INTRODUCTION

Avian pathogenic Escherichia coli (APEC) is the primary cause of colibacillosis in poultry and poses a significant challenge for the global poultry industry [1]. APEC infection can trigger systemic inflammation, organ damage, reduced growth performance, and increased mortality in broiler chickens [2]. These effects not only lead to economic losses from treatment costs and reduced carcass quality but also potentially threaten public health if APEC strains carry antibiotic resistance genes that can be transferred to zoonotic pathogens [3]. Within a One Health framework, APEC control must address animal health, food safety, environmental contamination, and the broader issue of antimicrobial resistance (AMR) [4].

For decades, antibiotics have been widely used as growth promoters and for treating infections in poultry [5]. However, growing concerns about AMR have spurred the development of safer, more effective, and sustainable natural alternatives [6]. Among natural resources, medicinal plants rich in bioactive compounds have attracted increasing attention as phytogenic feed additives. One such plant is Myrmecodia sp., locally known as sarang semut (ant nest), an epiphytic plant traditionally used in Indonesia and Papua New Guinea [7]. Myrmecodia sp. contains secondary metabolites, including flavonoids, tannins, saponins, and phenolic compounds, and has reported antibacterial, antioxidant, anti-inflammatory, and immunomodulatory activities [8]. Compared with other phytotherapeutic agents frequently investigated in poultry nutrition, Myrmecodia sp. has been less extensively studied in controlled APEC challenge models, particularly regarding its effects on biomarkers of organ function and lipid metabolism [9–11].

In cases of APEC infection, damage to organs such as the liver, kidneys, and intestines results from bacterial colonization and an excessive inflammatory response [12]. Disruption of these organs can impair lipid metabolism, reduce detoxification, and lower feed utilization efficiency [13]. APEC infection is also often accompanied by decreased meat quality, including changes in intramuscular fat content, color, pH, and water-holding capacity, ultimately reducing the market value of poultry products [14]. Therefore, interventions that maintain organ integrity, stabilize lipid metabolism, and preserve meat quality are necessary to support the production of healthy, high-quality broilers [3].

Preparation methods for Myrmecodia sp., including ethanolic extracts and aqueous infusions, may yield distinct phytochemical profiles due to differences in solvent polarity [15, 16]. Ethanol-based extracts are generally richer in flavonoids and phenolic compounds, whereas aqueous infusions may contain higher proportions of water-soluble constituents such as tannins and saponins. Understanding these preparation-dependent differences is essential for optimizing biological efficacy in poultry systems. Previous reports from the same experimental trial have described growth performance, immune responses, and phytochemical characteristics of Myrmecodia sp. supplementation under APEC challenge. However, a focused evaluation of biochemical liver and kidney markers, together with serum and meat lipid profiles, has not been comprehensively addressed [17]. The One Health perspective emphasizes that plant-based interventions may reduce antibiotic dependency while supporting animal health and food safety [18, 19].

Despite the growing body of research on phytogenic feed additives as alternatives to antibiotics in poultry production, significant gaps remain in understanding their organ-level and metabolic effects under infectious challenge conditions. Previous studies on Myrmecodia sp. have primarily focused on growth performance, immune modulation, and general antimicrobial activity, with limited emphasis on biochemical indicators of organ function. In particular, there is a lack of comprehensive evaluation of liver and kidney biomarkers, such as serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), blood urea nitrogen (BUN), and creatinine, in broiler chickens exposed to APEC. Although plant-derived compounds have been reported to influence lipid metabolism in poultry [20], studies investigating the specific effects of Myrmecodia sp. on serum lipid fractions, including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), as well as corresponding lipid composition in meat, remain scarce. Another important gap is the limited comparative assessment of different preparation methods of Myrmecodia sp., particularly ethanolic extracts versus aqueous infusions, which are known to yield distinct phytochemical profiles and may result in differential biological effects. In addition, most existing studies have not integrated these physiological and biochemical outcomes within a One Health framework, which is essential for addressing AMR and ensuring sustainable poultry production. Therefore, a systematic investigation linking organ function, lipid metabolism, and meat quality under APEC challenge conditions is critically needed.

Addressing these gaps, the present study aimed to comprehensively evaluate the effects of Myrmecodia sp. extract and infusion on key physiological and biochemical parameters in broiler chickens experimentally infected with APEC. Specifically, this study assessed the impact of different concentrations of Myrmecodia sp. extract and aqueous infusion on liver function biomarkers (SGPT and SGOT), kidney function biomarkers (BUN and creatinine), serum lipid profile (LDL and HDL), and meat lipid composition. Furthermore, this study compared the relative efficacy of extract and infusion preparations in mitigating APEC-induced physiological disturbances, thereby identifying the most effective formulation for improving animal health and product quality. By integrating these findings, the study also aimed to provide scientific evidence supporting the use of Myrmecodia sp. as a phytogenic alternative to antibiotic growth promoters, contributing to reduced antimicrobial use and aligning poultry production practices with the One Health approach.

MATERIALS AND METHODS

Ethical approval

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.

Study period and location

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.

Experimental design

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].

Preparation of Myrmecodia sp. extract

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].

Preparation of Myrmecodia sp. infusion

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].

Purification method for field isolate of E. coli O78

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].

Sample collection and blood analysis

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.

Measurement of HDL and LDL cholesterol in meat

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].

Observation variables

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

Statistical analysis

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)

RESULTS

Liver function enzymes (SGPT and SGOT)

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).

Administration of ant nest extract and herbal infusion of ant nests infected with Escherichia coli bacteria to broiler chickens showed a significant effect (p < 0.05) on SGPT and SGOT. SGPT = Serum glutamate pyruvate transaminase, SGOT = Serum glutamate oxaloacetate transaminase.

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.

Kidney function parameters (BUN and creatinine)

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.

Administration of ant nest extract and herbal infusion of ant nest infected with Escherichia coli bacteria to broiler chickens showed that it had a significant effect (p < 0.05) on BUN and creatinine levels. BUN = Blood urea nitrogen.

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.

Lipid profile (LDL and HDL) in serum

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).

Administration of ant nest extract and herbal infusion of ant nests infected with avian pathogenic Escherichia coli to broiler chickens showed a significant effect (p < 0.05) on serum LDL and HDL levels. LDL = Low density lipoprotein, HDL = High density lipoprotein.

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.

Lipid profile and cholesterol modulation in broiler meat

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).

Administration of ant nest extract and herbal infusion of ant nests infected with avian pathogenic Escherichia coli to broiler chickens showed a significant effect (p < 0.05) on serum LDL and HDL levels. LDL = Low density lipoprotein, HDL = High density lipoprotein.

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.

DISCUSSION

Hepatoprotective effects of Myrmecodia sp. in APEC-induced liver dysfunction

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.

Renoprotective function and improvement of kidney biomarkers

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].

Modulation of serum lipid metabolism

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.

Improvements in meat lipid composition and implications for meat quality

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].

Comparative performance of extract versus infusion

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].

One Health implications and relevance to AMR mitigation

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].

CONCLUSION

The present study demonstrated that supplementation with Myrmecodia sp. significantly mitigated the adverse physiological effects induced by APEC infection in broiler chickens. APEC challenge resulted in marked elevations in SGPT, SGOT, BUN, creatinine, and LDL levels, accompanied by a reduction in HDL levels, indicating hepatic dysfunction, renal impairment, and disrupted lipid metabolism. Administration of Myrmecodia sp., particularly in the 1% infusion form, effectively restored these parameters toward normal physiological ranges. The infusion treatment showed the lowest SGPT (64.59 U/L), SGOT (40.40 U/L), BUN (6.11 mmol/L), and LDL levels (39.65 mg/dL), along with improved HDL concentrations in both serum and meat, indicating superior hepatoprotective, renoprotective, and hypolipidemic effects compared with extract and antibiotic treatments.

From a practical perspective, these findings suggest that Myrmecodia sp., especially as an aqueous infusion, can serve as an effective phytogenic feed additive to enhance organ function, stabilize lipid metabolism, and improve meat lipid quality in broiler production systems under infectious stress. The observed improvements in meat HDL and LDL profiles further highlight its potential to enhance the nutritional quality and market value of poultry products, thereby supporting both animal health and consumer health within a One Health framework.

The major strength of this study lies in its comprehensive evaluation of organ function biomarkers, serum lipid profile, and meat lipid composition under a controlled APEC challenge model, and in its direct comparison of two preparation methods for Myrmecodia sp. (extract vs. infusion). This integrative approach provides novel insights into the physiological and metabolic responses to phytogenic supplementation and bridges the gap between animal health and product quality.

However, certain limitations should be acknowledged. The study was conducted under controlled experimental conditions, with a relatively small sample size and a single APEC strain, which may not fully reflect field variability. In addition, detailed mechanistic investigations at the molecular level, including gene expression and modulation of the gut microbiota, were not performed. The phytochemical composition was inferred from previous reports rather than quantified in the current experimental batch.

Future research should focus on large-scale field trials, dose optimization, and long-term safety evaluation of Myrmecodia sp. supplementation. Further studies are also needed to elucidate the molecular mechanisms underlying its hepatoprotective and lipid-modulating effects, including its interactions with genes, the gut microbiota, immune pathways, and lipids involved in lipid metabolism. Comparative studies with other phytogenic additives and economic feasibility assessments would further strengthen its application in commercial poultry production.

In conclusion, Myrmecodia sp., particularly in infusion form, represents a promising natural alternative to antibiotic growth promoters, capable of improving organ function, regulating lipid metabolism, and enhancing meat quality in broilers challenged with APEC. Its application aligns with sustainable poultry production strategies and contributes to AMR mitigation within the One Health paradigm.

DATA AVAILABILITY

The supplementary data can be available from the corresponding author upon reasonable request.

AUTHORS’ CONTRIBUTIONS

EFL and WPL: Conception and design of the study. MAA and EPH: Conducted the study and analyzed the results. ISH, and WT: Interpreted the results. MZR and FSD: Analysis and interpreted the data. ARK, FA, and SR: Study design, data interpretation, and drafted the manuscript. AM, MS, and ML: Methodology development, supervision, and critical revision of the manuscript. All authors have read and approved the final version of the manuscript.

COMPETING INTERESTS

The authors declare that they have no competing interests.

PUBLISHER’S NOTE

Veterinary World remains neutral with regard to jurisdictional claims in the published institutional affiliations.

ACKNOWLEDGMENTS

The authors express their sincere gratitude to the Indonesian Education Scholarship (BPI) Program (No. 01209/J5.2.3/BPI.06/9/2022) for financial support provided as part of the doctoral education funding scheme, administered by the Indonesian Endowment Fund for Education (LPDP) and the Center for Higher Education Funding and Assessment (PPAPT). The authors also acknowledge Universitas Airlangga and Universitas Islam Kadiri for their valuable institutional support.

REFERENCES

1. Boulbair I, Hu J, Hammoudi A, Zhang B, Aissat S, Wang X. Molecular characterization and antibiotic resistance of avian pathogenic Escherichia coli (APEC) isolates from broiler chickens in Algeria. Animals 2025;15(22):3324. [Google Scholar] | [Crossref]
2. Wei L, Liu X, Tan Z, Zhang B, Wen C, Tang Z. Chlorogenic acid mitigates avian pathogenic Escherichia coli-induced intestinal barrier damage in broiler chickens via anti-inflammatory and antioxidant effects. Poult Sci 2025;104(5):105005. [Google Scholar] | [Crossref]
3. Kathayat D, Lokesh D, Ranjit S, Rajashekara G. Avian pathogenic Escherichia coli (APEC): An overview of virulence and pathogenesis factors, zoonotic potential, and control strategies. Pathogens 2021;10(4):467. [Google Scholar] | [Crossref]
4. Pitt SJ, Gunn A. The One Health concept. Br J Biomed Sci 2024;81(1):12366. [Google Scholar] | [Crossref]
5. Nduku X, Stempa T, Lungu NS, Ndobeni TN, Mlambo V. Prospects for antibiotic-free poultry production in South Africa: An analysis of the enablers and stumbling blocks. One Health 2025;21(1):101144. [Google Scholar] | [Crossref]
6. Salam MA, Al-Amin MY, Salam MT, Pawar JS, Akhter N, Rabaan AA. Antimicrobial resistance: A growing serious threat for global public health. Healthcare (Basel) 2023;11(13):1946. [Google Scholar] | [Crossref]
7. Primasari A, Apriyanti E, Ambardhani N, Satari MH, Herdiyati Y, Kurnia D. Formulation and antibacterial potential of sarang semut (Myrmecodia pendans) against oral pathogenic bacteria: An in vitro study. Open Dent J 2022;16(Suppl 4):e1874210621121400. [Google Scholar] | [Crossref]
8. Daulay M, Syahputra M, Sari MI, Widyawati T, Anggraini DR. The potential of Myrmecodia pendans in preventing complications of diabetes mellitus as an antidiabetic and antihyperlipidemic agent. Open Vet J 2024;14(7):1607-1613. [Google Scholar] | [Crossref]
9. Hertiani T, Sasmito E, Sumardi, Ulfah M. Preliminary study on immunomodulatory effect of sarang-semut tubers Myrmecodia tuberosa and Myrmecodia pendens. OnLine J Biol Sci 2010;10(3):136-141. [Google Scholar] | [Crossref]
10. Dirgantara S, Insanu M, Fidrianny I. Medicinal properties of ant nest plant (Myrmecodia genus): A comprehensive review. Open Access Maced J Med Sci 2022;10(F):97-103. [Google Scholar] | [Crossref]
11. Widyawati T, Pase MA, Daulay M, Sumantri IB, Yusoff NA. Evaluation of Myrmecodia pendans water extracts on hematology profiles, liver, kidney function and malondialdehyde level in healthy volunteer. Pharmacogn J 2020;12(6):1489-1493. [Google Scholar] | [Crossref]
12. Khairullah AR, Afnani DA, Riwu KHP, Widodo A, Yanestria SM, Moses IB. Avian pathogenic Escherichia coli: Epidemiology, virulence and pathogenesis, diagnosis, pathophysiology, transmission, vaccination, and control. Vet World 2024;17(12):2747-2762. [Google Scholar] | [Crossref]
13. Chen M, He Y, Jia Y, Wu L, Zhao R. Liver transcriptome response to avian pathogenic Escherichia coli infection in broilers with corticosterone treatment. Poult Sci 2025;104(5):105020. [Google Scholar] | [Crossref]
14. Joseph J, Zhang L, Adhikari P, Evans JD, Ramachandran R. Avian pathogenic Escherichia coli (APEC) in broiler breeders: An overview. Pathogens 2023;12(11):1280. [Google Scholar] | [Crossref]
15. Sumardi, Hertiani T, Sasmito E. Ant plant (Myrmecodia tuberosa) hypocotyl extract modulates TCD4+and TCD8+cell profile of doxorubicin-induced immune-suppressed Sprague-Dawley rats in vivo. Sci Pharm 2013;81(4):1057-1069. [Google Scholar] | [Crossref]
16. Kumar A, Nirmal P, Kumar M, Jose A, Tomer V, Oz E. Major phytochemicals: Recent advances in health benefits and extraction method. Molecules 2023;28(2):887. [Google Scholar] | [Crossref]
17. Hartady T, Syamsunarno MRAA, Basyirasaniyanti B, Khairani S, Rosdianto AM. Spleen histopathological evaluation of broiler chickens challenged with Escherichia coli and its effect towards the combination of Javanese cardamom and turmeric herbs. Vet Sci 2025;12(10):975. [Google Scholar] | [Crossref]
18. Dardouri M, Mtibaa MM, Azaiez S, Khachroub AM, Mansour W. Herbs impact on poultry health and antimicrobial resistance: A scoping review with One Health perspective. BMC Vet Res 2025;21(1):499. [Google Scholar] | [Crossref]
19. Aminullah N, Mostamand A, Zahir A, Mahaq O, Azizi MN. Phytogenic feed additives as alternatives to antibiotics in poultry production: A review. Vet World 2025;18(1):141-154. [Google Scholar] | [Crossref]
20. Ding X, Giannenas I, Skoufos I, Wang J, Zhu W. The effects of plant extracts on lipid metabolism of chickens: A review. Anim Biosci 2023;36(5):679-691. [Google Scholar] | [Crossref]
21. Watts A, Wigley P. Avian pathogenic Escherichia coli: An overview of infection biology, antimicrobial resistance and vaccination. Antibiotics 2024;13(9):809. [Google Scholar] | [Crossref]
22. Lisnanti EF, Lokapirnasari WP, Al-Arif MA, Hestianah EP, Hidanah S, Moses IB. The ameliorative effects of Myrmecodia sp. as an antibiotic growth promoter in broiler chicken with avian pathogenic Escherichia coli infection. Open Vet J 2025;15(11):5858-5871. [Google Scholar] | [Crossref]
23. Ternent HE, Innocent GT, Filshie LM, Taylor DJ, Steele WB, McEwen SA. Frozen storage of Escherichia coli O157 in buffered peptone water and its detection on bovine carcasses. J Food Prot 2004;67(1):40-45. [Google Scholar] | [Crossref]
24. Farizqi MTI, Effendi MH, Adikara RTS, Yudaniayanti IS, Putra GDS, Khairullah AR. Detection of extended-spectrum β-lactamase-producing Escherichia coli genes isolated from cat rectal swabs at Surabaya Veterinary Hospital, Indonesia. Vet World 2023;16(9):1917-1925. [Google Scholar] | [Crossref]
25. Dameanti FNAE, Dacosta AO, Adrenalin SL, Fatmawati M, Permata FS, Siswanto HP, Ariyanti T. A comparative study on histopathological features of duodenum, jejunum, and ileum from broiler chicken with avian pathogenic Escherichia coli infection. J Indones Trop Anim Agric 2023;48(3):194-207. [Google Scholar] | [Crossref]
26. Kelly LM, Alworth LC. Techniques for collecting blood from the domestic chicken. Lab Anim (NY) 2013;42(10):359-361. [Google Scholar] | [Crossref]
27. Haedrich J, Stumpf C, Denison MS. Rapid extraction of total lipids and lipophilic POPs from all EU-regulated foods of animal origin: Smedes'method revisited and enhanced. Environ Sci Eur 2020;32(1):118. [Google Scholar] | [Crossref]
28. Fraga-Corral M, Otero P, Cassani L, Echave J, Garcia-Oliveira P, Carpena M. Traditional applications of tannin rich extracts supported by scientific data: Chemical composition, bioavailability and bioaccessibility. Foods 2021;10(2):251. [Google Scholar] | [Crossref]
29. Hasan MH, Zakaria H, Abdul Wahab I, Ponto T, Adam A. Myrmecodia platytyrea methanol tuber extract ameliorates hyperglycemia in STZ-induced diabetic Sprague-Dawley male rats. Indones J Pharm 2021;32(3):338-348. [Google Scholar] | [Crossref]
30. Wang J, Li H, Yang P, Chen X, Chen S, Deng L. Exploring the value of blood urea nitrogen-to-albumin ratio in patients with acute pancreatitis admitted to the intensive care unit: A retrospective cohort study. Front Nutr 2025;12(1):1435356. [Google Scholar] | [Crossref]
31. Sujono TA, Munawaroh R, Wijayanti PK, Lestari YP. The protective effect of sarang semut (Myrmecodia tuberose) tubers infusion on gentamicin-piroxicam induced nephrotoxicity in rats. Indones J Pharm 2014;25(2):91-97. [Google Scholar] | [Crossref]
32. Vyletelová V, Nováková M, Pašková Ľ. Alterations of HDL's to piHDL's proteome in patients with chronic inflammatory diseases, and HDL-targeted therapies. Pharmaceuticals 2022;15(10):1278. [Google Scholar] | [Crossref]
33. Hasan M, Wahab I, Mizaton H, Rasadah M. Efficacy of Myrmecodia platytyrae water extract in reducing cholesterol level in hypercholesterolemia-induced Sprague-Dawley rat. Malays J Med Biol Res 2020;7(2):69-80. [Google Scholar] | [Crossref]
34. Bjune K, Halvorsen PS, Wangensteen H, Leren TP, Bogsrud MP, Strøm TB. Flavonoids regulate LDLR through different mechanisms tied to their specific structures. J Lipid Res 2024;65(5):100539. [Google Scholar] | [Crossref]
35. Lisnanti EF, Lokapirnasari WP, Hestianah EP, Al Arif MA, Baihaqi ZA, Yulianto AB. Antibacterial alternatives using the potential of the ant nest plant (Myrmecodia spp.). Int J One Health 2024;10(1):148-152. [Google Scholar] | [Crossref]
36. Sidorova YS, Petrov NA, Markova YM, Kolobanov AI, Zorin SN. The influence of a high-cholesterol diet and forced training on lipid metabolism and intestinal microbiota in male Wistar rats. Int J Mol Sci 2024;25(10):5383. [Google Scholar] | [Crossref]
37. Ruiz-Capillas C, Herrero AM. Development of meat products with healthier lipid content: Vibrational spectroscopy. Foods 2021;10(2):341. [Google Scholar] | [Crossref]
38. Tan Z, Halter B, Liu D, Gilbert ER, Cline MA. Dietary flavonoids as modulators of lipid metabolism in poultry. Front Physiol 2022;13(1):863860. [Google Scholar] | [Crossref]
39. Dong S, Li L, Hao F, Fang Z, Zhong R, Wu J, Fang X. Improving quality of poultry and its meat products with probiotics, prebiotics, and phytoextracts. Poult Sci 2024;103(2):103287. [Google Scholar] | [Crossref]
40. Sun W, Shahrajabian MH. Therapeutic potential of phenolic compounds in medicinal plants-natural health products for human health. Molecules 2023;28(4):1845. [Google Scholar] | [Crossref]
41. Wallace RJ, Oleszek W, Franz C, Hahn I, Baser KH, Mathe A. Dietary plant bioactives for poultry health and productivity. Br Poult Sci 2010;51(4):461-487. [Google Scholar] | [Crossref]
42. Velazquez-Meza ME, Galarde-López M, Carrillo-Quiróz B, Alpuche-Aranda CM. Antimicrobial resistance: One Health approach. Vet World 2022;15(3):743-749. [Google Scholar] | [Crossref]
43. Folina A, Kakabouki I, Baginetas K, Bilalis D. Integration of bioresources for sustainable development in organic farming: A comprehensive review. Resources 2025;14(7):102. [Google Scholar] | [Crossref]