A high stocking density in broiler chicken rearing systems triggers heat and humidity accumulation, impairs air circulation and increases the risk of heat stress (Apalowo et al., 2024). This suppresses appetite and drinking activity and subsequently leads to slowed growth rates and decreased feed conversion efficiency (Sapsuha et al., 2023). Crowded conditions also increase competition for feed and water, creating unequal access and reducing growth homogeneity. This is accompanied by elevated pathogen exposure and accelerated disease transmission due to close proximity between animals. This combination of environmental stress, resource competition and microbial load directly causes gut health issues, including microbiota imbalance and decreased mucosal integrity, ultimately reducing production performance and physiological resilience of broiler chickens during critical rearing phases (Selvam et al., 2017; Bach et al., 2019;  Citra et al., 2025).

Synbiotics, a combination of prebiotics and probiotics, work synergistically to promote digestive health and improve livestock performance (Melara et al., 2022). Prebiotic components such as soluble fiber and polyphenols provide fermentable substrates for lactic acid bacteria (LAB). These bacteria produce organic acids that lower the lumen’s pH, making the intestinal environment less conducive to enteric pathogens (Abdul et al., 2023). Concurrently, probiotics enhance beneficial microbial colonization, yield bacteriocins and mild antimicrobial metabolites and improve microbial community balance (Loo et al., 2025). These combined effects enhance mucosal integrity through improved villus structure and strengthened tight junctions, reduce pathogen and endotoxin transport and eventually support digestibility, nutrient absorption and physiological resilience under challenging culture conditions (Chowdhury et al., 2025).

Purslane (Portulaca oleracea L.) has the potential to be used in broiler chickens because it contains various beneficial bioactive compounds, such as flavonoids, polyphenols, vitamins and other antioxidant compounds (Kumar et al., 2022). These compounds act as natural antioxidants that can ward off free radicals and reduce oxidative stress in the body, thereby promoting cell and tissue health in broiler chickens. Furthermore, the antioxidant and anti-inflammatory properties of purslane may preserve the integrity of the intestinal mucosa and bolster the immune system, which can ultimately improve the overall health and growth performance of broilers. With the right formulation and dosage in rations, it can be utilized as a natural feed supplement to support the healthier and more efficient production of broilers (Wang et al., 2021).

A review of the literature revealed no reports specifically looking at the administration of a synbiotic combining purslane extract and L. plantarum to broiler chickens raised at a high density. The majority of related studies still concentrate on the use of L. plantarum-based probiotics or prebiotics or synbiotics from other sources, as well as tests of purslane as a feed ingredient without probiotic combinations and without the context of cage density. Therefore, this study offers a clear novelty by examining the effects of a synbiotic consisting of purslane extract and L. plantarum to overcome the environmental stress and the performance reduction that frequently happens in high-density environments.

Synbiotic preparation
 
The synbiotic was prepared based on the method of Sapsuha et al., (2023). Briefly, purslane was air-dried and ground into flour. Purslane extract was formulated by extracting 1 kg of purslane flour using a maceration technique by soaking it in 4 L of 96% ethanol solution for 3 x 24 hours. During the maceration process, stirring was carried out twice, in the morning and afternoon. The filtrate was then filtered and evaporated using a rotary evaporator to produce purslane extract. Synbiotic preparation began by mixing 5 ml of purslane extract/100 ml of distilled water with 10 ml of L. plantarum (bacterial concentration 1 x 10⁹ cfu/ml). The resulting synbiotic was stored in the refrigerator until use.
 
In vivo experiment
 
This study was approved by the Animal Research Ethics Committee of the Faculty of Agriculture, Khairun University (No. 08/KEPH/PH/2024). A total of 410 unsexed Lohmann broiler chickens were housed together until day 14 on a commercial starter feed. On day 14, the broilers were weighed individually (average body weight 408.39±0.69 g) and selected for use in this study. The experiment was arranged in a completely randomized design with five treatment groups, with each treatment replicated five times: CONT- (negative control with a normal stocking density of 10 birds/m²), CONT+ (positive control with a high stocking density of 18 birds/m²), SYN0.5, SYN1.0 and SYN1.5 with a high stocking density of 18 birds/m². The feed was provided in mash form and formulated (Table 1) as a starter feed (days 14-21) and a finisher feed (days 22-35). The synbiotic was added since day 14 at 0.5, 1.0 and 1.5 ml/kg for SYN0.5, SYN1.0 and SYN1.5 treatment groups, respectively, while no synbiotic was administered to CONT- and CONT+ treatment groups. Feed and drinking water were provided ad libitum until day 35. All chickens were vaccinated with commercial Newcastle (ND) vaccine on day 4 via eye drops and on day 18 via drinking water and on day 12 with Gomboro vaccine via drinking water. Weight gain, feed consumption and feed conversion ratio (FCR) were recorded weekly.



On day 35, one rooster with a body weight close to the average body weight of the cage was taken and blood was collected from its wing vein and then placed in a tube (without coagulant) to produce blood serum after clotting at room temperature for approximately 2 hours. Roosters were selected for blood sampling to avoid physiological errors due to sex variation. For practical reasons, the previously blood-sampled chickens were slaughtered and their internal organs were removed. To measure the intestinal bacterial population, digesta was collected from the ileum and cecum and placed in sterile sample containers. Digesta was also collected from the duodenum, jejunum, ileum and cecum for pH measurement (using an electronic pH meter). For morphology assessment, intestinal sections approximately 2 cm from each section of the small intestine were taken and put in sample tubes containing 10% neutral buffered formalin. For histological analysis, duodenal, jejunal and ileal sections (5 μm each) were stained with hematoxylin and zeosin. Measurement of villus height and crypt depth was performed using an optical microscope equipped with a camera.

Agar (Merck KGaA) and incubated at 38°C for 24 hours under aerobic conditions. Coliform bacteria growing on the agar medium appeared red and bacterial colonies were then determined. Total lactic acid bacteria (LAB) were calculated on de Man, Rogosa and Sharpe agar (MRS; Merck KGaA), after which the samples were incubated anaerobically at 38°C for 48 hours (Sapsuha et al., 2023).

This study was conducted using a completely randomized design. Data were analyzed using ANOVA (SPSS 25.0). Duncan’s Multiple Range Test was used to distinguish statistically significant means (p<0.05).

Broiler performance
 
Broiler performance data are presented in Table 2. SYN1.5 treatment group (P<0.05) had greater daily weight gain than CONT⁺, CONT^- and SYN0.5 treatment groups, but did not differ from SYN1.0 treatment group. For daily feed consumption, it was the highest in SYN1.5 group (P<0.05) and feed efficiency was lower in CONT⁺, SYN1.0 and SYN1.5 groups (p<0.05) than in CONT^- and SYN0.5 groups.



Improving metabolism through supplemental feed such as synbiotics can indirectly increase the weight and quality of broiler carcasses since they contain both beneficial bacteria and bioactive substances. Numerous studies have indicated that feed additives, including synbiotics, can boost the immune system, lower cholesterol levels and enhance metabolic efficiency of broiler chickens (Sliżewska et al., 2020; Du et al., 2023). Purslane is rich in bioactive substances such as flavonoids, alkaloids, tannins, saponins and terpenoids, which have potential antioxidant, antibacterial and anti-inflammatory properties (Kumar et al., 2022; Atasoy and Özcan, 2022). When combined with L. plantarum, it can produce a synergistic effect on improving the performance and health status of broilers raised at a high density.

Administering the synbiotic of purslane extract and L. plantarum to broilers raised at the high density could improve final body weight and feed conversion ratio compared to the control group. This is because it had efficacy in increasing the colonization of lactic acid bacteria and preserving the integrity of intestinal villi, hence maintaining the efficiency of nutrient absorption despite the environmental stress caused by the high stocking density. In addition, the competitive activity of L. plantarum in the digestive tract effectively limited the proliferation of pathogenic bacteria such as Escherichia coli and Clostridium, which tend to increase under environmental stress conditions. The synergistic mechanism between the prebiotic fiber in purslane extract and the metabolic activity of L. plantarum also facilitated the production of short-chain fatty acids, which directly improved the immune system health and supported the growth performance of broiler chickens under high-density stress (Wang et al., 2021; Obianwuna et al., 2023).
 
Intestinal morphology of broilers
 
The average intestinal morphology of broiler chickens raised at the high density is provided in Table 3. The results indicated that the administration of the synbiotic to them significantly (p<0.05) increased the length of villi in the duodenum, jejunum and ileum, as well as the ratio of villus length to crypt depth in the jejunum and ileum. However, it had no significant effect (p>0.05) on crypt depth in all segments of the small intestine or the ratio of villus length to crypt depth in the duodenum.



Increased villus height and absorptive surface area observed in the treatment groups indicated that this supplementation effectively facilitated the regeneration of intestinal epithelial cells which were under stress by the high stocking density. The production of secondary metabolites from purslane extract was shown to reduce local inflammatory responses, thus maintaining intestinal morphological integrity against health disorders often associated with environmental stress and competition for space. Consistent with these findings, improvements in intestinal histomorphology were positively correlated with greater final body weight and better feed utilization when compared to the untreated groups (Salah et al., 2019).

Increasing the height of the villi and the absorptive surface area of the intestine in broiler chickens had a central role in improving intestinal health. Taller villi and a larger absorptive surface allowed for more efficient absorption of nutrients such as protein, fat and minerals, optimally meeting broilers’ nutritional needs. This condition promoted the growth of beneficial bacteria in the digestive tract, inhibited the colonization of pathogenic bacteria and reduced the risk of digestive disorders. Furthermore, healthy intestine with well-structured villi strengthened the local immune system, making them more resistant to disease and environmental stress. Ultimately, improved morphology of intestinal villi had a beneficial effect on production performance, including greater body weight gain and more efficient feed conversion (Hofmann et al., 2020; Wickramasuriya et al., 2022).
 
Gut microbial population
 
The average pH and intestinal microbial counts of broiler chickens raised at the high density are shown in Table 4. The results demonstrated that administering the synbiotic to broiler chickens significantly (p<0.05) decreased the pH in the ileum and cecum, but had no significant effect (p>0.05) on the pH of the duodenum and jejunum.



The data in Table 4 indicates that the administration of the synbiotic to broiler chickens raised at the high density significantly (p<0.05) increased lactic acid bacteria and decreased coliform bacteria in the ileum and cecum.

The performance improvements observed in this study indicated that the synbiotic supplementation of purslane extract and L. plantarum could mitigate the negative impacts of the high density by optimizing the intestinal microenvironment. The increased population of beneficial bacteria contributed to the stability of the intestinal mucosal morphology, which in turn facilitated more optimal nutrient absorption despite the challenges of the high-density environment (Li et al., 2023). The synbiotic could increase the population of beneficial microbes in the digestive tract of broiler chickens. It provided a selective substrate for beneficial bacteria, thereby stimulating their growth and improving their ability to compete with pathogenic bacteria (Piatek et al., 2020. The dominance of these beneficial microbes helped suppress the colonization and activity of harmful microorganisms, so it allowed for a more stable and healthy intestinal environment. This condition contributed to enhanced nutrient digestibility and absorption, strengthened the local intestinal immune system and reduced the risk of digestive disorders. Therefore, appropriate synbiotic supplementation could improve the intestinal ecology and support the overall production performance of broiler chickens.

Administering the synbiotic of purslane extract and L. plantarum at up to 1.5 ml/kg of feed could improve the performance, morphology and gut microbiota of broiler chickens raised at the high density.

This research was supported by the RIIM LPDP Grant and BRIN, grant number 148/IV/KS/11/2023 and 793/UN44/L1/KS.01/2023. We also thanks to the LPPM Universitas Khairun.
 
Declaration of competing interest
 
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

We, the authors, hereby declare that there are no conflicts of interest, whether financial“or non-financial, that could have influenced the research, preparation, or publication of our“manuscript. All co-authors have reviewed and approved this declaration and confirm that they have no“competing interests related to the work presented in the manuscript. This declaration is made truthfully and in accordance with the ethical standards of scientific “publication.