The experimental protocol involving broiler chickens was reviewed and approved by the Institutional Ethical Committee of Universitas Airlangga, Indonesia, under approval number 1.KEH.005.01.2024. All animal procedures were conducted in accordance with institutional guidelines for the care and use of animals in research and complied with internationally accepted principles of animal welfare and humane experimentation. The study design, handling procedures, oral challenge model, sample collection, and endpoint assessments were planned to minimize animal stress, pain, and unnecessary suffering throughout the experimental period. Birds were maintained under appropriate husbandry conditions, including adequate housing, ventilation, temperature, lighting, and provision of feed and water, and were monitored daily for general health status and clinical signs following the APEC challenge. All interventions were performed by trained personnel using standard operating procedures. Blood collection was performed carefully at the end of the experimental period using appropriate restraint techniques to minimize distress. No procedures beyond those approved by the ethics committee were performed during the study.

This study was conducted from January to August 2024. The research was carried out at the partner poultry farm of Universitas Islam Kadiri, located in Blabak, Pesantren District, Kediri City, East Java, Indonesia. Birds were housed in floor pens under standard commercial management conditions, with ad libitum access to feed and water. Temperature, ventilation, and lighting were maintained according to broiler management guidelines. Serum biochemical parameters were analyzed at OKTA SAINTIKA Laboratory in Malang, East Java, Indonesia. The APEC used in this study was an isolated and identified strain maintained at the Microbiology Laboratory, Faculty of Veterinary Medicine, Universitas Brawijaya.

The material used in this study consisted of Lohmann strain (MB 202) broiler chickens, a commercial strain produced by PT Japfa Comfeed Indonesia. A total of 56 male broilers were allocated to seven treatment groups, with each treatment replicated four times and two birds per replicate (n = 8 birds/treatment). The number of birds per treatment was determined based on comparable experimental studies evaluating biochemical parameters in broilers under infectious challenge models, in which similar group sizes have been reported to be adequate for detecting statistically significant differences.

The treatment groups were arranged as follows:

K–: Negative control (uninfected, untreated)

K+: Positive control (APEC-infected only)

P0: APEC-infected + zinc bacitracin (Antibiotic growth promoter [AGP] control; 0.5 g/kg feed)

P1: APEC-infected + Myrmecodia sp. extract 15% at 0.1% concentration in drinking water

P2: APEC-infected + Myrmecodia sp. extract 30% at 0.1% concentration in drinking water

P3: APEC-infected + Myrmecodia sp. infusion at 1% of total drinking water

P4: APEC-infected + Myrmecodia sp. infusion at 2% of total drinking water

Extracts and infusions were administered daily from day 8 to day 35. APEC infection was performed on day 21 via oral administration of a 10⁸ CFU/mL bacterial suspension prepared in sterile saline [21].

The tubers of the ant nest plant (Myrmecodia sp.) were cleaned, sliced, and dried in direct sunlight. The dried material was ground into a fine powder and macerated in 96% ethanol (Merck, Darmstadt, Germany) at a 1:10 (w/v) ratio for 24 h at 25°C. The mixture was filtered twice, and the filtrate was concentrated with a rotary evaporator (Heidolph, Schwabach, Germany) at 60°C to obtain a crude extract. The extract was stored at 4°C until further use [10]. The plant material was obtained from local collectors in Papua, Indonesia, and taxonomically authenticated before experimental use. The phytochemical profile of the Myrmecodia sp. batch used in this study, including flavonoids, phenolic compounds, tannins, and saponins, has been previously characterized and reported [22].

The infusion was prepared by boiling powdered Myrmecodia sp. in distilled water (Brataco, Jakarta, Indonesia). For the 1% infusion, 1 g of powdered simplicia was boiled in 100 mL of distilled water at 90°C for 15 min, then cooled and filtered through muslin cloth. For the 2% infusion, 2 g of powdered simplicia was processed using the same procedure with 100 mL of distilled water. All infusions were freshly prepared daily and administered via drinking water according to the respective treatment groups [22].

The field isolate, E. coli O78, was initially enriched in 0.1% Buffered Peptone Water (Oxoid, Hampshire, UK) and incubated at 37°C for 24 h, with turbidity indicating bacterial growth. The culture was then streaked onto Sorbitol MacConkey Agar (Oxoid, Hampshire, UK) and incubated under the same conditions. On Sorbitol MacConkey Agar, E. coli O78 colonies were round, nucleated, and colorless due to the strain’s inability to ferment sorbitol [23]. Colorless colonies were subcultured onto Eosin Methylene Blue Agar (Oxoid, Hampshire, UK) and incubated at 37°C for 24 h, where colonies with a characteristic green metallic sheen and dark centers confirmed E. coli growth [24].

Biochemical identification was further performed using Triple Sugar Iron Agar, Sulfide Indole Motility medium, Methyl Red–Voges Proskauer medium, citrate utilization medium, and urease medium (Oxoid, Hampshire, UK), confirming the isolate as E. coli O78. The confirmed isolate was then standardized to a 0.5 McFarland turbidity in sterile saline with a densitometer to prepare the inoculum for experimental infection [25].

At the end of the experiment (day 35), blood samples were collected from the brachial vein of one bird per replicate using both ethylenediaminetetraacetic acid (EDTA) and plain vacutainer tubes (BD, Franklin Lakes, NJ, USA). The samples were centrifuged at 1,000 × g for 15 min, after which the sera were separated and stored at –20°C until biochemical analyses were performed [26].

Serum biochemical parameters were analyzed at OKTA SAINTIKA Laboratory, Malang, East Java, Indonesia, using standard commercial reagent kits. The following assays were conducted:

SGPT and SGOT were measured spectrophotometrically using the Humazym-UV Test (Human Diagnostics, Wiesbaden, Germany) at a wavelength of 340 nm. BUN was determined using the enzymatic UV method at a wavelength of 578 nm. Creatinine was measured using the alkaline picrate method at a wavelength of 520 nm. LDL and HDL were analyzed using the enzymatic CHOD–PAP method at a wavelength of 500 nm.

Cholesterol levels in meat, specifically HDL and LDL, were measured using the chloroform extraction method. About 4 g of minced meat was extracted with chloroform (Merck, Darmstadt, Germany) in three 25 mL portions. The lipid layer was then evaporated to dryness, and the remaining fat residue was transferred into a test tube, covered with aluminum foil to protect it from light, and analyzed enzymatically using a photometric method. Cholesterol was quantified after enzymatic hydrolysis and oxidation, which yielded quinoneimine from hydrogen peroxide and aminophenazone in the presence of phenol and peroxidase [27].

The observed variables included serum levels of SGPT, SGOT, creatinine, BUN, LDL, and HDL, as well as LDL and HDL concentrations in meat.

All data were analyzed using analysis of variance under a completely randomized design. When significant effects were observed (p < 0.05), mean comparisons were conducted using Duncan’s multiple range test at the 5% level. Statistical analyses were conducted using IBM SPSS Statistics version 26.0 (IBM Corp., NY, USA)

The mean SGOT and SGPT values in APEC-infected broilers treated with Myrmecodia sp. extract or infusion are presented in Table 1. Administration of Myrmecodia sp. extract or infusion significantly affected both SGPT and SGOT levels (p < 0.05).

SGPT levels ranged from 64.59 to 157.60 U/L. The lowest value was observed in the negative control (K–) at 68.04 U/L, while the highest was in the positive APEC-infected control (K+) at 157.60 U/L. The AGP control (P0) also showed elevated SGPT levels (122.46 U/L). Treatments with Myrmecodia sp. extract reduced SGPT levels, particularly in P1 (15% extract; 139.51 U/L) and P2 (30% extract; 125.51 U/L). Infusion treatments showed more pronounced improvement, with P3 (1% infusion) recording the lowest SGPT value (64.59 U/L), followed by P4 (2% infusion; 104.61 U/L).

Similar trends were observed for SGOT activity. Compared with the AGP control (P0), both extract and infusion treatments reduced transaminase levels, indicating improved hepatic integrity. The 15% extract treatment (P1) also showed a measurable hepatoprotective effect.

The average BUN and creatinine levels of APEC-infected broilers treated with Myrmecodia sp. are shown in Table 2. Treatments significantly affected BUN levels (p < 0.05), which ranged from 6.11 to 25.48 mmol/L. The lowest BUN level was observed in K– (8.97 mmol/L), while K+ showed the highest value (25.48 mmol/L). The AGP control (P0) recorded 9.56 mmol/L. Among the treatments, P3 (1% infusion) showed the greatest reduction in BUN, indicating improved renal function.

Extract treatments resulted in BUN values of 16.68 mmol/L (P1) and 17.12 mmol/L (P2). Infusion treatments showed lower BUN levels, with P3 (1% infusion) at 6.11 mmol/L and P4 (2% infusion) at 15.82 mmol/L.

Creatinine levels also differed significantly among treatments (p < 0.05), ranging from 0.53 to 4.66 U/L. The lowest creatinine value was found in K– (0.53 U/L), while the highest occurred in K+ (4.66 U/L). The AGP control (P0) recorded 2.94 U/L. Extract treatments resulted in creatinine levels of 2.67 U/L (P1) and 1.46 U/L (P2). Infusion treatments yielded values of 2.05 U/L (P3) and 1.98 U/L (P4), indicating that infusion-based administration generally improved renal function. Overall, treated groups exhibited lower creatinine levels than the positive control group.

The effects of Myrmecodia sp. extract and infusion on serum LDL and HDL levels are summarized in Table 3. Both parameters were significantly affected by treatment (p < 0.05). Serum LDL levels ranged from 39.65 to 99.65 mg/dL. K– (55.85 mg/dL) had lower LDL values than K+ (99.55 mg/dL), while the AGP control (P0) recorded 79.17 mg/dL. Extract treatments showed LDL levels of 45.24 mg/dL (P1) and 43.52 mg/dL (P2). Infusion treatments produced the lowest LDL value in P3 (39.65 mg/dL), followed by P4 (46.62 mg/dL).

Serum HDL levels ranged from 22.13 to 71.78 mg/dL. K– had the highest HDL level (64.88 mg/dL), whereas K+ had the lowest (22.13 mg/dL). The AGP control (P0) recorded 71.78 mg/dL. Extract treatments resulted in HDL values of 28.58 mg/dL (P1) and 26.94 mg/dL (P2). Infusion treatments showed HDL levels of 68.26 mg/dL (P3) and 50.11 mg/dL (P4). The best overall lipid modulation was observed in P3 (1% infusion), which produced the lowest LDL and one of the highest HDL levels among treatment groups.

The effects of Myrmecodia sp. extract and infusion on meat LDL-C and HDL-C levels are summarized in Table 4. Myrmecodia sp. supplementation significantly improved both serum and meat lipid profiles in APEC-infected broilers (p < 0.05). Significant differences in meat LDL-C and HDL-C concentrations were observed among treatment groups (p < 0.05).

Meat LDL-C levels ranged from 50.01 mg/dL (P3) to 79.47 mg/dL (P0). The positive control (K+) recorded 74.88 mg/dL, while the negative control (K–) showed 55.03 mg/dL. Lower meat LDL-C values were observed in P2 (60.76 mg/dL), P3 (50.01 mg/dL), and P4 (59.44 mg/dL) compared with the positive control.

Meat HDL-C levels ranged from 15.52 ± 6.03 mg/dL (P0) to 60.68 ± 4.95 mg/dL (K–). The positive control (K+) recorded 27.80 ± 5.47 mg/dL. Among treatment groups, P3 (1% infusion) showed 56.10 ± 7.74 mg/dL, while P4 recorded 45.62 ± 8.74 mg/dL.

The results of this study demonstrate that Myrmecodia sp. extract and infusion were associated with improvements in broilers infected with APEC, as reflected in enhanced liver and kidney function, lipid metabolism, and meat lipid composition. Importantly, this study extends previous findings on growth and immune responses by providing biochemical evidence of organ-level recovery under conditions of infectious stress. These findings support the growing interest in phytogenic compounds as potential complementary strategies to reduce antibiotic dependence, thereby supporting efforts to reduce AMR within a One Health framework.

APEC infection caused a substantial increase in SGPT and SGOT levels, indicating hepatocellular damage from systemic inflammation and endotoxin exposure [3]. The significant increase in transaminases in the positive control group aligns with the well-documented pathogenesis of colibacillosis, in which oxidative stress, lipopolysaccharide-induced inflammation, and impaired hepatocyte membrane integrity contribute to liver dysfunction [13].

Administration of Myrmecodia sp., particularly as an infusion (P3), was associated with lower SGPT and SGOT activities than the positive control. These findings suggest a potential hepatoprotective role, possibly attributable to flavonoids, phenolic compounds, and tannins, which modulate inflammatory pathways [10]. The infusion group showed lower transaminase values, which may reflect differences in phytochemical solubility. Water-soluble compounds such as tannins and saponins have been reported to enhance antioxidant enzyme activity and reduce lipid peroxidation, thereby limiting hepatocellular leakage under acute infectious stress [28, 29]. These observations indicate that preparation-dependent phytochemical availability may influence the magnitude of hepatic protection during systemic bacterial challenge.

APEC infection similarly impaired renal function, as indicated by elevated BUN and creatinine levels in the positive control. These increases reflect reduced glomerular filtration, renal inflammation, and nephron stress caused by systemic bacterial infection [30].

Myrmecodia sp. supplementation was associated with reduced levels of renal biomarkers compared with the positive control, particularly in the 1% infusion group (P3). The reduction in BUN and creatinine suggests improved glomerular filtration and reduced inflammation-mediated renal damage. Flavonoids and phenolic antioxidants may contribute to preserving renal microvascular integrity and mitigating oxidative stress-induced tubular injury, which are commonly observed in systemic infections. These findings provide biochemical support for a renoprotective effect under infectious conditions [31].

APEC infection disrupted lipid homeostasis, characterized by increased LDL and decreased HDL levels. These alterations align with previous reports indicating that systemic inflammation impairs hepatic lipid regulation, promotes lipid peroxidation, and accelerates HDL particle degradation [32].

Both extract and infusion treatments improved the serum lipid profile relative to the positive control. Notably, the 1% infusion group exhibited lower LDL and higher HDL concentrations compared with infected controls. This enhancement in lipid metabolism is likely mediated through the combined antioxidant and anti-inflammatory actions of Myrmecodia sp., which support hepatic recovery and optimize lipid processing [33]. Flavonoids and tannins may also modulate key lipid regulatory enzymes, potentially reducing LDL synthesis and enhancing HDL stability [34]. Phytogenic treatments demonstrated comparable or favorable effects on lipid parameters compared with zinc bacitracin (P0), suggesting potential metabolic modulation beyond antimicrobial activity [35]. The relatively elevated HDL concentration observed in the zinc bacitracin group may reflect antibiotic-associated modulation of lipid metabolism, possibly through alterations in gut microbiota composition and bile acid metabolism, thereby influencing hepatic lipoprotein regulation. However, this effect requires further investigation to clarify its underlying mechanisms.

APEC infection negatively influenced meat lipid composition. Treatment with Myrmecodia sp. was associated with higher HDL-C and lower LDL-C in meat compared with infected controls. Treatment with Myrmecodia sp. effectively counteracted these changes. The 1% infusion group exhibited HDL-C values approaching those of the negative control. This indicates improved intramuscular lipid metabolism and reduced oxidative stress within muscle tissues [10].

Improved lipid profiles in meat may reflect systemic metabolic stabilization and reduced oxidative stress in muscle tissues [36]. Enhanced meat lipid profiles are particularly important from the perspectives of food safety and consumer health [37]. Higher HDL and lower LDL levels contribute to better nutritional quality of poultry products, supporting public health goals and improving marketability [38]. By linking biochemical organ recovery to measurable changes in meat lipid composition, this study provides an integrative perspective that links animal physiology to product quality, a connection that has been less emphasized in previous Myrmecodia-based studies. These findings suggest that phytogenic supplementation may contribute to both animal health and product quality [39].

Across multiple evaluated parameters, infusion treatments, particularly P3 (1% infusion), demonstrated consistently favorable outcomes compared with extract treatments. Ethanol-based extraction typically concentrates specific flavonoids and phenolic compounds, whereas aqueous infusion yields a phytochemical profile enriched in water-soluble constituents. The observed differences in biological responses are consistent with variations in phytochemical solubility and bioavailability, particularly the presence of tannins and saponins in aqueous preparations [40]. These results highlight that the preparation method is not merely a technical variable but a biologically relevant factor influencing therapeutic efficacy under infectious stress. These findings emphasize that preparation methods play a critical role in shaping physiological responses and optimizing the therapeutic potential of phytogenic compounds in poultry systems [41].

The findings of this study highlight the potential role of Myrmecodia sp. supplementation as a supportive strategy in broiler production systems aimated to reducing reliance on antibiotic growth promoters. The observed improvements in organ function biomarkers and lipid parameters indicate that phytogenic supplementation may help maintain physiological stability under infectious challenge conditions. Such strategies could contribute to reducing routine antibiotic use and, consequently, lowering selective pressure associated with AMR [35].

Within the One Health framework, these outcomes are aligned with international efforts to safeguard animal health while promoting food safety and responsible antimicrobial stewardship across animal, human, and environmental interfaces [42]. The use of locally available medicinal plants may further support sustainable livestock management practices and reduce dependence on synthetic pharmaceutical inputs [43].