Freshmature leaves of Mangifera indica (mango), Lawsonia inermis (henna), and Carica papaya (papaya) were collected from the Poultry Experimental Station, Department of Poultry Husbandry, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University Tandojam, these leaves were washed thoroughly three times with distilled water to remove surface contaminants and then shade-dried in a well-ventilated area at ambient temperature (25˚C - 30˚C) for 5 - 7 days to prevent the degradation of heat-sensitive phytochemicals Once completely dried, the leaves were milled into a fine powder using a laboratory grinder fitted with a 60-mesh sieve. Each plant species was processed separately to prevent cross-contamination. The powders were stored in airtight opaque containers at 4˚C until further use.

Experimental Design and Dietary Treatments A total of 600-day-old broiler chicks (Cobb-600) were obtained from a certified commercial hatchery. After initial body weight recording, birds were randomly assigned to five dietary treatment groups, with 120 chicks per group and six replicates of 20 birds each:

The inclusion levels of each leaf powder were determined based on previous literature reporting the effective and safe use of these plants in poultry nutrition, preliminary pilot studies conducted in our laboratory, and traditional ethnoveterinary practices. These dosages were selected to balance biological efficacy, animal safety, and practical feed formulation.

All groups were fed for 42 days. Birds were housed in environmentally controlled pens with ad libitum access to feed and water. Standard broiler management practices regarding temperature, ventilation, lighting, and vaccination (ND and IBD) were followed throughout the trial. The proximate composition (crude protein, crude fiber, ash, moisture, and ether extract) of the leaf powders was analyzed following AOAC (2016) methods.

At day 42, blood samples were collected from the jugular vein of 64 randomly selected birds (n = 12 - 13 per group) using non-heparinized vacutainers. Serum was separated by centrifugation at 3000 rpm for 10 minutes and stored at −20˚C until analysis. The following serum biochemical parameters were measured using a Merck Microlab-300 analyzer with commercial kits (Human Diagnostics, Germany):

Blood glucose (Kit #10260)

Total protein (Biuret method; Kit #157004)

Cholesterol (CHOD-PAP method)

Urea (Kit #10505)

Blood samples from 30 birds (n = 6 per group) were collected in heparinized vacutainers. Plasma was separated and stored at −20˚C. Concentrations of calcium (Ca), phosphorus (P), sodium (Na), potassium (K), and magnesium (Mg) were determined using atomic absorption spectroscopy (AAS) according to standard protocols.

Segments (2 cm) of the small intestine (duodenum, jejunum, ileum) were excised post-mortem, fixed in 10% neutral buffered formalin for 72 hours, dehydrated, embedded in paraffin, and sectioned at 5 µm thickness. Slides were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus CX31). Villus height, width, and crypt depth were measured using ImageJ software, and the villus height to crypt depth (VH: CD) ratio was calculated.

Malondialdehyde (MDA) levels in breast meat samples were assessed as a marker of lipid peroxidation using the thiobarbituric acid (TBA) assay). Briefly, 1 mL of tissue homogenate was mixed with TBA reagent and heated in a water bath at 95˚C for 60 minutes. The absorbance of the supernatant was measured at 532 nm, and MDA concentration was calculated using a standard curve.

The GPx activity was estimated by measuring the rate of oxidation of glutathione (GSH) in the presence of hydrogen peroxide (H₂O₂), based on the decrease in NADPH absorbance at 340 nm. Absorbance was recorded at 412 nm after 10 minutes of incubation, and enzyme activity was expressed in U/mL.

SOD activity was determined using a commercial assay kit (Human Diagnostics, Germany) as per the manufacturer’s instructions. Absorbance was read at 560 nm using a spectrophotometer, and results were expressed in U/mL.

Data were entered into Microsoft Excel and analyzed using SAS software (Version 9.4, SAS Institute Inc., Cary, NC, USA). Two-way ANOVA was used to evaluate the effects of treatment and replication. Means were compared using Tukey’s Honest Significant Difference (HSD) test. Results were considered statistically significant at p < 0.05. All data are presented as mean ± standard deviation (SD).

This section reports the levels of Malondialdehyde (MDA), Superoxide Dismutase (SOD), and Glutathione Peroxidase (GPx) in the breast muscle of broilers across different treatment groups ( Table 1 ).

Key: Values with different superscript letters within the same row differ significantly (p < 0.05). Group E = 3% mixed herbs per 100 kg feed.

Malondialdehyde (MDA) MDA levels were significantly (p = 0.0103) reduced in all supplemented groups compared to the control. Group E showed the lowest MDA concentration (1.24 ± 0.31 nmol/mL), indicating enhanced antioxidant protection. Group A and B showed the highest MDA levels, with no statistical difference between them. Intermediate values were observed in Groups C and D.

SOD activity was significantly higher (p = 0.0236) in the herbal mix group (E) than in the control and M. indica groups. Group E had the highest SOD activity (92.61 ± 10.08 U/mL), followed by Group D. Group B recorded the lowest activity.

GPx activity significantly increased across all treated groups compared to the control (p = 0.0226). Group E again showed the highest GPx activity (90.62 ± 12.08 U/mL). Group B recorded the lowest enzyme activity among treatments.

Blood MDA, SOD, and GPx levels were evaluated to assess systemic antioxidant responses ( Table 2 ).

Key: ^abcMean values in the same row that do not share a common letter differ significantly (p < 0.05). Legends: Group-A = Control, Group-B = 0.5% Mangifera indica, Group-C = 0.15% Lawsonia inermis, Group-D = 0.5% Carica papya, Group-E = 0.58% mixture of herbs g/100kg feed.

MDA, a biomarker of lipid peroxidation and oxidative stress, exhibited a significant reduction (p = 0.0103) in all supplemented groups compared to the control. The highest MDA levels were observed in the control group (Group A: 4.5 ± 0.2^a), while the mixed herbal supplementation group (Group E) demonstrated the lowest MDA concentration (1.24 ± 0.31^b), indicating enhanced antioxidant protection. Among individual herbal treatments, Carica papaya (Group D) showed markedly reduced MDA levels (3.2 ± 0.3^d), outperforming Mangifera indica (Group B: 3.8 ± 0.1^b) and Lawsonia inermis (Group C: 3.5 ± 0.2^c).

SOD activity, reflecting enzymatic defense against superoxide radicals, was significantly elevated in supplemented groups compared to the control (p = 0.0236). The control group (Group A) displayed the lowest SOD activity (45.2 ± 1.5^c), whereas the mixed herbal group (Group E: 57.6 ± 2.0^a) and Carica papaya group (Group D: 56.4 ± 1.7^a) exhibited the highest activity. Intermediate values were recorded for Mangifera indica (Group B: 52.3 ± 1.8^b) and Lawsonia inermis (Group C: 54.8 ± 2.1^ab).

GPx activity, critical for detoxifying hydrogen peroxide, increased significantly (p = 0.0226) in all supplemented groups relative to the control. The lowest GPx activity was observed in the control group (Group A: 28.5 ± 1.2^b), while the mixed herbal group (Group E: 38.0 ± 1.2^a) achieved the highest activity. All herbal treatments showed elevated GPx levels: Mangifera indica (Group B: 35.7 ± 1.5^a), Lawsonia inermis (Group C: 36.2 ± 1.4^a), and Carica papaya (Group D: 37.5 ± 1.3^a).

The antioxidant activity of broiler chicken meat was evaluated using the DPPH radical scavenging method across five treatment groups: one control and four experimental groups supplemented with Mangifera indica, Lawsonia inermis, Carica papaya leaf powders, and their mixture. Significant variation in antioxidant activity was observed among the groups (p < 0.05), with herbal supplementation markedly enhancing the meat’s antioxidant capacity as shown in Table 3 .

Key: Values with different superscript letters (a, b, c) within a column differ significantly (p < 0.05). Legends: Group-A = Control, Group-B = 0.5% Mangifera indica, Group-C = 0.15% Lawsonia inermis, Group-D = 0.5% Carica papya, Group-E = 0.58% mixture of herbs g/100kg feed.

The control group exhibited the lowest DPPH radical scavenging activity (25.34% ± 1.23%), significantly differing from all treatment groups (p = 0.003). This highlights the limited antioxidant potential of the basal diet without herbal supplementation.

Supplementation with 0.5% Mangifera indica leaf powder significantly improved antioxidant activity, achieving a DPPH value of 35.67% ± 1.45% (p = 0.003), indicating moderate antioxidant enhancement. In contrast, 0.15% Lawsonia inermis supplementation resulted in intermediate DPPH activity (30.12% ± 1.37%), which, while not significantly different from the control (p = 0.015), was lower than Mangifera indica and Carica papaya groups, suggesting a milder effect.

The 0.5% Carica papaya group demonstrated the highest antioxidant activity among single-herb treatments, with a DPPH value of 40.89% ± 1.56% (p = 0.001), underscoring its potent antioxidant properties.

The mixed herbal supplementation (0.58% combination of Mangifera indica, Lawsonia inermis, and Carica papaya) yielded the highest DPPH activity (45.23% ± 1.48%, p = 0.000), significantly outperforming all other groups. This synergistic effect highlights the superior efficacy of combining these herbs in enhancing meat quality through improved antioxidant activity.

The Level of blood lipid and total protein was evaluated across five groups, which included, one control and four treated groups supplemented with Mangifera indica, Lawsonia inermis, Carica papaya leaf powders, and their mixture.

All the lipid parameters (cholesterol, triglycerides, LDL, HDL, VLDL, and non-HDL) showed statistically significant differences between the groups (p < 0.0001). The Least Significant Difference (LSD) values indicate significant distinctions between groups for each lipid parameter shown in Table 4 .

Key: Different superscript letters within rows denote significant differences (p < 0.05).

Exhibited the highest cholesterol levels (99.66^a ± 0.57), significantly differing from the other groups. This group also showed moderate HDL and LDL levels but relatively lower VLDL (19.67^a ± 0.58). Group A had the second-lowest non-HDL level (37.33^c ± 0.55).

Had the lowest cholesterol (76.00^c ± 1.00) among all groups, with the highest lipid triglyceride level (126.67^a ± 0.57). The LDL level was the highest (90.33^b ± 0.47), but HDL was lower (15.40^c ± 0.53) than Groups A and C. Non-HDL levels were moderately low (35.33^d ± 1.15).

Presented cholesterol levels (99.00^a ± 1.22) comparable to Group A. The lipid triglyceride levels were lower than Group B but higher than Group D. LDL levels (78.33^b ± 0.57) were relatively lower than Group B but higher than Group A. Group C displayed the highest HDL levels (19.33^d ± 1.15) among all groups.

Showed the lowest cholesterol (56.00^d ± 0.43) and lipid triglyceride (108.33^d ± 0.66) levels. LDL levels (80.00^a ± 0.57) were moderately high, while VLDL was the lowest (11.40^d ± 0.28). Non-HDL levels (70.33^b ± 0.65) were significantly higher compared to Groups A, B, and C.

Exhibited moderate cholesterol (86.00^b ± 0.78) and triglyceride (117.33^b ± 0.33) levels. LDL levels (41.33^a ± 0.44) were the lowest, but this group had the highest VLDL levels (17.40^b ± 0.30) and the most elevated non-HDL levels (76.00^a ± 0.57) among all groups.

The supplementation of Mangifera indica, Lawsonia inermis, and Carica papaya leaf powders significantly influenced the lipid profiles and total protein levels in broiler chickens. Groups varied in cholesterol, HDL, LDL, VLDL, and non-HDL levels, with statistical significance indicating notable differences in lipid metabolism influenced by the supplementation.

The activities of blood enzymes, including LDH, AST, ALT, CK, and ALP, varied significantly among the treatment groups (p < 0.05). Herbal supplementation influenced these parameters compared to the control, indicating potential hepatoprotective and metabolic effects (as shown in Table 5 ).

Key: Enzymes are expressed in Units per Liter (U/L).

This group recorded the highest LDH (1175.3 ± 10.5 U/L), AST (234.07 ± 8.2 U/L), and ALT (22.59 ± 0.9 U/L) activities. CK (5561 ± 112 U/L) and ALP (2421 ± 45 U/L) levels were also elevated, representing baseline enzyme activity without supplementation.

Compared to the control, LDH (1150.2 ± 9.8 U/L), AST (220.45 ± 7.6 U/L), and ALT (20.75 ± 0.8 U/L) were reduced. CK (5400 ± 105 U/L) and ALP (2300 ± 43 U/L) were moderately lower, suggesting improved liver function.

This group showed the lowest enzyme activities among all treatments, with LDH (1130.5 ± 11.0 U/L), AST (210.30 ± 8.0 U/L), ALT (18.90 ± 0.7 U/L), CK (5300 ± 110 U/L), and ALP (2250 ± 40 U/L). These reductions indicate enhanced hepatic health and reduced tissue damage.

LDH (1180.0 ± 12.3 U/L) and AST (235.50 ± 8.5 U/L) were slightly higher than the control, while ALT (23.10 ± 1.0 U/L) was comparable. CK (5580 ± 115 U/L) was marginally higher, and ALP (2400 ± 47 U/L) remained close to control values, suggesting a moderate effect on enzyme activity.

This group exhibited intermediate enzyme activities with LDH (1165.8 ± 10.7  U/L), AST (225.90 ± 7.9 U/L), ALT (21.50 ± 0.8 U/L), CK (5450 ± 108 U/L), and ALP (2350 ± 42 U/L). These results indicate a balanced modulation of enzyme activity without excessive elevation.

The immune response of broiler chickens was evaluated through RBC count, IgM, IgG, Newcastle disease HI titer, and H:L ratio. Significant differences (p < 0.05) were observed among groups (as shown in Table 6 ), indicating that herbal supplementation enhanced immune function compared to the control.

Key: SRBC, Sheep red blood cell; H:L, heterophil:lymphocyte. Legends: Group-A = Control, Group-B = 0.5% Mangifer aindica, Group-C = 0.15% Lawsonia inermis, Group-D = 0.5% Carica papya, Group-E = 0.58% mixture of herbs g/100kg feed.

The control group exhibited the lowest immune indicators, with RBC (6.17 ±  0.12), IgM (2.34 ± 0.10 mg/dL), IgG (3.83 ± 0.14 mg/dL), HI titer (6.00 ± 0.18), and H:L ratio (0.786 ± 0.025).

This group showed improved values compared to the control, with RBC (6.50 ± 0.15), IgM (2.60 ± 0.12 mg/dL), IgG (4.10 ± 0.13 mg/dL), HI titer (6.50 ± 0.20), and H:L ratio (0.740 ± 0.022).

RBC (6.70 ± 0.13), IgM (2.75 ± 0.11 mg/dL), IgG (4.25 ± 0.15 mg/dL), HI titer (6.80 ± 0.19), and H:L ratio (0.730 ± 0.024) were further improved, showing a strong immune-enhancing effect.

This group also showed enhanced immune responses with RBC (6.40 ± 0.14), IgM (2.50 ± 0.10 mg/dL), IgG (3.95 ± 0.12 mg/dL), HI titer (6.30 ± 0.21), and H:L ratio (0.760 ± 0.026).

The highest immune responses were recorded in this group, with RBC (6.80 ± 0.16), IgM (2.85 ± 0.13 mg/dL), IgG (4.30 ± 0.14 mg/dL), HI titer (7.00 ± 0.22), and H:L ratio (0.720 ± 0.023), indicating the synergistic effect of combined herbal supplementation.