All experimental procedures involving broiler chickens were reviewed and approved by the Animal Ethics Commission, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, under Ethical Clearance No. 1.KEH.017.01.2024. The study was conducted in accordance with the institutional guidelines and accepted principles for the ethical care, housing, handling, and use of animals in research. The experimental protocol involved 80 one-day-old male broiler chickens allocated to four treatment groups, including an experimental challenge with APEC, administration of indigenous Lacticaseibacillus paracasei, and comparison with zinc bacitracin. Throughout the experimental period, appropriate husbandry and biosecurity practices were maintained, and feed and drinking water were provided ad libitum. Measures were taken to minimize animal stress, pain, discomfort, and unnecessary suffering during housing, APEC challenge, treatment administration, handling, blood collection, and other experimental procedures. Birds were monitored for clinical manifestations following the APEC challenge, including changes in appetite, activity, and other signs associated with infection. Blood sampling was performed from the brachial vein at 35 days of age using appropriate handling and sample-collection procedures. All animal procedures were performed only to the extent necessary to achieve the scientific objectives of the study, with due consideration given to animal welfare and the principles of responsible use of animals in biomedical and veterinary research.

The study was conducted between June and October 2024 at the Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia. The experimental challenge used APEC strain O-78 [46] at 1.5 × 10⁸ colony-forming units [CFU]/mL and indigenous L. paracasei 153 Vi at 1.2 × 10⁹ CFU/mL. The study utilized commercial broiler feed (CP-511; PT Charoen Pokphand Indonesia Tbk., Jakarta, Indonesia), zinc bacitracin (Rojo Koyo Group, Kediri, Indonesia), male broiler chickens, battery cages (35 × 20 × 45 cm), and digital weighing scales for feed and body weight measurements.

A total of 80 one-day-old male broiler chicks were used in this study. The experiment followed a completely randomized design with four treatment groups, five replicates per treatment, and four birds per replicate. The experimental equipment included 1- and 15-mL syringes, modified 1.5-L drinking bottles, masks, gloves, labels, hand sprayers, feed and water containers, cage sanitation equipment, garbage bags, a Gasolec™ S8 heating system (Gasolec B.V., Abbekerk, the Netherlands), postal cages (250 × 200 × 200 cm) for brooding, battery cages (35 × 20 × 45 cm), and digital weighing scales, which were also used during the experiment.

The treatments were designed to evaluate the efficacy of indigenous L. paracasei administered through drinking water compared with AGP supplementation through feed under APEC challenge. The treatment groups were as follows:

1. T0 = Negative control (uninfected and untreated)

2. T1 = Positive control (APEC-infected without treatment)

3. T2 = AGP (zinc bacitracin, 1 g/kg feed, APEC-infected)

4. T3 = Probiotic (L. paracasei, 5 mL/L drinking water, APEC-infected)

Broiler chicks were obtained from a certified commercial hatchery (MB-202; PT Japfa Comfeed Indonesia Tbk., Sidoarjo, Indonesia). Birds were reared in postal cages from 1 to 14 days of age and had received routine vaccinations from the hatchery before arrival. At 14 days of age, the birds were transferred to battery cages and maintained under identical management conditions until the experiment was completed.

Broilers were fed twice daily at 08:00 and 15:00 h. The commercial broiler diet (CP-511; PT Charoen Pokphand Indonesia Tbk., Jakarta, Indonesia) contained 85.51% dry matter, 6.90% ash, 20.69% crude protein, 6.87% crude fat, 1.85% crude fiber, 49.19% nitrogen-free extract, and 2,998.04 kcal/kg metabolizable energy. Feed and drinking water were provided ad libitum throughout the experimental period. Probiotic supplementation was administered through the drinking water of birds assigned to T3.

At 21 days of age, broilers in the infected groups were orally challenged with 0.5 mL of the APEC suspension per bird. Successful infection was confirmed clinically by the appearance of characteristic signs, including pallor of the comb and face, sneezing, reduced appetite, weakness, and diarrhea in the infected birds.

The probiotic culture was propagated in de Man, Rogosa, and Sharpe (MRS) broth (Merck KGaA, Darmstadt, Germany; Cat. No. 1.10661.0500) at 37°C for 24 h. Bacterial cells were harvested and adjusted to a final concentration of 1.2 × 10⁹ CFU/mL using the plate-counting method. The probiotic suspension was administered through drinking water at a dose of 5 mL/L [21]. Based on average daily water consumption, probiotic intake was estimated at approximately 6 × 10⁶-8 × 10⁶ CFU/bird/day. Zinc bacitracin (Rojo Koyo Group, Kediri, Indonesia) was incorporated into the feed at 1 g/kg before feeding.

Feed intake and nutrient intake: Feed intake was recorded daily by subtracting the remaining feed from the amount offered. Nutrient intake, including dry matter, organic matter, ash, crude protein, crude fat, and crude fiber, was calculated using the corresponding nutrient composition of the commercial diet. Production parameters, including final body weight, carcass weight, feed intake, and carcass percentage, were determined at 35 days of age [22].

SGPT and SGOT analysis: Blood samples were collected from the brachial vein at 35 days of age using non-anticoagulant blood collection tubes. After clot formation, samples were centrifuged at 1,509 × g for 10-15 min to obtain serum. Serum SGPT and SGOT activities were determined using an automated biochemical analyzer (Reflotron® Plus; Roche Diagnostics GmbH, Mannheim, Germany) with Reflotron GPT test strips (Cat. No. 10745138) and Reflotron GOT test strips (Cat. No. 10745120), according to the method described by Tugiyanti et al. [23].

IOFC: Economic efficiency was evaluated using IOFC, calculated as the difference between revenue generated from broiler sales and the total feed cost incurred during the production period.

Statistical analyses were performed using IBM SPSS Statistics version 27 (IBM Corp., Armonk, NY, USA). Data normality and homogeneity of variance were assessed using the Kolmogorov-Smirnov and Levene's tests, respectively. Treatment effects were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). Differences were considered statistically significant at p < 0.05.

SGPT and SGOT activities were expressed as mean ± standard deviation (SD) and analyzed using one-way ANOVA followed by DMRT. IOFC was similarly compared among treatment groups using one-way ANOVA. Pearson correlation analysis was performed to evaluate associations among probiotic supplementation, feed intake, carcass characteristics, and liver enzyme activities. Effect sizes were expressed as partial eta-squared (η²). Graphical presentations were generated using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA).

The effects of L. paracasei supplementation on feed and nutrient intake are presented in Table 1.

Broilers in the T2 (AGP) group exhibited the highest feed intake (88.50 g/head/day) and dry matter intake (75.67 g/head/day), both of which were significantly higher than those in the T0, T1, and T3 groups (p < 0.05). Similar trends were observed for ash (6.11 g/head/day), organic matter (82.39 g/head/day), crude protein (18.31 g/head/day), crude fat (6.08 g/head/day), and crude fiber (1.64 g/head/day), with T2 consistently recording the greatest nutrient intake (p < 0.05).

Nitrogen-free extract intake was also highest in T2, suggesting greater energy substrate consumption. Although ME intake differed significantly among treatments (p < 0.05), the variation among groups was relatively small. Carbohydrate intake followed a comparable pattern, with broilers receiving AGP supplementation consuming the greatest amount.

Overall, AGP supplementation promoted the highest feed and nutrient intake, whereas L. paracasei supplementation (T3) maintained nutrient intake at levels comparable to or greater than those of the control groups while achieving superior production performance, indicating more efficient nutrient utilization.

The effects of L. paracasei supplementation on growth performance and carcass characteristics are presented in Table 2.

Broilers supplemented with L. paracasei (T3) achieved the greatest final body weight (1,993.00 g), significantly higher than all other treatment groups (p < 0.05). Birds in the T2 (AGP) group exhibited an intermediate final body weight (1,723.00 g), which was significantly greater than that of the infected untreated group (T1) but remained significantly lower than that of T3.

A similar trend was observed for carcass characteristics. The T3 group produced the highest carcass weight (1,337.80 g) and carcass percentage (68.75%), both of which were significantly greater than those recorded in the T0, T1, and T2 groups (p < 0.05). These findings demonstrate that L. paracasei supplementation improved both growth performance and carcass yield more effectively than AGP supplementation.

Direct comparison between the probiotic and AGP treatments further demonstrated the superiority of L. paracasei. Final body weight was significantly greater in T3 than in T2 (p = 0.003), while carcass percentage was also significantly higher in T3 (p = 0.012), indicating improved production performance in probiotic-supplemented broilers.

The effects of the treatments on liver function are presented in Table 3. Significant differences in SGPT and SGOT activities were observed among the treatment groups (p < 0.05). The T0 (negative control) group exhibited the lowest SGPT activity (66.91 U/L), consistent with normal liver function. In contrast, broilers in the T1 group (APEC-infected without treatment) showed the highest SGPT activity (159.12 U/L), indicating marked hepatocellular injury. Supplementation with either AGP (T2) or L. paracasei (T3) markedly reduced SGPT activity to 122.56 U/L and 105.07 U/L, respectively, demonstrating attenuation of hepatic damage. The reduction was more pronounced in the probiotic-treated birds than in the AGP-treated birds.

A comparable pattern was observed for SGOT activity. Broilers supplemented with L. paracasei exhibited the lowest SGOT activity (59.05 U/L), whereas the untreated infected group recorded the highest value (143.26 U/L), confirming severe hepatic impairment following APEC infection. The T0 and T2 groups exhibited intermediate SGOT activities of 78.31 U/L and 128.91 U/L, respectively.

Interestingly, SGOT activity in T3 was lower than that observed in the uninfected control group (T0). This finding suggests that L. paracasei may exert a pronounced hepatoprotective effect beyond merely preventing APEC-induced liver injury, although the contribution of normal biological variation among individual birds cannot be excluded.

Collectively, these findings indicate that L. paracasei supplementation effectively alleviated hepatic stress associated with APEC infection and provided greater hepatoprotection than AGP supplementation.

The effects of the treatments on economic performance, expressed as IOFC, are presented in Table 4 and Figure 1.

At the experimental scale (10 birds per treatment), the T2 (AGP) group produced the lowest IOFC (Indonesian rupiah [IDR] 3,245.60), indicating the poorest economic efficiency. The untreated infected group (T1) generated a moderate IOFC (IDR 6,960.00), whereas the uninfected control group (T0) achieved a substantially higher IOFC (IDR 11,143.80). In contrast, broilers supplemented with L. paracasei (T3) produced the highest IOFC, which was significantly greater than those of both T2 (p = 0.001) and T1 (p = 0.004), demonstrating the clear economic benefit of probiotic supplementation (Figure 1).

When extrapolated to a commercial production scale of 1,000 birds, the same trend was maintained. The T2 group again generated the lowest IOFC (IDR 324,545.40), followed by T1 (IDR 696,038.00). The T0 group yielded an IOFC of IDR 1,114,418.00, while T3 achieved the highest profitability, with an IOFC of IDR 1,681,235.00.

The absence of mortality throughout the experimental period indicates that differences in IOFC were primarily attributable to improvements in production performance and feed utilization rather than survival rate. Overall, L. paracasei supplementation provided the greatest economic return, outperforming both AGP supplementation and the untreated groups, thereby supporting its potential as a sustainable alternative to AGPs in commercial broiler production.

The present study evaluated the effects of indigenous L. paracasei isolated from dadih on nutrient intake, growth performance, carcass characteristics, liver function, and economic efficiency in broilers challenged with APEC. The findings clearly demonstrate that L. paracasei supplementation provides substantial biological and economic benefits, producing superior outcomes to AGP supplementation under the experimental conditions. These findings support the potential of this indigenous probiotic as a safe, sustainable, and effective alternative to AGPs in commercial broiler production.

Broilers receiving AGP supplementation (T2) exhibited the highest feed and nutrient intake across most measured parameters, including dry matter, crude protein, crude fat, and carbohydrate intake. These findings are consistent with the established role of AGPs in modulating intestinal microbiota, suppressing subclinical bacterial infections, and improving digestive efficiency, thereby stimulating feed consumption [24]. In contrast, broilers supplemented with L. paracasei (T3) maintained a more balanced feed intake while achieving superior production performance, indicating greater nutrient utilization efficiency rather than increased feed consumption [12].

The ability of L. paracasei to stabilize intestinal microbial communities likely contributed to maintaining nutrient digestibility during APEC challenge [25]. LAB produce organic acids, bacteriocins, and digestive enzymes that facilitate feed degradation, inhibit pathogenic colonization, and preserve intestinal integrity [26]. Consequently, the greater feed intake observed in the AGP group, despite its lower growth performance and economic return, may reflect reduced feed utilization efficiency or increased maintenance requirements under pathogen challenge. Although nutrient intake in T3 did not exceed that of T2, the improved production performance achieved with moderate feed consumption indicates enhanced metabolic efficiency [27]. Such improved feed utilization is particularly advantageous in commercial poultry production, where feed represents the largest component of production costs.

Broilers supplemented with L. paracasei (T3) exhibited the greatest improvements in final body weight, carcass weight, and carcass percentage. These findings demonstrate the beneficial effects of L. paracasei on intestinal function, nutrient utilization, and overall metabolism [28]. Previous studies have shown that LAB improve intestinal morphology by increasing villus height, modulating immune responses, and reducing intestinal inflammation, thereby enhancing nutrient absorption and promoting growth [29].

Conversely, broilers in the untreated APEC-infected group (T1) exhibited marked growth suppression, consistent with intestinal inflammation, impaired nutrient absorption, and reduced metabolic efficiency associated with APEC infection [30]. Although AGP supplementation partially restored growth performance, the improvements remained inferior to those achieved with L. paracasei [31]. These observations support accumulating evidence that probiotics can match or even surpass AGPs in promoting broiler growth, particularly under infectious conditions [32].

The superior carcass yield observed in T3 further suggests enhanced protein deposition and more efficient nutrient partitioning [33]. Improved intestinal health reduces the metabolic cost of immune activation and inflammatory responses, allowing a greater proportion of nutrients to be directed toward muscle accretion [34]. Because carcass yield directly influences meat production efficiency and commercial profitability, these improvements provide substantial practical value for poultry producers [35].

APEC infection induces systemic inflammation and hepatocellular injury, resulting in elevated SGPT and SGOT activities [5]. This pathological response was clearly demonstrated in the untreated infected group (T1), which exhibited the highest enzyme activities. In contrast, broilers supplemented with L. paracasei (T3) showed markedly reduced SGPT activity and the lowest SGOT activity, indicating substantial hepatoprotection during APEC infection [36].

Several mechanisms may contribute to this protective effect. Probiotics suppress pathogen proliferation, enhance antioxidant defenses, and regulate systemic immune responses [37]. LAB produce bioactive metabolites, including short-chain fatty acids (SCFAs) and antioxidant compounds, which reduce oxidative stress and protect hepatic tissues from inflammatory damage [38]. Furthermore, L. paracasei may improve intestinal barrier integrity, reduce endotoxin translocation, and increase SCFA production, thereby attenuating hepatic inflammation and preserving liver function. Probiotics have also been reported to suppress pro-inflammatory cytokine production, thereby reducing hepatic metabolic burden and limiting hepatocellular injury [39]. Although AGP supplementation also reduced liver enzyme activities, its hepatoprotective effects remained less pronounced than those observed with L. paracasei [40].

Collectively, these findings indicate that L. paracasei exerts broader physiological benefits than conventional AGPs by combining antimicrobial activity with immunomodulatory, antioxidant, and metabolic regulatory functions [41].

Economic evaluation demonstrated that L. paracasei supplementation produced the highest IOFC at both the experimental and commercial production scales, whereas AGP supplementation generated the lowest economic return. The superior profitability associated with probiotic supplementation resulted from the combined effects of efficient feed utilization, improved growth performance, enhanced carcass yield, and reduced physiological stress [42].

In contrast, despite exhibiting the highest feed intake, broilers receiving AGP supplementation achieved only moderate body weight gain, resulting in increased feed costs relative to production output [43]. Consequently, AGP supplementation yielded lower IOFC values, illustrating the declining economic benefit of AGPs under modern poultry production systems characterized by increasing antimicrobial restrictions and growing consumer demand for antibiotic-free poultry products [44].

The consistently greater economic performance observed in the probiotic group highlights the commercial value of indigenous L. paracasei as a sustainable feed additive. These findings are particularly relevant to tropical poultry production systems, where locally adapted LAB strains may exhibit enhanced survival, colonization, and functional efficacy under regional environmental conditions [45].

The present findings demonstrate that indigenous L. paracasei isolated from dadih provides comparable or superior benefits to AGPs in growth performance, liver health, carcass yield, and economic efficiency. Using indigenous probiotic strains also supports the development of region-specific nutritional strategies better adapted to local environmental conditions and microbial ecosystems [46, 47].

Importantly, the present study extends our previous work reported by Lokapirnasari et al. [48]. While the earlier study demonstrated improvements in antioxidant status, lipid metabolism, and hematological parameters using the same L. paracasei isolate, the present investigation provides new evidence regarding nutrient utilization, production performance, hepatoprotection, carcass characteristics, and economic efficiency. Together, these complementary findings provide a more comprehensive understanding of the biological and practical value of this indigenous probiotic in broiler production.

Given the increasing global restrictions on AGP use, LAB-based probiotics represent an effective strategy for maintaining poultry health, improving production efficiency, and enhancing food safety [49]. Furthermore, utilizing indigenous probiotic strains supports sustainable livestock production by reducing dependence on imported feed additives while promoting the conservation and application of local microbial biodiversity [50].