic measurement of absorbance. SOD levels were expressed in units (U/ml). Malondialdehyde (MDA) levels were determined using a thiobarbituric acid (TBA) reactive substances assay. Each sample was vortexed, mixed with 8.1% sodium dodecyl sulfate, and incubated at room temperature for 10 min. The control samples were processed similarly. Subsequently, 20% acetic acid and 0.6% TBA were added, and the mixture was incubated in a water bath at 90-95 °C for 1 h. After incubation, a butanol/pyridine solution (15:1) was added to the supernatant, vortexed, and centrifuged. MDA concentrations were reported in nmol/mL.

Data were analyzed using one-way analysis of variance (ANOVA) based on a completely randomized design (CRD). The statistical model used was:

Y_ij = μ + τ_i + ϵ_ij

where Y_ij is the observed value, μ is the overall mean, τ_i​ is the treatment effect, and ϵ_ij​ is the experimental error. Prior to analysis, data normality was tested using the Shapiro-Wilk test, and homogeneity of variances was evaluated using Levene’s test. Differences among treatments were further analyzed using Duncan’s multiple range test when significant effects were observed (p<0.05). All statistical analyses were performed using SPSS version 25.0.

Dietary supplementation of encapsulated D. pentandra leaf extract significantly affected daily feed intake (DFI) (p<0.05), whereas no significant effects were observed on daily weight gain (DWG), final body weight, or feed conversion ratio (FCR) (Table 3). Broilers in the T2 group exhibited the highest feed intake, which increased by 6.1% compared to T0 (107.07 vs 100.89 g/day) and by 1.7% compared to T1 (107.07 vs 105.29 g/day). In contrast, no significant differences were observed in DWG (p = 0.737), final body weight (p = 0.647), or FCR (p = 0.504).

Most hematological variables, including erythrocytes, hemoglobin, hematocrit, MCV, MCH, MCHC, leukocytes, heterophils, lymphocytes, and H/L ratio, were not significantly affected by treatments (p>0.05) (Table 4). However, significant differences were observed in thrombocyte count and platelet distribution width (PDW). Thrombocyte count decreased significantly in T3 by 42.8% compared to T0 (15.80 vs 27.60 ×10³/µL; p = 0.003). Similarly, PDW values were reduced by 41.3% in T2 compared to T0 (8.00 vs 13.64; p = 0.010).

Serum protein profile data are presented in Table 5. No significant differences were observed in albumin (p = 0.401), SGOT (p = 0.495), or SGPT (p = 0.185) levels among treatments. Although SGPT levels in T3 were numerically higher (5.98 U/L) compared to T0 (2.70 U/L), this increase (+121.5%) was not statistically significant.

The relative weight of the bursa of Fabricius was significantly affected by treatment (p = 0.015), while spleen and thymus weights remained unchanged (p = 0.711 and P = 0.438, respectively) (Table 6). Broilers in T2 exhibited the highest bursa weight, which increased by 50.0% compared to T0 (0.06 vs 0.04%).

No significant differences were observed in superoxide dismutase (SOD) activity (p = 0.205) or malondialdehyde (MDA) levels (p = 0.883) among treatments (Figure 1). However, SOD activity showed an increasing trend in T3, with a 29.3% higher value compared to T0 (5.433 vs 4.203 U/mL). In contrast, MDA levels remained relatively stable across treatments, with only a slight increase of 7.4% in T3 compared to T0 (1.721 vs 1.602 nmol/mL). While encapsulated D. pentandra did not significantly alter antioxidant status, the numerical increase in SOD activity in T3 warrants further investigation to determine if a biologically relevant effect exists under different experimental conditions.

Data on daily weight gain (DWG), final body weight, and feed conversion ratio (FCR) observed in this study were within the normal range for broiler chickens, indicating that the applied high stocking density (17 birds/m²) did not markedly impair growth performance under the current experimental conditions. This suggests that the physiological stress induced was not severe enough to adversely affect growth-related parameters, which represents a limitation in fully evaluating the extract’s impact under strong stress. This finding agrees with previous reports by Chauhan et al. (2022) indicating that broilers can maintain productive performance under moderate stocking densities when environmental management and nutritional conditions are adequate. Consistent with this interpretation, the high-density control group (T1) showed no significant deterioration in DWG, final body weight, FCR, SGOT, or SGPT compared with the low-density control group (T0), further indicating that the stocking-density treatment did not impose a sufficiently severe physiological challenge under the present experimental conditions.

The higher daily feed intake (DFI) observed in the T2 group indicates that supplementation with encapsulated D. pentandra leaf extract may help maintain feed consumption under relatively challenging conditions. This response may be attributed to the biological activities of flavonoids, tannins, saponins, and phenolic compounds present in the extract (Saavedra-Molina et al., 2024). These phytochemicals have been reported to improve gastrointestinal health by enhancing intestinal integrity, modulating gut microbiota composition, reducing oxidative damage to intestinal epithelial cells, and stimulating digestive enzyme secretion (Kwon et al., 2023). Improved intestinal functionality may subsequently increase feed palatability and nutrient utilization efficiency, leading to higher feed consumption (Michalak et al., 2025).

Flavonoids, particularly quercetin derivatives identified in D. pentandra, are known to exert anti-inflammatory and antioxidant effects within the gastrointestinal tract, thereby preserving villus structure and absorptive capacity (Parasuraman et al., 2016). Likewise, saponins may suppress pathogenic microorganisms while promoting beneficial microbial populations, contributing to improved digestive health and nutrient assimilation (Zhang et al., 2023). Similar increases in feed intake following phytogenic supplementation with encapsulated Cosmos caudatus have been reported by (Agusetyaningsih et al., 2022)in broilers receiving polyphenol-rich plant extracts under environmental stress conditions.

Despite the increase in feed intake, no significant improvements were observed in DWG or FCR. This finding suggests that the increased feed consumption was not sufficient to produce measurable improvements in growth performance(Kavoi et al., 2016). One possible explanation is that the physiological challenge imposed by the experimental stocking density was relatively mild, limiting the opportunity for phytogenic supplementation to demonstrate growth-promoting effects (Li et al., 2024). Previous studies by (Li et al., 2019) have shown that the beneficial effects of phytogenic additives become more evident when broilers are exposed to severe oxidative, thermal, or management-related stressors. Therefore, future studies should evaluate the efficacy of encapsulated D. pentandra leaf extract under more pronounced stress conditions.

The hematological parameters, including erythrocytes, hemoglobin, hematocrit, and erythrocyte indices (MCV, MCH, MCHC), remained within normal physiological ranges and did not differ significantly among treatments. This indicates that neither high stocking density nor supplementation with encapsulated D. pentandra leaf extract adversely affected oxygen transport capacity or erythropoiesis. These findings are consistent with previous studies reporting that broilers can maintain normal hematological profiles under moderate stocking density when environmental and nutritional conditions are adequately managed(Rashid et al., 2016); (Tella, 2025).

The current results are consistent with those reported by (Rashid et al., 2016), who observed stable hematological profiles in broilers receiving antioxidant-rich phytogenic additives. Similarly, (Tella, 2025) reported that plant-derived bioactive compounds did not significantly alter erythrocyte-related parameters when birds were maintained under relatively stable environmental conditions.

Leukocyte counts and heterophil-to-lymphocyte (H/L) ratios also remained unaffected by treatments. Leukocyte values observed in this study were within the normal physiological range reported for healthy broilers, suggesting that birds maintained adequate immune surveillance and physiological homeostasis throughout the experimental period (Lebedev et al., 2024). The H/L ratio is considered one of the most reliable indicators of chronic stress in poultry because elevated corticosterone concentrations typically increase heterophil numbers while reducing circulating lymphocytes (Cotter, 2015). Generally, H/L ratios exceeding 0.5 are associated with significant physiological stress, whereas lower values indicate normal adaptation to environmental conditions (B, 2018). The low H/L ratios observed in the present study (0.03-0.05) therefore suggest that the birds did not experience substantial chronic stress despite being reared at a higher stocking density (Uysal et al., 2025).

In contrast, thrombocyte counts and PDW were significantly lower in T1, T2, and T3 compared to T0, with only minor numerical differences among the high-density groups. Thrombocytes play important roles in hemostasis and innate immune responses in avian species (Ferdous & Scott, 2023). The reduction in PDW may indicate a more homogeneous platelet population and reduced platelet activation status (Shubha Sangeetha et al., 2019). This effect may be associated with flavonoids present in D. pentandra leaves, which have been reported to possess antiplatelet and antioxidant properties through inhibition of thromboxane synthesis, suppression of oxidative stress-induced platelet activation, and modulation of intracellular calcium signaling pathways (Santoso et al., 2017); (Zverev, 2017).

Previous studies by (Santoso et al., 2017) have demonstrated that flavonoid-rich phytogenic supplements can influence platelet activity without inducing pathological changes in hematological status. Because thrombocyte counts remained within physiological limits, the observed reductions likely represent modulation of platelet function rather than suppression of thrombopoiesis (Vallance et al., 2019). Nevertheless, the biological significance of these findings requires further investigation, particularly regarding the potential anti-inflammatory and immunomodulatory properties of D. pentandra bioactive compounds.

Data on serum concentrations, including albumin, SGOT, and SGPT, did not differ significantly among treatments and remained within normal physiological ranges, indicating that liver function and protein metabolism were not adversely affected by high stocking density or dietary supplementation. Similar findings have been reported in broilers, where stable serum biochemical parameters reflect normal hepatic function and metabolic homeostasis under controlled rearing conditions (Punyatong et al., 2025)

These findings suggest that encapsulated D. pentandra leaf extract is physiologically safe and does not cause hepatotoxicity. The stability of liver enzyme activity may also reflect the protective role of antioxidant compounds in maintaining hepatocyte integrity, as flavonoids and polyphenols are known to mitigate oxidative damage and support liver function in poultry (Siyanova & Zalyubovskaya, 2025)

However, the absence of significant variation among treatments may also indicate that the experimental conditions did not impose sufficient metabolic or hepatic stress to challenge liver function in broilers. Under mild or non-stress conditions, hepatic biomarkers often remain stable, thereby limiting the ability to detect functional improvements from dietary interventions (Mukul Ray et al., 2020). As a result, the capacity of the extract to demonstrate hepatoprotective effects under stress conditions may not have been fully expressed. This highlights the need for future studies under more challenging physiological conditions to evaluate better the functional benefits of the extract on liver health.

Data on the relative weight of the bursa of Fabricius were significantly higher in the T2 group, indicating a positive effect of encapsulated D. pentandra leaf extract on the development of this primary lymphoid organ. The bursa of Fabricius is a primary lymphoid organ unique to avian species and serves as the principal site for B-lymphocyte proliferation, differentiation, and maturation (Szőcs et al., 2024). Enlargement of this organ is generally associated with enhanced humoral immune competence and improved antibody production capacity (Szőcs et al., 2024).

The observed increase may be attributed to the immunomodulatory activities of flavonoids, quercetin derivatives, tannins, and other phenolic compounds present in D. pentandra. These bioactive compounds have been reported to stimulate lymphocyte proliferation, regulate cytokine secretion, enhance macrophage activity, and protect immune cells from oxidative damage (Abubakar et al., 2019). (Ang et al., 2014), demonstrated that D. pentandra extract significantly stimulated splenocyte and thymocyte proliferation, supporting its immunostimulatory potential.

Furthermore, flavonoids may promote B-cell differentiation through modulation of intracellular signaling pathways involved in immune cell activation and cytokine production (Liu & Jiao, 2025). Enhanced antioxidant protection may also preserve the viability and functionality of developing lymphocytes within the bursa, thereby contributing to increased organ development (Ke et al., 2023). Similar improvements in lymphoid organ development and humoral immunity have been reported by (Ke et al., 2023)in broilers receiving phytogenic supplements rich in polyphenols and flavonoids.

Interestingly, the positive response was most evident in the T2 (0.5 g/kg), whereas the higher supplementation level T3 (1.0 g/kg) did not produce additional improvements. This finding suggests that moderate supplementation may provide optimal immunomodulatory stimulation, whereas excessive concentrations may not confer further benefits due to saturation of physiological response mechanisms. Similar dose-dependent responses have been reported for various phytogenic feed additives used in poultry nutrition.

The absence of significant changes in spleen and thymus weights suggests that systemic immune activation and T-cell development remained stable(Mehrzad et al., 2024). This observation may indicate that the birds were not exposed to severe immunological challenges during the experimental period (Mehrzad et al., 2024). Therefore, future studies employing pathogen challenge models or vaccination response assessments are warranted better to characterize the immunomodulatory properties of D. pentandra leaf extract.

Serum SOD activity and MDA levels are widely used indicators of antioxidant defense and oxidative stress in broilers, reflecting the balance between reactive oxygen species production and antioxidant capacity (Nawaz & Zhang, 2021). Data on SOD activity did not differ significantly among treatments, although a numerical increase was observed in supplemented groups. Similarly, MDA levels remained stable across all treatments, indicating that lipid peroxidation and oxidative damage were not markedly elevated.

These results suggest that broilers maintained oxidative balance under the given experimental conditions and that the endogenous antioxidant system was sufficient to prevent excessive oxidative stress. Similar findings have been reported in broilers under moderate environmental stress, where antioxidant biomarkers remain stable due to effective physiological adaptation (Cimpean et al., 2025). The absence of significant differences in SOD and MDA levels suggests that under the present experimental conditions, the broilers’ endogenous antioxidant system was sufficient, or the oxidative challenge was not severe enough to elicit a detectable antioxidative effect from the supplement. This limits the ability to clearly demonstrate the extract’s antioxidative potential in this specific context.

This condition represents an important limitation in evaluating the efficacy of antioxidant supplementation. When oxidative stress is not strongly induced, the measurable benefits of exogenous antioxidants may be minimal or undetectable, as also discussed in studies on antioxidant supplementation under low-stress conditions (Carvalho et al., 2022); (Ngestiningsih et al., 2019). Therefore, the absence of significant differences in SOD and MDA should not be interpreted as a lack of biological activity, but rather as a consequence of limited oxidative challenge within the experimental system.

In addition, the relatively short experimental duration and controlled environmental conditions may have further contributed to the stability of oxidative parameters. Previous studies have shown that stronger stressors, such as heat stress or higher stocking densities, are required to significantly elevate oxidative stress markers in broilers (Sharma et al., 2020). Future studies employing stronger stress models, such as higher stocking densities, heat stress, or longer rearing periods, are needed to assess better the antioxidative potential of encapsulated D. pentandra leaf extract.