Effects of Lacticaseibacillus paracasei and Monensin on Performance and Antioxidant Status in Eimeria tenella Infected Broilers

Coccidiosis, caused by Eimeria spp., is one of the most economically significant parasitic diseases in poultry, leading to substantial financial losses due to stunted growth, low feed conversion rates, and elevated mortality. Regarding these, Eimeria tenella is especially well-known for causing large cecal lesions and interfering with broiler nutrient absorption, which lowers production and compromises intestinal integrity (Mohsin et al., 2022). Complicating the burden of this disease are high rates of morbidity and mortality as well as the related expenses of chemotherapeutic control. The Ministry of Agriculture and Rural Affairs of the People’s Republic of China (PRC, 2023) indicated that the peak incidence of coccidiosis transpired from April to September 2023, comprising up to 90% of all documented cases, hence highlighting its global significance.

Anticoccidial medications, such as monensin, an ionophore that remains a cornerstone in the prevention of coccidiosis, are a major component of conventional control tactics. Monensin’s effectiveness has been mechanistically linked to its capacity to disrupt E. tenella’s life cycle by inducing autophagy and programmed cell death (Qi et al., 2020; Zhang et al., 2022). Nevertheless, the emergence of resistant Eimeria strains and the increasing consumer demand for antibiotic-free poultry production systems have given rise to concerns regarding the long-term reliance on ionophores. Alternative or complementary strategies to maintain intestinal health while maintaining production efficiency have been the subject of interest in response to these concerns.

Although monensin is often included in discussions about antibiotic use in animal husbandry, legally and according to pharmaceutical regulations, monensin is classified as an ionophore and is widely used as an antiprotozoal agent and growth promoter in the livestock industry, including in Indonesia and several other countries. Currently, efforts to reduce antibiotic use in the livestock industry are increasingly driven by concerns about antimicrobial resistance and its impact on human health. The use of monensin as an anticoccidial agent is a strategic step that is permitted and regulated at both national and international levels, distinguishing its use from antibiotics used in human medicine. In the context of this research, the use of monensin aims to reduce dependence on antibiotics during the coccidiosis control process, while also demonstrating that a combination of probiotics and ionophores can be a safe and effective approach to maintaining animal health without violating the principles of antibiotic-free animal product development.

Probiotics have emerged as potential biological alternatives. Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) received attention for its capacity to reduce oxidative stress, enhance immune responses, and modulate intestinal microbiota in broilers that have been infected with Eimeria tenella (Kang et al., 2023; Lima et al., 2020). Probiotic supplements have been shown to strengthen the integrity of the gut barrier and improve nutrient utilization by improving intestinal morphology, increasing the activity of antioxidant enzymes like glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and lowering malondialdehyde (MDA) levels (Mohsin et al., 2022; Wang et al., 2021). Probiotics enhance microbial balance by promoting beneficial taxa such as Lactobacillus and Bacteroides while inhibiting pathogenic bacteria including Clostridium, Escherichia, Shigella, and Salmonella, resulting in a healthier intestinal environment (Chen et al., 2024; Guo et al., 2025).

According to recent research, administering probiotics alongside anticoccidials like monensin may have additive effects. While L. paracasei improves gut health, antioxidant defense, and immunological modulation, monensin directly targets parasites by affecting ion homeostasis (El-Shall et al., 2024). The capacity for improved coccidiosis management without sacrificing growth performance is presented by this dual approach. There is, however, lacking of studies directly comparing L. paracasei and monensin, either separately or in combination, under standardized E. tenella challenge circumstances, especially during the finisher phase, when metabolic balance and growth efficiency are crucial for total production.

In order to determine if adding Lacticaseibacillus paracasei to drinking water, either alone or in combination with monensin, may improve the growth performance and oxidative stress status of broilers that have been experimentally challenged with E. tenella, this study was carried out. In comparison to infected controls, we predicted that L. paracasei would increase feed intake, body weight gain, and feed conversion ratio. We also predicted that, because of complementary parasite- and host-directed mechanisms, its combination with monensin would result in the largest increase in feed efficiency. Furthermore, it is anticipated that taking probiotic supplements will improve the antioxidant defense system, as seen by higher SOD activity and lower MDA levels (Mohsin et al., 2022; Qi et al., 2020; Wang et al., 2021). This study uses a standardized E. tenella challenge model to give a novel comparative evaluation of water-delivered L. paracasei and monensin, both separately and together. Through the integration of oxidative biomarkers and performance data, this study provides a cohesive view of how probiotic–ionophore interactions improve redox homeostasis and feed efficiency. The development of sustainable drug-reduced poultry production methods that strike a compromise between food safety, animal welfare, and efficacy is anticipated to be aided by these findings.

The study was carried out from July to October 2025 at the Universitas Airlangga Faculty of Veterinary Medicine in Surabaya, Indonesia. Floors, brooders, feeders, drinkers, and the raising facility were all cleaned, disinfected, and allowed to dry for three days prior to the arrival of day-old chicks (DOCs). Each of the five circular brooder pens (60 cm in height and 92 cm in diameter) was coated with around 8 cm of bedding made of rice husks and covered with a newspaper. The chicks were given sugar water and multivitamins for three days after they arrived to help them adjust. On day five of the vaccination protocol, the Newcastle Disease (ND) HB1 vaccine was given via eye drops, and on day fourteen, ND Lasotta. The temperature and humidity were recorded using thermometers and hygrometers (32–34 °C and 60–70% relative humidity, respectively), and brooding was kept under control by 25-Watt heat lamps. The following nutrients were included in the basal diet on a dry matter basis: 89.96% dry matter, 5.93% ash, 23.85% crude protein, 7.13% crude fat, 3.05% crude fiber, and 3,154.31 kcal/kg metabolizable energy.

A completely randomized design (CRD) was implemented, involving 125-day-old broiler chicks allocated into five treatments, each with five replicates and five birds per replicate. Probiotic L. paracasei added via drinking water at 4 mL/L starting from Day 1 until Week 6. Monensin incorporated into feed at 1 g/kg starting from Day 1 until Day 26. Treatment with Eimeria tenella performed on Day 21, with 10,000 oocysts per bird. The key measurement points in week 5 Body weight gain (BWG), feed intake (FI), and sampling for oxidative status are recorded. In week 6 Final body weight, blood collection for SOD and MDA assays, and necropsy.

The administration of monensin was limited to days 1 through 26, while the probiotic was provided continuously until the end of the experiment (week 6). E. tenella inoculation was performed on day 21, so monensin was discontinued five days after inoculation.

The rationale behind administering monensin during this period is based on industry regulations and common practices, where ionophore administration like monensin is often carried out during the early growth phase to effectively prevent and control coccidiosis infections while the parasite is proliferating in the gastrointestinal tract. Discontinuing monensin after weeks 3 or 4 is frequently practiced to reduce residue risk in meat and to comply with updated regulations regarding the use of antibiotics and ionophores in feed.

Additionally, stopping monensin on day 26 aims to evaluate residual effects and potential interactions with the probiotic during active parasitic stages and ongoing immune responses. In commercial and clinical settings, monensin usage protocols vary depending on the farm and region; however, generally, administering during early growth phases and ceasing before the end of the growth cycle is a common strategy to reduce residues and align with local regulations.

The treatments included the following.

• P0 (–) = negative control (uninfected, no probiotic or monensin)
• P0 (+) = positive control (infected with Eimeria tenella 1 × 10⁴ oocysts, no probiotic or monensin).
• P1 = monensin 1 g/kg feed + E. tenella infection (1 × 10⁴ oocysts)
• P2 = L. paracasei 4 mL/L drinking water + E. tenella infection (1 × 10⁴ oocysts)
• P3 = L. paracasei 4 mL/L drinking water + monensin 1 g/kg feed + E. tenella infection (1 × 104 oocysts)

Probiotic supplementation (L. paracasei) was administered through drinking water from day 1 to week 6, whereas monensin was mixed into the feed from days 1 to 26, following the manufacturer’s recommendation. Oral inoculation with Eimeria tenella was performed on day 21.

Data collection followed the guidelines set forth by the Association of Official Analytical Chemists (AOAC). Weekly records of growth performance parameters were kept, including body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR), and feed efficiency (FE). By dividing the total feed intake (g) by the BWG (g), the FCR was calculated; lower values denoted higher feed efficiency. BWG divided by the total feed intake was used to calculate FE, which was then expressed as a percentage. Using conventional humane methods, blood samples were taken from all broilers on day 42 before they were killed by exsanguination through the jugular, brachial (cutaneous ulnar), and ischiadic (sciatic) veins.

A completely randomized design was used to assess all of the data that was gathered. Prior to the analysis of variance (ANOVA), the normality and homogeneity of variance were confirmed. We employed SPSS Statistics v27 (IBM Corp., Armonk, NY, USA) to execute a one-way analysis of variance (ANOVA). Duncan’s multiple range test was employed for post-hoc comparisons where there were big disparities. Statistical significance was established at a value of P < 0.05.

The feed intake data for broilers is summarized in Table 1. Among the interventions, no significant differences (P > 0.05) were observed at week 4. Significant variations were observed at weeks 5 and 6 (P < 0.05). Broilers that were treated with Lacticaseibacillus paracasei (P2) or the combined treatment of L. paracasei + monensin (P3) exhibited a higher feed intake than the infected untreated control (P0 +) during week 5. Feed intake values at week 5 varied from 154.82 ± 14.85 g (P0 +) to 168.17 ± 1.48 g (P2). Differences among regimens were not statistically significant at week 6.

Table 1: Feed intake (g/bird/day) of broiler chickens under different treatments

a,b,c Different superscript letters within a row indicate statistically significant differences between treatment groups (P < 0.05).

The feed conversion ratio (FCR), which is a critical metric for feed efficacy, is presented in Table 2. Early infection or probiotic supplementation did not appear to have an impact on feed utilization at week 4. At this time, no substantial differences were observed among the treatments. Effects were significant at weeks 5 and 6 (P < 0.05). During weeks 5 and 6, the positive control group (P0 +) demonstrated the maximum FCR values (1.65 ± 0.08 and 1.78 ± 0.76, respectively), which suggests that the infection with E. tenella resulted in a decrease in efficiency. The lowest FCR was observed in P3 (1.38 ± 0.07) at week 6, followed by P2 (1.44 ± 0.12). Conversely, L. paracasei (P2) and the combination of L. paracasei and monensin (P3) enhanced feed efficiency. These values were substantially lower than those of the infected control, which serves as evidence of the advantageous effects of probiotic supplementation on nutrient utilization during coccidial challenges.

Table 3 presents the feed efficiency (FE) results. Statistically significant differences (P < 0.05) were observed between the groups at weeks 5 and 6. At week 6, the combined treatment group P3 had the highest FE (72.27 ± 4.12%), followed by P2 (69.51 ± 5.94%). Infected control group P0 + had the lowest efficiency (57.72 ± 3.29%). These data show that L. paracasei, when supplied alone or in combination with monensin, improved feed utilization efficiency compared to infected, untreated birds.

Table 2: Feed conversion ratio (FCR) of broiler chickens subjected to various treatments.

a,b,c Different superscript letters within a row indicate statistically significant differences between treatment groups (P < 0.05).

Table 3: Feed efficiency (%) of broiler chickens under different treatments.

a,b,c Different superscript letters within a row indicate statistically significant differences between treatment groups (P < 0.05).

The average daily body weight gain is presented in Table 4. Significant improvements (P < 0.05) were observed in the probiotic-supplemented groups compared to the infected controls. At week 5, BWG was highest in the L. paracasei group (P2; 88.14 ± 9.02 g/bird/day) and in the combined treatment (P3; 78.80 ± 8.28 g/bird/day). At week 6, P3 exhibited the greatest increase (115.14 ± 24.90 g bird⁻¹ day⁻¹), followed by P2 (93.83 ± 18.57 g bird⁻¹ day⁻¹), whereas P0 showed the lowest gain (42.65 ± 13.73 g bird⁻¹ day⁻¹).

Table 4: Average daily body weight gain (g/bird/day) of broilers under different treatments

a,b,c Different superscript letters within a row indicate statistically significant differences between treatment groups (P < 0.05).

The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) are summarized in Table 5. SOD activity, an indicator of antioxidant capacity, differed significantly among treatments (P < 0.05). The combined treatment group (P3) demonstrated the highest SOD activity (5.68 ± 0.02 U/mL at week 5) followed by the probiotic-only group (P2; 4.93 ± 0.05 U/mL), whereas the infected control (P0 -) showed the lowest (3.74 ± 0.01 U/mL).

Table 5: Superoxide dismutase (SOD) activity (U/mL) of broiler serum under different treatments.

a,b,c Different superscript letters within a row indicate statistically significant differences between treatment groups (P < 0.05).

MDA, a biomarker of lipid peroxidation and oxidative damage, also varied significantly among the groups (P < 0.05). The lowest MDA concentrations were observed in the combined treatment group, P3 (12.31 ± 0.21 µmol/L at week 5), In general, higher SOD and lower MDA levels indicate reduced oxidative stress and improved redox balance in birds receiving L. paracasei supplementation.

The purpose of this study was to explore the effects of the probiotic Lacticaseibacillus paracasei on growth performance, feed efficiency, and oxidative stress indicators in broilers that were challenged with Eimeria tenella. The probiotic was supplied either alone or in conjunction with the ionophore anticoccidial monensin. Probiotic supplementation was found to be beneficial for infected birds, as evidenced by the persistent observation of significant changes (P < 0.05) across a range of performance indicators and biochemical indices.

As shown in Table 1, broilers supplemented with L. paracasei (P2) or a combination of L. paracasei and monensin (P3) demonstrated significantly higher feed intake than the infected, untreated control group (P0+) during week 5 (P < 0.05). The improvement in feed intake might be attributed to the ability of probiotics to alleviate intestinal inflammation, enhance mucosal integrity, and promote appetite recovery after E. tenella infection. Previous studies have reported that E. tenella infection damages the intestinal epithelium and induces inflammation, thereby suppressing feed intake (Campos et al., 2022). Furthermore, local research by Yulianto et al. (2024) and Giannenas et al. (2014) reported that supplementation with probiotics counteracts these effects by restoring gut functionality and microbial balance. Thus, L. paracasei likely contributes to improved nutrient absorption and gastrointestinal comfort, enabling birds to maintain a higher feed intake even under parasitic stress.

The results of the feed conversion ratio and feed efficiency (Tables 2 and 3) indicated that the supplementation of L. paracasei, especially in conjunction with monensin (P3), significantly (P < 0.05) enhanced feed utilization efficiency relative to the infected control group. The lowest feed conversion ratio (FCR) of 1.38 ± 0.07 and the highest feed efficiency (FE) of 72.27 ± 4.12% were recorded in P3 during week 6, indicating potential additive effects of the probiotic and anticoccidial treatments. The enhancement of feed conversion ratio (FCR) is linked to improved nutrient digestibility and absorption, resulting from probiotic-induced changes in intestinal microbiota. This includes an increase in beneficial bacterial populations, such as Lactobacillus and Bacteroides, alongside a reduction in pathogenic taxa, including Clostridium, Escherichia–Shigella, and Salmonella (Chen et al., 2024; Guo et al., 2025).

These results are in accordance with reports that probiotics can enhance growth and FCR in poultry that have been infected with Eimeria spp. by preserving gut integrity and immune balance (Kang et al., 2023; Lima et al., 2020). The addition of monensin enhances this effect by reducing oocyst shedding and inhibiting parasite replication through the disruption of ion gradients (Qi et al., 2020). Study by Lokapirnasari et al. (2025) also demonstrated that L. paracasei improves economic efficiency and performance in pathogen-challenged broilers. Hence, similar to the findings of (El-Shall et al., 2024; Bozkurt et al., 2014), the combined treatment (P3) indicates a synergistic interaction between host-directed (probiotic) and parasite-directed (ionophore) mechanisms, resulting in superior feed utilization efficiency.

Groups P2 and P3 showed a significant (P < 0.05) improvement in average daily body weight gain compared to P0+ (Table 4). The maximum BWG (115.14 ± 24.90 g/bird/day) was seen in birds who received L. paracasei and monensin (P3) combined, indicating potential synergistic interaction in immune regulation and nutritional absorption. Probiotic supplementation improves growth by increasing the height of villus, increasing the activity of digesting enzymes, and promoting the production of antioxidant enzymes (Awais et al., 2019; Kang et al., 2023). Monensin is a supplement that works in conjunction with this by inhibiting the proliferation of E. tenella, minimizing tissue damage, and enhancing the retention of nutrients (Qi et al., 2020). Similarly, Yulianto et al. (2021) found that probiotic supplementation enhanced the production of immunological markers (ChIFN-γ and ChIL-10) in broilers infected with E. tenella, indicating an immunomodulatory mechanism behind the growth augmentation. These findings collectively demonstrate that L. paracasei, either alone or in combination, encourages recuperation and compensatory growth after a coccidial infection.

The L. paracasei supplemented groups (P2 and P3) had significantly (P < 0.05) lower MDA concentration and significantly (P < 0.05) higher SOD activity compared to the infected control, as shown in Tables 5 and 6. While lower MDA levels signify less oxidative stress and lipid peroxidation, higher SOD levels show improved antioxidant defense. The maximum SOD activity was obtained with combined treatment (P3), suggesting that ionophore and probiotic co-supplementation successfully reduced oxidative damage.

Table 6: Malondialdehyde (MDA) concentration (µmol/L) in the serum of broiler chickens under different treatments.

a,b,c Different superscript letters within a row indicate statistically significant differences between treatment groups (P < 0.05).

Biologically, the significant increase in SOD activity could be due to a stronger antioxidant response in the later stages of the experiment, either in response to oxidative stress caused by infection or to an improved immune system following treatment with probiotics and monensin. During this period, the chickens’ bodies may experience increased production of antioxidant enzymes as a defense mechanism to address oxidative damage that may have occurred due to Eimeria tenella infection, as well as active tissue repair processes.

In terms of experimental factors, these changes could also be due to variability in sampling, differences in treatment timing that affect the induction of defense mechanisms, or limitations in the measurement method. For example, if measurements were taken when the chickens were entering the recovery phase or experiencing increased immune activity, this could be reflected as a surge in SOD activity. The significant increase may also indicate that the antioxidant defense system is peaking, which is consistent with the decrease in MDA indicating improved oxidative status.

Given that E. tenella infection induces oxidative stress and disrupts redox balance homeostasis (Attia et al., 2023), the improved antioxidant profile of P2 and P3 underscores the probiotic’s functional role in maintaining cellular protection and metabolic balance during infection. Probiotic supplementation generally increases SOD activity in broiler chickens. For instance, a study found that broilers administered with a probiotic (Saccharomyces cerevisiae) had significantly higher SOD activity compared to the control group (Ogbuagu et al., 2018). The increased SOD activity in broilers given a combination of fisetin and probiotics. the addition of a probiotic mixture (Bacillus licheniformis, B. subtilis, and Clostridium butyricum) in drinking water resulted in an increasing trend in SOD levels (Yao et al., 2024).

Overall, significant (P < 0.05) improvements in feed intake, FCR/FE, BWG, and antioxidant capacity support the use of L. paracasei, especially in combination with monensin, as part of a sustainable coccidiosis control program in broilers. The additive effect observed in this study aligns with the trend of reducing reliance on anticoccidials alone and incorporating gut health-enhancing agents.

The improvements in performance, elevated SOD activity, and reduced MDA levels indicate enhanced oxidative status and metabolic efficiency in the treated chickens. The underlying mechanisms such as modulation of gut microbiota, intestinal integrity, and cytokine responses are supported by previous research; however, since these parameters were not directly measured in this study, these explanations remain as hypotheses. Further research involving direct measurement of microbiota composition, gut morphology, and cytokine levels is necessary to confirm these mechanistic pathways.

In conclusion, broilers challenged with Eimeria tenella showed significantly (P < 0.05) enhanced growth performance, feed efficiency, and antioxidant capacity when supplemented with Lacticaseibacillus paracasei, either alone or in combination with monensin. The best results were consistently obtained by the combined therapy (P3: L. paracasei + monensin), which included the lowest feed conversion ratio, the greatest body weight gain, and an improved antioxidant response, which was indicated by higher SOD and lower MDA levels. According to these results, the anticoccidial action of monensin in reducing parasite activity and the probiotic’s capacity to regulate gut microbiota and preserve intestinal integrity work in Interactive effect. In general, combining L. paracasei with traditional anticoccidials provides a interactive effect to enhance feed utilization, growth performance, and oxidative stability, bolstering its potential as a long-term substitute for efficient coccidiosis control and decreased antibiotic usage in commercial poultry production.

Although mechanisms such as increased beneficial gut microbiota and modified immune responses, including increased cytokines such as ChIFN-γ and ChIL-10, have been reported in previous studies as factors influencing chicken health and performance, these parameters were not directly measured in this study. Therefore, explanations regarding these mechanisms should be considered as possibilities and hypotheses based on previous research, rather than as conclusions based on the data obtained.

The authors would like to express their sincere gratitude to the Directorate of Research and Community Service (DPPM), Ministry of Higher Education, Science and Technology, Directorate General of Research and Development, for supporting this study. The authors also extend their appreciation to the Rector of Universitas Airlangga, the Head of the Institute for Research and Community Service (LPPM), and the Faculty of Veterinary Medicine, Universitas Airlangga, for providing the facilities and assistance that made this research possible.

This study is the first to demonstrate the synergistic effects of drinking water–delivered Lacticaseibacillus paracasei combined with monensin on growth performance, feed efficiency, and oxidative status in Eimeria tenella–challenged broilers. By integrating production parameters with systemic antioxidant biomarkers (SOD and MDA), this work provides novel evidence that a probiotic–ionophore strategy simultaneously enhances feed utilization and redox homeostasis, offering a practical approach to reduce reliance on anticoccidials while sustaining broiler productivity.

Conceptualization: LWP, ML. Data curation, Funding acquisition, Supervision and Project administration: LWP. Formal analysis: A-AMAA, SA, GR. Investigation: LM, AWH, BD, PAWW. Methodology: LWP, YAB. Resources: LM, ML. Validation: SA, YAB. Writing original draft: LWP, YAB, RZNA. Writing review and editing: LWP, ML, GR, SHI.

This research was funded by the Directorate of Research and Community Service (DPPM), Ministry of Higher Education, Science and Technology, Directorate General of Research and Development, Indonesia, through the Research Grant for Fiscal Year 2025 (Decree No. 0419/C3/DT.05.00/2025; Master Contract No. 059/C3/DT.05.00/PL/2025; Agreement No. 2379/B/UN3.LPPM/PT.01.03/2025)

All experimental procedures were approved by the Animal Ethics Commission of the Faculty of Veterinary Medicine, Universitas Airlangga (approval number: 1. KEH.71.05.2025).

No generative AI or AI-assisted technologies were used in the design of the study, data collection, data analysis, or interpretation of the results. Only standard statistical software and conventional writing tools were used during manuscript preparation. All text, tables, and conclusions were produced by the authors without assistance from generative AI systems.

The datasets used and/or analysed during the current study are standard.

The authors have declared no conflict of interest.